8 ISSN 0250-8362 ISSN 1 0 2 olume 40, December 2018 olume 40, December V Bulletin Dengue

Dengue Bulletin Volume 40, December 2018 O H W by . the the the and The also to 2 in on 6 are changing 3 review Bulletin 8 workers aspects. letters - management, management, 0 experts 5 and Dengue meetings 2 in-house information information infection, infection, 0 research related clinical to

N for annual S fever reviews other S updated the I international

all book strategies, strategies, subjected by and information are publish notes, control haemorrhagic haemorrhagic national/international national/international disseminate disseminate Asia strains provide of to short -reviewed o is T

publication peer DENV attempted attempted for South-East South-East articles, are fever//dengue Bulletin for new subjects. proceedings the and review received Office circulating of dengue -related patterns, of experts accepts managers, about Regional disciplines. objective DF/DHF status also manuscripts

on The All WHO The current epidemiological epidemiological information Bulletin editor programme published. professional respective From the Editor’s Desk

There is about a 30-fold increase in dengue incidence over the past 50 years and it is now regarded as one of the most important arboviral infections in the world. There are many reasons for this increase, including faster spread of the through global travel, spread of the vectors to new geographical locations, rapid urbanization, global warming and . About 52% of the population in the WHO South-East Asia Region is estimated to be at risk for dengue with 10 out of the 11 Member States (with the exception of the Democratic People's Republic of Korea) being endemic. All four serotypes of the virus are circulating in the Region. The Region has seen larger outbreaks of dengue in the past two years, with experiencing the largest outbreak ever recorded in 2017. Many other countries, including , and Myanmar, have also seen focal outbreaks of increasing magnitude. All these resulted in researchers engaging in the clinical features, management, vector biology and control of dengue. In line with this priority, the Dengue Bulletin is published every year, encouraging researchers to explore different aspects of the disease and contribute to the knowledge gap and evidence base for combating the rapid spread of this deadly disease. The 40th volume of Dengue Bulletin is in your hands. It consists of papers on a new GIS surveillance tool, clinical management, vector behaviour, and the role of knowledge, attitudes and practices in dengue control. We now invite contributions for volume 41. The deadline for the receipt of the manuscripts is 31 October 2019. Contributors are requested to kindly follow the instructions given at the end of the Bulletin during the preparation of their manuscripts. Contributions should either be accompanied by flash drives and sent to the Editor, Dengue Bulletin, WHO Regional Office for South-East Asia, Red Fort Capital Parsvnath Tower 1, Bhai Vir Singh Marg, Gole Market Sector 4, New 110 001 India, or by email as a file attachment to the Editor at [email protected]. Readers who want copies of the Dengue Bulletin may write to the same address or the WHO Country Representative in their country of residence. The pdf version will be available on the WHO Regional Office website.

Dr Ahmed Jamsheed Mohamed Regional Adviser Neglected Tropical Diseases Control and Editor, Dengue Bulletin World Health Organization Regional Office for South-East Asia New Delhi, India Dengue Bulletin V olume 40, December 2018 Dengue Bulletin, Volume 40, December 2018. ISSN 0250-8362 © World Health Organization 2019 Some rights reserved. This work is available under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 IGO licence (CC BY-NC-SA 3.0 IGO; https://creativecommons.org/licenses/by-nc-sa/3.0/igo). Under the terms of this licence, you may copy, redistribute and adapt the work for non- commercial purposes, provided the work is appropriately cited, as indicated below. In any use of this work, there should be no suggestion that WHO endorses any specific organization, products or services. The use of the WHO logo is not permitted. If you adapt the work, then you must license your work under the same or equivalent Creative Commons licence. If you create a translation of this work, you should add the following disclaimer along with the suggested citation: "This translation was not created by the World Health Organization (WHO). WHO is not responsible for the content or accuracy of this translation. The original English edition shall be the binding and authentic edition". Any mediation relating to disputes arising under the licence shall be conducted in accordance with the mediation rules of the World Intellectual Property Organization.. Suggested citation. Dengue Bulletin, Volume 40, December 2018. New Delhi: World Health Organization, Regional Office for South-East Asia; 2017. Licence: CC BY-NC-SA 3.0 IGO. Cataloguing-in-Publication (CIP) data. CIP data are available at http://apps.who.int/iris. Sales, rights and licensing. To purchase WHO publications, see http://apps.who.int/bookorders. To submit requests for commercial use and queries on rights and licensing, see http://www.who.int/about/licensing. Third-party materials. If you wish to reuse material from this work that is attributed to a third party, such as tables, figures or images, it is your responsibility to determine whether permission is needed for that reuse and to obtain permission from the copyright holder. The risk of claims resulting from infringement of any third-party-owned component in the work rests solely with the user. General disclaimers. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of WHO concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted and dashed lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers' products does not imply that they are endorsed or recommended by WHO in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by WHO to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall WHO be liable for damages arising from its use. Indexation: Dengue Bulletin is being indexed by BIOSIS and Elsevier's Bibliographic Databases including, EMBASE, Compendex, Geobase and Scopus Contents

Acknowledgements...... iii

1. Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala...... 1 Gayatri Lekshmy Kumar, Zinia T Nujum, Lalitha Kailas, Vijayakumar K

2. Utility of CDC DENV 1–4 real-time RT-PCR assay for the diagnosis of dengue ...... 15 Mohan K. Shukla, Pradip V. Barde, Neeru Singh

3. Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study...... 19 Heera Hassan, Reena John, Prithi Nair, MA Andrews

4. Scenario of dengue and in Pune district, Maharashtra, India during 2016: a retrospective study at an apex referral laboratory...... 33 Alagarasu K, Jadhav SM, Bachal RV, Bote M, Kakade MB, Ashwini M, Singh A, Parashar D

5. Circulation of dengue serotypes in four provinces of northern during 2013–2017...... 44 Punnarai Veeraseatakul, Jarurin Waneesorn, Kongphob Thilaogam, Yuddhakarn Yananto, Kotchakorn Intamul, Somkhid Thichak

6. Molecular identification of dengue virus and erythrovirus B19 in three towns of the State of Amazonas, during 2013–2018...... 53 Regina Maria Pinto de Figueiredo, Thiago Serrão Pinto, Kelry Mazurega de Oliveira Dinelly, Wellyngton do Nascimento Lopes, Luzia de Souza Granjeiro, Carlene Barroso Caripuna, Naylê de Oliveira Alves Mendes, Maria Itelvina Rodrigues de Souza, Anete Jane Cavalcante da Silva, Valcinei Silva Amorim, Victor Costa de Souza, Valdinete Alves do Nascimento, Felipe Gomes Naveca

7. Potential breeding sites of aegypti in Maldives...... 63 B. N. Nagpal, K. Vikram, S. K. Gupta, Sana Saleem, Nishan, Sushil Pant, Arvind Mathur, Ahmed Jamsheed Mohamed

Dengue Bulletin – Volume 40, 2018 i 8. Trends of dengue fever in Madhya Pradesh, India...... 72 Mrigenedra P. Singh, Sunil K. Chand, Arvind Jaiswal, Ramesh C. Dhiman

9. Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha...... 83 Animesha Rath, Rupenangshu K Hazra

10. Association between entomological indices, breeding of Aedes mosquitoes and container types in Delhi for the prevention and control of dengue...... 100 Babita Bisht, RoopKumari, Himmat Singh, BN Nagpal, AK Bansal, NR Tuli

11. Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector ...... 114 Nisha Sogan, Smriti Kala, Neera Kapoor, BN Nagpal

12. Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India...... 124 Rajalakshmi Anbalagan, Arpita Shukla, Kaviyarasan, Jayalakshmi Krishnan

13. Knowledge, attitude and practices for prevention and control of dengue fever among community members in North Delhi Municipal Corporation...... 137 Babita Bisht, Roop Kumari, BN Nagpal, Himmat Singh,Kumar Vikram, Sanjay Sinha, NR Tuli

14. A geostatistical study to prioritize dengue-affected areas for implementation of effective control by municipal corporations of Delhi, India...... 153 Sanjeev Kumar Gupta, Poonam Saroha, Kumar Vikram, NR Tuli, Himmat Singh, Rekha Saxena, Aruna Srivastava, BN Nagpal, MC Joshi

15. Reading levels of selected USA Federal Government dengue webpages...... 164 Jeffrey L. Lennon, Christopher M. Seitz

16. Unusual complications of dengue fever...... 168 Rajeev Upreti, Monica Mahajan, Ram Shankar Mishra

17. Instructions for contributors...... 173

ii Dengue Bulletin – Volume 40, 2018 Acknowledgements

The Editor, Dengue Bulletin, World Health Organization (WHO) Regional Office for South-East Asia, gratefully thanks the following for peer-reviewing the manuscripts submitted for publication.

1. Barde, Pradip 9. P.S. Indu Scientist ‘E’ Professor and Head National Institute of Research in Tribal Health Department of Community Medicine Jabalpur, India Thiruvananthapuram, Kerala 2. Biswas, Ashutosh 10. Ranjit, Manoranjan Professor, Department of Medicine Scientist ‘F’ All India Institute of Medical Sciences Regional Medical Research Centre New Delhi, India Bhubaneswar, Odisha 3. Chand, S.K. 11. Savargaonkar, Deepali Research Scientist Scientist ‘D’ National Institute of Malaria National Institute of Malaria Research Center Jabalpur, India Dwarka Sec 8, New Delhi, India 4. Das, Aparup 12. Sharma, R.S. Director Retd. Additional Director National Institute of Research in Tribal Health, National Centre for Disease Control Jabalpur New Delhi, India 5. Dash, A.P. 13. Shrivastav, Aruna Vice Chancellor Retd. Scientist ‘F’ Central University of Tamil Nadu National Institute of Malaria Research Center Thiruvarur, India Dwarka Sec 8, New Delhi, India 6. Hazra, R.K. 14. Singh, Himmat Scientist ‘E’ Scientist ‘D’ Regional Medical Research Centre National Institute of Malaria Research Center Bhubaneswar, Odisa Dwarka Sec 8, New Delhi, India 7. Joshi, P.L. 15. Srivastava, P.K. Retd. Former Director Retd. Additional Director National Vector Borne Disease Control National Vector Borne Disease Control Programme Programme Sham Nath Marg Delhi, India Delhi-110054, India 16. Tuli, N.R. 8. Kumar Vikram DHO Technical officer South Delhi Municipal Corporation National Institute of Malaria Research Center New Delhi, India Dwarka Sec 8, New Delhi, India

The quality and scientific standing of the Dengue Bulletin is largely due to the conscious efforts of the experts and also to the positive response of contributors to comments and suggestions.

The manuscripts were reviewed by Dr B.N. Nagpal and Dr Mohammad A. Jamsheed, with respect to format; content; conclusions drawn, including review of tabular and illustrative materials for clear, concise and focused presentation; and bibliographic references.

Dengue Bulletin – Volume 40, 2018 iii

Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala

Gayatri Lekshmy Kumar,# Zinia T Nujum, Lalitha Kailas, Vijayakumar K

Government Medical College, Thiruvananthapuram

Abstract Background The state of Kerala is hyperendemic for dengue. Infants and children are more likely than adults to develop severe clinical disease due to dengue. Few studies have been done to elucidate the risk factors for mortality among dengue-affected children in the state. We undertook this study to identify the risk factors for mortality in dengue-affected children admitted to Sri Avittom Thirunal (SAT) Hospital, Government Medical College, Thiruvananthapuram. Methods We conducted a retrospective study based on the case records of patients admitted with dengue fever to the paediatric wards of SAT Hospital during the period 2003–2013. It was an unmatched case–control design. The cases were children admitted with dengue fever who had died. Controls were children admitted with dengue who had been discharged from the hospital after recovering from their illness. A total of 661 cases of dengue fever were admitted in the years2003–2013 in the paediatric wards of SAT Hospital. Among those admitted, we studied 17 cases and 39 controls. Results Among children with dengue who had died, a higher percentage were boys. The mean age of cases was higher (7.77years) than that of controls (4.2[SD 3.7]years). The mean serum creatinine level in cases (0.8 [SD 0.3]mg/dL)was significantly higher than in controls (0.6 [SD 0.3]) mg/dL). The factors associated with mortality in dengue were the presence of comorbidities, being a referred case, history of drug intake and previous history of dengue. Following multivariable analysis, the factors independently associated with dengue mortality among children were the presence of comorbidity (75.9 [5.68−1014]), anorexia (20.81 [1.1−392.6]), altered sensorium(59.6 [4.4−808]) and raised serum creatinine(0.09 [0.01−0.79]). Conclusion Children with comorbidity, anorexia, altered sensorium and raised creatinine levels are at a higher risk of mortality from dengue. Such children should be prioritized for care in order to save their lives. More area-specific research is required in this direction.

Keywords: Comorbidity; creatinine levels; dengue mortality; paediatric; Thiruvananthapuram.

#E-mail: [email protected]

Dengue Bulletin – Volume 40, 2018 1 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala

Introduction

Dengue is an emerging tropical viral disease that causes widespread morbidity and mortality among affected individuals in high-risk areas. The majority of dengue viral infections are self-limiting, but complications result in high morbidity and mortality. Today, severe dengue has become a leading cause of hospitalization and death among children in high-risk areas.

The diagnosis of dengue viral infection is essentially clinical, although confirmation requires laboratory tests, including serology, non-structural protein 1 (NS1) antigen detection, polymerase chain reaction (PCR) and viral cultures. There are no specific anti dengue drugs and treatment is basically supportive; it consists of early recognition of complications and appropriate fluid therapy.

Burden of dengue

Globally, there were an average of 9221 deaths due to dengue per year between 1990 and 2013. Considering fatal and non-fatal outcomes together, dengue was responsible for 1.14 million (0.73–1.98 million) disability-adjusted life years in 20131.

The WHO South-East Asia Region contributes to 52% of dengue cases annually. India is one of the seven identified countries in this Region that regularly reports outbreaks of dengue fever/ dengue haemorrhagic fever (DF/DHF)2. Based on data from the National Vector Borne Disease Control Programme (NVBDCP), the number of cases reported in 2013 was 74 454 for dengue with 167 deaths. Paediatric cases of DHF have a high mortality. In 2015, Delhi recorded its worst outbreak since 2006 with over 15 000 dengue cases reported2. In 2013, Kerala had the largest number of cases (7938) and 29 deaths3. Kerala is now hyperendemic for dengue, with the presence of all four serotypes, high rates of co infection and local genomic evolution of viral strains4.

Infants and children 4–6 years of age are significantly more likely than adults to develop DHF/dengue shock syndrome (DSS) or manifestations of severe clinical illness. There have been isolated reports of mortality in paediatric DHF/DSS cases, which show that mortality is much higher in these cases than the cumulative mortality reported by the NVBDCP. Secondary prevention to reduce mortality through improved clinical case management has substantially lowered the mortality rate for severe dengue over the past two decades from 10–20% to <1%5,6. The first objective of the WHO Global Strategy 2012–2020 is to reduce the mortality due to dengue by 50% from 2010 levels7. The number of deaths is determined not only by factors that facilitate transmission but also by those that influence the severity of the disease and the ease of access to health care8. Organ involvement, shock, bacteraemia, comorbidities, haemorrhage and certain biochemical parameters have been identified as useful predictors of mortality in studies conducted in dengue-endemic countries, but there may be regional differences.

2 Dengue Bulletin – Volume 40, 2018 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala

Few studies have been done to elucidate the risk factors for morbidity and mortality among dengue-affected children in Kerala. Hence, we undertook this study to identify the factors associated with mortality due to dengue among children admitted to SAT Hospital, Government Medical College, Thiruvananthapuram.

Methods

We conducted a retrospective, unmatched case–control study based on the case records of patients admitted with dengue fever to the paediatric wards of Government Medical College, Thiruvananthapuram from 2003 to 2013. Data collection from case records spanned a duration of 4 months, starting in July 2013. We included all completed laboratory-confirmed case records from 2003 to 2013. The inpatient departments, intensive care units (ICUs) and records library of the Department of Paediatrics formed the study setting. Cases were children admitted with dengue fever during 2003–2013 who had died. Controls were children admitted with dengue who had improved and had been discharged from hospital after any duration. A total of 661 cases of dengue fever were admitted from 2003 to 2013. We identified 17 children who had died as cases and randomly included 39 children with dengue who had been admitted and recovered as controls.

Study variables

The sociodemographic variables studied were age, residence and sex of the patients. The weight of children in the control group was taken from the case records.

Disease related variables

The disease-related variables studied were duration of fever, presence of any comorbidities,* referral status, history of any drug intake and previous history of dengue. The symptoms and signs of dengue were fever, conjunctival congestion, retro-orbital pain, frontal headache, myalgia, backache, arthralgia, mucosal bleeding, lethargy, bleeding manifestations, type of bleed, rash, flushed face, anorexia, nausea, abdominal pain, restlessness, vomiting, cough, breathlessness, diarrhoea, periorbital oedema, pedal oedema, constipation, seizures and altered consciousness.

* Comorbidities obxserved were febrile seizures, diabetes insipidus, obesity, asthma, ventricular septal defect, hand, foot and mouth disease, protein–energy malnutrition and recurrent epistaxis.

Dengue Bulletin – Volume 40, 2018 3 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala

Investigation-related variables

We diagnosed dengue using anti dengue IgM, NS1 antigen and anti-dengue IgG antibody. We also did routine blood examination, platelet count, liver function tests and renal function tests. Other investigations included random blood sugar (RBS), reverse transcription polymerase chain reaction(RT-PCR), prothrombin time and international normalized ratio (PT/INR), activated partial thromboplastin time(aPTT), packed cell volume/haematocrit(PCV/ HCT) (and rise in PCV). Serum electrolytes tested were calcium, sodium, bicarbonate, potassium and creatine phosphokinase-muscle/brain (CPK-MB). We also estimated total protein and albumin, and did a routine urine examination. Other investigations were ECG (electrocardiogram),Widal and rapid malaria tests, as the case demanded.

Ethical clearance

We obtained clearance from the Institutional Ethics Committee. We sought permission from the heads of departments of the concerned institution and from the head of the records library. Confidentiality was maintained throughout the study.

Statistical data analysis

The clinical and laboratory data of cases and controls were entered in an Excel file and analyzed using the SPSS software. All qualitative variables were expressed as proportion and quantitative variables as means with standard deviations; the strength of association was studied using the odds ratio and confidence interval. The chi-square test and t-test were used to test for significance. After bivariable analysis, multivariable analysis was done using logistic regression to find the independent predictors of mortality.

Results

The mean time from the onset of symptoms to hospitalization among cases was 4.1 days (SD=1.67 days). The mean time from hospitalization to mortality was 3 (3.02) days. Most of the deaths occurred at night. Two of the children who had died had a history of dengue among relatives.

Comparison of baseline characteristics of cases and controls

Among the cases (n=17), 11 (64.7%) were boys and 6 (35.2%) were girls, whereas among the controls (n=39), 18 (46.1%) were boys and 21 (53.8%) were girls. The mean (SD) age

4 Dengue Bulletin – Volume 40, 2018 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala of children who had died due to dengue was higher (7.77 [3.88] years) compared to that of children who had recovered from dengue (4.2 [3.7] years). Most commonly affected age group was 2-3 years. Most of the deaths occurred among children in the age group of 5–10 years followed by those in the age group of 10–15 years (Figure 1). Dengue with warning signs was noted in 1 (5.9%) case and 17 (43.6%) controls. The mean weight of the children who had died due to dengue was higher (25.3 [12.1] kg) and fell into the unhealthy body mass index (BMI) range compared to the children who had recovered (12.7 [1.5] kg).

Figure 1: Age categories of cases and controls

30

25 en 20 ildr ch

of 15 24

er 9 mb 10

Nu 3 5 2 6 5 1 3 0 1 2 0–1yrs 2–3 yrs3–5 yrs5–10 yrs10–15 yrs Age of children cases controls

Comparison of clinical features of study participants

The mean duration of fever in cases was 4.7 (1.5) days and in controls it was 4.6 (2.1) days. Arthralgia and mucosal bleeding were noted in 11.8% of those who had died. Symptoms such as lethargy, anorexia, restlessness, abdominal pain and breathlessness predominated in dengue-affected children who had died compared to those who had recovered. Bleeding manifestations, seizures and altered sensorium were also more frequent in children who had died than in those who had recovered (Table 1). Severe dengue was observed in 16 (94.1%) cases and 2 (5.1%) controls. The presence of comorbidities, anorexia and altered sensorium were found to be significant predictors of death in children with dengue.

Dengue Bulletin – Volume 40, 2018 5 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala

Table 1: Baseline characteristics and clinical features associated with dengue mortality in children

Cases Controls Odds ratio (OR; Adjusted Variables P value (n=17) (n=39) 95%CI ) OR(95%CI) Boys 11 (64.7%) 18 (46.25%) 0.16 2.1(0.66–6.94) - Clinical features Arthralgia 2 (11.8%) 0 0.03 - - Mucosal bleed 2 (11.8%) 0 0.03 - - Lethargy 11 (64.7%) 4 (10.3%) 0.0001 16.0 (3.82−67.38) - Bleeding 12 (70.6%) 4 (10.3%) 0.0001 21.0 (4.8−91.2) - manifestation Anorexia 7 (41.2%) 1 (2.6%) 0.0001 26.6 (2.9− 242.1) 20.81 (1.1−392.6) Restlessness 5 (29.4%) 0 0.0001 - - Abdominal pain 7 (41.2%) 6 (15.4%) 0.04 - - Breathlessness 3 (17.6%) 1 (2.6%) 0.04 8.14 (1.0−84.93) - Seizures 6 (35.3%) 3 (7.7%) 0.01 6.5 (1.4−30.58) - Altered 9 (52.9%) 1 (2.6%) 0.0001 42.7 (4.72−386.7) 59.6( 4.4−808) sensorium Comorbidity 8 (47.1%) 1 (2.6%) 0.0001 33.7 (3.7−305.5) 75.9 (5.68−1014) Drug intake 11 (64.7%) 4 (10.3%) 0.0001 16.3 (3.82−67.38) - Referred cases 13 (76.5%) 9 (23.1%) 0.0001 10.8 (2.8−41.61) -

The mean pulse rate (119.41 [25.9]/min) was higher in those children who had died compared to those who had recovered (103.8 [19.4]/min). Urine output was relatively lower (1.85 [2.45] mL/kg/h) among cases compared to controls (4.38[10.6] mL/kg/h)]. The complications seen among cases were disseminated intravascular coagulation (DIC) in 8 (47.1%), acute respiratory distress syndrome (ARDS) in 5 (29.4%), renal failure in 3 (17.6%) and liver failure in 9 (52.9%). Ventilator support was given for 16 cases and 1control. The mean time to intubation was 12.9 (19.9) h and the mean duration of intubation was 37.5 (55.2) h.

Comparison of the findings of study participants

NS1 antigen positivity was significantly more common in cases than in controls (Table 2). Dengue IgG positivity was also more frequent in cases (7 [41.2%]) than in controls(11 [28.2%]). Dengue IgM was positive in 8 (47.1%) cases and 24 (61.5%) controls. Haemoconcentration (haematocrit >20) was seen more often in cases than in controls with a mean haematocrit value of 24. More cases had significantly greater thrombocytopenia than controls, with the lowest platelet count being significantly low (P<0.04). Among the haematological

6 Dengue Bulletin – Volume 40, 2018 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala investigations carried out, the mean blood urea level was significantly higher in cases (33.2 [15.7] mg/dL) compared to controls (15.7 [10.8]mg/dL), as was the mean serum creatinine level (Table 2). The mean serum sodium level in cases (128.8 [36.1]mg/dL)was significantly higher than in controls (99.6 [59.4] mg/dL). Cases had a significantly higher mean haemoglobin (13 [2] mg%) than controls (11.4 [1.7] mg%). The comparison of PT, INR andaPTT is shown in Table 2. Mean aspartate aminotransferase (AST) among cases was 784.53 mg/dL (1736 mg/dL) and among controls 225.18 (581) mg/dL. Mean alanine aminotransferase (ALT) values of cases and controls was 210 mg/dL and 89.31 mg/dL, respectively.

Table 2: Investigation and treatment-related characteristics associated with dengue

Investigations N (%) Adjusted OR P value OR (95%CI) done Cases Controls (95%CI) NS1 antigen 2.13 9 (52.9%) 4 (10.8%) 0.003 - positive (1.29−3.5) Dengue IgG 7 (41.2%) 11 (28.2%) 0.74 - - positive Dengue IgM 8 (47.1%) 24 (61.5%) 0.17 - - positive Blood urea 33.2 (15.7) mg/dL 15.7 (10.8) mg/dL 0.007 - - Serum 0.09 0.8 (0.3) mg/dL 0.6 (0.3) mg/dL 0.03 - creatinine (0.01−0.79) Serum 128.8 (36.1) mg/ 99.6 (59.4) mg/dL 0.03 - - sodium dL Haemoglobin 13 (2) mg% 11.4 (1.7) mg% 0.009 - - Clot formation 1.53 (0.5)s 2 s 0.002 - - time (CFT) Treatment given Crystalloids used 0.5% normal 0.25 4 (23.5) 0 0.002 - saline (0.15−0.40) 3%normal 0.25 4 (23.5) 0 0.002 - saline (0.156−0.40) Hydroxyethyl 0.013 14 (82.4) 2 (5.6) 0.001 - starch (HES) (0.002−0.084) Fresh frozen 0.200 8 (47.1) 0 0.001 - plasma (FFP) (0.111−0.359) 0.234 Platelets 6 (35.3) 0 0.001 - (0.140−0.393)

Dengue Bulletin – Volume 40, 2018 7 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala

Investigations N (%) Adjusted OR P value OR (95%CI) done Cases Controls (95%CI) 0.012 Packed RBCs 12 (70.6) 1 (2.6) 0.001 - (0.001−0.112) Sympathomimetics Dobutamine 5 (29.4%) 0 NA NA - Dopamine 13 (76.5%) 0 NA NA - Noradrenaline 10 (58.8%) 0 NA NA - 608 Adrenaline 16 (94.1%) 1 (2.6%) 0.001 - (35.78−10330) 11.69 Steroids 4 (23.5%) 1 (2.6%) 0.01 - (1.19−114.3) 56 Antibiotics 14 (82.4%) 3 (7.7%) 0.001 - (10.07−311.28) Antivirals 2 (11.8%) 0 NA NA -

The presence of any comorbidity, referred cases, history of drug intake and previous history of dengue were factors that were significantly associated with dengue mortality in children. The significant symptoms and signs in cases that had died were arthralgia, mucosal bleeding, lethargy, bleeding manifestations, anorexia, restlessness ,abdominal pain, breathlessness and altered sensorium (Table 2).

Treatment characteristics of the study participants

The patients were treated with crystalloids and colloids, as their condition required. Four cases (23.5%) received normal saline (0.5% and 3%). Hydroxyethyl starch (HES), fresh frozen plasma (FFP), platelet concentrate and packed red blood cells (RBCs) were required significantly more often by cases than controls (Table 2). Children who had died due to dengue were also administered steroids and antiviral drugs during the course of their treatment (Table 2).

Determinants of dengue mortality

Following multivariable analysis, the factors independently associated with dengue mortality among children were the presence of any comorbidity (P=0.001), anorexia (P=0.04), altered sensorium (P=0.003) and raised serum creatinine (P=0.03).

8 Dengue Bulletin – Volume 40, 2018 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala

Discussion

Few studies have examined the clinical features and outcomes of children affected by dengue in Kerala. We undertook an unmatched case–control study to identify the determinants of dengue mortality in children based on the case records of patients admitted with dengue fever in the paediatric wards of GMC hospital, Thiruvananthapuram from 2003 to 2013. We included a total of 56 study subjects of which 17 were cases (children who had died) and 39 were controls (children who had recovered).

We found that more boys had died than girls (Figure 1), as in some other studies11–13. However, one study found that girls had a higher risk of mortality (OR 1.57, 95% CI 1.1–2.2)14, and in another, adult women constituted 90% of those who died due to dengue15. The observed predominance of women in these studies may have been due to their relatively robust immune response, making them more prone to developing a greater inflammatory response or higher susceptibility to capillary permeability (15,16). This disparity in findings points to the need for more studies to elucidate an association between gender and dengue.

The mean age of the cases was 7.77years (SD=3.88). Though the most common age group affected by dengue was 2–3 years, mortality was highest in the age group of 5–10 years. Another study in Mumbai reported three deaths in 38 DHF/DSS cases with a mean age of 4.9 years17. In a study done in a reference hospital in north-east Brazil to study dengue infection in children and adolescents, the majority of children with dengue who required hospitalization were in the 10–15 years’ age group18.

We diagnosed dengue using anti dengue IgM, NS1 antigen and anti-dengue IgG antibody. Performing concurrent assays for dengue virus NS1, anti-dengue IgM and anti-dengue IgG along with platelet enumeration in the “dengue package” is immensely beneficial for patients, clinicians and public health officials19. NS1 antigen was positive significantly more often in cases (9 [52.9]%) than controls. The NS1 antigen circulates uniformly in all serotypes of the dengue virus at high levels during the first few days of illness20. Hence, it helps in early detection of the illness, especially within 4 days of onset compared to IgM, which reaches adequate levels for detection only in 5–10 days21. Studies claim that in addition to early diagnosis, the NS1 antigen may also be an indicator of disease severity22. Secondary dengue and severe dengue were more common among them compared to controls, as evidenced by the variation in positivity of dengue IgM and dengue IgG.

The presence of any comorbidity is usually associated with higher rates of mortality in dengue. The comorbidities observed in our study were febrile seizures, diabetes insipidus, obesity, asthma, ventricular septal defect (VSD), hand, foot and mouth disease (HFMD), protein–energy malnutrition (PEM) and recurrent epistaxis. Other studies have also found dengue patients with comorbidities to have a higher incidence of mortality22–25.

Dengue Bulletin – Volume 40, 2018 9 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala

Common reasons for referral among patients were decreased platelet count, poor general condition and seizures. Most patients usually seek primary medical care in private hospitals and are referred to government hospitals only when their condition worsens. This time lag results in loss of treatment opportunity, which is critical to their life.

The bleeding manifestations observed were haematemesis, melaena, bleeding from the gums, haematochezia and haematuria. Melaena was the most common symptom. Haemorrhagic complications such as epistaxis, gingival bleeding, gastrointestinal bleeding, haematuria and hypermenorrhoea, although rare, are important causes of death in dengue25.

Significant clinical signs associated with dengue mortality in children were neurological manifestations (lethargy, anorexia, restlessness, seizures and altered sensorium), respiratory manifestations (breathlessness), joint manifestations (arthralgia) and gastrointestinal manifestations (abdominal pain). Abdominal pain is one of the warning signs of dengue, according to the WHO 2009 case definition26. In another study, the clinical manifestations that were most significantly associated with dengue fever were myalgia, headache, skin rash and anorexia27.

In our study, complications noted in cases of mortality were ARDS, DIC, liver failure, renal failure and cardiac complications. These were similar to the findings of another study done in south India, in which severe refractory shock, DIC, ARDS, hepatic failure and neurological manifestations, singly or in combination, were the most common causes of death28.

In our study, blood urea and serum creatinine levels were higher in cases compared to controls. These abnormal renal function tests could be attributed to the failing renal function in severe dengue. Serum sodium level was also higher in cases than in controls. This observation was contrary to the findings of another study done in north India, in which out of 5 patients with significant hyponatraemia, 3 had a poor outcome29. The wide variation in the standard deviations of AST and ALT was due to extremes of values such as the high value of AST (7161mg/dL) among cases.

PT, INR and aPTT were raised in cases compared to controls. This finding points to activation of the coagulation system. In one study, PT and aPTT were significantly prolonged and fibrinogen levels significantly lower in patients with DSS as compared to patients with non-shock DHF30. The mean pulse rate of cases was higher than that of controls. The mean capillary filling time, respiratory rate and the urine output were lower in cases than in controls. The mean pulse pressure at the time of admission was significantly higher in cases (37.1 [18]mmHg) than controls (22.15 [10]mmHg). This could signify the stage prior to haemoconcentration following plasma leakage since a progressive decline was noted in the levels of pulse pressure following admission.

The mean haemoglobin was significantly higher in cases than in controls. This was contradictory to the findings of a study in which a haemoglobin value of less than 9 mg/ dL was associated with a fourfold higher risk of mortality and/or severity31. This could be

10 Dengue Bulletin – Volume 40, 2018 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala attributed to the small sample size of the study. Haemoconcentration and thrombocytopenia were greater in cases compared with controls in our study. The main pathogenic feature of dengue is an increase in vascular permeability leading to loss of plasma from the blood vessels, which causes haemoconcentration, low blood pressure and shock32. This may also be accompanied by haemostatic abnormalities such as thrombocytopenia, vascular changes and coagulopathy32.

Most of the deaths occurred at night. This could be due to the disproportionately less emergency care received at night compared to that during the day.

In our study, the mean time of onset of disease to hospitalization was 4.1 days (SD=1.67 days), and from hospitalization to mortality it was 3 days (SD= 3.02 days). Other studies found that the time from onset of fever to hospital admission ranged from a mean of 2.9 days to 4.7 days33–35. The median time from admission to a positive test result was 2.5 days and the median time from the onset of illness to death was 12 days15. The duration of stay of dengue patients from hospitalization to death can be considered as the window of opportunity available for the health professional to prevent death.

In a hyperendemic state such as Kerala, where prevalence among vulnerable groups such as children is high, control measures should be aimed at the key determinants of dengue mortality. Though the aggregated data were analyzed, the unique factors that could have played a role in the pathogenesis of deaths due to dengue could not be studied as it was beyond the scope of this study. Further, the study was retrospective and based on records. Since the sample size for the study was small, some results had wide confidence limits.

Conclusion and recommendations

The presence of any comorbidity (pre-existing disease) can augment the severity of dengue fever and hence should be addressed with equal importance in affected patients to prevent mortality. Raised creatinine levels can be a valuable biomarker of the risk of mortality. Deaths are commonly seen at night, and strengthening emergency health facilities could avert such deaths. More area-specific research in larger samples is required for this. Such studies also need to be replicated in other dengue-endemic areas for better decision-making and prioritization to prevent childhood mortality due to dengue.

Acknowledgements

We thank the house surgeons of the 2008 batch of Government Medical College, Thiruvananthapuram for their valuable help in data collection for the study in 2013. Above all, we thank and pray for those patients whose information was shared with us as a part of the study.

Dengue Bulletin – Volume 40, 2018 11 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala

Funding sources

We did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Conflicts of interest

None.

References

[1] Stanaway JD, Shepard DS, Undurraga EA, Halasa YA, CoffengLE, Brady OJ et al. The global burden of dengue: an analysis from the Global Burden of Disease Study 2013. Lancet Infect Dis. 2016;16:712–23. [2] Brady OJ, Gething PW, Bhatt S, Messina JP, Brownstein JS, Hoen AG et al. Refining the global spatial limits of denguevirus transmission by evidence-based consensus. PLoS Negl Trop Dis. 2012;6:e1760. [3] Cecilia D. Current status of dengue and chikungunya in India. WHO South East Asia J Public Health. 2014;3:22–6. [4] Anoop M, Aneesh I, Thomas M, Sairu P, Nabeel AK, Unnikrishnan R et al. Genetic characterization of dengue virus serotypes causing concurrent infection in an outbreak in Ernakulam, Kerala, South India. Indian J Exp Biol. 2010; 48:849–57. [5] Kalayanarooj S. Standardized clinical management: evidence of reduction of dengue haemorrhagic fever case fatality rate in Thailand. Dengue Bull. 1999; 23:10–17. WHO Regional Office for South-East Asia (http://www.who.int/iris/handle/10665/148661, accessed 6 March 2019). [6] Lan NT, Hung NT, Ha DQ, Phuong BTM, Lien LB,Tuan LAet al. Treatment of dengue haemorrhagic fever at Children’s Hospital No. 1, Ho Chi Minh City, , 1991–1996. Dengue Bull. 1998;22:99–106. [7] Global strategy for dengue prevention and control, 2012–2020. Geneva: World Health Organization; 2012. [8] Díaz-Quijano FA, Waldman EA. Factors associated with dengue mortality in Latin America and the Caribbean, 1995–2009: an ecological study. Am J Trop Med Hyg. 2012;86:328–34. [9] Bulugahapitiya D, Satarasinghe R. Preponderance of blood group B among dengue fever patients with serious complications in a tertiary care hospital. Ceylon Med J.2011;48:95–96 (http://doi.org/10.4038/ cmj.v48i3.3358, accessed 9March 2019). [10] Kalayanarooj S, Gibbons RV, Vaughn D, Green S, Nisalak A, Jarman RG et al. Blood group AB is associated with increased risk for severe dengue disease in secondary infections. J Infect Dis. 2007;195:1014–7. [11] Pun R, Pant KP, Bhatta DR, Pandey BD. Acute dengue infection in the western Terai region of . JNMA J Nepal Med Assoc. 2011;51:11–4. [12] Sah OP, Subedi S, Morita K, Inone S, Kurane I, Pandey BD. Serological study of dengue virus infection in Terai region, Nepal. Nepal Med Coll J. 2009;11:104–6.

12 Dengue Bulletin – Volume 40, 2018 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala

[13] Shah Y, Katuwal A, Pun R, Pant K, Sherchand SP, Pandey K et al. Dengue in western Terai region of Nepal. J Nepal Health Res Counc. 2012;10:152–5. [14] Anders KL, Nguyet NM, Chau NV, Hung NT, Thuy TT, Lien le B et al. Epidemiological factors associated with dengue shock syndrome and mortality in hospitalized dengue patients in Ho Chi Minh City, Vietnam. Am J Trop Med Hyg. 2011;84:127–34. [15] Halstead SB. Epidemiology. In: Gubler DJ, Kuno G,editors. Dengue and dengue haemorrhagic fever. London: CAB International; 1997:38. [16] Halstead SB, Nimmannitya S, Cohen SN. Observations related to pathogenesis of dengue hemorrhagic fever. IV. Relation of disease severity to antibody response and virus recovered. Yale J Biol Med. 1970;42:311–28. [17] Shah I, Deshpande GC, Tardeja PN. Outbreak of dengue in Mumbai and predictive markers for dengue shock syndrome. J Trop Pediatr. 2004;50:301–5. [18] Pires Neto Rda J, de Sá SL, Pinho SC, Pucci FH, Teófilo CR, Evangelista PD et al. Dengue infection in children and adolescents: clinical profile in a reference hospital in northeast Brazil. Rev Soc Bras Med Trop. 2013;46:765–8. [19] Karunakaran A, Ilyas WM, Sheen SF, Jose NK, Nujum ZT. Risk factors of mortality among dengue patients admitted to a tertiary care setting in Kerala, India. J Infect Public Health. 2014;7:114–20. [20] Bessof K, Delorey M, Sun W, Hunsperger E. Comparison of two commercially available dengue virus (DENV) NS1 capture enzyme-linked immunosorbent assays using a single clinical sample for diagnosis of acute DENV infection. Clin Vaccine Immunol. 2008;15:1513–8. [21] Datta S, Wattal C. Dengue NS1 antigen detection: a useful tool in early diagnosis of dengue virus infection. Indian J Med Microbiol.2010;28:107–10. [22] Lee IK, Liu JW, Yang KD. Clinical and laboratory characteristics and risk factors for fatality in elderly patients with dengue hemorrhagic fever. Am J Trop Med Hyg. 2008;79:149–53. [23] Yee-Sin L, Tun LT, Dale AF, Jenny GL, Helen MO, Rajmohan LN et al. Confirmed adult dengue deaths in : 5-year multi-center retrospective study. BMC Infect Dis. 2011;11:123 (http://dx.doi. org/10.1186/1471-2334-11-123, accessed 6 March 2019). [24] Tomashek KM, Gregory CJ, Rivera Sánchez A, Bartek MA, Garcia Rivera EJ, Hunsperger E et al. Dengue deaths in : lessons learned from the 2007 epidemic. PLoS Negl Trop Dis. 2012;6:e1614. [25] Ong A, Sandar M, Chen MI, Sin LY. Fatal dengue hemorrhagic fever in adults during a dengue epidemic in Singapore. Int J Infect Dis. 2007;11:263–7. [26] Comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. Revised and expanded edition. New Delhi: WHO Regional Office for South-East Asia; 2011. [27] de la C Sierra B, Kourí G, Guzmán MG. Race: a risk factor for dengue hemorrhagic fever. Arch Virol. 2007;152:533–42. [28] Kamath SR, Ranjit S. Clinical features, complications and atypical manifestations of children with severe forms of dengue hemorrhagic fever in South India. Indian J Pediatr. 2006;73:889–95. [29] Dhooria G, Bhat D, Bains H. Clinical profile and outcome in children of dengue hemorrhagic fever in North India. Iran J Pediatr. 2008;18:222–8.

Dengue Bulletin – Volume 40, 2018 13 Risk factors of mortality among children with dengue in a tertiary care centre, Thiruvananthapuram, Kerala

[30] Nguyen TH, Lei HY, Nguyen TL, Lin YS, Huang KJ, Le BL et al. Dengue hemorrhagic fever in infants: a study of clinical and cytokine profiles. J Infect Dis. 2004;189:221–32. [31] Villar-Centeno LA, Diaz-Quijano FA, Martinez-Vega RA. Biochemical alterations as markers of dengue hemorrhagic fever. Am J Trop Med Hyg. 2008;78:370–4. [32] Dengue haemorrhagic fever: diagnosis, treatment, prevention and control, second edition. Geneva: World Health Organization; 1997. [33] Communicable diseases surveillance in Singapore. Singapore: MoH; 2008. [34] Halstead SB. Neutralization and antibody-dependent enhancement of dengue . Adv Virus Res.2003;60:421–67. [35] Halstead SB, Rojanasuphot S, Sangkawibha N. Original antigenic sin in dengue. Am J Trop Med Hyg.1983;32:154–6.

14 Dengue Bulletin – Volume 40, 2018 Utility of CDC DENV 1–4 real-time RT-PCR assay for the diagnosis of dengue

Mohan K. Shukla, Pradip V. Barde,# Neeru Singh

National Institute of Research in Tribal Health (NIRTH), Jabalpur, Madhya Pradesh, India

Abstract Prompt diagnosis is essential for patient care, and early outbreak warnings are important for activating epidemic control systems. The Centers for Disease Control and Prevention (CDC) has developed the DENV 1–4 real-time reverse transcription polymerase chain reaction (RT-PCR) multiplex assay (qRT-PCR) for the detection of dengue virus (DENV). We compared this assay with conventional RT-PCRs (cRT-PCR) and found the CDC real-time RT-PCR kit to be better than conventional RT-PCRs in meso-endemic and epidemic areas. The CDC DENV 1–4 real-time RT- PCR multiplex assay can be performed in lesser time (223 [+8.4] min) than conventional RT-PCRs (499.5 [+7.7] min). Serotype identification is important for surveillance and the CDC kit detects the serotype simultaneously with DENV. Thus, this test would be a useful tool for both DEN surveillance and serotype monitoring.

Keywords: Dengue; real-time RT-PCR; conventional RT-PCR.

Introduction

Dengue (DEN) is a re-emerging infection. An estimated 390 million cases are reported globally every year1. Dengue virus (DENV) has four serotypes – DENV 1–4, which are classified into several genotypes that differ by 5–6% at the nucleotide level2. All four serotypes are capable of causing an array of symptoms, ranging from mild disease (classical dengue fever) to severe disease (haemorrhagic fever/shock syndrome)3. DENV viraemia is relatively high at the onset of symptoms, which makes reverse transcription polymerase chain reaction (RT-PCR) a reliable test for the early diagnosis of dengue. In addition, RT-PCR may allow serotype identification, which has implications for surveillance. Detection of IgM antibody and NS1 DENV soluble antigen by enzyme-linked immunosorbent assay (ELISA) in serum samples has the limitations of delayed diagnosis (fifth day after onset) and inability to identify the serotype. Conventional RT-PCRs (cRT-PCR) developed for the detection of DENV-specific nucleic acid in the acute phase can detect the serotype, although with limitations on the number of samples that can be handled owing to problems such as cross-contamination,

#E-mail: [email protected]

Dengue Bulletin – Volume 40, 2018 15 Comparison of dengue multiplex real-time RT-PCR with conventional RT-PCR multiple reactions and longer time (6–8 hours) for serotyping. The US Centers for Disease Control and Prevention (CDC) has developed the DENV 1–4 real-time RT-PCR multiplex assay (qRT-PCR) to overcome these hurdles, as it can diagnose DENV and its serotype4. We tested this assay for DEN diagnosis in Madhya Pradesh (MP), India and compared its utility for early diagnosis with cRT-PCR.

Methods

Serum samples collected from patients suspected to have DEN from different districts of MP and Chhattisgarh were referred to the Virology Laboratory of the ICMR-National Institute of Research in Tribal Health (NIRTH), Jabalpur, for serological and molecular diagnosis of DEN5.

One hundred and two serum samples collected during the acute phase of illness, received between April and November 2014 from various districts of MP, were subjected to molecular tests by cRT-PCR as described by Lanciotii et al. with a few modifications, and qRT-PCR was done as per the manufacturer’s instructions4,6. The samples collected during outbreak(s) within the study period were also included in the study7. Two trained technicians performed both the tests following standard operating procedures; the tests were monitored and recorded using a stopwatch.

RNA samples used for both the tests were extracted using the viral RNA extraction kit (Qiagen, Germany Cat. No. 52906) following the manufacturer’s protocol. For cRT-PCR, SuperScript® III (Cat. No. 12574-026, Invitrogen, USA) and for qRT-PCR, SuperScript® III system (Cat. No. 11732-020, Invitrogen, USA) were used.

The timings were noted for all the steps. The cRT-PCR was performed in steps, i.e. reaction set-up for one-step RT-PCR, run time in thermal cycler (ABI GeneAmp® PCR System 9700), electrophoresis, nested PCR, run time in thermal cycler, and electrophoresis and gel analysis. On the other hand, the steps performed in qRT-PCR were qRT-PCR set-up, run time in thermal cycler (ABI 7500 PCR System) and data analysis. In general, at a time, four samples were subjected to cRT-PCR and 10–13 samples for qRT-PCR. The data were analysed using appropriate statistical tests.

The analysis of time taken for the test up to serotyping by both the technicians for qRT- PCR was 223 (+8.4) min compared to 499.5 (+7.7) min for cRT-PCR. This difference was statistically significant (P<0.005). Although setting up of the qRT-PCR reaction took 43.5 (+4.9) min compared to 23 (+1.4) min for cRT-PCR, it may be noted that the cRT-PCR (without serotyping) time (302 [+2.1 min]) was longer than the qRT-PCR time (with serotyping) (223 [+8.4 minutes]) (Figure 1).

All 94 DENV-positive cases (DENV 1 [n=8], DENV 2 [n=8] and DENV 3 [n=78]) were detected using these tests, which included three cases of coinfection of DENV-2 with DENV-1 and DENV-3. The results of serotyping by both the methods were similar.

16 Dengue Bulletin – Volume 40, 2018 Comparison of dengue multiplex real-time RT-PCR with conventional RT-PCR

Results and discussion our findings show that both qRT-PCR and cRT-PCR can detect serotypes with equal efficiency, but the former has a distinct advantage due to its shorter detection time. Early diagnosis of DENV along with detection of the serotype is crucial as the infecting serotype is an important factor in determining severity8. Further, in an area where more than one serotype is circulating, there is a possibility of multiple serotype infection, which is more severe8. Thus, a test that can diagnose dengue along with the serotype in a relatively short time helps in better patient management. It also helps in curbing outbreaks by quick and reliable detection of etiology along with the cause of transmission.

We suggest that the qRT-PCR test be standardized for detection of viral RNA from mosquitoes caught from the field. This will help in incriminating the vector, which will allow health authorities to control specific vector(s).

The CDC assay has an internal control mechanism to detect human ribonucleoprotein complex (RNP); it is able to distinguish non-reactive samples from those with PCR inhibitors. If the chances of contamination are minimized, this assay can give a reliable diagnosis4.

Some limitations of this test include the inability to quantitatively detect DENV and the absence of chikungunya virus (CHIKV) detection primer probes in the panel. Despite these limitations, the CDC DENV 1–4 RT-PCR assay enables the diagnosis of DEN with DENV serotyping in a shorter time and permits handling of a larger number of samples, giving it an advantage over cRT-PCR. The simplicity in performing the test in an equipped laboratory makes the CDC DENV 1–4 RT-PCR assay beneficial for DEN surveillance and serotype monitoring.

Figure 1: Graph depicting the time taken for conducting two assays. The X-axis denotes the steps involved in the test and the Y-axis denotes the time in minutes. The standard deviation in the operators is given in brackets.

Dengue Bulletin – Volume 40, 2018 17 Comparison of dengue multiplex real-time RT-PCR with conventional RT-PCR

Acknowledgments

The authors are thankful to the Secretary, Government of India, DHR, MoH&FW, and the Director General, ICMR for financial support under the Viral Diagnostic Network project (no. VIR/43/2011-ECD-1). The authors also thank CDC, Puerto Rico, USA for providing CDC DENV 1–4 real-time RT-PCR assay kits gratis, and Dr Jorge Munoz, CDC, Puerto Rico, USA for a critical review and suggestions on the manuscript. The authors thank Dr S Rajasubramaniam, Scientist “E”, NIRTH, Jabalpur for critical comments on the manuscript and assistance with language.

References

[1] Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL et al. The global distribution and burden of dengue. Nature. 2013;496(7446):504–7. [2] Rico-Hesse R. Molecular evolution and distribution of dengue viruses type 1 and 2 in nature. Virology. 1990;174(2):479–93. [3] Rico-Hesse R. Dengue virus evolution and virulence models. Clin Infect Dis. 2007;44(11):1462–6. [4] Santiago GA, Vergne E, Quiles Y, Cosme J, Vazquez J, Medina JF et al. Analytical and clinical performance of the CDC real time RT-PCR assay for detection and typing of dengue virus. PLoS Negl Trop Dis. 2013;7(7):e2311. doi: 10.1371/journal.pntd.0002311. [5] National Institute for Research in Tribal Health. In: Indian Council for Medical Research [website] (http://www.nirth.res.in, accessed 9 March 2019). [6] Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol. 1992;30(3):545–51. [7] Barde PV, Kori BK, Shukla MK, Bharti PK, Chand G, Kumar G et al. Median Outbreak of Dengue virus 1 genotype III in rural central India. Epidemiol Infect. 2015;143(2):412–18. [8] Vaughn DW, Green S, Kalayanarooj S, Innis BL, Nimmannitya S, Suntayakorn S et al. Dengue viremia titer, antibody response pattern, and virus serotype correlate with disease severity. J Infect Dis. 2000;181(1):2–9.

18 Dengue Bulletin – Volume 40, 2018 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study

Heera Hassan,a# Reena John,b Prithi Nair,b MA Andrewsc

aDepartment of Microbiology, Govt Medical College, Thiruvananthapuram bDepartment of Microbiology, Govt Medical College, Thrissur cDepartment of Medicine, Govt Medical College, Thrissur

Abstract Dengue is endemic in India and exacts a high economic burden on both governments and individuals. The objective of this study was to detect dengue virus in clinically suspected fever cases as per WHO dengue case definition by reverse transcriptase polymerase chain reaction (RT-PCR), and to serotype the virus to find out the currently circulating serotypes of dengue virus in Thrissur district of Kerala. Additionally, a commercially available non-structural protein 1 (NS1) rapid card test was also evaluated. Serum samples from 102 probable dengue cases were collected during the pre-monsoon showers and subjected to NS1 rapid card test, RT-PCR and IgM enzyme-linked immunosorbent assay (ELISA). NS1 rapid card test was found to have a sensitivity of 63.38% and a specificity of 83.87%. Samples positive by RT-PCR were further serotyped using the same method. Seventy-one samples were confirmed to have dengue infection; 40 were positive for dengue virus (DENV). All the four serotypes of DENV were found. DENV-2 was the most prevalent serotype. This is the first case report of DENV-4 infection from Kerala. Coinfection with DENV-1 and DENV-4 was detected in one patient. Co-circulation of all the DENV serotypes provides a favourable environment for micro-evolution of new serotypes. The study finding can help the National Vector Borne Disease Control Programme (NVBDCP) and the state health department of Kerala to prioritize efforts to control dengue by effective vector control tools.

Keywords: Dengue virus; RT-PCR; IgM ELISA; NS1antigen; coinfection.

Introduction

Member States of the WHO-South East Asia Region and Western Pacific Region bear nearly 75% of the global disease burden due to dengue. Of the 11 countries in the South-East Asia Region, 10 countries, including India, are endemic for dengue1. In 2012, the Region reported 0.29 million cases, out of which India contributed 20%. Circulation of several serotypes has also been reported from these countries1,2. Co-circulation of multiple dengue serotypes coupled with increased human activity increases the likelihood of genetic changes,

#E-mail: [email protected]

Dengue Bulletin – Volume 40, 2018 19 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study leading to diversity in virus populations. Genetic recombination, natural selection and genetic bottlenecks have been implicated as factors that may lead to the emergence of new serotypes, such as the dengue virus serotype 5 (DENV-5), which follows a sylvatic cycle and was isolated in October 2013. Discovery of newer sylvatic strains in future may impede the dengue vaccine initiative3. The role of host immune status in disease severity with respect to host immune response and host genetics requires more research at the molecular level. More epidemiological studies are required to study disease severity with respect to the infecting serotypes and genotypes, sequence of infecting serotypes during primary and secondary infections, and time interval between primary and secondary infections4. Repeated infections with multiple serotypes of the virus lead to complications and hence circulation of multiple serotypes is associated with an increased risk of complications of dengue in susceptible individuals. Estimation of the geographical distribution and corresponding burden of dengue contributes to its global burden in terms of morbidity and mortality, and provides an idea of how to control dengue with the limited resources that may be available and to evaluate the impact of such activities.

Currently, these data can also be used for reliably scoping vaccine demand and delivery strategies. All the four serotypes of DENV have been reported from India5. Cyclic dengue epidemics have been reported mostly from the central and southern districts of Kerala since 20016–9 and are on the increase. Most of the reported cases are patients admitted with complications and hence represent only the tip of the iceberg. We aimed to bridge the gap in the literature regarding the circulating DENV serotypes in Thrissur, Kerala. This area’s climate, vegetation and other favourable conditions (coconut shells used for latex collection in rubber plantations, cocoa pods) could account for the increased vector survival and multiplication. Local human movement10 allows virus spread to urban areas. As expected, most of the cases that would otherwise be treated as mere fever cases and dismissed as outpatients (due to lack of severity) proved to be dengue. The efficiency in detecting cases of a commercially available NS1 rapid card test that was being used in the hospital was also evaluated. The presence of 102 cases in a tertiary care centre just during the pre-monsoon showers indicates the possibility of an ongoing dengue endemic in the area in the form of a sylvatic cycle or an epidemic cycle with periodic remissions and exacerbations.

Materials and methods

We conducted a cross-sectional study for a period of 1 year. Sample collection commenced in the month of April 2015. Though the monsoon showers were expected in June, the area witnessed pre-monsoon showers in the first week of April 2015. The population selected for study were paediatric and adult patients who were clinically diagnosed as probable cases of dengue or who presented with complications of dengue as per the WHO 2009 case definitions or 1997 case definitions, but within 7 days of fever. Patients presenting with viral fever-like symptoms (probable dengue fever or classical dengue fever) to the Medicine and Paediatric OPD were treated as outpatients. Patients who presented with complications such as decreased platelet counts, hemorrhage or shock were admitted in the Medicine or Paediatric ICU and treated as inpatients. Patients with fever for more than 7 days, those

20 Dengue Bulletin – Volume 40, 2018 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study classified as having fever of unknown origin who did not satisfy the WHO dengue case definition, those with fever of known etiology and neonates less than 28 days of age were excluded from the study.

The sample size was calculated from a previous study8 using the formula 4pq/d2, where p was the proportion of cases that were positive by reverse transcriptase polymerase chain reaction (RT-PCR)(49.3%), q was 100–p and d was 20% of p. A minimum of 102 fever cases was studied. Blood samples of outpatients were collected in the blood collection centre and in person from patients admitted to the ICU. The samples were subjected to centrifugation and the separated serum was used for further tests. The tests used were (see colour plate 1): NS1 rapid card test (CTK Biotech – CE OnSite; catalogue no: R0063C10 ), IgM capture enzyme-linked immunosorbent assay (ELISA) (National Institute of Virology kit) for dengue and RT-PCR. The first two tests were done in our institution and RT-PCR was done at the National Institute of Virology unit in the Alappuzha district of Kerala. The minimum sample size was completed in June 2015, in a matter of just 3 months.

Results

The sample size of 102 cases, intended to be reached over a period of 1year, was completed in a period of 3 months. This highlights the heavy incidence of dengue cases in the area.

Eleven patients were <15 years of age, 32 patients were in the age group of 16–30 years, 28 patients were in the age group of 31–45 years, 21 were in the age group of 46–60 years and 10 were above 60 years (Figure 1).

Figure 1: Age group-wise distribution of probable dengue cases

40

32 30 28

21 20

11 10 Number of cases 10

0 <15 years 16–30 years 31–45 years 46–60 years >60 years Age group ranges

Dengue Bulletin – Volume 40, 2018 21 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study

Of the 102 cases, 40(39%) were hospitalized due to complications, the rest were outpatients (Figure 2).

Figure 2: Outpatient and inpatient distribution of probable dengue cases

39% Inpatients=40 61% Outpatients-62

Table 1 shows that 42.2% of the total cases were outpatients without severe disease but diagnosed as dengue, in contrast to the usually reported severe dengue cases who get admitted and form only 27.5% of the total.

Table 1: The percentage of laboratory-confirmed cases among inpatients and statistically missed/uncounted outpatients who met the WHO clinical diagnostic criteria for dengue

Dengue positive Dengue negative Total Types of cases Number % Number % Number % Outpatients 43 42.2 19 18.6 62 60.8 Inpatients 28 27.5 12 11.8 40 39.2 Total 71 69.6 31 30.4 102 100.0

Out of the total 102 cases,32 cases presented within1–4 days of illness, of whom 18 were RT-PCR positive (56.25%). The remaining70 cases (68.6%) presented within 5–7 days of illness, out of whom 22 were RT-PCR positive (31.43%). A total of 40 cases were found to be positive for DENV with RT-PCR (Figure 3).

22 Dengue Bulletin – Volume 40, 2018 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study

Figure 3: RT-PCR* positivity with respect to day of presentation

80 70

60 32 40 18 22 20

0 1–4 days of fever 5–7 days of fever onset onset Number of probable dengue cases Confirmed RT-PCR positives

*Reverse-transcriptase polymerase chain reaction

One patient was coinfected with DENV-1 and DENV-4(see colour plate 3), which was accounted for separately. Hence, 41 dengue viruses were serotyped in 40 cases (Figure 4).

Figure 4: Percentage and number of dengue virus serotypes

5% 3%

29%

63%

DENV1=12 DENV2=26 DENV3=1 DENV4=2

DENV: dengue virus

Dengue Bulletin – Volume 40, 2018 23 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study

Out of the 32 cases that presented within1–4 days of illness, 20 were NS1 positive (62.5%) and 8 were IgM positive (25%). Of the 70 cases that presented within5–7 days of illness, 30 were NS1 positive (42.85%) and 35 were IgM positive (50%) (Figure 5).

Figure 5: NS1 rapid card positivity and IgM ELISA positivity with respect to the day of presentation

80 70 70

60

50

40 32 35 30 30 20 20 8 10

0 1–2 days of fever onset 5–7 days of fever onset

Number of probable dengue cases no: of NS1 positives No: of IgM positives

NS1: non-structural protein1 IgM: immunoglobulin M

Table 2: Evaluation of NS1 rapid card test with respect to confirmatory RT-PCR results over 1–7 days

RT-PCR + RT-PCR– NS1+ 31 20 NS1– 9 42

Sensitivity = true positives/diseased x 100= 31/40 x100= 77.5% Specificity = true negative/non-diseased x 100= 42/62 x100= 67.74% NS1: non-structural protein1 RT-PCR: reverse transcriptase polymerase chain reaction

24 Dengue Bulletin – Volume 40, 2018 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study

The sensitivity of the NS1 rapid card test was 83.33% at 1–4 days of onset of fever and declined to 72.72% at 5–7 days of presentation. The specificity of the test was 64.28% at 1–4 days of onset of fever while it increased to 70.83% at 5–7 days (Figure 6a).

Figure 6a: Evaluation of NS1 rapid card test with respect to the day of presentation (considering RT-PCR only as the confirmatory test)

90.00% 83.33% 80.00% 72.72% 70.83% 70.00% 64.28% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% 1–4 days of fever onset 5–7 days of fever onset NS1 sensitivity NS1 specificity

NS1: non-structural protein1 RT-PCR: reverse transcriptase polymerase chain reaction

Table 3: Evaluation of NS1 card test with respect to confirmatory dengue tests (IgM ELISA or RT-PCR)

Confirmed Dengue results Test Result Positive Negative Total (N= 71) (N=31) 102 Positive 45 5 50 Rapid NS1 Negative 26 26 52 Sensitivity of NS1 rapid test= true positives/diseased x 100= 45/71x100=63.38% Specificity of NS1 test= true negative/non-diseased x 100= 26/31x100=83.87% NS1: non-structural antigen 1 RT-PCR: reverse transcriptase polymerase chain reaction IgM: immunoglobulin M

Dengue Bulletin – Volume 40, 2018 25 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study

Figure 6b: Evaluation of NS1 rapid card test with respect to the time of presentation (considering RT-PCR or IgM positive as confirmatory tests)

100.00% 90.40% 90.00% 77.27% 80.00% 70% 70.00% 57.14% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% 1–4 days of fever onset 5–7 days of fever onset

Sensitivity of NS1 rapid test Specificity of NS1 rapid test

NS1: non-structural antigen 1 RTPCR: reverse transcriptase polymerase chain reaction IgM: immunoglobulin M

Discussion

In India, dengue is reported to be predominantly a disease of young adults. Studies in Delhi spanning the period 1999–2006 showed a consistent pattern with the peak number of confirmed cases occurring in 21–30 year olds, generally followed by adolescents (11–20-year- old group)11–17. Our findings were similar.

Most of the studies done on dengue fever are hospital based and concentrate only on inpatients and the association of their clinical profile with the dengue virus serotype11,18–22. In our study, 62 (60.8%) were outpatients requiring no hospitalization. In the outpatient category, 43 cases (42.2%) were confirmed to have dengue infection. This gives an idea of the extent of potentially infective sources that can result in spread to other individuals. Use of personal protective measures to protect against mosquito bites in such patients is also not monitored. Forty patients (39.2%) were hospitalized, mainly due to a decreasing platelet count. Twenty-eight patients (27.5%) in the inpatient category were confirmed to have dengue infection.

In our study, 70 cases (68.6%) presented on days 5–7 of illness, probably because our institution is a tertiary care centre, under which there are many peripheral health centres and also because of the location of the institute in a rural area, Mulangunnathukavu, which

26 Dengue Bulletin – Volume 40, 2018 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study is more than 10 km from Thrissur city. Out of the 32 cases that presented on days 1–4 of illness, 18 were RT-PCR positive (56.25%). Out of the 70 cases that presented on days 5–7 days of illness, 22 were RT-PCR positive (31.43%). A total of 40 cases were confirmed to have dengue with RT-PCR. MAC ELISA was also done to confirm dengue infection. Though antibody detection is recommended by the NVBDCP only after 5 days of fever, the history given by patients regarding the onset of fever may not always be reliable. Hence the test was performed for all cases. IgM was present in 8 cases during days 1–4 of fever onset; 12 cases were both RT-PCR and IgM positive, depicting the seroconversion stage. Thirty-one cases were RT-PCR negative but IgM positive, showing that the body had started clearing the virus by producing antibodies. The total number of confirmed dengue cases according to the NVBDCP guidelines was thus 71.

The predominant serotype found in the study was DENV-2, which was detected in 26 cases (63%), followed by DENV-1 – 12 cases (29%). Of the total cases that were RT-PCR positive, 2 cases were infected with DENV-4 (5%) and 1 case with DENV-3 (3%). The data show co-circulation of all serotypes, but predominantlyDENV-2 and DENV-1. No further study was done to detect other flaviviruses among cases that were RT-PCR negative. There is only one study on dengue in Thrissur (Trichur) in the literature and it was done in 1974 when only DENV-2 had been detected in the area23. An outbreak in Ernakulam district in 2008 showed the presence of DENV-1, DENV-2 and DENV-3 in the area8. Another study done in Thiruvananthapuram by Sheeba et al in 2008–2009 found that the serotype involved in the outbreak was DENV-19. A study in Tamil Nadu in 2003 confirms the presence of DENV-3 there24. Studies in Karnataka in 1970–1971 and in 1993 suggest the presence of DENV-1 and DENV-225,26. All the four serotypes were involved in the outbreak in Delhi in 200616. DENV-1 was isolated in 1956 at Vellore. There have been occasional reports of circulation of DENV-4, though it is not the predominant type in India27. There are no previous reports of the presence of DENV-4 in Kerala.

Dengue has been reported in various parts of the world, mainly in tropical and subtropical areas. The dengue serotypes circulating in Singapore are DENV-1, DENV-2 and DENV-3, with sporadic reports of DENV-428. Africa seems to have the highest case load and all four serotypes circulate in the continent. In the Caribbean islands, DENV-1 and DENV-2 have been implicated in outbreaks29.

In our study, one case was coinfected with DENV-1 and DENV-4 (colour plate 2). Coinfection with DENV-1, DENV-2 and/or DENV-3 has been reported in Ernakulam district8. Nine cases with concurrent infection were reported from Delhi in August–September 2006. Coinfection with DENV-1 and DENV-3 serotypes constituted 4 of the 9 total concurrent infections16. DENV-1 and DENV-4 coinfection has also been reported from Brazil30.

The NS1 rapid card test was evaluated with respect to RT-PCR for antigen detection (considering that only RT-PCR-positive cases are regarded as dengue-positive cases), and the sensitivity and specificity were found to be 77.5% and 67.74%, respectively (Table 2). The sensitivity for those who reported in 1–4 days and 5–7 days of onset of fever was

Dengue Bulletin – Volume 40, 2018 27 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study

83.33% and 72.72%, respectively, and the specificity was 64.28% and 70.83%, respectively. However, such a comparison does not include all the diseased population, as some would have already seroconverted. When the total diseased population is taken as those who are either or both RT-PCR and IgM positive, the sensitivity and specificity of the NS1 card test for those with onset of fever of 1–7­ days is 63.38% and 83.87%, respectively (Table 3). The sensitivity and specificity of the NS1 rapid detection kit in our study is comparable to that reported in other studies. In a study from Sri Lanka, the sensitivity and specificity of the dengue Duo kit NS1 rapid test alone were 61.07% and 96.32%, respectively31. High sensitivity (71%) and specificity (99%) has been reported in an article evaluating a rapid detection kit in Singapore32. In another study carried out by different reference laboratories in Thailand, the sensitivity of NS1 rapid detection kits was found to range between 71.9% and 79.1%, and the specificity from 95% to 100%33. Though in our study the sensitivity of the NS1 rapid test falls within this range, the specificity is much lower for this kit compared to the other studies (see Figure 6b).

Our study had some limitations. As our hospital is a tertiary care centre, most of the cases (mostly the most serious referred ones) would reach here only after 7 days and therefore the data cannot reliably be extrapolated to the actual population. Since the treatment of uncomplicated dengue fever is usually symptomatic as in any other viral fever, most physicians do not insist on etiological confirmation. Evaluation and validation of different NS1 rapid detection kits could not be done, which would have provided a better recommendation of the most sensitive NS1 rapid detection kit for implementation of the NVBDCP in the state.

Conclusion

zz Out of the 71 dengue infections, 40 cases were confirmed to have DENV in their serum by RT-PCR.

zz DENV-2 was the most prevalent serotype (63%).

zz NS1 antigen rapid test is sensitive within 4 days of fever (77.27%).

zz NS1 antigen rapid test is specific after 5 days of fever (90.4%). These data can provide support to the NVBDCP and state vector-borne disease control programmes to prioritize efforts to control dengue with effective vector control tools.

The study could not be conducted beyond the minimum required sample size (which was attained in 3 months) due to various technical and financial constraints, and hence may not represent the situation over 1 year.

zz Both NS1 antigen and IgM antibody tests should be performed simultaneously so that the maximum number of dengue cases can be detected.

zz More studies are required to find an association between DENV serotypes and the severity of the disease.

28 Dengue Bulletin – Volume 40, 2018 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study

Colour plate 1: Flow diagram to show the sequential analysis of the study on dengue virus and its serotypes

Colour plate 2: Gel electrophoresis picture of representative samples using Genesnap Software

Dengue Bulletin – Volume 40, 2018 29 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study

zz A field study is required to find out the real prevalence of DENV and its serotypes in the area. This will help in understanding if a vaccination programme is required to reduce morbidity and mortality.

zz Rigorous mosquito control measures and health education have to be implemented to decrease incidence.

zz A system for continuous surveillance of dengue cases and its serotypes is necessary to understand the circulating serotypes in the area and also to be on the lookout for detec- tion of new serotypes.

Acknowledgments

The authors are thankful to Dr B. Anukumar and the staff of the National Institute of Virology unit, Alappuzha, Kerala for their support to this study. The authors are also thankful to Dr Purushothaman, Head of the Department of Paediatrics, Government Medical College, Thrissur for his cooperation.

Declaration

The authors state that this article has not been published earlier and will not be submitted for publication elsewhere if accepted for publication in the WHO Dengue Bulletin.

References

[1] Comprehensive guidelines for prevention and control of dengue and dengue hemorrhagic fever, revised and expanded edition. New Delhi: WHO Regional Office for South-East Asia; 2011. [2] World Health Organization and the Special Programme for Research and Training in Tropical Diseases (TDR). Dengue guidelines for diagnosis, treatment, prevention and control: new edition. Geneva: World Health Organization; 2009. [3] Mustafa MS, Rasotgi V, Jain S, Gupta V. Discovery of fifth serotype of dengue virus (DENV-5): a new public health dilemma in dengue control. Med J Armed Forces India. 2015;71:67–70. [4] Kalichamy A. Genetics of susceptibility to severe dengue virus infections: an update and implications for prophylaxis, prognosis and therapeutics. Dengue Bull. 2016;39:1–18. [5] Chaturvedi UC, Nagar R. Dengue and dengue hemorrhagic fever: Indian perspective. J Biosci. 2008;33:429–41. [6] Kumar NP, Jayakumar PR, George K, Kamaraj T, Krishnamoorthy K, Sabesan S et al. Genetic characterization of dengue viruses prevalent in Kerala State, India. J Med Microbiol. 2013;62:545–52. [7] Anoop M, Mathew AJ, Jayakumar B, Issac A, Nair S, Abraham R et al. Complete genome sequencing and evolutionary analysis of dengue virus serotype 1 isolates from an outbreak in Kerala, South India. Virus Genes. 2012;45:1–13. doi: 10.1007/s11262-012-0756-3.

30 Dengue Bulletin – Volume 40, 2018 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study

[8] Anoop M, Issac A, Mathew T, Philip S, Kareem NA, Unnikrishnan R et al. Genetic characterization of dengue virus serotypes causing concurrent infection in an outbreak in Ernakulam, Kerala, South India. Indian J Exp Biol. 2010;48:849–57. [9] Sheeba PM, Jose R, Sreekumar E, Vimalraj AN, Kurian S, Bai JTR. A comparative study of dengue syndromes in a tertiary care centre. Academic Medical Journal of India. 2014;2:60–6. [10] Stoddard ST, Forshey BM, Morrison AC, Paz-Soldan VA, Vazquez-Prokopec GM, Astete H et al. House-to- house human movement drives dengue virus transmission. Proc Natl Acad Sci U S A. 2013;110:994–9. [11] Pandey A, Diddi K, Dar L, Bharaj P, Chahar HS, Guleria R et al. The evolution of dengue over a decade in Delhi, India. J ClinVirol. 2007;40:87–8. [12] Gupta E, Dar L, Narang P, Srivastava VK, Broor S. Serodiagnosis of dengue during an outbreak at a tertiary care hospital in Delhi. Indian J Med Res. 2005;121:36–8. [13] Gupta E, Dar L, Kapoor G, Broor S. The changing epidemiology of dengue in Delhi, India. Virol J. 2006;3:92. [14] Chakravarti A, Kumaria R, Berry N, Sharma VK. Serodiagnosis of dengue infection by rapid immunochromatography test in a hospital setting in Delhi, India, 1999–2001. Dengue Bull. 2002;26:107–12. [15] Singh NP, Jhamb R, Agarwal SK, Gaiha M, Dewan R, Daga MK et al. The 2003 outbreak of dengue fever in Delhi, India. Southeast Asian J Trop Med Public Health. 2005;36:1174–8. [16] Bharaj P, Chahar HS, Pandey A, Diddi K, Dar L, Guleria R et al. Concurrent infections by all four dengue virus serotypes during an outbreak of dengue in 2006 in Delhi, India.Virol J. 2008;5:1. [17] Rai S, Chakravarti A, Matlani M, Bhalla P, Aggarwal V, Singh N et al. Clinico-laboratory findings of patients during dengue outbreak from a tertiary care hospital in Delhi. Trop Doct. 2008;38:175–7. [18] Daniel R, Rajamohanan, Aby Philip AZ. A study of clinical profile of dengue fever in Kollam, Kerala, India. Dengue Bull. 2005;29:197–202. [19] Sharma S, Sharma SK, Mohan A, Wadhwa J, Dar L, Thulkar S et al. Clinical profile of dengue hemorrhagic fever in adults during 1996 –outbreak in Delhi, India. Dengue Bull. 1998;22:20–30. [20] Acharya SK, Buch P, Irshad M, Gandhi BM, Joshi YK, Tandon BN. Outbreak of dengue fever in Delhi. Lancet. 1988;2:1485–6. [21] Singh NP, Jhamb R, Agarwal SK, Gaiha M, Dewan R, Daga MK et al. The 2003 outbreak of dengue fever in Delhi, India. Southeast Asian J Trop Med Public Health. 2005;36:1174–8. [22] Sinha N, Gupta N, Jhamb R, Gulati S, Kulkarni AV. The 2006 dengue outbreak in Delhi, India. J Commun Dis. 2008;40:243–8. [23] Sreenivasan MA, Rodrigues FM, Venkateshan CN, Jayaram Panikar CK. Isolation of dengue virus from Trichur district (Kerala State). Indian J Med Res.1979;69:538–41. [24] Paramasivan R, Thenmozhi V, Hiriyan J, Dhananjeyan K, Tyagi B, Dash AP. Serological and entomological investigations of an outbreak of dengue fever in certain rural areas of Kanyakumari district, Tamil Nadu. Indian J Med Res. 2006;123:697–701. [25] Padbidri VS, Adhikari P, Thakare JP, Ilkal MA, Joshi GD, Pereira P et al. The 1993 epidemic of dengue fever in Mangalore, Karnataka state, India. Southeast Asian J Trop Med Public Health. 1995;26:699–704.

Dengue Bulletin – Volume 40, 2018 31 Detection and serotyping of dengue virus in a tertiary care centre in Thrissur, Kerala: a cross-sectional study

[26] George S, Soman RS. Studies on dengue in Bangalore city: isolation of virus from man and mosquitoes. Indian J Med Res. 1975;63:396–401. [27] Gupta N, Srivastava S, Jain A, Chaturvedi UC. Dengue in India. Indian J Med Res. 2012;136:373–90. [28] Yung CF, Lee KS, Thein TL, Tan LK, Gan VC, Wong JG et al. Dengue serotype-specific differences in clinical manifestation, laboratory parameters and risk of severe disease in adults, Singapore.Am J Trop Med Hyg. 2015;92:999–1005. [29] Dengue epidemiology (http://www.denguevirusnet.com/epidemiology.html, accessed 9 March 2019). [30] Colombo TE, Vedovillo D, Mondini A, Reis AFN, Cury AAF, de Oliveira FH et al. Co-infection of dengue virus by serotypes 1 and 4 in patient from medium sized city from Brazil. Rev. Inst. Med. trop. S. Paulo [online]. 2013, vol.55, n.4, pp.275–81(http://dx.doi.org/10.1590/S0036-46652013000400009, accessed 9March 2019). [31] Tissera HA, Kaluarachchi DP, Jayasena TD, Amarasinghe A, de Silva AM, Weerakoon B et al. Evaluation of sensitivity and specificity of commercially available dengue rapid test kit in two hospitals in Colombo, Sri Lanka. Dengue Bull. 2014;38:84–95. [32] Zhang H, Li W, Wang J, Peng H, Che X, Chen X et al. NS1-based tests with diagnostic utility for confirming dengue infection: a meta-analysis. Int J Infect Dis. 2014;26:57–66. [33] Pal S, Dauner AL, Mitra I, Forshey BM, Garcia P, Morrison AC et al. Evaluation of dengue NS1 antigen rapid tests and ELISA kits using clinical samples. PLoS One. 2014;9:e113411.

32 Dengue Bulletin – Volume 40, 2018 Scenario of dengue and chikungunya in Pune district, Maharashtra, India during 2016: a retrospective study at an apex referral laboratory

Alagarasu K,# Jadhav SM, Bachal RV, Bote M, Kakade MB, Ashwini M, Singh A, Parashar D

Dengue/Chikungunya Group, ICMR-National Institute of Virology, 20A, Dr Ambedkar Road, Pune-411001, Maharashtra, India

Abstract Dengue and chikungunya are the most common vector-borne diseases that pose a threat to public health in India. There is a lack of data on the demographic and clinical profile of these two diseases from different regions of India. Such data would help in comparing the disease scenario between different years. Hence, we carried out a retrospective study to analyse the available demographic and clinical data on dengue and chikungunya cases referred to an apex referral laboratory in Pune district, Maharashtra during 2016 and make it available in the public domain. The clinical profile of the dengue/chikungunya cases was also analysed to identify the symptoms that would aid in the differential diagnosis of dengue and chikungunya. In 2016, among the total of 4457 samples tested, 40% were positive for chikungunya while 11.2% were positive for dengue and 7.3% were positive for both dengue and chikungunya. Among the 408 samples tested for chikungunya viral RNA, 49.8% samples were positive while from the 291 samples tested for dengue virus RNA, 13 samples were positive. Dengue virus type 2 was the predominant serotype observed. Chikungunya cases had a higher mean age and was seen more often in women compared to dengue. Multivariate analysis revealed that age above 30 years, female gender, body pain, oedema and joint pain were associated with chikungunya, while eye pain was associated with dengue. Our study suggests that chikungunya infections were dominant in 2016 in Pune district, Maharashtra. The combination of age above 30 years, female gender, body pain, oedema and joint pain may aid in the differential diagnosis of chikungunya and dengue.

Keywords: Chikungunya; dengue; coinfections; joint pain; rash.

Introduction

Dengue (DEN) and chikungunya (CHIK) are the two dominant vector-borne viral diseases in India. The viruses responsible for these two diseases are transmitted by the Aedes mosquito and pose a huge threat to public health. Infection with the dengue virus (DENV) results in

#E-mail: [email protected]

Dengue Bulletin – Volume 40, 2018 33 Scenario of dengue and chikungunya in Pune district, Maharashtra: a retrospective study diverse clinical manifestations ranging from dengue fever (DF), a mild form of the disease, to dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS), which are severe forms of the disease1. Infection with the chikungunya virus (CHIKV) leads to chikungunya fever (CHIKF) which is characterized by fever and severe joint pains and, in many cases, CHIKV is known to induce persistent arthritis2. In 2016 alone, 129 166 cases of DEN and 64 057 cases of CHIK were reported from India through the National Vector Borne Disease Control Programme (NVBDCP)3,4. NVBDCP collects data through its sentinel surveillance centres, which provide the data on the number of positive cases. The number of cases reported by NVBDCP accounts for only the tip of the iceberg and a large number of cases go unreported5. The Indian Council of Medical Research (ICMR)-National Institute of Virology (NIV) from Pune, Maharashtra is an apex referral laboratory of NVBDCP and provides information related to serotypes/genotypes of DENV and CHIKV circulating in different geographical regions to the NVBDCP. Based on the need and as a service to the public, NIV also provides a diagnosis for suspected DEN and CHIK cases referred from the surrounding private and public hospitals in Pune and to primary health care centres from Pune district. In 2016 alone, ICMR-NIV provided a diagnosis for more than 4000 suspected cases of DEN/ CHIK. No information about the demographic and clinical profile of DEN and CHIK cases is available in the public domain, particularly from this region of India. There is a need to record and publish data on the number of DEN/CHIK cases tested, their occurrence, and the demographic and clinical profile of cases available in public health laboratories/hospitals. Such data would be valuable for comparing the scenario of DEN and CHIK in different years and help in formulating policies related to the diagnosis and control of these infections. We undertook a retrospective study to analyse the available demographic and clinical data on DEN and CHIK cases referred to ICMR-NIV during 2016 and make it available in the public domain. Moreover, the symptoms of DEN and CHIK mimic each other and pose problems in clinical diagnosis when both viruses are circulating during the same period. In view of this, we also analysed the clinical profile of the DEN/CHIK cases to identify the symptoms that would aid in clinical diagnosis, if any, which were specifically associated with DEN or CHIK.

Methods

ICMR-NIV receives samples from suspected cases of DEN/CHIK from the local hospitals in and around Pune, and also from primary health care centres located in Pune and the surrounding districts for diagnosis by the IgM capture enzyme-linked immunosorbent assay (ELISA) method (MAC ELISA). All the samples received for diagnosis are accompanied by a case history sheet filled by the treating physician. The symptoms that were present during the visit to hospital were filled and laboratory parameters, if available, were also entered.

A subset of samples within 5–6 days post onset of illness and negative by MAC ELISA are tested by multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) for simultaneous detection of DENV and CHIKV6. The samples that are positive for DEN by

34 Dengue Bulletin – Volume 40, 2018 Scenario of dengue and chikungunya in Pune district, Maharashtra: a retrospective study real-time RT-PCR are further tested by semi-nested two-step RT-PCR for detection of the serotype of DEN as described earlier7.

Our study was retrospective and was approved by the Institutional Human Ethics Committee. All the case history sheets of the samples received during 2016 were used for retrieving the clinical data. The results of MAC ELISA and molecular tests, if done, were also retrieved from the ICMR-NIV records. The data were entered in a template prepared using Epi Info version 7.

Statistical analysis was performed using Epi Info version 7 and SPSS version 18. Demographic and clinical characteristics were compared between four groups: DEN, CHIK, dual positives and dual negatives. We used Pearson chi-square or Fisher exact test for analysing the categorical variables and Student t-test for continuous variables. To identify independent predictors for DEN or CHIK positivity, we used univariate logistic regression on all the demographic characteristics and clinical symptoms. All significant variables were included in multivariate logistic regression with the step-wise backward elimination method and odds ratios (ORs) with 95% confidence intervals were reported for the significant variables. P<0.05 was considered as statistically significant.

Results

Samples and demographic characteristics of the patients

During 2016, a total of 4457 samples were tested, among which 1784 samples were positive for CHIK, 501 for DEN, 326 were dual positives (positive for both DEN and CHIK) and 1452 samples tested negative. The remaining samples (n = 394) gave equivocal results (Figure 1). The number of samples referred to the laboratory and the number of samples that tested positive for either CHIK or DEN or both started increasing gradually from week 15, peaked between weeks 34 and 40 and started decreasing gradually thereafter. The number of DEN and dual-positive cases peaked in the month of August while CHIK cases peaked in the month of September 2016 (Figure 2 ).

Chikungunya viral RNA positivity and dengue virus serotype distribution

Among the IgM negative samples, 408 samples were tested for CHIKV RNA, 203 samples were positive, while from the 291 samples tested for DENV RNA 13 samples were positive. Among the 13 samples, 6 were DENV-2, while 4 were positive for DENV-3, and 2 and 1 were positive for DENV-4 and DENV-1, respectively. We also retrieved the data on serotyping for earlier years (2009–2015), which showed a dominance of DENV-2 from 2013 onwards (Table 1).

Dengue Bulletin – Volume 40, 2018 35 Scenario of dengue and chikungunya in Pune district, Maharashtra: a retrospective study

Figure 1: Percentage positivity for dengue and chikungunya during 2016

Percentage positivity

Chikungunya 32.6 40 Dengue Dual Positives Equivocals 8.8 Negative 7.3 11.2

Figure 2: Week wise samples positive for dengue and chikungunya for the year 2016

36 Dengue Bulletin – Volume 40, 2018 Scenario of dengue and chikungunya in Pune district, Maharashtra: a retrospective study

Table 1: Distribution of dengue virus serotypes from 2009 to 2016

Year Positive/Tested DENV1 DENV2 DENV3 DENV4 2009 31/323 07 10 13 01 2010 37/411 06 15 15 01 2011 1/34 0 0 01 0 2012 28/204 05 11 12 0 2013 103/320 12 50 37 04 2014 73/282 07 33 09 24 2015 26/86 01 13 05 07 2016 13/291 01 06 04 02

Comparison of demographic and clinical characteristics between patients with dengue, chikungunya, those who were dual positive and dual negative

To compare the demographic and clinical characteristics between different groups of patients, we selected samples that were tested for both DEN and CHIK. From the samples tested for both DEN and CHIK during 2016, 2041 samples with definite results were included for further analysis while those with equivocal results were excluded. Out of the 2041 samples selected for analysis, 836 samples were positive for CHIK, 221 samples were positive for DEN, 326 were dual positives and 658 samples were negative for both viruses.

The mean age of patients with CHIK (38±16 years) was higher than that of those with DEN (30.2±14.6 years), dual-positive and dual-negative groups (P<0.0001). More subjects above 30 years of age were in the CHIK group as compared to the other groups. The proportion of men was higher among DEN cases (61.5%) as compared to CHIK (43.8%) and other groups (P<0.0001) (Table 2).

With regard to the clinical symptoms at presentation, joint pain, rash and oedema were observed more frequently in CHIK patients as compared to those with DEN only and dual- negative cases. Body pain and chills were present more often in CHIK than in DEN patients. Eye pain and nasal congestion were less frequently observed in CHIK compared to DEN patients and dual-negative patients (P<0.05) (Table 2). Among the rare symptoms related to bleeding manifestations and DHF, the frequency of the different symptoms were not different between the study groups except for effusion, which was higher among DEN cases (Table 3).

Dengue Bulletin – Volume 40, 2018 37 Scenario of dengue and chikungunya in Pune district, Maharashtra: a retrospective study

Table 2: Common clinical symptoms in chikungunya, dengue, dual-positive and -negative cases during 2016

Chikungunya+ Chikungunya Dengue Negative Dengue P value n = 836 n = 221 n = 658 n = 326

Age (mean±SD)* 38±16 30.2±14.6 32.9±16.4 32.8±17.3 <0.001 Age above 30 years* 535 (64.7) 94 (42.7) 157 (48.9) 310 (47.3) <0.001 Males (%) 366 (43.8) 136 (61.5) 152(46.6) 317 (48.2) <0.001 Fever 795 (95.1) 206 (93.2) 306 (93.9) 614 (93.3) 0.461 Head ache 657 (78.6) 165 (74.7) 260 (79.7) 527 (80.1) 0.373 Body pain 752 (86.0) 173 (78.3) 268 (82.2) 543 (82.5) <0.001 Chills 497 (59.4) 110 (49.8) 188 (57.7) 359 (54.6) 0.042 Joint pain 759 (90.8) 161 (72.8) 276 (84.7) 487 (74.0) <0.001 Rash 277(33.1) 61 (27.6) 97 (29.8) 115 (17.5) <0.001 Nausea/vomiting 272 (32.5) 75 (33.9) 100 (30.7) 175 (26.6) 0.055 Eye pain 247 (29.5) 78 (35.3) 96 (29.5) 248 (37.7) 0.004 Nasal congestion 39 (4.7) 15 (6.8) 17 (5.2) 56 (8.5) 0.018 Sore throat 96 (11.5) 21 (9.5) 34 (10.4) 99 (15.0) 0.05 Edema 114 (13.6) 11 (5.0) 33 (10.1) 30 (4.6) <0.001 Abdominal pain 91 (10.9) 24 (10.9) 42 (12.9) 95 (14.4) 0.183 Diarrhea 69 (8.2) 10 (4.5) 24 (7.4) 35 (5.3) 0.067

*For chikungunya n = 827, for dengue n =220, for dual positives n = 321 and dual negatives n = 655.

38 Dengue Bulletin – Volume 40, 2018 Scenario of dengue and chikungunya in Pune district, Maharashtra: a retrospective study

Table 3: Infrequent clinical symptoms in chikungunya, dengue, dual-positive and -negative cases during 2016

Chikungunya+ Symptoms/laboratory Chikungunya Dengue Negative dengue P value parameters n = 836 n =221 n = 658 n = 326 Bleeding gums 11 (1.3) 2 (0.90) 0 3 (0.46) 0.087 Cold clammy sweat 3 (0.36) 2 (0.90) 1 (0.31) 0 0.685 Effusion 2 (0.24) 3 (1.4) 0 0 0.004 Epistaxis 1 (0.12) 2 (0.90) 1 (0.31) 2 (0.30) 0.297 Haematemesis 2 (0.24) 1 (0.45) 2 (0.61) 2 (0.30) 0.785 Haematuria 2 (0.24) 1 (0.45) 2 (0.61) 2 (0.30) 0.785 Melaena 10 (1.2) 4 (1.8) 3 (0.92) 2 (0.30) 0.151 Petechiae 31 (3.7) 11 (5.0) 13 (4.0) 18 (2.7) 0.422 Purpura/ecchymoses 6 (0.72) 2 (0.90) 4 (1.2) 2 (0.30) 0.395 Jaundice 11 (1.3) 5 (2.3) 5 (1.5) 3 (0.46) 0.128 Lethargy 3 (0.36) 2 (0.90) 0 4 (0.61) 0.38 Restlessness 3 (0.36) 3 (1.4) 1 (0.31) 5 (0.76) 0.288

Univariate and multivariate analysis of clinical symptoms associated with dengue and chikungunya

Univariate followed by multivariate analyses showed that age above 30 years, body pain, oedema, joint pain, nausea/vomiting, melaena and rash were positively associated, while eye pain and nasal congestion were negatively associated with DEN- and CHIK-like illness as compared to dual-negative cases (Table 4).

When CHIK cases were compared with CHIK-negative cases excluding DEN, age above 30 years, oedema, joint pain, nausea/vomiting and rash were positively associated while eye pain and nasal congestion were negatively associated with CHIK (Table 4).

When DEN cases were compared with DEN-negative cases excluding CHIK, rash was positively associated while chills and female gender were negatively associated with DEN (Table 4).

When CHIK cases were compared with DEN cases, age above 30 years, female gender, body pain, oedema and joint pain were positively associated with CHIK while eye pain was associated with DEN (Table 4).

Dengue Bulletin – Volume 40, 2018 39 Scenario of dengue and chikungunya in Pune district, Maharashtra: a retrospective study

CI) 0.5 2.2 1.68 1.93 1.84 2.45 3.34 aOR (95% (1.78–3.37) (1.06–2.67) (0.93–4.04) (1.11–4.35) (0.35–0.72) (1.33–2.54) (2.16–5.17) only DEN positive NS RM RM RM RM 0.030 0.080 0.020 Only CHIK positive vs P value <0.001 <0.001 <0.001 <0.001

0.7 1.8 1.41 0.59 0.43 5.01 (0.51–0.98) (0.34–0.56) (0.43–0.81) (0.99–2.00) (1.23–2.62) (0.89–28.11) aOR (95% CI) both negative Only DEN positive vs RM RM RM RM RM RM 0.060 0.040 0.070 P value <0.001 <0.001 <0.001

4.54 1.31 0.38 1.88 2.23 3.59 2.22 (1.5–2.36) (1.42–3.51) (2.62–4.91) (0.23–0.61) (1.01–1.72) (1.67–2.96) (0.91–22.76) aOR (95% CI) both negative Only CHIK positive vs RM RM RM RM RM 0.070 0.040 P value <0.001 <0.001 <0.001 <0.001 <0.001

2.1 0.5 1.38 0.45 1.97 2.28 4.53 1.29 (1–20.58) (0.4–0.62) (0.3–0.67) (1.14–1.68) (1.28–3.04) (1.78–2.92) (1.02–1.63) (1.62–2.71) aOR (95% CI) Any positive vs both negative RM RM RM RM 0.050 0.030 P value <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 Table 4: Multivariate logistic regression analysis of clinical symptoms associated with groups Table characters Symptoms/ demographic Age above 30 years Rash Nasal congestion Body pain Chills Oedema Eye pain Female Joint pain Melaena Diarrhoea Nausea/vomiting aOR: adjusted odds ratio; 95%CI: 95% confidence interval; RM: variables removed by model

40 Dengue Bulletin – Volume 40, 2018 Scenario of dengue and chikungunya in Pune district, Maharashtra: a retrospective study

Discussion

The results suggest that CHIK infections dominated in 2016 while DEN infections occurred at low levels. Though the number of DEN and CHIK cases were equal in the month of June, thereafter the number of DEN cases gradually dropped while CHIK gained predominance. NVBDCP data suggest that though both DEN and CHIK cases have been reported from Maharashtra since 2010, a CHIK outbreak was larger only during 2010 followed by a reduction in the number of cases during subsequent years. In contrast, large numbers of DEN cases have been reported in Maharashtra since 20133. Our data show that DENV-2 was the dominant serotype during these years. Hence, is possible that the exposure levels to DENV-2 might have increased the herd immunity against the serotype while the number susceptible to CHIK might have increased. This might be the reason for the larger number of CHIK cases and reduced numbers of DEN cases in 2016.

In our study, large numbers of CHIK cases were observed in the month of September while large numbers of DEN were observed in August. Our earlier five-year study from 2005 to 2010 found larger numbers of DEN cases in the month of October8. In Maharashtra, mosquito-borne diseases usually coincide with the rainfall, which occurs mainly from June to October (south-west monsoon)9.

During 2016, 7.3% of the samples were positive for both CHIK- and DEN-specific IgM antibodies. IgM antibodies against DEN are known to persist from 3 to 6 months, while CHIK-specific IgM antibodies persist for longer periods10,11. Moreover, coinfections are usually characterized by the presence of either both viral antigens or viral RNA in the sample. This suggests that it is possible that these individuals might have coinfection or might have got sequential infections with CHIK followed by DEN or vice versa during the outbreak season. DEN and CHIK coinfection has been reported to be associated with severe disease12. In our study, the demographic characteristics and clinical symptoms between DEN cases and CHIK cases were not different, suggesting that half the subjects might have been infected with DEN first while the other half might have been infected with CHIK first. A higher frequency of coinfection has been reported in the Indian setting13.

Investigation of demographic data showed that more women and patients aged 30 years and above were in the CHIK-affected group as compared to the other groups. Studies done in and Trinidad have shown a similar demographic pattern14,15. Our five-year study reported that the 21–30 years age group was the most affected by DEN8. Studies have shown that women exhibit a greater pro-inflammatory cytokine response on exposure to bacterial endotoxin16. It is possible that CHIK infection in women may be associated with a greater inflammatory response resulting in severe symptoms as compared to men and hence a higher proportion of women with CHIK infection visit clinics17. Together with gender, age above 30 years might also influence the inflammatory response. However, this needs further investigation.

Dengue Bulletin – Volume 40, 2018 41 Scenario of dengue and chikungunya in Pune district, Maharashtra: a retrospective study

Comparison of clinical symptoms between different groups of patients showed an increased frequency of cases with body pain, chills, joint pain, rash and oedema among CHIK-infected patients as compared to DEN cases. Various studies have also shown the association of joint pain and rash with CHIK infection, while there are also contradictory reports regarding chills, body pain and oedema14,15,18,19. Rash and arthralgia combined with other symptoms or laboratory parameters have been reported to be good predictors of CHIK20. In our study, in multivariate analysis, age above 30 years, female gender, oedema, body pain and joint pain were positively associated, and eye pain was negatively associated with CHIK compared to DEN cases. These demographic characteristics and symptoms in combination can aid in the differential diagnosis of DEN and CHIK.

References

[1] Alagarasu K. Genetics of susceptibility to severe dengue virus infections: an update and implications for prophylaxis, prognosis and therapeutics. Dengue Bull. 2016;39:1–18. [2] Amdekar S, Parashar D, Alagarasu K. Chikungunya virus-induced arthritis: role of host and viral factors in the pathogenesis. Viral Immunol. 2017;30:691–702. [3] National Vector Borne Disease Control Programme. Dengue cases and deaths in the country since 2010 (http://www.nvbdcp.gov.in/den-cd.html, accessed 9 March 2019). [4] National Vector Borne Disease Control Programme. Clinically suspected chikungunya fever cases since 2010 (http://www.nvbdcp.gov.in/den-cd.html, accessed 9 March 2019). [5] Shepard DS, Halasa YA, Tyagi BK, Adhish SV, Nandan D, Karthiga KS et al. Economic and disease burden of dengue illness in India. Am J Trop Med Hyg. 2014;91:1235–42. [6] Cecilia D, Kakade M, Alagarasu K, Patil J, Salunke A, Parashar D et al. Development of a multiplex real-time RT-PCR assay for simultaneous detection of dengue and chikungunya viruses. Arch Virol. 2015;160:323–27. [7] Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol. 1992;30:545–51. [8] Cecilia D. Current status of dengue and chikungunya in India. WHO South East Asia J Public Health. 2014;3:22–26. [9] Shil P, Kothawale DR, Sudeep AB. Rainfall and chikungunya incidences in India during 2010–2014. Virusdisease. 2018;29:46–53. [10] Prince HE, Matud JL. Estimation of dengue virus IgM persistence using regression analysis. Clin Vaccine Immunol. 2011;18:2183–5. [11] Pierro A, Rossini G, Gaibani P, Finarelli AC, Moro ML, Landini MP et al. Persistence of anti-chikungunya virus-specific antibodies in a cohort of patients followed from the acute phase of infection after the 2007 outbreak in Italy. New Microbes New Infect. 2015;7:23–5.

42 Dengue Bulletin – Volume 40, 2018 Scenario of dengue and chikungunya in Pune district, Maharashtra: a retrospective study

[12] Chahar HS, Bharaj P, Dar L, Guleria R, Kabra SK, Broor S. Co-infections with chikungunya virus and dengue virus in Delhi, India. Emerg Infect Dis. 2009;15:1077–80. [13] Saswat T, Kumar A, Kumar S, Mamidi P, Muduli S, Debata NK et al. High rates of co-infection of dengue and chikungunya virus in Odisha and Maharashtra, India during 2013. Infect Genet Evol. 2015;35:134–41. [14] Mohd Zim MA, Sam IC, Omar SF, Chan YF, AbuBakar S, Kamarulzaman A. Chikungunya infection in Malaysia: comparison with dengue infection in adults and predictors of persistent arthralgia. J Clin Virol. 2013;56:141–5. [15] Sahadeo N, Mohammed H, Allicock OM, Auguste AJ, Widen SG, Badal K et al. Molecular characterisation of chikungunya virus infections in Trinidad and comparison of clinical and laboratory features with dengue and other acute febrile cases. PLoS Negl Trop Dis. 2015;9:e0004199. [16] Wegner A, Benson S, Rebernik L, Spreitzer I, Jäger M, Schedlowski M et al. Sex differences in the pro‑inflammatory cytokine response to endotoxin unfold in vivo but not ex vivo in healthy humans. Innate Immun. 2017;23:432–9. [17] Delgado-Enciso I, Paz-Michel B, Melnikov V, Guzman-Esquivel J, Espinoza-Gomez F, Soriano-Hernandez AD et al. Smoking and female sex as key risk factors associated with severe arthralgia in acute and chronic phases of chikungunya virus infection. Exp Ther Med. 2018;15:2634–42. [18] Velasco JM, Valderama MT, Lopez MN, Chua D Jr, Latog R 2nd, Roque V Jr et al. Chikungunya virus infections among patients with dengue-like illness at a tertiary care hospital in the , 2012– 2013. Am J Trop Med Hyg. 2015;93:1318–24. [19] Reller ME, Akoroda U, Nagahawatte A, Devasiri V, Kodikaarachchi W, Strouse JJ et al. Chikungunya as a cause of acute febrile illness in southern Sri Lanka. PLoS One. 2013;8:e82259. [20] Laoprasopwattana K, Kaewjungwad L, Jarumanokul R, Geater A. Differential diagnosis of Chikungunya, dengue viral infection and other acute febrile illnesses in children. Pediatr Infect Dis J. 2012;31:459–63.

Dengue Bulletin – Volume 40, 2018 43 Circulation of dengue serotypes in four provinces of northern Thailand during 2013–2017

Circulation of dengue serotypes in four provinces of northern Thailand during 2013–2017

Punnarai Veeraseatakul,# Jarurin Waneesorn, Kongphob Thilaogam, Yuddhakarn Yananto, Kotchakorn Intamul, Somkhid Thichak

Regional Medical Sciences Center 1 Chiang Mai, Department of Medical Sciences, Ministry of Public Health, 191 M.8 T. Donkaew, Maerim District, Chiang Mai 50180, Thailand

Abstract Dengue virus infection is an infectious disease with epidemic potential caused by dengue virus. The virus has 4 serotypes – dengue-1, dengue-2, dengue-3 and dengue-4. It is a major health problem in many parts of Thailand, including four provinces in the northern part; Chiang Mai, Lamphun, Lampang and Mae Hong Son. We aimed to determine the dengue serotypes during 2013–2017 in these four provinces through laboratory testing. A total of 1170 samples from suspected dengue patients in the acute phase were tested for serotypes by real-time-polymerase chain reaction (real time-PCR) or reverse transcriptase (RT)-PCR. Seven hundred and two samples were found positive for dengue RNA, of which 41.2% were of dengue-1, 23.8% of dengue-2, 18.9% of dengue-3 and 16.1% of dengue-4. Over these 5 years, the circulation of predominant dengue serotypes has changed, from dengue-1 in 2013 and 2014 to dengue-3 in 2015, dengue-4 in 2016 and back to dengue-1 in 2017. Our study indicated that all four serotypes were circulating and the predominant serotypes were not stable in northern Thailand. This information will be beneficial to the surveillance system for prevention and control of dengue virus infection.

Keywords: Dengue serotypes; northern Thailand.

Introduction

Dengue is a mosquito-borne viral infection caused by four distinct serotypes; dengue-1 (DENV-1), dengue-2 (DENV-2), dengue-3 (DENV-3) and dengue-4 (DENV-4)1. The epidemiology of dengue in Thailand is characterized by a 2-year cycle2; the incidence rates per 100 000 population increase within each cycle. High incidence rates of 141.78, 183.59, 246.34 and 223.85 per 100 000 population were reported by the Bureau of Epidemiology, Ministry of Thai Public Health in 2008, 2010, 2013 and 2015, respectively3–6. Gubler et al.7 and Lam et al.8 reported that virological surveillance based on the isolation and identification of dengue viruses infecting the human population provides an important means of early

#E-mail: [email protected]

44 Dengue Bulletin – Volume 40, 2018 Circulation of dengue serotypes in four provinces of northern Thailand during 2013–2017 detection of any change in the prevalence of dengue virus serotypes. Each serotype is capable of causing dengue epidemics in tropical and subtropical regions of the world, and has characteristics that affect the nature of the dengue epidemic and disease severity. Increased disease severity has been associated with all combinations of sequential infections, but a larger percentage of cases with the sequential infection combination of DENV-1 followed by DENV-2 have led to increased disease severity (9,10). We aimed to determine the circulation of the four different dengue virus serotypes in northern Thailand, and report on the details and findings of dengue infection in four provinces. Identification of the dengue serotypes circulating in various geographical locations has an important implication on dengue control and prevention.

Materials and methods

Specimens Serum samples were collected from suspected dengue patients in hospitals from four provinces of northern Thailand – Chiang Mai, Lamphun, Lampang and Mae Hong Son (Figure 1) during 2013–2017. Samples that were seropositive for dengue infection by antigen11 or antibody immunoglobulin (Ig)M/IgG tests12 were selected and subsequently subjected to further testing in our study. A total of 1170 seropositive samples taken during the acute stage of illness were sent for dengue serotype examination at the Regional Medical Sciences Center 1, Chiang Mai, Thailand (RMSC 1 CM).

Figure 1: Map of four provinces of northern Thailand

Dengue Bulletin – Volume 40, 2018 45 Circulation of dengue serotypes in four provinces of northern Thailand during 2013–2017

Viral RNA extraction

Dengue viral RNA was extracted using the QIAamp viral RNA Mini Kit, Cat. No. 52904 (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions13. The eluted RNA was kept at –70 °C until use.

RT-PCR

During 2013–2014, laboratory testing for dengue serotype was performed using two-step conventional reverse transcriptase polymerase chain reaction (RT-PCR) according to the protocol previously described by Yenchitsomanus et al.14. Dengue serotypes were identified by the size of the resulting bands – 504 bp for DENV-1, 346 bp for DENV-2, 196 bp for DENV-3 and 145 bp for DENV-4.

Real-time PCR

During 2015–2017, laboratory testing for dengue serotype was performed by the abTES (AITbiotech, Singapore)15according to the protocol previously described by Saengsawang et al.16. Real-time PCR was performed using the CFX96 Realtime thermocycler (Bio-Rad Laboratories, Hercules, CA, USA).

Results

The number of seropositive samples taken during the acute phase of illness and percentage of dengue serotypes in the four provinces from 2013 to 2017 are shown in Table 1. A total of 1170 serum samples were analysed, of which 702 samples were positive for dengue RNA with an average positivity rate of 60.0% by RT-PCR or real-time PCR, and were differentiated into four serotypes. We found all four serotypes throughout the 5 years. DENV-1 was the most predominant serotype (289 samples, 41.2%) followed by DENV-2 (167 samples, 23.8%), DENV-3 (133 samples, 18.9%) and DENV-4 (113 samples, 16.1%).

46 Dengue Bulletin – Volume 40, 2018 Circulation of dengue serotypes in four provinces of northern Thailand during 2013–2017

Table 1: Summary of dengue serotypes in 4 provinces of northern Thailand, 2013–2017

Seropositive Positive Dengue serotype Year Province acute dengue DENV-1 DENV-2 DENV-3 DENV-4 sample by PCR (%) (%) (%) (%) 2013 Chiang Mai 190 150 58 (38.7) 63 (42.0) 25 (16.7) 4 (2.7) Lamphun 28 13 2 (15.4) 11 (84.6) 0 (0) 0 (0) Lampang 49 43 18 (41.9) 13 (30.2) 12 (27.9) 0 (0) Mae Hong 75 48 35 (72.9) 5 (10.4) 4 (8.3) 4 (8.3) Son 254 113 Total 342 92 (36.2) 41 (16.1) 8 (3.1) (74.3) (44.5) 2014 Chiang Mai 45 16 14 (87.5) 0 (0) 2 (12.5) 0 (0) Lamphun 23 9 4 (44.4) 0 (0) 4 (44.4) 1 (11.1) Lampang 13 2 2 (100) 0 (0) 0 (0) 0 (00 Mae Hong 124 73 63 (86.3) 10 (13.7) 0 (0) 0 (0) Son 100 Total 205 83 (83.0) 10 (10.0) 6 (6.0) 1 (1.0) (48.8) 2015 Chiang Mai 126 81 10 (12.3) 6 (7.4) 26 (32.1) 39 (48.1) Lamphun 18 8 0 (0) 0 (0) 6 (75.0) 2 (25.0) Lampang 25 13 1 (7.7) 0 (0) 7 (53.8) 5 (38.5) Mae Hong 103 70 12 (17.1) 6 (8.6) 36 (51.4) 16 (22.9) Son 172 Total 274 23 (13.4) 12 (7.0) 75 (43.6) 62 (36.0) (62.3) 2016 Chiang Mai 60 18 1 (5.6) 4 (22.2) 4 (22.2) 9 (50.0) Lamphun 15 3 1 (33.3) 0 (0) 1 (33.3) 1 (33.3) Lampang 16 2 0 (0) 2 (100) 0 (0) 0 (0) Mae Hong 86 33 2 (6.1) 10 (30.3) 4 (12.1) 17 (51.5) Son 56 Total 177 4 (7.1) 16 (28.6) 9 (16.1) 27 (48.2) (31.6)

Dengue Bulletin – Volume 40, 2018 47 Circulation of dengue serotypes in four provinces of northern Thailand during 2013–2017

Seropositive Positive Dengue serotype Year Province acute dengue DENV-1 DENV-2 DENV-3 DENV-4 sample by PCR (%) (%) (%) (%) 2017 Chiang Mai 68 59 23 (39.0) 27 (45.8) 1 (1.7) 8 (13.6) Lamphun 13 3 2 (66.7) 0 (0) 1 (33.3) 0 (0) Lampang 11 4 1 (25.0) 3 (75.0) 0 (0) 0 (0) Mae Hong 80 54 40 (74.1) 7 (13.0) 0 (0) 7 (13.0) Son 120 Total 172 66 (55.0) 37 (30.8) 2 (1.7) 15 (12.5) (69.8) Total 702 289 167 133 113 1,170 (60.0) (41.2) (23.8) (18.9) (16.1)

The circulation of dengue serotypes in four provinces of northern Thailand from 2013 to 2017 is shown in Figure 2. From 2013 to 2014, the predominant serotype was DENV-1 (44.5% and 83.0%, respectively), followed by serotype DENV-2 (36.2% and 10.0%, respectively). In 2015, the predominant serotype had changed to DENV-3 (43.6%), followed by DENV-4 (36.0%). In 2016, the predominant serotype had changed to DENV-4 (48.2%), followed by DENV-2 (28.6%). In 2017, the predominant serotype had changed again to DENV-1 (55.0%), followed by DENV-2 (30.8%).

Figure 2: The circulation of dengue serotypes in 4 provinces of northern Thailand by year from 2013–2017

100%

80% DENV-4

60% e l p m a S DENV-3 % 40%

DENV-1 DENV-2 20%

0% 2013 2014 2015 2016 2017 DENV-1 DENV-2 DENV-3 DENV-4 Year

48 Dengue Bulletin – Volume 40, 2018 Circulation of dengue serotypes in four provinces of northern Thailand during 2013–2017

Circulation of the predominant dengue serotypes by province from 2013 to 2017 is shown in Figure 3. We analysed the data in each province that had more than 5 samples positive for dengue RNA. Data from Chiang Mai province showed that the predominant serotype was DENV-1 during 2013 and 2014 (38.7% and 87.5%), DENV-4 in 2015 and 2016 (48.1% and 50.0%), and DENV-2 in 2017 (45.8%) (Figure 3A). In Lamphun province, the predominant serotypes were DENV-2 in 2013 (84.6%), DENV-1 (44.4%) and DENV-3 (44.4%) in 2014, and DENV-3 (75.0%) in 2015 (Figure 3B). In Lampang province, the predominant serotypes were DENV-1 in 2013 (41.9%) and DENV-3 (53.8%) in 2015 (Figure 3C). In Mae Hong Son province, DENV-1 was the predominant serotype during 2013 and 2014 (72.9% and 86.3%, respectively), DENV-3 was predominant in 2015 (51.4%) and DENV-4 in 2016 (51.5%). In 2017, DENV-1 was predominant (74.1%; Figure 3D).

Figure 3: Circulation of dengue serotypes by province. (A) Chiang Mai province, (B) Lamphun province, (C) Lampang province and (D) Mae Hong Son province

Dengue Bulletin – Volume 40, 2018 49 Circulation of dengue serotypes in four provinces of northern Thailand during 2013–2017

Discussion

Throughout the 5-year period, the data from our study showed that all four serotypes were circulating in four provinces of northern Thailand in different proportions. The predominant serotype in circulation each year or every 2–3 years changed to other serotypes, as in the last 10-year period of study17. In 2013, according to the Annual epidemiology surveillance report 2013, Ministry of Thai Public Health, the number of dengue cases was high with an incidence rate of 246.34 per 100 000 population4 and we found that DENV-1 and DENV-2 were predominant (44.5% and 36.2%, respectively), whereas DENV-3 and DENV-4 were less prevalent, as previously reported (18-19). In 2014, DENV-1 and DENV-2 were the predominant serotypes (83.0% and 10.0%, respectively). Although the report of the Thai National Institute of Health found an upward trend of DENV-3, the incidence rate was not high (51.17 per 100 000 population)5. In 2015, there was an outbreak in many parts in Thailand, in which 146 082 cases of dengue were reported. In this region, we found the proportion of DENV-3 and DENV-4 had switched to a high prevalence during the same period, while the prevalence of DENV-1 and DENV-2 was low, as per the Annual epidemiology surveillance report 20156. The predominant serotype DENV-3 may be emerging as the cause of periodic dengue infection and affecting the non-immune population. In the past 20-year period in Thailand, the serotype DENV-3 has been reported to be predominant during the outbreaks in Bangkok in 1997 and 1998, with the number of dengue cases reported being 99 410 and 127 189 cases, respectively20. In 2016, the predominant serotype changed to DENV-4 and switched again to DENV-1 in 2017. The number of dengue cases was relatively low in these two years.

Our spatial study showed the circulation of dengue serotypes in four provinces of northern Thailand from year to year, and indicated the emergence of DENV-3 in the northern region of Thailand in 2015. This was accompanied by an increase in the total number of dengue cases reported. The circulation of dengue serotypes in Thailand and other countries is dynamic and many factors, such as the movement and number of the human and vector-borne population, environment, social life and public health organization, may have undergone dynamic changes. This information should be beneficial for long-term dengue surveillance, and future work can focus on , an emerging mosquito-borne virus, and surveillance of multiple arboviruses co-circulating in Thailand.

Acknowledgements

The authors would like to thank Mr Sangkom Vittayanan, the Director of the Regional Medical Sciences Center 1 Chiang Mai for all support. We are also grateful to staff of the clinical pathology section for all their support and cooperation.

50 Dengue Bulletin – Volume 40, 2018 Circulation of dengue serotypes in four provinces of northern Thailand during 2013–2017

References

[1] Gubler DJ. Dengue and dengue hemorrhagic fever. Clin Microbiol Rev. 1998;11:480–96. [2] Dengue haemorrhagic fever. Diagnosis, treatment, prevention and control, secondedition. Geneva: World Health Organization; 1997. (http://whqlibdoc.who.int/publications/1997/9241545003_eng. pdf, accessed 13 March 2019). [3] Ministry of Public Health. Annual epidemiological surveillance report 2003–2012. Nonthaburi: Bureau of Epidemiology, Department of Disease Control. (http://www.boe.moph.go.th/Annual/Total_Annual. html, accessed 23 March 2019). [4] Ministry of Public Health. Annual epidemiological surveillance report 2013. Nonthaburi: Bureau of Epidemiology, Department of Disease Control. (http://www.boe.moph.go.th/Annual/Total_Annual. html, accessed 23 March 2019). [5] Ministry of Public Health. Annual epidemiological surveillance report 2014. Nonthaburi: Bureau of Epidemiology, Department of Disease Control. (http://www.boe.moph.go.th/Annual/Total_Annual. html, accessed 23 March 2019). [6] Ministry of Public Health. Annual epidemiological surveillance report 2015. Nonthaburi: Bureau of Epidemiology, Department of Disease Control. (http://www.boe.moph.go.th/Annual/Total_Annual. html, accessed 23 March 2019). [7] Gubler DJ. The global emergence/resurgence of arboviral diseases as public health problems. Arch Med Res. 2002;33:330–42. [8] Lam SK. Two decades of dengue in Malaysia. Trop Med. 1994;35:195–200. [9] Sangkawibha N, Rojanasuphot S, Ahandrik S, Viriyapongse S, Jatanasen S, Salitul V et al. Risk factors in dengue shock syndrome: a prospective epidemiologic study in Rayong, Thailand. I. The 1980 outbreak. Am J Epidemiol. 1984;120:653–69. [10] Guzman MG, Kouri G, Bravo J, Soler M, Martinez E. Sequential infection as risk factor for dengue haemorrhagic fever/dengue shock syndrome (DHF/DSS) during the 1981 dengue haemorrhagic Cuban epidemic. Mem Inst Oswaldo Cruz. 1991;86:367. [11] Hunsperger EA, Yoksan S, Buchy P, Nguyen VC, Sekaran SD, Enria DA et al. Evaluation of commercially available diagnostic tests for the detection of dengue virus NS1 antigen and anti-dengue virus IgM antibody. PLoS Negl Trop Dis. 2014;8:e3171. [12] Innis BL, Nisalak A, Nimmannitya S, Kusalerdchariya S, Chongswasdi V, Suntayakorn S et al. An enzyme-linked immunosorbent assay to characterize dengue infections where dengue and Japanese encephalitis co-circulate. Am J Trop Med Hyg. 1989;40:418–27. [13] QIAGEN. QIAamp® viral RNA mini handbook, March 2018 (www.qiagen.com, accessed 13 March 2019).

Dengue Bulletin – Volume 40, 2018 51 Circulation of dengue serotypes in four provinces of northern Thailand during 2013–2017

[14] Yenchitsomanus PT, Sricharoen R Jaruthasana l, Pattanakitsakul SN, Nitayaphan S, Mongkolsapaya J et al. Rapid detection and identification of dengue viruses by polymerase chain reaction (PCR). Southeast Asian J Trop Med Public Health. 1996;27:228–36. [15] AITbiotech. abTESTMDEN 5 qPCR kit package insert. AITbiotech, The Rutherford, Singapore: 2015. [16] Saengsawang J, Nathalang O, Kamonsil M, Watanaveeradej V. Comparison of two commercial real-time PCR assays for detection of dengue virus in patient serum samples. J Clin Microbiol. 2014;52:3781–3. [17] Veeraseatakul P, Saosathan S, Chutipongvivate S. Pattern of dengue serotypes in four provinces of northern Thailand from 2003–2012. Dengue Bull. 2014;38:11–19. [18] Kalayanarooj S, Vangveeravong M, Vatcharasaevee V. Guidelines for diagnosis and treatment of dengue hemorrhagic fever. In: The celebration of Her Majesty the Queen’s 80th Birthday Anniversary, second edition. Bangkok: Ministry of Public Health, Thailand Press; 2013. [19] Chutipongvivate1 S, Prompunjai Y. Spatial circulation of dengue serotypes in eastern Thailand during 2012–2015. Int J Trop Dis Health. 2016;20:1–10. [20] Nisalak A, Endy TP, Nimmannitya S, Kalayanarooj S, Thisayakorn U, Scott RM et al. Serotype-specific dengue virus circulation and dengue disease in Bangkok, Thailand from 1973 to 1999. Am J Trop Med Hyg. 2003;68:191–202.

52 Dengue Bulletin – Volume 40, 2018 Molecular identification of dengue virus and erythrovirus B19 in three towns of the State of Amazonas, Brazil during 2013–2018

Regina Maria Pinto de Figueiredo,a# Thiago Serrão Pinto,a Kelry Mazurega de Oliveira Dinelly,a Wellyngton do Nascimento Lopes,a Luzia de Souza Granjeiro,b Carlene Barroso Caripuna,b Naylê de Oliveira Alves Mendes,b Maria Itelvina Rodrigues de Souza,c Anete Jane Cavalcante da Silva,c Valcinei Silva Amorim,c Victor Costa de Souza,d Valdinete Alves do Nascimento,d Felipe Gomes Navecad

aGerência de Virologia, Fundação de Medicina Tropical Doutor Heitor Vieira Dourado, Manaus, Amazonas, Brazil. bSecretaria Municipal de Saúde-SEMSA, Itacoatiara, AM, Brazil cSecretaria Municipal de Saúde-SEMSA, Tefé, AM, Brazil dLaboratório de Ecologia de Doenças Transmissíveis na Amazônia, Instituto Leônidas e Maria Deane - Fiocruz Amazônia, Manaus, Amazonas, Brazil

Abstract Acute febrile syndrome may be caused by different pathogens, including viruses. We used nucleic acid amplification techniques for detection of dengue virus (DENV) and erythrovirus B19 (B19V) on the serum samples of patients who attended hospital between 2013 and 2018 in the municipalities of Itacoatiara, Manacapuru and Tefé in Amazonas state, Brazil. We identified DENV-4 as the most frequent DENV serotype in the three cities. In addition, 34 patients were infected with B19V. Nucleotide sequence analysis identified genotype I for B19V and genotype Asian/American, genotype III, genotypes I and II for DENV2, DENV3 and DENV4, respectively. In summary, our data showed the circulation of different DENV serotypes/genotypes and B19V genotype I among patients suspected of arboviral infections in three different cities in the countryside of Amazonas state, Brazil. This result strengthens the necessity for differential diagnosis of acute febrile illness, even in remote areas of the Amazon rainforest.

Keywords: Dengue virus; erythrovirus B19; Amazonas; Brazil; PCR.

#E-mail: [email protected]

Dengue Bulletin – Volume 40, 2018 53 Molecular identification of dengue virus and erythrovirus B19 in State of Amazonas, Brazil

Introduction

Dengue virus has four antigenically, but distinct genetically, related serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). Each serotype is composed of phylogenetically distinct groups that have been classified into “genotypes” or “subtypes”1. They belong to the genus Flavivirus (family Flaviviridae), have a wild cycle present in Asia and Africa between non- human primates and other species of the Aedes mosquito and a well-established urban cycle between Ae. aegypti, its main vector, and humans2,3. This contributes to DENV being considered the most well-distributed arbovirus, with high-impact epidemics, and an important cause of morbidity and mortality4.

The prevalence of dengue is highest in the tropical areas of Asia and the Americas. An estimated 500,000 people with severe dengue require hospitalization each year, and about 2.5% of those severely affected die. Until epidemiological week 25 (1/1/2017 to 06/24/2017) of 2017, there were 192,123 probable cases of dengue in Brazil. The Amazonas State registered 3,063 probable cases, 3 cases of severe dengue fever and 11 dengue cases with confirmed alarm signs5.

However, in spite of this situation, it has been observed in recent years that dengue can be easily confused with other viral diseases. Oropouche virus (OROV) was detected in samples from Tefé negative for DENV6, studies in Manaus demonstrated IgM antibodies to erythrovirus B19 (B19V) in samples from patients with exanthematic disease, who tested negative for dengue during the 1998 epidemic7. Over the past years, B19V has been detected in dengue-negative samples. B19V is a single-stranded DNA virus that belongs to the Parvoviridae family, genus Erythrovirus, which is primarily transmitted by the respiratory route and is responsible for childhood infections that cause erythema infectiosum8. Only one serotype and three genotypes are recognized9,10.

In this study, we investigated the circulation of DENV and B19V in patients treated in Tefé, Manacapuru and Itacoatiara cities of Amazonas state. From January 2013 to January 2018, serum samples were collected from cases of acute febrile syndrome (0–5 days after symptom onset) and submitted to viral investigation.

Materials and methods

Study area

Tefé city (03°21’14” S; 64°42’39” W) is a municipality in the Meso region of the Amazonian Centre, and is 523 km away from Manaus (Figure 1) Its population, according to estimates by the Brazilian Institute of Geography and Statistics (IBGE)11, is 62 230 inhabitants. The city lies on the river Tefé, which is one of the tributaries of the river Solimões on its right bank.

54 Dengue Bulletin – Volume 40, 2018 Molecular identification of dengue virus and erythrovirus B19 in State of Amazonas, Brazil

The main source of income of the city is local commerce and agriculture, since several types of foodstuff are sold to other cities, including the capital, Manaus11.

Itacoatiara city (03º08’35” S; 58º6’39” W) is located to the east of Manaus, approximately 270 km distant from Manaus (Figure 1), with 86 839 inhabitants. It is considered to have the largest agricultural area and livestock in the northern region of Brazil, with 8891.9 km² and is one of the largest tourist destinations in the Amazon11.

Manacapuru city (03°17’59” S; 60°37’14” W); is located to the south of Manaus, approximately 84 km away (Figure 1). It occupies an area of 7329.234 km²; the estimated population is 94 175 inhabitants who have agriculture and livestock as main the economic activities11.

Figure 1: Map of the state of Amazonas located in the northern region of Brazil; in green the city of Itacoatiara, in red Manacapuru and orange Tefé

Adapted from IBGE11

Sample collection, molecular testing and sequencing analysis

We analysed 925 serum samples by reverse transcriptase polymerase chain reaction (RT-PCR); 195 serum samples from Itacoatiara (2014–2018), 103 samples from Manacapuru (2013) and 627 from Tefé (2013–2018). We selected patients based on the spontaneous demand of the health units of each municipality. Selected patients were those who were clinically

Dengue Bulletin – Volume 40, 2018 55 Molecular identification of dengue virus and erythrovirus B19 in State of Amazonas, Brazil diagnosed with acute febrile syndrome, with fever (measured or not) lasting for up to 2 weeks, accompanied by two or more of the following non-specific symptoms: headache, myalgia, arthralgia, retro-orbital pain, photophobia, asthenia, anorexia and general malaise. Patients were excluded if there was any restriction to peripheral venous blood collection. Patients were informed about the study and signed a consent form approved by the Ethics Committee of the Tropical Medicine Foundation Dr Heitor Vieira Dourado (FMT-HVD), number 700.915.

RNA extraction was performed using the QIAamp Viral RNA Mini Kit (QIAGEN), following the manufacturer’s instructions. After RNA extraction, reverse transcription (RT) for complementary DNA synthesis (cDNA) was performed with the AccessQuick™ RT-PCR system (Promega) kit, according to the manufacturer’s recommendations. Primers used for the PCR were selected from the conserved regions of the genes encoding the capsid/pre- membrane (C/PrM)12 structural proteins and the gene encoding the non-structural protein (NS)5 of the dengue viruses13,14. Serum samples from patients in Tefé, collected in 2013 and which tested negative for dengue by the PCR assay described above, were further tested by broad-range real-time PCR for dengue viruses, which does not discriminate between the DENV serotypes15.

Clinically screened and DENV-negative samples were submitted to a second nucleic acid extraction, DNA PureLink viral RNA/DNA Mini-Kit (Invitrogen, Carlsbad, CA). These samples were screened according to age and symptomatology. Samples from adult individuals were tested according to medical request, and we considered age groups according to the IBGE11. PCR reaction was then performed with PVP1 and PVP2 primers16 for 36 cycles consisting of 1 min at 94 °C, 1 min at 60 °C, and 1 min at 72 °C, and a final extension of 7 min at 72 °C. A second round of amplification was performed with 5 μL (diluted 1/100) of the first amplicon with primers PVP2 and PVP316.

Amplicons of DENV (C/prM and NS5 regions) and B19V were purified and sequenced in both directions, using the BigDye v3.1 Terminator Cycle Sequence Kit (Applied Biosystems, USA) at the genomics platform of Instituto Leônidas e Maria Deane (ILMD), Fiocruz Amazônia. The nucleotide sequences were submitted to the BLASTweb server (http://blast.ncbi.nlm. nih.gov/Blast.cgi)17 using the MegaBLAST algorithm, optimized for highly similar sequences.

Results

A total of 83 samples were positive for DENV by RT-PCR, 19 from Manacapuru, 21 from Itacoatiara and 43 from Tefé. DENV-2, DENV-3 and DENV-4 were present in Manacapuru, DENV-1, DENV-2 and DENV-4 in Itacoatiara, while all the serotypes were found in Tefé (Graph 1). Three patients reported travelling within 15 days of the onset of symptoms; 1 to Manaus and 2 to nearby municipalities. Patients with DENV had the most frequent symptoms: fever, headache, bone pain and ocular pain. Regarding gender, both men and women were

56 Dengue Bulletin – Volume 40, 2018 Molecular identification of dengue virus and erythrovirus B19 in State of Amazonas, Brazil infected. Considering the distribution of DENV by age group, it was observed that children and young people were more affected (Graph 1).

Of the 282 samples from Tefé collected during the year 2013, which were negative for dengue by RT-PCR, 71 were positive for DENV when tested by the real-time PCR technique.

Graph 1: Distribution of DENV serotypes by municipality and by age group during 2013–2018

39 33 28

1614 11 8 4 5 2 1 3 1 1 DENV1 DENV2 DENV3 DENV4 0-20 21-50 51-85 N.I. Itacoatiara 2 3 16 Manacapuru 1 4 14 Tefé 1 1 8 33 Age Group 28 39 11 5

In graph 1 the numbers on the bars indicate the total of each serotype of dengue by location and the number of positive cases for each age group. N.I. = not informed

Among the samples that were negative for DENV, 99 were randomly chosen and tested for B19V; of these, 34 were positive by PCR. Manacapuru, Itacoatiara and Tefé presented 18, 5 and 11 patients infected with B19, respectively (Graph 2). This virus was detected from January to August, with the highest frequency between February to May. These months, when there are frequent rains in the Amazon region, coincided with the period when there was greater frequency of dengue. However, a statistical test was not applied to confirm this observation. These patients presented with fever, headache, bone pain and ocular pain as the most frequent symptoms (Graph 2); only 3 patients had exanthema at the time of sample collection. Their ages ranged from 1 to 63 years (Graph 2). Only 1 patient from Manacapuru had a travel history to Manaus within 15 days of the onset of symptoms, and 3 patients reported working in a rural area – 1 patient from Itacoatiara and 2 from Manacapuru.

The sequences obtained with identities of 98–99% confirmed the results of genotyping of DENV (Table 1). Analysis of the genes encoding the VP2 and VP3 proteins of B19V showed that the sequences obtained were related to the genotype 1 lineage A1 (Table 1). These nucleotide sequences have been deposited at GenBank under the accession numbers MG973208 to MG973215.

Dengue Bulletin – Volume 40, 2018 57 Molecular identification of dengue virus and erythrovirus B19 in State of Amazonas, Brazil

Graph 2: Distribution of symptoms in cases of febrile illness due to B19V and positive cases by age group and municipality

30 29 26

18 15 15 11 8 5 3 4 2 2 2 1 1 … E a é u 0 0 0 R R A A A G G N N f r I I r I I E A H e a u - 2 - 7 - 5 N N M i G G A A L I V I T t C p 0 1 1 E L L P P E T a U

a

A 5 2 D I T F A A o c E Y E C N Y D R c a M E N D a T A O A n L t M O I E a R X O O B V E A B H B M Age Group Localities

In graph 2, the numerous bars indicate the total number of patients with fever, headache, etc.; the number of positive cases for each age group and by location during 2013–2018

Table 1: Results of molecular methods used for identification and genotyping of dengue virus serotypes and erythrovirus B19V

RT-PCR Collection year Samples Virus Target Genotyping

TF18; 40; 45; 73; 122; 191; M38 2013 DENV-4 C/prM GIIa

TF89; TF98; M34; M45 2013 DENV-4 NS5 GIIa

TF267 2017 DENV-2 C/prM Asian / AmericanGIIa

TF180; 189; 195; 197; 203 2016 DENV-3 C/prM GIIIa

ITA63 2015 DENV-4 NS5 GIIa ITA112 2017 DENV-4 C/prM GIa TF111; TF175; 195; 230; 231; 2013 235; 311; 320 TF241 2016 B19V VP2/ G1a VP3

58 Dengue Bulletin – Volume 40, 2018 Molecular identification of dengue virus and erythrovirus B19 in State of Amazonas, Brazil

RT-PCR Collection year Samples Virus Target Genotyping

TF236 2016 B19V VP2/ ND TF266 2017 VP3

ITA 14; ITA54; ITA75; ITA78; 2015 B19V VP2/ ND ITA 85 VP3 M09; M15; M17; M18; M22; 2013 B19V VP2/ ND M23; M26; M35; M36 VP3 M43; M50; M55; M73; M75; M76; M77; M82; M94

TF= Tefé; M= Manacapuru; ITA= Itacoatiara aThe genotypes presented were those with the highest probability, despite the small size of the PCR fragment sequenced. ND = test not done

Discussion

In our study, the DENV serotypes (DENV-1–4) in Tefé, Manacapuru and Itacoatiara were identified and characterized and DENV-4 was the most frequent. This DENV serotype was first detected in Manaus from autochthonous cases tested in 200818 and was later identified in the states of Roraima19, São Paulo20 and Rio de Janeiro21. At present, all four dengue serotypes are endemic throughout the country.

The nucleotide sequencing allowed the identification of Asian/American genotype for DENV-2, III for DENV-3, and I and II for DENV-4. With the exception of the DENV-4 genotype II, the others are frequently associated with severe manifestations of dengue22. In our study, patients did not show signs of severe dengue and fully recovered.

Analyses of the epidemiological files and medical records point to fever, headache, bone pain and ocular pain as the most frequent symptoms, and the absence of severe manifestations of dengue. There are many host and viral factors that can allow the development of severe dengue, including autoimmune diseases, diabetes and hypertension23,24. Epidemiological analyses have also shown that both men and women are infected with DENV, and children and young adults were more affected by DENV infection, as observed in previous researches conducted in Amazonas and other Brazilian states25.

In our study, 71 samples were negative by conventional RT-PCR yet positive when tested by real-time PCR, which is expected, since RT-qPCR is considered more sensitive, allowing high-yield screening, as well as delivering faster results. This indicates that it is an important diagnostic tool in regions where infections with different etiological agents can occur15.

Dengue Bulletin – Volume 40, 2018 59 Molecular identification of dengue virus and erythrovirus B19 in State of Amazonas, Brazil

We used a nested-PCR assay to investigate B19V DNA in plasma samples of patients with acute febrile syndrome in Itacoatiara, Manacapuru and Tefé, mid-size cities in the countryside of Amazonas state, Brazil. Among the samples tested, we found 29 B19V-positive children from 1 to 15 years of age. Other Brazilian studies conducted in different regions found the majority of positive cases in this same age group26. B19V-specific antibodies were detected in patients of Amazonas state for the first time among samples collected between January 1999 and December 2003, mostly in patients under 15 years7. B19 infections occurred in the period with the largest number of dengue cases as observed in other Brazilian regions27, but in other regions, dengue outbreaks occur during the summer months from November to April28, while the Amazon is marked by months of intense rainfall from December to May, followed by less rainy periods but with high temperatures. The nucleotide sequence analysis of the B19V samples from Tefé showed that the eight sequences are highly similar, at least over the region analyzed.

Our results highlights the importance of differential diagnosis, especially in remote areas such as the Amazon region where dengue, Zika, chikungunya and other lesser known arboviruses such as Mayaro and Oropouche co-circulate29. Therefore, differential diagnosis may improve the control measures against different pathogens co-circulating in a specific region.

Acknowledgements

We are grateful to Hospital Lazaro Reis of Manacapuru and Secretaria Municipal de Saúde- SEMSA of Manacapuru, Amazonas, Brazil for their support during the development of this project. Many thanks to Dr Jorge Luiz Lozano for comments and suggestions.

This study was funded by the Fundação de Amparo à Pesquisa do Estado do Amazonas – FAPEAM (www.fapeam.am.gov.br, call 001/2013 – PPSUS) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (http://www.cnpq.br).

References

[1] King CC, Chao DY, Chien LJ, Chang GJ, Lin TH, Wu YC et al. Comparative analysis of full genomic sequences among different genotypes of dengue virus type 3. Virology J. 2008;5:63. [2] Chen R, Vasilakis N. Dengue – quo tu et quo vadis? Viruses. 2011;3:1562–608. [3] Moreli ML, da Costa VG. A systematic review of molecular diagnostic methods for the detection of arboviruses in clinical specimens in Brazil and the importance of a differential diagnosis. Virology Discovery. 2013;1:1–8. [4] Dengue and severe dengue. In: World Health Organization [website]. 2018 (http://www.who.int/ mediacentre/factsheets/fs117/en, accessed 12 March 2019).

60 Dengue Bulletin – Volume 40, 2018 Molecular identification of dengue virus and erythrovirus B19 in State of Amazonas, Brazil

[5] Secretaria de Vigilância em Saúde – Ministério da Saúde. Boletim Epidemiológico. 2017;48(20):1–10 (http://portalsaude.saude.gov.br/images/pdf/2017/janeiro/15/svs2016-be003-dengue-se52.pdf, acessed 18 March 2019). [6] Naveca FG, Nascimento VA, Souza VC, de Figueiredo RMP. Human orthobunyavirus infections, Tefé, Amazonas, Brazil. PLoS Curr. 2018;10. pii: ecurrents.outbreaks.7d65e5eb6ef75664da68905c5582f7f7. [7] Figueiredo RM, Lima ML, Almeida TM, Bastos Mde S. Occurrence of parvovirus B19 in Manaus, AM. [Article in Portuguese]. Rev Soc Bras Med Trop. 2005;38:396–8. [8] Anderson MJ, Jones SE, Fisher-Hoch SP, Lewis E, Hall SM, Barlett CR et al. Human parvovirus, the cause of erythema infectiosum (fifth disease)? Lancet. 1983;1:1378. [9] Servant A, Laperche S, Lallemand F, Marinho V, De Saint Maur G, Meritet JF et al. Genetic diversity within human erythroviruses: identification of three genotypes. J Virol. 2002;76:9124–34. [10] Qiu J, Söderlund-Venermo M, Young NS. Human Parvoviruses. Clin Microbiol Rev. 2017;30:43–113. [11] Instituto Brasileiro de Geografia e Estatística-IBGE [website] (http://www.ibge.gov.br, accessed 23 March 2019). [12] Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol. 1992;30:545–51. [13] Bronzoni RV, Moreli ML, Cruz AC, Figueiredo LT. Multiplex nested PCR for Brazilian Alphavirus diagnosis. Trans R Soc Trop Med Hyg. 2004;98:456–61. [14] De Morais Bronzoni RV, Baleotti FG, Nogueira RMR, Nunes M, Figueiredo LTM. Duplex reverse transcription-PCR followed by nested PCR assays for detection and identification of Brazilian alphaviruses and flaviviruses. J Clin Microbiol. 2005;43:696–702. [15] Gurukumar KR, Priyadarshini D, Patil JA, Bhagat A, Singh A, Shah PS et al. Development of real time PCR for detection and quantitation of dengue viruses. Virol J. 2009;6:10. [16] de Mendonça MC, de Amorim Ferreira AM, dos Santos MG, de Barros JJ, von Hubinger MG, dos Santos Silva Couceiro JN. Heteroduplex mobility assay and single-stranded conformation polymorphism analysis as methodologies for detecting variants of human erythroviruses. J Virol Methods. 2008;148:40–7. [17] Zhang Z, Schwartz S, Wagner L, Miller W. A greedy algorithm for aligning DNA sequences. J Comput Biol. 2000;7:203–14. [18] Figueiredo RM, Naveca FG, Bastos MS, Melo MN, Viana SS, Mourão MP et al. Dengue type 4 virus in Manaus, Brazil. Emerg Infect Dis. 2008;14:667–9. [19] Temporao JG, Penna GO, Carmo EH, Coelho GE, do Socorro Silva Azevedo R et al. Dengue virus serotype 4, Roraima state, Brazil. Emerg Infect Dis. 2011;17:938–40. [20] Rocco IM, Silveira VR, Maeda AY, Silva SJS, Spenassatto C, Bisordi I et al. First isolation of dengue 4 in the state of São Paulo, Brazil, 2011. Rev Inst Med Trop Sao Paulo. 2012;54:49–51. [21] Campos R de M, Veiga CS, Meneses MD, de Souza LM, Fernandes CA, Malirat V et al. Emergence of dengue virus 4 genotypes II b and I in the city of . J Clin Virol. 2013;56:86–8.

Dengue Bulletin – Volume 40, 2018 61 Molecular identification of dengue virus and erythrovirus B19 in State of Amazonas, Brazil

[22] Mahboob A, Iqbal Z, Javed R, Taj A, Munir A, Saleemi MA. Clinical characteristics of patients with dengue fever: report of 48 patients in 2010. J Ayub Med Coll Abbottabad. 2010;22120–3. [23] Teixeira MG, Barreto ML, Guerra Z. Epidemiology and preventive measures of dengue. Informe Epidemiológico do SUS. 1999;8:5–33. [Article in Portuguese] [24] Whitehorn J, Simmons CP. The pathogenesis of dengue. Vaccine. 2011;29:7221–8. [25] Figueiredo RMP, Mourão MPG, Itapirema EF, de Matos AKM, Melo MN, Fonseca IS et al. Identification of dengue infection by different methods in Manaus, Amazonas, Brazil, during 1998–2012. Dengue Bull. 2013;37:36–45. [26] Wermelinger MC, Oelemann WM, Lima de Mendonça MC, Naveca FG, von Hubinger MG. Detection of human parvovirus B19 infection: a study of 212 suspected cases in the state of Rio de Janeiro, Brazil. J Clin Virol. 2002;25:223–30. [27] Di Paola N, Mesquita FS, Oliveira DBL, Villabona-Arenas CJ, Zaki Pour S, de Sousa-Capra C et al. An outbreak of human parvovirus B19 hidden by dengue fever. Clin Infect Dis. 2019;68:810–17. [28] Villabona-Arenas CJ, de Oliveira JL, de Sousa-Capra C, Balarini K, Pereira da Fonseca CR, Zanotto PM. Epidemiological dynamics of an urban dengue 4 outbreak in Sao Paulo, Brazil. Peer J. 2016;4:e1892. [29] Naveca FG, Nascimento VAD, Souza VC, Nunes BTD, Rodrigues DSG, Vasconcelos PFDC. Multiplexed reverse transcription real-time polymerase chain reaction for simultaneous detection of Mayaro, Oropouche, and Oropouche-like viruses. Mem Inst Oswaldo Cruz. 2017;112:510–13.

62 Dengue Bulletin – Volume 40, 2018 Potential breeding sites of Aedes aegypti in Maldives

B. N. Nagpal,a K. Vikram,b S. K. Gupta,b# Sana Saleem,c Nishan,c Sushil Pant,d Arvind Mathur,d Ahmed Jamsheed Mohameda

aWHO Regional Office for South-East Asia, New Delhi bNational Institute of Malaria Research, New Delhi cPublic Health Unit, Health Protection Unit Ministry of Health Maldives dWHO Country office for Maldives

Abstract Background and objective. Maldives witnessed the first outbreak of dengue in 1979 and intermittent outbreaks in 1988, 1998, 1999, 2006 and 2011. We conducted an entomological survey during October 2015 in seven islands, namely, A. Dh. Mahibadhoo, A. Dh. Maamigili, K. Huraa, K. Himmafushi, Hulhumale, Vilimale and Malé with the help of the health authorities of Maldives to ascertain the current status of dengue and to collect data on the distribution of the dengue vector. Methods. The seven islands were selected for the entomological survey on the basis of dengue cases recorded earlier. Houses, mosques, hospitals, schools, rehabilitation centres, police stations, parks, construction sites, unused boats lying on beaches, etc. were examined to look for breeding of Aedes mosquitoes. All kinds of water-holding containers, i.e. domestic containers (such as overhead tanks, rainwater harvesting tanks, curing tanks, coolers and flowerpots) and peridomestic containers (artificial fountains, tree holes, brick moulds, solid wastes such as disposable plastic/ thermocol glasses, cups and bottles) were searched for the presence or absence of Aedes larvae and/or pupae. All live mosquito larvae and pupae collected as per WHO standard methods were identified morphologically and larval indices were calculated. Results. A total of 30 distinct breeding habitats of Aedes mosquitoes were identified. Aedes breeding was recorded from every island and both the species of Aedes, i.e. Aedes aegypti and Aedes albopictus, were identified. Ae. aegypti was found breeding in domestic containers such as storage tanks, buckets and other plastic containers, whereas Ae. albopictus was found breeding in natural habitats such as tree holes, solid waste, unused boats, coconut shells, and tarpaulins in open areas. The maximum breeding was recorded from Villmale followed by Hulhumale and Malé, and minimum from Hurra Island. The overall larval indices, i.e. house index (HI), container index (CI), Breteau index (BI) and pupal index (PI), were calculated as 70, 59.7, 273 and 221, respectively. Conclusion. We observed how different types of water-holding containers, including containers for domestic use and discarded containers, contribute to the breeding of Ae. aegypti in Maldives. Dengue cases were reported from the islands where Aedes bred. Measures for disease control need to be vigorously implemented during the pre-monsoon season to prevent disease outbreak in the transmission season. For efficient control of dengue, appropriate community interventions are required. Keywords: Dengue, Maldives, entomological surveillance, control strategies.

#E-mail: [email protected]

Dengue Bulletin – Volume 40, 2018 63 Potential breeding sites of Aedes aegypti in Maldives

Introduction

Dengue infection is among the fastest emerging of all vector-borne diseases, posing a serious threat to 2.5 billion people worldwide. In the past 50 years, the incidence of dengue infection has increased 30-fold, affecting more than 120 tropical and subtropical countries. WHO estimated that, each year, 50–100 million new dengue infections occur globally1,2. Dengue fever (DF)/dengue haemorrhagic fever (DHF) is endemic in 10 of the 11 countries of the WHO South-East Asia Region except the Democratic People’s Republic of Korea; approximately 50% of the regional population are thus at risk.

Maldives witnessed the first outbreak of dengue in 1979 and, since then, it has experienced intermittent outbreaks in 1988, 1998, 1999, 2006 and 2011. In 2004, Maldives became a dengue-endemic country with periodic outbreaks of dengue during the rainy season. In 2006, a total of 2768 dengue cases and 10 deaths were reported in Maldives with a 52% increase in the number of people infected. The outbreak of dengue in 2011 was the worst, with 2909 cases and 12 deaths.

Dengue is caused by any of the four dengue virus (DENV) serotypes, i.e. DEN-1, DEN-2, DEN-3 and DEN-4, which are mainly transmitted by Aedes aegypti and Aedes albopictus. Ae. aegypti is known to be the principal dengue vector in almost all countries. In Maldives, the presence of both these vectors has been recorded earlier. They are found to breed in rainwater tanks, cemented tanks, various types of containers such as plastic trays, tree holes, overhead tanks and empty coconut shells. Environmental factors, change in climatic conditions, increased population density and water storage practices not only influence vector dynamics but also serve as potential risk factors for transmission of dengue3,4. Maldives has a tropical monsoon climate with temperatures ranging between 24 °C and 33 °C (75.2 °F and 91.4 °F), and average relative humidity between 76.9% and 82.2% throughout the year. The annual rainfall averages 2540 mm (100 inches) in the north and 3810 mm (150 inches) in the south5.

In the absence of a vaccine for dengue, there is an urgent need to focus on strengthening dengue prevention and control programmes, referred to as vector control. This survey aimed to study the distribution and abundance of the dengue vector, and identify its preferred breeding sites in Maldives so that dengue prevention and control strategies can be formulated.

Methodology

Study site

Maldives is a small country consisting of 1192 coral islands grouped in a double chain of 26 atolls, spread over approximately 90 000 sq.km (35 000 sq. miles). It lies between latitudes 1 °S and 8 °N, and longitudes 72 °E and 74°E. It had a population of about 397 397

64 Dengue Bulletin – Volume 40, 2018 Potential breeding sites of Aedes aegypti in Maldives in the year 2013 dispersed over 194 inhabited islands6. These islands are located about 200 km south-west of India. The population of Malé is 133 019.

During October 2015, a short entomological survey was conducted with the help of health authorities in Maldives to ascertain the status of dengue and collect data on the distribution and abundance of the dengue vector in seven islands, namely, A. Dh. Mahibadhoo, A. Dh. Maamigili, K. Huraa, K. Himmafushi, Hulhumale, Vilimale and Malé.

Entomological surveillance and sample collection

In this study, houses, mosques, hospitals, schools, rehabilitation centres, police stations, parks, construction sites, unused boats lying on beaches, etc. were surveyed for breeding of Aedes. Both immature and mature stages of Aedes mosquitoes were collected by the dipping and pipetting methods as per WHO standards. Adult Aedes mosquitoes were examined and collected with the help of aspirators and flashlights. All kinds of vector-breeding habitats in the study areas, such as discarded tyres, metal/plastic drums, cement tanks, metal containers, plastic buckets, plastic pots, flowerpots, mud pots, and miscellaneous small, discarded items such as tin cans, jars and plastic food containers were searched for the presence or absence of Aedes larvae and/or pupae. All live mosquito larvae and pupae collected were reared until adult mosquitoes emerged, which were identified using standard identification keys.

In the larval survey, different indices were used to record density levels of Ae. aegypti, such as house index (HI), container index (CI), Breteau index (BI) and pupal index (PI)2.

Results

A total of 100 houses were surveyed for the presence of breeding of Aedes mosquitoes and, of these, 70 were found positive for mosquito larvae/pupae. Overall, 457 containers were inspected, of which 273 containers were found positive for vector breeding. The commonly used larval indices, i.e. HI, CI, BI and PI, were calculated as 70, 59.7, 273 and 221, respectively. Island-wise HI, CI and PI were calculated. The minimum HI was recorded from K. Hurra Island (40.0) and maximum from K. Himmafushi (90.0), whereas the minimum CI was recorded from K. Hurra island (10.7) and maximum from Hulhumale island (85.5). The minimum PI was recorded from K. Hurra (0) and maximum from Hulhumale (528.6) (Table 1).

Our study indicates that breeding of Aedes mosquitoes was recorded from all the seven islands surveyed and both the species of Aedes, i.e. Ae. aegypti and Ae. Albopictus, were identified. The following 30 distinct breeding habitats of Aedes mosquitoes were identified: (i) tarpaulins, (ii) solid waste, (iii) drums, (iv) artificial fountains, (v) plastic storage tanks, (vi) wells, (vii) buckets, (viii) earthen pots, (ix) cemented tanks, (x) tree holes,

Dengue Bulletin – Volume 40, 2018 65 Potential breeding sites of Aedes aegypti in Maldives

(xi) plastic containers, (xii) plastic drums, (xiii) unused water tanks, (xiv) toilet seats (unused), (xv) rainwater harvesting pipes, (xvi) unused boats, (xvii) unused cemented tanks, (xviii) lower surface of the tanks (placed upside down), (xix) perforated pipes, (xx) discarded drinking water bottles, (xxi) iron frames, (xxii) tyre dumps, (xxiii) unused vehicles, (xiv) moulds of bricks, (xv) wooden cabinets, (xvi) stored road dividers, (xvii) coconut shells, (xviii) shafts of lift areas, (xix) plastic dustbins, and (xxx) discarded bottles.

Table 1: Island-wise entomological indices of Aedes aegypti

Dengue Contain- House Container Pupal Houses House Container No. of cases Island ers index index index checked positive positive pupae (till Oct checked (HI) (CI) (PI) 2015) A. Dh. 19 11 48 33 45 57.9 68.8 236.8 12 Mahibadhoo A.Dh. 17 14 55 42 35 82.4 76.4 205.9 28 Maamigili K. Huraa 20 8 122 13 0 40.0 10.7 0.0 2 K. Himmafushi 10 9 80 62 35 90.0 77.5 350.0 2 Hulhumale 7 6 55 47 37 85.7 85.5 528.6 252 Villimale 20 17 72 58 52 85.0 80.6 260.0 7 Malé 7 5 25 18 17 71.4 72.0 242.9 400

Ae. aegypti was found breeding in domestic containers such as storage tanks, buckets and other plastic containers whereas Ae. albopictus was found breeding in natural habitats such as tree holes, solid waste, unused boats, coconut shells, tarpaulins in open areas, etc.

Island-wise breeding habitats

A.Dh. Mahibadhoo. On this island, water storage tanks (cemented and plastic), solid waste (glass and plastic water bottles and small containers), artificial fountains and wells were found positive for breeding of Aedes during the survey. The maximum breeding was recorded from solid waste followed by water storage tanks.

A. Dh. Maamigili. On A.Dh. Maamigili island, tree holes, plastic containers (buckets, drums), unused water storage tanks (plastic), solid waste (junk), artificial fountains and wells were found positive for breeding of Aedes during the survey. The maximum breeding was recorded from water storage containers inside houses and unused water storage containers outside houses.

K. Huraa. On this island, discarded toilet seats, water tanks, rainwater harvesting pipes, solid waste and barrels were found positive for breeding of Aedes during the survey. Maximum breeding was recorded from discarded toilet seats.

66 Dengue Bulletin – Volume 40, 2018 Potential breeding sites of Aedes aegypti in Maldives

K. Himmafushi. Discarded toilet seats, water tanks, rainwater harvesting pipes, solid waste and barrels were found positive for breeding of Aedes during the survey. The maximum breeding was recorded from discarded toilet seats.

Hulhumale. Before starting the survey in Hulhumale, a meeting was conducted with the Director of Constructions to identify the ongoing construction sites. During the survey, tyre dumps, tarpaulins, unused water storage tanks, unused vehicles and plastic containers were

Figure 1: Aedes larvae breeding sites: (a) rainwater harvesting tanks, (b) plastic storage tanks, (c) artificial fountains, (d) toilet seats (unused), (e) empty coconut shells, (f) brick moulds, (g) tree holes, (h) discarded drinking water bottles, (i) solid waste, (j) unused water tanks, (k) unused boats, and (l) tyre dumps

Dengue Bulletin – Volume 40, 2018 67 Potential breeding sites of Aedes aegypti in Maldives found positive for breeding of Aedes. The maximum breeding was recorded from containers and water storage tanks followed by drums and solid waste at construction sites. In October 2015, a total of 82 cases of dengue were reported by the Public Health Department of Maldives in Hulhumale.

Malé. Construction work was ongoing in Malé island as well. Moulds of bricks, wooden cabinets, stored road dividers, water storage cemented tanks and solid waste at construction sites were found positive for dengue. Malé reported the largest number of dengue cases, i.e. 102 in June 2015 and 170 in July 2015.

Villimale. Solid waste (plastic buckets), coconut shells, shafts of lift areas, tyre dumps, plastic dustbins and unused boats were found positive for breeding of Aedes at Villimale during the survey.

Discussion

Dengue is a vector-borne disease related to environmental conditions, particularly to the domestic habits of the population. It is well known that Ae. aegypti and Ae. albopictus act as vectors for dengue. Larvae of these two species were found in clean and clear water in various types of artificial and natural containers7. Larvae of Ae. aegypti and Ae. albopictus were collected from 30 different breeding sites, which points to lack of entomological surveillance. Ae. albopictus was found to be predominant in outdoor areas during the entomological survey, which is consistent with the findings of the study by Rao, which found that Ae. albopictus breeds in containers and in both natural and artificial habitats8, whereas Ae. aegypti was collected mainly from domestic areas.

Water storage tanks and containers, rainwater harvesting pipes and tanks, discarded toilet seats, solid waste and discarded water bottles were the common breeding sites, whereas moulds of bricks, stored road dividers, shafts of lift areas, tarpaulins and wooden cabinets were the uncommon breeding sites in Maldives. These findings are different from the sites reported by Sharma et al. from Lakshadweep Island, in which cemented tanks, used tyres, bird pots, tree holes, etc., as the preferred containers for breeding of Aedes mosquitoes9. In Maldives, rainwater harvesting is a common practice due to scarcity of water, which provides favourable opportunities for Aedes mosquitoes to breed and proliferate. Studies from India have shown that due to irregular water supply and inadequate water shortage facilities, residents store water in various types of containers over prolonged periods of time and these containers act as major sites for mosquito breeding3,4,10. Plastic water storage tanks of 2000–5000 L capacity were common breeding sites on every island, which are perhaps mother foci/key containers for Aedes mosquitoes.

Artificial containers such as plastic containers and discarded water bottles that can hold water for a long time may act as preferred breeding sites for Aedes mosquitoes. Such probable breeding sites provide a means of storing potable water in communities where the supply

68 Dengue Bulletin – Volume 40, 2018 Potential breeding sites of Aedes aegypti in Maldives of pipe water is absent or scarce3. The types of containers, water quality and conditions of water containers are also important for mosquito breeding11.

Our results show that the entomological indices were above the critical level in most of the areas surveyed. There was high infestation of artificial water containers by larvae of Aedes mosquitoes, which may lead to an outbreak of dengue. These findings should caution the health authorities as well as communities to take timely and necessary control measures to prevent future outbreaks of dengue.

In Maldives, very few entomological studies have been carried out to know the breeding sites of Aedes mosquitoes, which were collected from different sources such as rainwater tanks, cemented construction areas, agricultural pits12, a variety of small containers, tree holes, rooftop water tanks, empty coconut shells and plastic trays kept below flowerpots13. Although these species have great epidemiological importance, not many surveys have been carried out to focus on larval distribution, their breeding habitats as well as Aedes larval indices. This study aimed to find the actual distribution of dengue vectors and their breeding habitats in Maldives.

Appropriate community interventions are needed for efficient control of dengue. Some key factors for community participation are to educate people on the breeding habitats of Aedes mosquitoes, how to reduce larval source activities and motivate people to shoulder responsibility for controlling mosquitoes in and around their homes. For integrated development of community-based vector control programmes, both the larval ecology of Ae. aegypti and sociological importance of the various container habitats should be considered.

Conclusion

In Maldives, an upsurge in dengue cases has been observed in the past few years, with frequent occurrence of dengue outbreaks. Our study observed how different types of water-holding containers, including containers for domestic use and discarded containers, contribute to the breeding of Ae. aegypti, which may act as a vector for transmission of dengue. Dengue cases were also reported from islands where Aedes breed. Disease control measures need to be implemented vigorously during the pre-monsoon season to prevent outbreaks in the transmission season.

Places with any larval indices (HI, BI, CI, PI) more than 10 are at high risk for dengue transmission. Of these indices, PI is the key predictor of an outbreak of dengue as a single pupa after emergence can transmit the disease. Except K. Hurra island, all the indices recorded were high, especially PI. On the basis of entomological indices, Hulhumale island was found to be at the highest risk for an outbreak of dengue compared to the other islands.

An effective vector control programme should be established in Maldives by using the knowledge gained from this study to prevent outbreaks of dengue in future.

Dengue Bulletin – Volume 40, 2018 69 Potential breeding sites of Aedes aegypti in Maldives

Recommendation

To formulate appropriate control strategies, surveys should be carried out in each island during the non-transmission season to effectively identify mother foci/key containers.

Conflicts of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Acknowledgments

The authors are thankful to Dr Ahmed Jamsheed Mohamed from the WHO Regional Office for South-East Asia for giving support to conduct entomological surveillance in Maldives. Thanks are also due to Dr Arvind Mathur (WHO Representative), Dr Sathu and Dr Igo from the WHO Country Office Maldives for their kind cooperation and support to the study. I am also thankful to Ms Maimoona, Dr Nazla, Dr Asma and Dr Nishan from the Ministry of Health, Maldives for arranging the field visits and providing the necessary reports, data and other material on dengue in Maldives. Special thanks are due to Mr Faisal, Mr Shiyam, Rashy and other field staff of the different islands, who helped in surveillance in difficult situations. This acknowledgement would remain incomplete without acknowledging the Indian Council of Medical Research, Ministry of Health and Family Welfare and Director, National Institute of Malaria Research, India for granting permission to Dr. B.N. Nagpal to visit Maldives for this study.

References

[1] Dengue and severe dengue, Fact sheet, updated April 2017. In: World Health Organization [website] (http://origin.who.int/mediacentre/factsheets/fs117/en/, accessed 8 March 2019). [2] Dengue haemorrhagic fever: diagnosis, treatment, prevention and control, second edition. Geneva: WHO; 1997. [3] Vikram K, Nagpal BN, Pande V, Srivastava A, Saxena R, Singh H et al. Detection of dengue virus in individual Aedes aegypti mosquitoes in Delhi, India. J Vector Borne Dis. 2015;52:129–33. [4] Vikram K, Nagpal BN, Pande V, Srivastava A, Gupta SK, Anushrita et al. Comparison of Ae. aegypti breeding in localities of different socio-economic groups of Delhi, India. Int J Mosq Res. 2015;2:83–8. [5] Climate Male: climate data 1981–2019 (http://www.tutiempo.net/en/Climate/Male/435550.htm, accessed 9 March 2019). [6] World Population Review (http://worldpopulationreview.com/countries/maldives-population/) [7] Rattanarithikul R, Panthusiri P. Illustrated keys to the medically important mosquitos of Thailand. Southeast Asian J Trop Med Public Health. 1994;25 (Suppl. 1):1–66.

70 Dengue Bulletin – Volume 40, 2018 Potential breeding sites of Aedes aegypti in Maldives

[8] Rao BB. Larval habitats of Aedes albopictus (Skuse) in rural areas of Calicut, Kerala India. J Vector Borne Dis. 2010;47:175–7. [9] Sharma SK, Hamzakoya KK. Geographical spread of Anopheles stephensi, vector of urban malaria, and Aedes aegypti, vector of dengue/DHF, in the Arabian Sea Islands of Lakshadweep, India. Dengue Bull. 2001;25:88–91. [10] Bhat MA, Krishnamoorthy K. Entomological investigation and distribution of Aedes mosquitoes in Tirunelveli, Tamil Nadu, India. Int J Curr Microbiol App Sci. 2014;3:253–60. [11] Chen CD, Lee HL, Stella-Wong SP, Lau KW, Sofian-Azirun M. Container survey of mosquito breeding sites in a university campus in Kuala Lumpur, Malaysia. Dengue Bull. 2009;33:187–93. [12] Shaheem I, Afeef A. Dengue and dengue haemorrhagic fever and its control in Maldives. Dengue Bull. 1999;23:30–3. [13] Prasittisuk C, Andjaparidze AG, Kumar V. Current status of dengue/dengue haemorrhagic fever in WHO South-East Asia Region. Dengue Bull. 1998;22:1–15.

Dengue Bulletin – Volume 40, 2018 71 Trends of dengue fever in Madhya Pradesh, India

Trends of dengue fever in Madhya Pradesh, India

Mrigenedra P. Singh,a Sunil K. Chand,a# Arvind Jaiswal,a Ramesh C. Dhimanb

aICMR-National Institute of Malaria Research Field Unit, NIRTH Campus, Nagpur Road, Post Garha, Jabalpur 482003, Madhya Pradesh, India bICMR-National Institute of Malaria Research, Department of Health Research, Sector 8, Dwarka, New Delhi 110077, India

Abstract Background. Dengue is the most important arboviral disease. Socioeconomic factors demographic transition, urbanization and climatic factors that affect vector densities are important drivers of dengue transmission. We present the longitudinal trend of dengue fever in rural and urban habitats of central India, and an analysis of the determinants of disease transmission in the community. Methodology. Morbidity data on dengue fever were collected from the state programme office and disease sentinel sites of Madhya Pradesh (MP). Data from the worst-affected districts were analysed to determine the risk factors for infection. A breeding survey was also carried out in the worst-affected villages of district Mandla and Narsinghpur. Results. Out of 51 districts in MP, dengue fever cases were reported from 48 districts during the period 2010–2015, of which 11 districts were the most affected. An increasing trend of dengue morbidity was observed during this period, which was statistically significant (P=0.001). A high incidence was reported among the overall urban population compared with the rural population. However, in districts Mandla and Narsinghpur, significantly more cases of dengue fever were reported from the rural than the urban population (P=0.0118). Dengue fever was more prevalent in males and those in the young active age group (15–35 years). Incidence was lowest among children ≤5 years and those >50 years old. Conclusions. Dengue is on rise in MP. Storage of water in open containers is the main risk factor. There is an urgent need for awareness programmes to motivate people to better manage water storage to keep mosquitoes from breeding in rural and urban areas.

Keywords: Dengue, Aedes aegypti, Aedes albopictus, Madhya Pradesh.

Introduction

Dengue is a re-emerging mosquito-borne viral disease, transmitted by Aedes aegypti and Ae. albopictus1. Socioeconomic factors, demographic transition, urbanization and climatic

#E-mail: [email protected]

72 Dengue Bulletin – Volume 40, 2018 Trends of dengue fever in Madhya Pradesh, India factors are important drivers of dengue transmission2. The global burden of the disease is uncertain due to lack of surveillance. However, 2.2 – 3.34 million cases of dengue fever were reported annually during 2010 and 2016 from different regions of the world, with a sharp increase in the number of cases and explosive outbreaks in recent years3. Another estimate of the global burden of dengue morbidity reported 58.4 million (23.6–121.9 million) apparent cases in 20134. The Americas, South-East Asia and Western Pacific regions are the most seriously affected3. Most countries rely on a passive surveillance system, and therefore infections resulting in less severe dengue disease or unusual clinical presentations are likely to go undiagnosed etiologically5.

In recent years, a large increase in dengue cases has been observed in several states of India, including Delhi, Punjab, Haryana and West Bengal6,7. Several epidemiological studies of dengue fever outbreaks have been conducted from southern India8–13. Dengue fever outbreaks in rural populations have also been investigated in the states of Maharashtra14,15, Haryana16,17, Uttar Pradesh18, West Bengal19,20, Madhya Pradesh (MP) and Chhattisgarh21,22.

Due to water shortages, people tend to store water in whatever manner possible. The use of containers to store water for domestic purposes, especially as they are often uncovered, as well as unplanned urbanization, household waste, inadequate water supply and a poor sewerage system create favourable conditions for breeding of Aedes mosquitoes23,24. Rural areas with a low population density also experience severe epidemics25,26 but the data from rural areas are scarce.

We present the longitudinal trend of dengue fever during 2010 to 2015 in rural and urban areas of MP. The study also stratifies the distribution of dengue cases by year, age, sex, area of residence and seasons.

Materials and methods clinically suspected cases were tested for dengue infection at the health facility by IgM antibody capture enzyme-linked immunosorbent assay (MAC-ELISA, NS1 ELISA) as per the national guidelines. It is mandatory to report all confirmed dengue cases diagnosed and treated at any health facility to the disease sentinel sites of the district concerned. District-wise data on dengue fever for the period 2010–2015 were procured from the State Programme Office, Bhopal (MP) and patients’ addresses, date the infection was diagnosed, age and sex data from the 11 high-incidence districts (Figure 1) were collected. These were reported to the official passive surveillance and reporting system at sentinel sites. Larval surveys were carried out in the affected villages of Narsinghpur and Mandla districts from December 2015 to February 2016 in the post-outbreak period. Vector-breeding indices such as house index (HI), container index (CI), Breteau index (BI) and pupal index (PI) were calculated followed the National Vector Borne Disease Control Programme (NVBDCP) guidelines27 described below.

Dengue Bulletin – Volume 40, 2018 73 Trends of dengue fever in Madhya Pradesh, India

Figure 1: Map of Madhya Pradesh, India showing districts with a high incidence of dengue fever

HI (percentage of houses infected with larvae and/or pupae) = (number of houses infected /number of houses inspected) x 100

CI (percentage of water-holding containers infected with larvae and/or pupae) = (number of positive containers/number of containers inspected) x 100

BI (number of positive containers per 100 houses inspected) = (number of positive containers/number of houses inspected) x 100

PI (number of pupae per 100 houses) = (number of pupae/number of houses inspected) x 100.

Morbidity data were coded numerically and stratified by area of residence (urban and rural), age groups (≤5 years, 6–14 years, 15–25 years, 26–35 years, 36–50 years and >50 years), sex (male and female) and seasons (monsoon [June, July, August], post monsoon [September, October, November], spring [December, January, February] and summer [March, April, May]). The cumulative incidence rate per 100 000 person-years was calculated and odds ratios (ORs) with 95% confidence intervals (CIs) were used to analyse the difference in incidence rates between areas of residence, age groups, sex and seasons. Non-parametric trend analysis was performed to test the trend of dengue morbidity over the period. Statistical analysis was done in R 3.3.2 for Windows.

74 Dengue Bulletin – Volume 40, 2018 Trends of dengue fever in Madhya Pradesh, India

Results

Out of 51 districts in MP, dengue fever cases were reported from 48 districts during the period 2010–2015 (Figure 2). During 2013–2015, several outbreaks of dengue fever were observed in 11 districts of MP (Table 1). An increasing trend in dengue morbidity was observed (Figure 3), which was statistically significant (P=0.001). Overall, there was a higher incidence of dengue fever in urban populations when compared with rural populations (OR 4.3; 95% CI: 2.1–10.2; P<0.0001). However, in Mandla and Narsinghpur districts, significantly more cases of dengue fever were reported from rural than urban areas (OR 2.0; 95% CI: 1.1–3.7; P=0.0118).

Further, age- and sex-wise analysis of the data from these high-incidence districts showed that more dengue cases occurred among men than women (65.5% vs 34.5%; 95% CI: 64.2–66.9). The mean age of infected persons was 24.1 ± 15.1 years (range 6 months to 87 years). The highest rate of infection was observed among young active age groups of 15–25 years (30%; 95% CI: 28–32) and 26–35 years (26.0%; 95% CI: 24.2–27.9), followed by 36–50 years (18.8%; 95% CI: 17.2–20.5). However, lower rates of infection were observed among children aged 6–14 years (12.7%; 95% CI: 11.3–14.2) and individuals >50 years old (7.4%; 95% CI: 6.4–8.6). Infection rates were lowest among children ≤5 years (5.0%; 95% CI: 4.2–6.1).

Figure 2: Pie chart showing the distribution of dengue fever cases in the districts of Madhya Pradesh, India from 2010 to 2015

Dengue Bulletin – Volume 40, 2018 75 Trends of dengue fever in Madhya Pradesh, India

Figure 3: Trends of dengue fever cases in 11 high-incidence districts of Madhya Pradesh, India

Table 1: Incidence of dengue fever in 11 highly effected districts of Madhya Pradesh, India (year 2010–2015)

Year Population Total District (32) 2010 2011 2012 2013 2014 2015 N (ci) N (ci)* N (ci) N (ci) N (ci) N (ci) N (ci)

Ashoknagar 691 387 (R)** 0 0 0 0 4 (0.58) 40 (5.79) 44 (6.36)

153 684 (U) 0 1 (0.65) 0 0 6 (3.90) 78 (50.75) 85 (55.31)

845 071 (T) 0 (0) 1 (0.12) 0 (0) 0 (0) 10 (1.18) 118 (13.96) 129 (15.26)

Bhopal 454 010 (R) 0 0 0 0 0 0 0

191 7051 (U) 57 (2.97) 10 (0.52) 114 (5.95) 158 (8.24) 706 (36.83) 223 (11.63) 1 268 (66.14)

2 371 061 (T) 57 (2.40) 10 (0.42) 114 (4.81) 158 (6.66) 706 (29.78) 223 (9.41) 1 268 (53.48)

Chhindwada 158 5739 (R) 0 0 0 38 (2.40) 42 (2.65) 0 80 (5.04)

505 183 (U) 1 (0.20) 0 0 79 (15.64) 31 (6.14) 2 (0.40) 113 (22.37)

2 090 922 (T) 1(0.05) 0 (0) 0 (0) 122 (5.60) 73 (3.49) 2 (0.10) 193 (9.23)

76 Dengue Bulletin – Volume 40, 2018 Trends of dengue fever in Madhya Pradesh, India

Year Population Total District (32) 2010 2011 2012 2013 2014 2015 N (ci) N (ci)* N (ci) N (ci) N (ci) N (ci) N (ci)

Gwalior 758 244 (R) 4 (0.53) 2 (0.26) 2 (0.26) 23 (3.03) 13 (1.71) 62 (8.18) 106 (13.98)

1 273 792 (U) 24 (1.88) 16 (1.26) 23 (1.81) 150 (11.78) 150 (11.78) 404(31.72) 767 (60.21)

2 032 036 (T) 28 (1.38) 18 (0.89) 25 (1.23) 173 (8.51) 163 (8.02) 466 (22.93) 873 (42.96)

Indore 848 988 (R) 0 0 0 0 2 (0.24) 0 2 (0.24)

2 427 709 (U) 1(0.04) 2 (0.08) 0 11 (0.45) 158 (6.51) 155 (6.38) 327 (13.47)

3 276 697 (T) 1 (0.03) 2 (0.06) 0 (0) 11 (0.34) 160 (4.88) 155 (4.73) 329 (10.04)

Jabalpur 1 023 255 (R) 7 (0.68) 1 (0.10) 0 8 (0.78) 37 (3.62) 9 (0.88) 62 (6.06)

1 440 034 (U) 9 (0.62) 6 (0.42) 2 (0.14) 36 (2.50) 114 (7.92) 32 (2.22) 199 (13.82)

2 463 289 (T) 16 (0.69) 7 (0.28) 2 (0.08) 44 (1.79) 151 (6.13) 41 (1.66) 261 (10.60)

Mandla 924 716 (R) 0 0 0 282 (30.50) 80 (8.65) 0 362 (39.15)

130 189 (U) 0 0 0 35 (26.88) 1 (0.77) 3 (2.30) 39 (29.96)

1 054 905 (T) 0 (0) 0 (0) 0 (0) 317 (30.05) 81 (7.68) 3 (0.28) 401 (38.01)

Morena 1 495 508 (R) 0 0 0 28 (1.87) 0 12 (0.80) 40 (2.67)

470 462(U) 14 (2.98) 3 (0.64) 0 89 (18.92) 11 (2.34) 76 (16.15) 193 (41.02)

1 965 970 (T) 14 (0.71) 3 (0.15) 0 (0) 117 (5.95) 11 (0.56) 88 (4.48) 233 (11.85)

Narsinghpur 888 314 (R) 0 26 (2.93) 64 (7.20) 28 (3.15) 191 (21.50) 10 (1.13) 319 (35.91)

203 540 (U) 2 (0.98) 0 0 3 (1.47) 15 (7.37) 6 (2.95) 26 (12.77)

1 091 854 (T) 2 (0.18) 26 (2.38) 64 (5.86) 31 (2.84) 206 (18.87) 16 (1.19) 345 (31.60)

Sagar 1 669 662 (R) 0 0 0 0 38 (2.28) 34 (2.04) 72 (4.31)

708 796 (U) 1 (0.14) 0 0 1 (0.14) 73 (10.30) 44 (6.21) 119 (16.79)

2 378 458 (T) 1 (0.04) 0 (0) 0 (0) 1 (0.04) 111 (4.67) 78 (3.28) 191 (8.03)

Shivpuri 1 430 627 (R) 0 0 0 11 (0.77) 4 (0.28) 4 (0.28) 19 (1.33)

295 423 (U) 2 (0.68) 2 (0.68) 0 38 (12.86) 70 (23.69) 514 (173.99) 626 (211.90)

1 726 050(T) 2 (0.12) 2 (0.12) 0 (0) 49 (12.84) 74 (4.29) 518 (30.01) 645 (37.37)

Total 11 770 450 (R) 11 (0.09) 29 (0.25) 66 (0.56) 418 (3.55) 411 (3.49) 171 (1.45) 1 106 (9.40)

952 5863 (U) 111 (1.17) 40 (0.42) 139 (1.46) 600 (6.30) 1 335 (14.01) 1 537 (16.14) 3 762 (39.49)

21 296 313 (T) 122 (0.57) 69 (0.32) 205 (0.96) 1 018 (4.78) 1 746 (8.20) 1 708 (8.02) 4 868 (22.86)

* ci: cumulative incidence (/100 000 person years); ** (R): rural; (U): urban; (T): total

Dengue Bulletin – Volume 40, 2018 77 Trends of dengue fever in Madhya Pradesh, India

Figure 4: Month-wise distribution of dengue fever cases in 11 high-incidence districts of Madhya Pradesh, India

Most cases were recorded during the monsoon (June–August) and post-monsoon seasons (September–November). More than 70% of the cases were reported between September and November (Figure 4). Only 6% of cases were reported during December to March.

The results of the vector breeding survey in two districts, namely, Mandla and Narsinghpur, showed that the HI was 17.5, CI 21.5, BI 12.2 and PI 39.7. The larvae were transported to the laboratory and when adult mosquitoes emerged, 74% belonged to the Aedes spp. and 36% to the Culex spp. Notably, among Aedes, Ae. aegypti accounted for 94.6%, Ae. albopictus 3.6% and Ae. vittatus 1.8%.

Discussion and conclusion dengue fever cases in India are showing an rising trend continuously since 2010 except for a little dip in 2014 (Figure 5). The largest number of cases was reported in this period from West Bengal, Delhi, Maharashtra, Odisha, Karnataka and Gujarat. MP ranks fourteenth countrywide in the number of dengue cases reported during this time period7.

This epidemiological investigation showed that dengue was more prevalent in males and young active age groups, which meant that the most active population appear to be at a higher risk of infection. A similar observation was reported in another outbreak investigation conducted in the rural areas of West Bengal28. Most of the cases were reported during the monsoon and post-monsoon seasons. However, a few outbreaks from the Mandla (June 2013) and Narsingpur (April–May 2014) districts of MP were reported by the National Institute for Research in Tribal Health Jabalpur29,30.

78 Dengue Bulletin – Volume 40, 2018 Trends of dengue fever in Madhya Pradesh, India

Figure 5: Box plot showing the number of dengue cases in India during 2010–2015

Erratic water supply appears to be a precursor of dengue infection. The vector breeding indices during the post-outbreak period are considerably high. Small-to-large water containers in households, such as cement cisterns and tanks, mud/metal pots, etc. were very common, which favoured breeding of vector mosquitoes. These containers are mostly kept uncovered for sanitation, bathing and use by cattle. Newly constructed buildings and concrete roads in the villages indicate urbanization. People store large amounts of water to manage the irregular water supply mainly in the post-monsoon and summer seasons.

The practice of observing “dry day” once a week as in Maharashtra should be carried out. On this day, all the water pots are emptied, cleaned and refilled with fresh water to get rid of potential mosquito breeding sites6.

To control the spread of dengue fever, urgent preventive action is required. There is an urgent need for an awareness programme to motivate people to better manage water storage to prevent mosquitoes from breeding in rural and urban areas. Evidence-based community mobilization programmes can add to the effectiveness of vector control efforts31. There is also a need for better clinical and entomological surveillance.

Dengue Bulletin – Volume 40, 2018 79 Trends of dengue fever in Madhya Pradesh, India

The limitation of this study is the non-availability of data on other potential risk factors, such as socioeconomic status, occupation, time spent outdoors during daytime hours, environmental factors, use of antivector measures, etc. Such data are not routinely collected at sentinel sites by the programme. Therefore, there is a need to undertake a prospective study to investigate all the potential exposure variables responsible for the transmission of disease in the community.

Ethical statement

No human subjects were involved in this investigation. The samples were collected and cases diagnosed and treated by state health authorities as part of a routine surveillance system. The analysis is based on secondary data sources from state health authorities. Therefore, no ethical issues were involved.

References

[1] Schaffner F, Mathis A. Dengue and dengue vectors in the WHO European Region: past, present, and scenarios for the future. Lancet Infect Dis. 2014;14:1271–80. [2] Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL et al. The global distribution and burden of dengue. Nature. 2013;496:504–7. [3] Dengue and severe dengue. In: World Health Organization [website]. 13 September 2018 (http:// www.who.int/mediacentre/factsheets/fs117/en/, accessed 13 March 2019). [4] Stanaway JD, Shepard DS, Undurraga EA, Halasa YA, Coffeng LE, Brady OJ et al. The global burden of dengue: an analysis from the Global Burden of Disease Study 2013. Lancet Infect Dis. 2016;16:712–23. [5] Ooi EE, Gubler DJ. Dengue in : epidemiological characteristics and strategic challenges in disease prevention. Cad Saude Publica. 2009;25 Suppl 1:S115–24. [6] Travasso C. Dengue cases in India doubled in 2014–15. BMJ. 2015;351:h6676. [7] NVBDCP. Dengue cases and deaths in the country since 2010 (http://nvbdcp.gov.in/den-cd.html, accessed 16 May 2016). [8] Kalappanvar NK, VinodKumar CS, Basavarajappa KG, Chandrasekhar G, Sanjay D. Outbreak of dengue infection in rural Davangere, Karnataka. Asian Pac J Trop Med. 2013;6:502–3. [9] Samuel PP, Thenmozhi V, Tyagi BK. A focal outbreak of dengue fever in a rural area of Tamil Nadu. Indian J Med Res. 2007;125:179–81. [10] Arunachalam N, Murty US, Kabilan L, Balasubramanian A, Thenmozhi V, Narahari D et al. Studies on dengue in rural areas of Kurnool District, Andhra Pradesh, India. J Am Mosq Control Assoc. 2004;20:87–90.

80 Dengue Bulletin – Volume 40, 2018 Trends of dengue fever in Madhya Pradesh, India

[11] Tewari SC, Thenmozhi V, Katholi CR, Manavalan R, Munirathinam A, Gajanana A. Dengue vector prevalence and virus infection in a rural area in south India. Trop Med Int Health. 2004;9:499–507. [12] Abdul Kader MS, Kandaswamy P, Appavoo NC, Anuradha. Outbreak and control of dengue in a village in Dharmapuri, Tamil Nadu. J Commun Dis. 1997;29:69–71. [13] Norman G, Theodre A, Joseph A. An insular outbreak of dengue fever in a rural south Indian village. J Commun Dis. 1991;23:185–90. [14] Mehendale SM, Risbud AR, Rao JA, Banerjee K. Outbreak of dengue fever in rural areas of Parbhani district of Maharashtra (India). Indian J Med Res. 1991;93:6–11. [15] Batra P, Saha A, Chaturvedi P, Vilhekar KY, Mendiratta DK. Outbreak of dengue infection in rural Maharashtra. Indian J Pediatr. 2007;74:794–5. [16] Kumar A, Sharma SK, Padbidri VS, Thakare JP, Jain DC, Datta KK. An outbreak of dengue fever in rural areas of northern India. J Commun Dis. 2001;33:274–81. [17] Jamaluddain M, Saxena VK. First outbreak of dengue fever in a typical rural area of Haryana state in northern India. J Commun Dis. 1997;29:169–70. [18] Tripathi P, Kumar R, Tripathi S, Tambe JJ, Venkatesh V. Descriptive epidemiology of dengue transmission in Uttar Pradesh. Indian Pediatr. 2008;45:315–8. [19] Gilotra SK, Bhattacharya NC. Mosquito vectors of dengue—chikungunya viruses in a rural area near Calcutta. Bull Calcutta Sch Trop Med. 1968;16:41–2. [20] Banerjee S, Aditya G, Saha GK. Household wastes as larval habitats of dengue vectors: comparison between urban and rural areas of Kolkata, India. PLoS One. 2015;10:e0138082. [21] Barde PV, Kori BK, Shukla MK, Bharti PK, Chand G, Kumar G et al. Maiden outbreaks of dengue virus 1 genotype III in rural central India. Epidemiol Infect. 2015A;143:412–8. [22] Barde PV, Shukla MK, Kori BK, Chand G, Jain L, Varun BM et al. Emergence of dengue in tribal villages of Mandla district, Madhya Pradesh, India. Indian J Med Res. 2015B;141:584–90. [23] Barreto ML, Teixeira MG. Dengue fever: a call for local, national, and international action. Lancet. 2008;372:205. [24] Gubler DJ. Cities spawn epidemic dengue viruses. Nat Med. 2004;10:129–30. [25] Cummings DA, Irizarry RA, Huang NE, Endy TP, Nisalak A, Ungchusak K et al. Travelling waves in the occurrence of dengue haemorrhagic fever in Thailand. Nature. 2004;427:344–7. [26] Chareonsook O, Foy HM, Teeraratkul A, Silarug N. Changing epidemiology of dengue hemorrhagic fever in Thailand. Epidemiol Infect. 1999;122:161–6. [27] NVBDCP. Guidelines for integrated vector management for control of dengue/ dengue haemorrhagic fever (http://nvbdcp.gov.in/Doc/dengue_1_.%20Director_Desk%20DGHS%20meeting%20OCT%20 06.pdf, accessed 16 May 2016). [28] Biswas DK, Bhunia R, Basu M. Dengue fever in a rural area of West Bengal, India, 2012: an outbreak investigation. WHO South East Asia J Public Health. 2014;3:46–50.

Dengue Bulletin – Volume 40, 2018 81 Trends of dengue fever in Madhya Pradesh, India

[29] Annual report 2013–14. Jabalpur, MP: Regional Medical Research Centre for Tribals, Indian Council of Medical Research (http://www.nirth.res.in/publications/annual_report/annual_report_2013-14.pdf, accessed 13 March 2019). [30] Annual report 2014–15. Jabalpur, MP: National Institute for Research in Tribal Health, Indian Council of Medical Research (http://www.nirth.res.in/publications/annual_report/2014-15.pdf, accessed 13 March 2019). [31] Andersson N, Nava-Aguilera E, Arosteguí J, Morales-Perez A, Suazo-Laguna H, Legorreta-Soberanis J et al. Evidence based community mobilization for dengue prevention in and (Camino Verde, the Green Way): cluster randomized controlled trial. BMJ. 2015;351:h3267. [32] Census 2011. Government of India, Ministry of Home Affairs. Office of the Registrar General and Census Commissioner, India. 2011 Census Data (http://censusindia.gov.in/pca/pcadata/Houselisting- housing-MP.html, accessed 13 March 2019).

82 Dengue Bulletin – Volume 40, 2018 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha

Animesha Rath,ab Rupenangshu K Hazrab#

aICMR-Regional Medical Research Centre, Bhubaneswar – 751023, India bSchool of Biotechnology, Kalinga Institute of Industrial Technology, Bhubaneswar – 751024, India

Abstract Aedes albopictus is an indigenous species in India and is believed to have originated in the tropical forest of South-east Asia. In general, it breeds outdoors. It is more prevalent in periurban areas and is associated with dengue fever. We undertook a study in selected districts of Odisha to identify the distribution of various breeding sources. The usefulness of indices in the immature stage of the mosquito was assessed to identify risk-prone areas for transmission of dengue and chikungunya. The results showed predominance of Ae. albopictus in all the districts studied in Odisha. Cuttack district accounted for the highest proportion of positive containers (15.7%) followed by Balasore (13.7%), Bhadrak and Jajpur (12.1%). Considering the various indices used for the prediction of dengue, Cuttack, Jajpur, Bhadrak and Balasore districts were found to be at high endemic risk. Earthen pots were found to be the most preferred breeding habitat with the highest container productivity of 29.6 and associated risk factor of 25.67. There was a significant difference in the mean larval development time in subpopulations of Ae. albopictus from the study districts. In all the subpopulations from the study districts, female mosquitoes emerged approximately a day after the emergence of male mosquitoes. A significant difference was also observed in the fecundity values, in terms of the total number of eggs, as long-living females could lay a larger number of eggs during their lifetime (P=0.00068). The higher values of adult indices indicated the high risk association of this species of mosquito with the spread of dengue and likelihood of an epidemic. This study updates the current status of preferred breeding habitats and vector distribution in different districts of Odisha. The differences in Ae. albopictus subpopulations reported here indicate a higher risk for acquiring dengue among the human populations settled in dengue-endemic districts of Odisha.

Keywords: Aedes albopictus; breeding habitats; indices; dengue; Odisha.

Introduction

Aedes albopictus is an indigenous species in India believed to have originated in the tropical forests of South-east Asia1. In general, Ae. albopictus is an outdoor breeder that is more prevalent in periurban areas, and is associated with dengue fever. Sumodan reported that in rubber plantations where tapping had been suspended, sap-collecting containers accumulate

#E-mail: [email protected]

Dengue Bulletin – Volume 40, 2018 83 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha rainwater and act as mosquito-breeding sites2. Discarded plastic teacups have become new breeding sites for Ae. albopictus3. Changing climatic conditions closely affect the density of vector mosquitoes4 and thus play a major role in creating new breeding habitats.

Clear water with a low oxygen partial pressure, such as rainwater, provides a potential breeding site for the Aedes mosquito5. Therefore, the practice of storing rainwater for domestic use provides a suitable breeding site for these mosquitoes. Christophers6 also claimed that a dry climate and domestic habits that lead to storage of water may be favourable for mosquito breeding, which later become “domesticated” and change their habitat in accordance with human movement and development5. Progressive changes such as urbanization, networks of trunk roads, and transportation of used tyres are considered to be important socioeconomic factors that aid in the spread of Ae. albopictus7. Consequently, the proximity of an area/ township to interstate highways leads to the spread of Ae. albopictus8.

Besides the factors that help in the spread of Ae. albopictus, there are also several factors that discourage it. These include abnormally dry or cold weather and proper handling and disposal of discarded materials such as tyres9. Most importantly, the individual features of each location seem to affect the biological characteristics of Ae. albopictus10. Variations in vectorial competence have been demonstrated in different subpopulations and confirmed through the use of molecular markers11. Both biotic and abiotic elements are responsible for the differential response in the Aedes subpopulation. Thus, a comparative analysis of geographically separated subpopulations is crucial for studies on the behavioural attributes of the Aedes mosquito.

Presently, control of the vector population is the best option for reducing the transmission of dengue and preventing outbreaks. For effective implementation of control programmes, studies on the ecological features of Ae. albopictus are necessary. Adequate information on vector distribution and abundance is a critical factor for predicting the risk of disease transmission12,13. Periodic monitoring of vector density, along with identifying and evaluating the productivity of important containers, are requisites of an effective vector surveillance programme14,15. Identification of potential breeding habitats and destruction of breeding sources are the key essentials for effectively controlling of the vector subpopulation below a threshold level16,17.

The re-emergence of dengue and chikungunya has led to exploration of the distribution of Aedes mosquitoes in Odisha18–21 with special reference to Ae. Albopictus, as it is currently the predominant vector in Odisha. We investigated the distribution of various breeding sources, and assessed usefulness of immature-stage indices to identify risk-prone areas for the transmission of dengue and chikungunya. We also identified thresholds that were later utilized for control-based approaches. Our study updates existing knowledge on the preferred breeding habitats and vector distribution in different districts of Odisha.

84 Dengue Bulletin – Volume 40, 2018 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha

Materials and methods

Study area

We carried out an entomological survey for a period of 1 year, from January 2015 to December 2015, in the state of Odisha (17.49 °N latitude to 22.34 °N latitude, 81.27 °E longitude to 87.29 °E longitude). The state has a tropical climate with temperatures ranging from 35 °C to 46 °C during summers, and 3 °C to 14 °C during winters, and annual rainfall of 100–200 cm. Our study focused on 10 districts of Odisha. Cuttack, Bhadrak, Balasore, Jagatsinghpur, Kendrapara and Jajpur have a history of dengue epidemics, whereas Khordha, Puri, Kalahandi and Dhenkanal are emerging as dengue-affected locales. We searched domestic, peridomestic and natural breeding spots at various locations for the immature (larvae and pupae) and adult forms of Aedes mosquitoes. We investigated distribution dynamics and breeding sources from randomly selected houses in each district. Finally, we compared rainfall data with that of the Breteau index (BI) to assess the impact of rainfall on the spread of dengue.

Collection of immature forms

Collection of larvae and pupae

All water-containing receptacles, both indoors and outdoors, were searched for breeding habitats in random locations of the study districts. Larvae and pupae were collected on a monthly basis to find a probable relationship between rainfall and the number of immature forms collected. These were collected using dippers and pipettes, and were transferred into plastic containers and properly labelled with the source of breeding, place and date of collection. Entomological indices for the larvae and pupae survey, such as the house index (HI), container index (CI), Breteau index (BI), pupal index (PI) and pupal container index along with container positivity were calculated following the WHO standard mathematical formulae. The values of HI >4, CI >3 and BI >5 were considered to be indicators of epidemic risk22. The collected immature forms were transferred to the laboratory and reared in enamel trays till they emerged as adult mosquitoes. The emerged adults were then identified and reared in separate colonies to assess the bionomics of each colony. The risk factor (RF) associated with each type of breeding container was also calculated to determine how attractive or repulsive the breeding site was22,23.

______No. of positive containers of type X/total no. of positive breeding habitats RF = x Breeding habitats of type X/potential breeding habitats

An RF value greater than 1 indicates that a particular container type was attractive whereas a value less than 1 indicates that it was not.

Dengue Bulletin – Volume 40, 2018 85 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha

Figure 1: Various breeding habitats identified in the study areas. (a) Cement tanks, (b) coconut shells, (c) tree holes, (d) earthen pots, (e) rubber tyres, (f) aluminium containers

Collection of adult mosquitoes

Adult mosquitoes were collected following the standard protocol of Chan et al.24. Adults were collected simultaneously with the immature larvae, around the same locations. Oral aspirators were used to collect the adult mosquitoes at both indoor and outdoor locations. The collected adults were transferred to test-tubes that were plugged with cotton, and later identified following the standard key25. Collection of resting adult Aedes mosquitoes is particularly difficult due to their low density and the wide variety of resting places. Unlike immature forms, the parameters for collection of adult mosquitoes are mainly based on density indices such as adult density (AD: total number of Aedes mosquitoes per 100 houses examined), adult house index (AHI: percentage of houses found positive for Aedes mosquitoes) and resting rate (RR: total number of female Aedes mosquitoes per 100 houses examined)26.

Mosquito rearing

Mosquito larvae brought to the laboratory were transferred to water-filled enamel trays and fed with yeast powder. Pupae were transferred to enamel bowls containing water and covered with cloth and double net cages till they emerged as adults. A 10% glucose solution soaked in cotton pads was provided to feed the emergent mosquitoes. Emerged females were fed with blood meals of restrained rabbit for 1 hour thrice a week. Enamel bowls with moist

86 Dengue Bulletin – Volume 40, 2018 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha filter paper were placed inside the cages for oviposition. Separate colonies were maintained for each collection site.

A sufficient number of eggs from each colony were induced to hatch by immersing them in water. Four batches of 25 first instar larvae were raised and followed to generate a subpopulation from each locality. The bionomics of the subpopulation was then estimated through horizontal life tables.

Survival, development time, emergence, and sex ratio of larvae and pupae

The number of days spent in each immature stage, along with the number of surviving larvae (at each instar) and pupae was recorded daily. Survival (lx) was represented as the percentage of immature forms (larvae/pupae) that reached the next developmental stage. The numbers of emerged adults as well as the sex ratio of the emergents were also recorded on a daily basis27.

Adult longevity and fecundity

The emerged adult mosquitoes from various batches were placed inside cloth and double net cages where the number of dead and living males and females was recorded every day. The adults were maintained with a 10% sucrose solution soaked in a cotton pad and placed inside the cages. Two days after emergence, the females were fed on blood from a restrained rabbit. The process of feeding was repeated thrice a week for 1 hour every time. After each feed , enamel bowls provided with moist filter paper were placed in each cage as ovitraps. The fecundity of the females as well as longevity of males and females was estimated through this process. Fecundity was expressed as number of eggs laid by a single female in a single day and recorded daily.

Data analysis

The survival and development time of immature forms along with longevity, oviposition period, and fecundity of adults were analysed with the analysis of variance (ANOVA) and significance was calculated by the Duncan test. Student t-test was used to find the proportional difference in sex ratio in each batch28 whereas ANOVA was used to determine the sex ratio among subpopulations.

The rainfall data for 1 year in all the studied areas was later compared with that of the BI to find the relationship between vector abundance and rainfall. The rainfall data were obtained from http://www.odisha.gov.in/disaster/src/RAINFALL/RAINFALL1/RAINFALL.html.

Dengue Bulletin – Volume 40, 2018 87 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha

Results

A total of 2237 houses were surveyed during the study period, of which 481 houses were found to contain the Aedes species, with 553 breeding containers that were positive. Cuttack district accounted for the highest proportion of containers found positive (15.7%), followed by Balasore (13.7%), Bhadrak and Jajpur (12.1%). In all, 1196 pupae were collected from the positive breeding containers. Ae. albopictus (46.15%) was the dominant species in all the locations (Figure 2). Larval and pupal indices of the positive breeding containers were calculated to evaluate the distribution pattern of the Aedes species. Aedes indices higher than the standard values indicate the risk of dengue in the particular area. In our study, all the districts were found to be at a high risk of dengue except Khordha, Puri and Dhenkanal, which are at marginal risk compared to the other districts. Considering especially the various indices used for the prediction of dengue, Cuttack, Jajpur, Bhadrak and Balasore districts were found to be at high endemic risk. Jajpur ditrict recorded highest HI (38.69) and CI (16.92) values whereas the highest BI (37.5) and PI (80.6) values were recorded from Cuttack district (Table 1).

Figure 2: Distribution of the Aedes species in the study districts

200 Composition of emerged adults

180

160

140

120

100

80 Number of mosquitoes

60

40

20

0 i l k ri ra ar ur da nd na ra ack pu r Pu jp pa ur sw tt ha ad Ja ra nka la Kh ngh Cu i e Bh nd Bale ts Ka Dh Ke ga Ja Toxorhynchytes Cx. vishnui Cx. quinquefaciatus Ae. vittatus Ae. albopictus Ae. aegypti

88 Dengue Bulletin – Volume 40, 2018 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha (PI) index 18.99 53.46 38.60 61.11 80.60 69.71 54.71 79.49 44.14 56.28 28.29 Pupal 13.50 24.72 15.79 20.37 37.50 27.80 34.08 24.36 29.28 33.67 10.73 Breteau index (BI) 6.57 9.02 4.94 11.20 10.97 14.17 11.55 12.65 11.83 16.92 12.17 index (CI) Container (HI) 5.06 8.33 index 21.50 16.67 30.17 34.02 25.11 20.09 38.69 26.13 11.71 House 45 98 58 88 132 187 168 122 186 112 1196 Number of pupae 44 32 87 67 76 57 65 67 22 36 553 Positive Positive containers Aedes breeding habitats in the surveyed districts of Odisha 401 487 614 580 601 482 534 396 445 399 Total Total 4939 inspected containers 36 12 70 82 56 47 58 77 24 19 481 houses Positive Positive 216 237 232 241 223 234 222 199 205 228 Total Total 2237 houses inspected Table 1: The larval and pupal indices of Table District Total Kalahandi Dhenkanal Cuttack Bhadrak Balasore Jagatsinghpur Kendrapara Jajpur Khordha Puri

Dengue Bulletin – Volume 40, 2018 89 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha

Among the breeding containers found positive, earthen pots were found to be most conducive breeding habitat with the highest container productivity of 29.6 and an associated RF of 25.67. Other than earthen pots, cement tanks and rubber tyres are also considered important breeding habitats, with RF values of 20.9 and 15.22, respectively. Earthen pots were found to be the most conducive breeding habitats in all the study districts except in Jagatsinghpur, Puri and Kalahandi, where cement tanks were found to be the most attractive breeding sites (Figure 3). Coconut shells followed by grinding stones were found to be less attractive, with RF values of 2.01 and 3.18, respectively (Table 2).

Figure 3: District-wise productivity of containers

18 Breeding containers 16

14 I)

(C 12 y it iv 10

oduct 8 pr r

ne 6 ai 4 Cont

2

0 i l r k k ri ar ur ra da na ra nd ac pu Pu jp ur pa sw tt ka ha ad Ja gh le ra Kh la Cu en in Bh Ba nd ts Ka Dh Ke ga Ja Steel/Aluminium utensils Earthen pots/flowers pots Tree holes Plastic tanks Tyres Coconut shells Grinding stones Plastic buckets Cement tanks

On comparing the relationship of rainfall with the BI, a weak correlation was found (r=0.4951, P=0.14568), indicating the absence of a dominant effect of rainfall on the prevalence of immature forms in all the breeding sites (Supplementary table).

90 Dengue Bulletin – Volume 40, 2018 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha (RF) 8.19 3.18 8.70 4.60 2.01 11.54 25.67 15.22 20.90 100.00 Risk factor 0.04 0.08 0.08 0.22 0.09 0.06 0.06 0.13 0.09 Pupal container index (PCI) 7.27 5.69 9.36 7.19 4.52 3.76 29.60 14.72 17.89 Container productivity 87 68 86 54 45 112 354 176 214 1196 Number of pupae 7 56 17 11 78 38 11 553 218 117 pupae Number of containers with 78 712 453 138 822 631 176 917 4939 1012 examined Total containers Total Table 2: Container productivity and pupal container index in the surveyed districts of Odisha Table Container type Total Plastic tanks Steel/aluminium utensils Grinding stones Earthen pots/flowers pots Tyres Plastic buckets Coconut shells Tree holes Tree Cement tanks

Dengue Bulletin – Volume 40, 2018 91 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha

Supplementary Table: Cumulative rainfall and Breteau index during the study period in the surveyed districts of Odisha

District Cumulative rainfall from Jan–Dec 2015 (mm) Breteau index (BI) Cuttack 97.98 37.5 Bhadrak 96.5 27.8008 Baleswar 99.2 34.0807 Jagatsinghpur 93.63 24.359

Kendrapara 100.41 29.2793

Jajpur 104.96 33.6683 Khurda 83.39 10.7317 Puri 94.48 15.7895 Kalahandi 113.94 20.3704 Dhenkanal 85.11 13.5021

All the immature forms (larvae/pupae) as well as adults collected were reared in the laboratory for analysis of larval/pupal survival and developmental time, along with adult longevity and fecundity. A significant difference in mean larval development time was found between the Ae. albopictus subpopulations from the study districts. The lowest mean larval developmental time was recorded for Ae. albopictus from Cuttack district (4.68 days), which differed significantly from that of Dhenkanal district, which had the highest developmental time of 9.79 days. A similar trend was observed for the pupal developmental time in the surveyed districts. Larval and pupal survivals were 97.3% and 99.3%, respectively, from Cuttack district, which was the highest recorded value among all the study subpopulations (Table 3).

The ratio for emergence of males and females in the study districts did not differ significantly, except for the Ae. albopictus subpopulation from Cuttack district where a relatively larger number of females emerged in comparison to males. Females emerged approximately a day after the males in all the Ae. albopictus subpopulations. The longevity of females was higher than those of males, varying from approximately 48.2 + 26.8 days. Males lasted from approximately 25.3 + 14.3 days. The longer lifespan of females led to longer oviposition time, which varied from 21.2 to 35.2 days (Table 4).

Daily fecundity did not vary significantly among the study subpopulations. Values ranged from approximately 11 eggs/female/day for the Cuttack subpopulation to 7 eggs/female/day for the Dhenkanal subpopulation. However, there was a significant difference in the fecundity values in terms of the total number of eggs, as females with a longer lifespan could lay more eggs during their lifetime (P=0.00068).

92 Dengue Bulletin – Volume 40, 2018 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha 3.38±0.17 9.79±0.25 2.98±0.17 3.28±0.17 3.43±0.48 76.5±1.29 71.0±1.63 Dhenkanal 13.07±0.20 63.1±5.6 36.9±5.6 8.33±0.28 9.45±0.31 35.22±1.7 6.87±0.54 Dhenkanal 1511±321.2 14.32±26.54 26.81±21.11 Kalahandi 2.38±0.10 6.39±0.22 1.98±0.33 2.23±0.09 2.03±0.39 8.62±0.18 96.5±0.58 90.25±2.22 43.5±3.9 56.5±3.9 Kalahandi 7.05±0.18 8.14±0.11 22.45±4.3 10.18±0.87 21.19±21.39 44.22±19.87 2459.9±817.66 Ae. albopictus in the Puri 3.18±0.25 9.04±0.15 2.88±0.26 3.08±0.13 2.98±0.13 Puri 12.12±0.13 88.25±1.26 73.25±0.96 7.2±1.1 58.3±7.5 41.7±7.5 8.01±0.06 9.04±0.76 30.58±3.9 31.25±17.65 16.33±12.25 1599±543.18 Khordha 3.30±0.08 9.33±0.18 2.95±0.31 3.15±0.13 3.08±0.25 87.0±2.58 12.48±0.15 71.75±1.26 Khordha 60.2±1.8 39.8±1.8 8.17±0.18 9.33±0.76 33.65±8.3 7.01±0.98 29.88±17.65 16.10±12.22 1543±431.66 Jajpur 81±2.16 2.70±0.35 7.65±0.13 2.45±0.30 2.68±0.17 2.50±0.35 93.5±0.58 10.33±0.13 Jajpur 51.4±6.4 8.1±0.76 48.6±6.4 6.99±0.23 8.88±0.34 29.33±6.1 1765±234.1 34.36±18.65 17.65±16.52 Ae. albopictus adults in the study districts of Odisha. 93±0.82 2.80±0.16 8.18±0.08 2.65±0.19 2.75±0.32 2.73±0.22 76.5±1.29 Kendrapara 10.93±0.06 7.6±1.5 54.7±1.8 45.3±1.8 7.89±0.01 8.63±0.16 29.98±5.3 Kendrapara 33.56±18.99 16.54±15.12 1632±652.26 2.1±0.08 2.20±0.08 5.88±0.25 1.70±0.12 7.98±0.22 1.98±0.33 93.75±1.70 97.25±0.96 Jagatsinghpur 41.2±5.2 58.8±5.2 6.88±0.12 8.13±0.14 9.83±0.98 48.17±23.66 23.35±11.14 2176±613.12 Jagatsinghpur 23.22±15±7.89 Balasore 2.5±0.24 2.55±0.39 7.28±0.13 2.30±0.32 9.78±0.11 2.43±0.31 85.25±4.19 93.25±0.96 Balasore 7.66±0.08 8.57±0.12 8.76±0.76 1876±187 49.67±2.33 50.33±2.33 27.33±4.98 39.65±16.52 19.43±11.09 Bhadrak 2.43±0.69 7.04±0.08 2.35±0.31 2.28±0.59 9.39±0.06 2.33±0.61 95.75±0.5 Four replicates were performed for each subpopulation. Four 88.75±2.22 Bhadrak 48.6±4.8 51.4±4.8 7.63±0.11 8.44±0.26 9.56±0.65 26.85±5.33 41.88±11.43 19.76±12.01 2098±546.11 Cuttack 2.05±0.13 4.68±0.45 1.95±0.13 1.18±0.22 6.63±0.41 1.45±0.46 97.25±0.96 99.25±0.96 study districts of Odisha. Four replicates were performed for each subpopulation. study districts of Odisha. Four Cuttack 37.9±2.4 62.1±2.4 6.13±0.08 7.98±0.19 11.32±1.2 21.22±2.68 25.26±8.77 46.33±20.11 2314±345.61 Pupa Pupa Larva Larvae 4 Larvae 3 Total Larva Total Larvae 1+2 Larva+pupa Male Male Male Female Female Female Table 4: Mean and standard deviation of longevity fecundity Table Developmental time (days) Immature stage survival (%) Table 3: Mean and standard deviation of development time survival larvae pupae Table Sex ratio (%) Emerging time (days) Daily fecundity no. of Total eggs Oviposition time (days) Longevity (days)

Dengue Bulletin – Volume 40, 2018 93 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha

The adult densities along with indices for immature forms were found to play a pivotal role in evaluating the risk of dengue in the surveyed districts. Higher values of adult indices indicated association of the species with a high risk for the spread of dengue and the risk of an epidemic.

Discussion

Our study revealed a differential distribution pattern of the Aedes species in endemic districts of Odisha. In all the study districts of Odisha, Ae. albopictus was the predominant species in comparison with other Aedes species, specifically Ae. aegypti. Larval surveys are economically viable and easy to operate; they helped in understanding the current distribution of Ae. albopictus in Odisha. The extent and intensity of breeding are calculated from the HI and the CI, respectively. The information from both the indices helps to obtain the BI, making it an excellent risk indicator for dengue outbreaks29. Known for the intensity of transmission, PI values are considered a better alternative indicator of the abundance of adult mosquitoes30. In this study, the values of the Aedes indices (CI >3, BI >4 and HI >5) indicated that the population density of Aedes and the number of potential breeding habitats was high enough to cause dengue outbreaks in the districts under study.

Water stored in different containers in and around residential areas constituted the major breeding habitats of Aedes mosquitoes. The findings of the survey of immature (larvae/ pupae) subpopulations indicated the potential breeding habitats and the risk of dengue outbreaks in the surveyed districts. Earthen pots and cement tanks were the most frequent and conducive breeding habitats in all the surveyed districts. This finding is in contrast to those from other parts of the world31–34, including Malaysia16,35–37 where plastic containers were the most conducive breeding habitats.

In this study, the highest RF was observed for earthen pots compared to other habitats. In another study, CI values have been reported for natural containers (44.4%) and tyres (37.9%)36. Plastic containers accounted for The highest proportion of all immature forms were collected from plastic containers, which although abundant in number, did not pose a high risk for dengue. In Sri Lanka, tyres had similar RF patterns for vectors associated with dengue. However, leaf axils were found to be non-conducive breeding habitats (RF=0.64) for Ae. albopictus23. In another study, Morrison et al.38 stated that, despite the abundance of plastic containers, targeting them in a source-destruction programme is inappropriate due to a low infestation rate of 3.6% as compared to the other containers.

The bionomics study of Ae. albopictus revealed that the subpopulations found in the Cuttack district had shorter developmental times than those in remaining districts. The subpopulation of Jagatsinghpur stood second in terms of developmental time and survival rate. The different survival rates of larvae/pupae among the subpopulations studied may be attributed to the wide-scale immigration from diverse regions, leading to differences in haplotype composition. The mean number of eggs laid by Ae. albopictus females in

94 Dengue Bulletin – Volume 40, 2018 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha Resting rate (RR) 60.75101 41.35021 72.22222 58.33333 55.12195 64.32161 62.61261 61.11111 60.98655 59.75104 72.84483 16.0804 21.18909 6.751055 35.18519 9.649123 9.268293 12.61261 35.47009 19.73094 23.23651 42.24138 index (AHI) Adult house Adult 91.2037 79.8995 72.73134 44.72574 64.03509 58.04878 69.81982 77.35043 70.40359 75.51867 96.98276 density (AD) 106 197 146 119 159 155 181 157 182 225 Total 1627 98 156 133 113 128 139 143 136 144 169 1359 Females 8 6 Ae. albopictus collected Ae. albopictus in the surveyed districts of Odisha 41 13 31 16 38 21 38 56 268 Males 16 76 22 19 32 28 83 44 56 98 474 houses Positive Positive 237 216 228 205 199 222 234 223 241 232 2237 Table 5: Indices of adults Table inspected Total houses Total District Total Dhenkanal Kalahandi Puri Khordha Jajpur Kendrapara Jagatsinghpur Balasore Bhadrak Cuttack

Dengue Bulletin – Volume 40, 2018 95 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha the Cuttack district was the highest, at 11.32 eggs/female/day. The longer survival rate of female mosquitoes in the Cuttack district make them biologically more fit than the other subpopulations and thus increase their chances of transmitting arboviral diseases more efficiently than the other subpopulations. Owing to this, intense control measures are necessary to check the spread of arboviral diseases specifically targeting Ae. albopictus.

The density indices of adults are more accurate and applicable measures for entomological studies to detect the risk of dengue and identify dengue-prone areas, as only female mosquitoes can transmit the dengue virus. The number of adults collected and, in particular, the number of females per area or habitat is considered an index by Rodrigues– Figueroa et al.39 and can best predict dengue occurrence in an area. Focks40 adds that the technique used to collect adult mosquitoes is extremely important in scientific investigations. The use of oviposition traps41 that have a low operating cost indirectly estimate the female population40,42. Further, Regis et al.43stated the egg density index can be used to identify areas with a high concentration of mosquitoes.

In summary, our study found that Cuttack district is at the highest risk of a dengue outbreak. The district has the highest BI and PI values, which that are considered to be the best indicators of a possible outbreak. Along with this, the mosquito subpopulation from the district shows the lowest developmental time, highest survival rates and high fecundity of female Ae. Albopictus, indicating the biological fitness of this subpopulation. We also found that, along with districts that are historically endemic for dengue, others are emerging, especially Kalahandi district. This district also has a predominance of dengue vectors, mainly Ae. albopictus, as seen by the higher values of indices associated with immature forms, along with parameters for bionomical and adult density. Thus, the superiority of the subpopulation indicates the high risk associated with dengue outbreaks and further emphasizes the need to re-evaluate and implement proper control strategies to abolish potential transmission of the vector population.

The response of subpopulations of mosquitoes that belong to the same species is affected by their genetic composition as well as environmental factors. Our study clearly showed differences in the life statistics of subpopulations of Ae. albopictus. These differences could be genetic, and would need to be evaluated in further studies. The differences in the subpopulations of Ae. albopictus reported in our study indicate a higher risk of acquiring dengue among human populations settled in the dengue-endemic districts of Odisha, as passive dispersion of Ae. albopictus takes place through constant vehicular movement leading to the circulation of different serotypes of the dengue virus.

Besides targeting key breeding containers, it is also essential to survey and target storm drains and drainage lines, which facilitate the breeding of Ae. albopictus in some areas. However, further studies are required to better understand how larval competition and type of container can affect the life history traits and abundance of this vector species. The influence on adult mosquitoes of environmental variables, and the quality and quantity of resources in containers should be studied. Interspecies mating studies between Ae. albopictus and Ae. aegypti should be carried out in the field to investigate the distribution of these species. Furthermore, the vectorial capacity of these should be studied in areas where they coexist.

96 Dengue Bulletin – Volume 40, 2018 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha

Acknowledgements

We are extremely grateful to the Director, RMRC, for providing a platform for the research work. We are also grateful to Ms Ipsita Mohanty, Mrs Nitika Pradhan, Ms Barsa Baisalini Panda, Ms Santoshini Dash and insectarium staff of the medical entomology division of RMRC for their immense support during the course of this study.

Disclosure

The authors declare no conflicts of interest.

Ethical approval

The Institutional Animal Ethics Community (IAEC) approved the feeding of adult mosquitoes on restrained rabbit blood.

References

[1] Smith CEG. The history of dengue in tropical Asia and its probable relationship to the mosquito Aedes aegypti. J Trop Med. 1956;59(10):243–51. [2] Sumodan PK. Potential of rubber plantations as breeding source for Aedes albopictus in Kerala, India. Dengue Bull. 2003;27:197–8. [3] Hiriyan J, Tewari SC, Tyagi BK. Aedes albopictus (Skuse) breeding in plastic cups around tea-vendor spots in Ernakulam City, Kerala state, India. Dengue Bull. 2003;73:195–6. [4] Vezzani D, Velásquez SM, Schweigmann N. Seasonal pattern of abundance of Aedes aegypti (Diptera: Culicidae) in City, . Mem Inst Oswaldo Cruz. 2004;99(4):351–6. [5] Lo EKC, Narimah A. Epidemiology of dengue disease in Malaysia. Journal of Malaysian Society of Health. 1984;4:27–35. [6] Christophers SR. Aedes aegypti (L.), the yellow fever mosquito: its life history, bionomics and structure. London: Cambridge University Press; 1960:739. [7] Dutta PD, Khan SA, Sharma CK, Doloi PI, Hazarika NC, Mahanta J. Distribution of potential dengue vectors in major townships along the national highways and trunk roads of northeast India. Southeast Asian J Trop Med Public Health. 1988;29(1):173–6. [8] Moore CG, Mitchell CJ. Aedes albopictus in the : ten-year presence and public health implications. Emerg Infect Dis. 1997;3(3):329–34. [9] Kobayashi M, Nihei N, Kurihara T. Analysis of northern distribution of Aedes albopictus (Diptera: Culicidae) in Japan by geographical information system. J Med Entomol. 2002;39(1):4–11. [10] Rodhain FR, Rosen L. Mosquito vectors and dengue virus-vector relationships. In: Dengue and dengue hemorrhagic fever. New York: CAB International; 1997:45–60.

Dengue Bulletin – Volume 40, 2018 97 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha

[11] Beerntsen BT, James AA, Christensen BM. Genetics of mosquito vector competence. Microb Mol Biol Rev. 2000;64:115–37. [12] Juliano SA, Lounibos LP. Ecology of invasive mosquitoes: effects on resident species and on human health. Ecol Lett. 2005;8(5):558–74. [13] Kweka EJ, Munga S, Himeidan Y, Githeko AK, Yan G. Assessment of mosquito larval productivity among different land use types for targeted malaria vector control in the western Kenya highlands. Parasit Vectors. 2015;8:1–8. [14] Britch SC, Linthicum KJ, Anyamba A, Tucker CJ, Pak EW; Mosquito Surveillance Team. Long-term surveillance data and patterns of invasion by Aedes albopictus in Florida. J Am Mosq Control Assoc. 2008;24(1):115–20. [15] Valenca MA, Marteis LS, Steffler LM, Silva AM, Santos RL. Dynamics and characterization of Aedes aegypti (L.) (Diptera: Culicidae) key breeding sites. Neotrop Entomol. 2013;42(3):311–6. doi: 10.1007/ s13744-013-0118-4. [16] Saifur RGM, Ahmad AH, Dieng H, Salmah MRC, Saad AR, Satho T. Temporal and spatial distribution of dengue vector mosquitoes and their habitat patterns in Penang Island, Malaysia. J Am Mosq Control Assoc. 2013;29(1):33–43. [17] Bowman LR, Donegan S, McCall PJ. Is dengue vector control deficient in effectiveness or evidence? Systematic review and meta-analysis. PLoS Negl Trop Dis. 2016;10(3):e0004551. doi: 10.1371/journal. pntd.0004551. [18] Das B, Swain S, Patra A, Tripathy HK, Mohapatra NM, Kar SK et al. Development and evaluation of a single step multiplex PCR to differentiate the aquatic stages of morphological similar Aedes (subgenus: Stegomyia) species. Trop Med Int Health. 2012;17(2):235–43. doi: 10.1111/j.1365-3156.2011.02899.x. [19] Das B, Sahu A, Das M, Patra A, Dwibedi B, Kar SK et al. Molecular investigations of chikungunya virus during outbreaks in Odisha, eastern India in 2010. Infect Genet Evol. 2012;12(5):1094–101. doi: 10.1016/j.meegid.2012.03.012. [20] Das B, Das M, Dwibedi B, Kar SK, Hazra RK. Molecular investigations of dengue virus during outbreaks in Odisha state, eastern India from 2010 to 2011. Infect Genet Evol. 2013;16:401–10. doi: 10.1016/j.meegid.2013.03.016. [21] Das B, Hazra RK. Entomological investigations with special attention to pupal indicators of Aedes vectors during outbreaks of dengue in coastal Odisha, India. J Vector Borne Dis. 2013;50(2):147–50. [22] Favier C, Degallier N, Vilarinhos Pde T, de Carvalho Mdo S, Yoshizawa MA, Knox MB. Effects of climate and different management strategies on Aedes aegypti breeding sites: a longitudinal survey in Brasilia (DF, Brazil). Trop Med Int Health. 2006;11(7):1104–18. [23] Weeraratne TC, Perera MDB, Mansoor MM, Karunaratne SP. Prevalence and breeding habitats of the dengue vectors Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in the semi-urban areas of two different climatic zones in Sri Lanka. Int J Trop Insect Sci. 2013;33(4):216–26. [24] Chan YC, Ho BC, Chan KL. Aedes aegypti (L.) and Aedes albopictus (Skuse) in Singapore City: 5. Observations in relation to dengue haemorrhagic fever. Bull World Health Organ. 1971;44(5):651. [25] Barraud PJ. Diptera, Vol V, Family Culicidae. Tribe Megarhinini and Culicini (The fauna of British India, Ceylon and Burma). London: Taylor and Francis; 1934:1–463. [26] Siregar FA, Makmur T. Survey on Aedes mosquito density and pattern distribution of Aedes aegypti and Aedes albopictus in high and low incidence districts in north Sumatera province. IOP Conference Series. Earth Environ Sci. 2018;130:012018.

98 Dengue Bulletin – Volume 40, 2018 Biology and bionomical aspects of invasive Aedes albopictus circulating in Odisha

[27] Gómez C, Rabinovich JE, Machado-Allison CE. Population analysis of Culex pipens fatigans Wied. (Diptera: Culicidae) under laboratory conditions. J Med Entomol. 1997; 13(4–5):453–63. [28] Steel RGD, Torrie JH. Bioestadística: principios y procedimientos, second edition. México: McGraw- Hill; 1988. [29] Tun-Lin W, Kay BH, Barnes A, Forsyth S. Critical examination of Aedes aegypti indices: correlations with abundance. Am J Trop Med Hyg. 1996;54(5):543–7. [30] Wai KT, Arunachalam N, Tana S, Espino F, Kittayapong P, Abeyewickreme W et al. Estimating dengue vector abundance in the wet and dry season: implications for targeted vector control in urban and peri-urban Asia. Pathog Glob Health. 2012;106(8):436–45. doi: 10.1179/2047773212Y.0000000063. [31] Chareonviriyaphap T, Akratanakul P, Nettanomsak S, Huntamai S. Larval habitats and distribution patterns of Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse), in Thailand. Southeast Asian J Trop Med Public Health. 2003;34(3):529–35. [32] Banerjee S, Aditya G, Saha GK. Household wastes as larval habitats of dengue vectors: Comparison between urban and rural areas of Kolkata, India. PLoS One. 2015;10(10):e0138082. doi: 10.1371/ journal.pone.0138082. [33] Verna TN. Species composition and seasonal distribution of mosquito larvae (Diptera: Culicidae) in Southern New Jersey, Burlington County. J Med Entomol. 2015;52(5):1165–9. doi: 10.1093/jme/tjv074. [34] Dhar-Chowdhury P, Haque CE, Lindsay R, Hossain S. Socioeconomic and ecological factors influencing Aedes aegypti prevalence, abundance, and distribution in Dhaka, . Am J Trop Med Hyg. 2016;94(6):1223–33. doi: 10.4269/ajtmh.15-0639. [35] Rozilawati H, Tanaselvi, K, Nazni WA, Mohd Masri S, Zairi J et al. Surveillance of Aedes albopictus Skuse breeding preference in selected dengue outbreak localities, peninsular Malaysia. Trop Biomed. 2015;32(1):49–64. [36] Faiz M, Nazri CD, Chua ST. Spatial and temporal distribution of Aedes (Diptera: Culicidae) mosquitoes in Shah Alam. Trop Biomed. 2017;34(1):1–9. [37] Maimusa HA, Ahmad AH, Kassim NFA, Ahmad H, Dieng H, Rahim J. Contribution of public places in proliferation of dengue vectors in Penang Island, Malaysia. Asian Pac J Trop Biomed. 2017;7(3):183–7. [38] Morrison AC, Gray K, Getis A, Astete H, Sihuincha M, Focks D et al. Temporal and geographic patterns of Aedes aegypti (Diptera: Culicidae) production in Iquitos, . J Med Entomol. 2004;41(6):1123–42. [39] Rodriguez-Figueroa L, Rigau-Perez JG, Suarez EL, Reiter P. Risk factors for dengue infection during an outbreak in Yanes, Puerto Rico in 1991. Am J Trop Med Hyg. 1995;52(6):496–502. [40] Focks DA. A review of entomological sampling methods and indicator for dengue vectors. Geneva: World Health Organization; 2003:40. [41] Fay RW, Eliason DA. A preferred oviposition site as a surveillance method for Aedes aegypti. Mosq News. 1966;26(4):531–5. [42] Morato VCG, Teixeira MG, Gomes AC, Bergamaschi DP, Barreto ML. Infestation of Aedes aegypti estimated by oviposition traps in Brazil. Rev Saude Publica. 2005;39(4):553–8. [43] Regis L, Monteiro AM, Melo-Santos MAV, Silveira JC, Furtado AF, Acioli RV et al. Developing new approaches for detecting and preventing Aedes aegypti population outbreaks: basis for surveillance, alert and control system. Mem Inst Oswaldo Cru. 2003;103(1):50–9.

Dengue Bulletin – Volume 40, 2018 99 Association between entomological indices and container types for the prevention and control of dengue

Association between entomological indices, breeding of Aedes mosquitoes and container types in Delhi for the prevention and control of dengue

Babita Bisht,a# RoopKumari,b Himmat Singh,c BN Nagpal,c AK Bansal,a NR Tulid

aNorth Delhi Municipal Corporation; bNational Centre for Disease Control cNational Institute of Malaria Research; dSouth Delhi Municipal Corporation

Abstract Worldwide, dengue is a problem of urban and suburban areas. It is transmitted by a typical species of the Aedes mosquito that breeds largely in containers. Almost all states in India report cases of dengue every year. Delhi has been reporting a large number of cases of dengue since 1996, but in 2015, there was an upsurge, with more than 15 000 cases. We conducted a study in the city zone of the North Delhi Municipal Corporation (NDMC) for the years 2013–2015 to identify key container types that serve as mother foci for breeding of mosquitoes in adverse climatic conditions, and help in the distribution of mosquitoes to other breeding sites under conducive environmental conditions. Entomological (larval) surveillance was conducted in selected households and public places for all possible types of containers. We found that there is a seasonal pattern of breeding in different types of containers. If targeted efforts are made during the relatively dry summer season to control breeding of dengue-causing mosquitoes in specific containers, transmission will be considerably reduced during the conducive rainy season.

Keywords: Vector; transmission; breeding containers; mother foci.

Introduction

Dengue is one of the most rapidly increasing mosquito-transmitted infections. It has been identified as an emerging disease in the South-East Asia Region1. Of the 2.5 billion people living in endemic areas worldwide, 1.8 billion people live in the Asia Pacific region. Annually, 50–100 million cases are reported globally1–3. WHO coined the term “break-bone fever” for dengue because of the symptoms of myalgia and arthralgia4.

In India, the first epidemic was reported in 1780 from Madras (now Chennai) and thereafter from Calcutta (now Kolkata) in 1963–19645. The dengue virus was first isolated

#E-mail: [email protected]

100 Dengue Bulletin – Volume 40, 2018 Association between entomological indices and container types for the prevention and control of dengue in India in 19456. With the first outbreak in 1988, and then in 19967, dengue has emerged as a major public health challenge in Delhi. In the 1996 outbreak of dengue haemorrhagic fever (DHF)/dengue shock syndrome (DSS), a total of 8900 cases were reported in Delhi, with a death rate of 4.2%8,9. Subsequent outbreaks of dengue were reported in 2003, 2006, 2010, 2013 and 2015. Delhi recorded the worst outbreak of dengue in 2015 with over 15 000 cases10. The notification of such a large number of cases of dengue in Delhi can be attributed to improved surveillance, migration of people to Delhi for economic activities, and more people seeking health care. Investigation and assessment of the epidemiology of dengue infection and the proportion of dengue infections in Delhi that were asymptomatic and symptomatic showed that 10.6% of dengue infections were either primary or secondary11.

The city zone of Delhi comprises the walled city of Delhi with an area of 25 sq.km and a population of 579 724, leading to a population density of 23 189 persons per sq.km. The large number of cases of dengue in this zone can be attributed to the high population density and various sociocultural practices adopted by the community. In areas of high population density, many people may be exposed, even when the indices associated with dengue are low, as has been observed by Sanchez et al.12.

This study aimed to identify the types of containers responsible for breeding dengue- causing mosquitoes and to analyse the breeding potential of different containers. This would help in planning effective strategies for vector surveillance and control in Delhi throughout the year.

Materials and methods

Study area

The city zone falls under the North Delhi Municipal Corporation (NDMC) and has seven municipal wards: Daryaganj (79), Chandni Chowk (80), Minto Road (81), Kucha Pandit (82), Sadar Bazar (83), Turkman Gate (84) and Ballimaran (86), each represented by an elected municipal councillor. This particular city zone was selected because it is the smallest zone with a large number of factors that contribute to dengue cases, such as a high population density (23 189 persons per sq.km), conducive geographical terrain with the Yamuna river flowing along its entire length, sociocultural practice of storing water in small containers, and the presence of several public institutions where mosquito breeding usually occurs in many unattended discards, which contribute to increase in overall breeding. There is always the fear of an outbreak of dengue in these municipal wards. Therefore, all the seven wards of this city zone were selected as the area for the study and data collection (Figure 1 and Figure 2).

Dengue Bulletin – Volume 40, 2018 101 Association between entomological indices and container types for the prevention and control of dengue

Figure 1: Study area in Delhi city zone

Figure 2: Ward-wise population of the city zone (as per Census 2011)

70000 60000 50000 n o i t

a 40000 l u

p 30000 o P 20000 10000 0 Daryaganj Chandni Minto Kucha Sadar Turkman Ballimaran Chowk Road Pandit Bazar Gate City zone

102 Dengue Bulletin – Volume 40, 2018 Association between entomological indices and container types for the prevention and control of dengue

Data collection

Random samples were collected for entomological surveillance. Besides a random selection of houses, all institutions, such as hospitals, colleges, schools and government offices, falling under the jurisdiction of the city zone were included in the study. Containers selected for the study were desert coolers, overhead tanks, water storage containers, waste articles, and water pots for birds. Our study was based on the hypothesis that breeding of the Aedes mosquito varies in different containers during different months.

We carried out entomological surveillance throughout the year. There were two teams under the supervision of an entomologist, with one domestic breeding checker and one field worker in each team. The data collected were analysed for entomological indices, i.e. house index(HI) = (% of houses with larvae and/or pupae); container index (CI) = (% of water- holding containers with larvae or pupae); Breteau index (BI) = (no. of positive containers per 100 houses inspected); and pupal index (PI) = (no. of pupae per 100 houses inspected).

The teams collected data on a daily basis from January 2013 to December 2015. Each positive container was either emptied or, if this was not possible, treated with an adequate dose of temephos (1% granules as supplied to the NDMC) per litre of water during data collection in households.

Data analysis

Entomological parameters were tabulated (Table 2) and appropriate statistical tools were used to analyse the data and examine the correlation between entomological indices associated with dengue and rainfall.

Results

From 2013 to 2015, a total of 32 507 houses and 44 935 containers were checked. The average HI and CI were 5.7% and 6.5%, respectively. In 2013, 2014 and 2015, an HI of 6.6%, 3.4% and 6.2% and a CI of 7.1%, 4.3% and 7.7% were reported, respectively. A large number of dengue cases (159) reported in 2015 in the city zone can be attributed to the high CI detected in 2015. Similarly, the BI was found to be highest (10.9) in 2015, followed by the year 2013, whereas it was close to 9.0 when all years were pooled (Table 2).

Dengue Bulletin – Volume 40, 2018 103 Association between entomological indices and container types for the prevention and control of dengue

Table 2: Year-wise number of houses and containers checked in the city zone

No. of Positive House Containers Positive Container Breteau Year houses houses index checked containers index index checked 2013 10 487 702 6.6% 14 701 1054 7.1% 10.05 2014 10 466 357 3.4% 14 004 609 4.3% 5.82 2015 11 554 826 6.2% 16 230 1260 7.7% 10.91 Total 32 507 1885 5.7 44 935 2923 6.5 8.99

Ward-wise data were analysed to study which ward reported high entomological indices and its relation to the number of dengue cases. The highest HI was 8.2% in ward no. 81 (Minto Road) followed by 6.9% in ward no. 80 (Chandni Chowk) and 5.9% in ward no. 79 (Daryaganj). The CI followed the same pattern; the highest CI of 9% was reported in ward no. 81 (Minto Road). This corresponded to the maximum number of dengue cases reported from the ward. Ward no. 81 has a large number of educational institutes and government offices, indicating that stakeholders there can contribute significantly to controlling dengue transmission by making concerted efforts.

Figure 3: Month-wise container index (CI) from 2013 to 2015

14.00% ) I 12.00% 2013 2014 2015 C (

x

e 10.00% d n i

8.00% r e

n 6.00% i a t

n 4.00% o

C 2.00% 0.00% JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC 2013 1.80% 5.60% 2.90% 5.10% 7% 6% 7.90% 10.30% 9.90% 9.30% 5.10% 3% 2014 1.60% 5.80% 4.10% 4.30% 4.80% 4.40% 3.60% 6.90% 5.40% 3.90% 2.10% 2.20% 2015 2.20% 5.10% 4.20% 6.10% 7.10% 6.80% 11.40% 13.10% 8.50% 8.30% 3.50% 3.10%

Months

104 Dengue Bulletin – Volume 40, 2018 Association between entomological indices and container types for the prevention and control of dengue 46 776 386 691 3.72 6.50 3.41 7.05 5.96 6237 1349 9800 2923 1024 12.44 Total 10371 44935 17178 0 0

0 0 9 28 48 66 37 842 944 3.33 1.95 0.00 0.00 0.95 Dec 1900 6 0 31 14 56 164 786 101 526 107 3.94 3.66 3.65 0.00 2.66 4.14 Nov 2929 1352 4 72 46 763 811 125 114 116 352 Oct 5.67 9.44 7.13 3.20 7.44 6.81 4936 1533 1704 7 33 865 121 850 134 127 156 444 Sep 3.88 8.16 5.22 6.49 9.55 5441 1958 1634 13.99 8 40 981 157 867 175 222 179 606 4.61 4.57 Aug 5767 2078 1666 16.00 10.51 10.68 10.74 8 28 Jul 966 131 871 142 133 110 410 3.21 5.63 7.80 8.23 6.63 5255 1616 1660 13.56 7 19 26 99 942 108 868 122 838 259 Jun 2.19 5.74 5.87 3.10 6.02 4415 1645 11.46 8 98 20 27 92 870 845 146 418 245 2.37 5.48 6.35 6.46 5.82 3861 1582 May 11.26 2 83 28 17 55 686 996 106 317 185 Apr 2.81 1.89 5.19 5.36 3.77 3565 1460 12.10 0

2 5 0 49 97 99 43 965 211 5.08 2.06 3.81 2.37 3.25 Mar 2598 1325 0

0 0 0 37 68 60 84 868 121 Feb 4.26 0.00 5.49 0.00 6.95 2204 1208 0

0 0 9 0 28 37 48 66 Jan 944 842 3.33 0.00 1.95 0.00 0.95 1900 Table 3: Month-wise breeding of dengue-causing mosquitoes in different types of containers from 2013 to 2015 Table Containers/months Coolers checked No. positive CI Water storage containers Water checked CI No. positive Overhead tanks checked Total positive Total Total containers checked Total CI No. positive Water pots for birds Water checked No. positive Domestic waste containers checked No. positive Overall CI CI CI

Dengue Bulletin – Volume 40, 2018 105 Association between entomological indices and container types for the prevention and control of dengue

During the non-transmission season, a peak in CI (CI of 5.6 in 2013, 5.8 in 2014 and 5.1 in 2015) was reported in the month of February in all the three years of the study. This may be attributed to breeding detected in mother foci containers. Breeding indices showed a rising trend from July onwards following the monsoons, which declined only in November. The highest CI of 13.1 was reported in August 2015 followed by 11.4 in July 2015. From the programme implementation perspective, there is a need to strengthen surveillance and vector control in February by destroying mother foci. The highest CI during the monsoon period may be attributed to favourable climatic conditions and multiple breeding sites created by water stagnation in waste containers lying in domestic and peridomestic areas (Figure 3).

Overhead tanks and ground water storage containers were found to be positive throughout the year, although the positivity was low (CI was 5.9 in water storage tanks and 3.7 in overhead tanks). Although coolers showed the highest CI positivity of about 12.4, they were found positive during the transmission period (July to October).

Other sites with a high CI included water storage containers and waste containers. During the monsoon period, a CI of 8–10 was observed in waste containers lying in the open. A CI of 5.6 was reported in the overhead tanks during the transmission season, while during the pre-transmission period, it was about 3–5 (Table 3). The correlation between the number of containers checked and those found positive ranged from 70.8% to 92.2% (r2), except in the case of overhead tanks, where the correlation was only 2%, as they are usually constant in number for each house checked.

Figure 4: Monthly rainfall and container index (CI) variation in the study area

14 300 ) ) I 12 CI AVG. Rainfall 250 m C ( c

( x 10 l l e 200 a d f n n i 8 i l

r 150 a r e

n 6 e i g a t

100 a r n 4 e o v C 2 50 A 0 0 JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC Months

The CI was found to be maximum during the months of August and September, which showed a positive association with the average rainfall in Delhi. However, the CI was seen to increase from January onwards and peak during the rains due to addition of solid waste breeding grounds after the rains in peridomestic areas (Figure 4).

106 Dengue Bulletin – Volume 40, 2018 Association between entomological indices and container types for the prevention and control of dengue

Figure 5: Seasonal variations in breeding in containers and their types

50 Water pots for birds Domestic waste containers 45 4.5 Water storage containers 40 Overhead tanks 10.6 5.2 Desert coolers x 35 5.6 e

d 3.2 n 30 6.4 i

r 5.4 8.2 e 10.7 7.4 n 25 1.8 5.7 i a

t 6.4 5.3 9.5 n 20 3.1 6.6 o 4.6 6.8 C 3.7 5.8 6 15 0 3.2 3.8 0 0 2.8 2.3 2.1 5.6 2.6 10 2 0 2.3 4.1 0 6.9 13.5 16 13.9 5 0 3.2 12 11.2 11.4 3.9 0 0.9 9.4 0.9 3.3 4.2 5 3.6 3.3 0 0 0 0 0 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Months

Obviously, when all the container types were pooled, there was an tremendous increase in the number of containers in which mosquitoes were breeding, as well as those that were found positive from April to October. It is noteworthy that the overhead containers played a vital role in the maintenance of breeding throughout the year, even during the non-transmission season of November–March. This was similar in the case of water storage containers in peridomestic areas.

The Aedes mosquito breeds mainly in domestic and peridomestic settings, in clean water collected in containers such as desert coolers, uncovered overhead tanks/water storage containers, old tyres or any other waste article that can hold water. During the rainy season, unattended or waste articles lying on rooftops or in open areas get filled with rainwater and provide ideal conditions for breeding (Figure 5).

Table 4: Correlation between rainfall and entomological indices

No. of House positive Container Breteau Pupal Rainfall index container index (CI) index (BI) index(PI) (HI) types Correlation (r) 0.76 0.87 0.89 0.91 0.84 Per cent correlation (r2) 0.582 0.76 0.79 0.84 0.70

Dengue Bulletin – Volume 40, 2018 107 Association between entomological indices and container types for the prevention and control of dengue

Rainfall has a considerable impact on dengue-associated indices; almost all of them were found to be highly correlated with rainfall. As rainfall increases, indices for all container types increase (58%; r2= 0.58), as shown in Table 4. The data correlating rainfall with other entomological indices were not adjusted for 0–15 days to allow for the lag phase caused by the number of day sit takes for the mosquito to breed after the rains.

Discussion

In 2015, a large number of dengue cases were reported from ward no. 81 (Minto Road), which was also one of the areas included in our study. Our findings for this area also showed high breeding indices in this area in 2015, which can also be attributed to the heavy and intermittent rains as well as absence of sufficient measures to control the transmission of dengue by various institutions. Desert coolers of hostels were the most common containers that were found positive. Hostel wardens and caretakers were trained to identify larvae and implement the necessary measures (such as emptying the tanks of coolers) to control the breeding of the dengue-causing mosquitoes. In coolers that could not be emptied, 10 g of temephos granules were added to prevent mosquito breeding. It was made clear that the containers either needed to be emptied or temephos granules used when they could not be emptied.

Breeding was reported in overhead tanks both during non-transmission and transmission periods. Overhead tanks acted as mother foci during the pre-transmission season. The positivity rate of water pots for birds was 5.2–5.7%. We also observed a shift in breeding foci from desert coolers to other containers, such as waste containers lying in the open, water storage containers and water pots for birds during the transmission season. This fact was also supported by another study conducted in Delhi by Kumar et al. (2015) on the breeding of Ae. aegypti in various socioeconomic groups13. Six types of water containers were found to be infested with Ae. aegypti: (i) overhead tanks, (ii) curing tanks, (iii) plastic containers/ drums for water, (iv) solid waste, (v) coolers and (vi) water pots for birds. In all the localities surveyed during the transmission season, solid waste containers were the most preferred breeding sites followed by curing tanks, plastic containers, overhead tanks, coolers and water pots for birds. In the non-transmission season, overhead tanks and curing tanks were found to be the primary and the most preferred breeding containers followed by coolers and plastic containers. In a similar study in Tiruchirappalli district, Tamil Nadu by Rajesh et al., among all types of containers surveyed, cement cisterns (59.25%), mud pots (53.84%), unused tyres (42.85%), unused wells (33.33%), plastic containers and vessels (25%) were positive for mosquito larvae14.

Sanchez et al. studied the larval indices of Ae. aegypti and risk of dengue epidemics. They observed that the HI and BI at the municipality level were 1.53 and 1.73, respectively, but all areas with dengue cases had a BI <1 and, after the outbreak, the indices returned to the average values of <1 at all levels of measurement12,15. In a study on container-breeding

108 Dengue Bulletin – Volume 40, 2018 Association between entomological indices and container types for the prevention and control of dengue mosquitoes with special reference to Ae. aegypti and Ae. albopictus in Thiruvananthapuram district, it was observed that Aedes was breeding in 87% of the containers. The most common species of Aedes was Ae. albopictus. About 86% (60/70) of the clusters were positive for Ae. Albopictus and 11% (8/60) for Ae. aegypti. The distribution of Ae. aegypti was highest in unused tyres, followed by grinding stones, tarpaulins, thermocol and metal containers. In a study on the seasonal fluctuation of Ae. aegypti in different localities of Delhi, water coolers and tyres were found to be the preferred breeding habitats of the Aedes mosquito in Delhi16. Ae. aegypti, being hygroscopic, showed a phenomenon of moving to the mother foci in the central areas of the city, which are somewhat humid even in the dry season, and then spread out during the wet season.

A study from Dehradun reported that the maximum breeding was found in plastic containers followed by tin containers, earthen pots, desert coolers and cement tanks. A study showed that preference for type of water storage container is different in different socioeconomic groups due to which altered breeding is being observed; in low-income group (LIG) colonies, the preference is for earthen pots; in middle-income group (MIG) colonies, for desert coolers and in high-income group (HIG) colonies, plastic and tin containers were the primary breeding containers for Ae. aegypti17.

In a study on entomological surveillance and its significance during a dengue outbreak in the district of Tirunelveli in Tamil Nadu18, HI, CI and BI were 48.2, 28.6 and 48.2, respectively, before an entomological intervention; after the intervention, these indices were considerably reduced and HI values were 10.2, 5.2, 2.5 and 1.6 in the 2nd, 3rd, 4th and 5th weeks, respectively; the CI values were 2.3, 0.9, 0.4 and 0.3, respectively; and BI values were 12.9, 6.2, 2.8 and 1.6, respectively.

Studies have shown that there is a lag phase between breeding and rainfall in case of breeding mosquitoes such as malaria, as these are vectors that breed in open waterbodies, ditches and ponds due to the soil–water saturation factor19. However, in our study, the lag phase seemed to have little significance as there was a positive correlation between all entomological indices with rainfall without any lag phase. A study on the seasonal planning of vector control of Ae. aegypti and Ae. albopictus in a highly dengue-endemic area of India reported that four key containers (plastic drums, cement tanks, overhead tanks and desert coolers) harboured 86% of Aedes pupae in Delhi and, except desert coolers, all the others form a perennial source of breeding. Therefore, an intervention successfully targeting these containers would be effective in considerably controlling a dengue outbreak20.

In the entire city zone under consideration, breeding of Aedes was recorded in water storage containers. Due to scarcity of water, these containers are not fully dried and more water is filled over the left-over water in these containers; this provides an ideal larval habitat for mosquitoes to breed. Similar observations were also reported by a study conducted in Tiruchirappalli district, Tamil Nadu, India14 and the Philippines21. A study conducted in Kuala Lumpur, Malaysia showed that plastic containers were the containers of choice for mosquito breeding (30%). Community mobilization is required to eliminate containers made

Dengue Bulletin – Volume 40, 2018 109 Association between entomological indices and container types for the prevention and control of dengue

Common breeding sites

A: unused/discarded coolers; B: overhead plastic tanks without lids; C: bird pots; D: discarded tyres of artificial materials, both indoors and outdoors. It is vital for dengue-control programmes to recommend “search-and-destroy” campaigns in the community and include these unrecognized containers as well22,23.

A study from also reported that Ae. aegypti prefer breeding in synthetic water containers in peridomestic or domestic settings. Health communication must stress the importance of managing all water-holding containers, irrespective of location (indoor/ outdoor), with special emphasis on the management of these containers during the rainy season24,25.

A specific strategy formulated to address the problem of unrecognized and recognized containers with the highest proportion positive for Aedes larvae will help in the prevention and control of dengue. Studies have shown that there is a considerable impact of climatic factors on breeding sites. By targeting these specific breeding sites, the breeding of mosquitoes can be controlled23.

110 Dengue Bulletin – Volume 40, 2018 Association between entomological indices and container types for the prevention and control of dengue

The city zone comprises the walled city of Delhi with a high population density of 23 189 persons per sq.km. Despite a low CI, there is the possibility of a large outbreak due to the large congregation and the biting habits of Aedes. Well-equipped entomological teams were able to curtail breeding by emptying coolers and putting adequate amounts of temephos granules in desert coolers and other containers that could not be emptied due to scarcity of water. For control in other public places such as hospitals, parks and offices, intersectoral coordination meetings were organized to seek the participation of all stakeholder departments in preventing and controlling mosquito breeding.

Conclusion

Dengue, a viral illness, has no specific treatment or vaccine available till date. Thus, reduction of infection and control at source remains the best strategy for prevention of local transmission. Entomological surveillance is an appropriate tool to identify key containers and the period during which mosquitoes breed. We found desert coolers to be the most preferred breeding containers, and that maximum breeding occurred in coolers from April to October. Overhead tanks did not show any variation in breeding, as breeding was reported as early as January and February, thereby indicating that overhead tanks are mother foci and, under favourable climatic conditions, there is a shift of larval breeding from overhead tanks to other domestic containers, with further spread outdoors during the monsoon season.

During the pretransmission season, overhead tanks need to be targeted for prevention and control of dengue. Desert coolers become egg banks if they are not cleaned after the season is over and become breeding sites for mosquitoes during the transmission season. Therefore, an integrated vector management approach should focus on checking the positivity of various dengue-associated indices indifferent containers all through the year. This can be augmented by community participation involving all stakeholders, public sector offices, schools and other departments, and introducing “search-and-destroy” campaigns for better control of dengue.

Acknowledgments

We wish to express our gratitude to Shri PK Singh, the then Additional Commissioner (Health) for always being supportive of our academic activities. Our sincere thanks to all the deputy health officers of the city zone where the study was conducted, especially Dr SB Singh, for encouraging our entomological team to collect and compile the data. We extend our heartfelt thanks to Gaurav from our team whom we lost during data collection.

Dengue Bulletin – Volume 40, 2018 111 Association between entomological indices and container types for the prevention and control of dengue

References

[1] Comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever, revised and expanded edition. New Delhi: World Health Organization, Regional Office for South-East Asia;2011 (http://www.searo.who.int/entity/vector_borne_tropical_diseases/documents/SEAROTPS60/ en/, accessed 24 November 2018). [2] Halsead SB. Pathogenesis of dengue: challenges to molecular biology. Science. 1988;239(4839):476–81. [3] Stanaway JD, Shepard DS, Undurraga EA, Halasa YA, Coffeng LE, Brady OJ et al. The global burden of dengue: an analysis from the Global Burden of Disease study 2013. Lancet Infect Dis. 2016;16(6):712– 23. [4] Packard RM. “Break-bone” fever in Philadelphia, 1780: reflections on the history of disease. Bull Hist Med. 2016;90(2):193–221. [5] Gupta N, Srivastava S, Jain A, Chaturvedi UC. Dengue in India.Indian J Med Res. 2012;136(3):373–90. [6] Sabin AB. Research on dengue during World War II. Am J Trop Med Hyg. 1952;1(1):30–50. [7] Kabra SK, Jain Y, Pandey RM, Madhulika, Singhal T, Tripathi Pet al. Dengue haemorrhagic fever in children in the 1996 Delhi epidemic. Trans R Soc Trop Med Hyg. 1999;93(3):294–8. [8] Broor S, Dar L, Sengupta S, Chakraborty M, Wali JP, Biswas A. Recent dengue epidemic in Delhi, India. In: Saluzzo JE, Dodet B, editors. Factors in the emergence of arbovirus diseases. Paris: Elsevier; 1997:123–7. [9] Sharma PL, Sood OP, editors. Round table conference series—dengue outbreak in Delhi: 1996. Gurgaon, India: Ranbaxy Science Foundation; 1996;1–3. [10] Surveillance data on notification of dengue cases released by MIS cell of South Delhi Municipal Corporation. [11] Vikram K, Nagpal BN, Pande V, Srivastava A, Saxena R, Anvikar A et al. An epidemiological study of dengue in Delhi, India. Acta Trop. 2016;153:21–7. [12] Sanchez L, Vanlerberghe V, Alfonso L, Marquetti M del C, Guzman MG, Bisset J et al. Aedes aegypti larval indices and risk for dengue epidemics emerging infectious diseases. Emerg Infect Dis. 2006;12(5):800–6.. [13] Kumar V, Pande V, Srivastava A, Gupta S, Singh H, Saxena R et al. Comparison of Ae. aegypti breeding in localities of different socio-economic groups of Delhi, India. Int J Mosq Res. 2015;83(22):83–8. [14] Rajesh K, Dhanasekaran D, Tyagi BK. Survey of container breeding mosquito larvae (dengue vector) in Tiruchirappalli district, Tamil Nadu, India. J Entomol Zool Stud. 2013;1(6):88–91. [15] Vijayakumar K, Sudheesh Kumar TK, Nujum ZT, Umarul F, Kuriakose A.A study on container breeding mosquitoes with special reference to Aedes (Stegomyia) aegypti and Aedes albopictus in Thiruvananthapuram district, India. J Vector Borne Dis. 2014;51(1):27–32. [16] Sharma RS, Kaul SM, Sokhay J. Seasonal fluctuations of dengue fever vector, Aedes aegypti (Diptera: culicidae) in Delhi, India. Southeast Asian J Trop Med Public Health. 2005;36(1):186–90. [17] Singh S, Vandna, Rahman A. Contribution of Aedes aegypti breeding by different income group communities of Dehradun city, Uttarakhand, India. Biol Forum. 2013;5(1):96–9.

112 Dengue Bulletin – Volume 40, 2018 Association between entomological indices and container types for the prevention and control of dengue

[18] Basker P, Kannan P, Porkaipandian RT, Saravanan S, Sridharan S, Kadhiresan M. Study on entomological surveillance and its significance during a dengue outbreak in the district of Tirunelveli in Tamil Nadu, India. Osong Public Health Res Perspect. 2013;4(3):152–8. [19] Baeza A, Bouma MJ, Dobson AP, Dhiman R, Srivastava HC, Pascual M. Climate forcing and desert malaria: the effect of irrigation. Malar J.2011;10:190. [20] Roop K, Priya S, Sunita P, Mujib M, Kanhekar LJ, Venkatesh S. Way forward for seasonal planning of vector control of Aedes aegypti and Aedes albopictus in a highly dengue endemic area in India. Austin J Infect Dis. 2016;3(1):1022. [21] Edillo FE, Roble ND, Otero ND 2nd.The key breeding sites by pupal survey for dengue mosquito vectors, Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse), in Guba, City, Philippines. Southeast Asian J Trop Med Public Health. 2012;43(6):1365–74. [22] Salamat MSS, Cochon KL, Crisostomo GCC, Gonzaga PBS, Quijano NA, Torio JF Entomological survey of artificial container breeding sites of dengue vectors in Batasan Hills, Quezon City. Acta Medica Philippina. 2013;47(3):63–8. [23] Mahmud MAF, Mutalip MH, Lodz NA, Shahar H. Study on key Aedes spp. breeding containers in dengue outbreak localities in Cheras district, Kuala Lumpur. Int J Mosq Res. 2018;5(2):23–30. [24] Naish S, Dale P, Mackenzie JS, McBride J, Mengersen K, Tong S .Climate change and dengue: a critical and systematic review of quantitative modelling approaches. BMC Infect Dis. 2014;14:167. [25] Lin CH, Schiøler KL, Ekstrøm CT, Konradsen F. Location, seasonal, and functional characteristics of water holding containers with juvenile and pupal Aedes aegypti in Southern Taiwan: a cross-sectional study using hurdle model analyses. PLoSNegl Trop Dis. 2018;12(10):e0006882. doi: 10.1371/journal. pntd.0006882. eCollection 2018 Oct.

Dengue Bulletin – Volume 40, 2018 113 Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector Aedes aegypti

Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector Aedes aegypti

Nisha Sogan,a Smriti Kala,b Neera Kapoor,c BN Nagpald#

aNational Institute of Malaria Research (NIMR), New Delhi bInstitute of Pesticide Formulation Technology (IPFT), Gurugram cIndira Gandhi National Open University (IGNOU), New Delhi dWHO Regional Office for South-East Asia, New Delhi

Abstract Oils have been used for the control of diseases and vectors. We attempted to develop a neem- (Azadirachta indica) and karanj oil (Pongamia pinnata)-based nanoemulsion in various combinations (1:0, 0:1, 1:1, 1:2, 2:1) to control the larval stages of the dengue vector. We followed thelow-energy emulsification method to develop oil-in-water nanoemulsions. All the nanoemulsions (F1, F2, F3, F4 and F5) developed were found to be stable, with the droplet size ranging from 83.2 nm to 97nm in diameter. Nanoemusions were characterized through the technique of dynamic light scattering using a Nano-ZS Zetasizer and electron microscopy using transmission electron microscopy (TEM), and were found to be spherical. The WHO larval susceptibility test method was followed to evaluate the larvicidal efficacy of the nanoemulsions against IV instar larvae of Aedes aegypti. The results of larvicidal bioassays revealed that nanoemulsion F3 (1:1 neem oil and karanj oil) had better larvicidal activity than the other nanoemulsions (F1, F2, F4 and F5)due to synergistic interaction,

with an LC50of 2.06 ppm. A nanoemulsion of neem and karanj oils has the potential to act as a larvicide against Ae. aegypti.

Keywords: Nanoemulsion; Aedes aegypti; neem oil; karanj oil; synergisticactivity; LC50.

Introduction

Dengue is the most rapidly spreading mosquito-borne arboviral disease in the world. Around 2.5 billion people in 100 countries are at risk of acquiring dengue viral infection, with more than 50 million new infections being projected annually. Around 500 000 cases of DHF and 20 000 cases require hospitalization, with 25 000 deaths, mainly in children1–3.

#E-mail: [email protected]

114 Dengue Bulletin – Volume 40, 2018 Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector Aedes aegypti

Currently, dengue is endemic in more than 100 countries in WHO’s African, Eastern Mediterranean, South-East Asia and Western Pacific regions, and the Region of the Americas4. The South-East Asia and Western Pacific regions are the most seriously affected, contributing to 75% of the global burden of dengue5. In India, all the states and Union Territories are endemic for dengue6. According to the National Vector Borne Disease Control Programme (NVBDCP) report 2014–2015, India accounts for 20% of the total cases of dengue in South- East Asia7.

The vector for dengue is Aedes aegypti, which bites during the day and has a high vectorial capacity for transmission of viruses such as dengue (DENV), chikungunya (CHIKV), Zika (ZIKV) and yellow fever (YFV), resulting in diseases with epidemic potential such as dengue, chikungunya and Zika virus disease. Till now, no specific treatment is available for dengue, so vector control and surveillance become an important strategy for the prevention of dengue and DHF. One of the convenient ways to control the vector population is to use larvicides8. Control of the dengue vector relies on synthetic insecticides (e.g.temephos, fenthion, etc.) and insect growth regulators (e.g. diflubenzuron, methoprene, etc.). Extensive use of synthetic insecticides has led to insecticide resistance in Ae. aegypti and negative effects on humans and the environment on account of their non-biodegradability, persistent residues and toxicity towards non-target organisms9–11. In this context, botanical insecticides provide sustainable, eco-friendly options to control the vector population.

Neem and karanjtrees are widely foundin India and have medicinal properties12,13. Neem oil is obtained from the seeds of the neem kernel, and contains phytochemicals such as azadirachtin, nimbin and salannin. These pytochemicals affect the biochemical and physiological processes of insects and thus retard their growth and development14. Neem-based products are reported to have a low toxicity to birds, fish and mammals, and the chances of development of resistance are low due to the multiple modes of action on insects15. Neem oil has been reported to have larvicidal activity against a variety of insects, including mosquito larvae16. Neem oil formulations have been reported to have antifeedant, ovicidal, larvicidal, insect growth regulatory and repellent activity against insect pests17. Neem oil has shown activity as a potential larvicide in the form of a nanoemulsion18.

Karanj oil is extracted from the seeds of karanja (Pongamia pinnata Linn). The P. pinnata L plant has been documented to have anti-inflammatory, antiplasmodial, antinonciceptive, antihyperglycaemic, antilipidoxidative, antidiarrhoeal, antiulcer, antihyperammonic and antioxidant activities in traditional systems of medicine such as Ayurveda and Unani19. Due to the presence of toxic flavonoids, such as karanjin, pongapin and pongaglabrin, the oil is considered to be inedible. However, after detoxification, the meal can be used as a feed ingredient for cattle20. Karanj cake has been reported to have nematicidal activity and also improves soil fertility21. Phytochemicals such as flavonoids, chalcone, steroids and terpenoids in karanj oil are reported to be defensive in nature22.

Dengue Bulletin – Volume 40, 2018 115 Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector Aedes aegypti

The synergistic activity of neem and karanjhas been reported previously21,23. A nanoemulsion of neem oil (NO) and karanj oil (KO) has not been reported previously in the literature. We have developed a neem oil- and karanj oil-based nanoemulsion and evaluated its larvicidal activity against Ae. aegypti.

Materials and methods

Chemicals

Neem oil and karanj oil were supplied by a local supplier. The surfactants used in our study were Tween-80 ( supplied by Finar, synthesis grade) and vegetable oil ethoxylate (VO-2000 supplied by Solvay, commercial grade). Antifreezing agent (propylene glycol), industrial grade, was supplied by Supreme Surfactant Limited, Sonipat, Haryana.

Mosquito larvae

Ae. aegypti larvae were collected from Delhi National Capital Region (NCR) and maintained atroom temperature of 25 + 2oC with a relative humidity (RH) of 60–70% in dechlorinated tap water in an enamel bowl. Larvae were provided with a feed of dog biscuit and yeast (3:1).

Synthesis of the nanoemulsions

Various combinations of the surfactants were screened for the stability of nanoemulsions; 3:1 (Tween-80:VO-2000) was found to be stable and was used to formulate nanoemulsions with different ratios of NO and KO.

An oil-in-water nanoemulsion was formulated according to the low-energy emulsification method24. Surfactants, an antifreezing agent and the oils were pooled together and constituted the oily phase, while distilled water constituted the aqueous phase. The aqueous phase was added slowly through the oily phase under magnetic stirring (1100 rpm) for 30 min and allowed to equilibrate at 25 °C for 24 h followed by visual inspection. The final mass was kept constant (25 g) for all the formulations, constituted by 75% (w/w) of distilled water, 10% (w/w) of NO:KO (1:0, 0:1, 1:1, 1:2, 2:1), 5% (w/w) propylene glycol and 10% (w/w) of surfactant mixture 3:1 (Tween-80:VO).

Characterization of the nanoemulsions

Nanoemulsions were characterized on the basis of droplet size and polydispersity index (PDI). The Z-average diameter (mean droplet size) and PDI of nanoemulsions were measured using the Nano-ZS Zetasizer (Malvern, UK). Each sample was diluted with distilled water for analysis.

116 Dengue Bulletin – Volume 40, 2018 Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector Aedes aegypti

The morphology and size of the nanoemulsions were investigated by TEM images using the Phillips, Technai Fe 12 instrument at the Indian Agricultural Research Institute (IARI), Pusa, New Delhi. A drop of nanoemulsion was placed on a carbon-coated copper grid and allowed to completely dry and images were recorded.

Stability of the nanoemulsions

The nanoemulsions developed were subjected to centrifugation at 3500 rpm for 15 min.

Thermodynamic stability was checked by storing the formulated emulsion at a temperature of 40 ºC and at room temperature (25 ºC) for 1 month.

Larvicidal activity of nanoemulsions against Ae. aegypti

IV instar larvae of Ae. aegypti mosquitoes were treated with different concentrations of nanoemulsions, following the standard larval susceptibility test method of WHO25. Twenty larvae (in a 250mL beaker) at the early IV instar stage were exposed to different concentrations, i.e. 0.5, 2, 4, 6, 8 and 16 ppm of nanoemulsions (F1,F2,F3,F4 and F5). The final concentration of surfactants was 0.075% for Tween-80 and 0.025% for VO-2000. The final concentration of propylene glycol was 0.05%. Three replicates were maintained for each concentration. During the exposure period, no food was offered to the larvae. At exposure periods of 24 h, the percentage mortality was calculated. A set-up containing distilled water, surfactants and propylene glycol was maintained as control. Mortality was also observed. Moribund larvae that did not show any movement on tapping with a needle were counted as dead. The mortality in controls was calculated using the Abbott formula whenever required26.

Statistical analysis

Lethal concentrations (LC50 and LC90) with fiducial limits at 95% confidence level, chi-square and degree of freedom (Df) were calculated according to Probit analysis using SPSS (Statistical Package for the Social Sciences) software version 22.

Results

All the formulations–F1, F2, F3, F4 and F5–were found to be stable at room temperature and at 40ºC. The centrifugation test did not affect the stability of the nanoemulsions; no phase separation was observed. Formulations were found to be spherical with droplet sizes of 91, 87, 83.2, 94 and 97 nm, and PDI of 0.25, 0.27, 0.23, 0.29, 0.24 for F1, F2, F3, F4 and F5, respectively (Figure 1).

Dengue Bulletin – Volume 40, 2018 117 Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector Aedes aegypti

Figure 1: Transmission electron micrograph (TEM) image of nanoemulsion F3 (1NO:1KO)

Nanoemulsion droplets

Arrows denote nanoemulsion droplets

Among the various combinations developed, F3 (1:1NO:KO)showed the maximum synergistic activity, with an LC50 of 2.06ppm. TheLC50of F5 (2NO:1KO) was 4.082 ppm, of F4 (1NO:2KO) 4.538 ppm, F1 (1NO:0KO) 3.70 ppm and F2 (0NO:1KO) 5.626 ppm (Table 1 and Figure 2). In the control group no mortality was observed. This clearly indicated that Tween-80, propylene glycol and vegetable oil ethoxylate (VO-2000) had no toxic effects on larvae.

Table 1: Larvicidal activity of NO and KO nanoemulsion and their synergistic combination of NO:KO against Ae. aegytpi

Formulation LC50*(LCL–UCL) LC90(LCL–UCL) Chi-square Df F1 3.70(2.8–4.2) 12.04(8.28–22.23) 2.614 4 F2 5.626(3.975–8.76) 30.277(16.578–94.219) 1.142 4 F3 2.06 (1.5–2.7) 6.56(4.46–12.990) 3.405 3 F4 4.538(3.367–6.346) 17.860(11.416–38.282 0.1483 4 F5 4.082(3.096–5.520 13.811(9.321–26.482) 4.438 4

* Values are based on three replicates. * LCL and UCL are upper and lower fiducial limits at 95% confidence level. Df is degree of freedom.

118 Dengue Bulletin – Volume 40, 2018 Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector Aedes aegypti

Figure 2: Larvicidal activity of NO and KO nanoemulsion and their synergistic combination (NO:KO) against Ae. aegytpi

6

5

4 LC 50 3

2

1

0 F1(Neem oil) F2(karanj oil) F3 (1:1)Neem oil F4 (1:2) Neem oil F5 (2:1) Neem oil and Karanj oil and Karanj oil and Karanj oil Nanoemulsions

Discussion

Insecticides of botanical origin, including plant extracts and oils, are attractive alternatives to synthetic insecticides because they contain a mixture of active biomolecules, many of which are selective with little or no harmful effects on non-target organisms and the environment10,27. Botanical insecticides are biodegradable and insects rarely develop resistance against them because a number of complex phytochemicals are involved in a synergistic way to execute such responses. Thus, the use of botanicals (oil-based insecticides) can also restrict the long- term negative effects of synthetic insecticides on the environment28.

The PDI of all the formulations were in range of 0.23–0.29,which is a characteristic of the high fidelity of the system29. Selection of the optimal blend of surfactant is also an important factor in stabilization of the nanoemulsionbecause of the strong repulsive force that prevents flocculation and coalescence between the nanodroplets30 Among the various combinations developed so far, F3 (1:1 NO:KO)showed the maximum synergistic activity compared tothe other combinations of neem and karanj oil, i.e. F1 (1NO:0 KO), F2 (0NO:1KO), F4 (1NO:2KO) and F5 (2NO:1KO). The observed efficacy might be due to synergistic activity among the various phytochemicals present in neem oil and karanj oil. The findings of our research study are in agreement with previous studies that have reported synergistic activity between NO and KO21,23.

The observed efficacy of the nanoemulsion was due to the small size of the droplet, which increases the area of contact between the insecticidal oils and larvae, thus increasing the efficacy of the nanoemulsion18.

Dengue Bulletin – Volume 40, 2018 119 Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector Aedes aegypti

Nanoemulsions are a new generation of formulations that are in vogue. Their droplet size ranges from 100 nm to 600 nm31. Nanoemulsions possess various characteristics such as stability, long shelf-life, low viscosity and transparency, which render them useful for various industrial applications such as drug delivery, cosmetics and pesticide delivery32,33.

There are various reports on the larvicidal efficacy of nanoemulsions. An oil-in-water emulsion of rosemary essential oil has resulted in 90% mortality in Ae. aegypti34. Neem oil nanoemulsion has been shown to be effective against Culex quinquefasciatus18.

Botanical insecticides such as NO- and KO-based nanoemulsions may be used as potential larvicides. NO- and KO-based nanoemulsionis are biodegradable and environmentfriendly. Insecticide resistance and pest resurgence can be suppressedwith the use of an NO- and KO- based nanoemulsion, because of the array of complex biomolecules involved synergistically against the target pest.

Our research work was a laboratory study; field application will give a clearer idea about how the nanoemulsion system works in natural environmental conditions. The residual efficacy of the nanoemulsion can also be evaluated.

Conclusion

The non-judicious use of insecticides has led to insecticide resistance and negative effects on the environment, including on humans. In this regard, botanical insecticides with proven activity can provide sustainable eco-friendly options. Our research study was focused on the development and characterization of a stable nanoemulsion synergist (NO and KO). Larvicidal activity was enhanced with a synergistic formulation F3 (1NO:1KO).

Acknowledgements

The authors acknowledge guidance from Dr Adarsh Shanker and the Director, NIMR for providing all necessary facilities.

Conflicts of interest

The authors declare no conflicts of interest.

Ethical statement, involvement of human/animal subjects in the study

Not applicable

120 Dengue Bulletin – Volume 40, 2018 Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector Aedes aegypti

Funding source

We did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References

[1] Halstead SB. Dengue. Curr Opin Infect Dis. 2002;15:471–6. [2] Halstead SB. Dengue. Lancet. 2007;370:1644–52. [3] Chaturvedi UC, Shrivastava R. Dengue haemorrhagic fever: a global challenge. Indian J Med Microbiol. 2004;22:5–6. [4] Dengue and severe dengue. In: World Health Organization [website]. 2018 http://www.who.int/ mediacentre/factsheets/fs117/en, accessed 12 March 2019). [5] Aguiar M, Rocha F, Pessanha JE, Mateus L, Stollenwerk N. Carnival or football, is there a real risk for acquiring dengue fever in Brazil during holidays seasons? Sci Rep. 2015;5:8462. [6] Kumar PSS, Arjun MC, Gupta SK,Nongkynrih B. Malaria, dengue and chikungunya in India – an update. Indian J Med Spec.2018;9:25–9. [7] NVBDCP National Vector Borne Disease Control Programme (nvbdcp.gov.in/Doc/Annual-report- NVBDCP-2014-15.pdf). [8] Sogan N, Kapoor N, Kala S, Patanjali PK, Nagpal BN, Vikram K et al. Larvicidal activity of castor oil nanoemulsion against malaria vector Anopheles culicifacies. Int J Mosq Res. 2018;5:1–6. [9] Tiwary M, Naik SN, Tewary DK, Mittal PK, Yadav S. Chemical composition andlarvicidal activities of the essential oil of Zanthoxylum armatum DC (Rutaceae) against three mosquito vectors. J Vector Borne Dis. 2007;44:198–204. [10] Benelli G. Plant-mediated biosynthesis of nanoparticles as an emerging tool against mosquitoes of medical and veterinary importance: a review. Parasitol Res. 2016a;115:23–4 (http://dx.doi.org/10.1007/ s00436-015-4800-9, accessed 12 March 2019). [11] Benelli G. Plant-mediated synthesis of nanoparticles: a newer and safer tool against mosquito-borne diseases? Asian Pac J Trop Biomed. 2016b;6:353–4. [12] Kirtikar KR, Basu BD, editors. Indian medicinal plants, second edition. Allahabad: Lalitha Mohan Basu; 1935,536. [13] Muthu C, Ayyanar M, Raja N,I gnacimuthu S. Medicinal plants used by traditional healers in Kancheepuram district of Tamil Nadu, India. J Ethnobiol Ethnomed. 2006;2:43. [14] Dua VK, Pandey AC, Raghavendra K, Gupta A, Sharma T, Dash AP. Larvicidal activity of neem oil (Azadirachta indica) formulation against mosquitoes. Malar J. 2009;8:124. [15] Chaudhary S, Kanwar RK, Sehgal A, Cahill DM, Barrow CJ, Sehgal R et al. Progress on Azadirachta indica based biopesticides in replacing synthetic toxic pesticides. Front Plant Sci. 2017;8:610.

Dengue Bulletin – Volume 40, 2018 121 Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector Aedes aegypti

[16] Su T, Mulla MS.Antifeedancy of neem products containing azadirachtin against Culex tarsalis and Culex quinquefasciatus (Diptera: Culicidae). J Vector Ecol.1998;23:114–22. [17] Schmutterer H. Properties of natural pesticides from the neem tree, Azadirachta indica. Ann Rev Entomol.1990;35:271–97. [18] Anjali CH, Sharma Y, Mukherjee A, Chandrasekaran N. Neem oil (Azadirachta indica) nanoemulsion—a potent larvicidal agent against Culex quinquefasciatus. Pest Manag Sci. 2012;68:158–63. [19] Nguyen-Pouplin J, Tran H, Tran H, Phan TA, Dolecek C, Farrar J et al. Antimalarial and cytotoxic activities of ethnopharmacologically selected medicinal plants from South Vietnam. J Ethnopharmacol. 2007;109:417–27. [20] Mandal B, Ghosh Majumdar S, Maity CR. Protease inhibitors and in vitro protein digestibility of defatted seed cakes of akashmoni and karanja. J Am Oil Chem Soc. 1985;62:1124–6. [21] Pant M, Dubey S, Raza SK, Patanjali PK. Encapsulation of neem and karanja oil mixture for synergistic as well as larvicidal activity for mosquito control. J Sci Ind Res. 2012;71:348–52. [22] Pavela R. Possibilities of botanical insecticide exploitation in plant protection. Pest Technology. 2007;1:47–52. [23] Shanmugasundaram R, Jeyalakshmi T, Dutt MS, Murthy PB. Larvicidal activity of neem and karanja oil cakes against mosquito vectors, Culex quinquefasciatus (Say), Aedes aegypti (L.) and Anopheles stephensi (L.). J Environ Biol. 2008;29:43–5. [24] Pant M, Dubey S, Patanjali PK, Naik SN, Sharma S. Insecticidal activity of eucalyptus oil nanoemulsion with karanja and jatropha aqueous filtrates. Int Biodeterior Biodegradation. 2014;91:119–27. [25] Guidelines for laboratory and field testing of mosquito larvicides.Geneva: WHO Communicable Disease Control, Prevention and eradication, WHO Pesticide Evaluation Scheme; 2005 (https://apps.who.int/ iris/bitstream/handle/10665/69101/WHO_CDS_WHOPES_GCDPP_2005.13.pdf;jsessionid=D3FCE ACE0A06BDD0379A97F469F9A164?sequence=1, accessed 12 March 2019). [26] Abbott WS. A method for computing the effectiveness of the insecticide. J Econ Entomol. 1925;18:265– 72 (https://doi.org/10.1093/jee/18.2.265a, accessed 12 March 2019). [27] Govindarajan M, Rajeswary M, Veerakumar K, Muthukumaran U, Hoti SL, Mehlhorn Het al. Novel synthesis of silver nanoparticles using Bauhinia variegata: a recent eco friendly approach for mosquito control. Parasitol Res. 2016;115:723–33. [28] Maurya P, Sharma P, Mohan L,Verma MM, Srivastava CN. Larvicidal efficacy of Ocimum basilicum extracts and its synergistic effect with neonicotinoid in the management of Anopheles stephensi. Asian Pac J Trop Dis. 2012;2:110–16. [29] Jafari SM, Assadpoor E, He Y, Bhandari B. Re-coalescence of emulsion droplets during high-energy emulsification. Food Hydrocoll. 2008;22:1191–202. [30] Sugumar S, Singh S, Mukherjee A, Chandrasekaran N. Nanoemulsion of orange oil with nonionic surfactant produced emulsion using ultrasonication technique: evaluating against food spoilage yeast. Appl Nanosci. 2016;6:113–20. [31] Solans S, Esquena J, Forigianini A, Uson N, Morales D, Izquierds P et al. Absorption and aggregation of surfactants in solution. In: Mittal KL, Dinesh OS, Editors. Nano-emulsion: formation properties and applications. New York: Marcel Dekker; 2003:525–54.

122 Dengue Bulletin – Volume 40, 2018 Neem-and karanj oil-based nanoemulsion for control of larval stages of the dengue vector Aedes aegypti

[32] Bouchernal K, Brianeon S, Perrier E, Fessi H. Nano-emulsion formulation using spontaneous emulsification, solvent oil and surfactant optimization. Int J Pharm. 2004;280:241–51. [33] Sonneville-Aubrun O, Simonnet JT, L’Alloret F. Nanoemulsions: a new vehicle for skincare products. Adv Colloid Interf Sci. 2004;108:145–9. [34] Duarte JL, Amado JRR, Oliveira AEMFM, Cruz RAS, Ferreira AM, Souto RNP et al. Evaluation of larvicidal activity of a nanoemulsion of Rosmarinus officinalis essential oil. Braz J Pharmcogn. 2015;25:189–92.

Dengue Bulletin – Volume 40, 2018 123 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India

Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India

Rajalakshmi Anbalagan,a Arpita Shukla,a Kaviyarasan,b Jayalakshmi Krishnanc#

aDepartment of Life Sciences, Central University of Tamil Nadu, Thiruvarur, India bDepartment of Epidemiology and Public Health, Central University of Tamil Nadu, Thiruvarur, India cDepartment of Life Sciences, Central University of Tamil Nadu, Thiruvarur, India

Abstract Dengue is one of the most important arboviral diseases. It is transmitted to humans through the bite of infected female mosquitoes of the species Aedes aegypti and Ae. albopictus. Our study was carried out to survey the breeding habitats of mosquitoes that cause dengue and other mosquito vectors from different blocks of Nagapattinam district, Tamil Nadu. A total of 599 containers were inspected, among which 32 containers were positive for Aedes vectors. The breeding habitats were classified as: (1) container breeding, and (2) non-container breeding. Our study concludes that Ae. albopictus was the most abundantly present species followed by Culex quinquefasciatus in Nagapattinam district. Further, Cx. tritaeniorhynchus, Anopheles subpictus, An. barbirostris, Ae. aegypti, Cx. gelidus and Lutzia fuscana were also found. Our study identified various mosquito breeding sites and various vector and non-vector species in Nagapattinam district, Tamil Nadu, India.

Keywords: Field surveillance; dengue vectors; Nagapattinam district; breeding sites, identification.

Introduction

Dengue is one of the many vector-borne diseases (VBDs) and affects 390 million people worldwide. It is transmitted to humans through the bite of an infected female mosquito of the species Ae. aegypti and Ae. albopictus1. Dengue is spread by four serotypes of the dengue virus – DENV-1, DENV-2, DENV-3 and DENV-42. Diseases caused by Aedes vector mosquitoes, such as dengue, Zika fever, chikungunya and yellow fever are emerging and re-emerging globally3. Dengue is reported to be prevalent in more than 100 countries in the Americas, Eastern Mediterranean, Western Pacific, Africa and South-East Asia regions. Further, many European countries have reported the presence of autochthonous cases of

#E-mail: [email protected]

124 Dengue Bulletin – Volume 40, 2018 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India dengue1. The disease burden of dengue and its consequences is reflected in an economic loss of US$ 9 billion annually4.

Ae. aegypti is more common in human dwellings and breeds in forest tree holes. Ae. albopictus (Asian tiger mosquito) is also found within permanent or temporary water containers around urban areas and rural villages4. Dengue vectors have a worldwide distribution and breed in a variety of natural or artificial permanent and temporary water bodies, with a variety of oviposition habitats such as groundwater sites (pools, rivers and lakes) and container sites (bottles, cups and tyres)5.

Dengue is an emerging and re-emerging disease in India and globally. India, being a tropical country, has reported outbreaks of dengue in various years in various states8. Dengue outbreaks have also been reported in the Nagapattinam district of Tamil Nadu. According to reports from the National Vector Borne Disease Control Programme (NVBDCP), Tamil Nadu experienced a severe dengue epidemic in 2016, with 23 294 cases. Dengue is associated with urban settlements and the incidence rate of dengue in rural areas is also on the increase, often more than in urban areas. Thus, we aimed to assess dengue vector surveillance in the urban and suburban areas of Nagapattinam district of Tamil Nadu, by trying to understand the entomological indices and biodiversity of various vector and non-vector mosquitoes.

Materials and methods

Study area

A mosquito larval survey was conducted from January 2018 to March 2018. The study was carried out in the most affected areas of Nagapattinam district, Tamil Nadu. The blocks chosen were Keelaiyur, Thalainayar, Sembanarkoil, Kuttalam, Thirumarugal, Mayiladuthurai, Keelvelur and Nagapattinam. The mosquitoes emerged from immatures in different blocks were shown in (Table 1 and Figure 1). Nagapattinam is a shoreline front district of Tamil Nadu; on the eastern coast of Bay of Bengal, the district capital Nagapattinam lies between latitude 10.7906 degrees north and longitude 79.8428 degrees east (Figure 2).

Dengue Bulletin – Volume 40, 2018 125 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India

Table 1: Emerged mosquitoes from collected immature forms (block-wise) Culex quinquefasciatus was found to be high in Nagapattinam (urban area), Aedes albopictus was found to be high in Sembanarkoil block and Anopheles subpictus was recorded from Kuttalam block.

No. of emerged Sl. no. Name of the block Species of mosquitoes adult mosquitoes 1 NAGAPATTINAM Aedes aegypti 6 Culex quinquefasciatus 189 Anopheles subpictus 14 2 KEELVELUR Culex tritaeniorhynchus 3 Culex quinquefasciatus 6 3 TALAINAYAR Culex tritaeniorhynchus 15 4 SEMBANARKOIL Aedes albopictus 54 Lutzia fuscana 7 Culex gelidus 17 Culex quinquefasciatus 2 Culex tritaeniorhynchus 13 5 KUTTALAM Anopheles subpictus 28 Aedes albopictus 9 Culex quinquefasciatus 3

6 THIRUMARUGAL Aedes albopictus 10 Aedes aegypti 13 Culex tritaeniorhynchus 23 7 MAYILADUTHURAI Culex quinquefasciatus 7 Culex tritaeniorhynchus 11 Anopheles barbirostris 3 Anopheles subpictus 2 8 KEELAIYUR 0

126 Dengue Bulletin – Volume 40, 2018 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India

Figure 1: Emerged mosquitoes from collected immature forms (block-wise)

Nagapattinam block had the highest number of Culex quinquefasciatus and Sembaranarkoil had highest number of Aedes albopictus. Thirumarugal block had an almost equal number of Aedes albopictus and Aedes aegypti species followed by Culex tritaeniorhynchus.

Figure 2: Sampling places in various blocks of Nagapattinam District

Source: http://valanadu.in/about.php Eight blocks were chosen from Nagapattinam district, which contains various villages. Using GPS, the latitude and longitude were recorded. The pH of water was recorded and mosquito larvae were collected.

Dengue Bulletin – Volume 40, 2018 127 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India 4.00 0.00 0.00 0.00 0.00 2.57 4.00 8.00 3.67 0.00 20.00 42.24 positive positive container Pupae per 6.00 0.00 0.00 0.00 0.00 17.20 16.43 22.50 25.00 16.67 140.00 243.80 positive positive container Larval per per 0.52 0.00 0.00 0.00 0.00 0.68 0.31 0.36 1.43 0.34 0.00 3.64 Pupae container per 2.23 0.09 0.00 0.00 0.00 4.34 1.73 1.14 1.56 0.00 10.00 21.09 Larval container (%) 1.52 0.00 0.00 0.00 7.69 4.55 7.14 9.38 0.00 12.99 26.42 69.67 Positive Container 0 0 0 0 8 8 0 40 36 20 11 123 Pupae Collected 6 0 0 0 0 45 25 50 172 230 140 668 larvae Collected 1 0 0 0 2 1 1 3 0 10 14 32 Positive Container Container positivity, larval and pupal indices 2: Container positivity, Table 77 66 53 53 26 22 14 32 23 120 113 599 Container examined Total Pit Tyre Tree hole Tree Sintex tank Paddy field Paddy Cement Tank Cement Tank Polluted Well Polluted Breeding Sites Breeding Plastic buckets Irrigation canal Irrigation Plastic container Plastic Type of Container Drainage (Ditches) Type of Non Container 1 2 3 4 5 6 7 8 9 10 11 Sl.no. Container Positivity = Number of positive containers (infested)/Total number of containers inspected*100 containers of number (infested)/Total containers positive of Number = Positivity Container examined collected/container Pupae = container per Pupae examined container positive collected/ Pupae = container positive per Pupae A total of 599 containers was examined included natural and artificial breeding sites; 32 were found to be positive. Among the positive sites, 88 larvae and 123 pupae were collected.

128 Dengue Bulletin – Volume 40, 2018 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India

Mosquito sampling

Sampling was carried out between January and March 2018. Samples were collected from 599 natural and artificial water bodies at ground level (pits, ditches, channels) in public spaces of cities, roadside areas, houses, agricultural fields and coastal areas (Table 2). Sample sites were chosen randomly where positive cases of dengue had been reported. In general, 20–30 samples were collected from each site with a standard 350 mL dipper. When no larvae or pupae were detected, additional samples (up to 70) were collected to minimize false-negative results. The mosquito larvae were collected in plastic containers and mosquito rearing nets of about 0.55 mm mesh size into labelled containers and transferred to the laboratory for further analysis.

Rearing and identification of mosquitoes

The collected larvae were transported to the Vector Biology Research Laboratory (VBRL), at the Department of Life Sciences, Central University of Tamil Nadu, in separate rearing trays. The emerged mosquitoes were collected and identified using taxonomic keys according to species and sex. The entomological indices of larvae are given as house index (HI), container index (CI), Breateau index (BI), pupal index (PI) and adult premise index (API).

Calculation of entomological indices No. of houses positive for Aedes larvae HI= X 100 No. of houses searched

No. of pupae PI= X 100 No. of houses inspected

No. of positive containers CI= X 100 No. of containers searched

No. of positive containers BI= X 100 No. of houses searched

No. of houses positive for adult female mosquitoes API= X 100 No. of houses inspected

Dengue Bulletin – Volume 40, 2018 129 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India

Results

Out of 238 houses surveyed, 16 were found to support the breeding of Aedes mosquito species. Out of the 429 types of containers examined, 11 containers were reported to be positive for the presence of immature larvae. In continuation of this, 170 types of non- containers were examined, of which 21 were reported positive for the presence of immature larvae (Table 2). The larval indices were analysed in terms of HI, CI, BI and PI (Table 3). Among the entomological indices, high PI was noted followed by high BI in all the blocks examined (104.08 and 88.02, respectively). The HI and CI were found to be at the same level (42.22 and 42. 08, respectively) in all the blocks. The API percentage was also high (84.53%) in the selected blocks.

The reared mosquitoes were identified for sex, and females were found to constitute 57.3% of the total in all the blocks of Nagapatinam district. Among the 57% female emerged mosquitoes, 31% belonged to the Cx. quinquefasciatus species, 10% Ae. albopictus, 7% Cx. tritaeniorhynchus and 1% Ae. aegypti, followed by other species such as Lt. fuscana, Cx. gelidus, An. subpictus and An. barbirostris (Table 4 and Figure 3). We found substantial differences in the distribution of vectors between the blocks. The block-wise data showed that urban areas of Nagapattinam block had the highest number of Cx. quinquefasciatus and Sembaranarkoil had the highest number of Ae. albopictus.

Figure 3: Composition of the emerged adult female mosquitoes

35 31 Aedes aegypti

s 30 Aedes albopictus e o t i Culex quinquefasciatus u

q 25 s Culex tritaeniorhynchus o m

Lutzia fuscana t

l 20 u Culex gelidus d a

e Anopheles subpictus l 15 a

m 10 Anopheles barbirostris e F

10 f 7 o

% 4 5 3 1 1 0 0 Species of mosquitoes

130 Dengue Bulletin – Volume 40, 2018 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India PI 0.00 0.00 0.00 0.00 0.00 22.22 14.29 67.57 BI 0.00 8.89 6.67 8.57 4.35 15.00 14.81 29.73 CI 0.00 4.69 4.55 6.56 2.99 4.76 2.60 15.94 HI 0.00 6.67 0.00 0.00 2.50 0.00 11.43 21.62 0 0 0 0 5 0 40 10 25 Aedes infestation Pupae Larval indices(%) 0 95 45 45 62 25 668 245 151 Larvae 0 4 4 2 3 6 2 32 11 Positive

Containers 46 88 61 67 63 69 77 599 128 Examined 0 3 0 0 4 1 8 0 16 Positive Houses 23 45 27 30 35 40 37 46 283 Inspected Table 3: Entomological indices used to assess the level of Table Block name Keelaiyur Kuttalam Keelvelur Talainayar Thirumarugal Nagapattinam Sembanarkoil Mayiladuthurai S. no. Total 8 2 6 7 3 1 4 5 HI: house index; CI: container BI: Breateau Pl: pupal pndex Sembanarkoil block showed a higher HI followed by Thirumarugal. CI, BI and PI were found be high in Sembanarkoil.

Dengue Bulletin – Volume 40, 2018 131 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India

Discussion

Knowledge of the distribution of mosquitoes is an important part of a vector control programme and should be continued in order to implement such programmes9. We carried out this study to detect dengue-prone areas in Nagapattinam district, Tamil Nadu, by using entomological surveillance techniques. In our study, eight species of vector and non-vector mosquitoes were identified during the post-monsoon season, such as An. subpictus, An. barbirostris, Ae. aegypti, Ae. albopictus, Lt. fuscana, Cx. quinquefasciatus, Cx. tritaeniorhynchus and Cx. gelidus (Figure 4, Table 1). In line with our reports in Tamil Nadu, the previous work done at various districts other than Nagapattinam showed the presence of various vector and non-vector mosquitoes10.

Figure 4: Species composition of collected species

1% 4% 4% 10% 2% 17%

15%

47%

Aedes aegypti Aedes albopictus Culex quinquefasciatus Culex tritaeniorhynchus Lutzia fuscana Culex gelidus Anopheles subpictus Anopheles barbirostris

The major breeding sources recorded from various places included pits (26.42%), followed by tyres (12.99%), irrigation canals (9.38%), paddy fields (7.69%), drains (7.14%), polluted wells (4.55%) and plastic water storage tanks (1.52%) (Figure 5). Containers used for rainwater storage might be offering potential breeding sites for Aedes larvae in coastal regions. All the above-mentioned breeding sites were found to be inhabited by Cx. quinquefasciatus followed by Ae. albopictus, Cx. tritaeniorhynchus, An. subpictus, An. barbirostris, Ae. aegypti, Cx. gelidus and Lt. fuscana. Among the containers tested, the largest number of larvae were collected from pits followed by tyres. Thus, mosquito species were observed to breed in artificial containers filled with stagnant water, artificial pits and irrigation canals retained for a long duration without disturbing the breeding source. Climatic factors, housing structures, the types of used water containers varied in different areas of Nagapattinam. The selected study sites were densely populated urban areas and less populated rural areas. Both the

132 Dengue Bulletin – Volume 40, 2018 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India 0 0 0 0 0 0 0 % 0 0 0 0 0 0 0 F 0 0 0 0 0 0 % 0.69 0 0 0 0 0 3 0 M An. barbirostris An. 0 0 0 0 0 % 2.99 1.15 F 0 0 0 0 0 5 13 0 0 0 0 0 % 5.52 0.46 An. subpictus An. 0 0 0 0 0 2 M 24 0 0 0 0 0 % 1.8 0.7 8 0 0 3 0 0 0 F 0 0 0 0 0 % 0.9 0.5 Cx. gelidus 4 0 0 2 0 0 0 M 0 0 0 0 0 0 0 % 2 0 F 0 1 0 0 0 0 0 0 0 0 0 % 0.9 0 0 0 4 0 0 0 M Lutzia fuscana Lutzia 0 0 0 0 % 1.15 3.45 2.07 5 0 0 0 0 9 F 15 0 0 0 % 4.37 1.38 0.46 2.07 6 0 0 0 2 9 M 19 Cx. tritaeniorhynchus 0 0 0 % 10.6 0.69 19.1 0.46 F 0 3 0 2 0 46 83 0 0 0 % 5.29 0.92 10.3 0.23 0 4 0 1 0 M 23 45 Cx. quinquefasciatus 0 0 0 0 0 % 0.7 9.7 4: Species composition of the emerged mosquitoes

0 0 F 0 0 3 0 42 0 0 0 0 1 0 6 % Table 0 0 0 0 3 0 M 25 Ae. albopictus Ae. 0 0 0 0 1 0 0 % 0 0 F 0 0 6 0 0 0 0 0 0 3 0 0 % 0 0 0 0 0 0 Ae. aegypti Ae. 13 M containers Type of Non Container Pit Paddy fieldPaddy Drainage (Ditches) Polluted Well Polluted Type of Container Tyre Sintex tank Irrigation canal Irrigation 3 4 7 6 5 1 2 Sl. no The sex ratio of the collected specimens in emerged mosquitoes – 57% were female and 43% male mosquitoes.

Dengue Bulletin – Volume 40, 2018 133 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India areas have a shortage of a regular water supply, due to which people store water in various containers. Stagnant water is a favourable place for breeding of the dengue and other vectors11. The presence of Ae. albopictus and Ae. aegypti in the selected study areas are a high risk for dengue outbreaks.

Figure 5: Mosquito larvae breeding in various habitats

(A) Canal (B) Discarded tyre (C) Paddy field

(D) Stagnant water (E) Discarded tyre (F) Waster water in pit

134 Dengue Bulletin – Volume 40, 2018 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India

(G) Pit (H) Sintex tank (I) Pool

(J) Irrigation canal (K) Polluted well

Dengue Bulletin – Volume 40, 2018 135 Study of dengue vector breeding habitats and entomological indices in Nagapattinam district, Tamil Nadu, India

References

[1] Aranda C, Martínez MJ, Montalvo T, Eritja R, Navero-Castillejos J, Herreros E et al. Arbovirus surveillance: first dengue virus detection in local Ae. albopictus mosquitoes in Europe, Catalonia, Spain, 2015. Euro Surveill. 2018;23. [2] Guzman MG, Harris E. Dengue. Lancet. 2015;385:453–65. [3] Wilder-Smith A, Gubler DJ, Weaver SC, Monath TP, Heymann DL, Scott TW. Epidemic arboviral diseases: priorities for research and public health. Lancet Infect Dis. 2017;17:e101–e106. [4] Shepard DS, Undurraga EA, Halasa YA, Stanaway JD. The global economic burden of dengue: a systematic analysis. Lancet Infect Dis. 2016;16:935–41. [5] European Centre for Disease Prevention and Control (ECDC). Ae. albopictus. Factsheet for experts. Stockholm: ECDC (https://ecdc.europa.eu/en/disease-vectors/facts/mosquito-factsheets/aedes- albopictus, accessed 11 March 2019). [6] La Ruche G, Souarès Y, Armengaud A, Peloux-Petiot F, Delaunay P, Desprès P et al. First two autochthonous dengue virus infections in metropolitan , September 2010. Euro Surveill. 2010;15:19676. [7] National Vector Borne Disease Control Programe (http://www.nvbdcp.gov.in/index4.php?lang=1&le vel=0&linkid=431&lid=3715) [8] Gupta E, Ballani N. Current perspectives on the spread of dengue in India. Infect Drug Resist. 2014;7:337–42. [9] Basker P, Kolandaswamy KG. Study on the behavior of dengue viruses during outbreaks with reference to entomological and laboratory surveillance in the Cuddalore, Nagapattinam, and Tirunelveli districts of Tamil Nadu, India. Osong Public Health Res Perspect. 2015;6:143–58. [10] Victor TJ, Malathi M, Asokan R, Padmanaban P. Laboratory-based dengue fever surveillance in Tamil Nadu, India. Indian J Med Res. 2007;126:112–5. [11] Praveen G, Tyagi BK, Joe N, Thakur MB. An outbreak investigation of dengue fever in the coastal areas of Nagapattinam, Thiruvarur and Thanjavur districts in Tamil Nadu, India during 2012. International Journal of Mosquito Research. 2016;3:39–47.

136 Dengue Bulletin – Volume 40, 2018 Knowledge, attitude and practices for prevention and control of dengue fever among community members in North Delhi Municipal Corporation

Babita Bisht,a# Roop Kumari,b BN Nagpal,c Himmat Singh,c Kumar Vikram,c Sanjay Sinha,a NR Tulid

aNorth Delhi Municipal Corporation bNational Centre for Disease Control, Delhi cNational Institute of Malaria Research dSouth Delhi Municipal Corporation, Delhi

Abstract Dengue has established its endemicity in Delhi. The gap between outbreaks is decreasing and the disease poses a major public health challenge. With no specific treatment and vaccination available, source reduction remains the strategy of choice for dengue prevention. Civic bodies have engaged domestic breeding checkers for vector surveillance and providing health education. Even though Delhi has a literacy rate of more than 86%, dengue prevention remains a challenge. We decided to carry out an in-depth knowledge, attitude and practices (KAP) study to know the attitude of citizens and the practices adopted by them for dengue prevention. We conducted a descriptive cross-sectional study in six zones of the North Delhi Municipal Corporation (NDMC). About 65.75% of people related their source of knowledge to television and 64.5% to health personnel; 70.75% of the respondents knew that dengue is transmitted by the bite of a mosquito. However, we observed a gap in knowledge about common breeding places and the practices that need to be adopted by respondents for preventing dengue. Hence, there is a need to address the behaviour of community members. Although people have adequate knowledge, they do not adopt suitable practices to prevent dengue. There is thus a need for impact analysis and new tools and strategies that can be used for effective communication to seek community participation.

Keywords: Dengue, KAP, Prevention & Control.

Introduction

Dengue fever is an arboviral illness transmitted by the bite of an infected female Aedes mosquito. Dengue predominantly affects the tropical and subtropical regions with a recent transition from urban and semi-urban areas to rural areas. It has been estimated

#E-mail: [email protected]

Dengue Bulletin – Volume 40, 2018 137 KAP for prevention and control of dengue fever among community members in NDMC that approximately 2.5 billion people in tropical and subtropical countries are at risk of getting dengue fever. WHO has reported an increase in both the number of countries reporting dengue cases as well as the number of cases. Before 1970, only nine countries had experienced severe dengue epidemics whereas now dengue is endemic in more than 100 countries across all regions of WHO, especially the South-East Asia and the Western Pacific regions1. Epidemics of dengue are increasing in frequency. During epidemics, infection rates among those who have not been previously exposed to the virus are often 40–50% and can also reach 80–90%. This is the reason why a change in the dengue virus (DENV) serotype results in an increase in morbidity and mortality due to dengue. An estimated 50 million dengue infections occur worldwide annually and approximately 500 000 people with dengue haemorrhagic fever (DHF) require hospitalization each year.

In India, the first proven epidemic of dengue fever occurred in Calcutta in 1963–642. Dengue fever is emerging as a major public health challenge in Delhi. The epidemiology of dengue in Delhi has revealed a transition in the age group of those affected, from childhood to adulthood, as well as a change in spread from urban to rural settings. In Delhi, outbreaks have been reported in 2006, 2010, 2013 and 2015, showing that the gap between outbreak years is decreasing. This is a cause for concern, as all the four serotypes of DENV are circulating in Delhi. The erstwhile Municipal Corporation of Delhi (MCD) and now the three Delhi Municipal Corporations (DMCs) following trifurcation of the MCD in 2012, have the mandate to prevent and control vector-borne diseases in the areas under their jurisdiction. In the absence of specific treatment and a vaccine for dengue, control of mosquito breeding by source reduction remains the only strategy to prevent transmission. Even after a vaccine becomes available, source reduction will continue to be the key strategy.

Dengue transmission has been attributed to the behavioural patterns of citizens. Despite a high literacy rate of approximately 86%3, Delhi is struggling to contain outbreaks of dengue. A few studies on entomological and community knowledge, attitudes and practices (KAP) were carried out to assist the Municipal Corporation of Delhi (MCD) to better implement vector control activities in the city4,5. A number of community sensitization activities are being carried out by the three DMCs but despite possessing adequate knowledge and/or the ability to comprehend the messages on prevention and control of dengue, citizens have not adopted the practice of taking control measures by modifying their behaviour. We carried out a study to assess the knowledge, attitude and practices (KAP) of community members towards prevention and control of dengue. A major limitation of control programmes is that community members still feel that dengue prevention is the responsibility of the government. Their perception is that mosquitoes that transmit the dengue virus breed in drains and solid waste materials, and therefore the civic authorities must target cleaning of drains and managing solid waste properly before targeting citizens. The success of the National Vector-Borne Disease Control Programme (NVBDCP) will depend on social mobilization and behavioural change, and support from all stakeholders.

138 Dengue Bulletin – Volume 40, 2018 KAP for prevention and control of dengue fever among community members in NDMC

Methodology

Study design

We carried out a cross-sectional descriptive study in 2016. It was a community-based survey through a structured questionnaire.

Study area

We conducted the study in all the six zones of North Delhi Municipal Corporation (NDMC) – City zone, Sadar Paharganj zone, Karol Bagh zone, Narela zone, Civil Lines zone and Rohini Zone. The study population was taken from the three wards that had the highest cases of dengue reported in the past 3 years. In this community-based survey, we assessed the KAP of community members. The sample size was calculated using the following formula6:

N = Z2 X p(1-p) ______d2 Where N is the population size; Z is the z-score; d is the margin of error and p is the population proportion

The community members selected for this study were from both the sexes and above 18 years of age. All respondents were assured of confidentiality of the information provided by them and their consent was taken. The questionnaire was translated into Hindi for respondents who could not read English. The questionnaire was pretested to check that it included all the important components, i.e. the sociodemographic profile of the respondents, knowledge about dengue symptoms, signs and transmission modes, sources of information, attitudes towards dengue, practices adopted for the prevention of dengue, and services provided by the municipal corporation. To study the attitude of the respondents, six variables were identified and five options were given to the respondents. The five options were – strongly agree, agree, not sure, disagree, strongly disagree6. These options were scored from 5 to 1, respectively. After collating the data, SPSS was used for analysis.

Dengue Bulletin – Volume 40, 2018 139 KAP for prevention and control of dengue fever among community members in NDMC

Results

Table 1: Sociodemographic characteristics of the respondents

Parameters Frequency (N = 400) Percentage (%) Sex Female 164 41 Male 236 59 Age (in years) 18–29 45 11.25 30–44 96 24 45–59 138 34.5 >59 121 30.25 Marital status Unmarried 61 15.25 Married 339 84.75 Level of education Illiterate 28 7 Primary school 91 22.75 Secondary school 112 28 Graduate 114 28.5 Postgraduate 40 10 Professional 15 3.75 Family income per month (in Rs) <3000 32 8 3000–10 000 106 26.5 >10 000 262 65.5 Past incidence of dengue in family/neighbourhood Yes 168 42 No 232 58

Table 1 shows that 59% of the respondents were men while 41% were women. We tried to include an almost equal number of participants from both the sexes but since women were generally hesitant to answer the questionnaire, they asked the men available at home to answer. Twenty-four per cent of the respondents were in the age group of 30–44 years and 34.5% were in the age group of 45–59 years. About 84.75% were married. Seven per

140 Dengue Bulletin – Volume 40, 2018 KAP for prevention and control of dengue fever among community members in NDMC cent of the respondents were illiterate and 28% had graduation-level qualifications. Illiterate respondents were mainly from the cluster areas of the various zones while 42% had had dengue cases in their family or neighbourhood.

Table 2: Source of information on dengue fever

Parameters Frequency (N) Percentage (%) Had ever received information on dengue Yes 324 81 No 76 19 Source of information Television 263 65.75 Radio 145 36.25 Newspapers 185 46.25 Banners 28 7 Handbills 167 41.75 Friends & neighbours 118 29.5 Health personnel 258 64.5 Others 36 9

Figure 1: Sources of information as reported by the respondents

70 65.75 64.5

60

50 46.25 41.75 ge 40

ta 36.25 en

rc 29.5 30 Pe

20

9 10 7

0 r l s o rs on pe di od s nd si th pa onne ie Ra nne vi ndb ill s rs Fr le Ba ws me Ha pe r Te

Ne he th al Ot He

Dengue Bulletin – Volume 40, 2018 141 KAP for prevention and control of dengue fever among community members in NDMC

Eighty-one per cent of the respondents were aware of dengue fever. When they were asked about the source of their knowledge, 65.75% related it to television and 64.5% said that breeding checkers from the MCD had provided them with the necessary information during their visits. Other sources of information were the radio (36.25%), handbills (41.75%) and friends (29.5%). About 35% had information from more than two sources.

Most of the respondents (77.25%) knew that dengue is transmitted by mosquitoes, There was sizable percentage (21%) of people who believed that dengue is spread by house flies. About 68.25% respondents knew that dengue is associated with fever, and headache and muscle pains were common symptoms, whereas a few (16.25%) were aware of the bleeding manifestations of dengue. Twenty-six per cent of the respondents were aware of more than one symptom of dengue (Table 3, Figure 2).

Table 3: Knowledge regarding transmission of dengue fever and its symptoms

Parameters Frequency (N) Percentage (%) Symptoms of dengue fever Fever 273 68.25 Headache 122 30.5 Joint/muscular pain 140 35 Pain in abdomen 48 12 Nausea/vomiting 11 2.75 Bleeding 65 16.25 Don’t know 10 2.5

Figure 2: Knowledge regarding mode of transmission of dengue fever

90 77.25 80

70

60

t 50 en rc 40 Pe

30 21 20 5.25 10 2.5 0.5

0 Mosquito bite Houseflies Drinking dirty waterUnhygienic food Don’t know

142 Dengue Bulletin – Volume 40, 2018 KAP for prevention and control of dengue fever among community members in NDMC

Table 4: Knowledge about the characteristics of the vector

Parameters Frequency (N) Percentage (%) Common breeding sites* Desert coolers 277 69.25 Overhead tanks 142 35.5 Jars/containers for water storage 176 44 Flower pots/bottles with money plants/decorative 95 plants 23.75 Water pots for birds 71 17.75 Waste articles (e.g. plastic cups/bottles) 102 25.5 Used tyres/waste containers lying in open areas 88 22 Plants/vegetation 65 16.25 Drains/dirty water 132 33 Don’t know 48 12 Most frequent time of mosquito bite Day time 180 45 Night/evening 130 32.5 Any time 62 15.5 Don’t know 28 7

Desert coolers were reported as the most common breeding habitat of the vector (69.25%), followed by jars/containers for storing water (44%), overhead tanks (35.5%), and waste articles such as disposable cups, plastic bottles and cans (25.5%). Thirty-three per cent of the respondents were of the opinion that mosquitoes that transmit dengue breed in dirty water or drains, while 22% said that the Aedes mosquito may also breed in tyres and waste articles lying in the open (Table 4). During discussions, the respondents reported that the mosquito also breeds in clean water collections. Twelve per cent of the respondents did not know common breeding sites, while 9% reported more than two mosquito-breeding sites. Regarding the biting habits of the Aedes mosquito, 45% were aware that it bites during the day, while 15% reported that it could bite through the day (Table 4).

Dengue Bulletin – Volume 40, 2018 143 KAP for prevention and control of dengue fever among community members in NDMC

Table 5: Attitudes of respondents towards dengue fever

Strongly Not Strongly Variables Agree Disagree Mean* agree sure disagree

Dengue fever can be F (N) 134 193 16 46 11 3.98 prevented % 33.5 48.25 4 11.5 2.75 Dengue mosquito breeds F (N) 62 178 31 61 68 3.26 inside houses % 15.5 44.5 7.75 15.25 17 Dengue mosquito breeds F (N) 40 86 32 178 64 2.65 in drains % 10 21.5 8 44.5 16 Elimination of breeding F (N) 101 221 9 45 24 is the responsibility of 3.82 government staff % 25.25 55.25 2.25 11.25 6 Fogging is the only F (N) 85 213 7 63 32 method to prevent 3.64 breeding of mosquitoes % 21.25 53.25 1.75 15.75 8

Any person can get F (N) 87 145 82 54 32 3.66 dengue fever % 21.75 36.25 20.5 13.5 8

*Statistical mean calculated by SPSS. F frequency

Table 5 shows that 81.75% of the respondents strongly agreed/agreed that dengue is a preventable disease. The statistical mean for all respondents was 3.98, which is higher than the reference mean of 3, suggesting that respondents agree that dengue is preventable. Sixty per cent of the respondents believed that the mosquito that transmits dengue breeds inside houses (mean 3.26), 31.5% believed that it breeds in drains, whereas 60.5% did not agree with this. Regarding involvement of the community and the roles and responsibilities of government agencies, 80.75% of the respondents believed that it is the duty of the concerned government agency to control mosquito breeding and prevent transmission of dengue, and the higher mean value (3.82) associated with it supports this statement. This belief inhibits the community from taking control measures and participating in dengue prevention. A large proportion of respondents (74.25%) felt that fogging is the only way to prevent dengue; the statistical mean of 3.64 supports the statement. When asked about vulnerability to dengue infection, 58% of the respondents agreed that anyone can get dengue infection.

144 Dengue Bulletin – Volume 40, 2018 KAP for prevention and control of dengue fever among community members in NDMC

Table 6: Practices to prevent and control dengue

Preventive measures Frequency (N) Percentage (%) Do you empty and dry your water cooler every week? 197 49.25 Do you check the overhead tanks every week to see if the 75 18.75 lid is well covered? Do you keep water storage containers covered? 117 29.25 Do you change the water in the pots for birds every day? 107 26.75 Do you change the water in flower vases/decorative plants 76 19 every week? Do you dispose of solid waste such as plastic cups and 114 28.5 bottles in municipal bins? Do you keep used tyres/waste containers under cover? 48 12 Do you check for any stagnation of water around your 55 13.75 house? Do you drain off the water collected in the plates kept 9 2.25 under refrigerators/pots at least once in a week? During the rainy season, do you check for water stagnation 50 12.5 on your roof every week? None 15 3.75

Table 6 shows the practices adopted by respondents for the prevention and control of dengue fever. Of the respondents, 49.25% reported that they empty and dry desert coolers every week,12.5% check their rooftops for water stagnation and 18.75% confirmed that they keep overhead tanks covered with a lid. Nearly one third (29.25%) keep water storage containers covered, 28.5% dispose of solid waste such as disposable cups and plastic bottles in municipal bins, and 26.75% change water in the bird pots daily. This practice may be attributed to religious beliefs. Whereas 19% of the respondents change the water in flower vases/decorative plants every week, 3.75% denied of taking any measures to prevent and control mosquito breeding.

When the statistical correlation between knowledge and practice was done, it was found to be positively correlated (r=0.847; at a 0.01 level of significance).

Dengue Bulletin – Volume 40, 2018 145 KAP for prevention and control of dengue fever among community members in NDMC

Figure 3: Graphical representation of knowledge and practices 100 90 80 70 60

ge 50 ta

en 40 rc 30 Pe 20 10 0 . ) r s s s s e e e ts th en .g iv te st es er rd ow nk ea ag tl an wi (e at bi

op ta ar kn

or wa wa pl ool

ot r or t r es es in st c s/ fo ad /b tl cl n’ ec

fo rt g re ti ot ps ts s he in /d se ty Do ar

ly er er ts cu po De rs ts/b in c ed r an ste Ov ti ta te ne pl po Us

as ai y Wa on er pl Wa /c rs ow cont Fl mone Ja

Knowledge on Breeding Sites Practice for prevention

There is a gap between knowledge and practices adopted by respondents to prevent dengue (Figure 3). Regarding water pots for birds, 17.75% of the respondents were aware that these act as breeding habitats for mosquitoes, whereas 26.75% reported that they clean these water pots daily before filling them. This gap can be attributed to religious practices and rituals. They feel that water being served to birds should be clean and offered in clean containers. Similarly, 25.5% of the respondents reported that they knew that breeding also occurs in solid waste, whereas 28.5% reported that they dispose of such solid waste in municipal bins.

Table 7: Personal protective measures for prevention of dengue

Preventive measures Frequency (N) Percentage (%) Mosquito spray 124 31 Mat/coil/liquid vaporizer 294 73.5 Mosquito net for sleeping 68 17 Wear clothes that cover the full body to prevent 153 38.25 mosquito bites Wire mesh on windows and doors 176 44 Use of smoke to drive mosquitoes away 12 3 None 57 14.25

146 Dengue Bulletin – Volume 40, 2018 KAP for prevention and control of dengue fever among community members in NDMC

Figure 4: Activities undertaken by respondents for personal protection against dengue

None 14.25

Use of smoke to drive mosquitoes away 3

Wire mesh on windows and doors 44

Wear clothes that cover the full body to prevent 38.25 mosquito bites

Mosquito net for sleeping 17

Mat/coil/liquid vaporizer 73.5

Mosquito spray 31

020406080 Percentage

On enquiring about personal protective measures taken by the respondents against dengue, 73.5% informed that they use mats/coils/liquid vaporizers, 44% have wire meshes for the windows and doors of their houses, 38.25% wear full-sleeved clothes to cover their bodies, and encouraged their children to wear full-sleeved clothes when they received such directions from school. Thirty-one per cent carry out some type of spraying of their houses, and 17% use mosquito nets as a personal protective measure. Use of repellents/vaporizers/ coils is high due to the marketing techniques of multinational companies (Table 7; Figure 4).

Table 8: Control measures undertaken by the Department of Health

Control measures Frequency (N) Percentage (%) Has any domestic breeding checker 202 50.5 visited your house? Do they organize any awareness 147 36.75 campaigns on prevention of dengue? Does the Department of Health carry out 133 33.25 spraying/fogging in your house?

To evaluate the activities carried out by the staff of the Department of Public Health, the questionnaire included common activities performed by the staff. About 50.5% of the respondents informed that some official had visited their house to check the coolers occasionally; 36.75% informed that staff of the MCD also provides health education, and 33.25% informed that, at times, MCD staff carry out spraying/fogging in their houses (Table 9).

Dengue Bulletin – Volume 40, 2018 147 KAP for prevention and control of dengue fever among community members in NDMC

Discussion

Dengue is emerging as a major public health challenge in Delhi. Community participation has been identified as a key strategy for the prevention and control of dengue fever, as the mosquitoes that transmit dengue breed in domestic and peridomestic settings. A study conducted in 2010 in slum/unauthorized areas of Delhi found that only 40.62% of respondents were aware that dengue is transmitted by a mosquito bite, while 56.25% were not4. Our study shows that 77.25% of the respondents were aware that mosquito is the vector that transmits dengue. However, a hospital-based study in Delhi reported that 89% of respondents from urban areas were aware that mosquito is the vector that transmits dengue5, perhaps because of better health education activities by civic bodies and other health agencies. Messages on television and interpersonal communication by health personnel (domestic breeding checkers) have led to improved knowledge of dengue. In a study carried out in the rural areas of central India, 76.58% of the respondents were aware of the fact that mosquito is the vector for dengue transmission6. A study in Male, Maldives, showed that 46% of respondents had poor knowledge, 41% moderate and 13% high knowledge about dengue7.

In our study, 65.75% of the respondents reported that television was the main source of their knowledge. This fact was supported by a study in Puerto Rico and Malaysia8,9. In a study conducted in 2005 in a resettlement colony of Delhi, 59% of respondents reported that television was the most important source of their information10. The various municipal corporations of Delhi have engaged domestic breeding checkers to check domestic breeding and impart health education to residents on common breeding sites and measures to prevent these.

Our study showed that health personnel played a significant role in imparting information (64.5%). Other studies in Delhi also support this fact10. In a similar study carried out in Thailand, 52.5% of the respondents agreed that health personnel were their source of information11.

A significant percentage of respondents (71.92%) was aware that fever was the main symptom of dengue. Another study also reported similar findings10. However, in a study conducted by Ahmed et al. in 2008, 41.9% of the respondents reported bleeding as a common symptom12 whereas in our study, 16.25% were aware of the bleeding manifestations of dengue.

Seventy per cent of the respondents reported that desert coolers were the most common breeding site, whereas in other studies it was considerably less – 11.22% and 42.4%5,6. This variation in the findings can be attributed to the improved knowledge of respondents over the years. Respondents were also aware of other common breeding sites such as jars/ containers for storing water (44%) and waste articles such as disposable cups, plastic bottles, cans (25.5%).

148 Dengue Bulletin – Volume 40, 2018 KAP for prevention and control of dengue fever among community members in NDMC

Thirty-three per cent of the respondents were of the opinion that mosquitoes that transmit dengue breed in dirty water collections or drains. This perception often poses a challenge during field surveys as citizens often complain about the threat of dengue due to choked or overflowing drains/sewers. This perception needs to be altered and addressed through health awareness campaigns. Waste articles/waste tyres stored by junk dealers and tyre repair shops were also implicated as breeding sites by respondents. Breeding sites of Aedes may vary from city to city due to different sociocultural practices. There is a need to target these favoured breeding sites in the action plans to control dengue. Strong administrative will is needed to include source reduction of mosquito breeding into action plans of the engineering and sanitation departments. As the Aedes mosquito bites during the day, when most people are outdoors for different educational/commercial/economic activities, preventive measures should be taken at workplaces, schools and other institutions.

A significant percentage of the respondents (81.75%) accepted that dengue is a preventable disease. This was also proved statistically. Regarding involvement of the community and roles and responsibilities of government agencies, 80.75% of the respondents felt that it is the duty of the concerned government agency to control breeding of mosquitoes and prevent dengue. This attitude stops them from taking control measures in and around their houses/ workplaces. In contrast, a study from Brazil reported that 75.3% of the respondents felt it is the responsibility of citizens but fogging is the responsibility of the government13,14. In our study, 74.25% of the respondents felt that fogging is the only way to prevent dengue. These findings need to be addressed while formulating an action plan in Delhi.

Our study showed that people are aware of personal protective measures against mosquito bite (73.5%). Other studies also support this finding15.

Our study showed that there was a significant gap between knowledge about dengue in the community and the practices adopted by the community. This shows a need to address this gap by behaviour modification of citizens. There is a need to reduce garbage by providing sufficient bins and educating citizens to not litter. Timely and proper disposal of solid waste from all sites should be done to prevent collection of rainwater. Guidelines have also been developed for the Communication for Behavioural Impact (COMBI) model regarding the need for adopting appropriate, sustainable behavioural changes, interventions, and planning sustainable interventions to address behavioural issues related to the prevention and control of dengue16. In a study carried out in Amritsar in Punjab, high knowledge was reported among respondents (98.8%) but preventive measures were not being practised satisfactorily17. There is a need for making people living in villages and slums aware of the different preventive practices and reduce the gap that exists between their knowledge and application; this was also reported in a study conducted among the rural areas and the slums of north India18. Prevention of mosquito breeding can be linked to other national campaigns such as the Swachh Bharat Mission19. Areas should be stratified using epidemiological, entomological and behavioural risk indicators in order to develop and deliver an intervention mix that will respond to the priority risk indicators of that area20. In another study in the slums of Delhi,

Dengue Bulletin – Volume 40, 2018 149 KAP for prevention and control of dengue fever among community members in NDMC it was suggested that housewives be targeted to improve their awareness as they are the ones mostly responsible for water storage and cleanliness of the house and surroundings. It was also suggested that messages spread by word of mouth have a greater impact; this should be taken care of by municipal health workers when they go door-to-door to provide information on the breeding sites of mosquitoes and how to prevent dengue infection21.

Conclusion

Dengue has established its endemicity in Delhi. Despite various measures taken by the civic bodies, it poses a major public health challenge. There is a positive correlation between knowledge and practices but a gap has been observed in the practices adopted by respondents to prevent dengue. It is the responsibility of the house owner/ person occupying the premises to take measures to prevent and control mosquito breeding in and around the premises, and not depend only upon domestic breeding checkers. The behaviour of community members should be addressed so that they adopt these practices as a part of their regular routine. Existing mechanisms for communication should be evaluated through impact analysis, and new tools and strategies used for effective communication to enhance community participation. Capacity-building of domestic breeding checkers and other field staff will help in effective communication with citizens. Electronic/social media could also be used frequently as a source of information dissemination. Proper management of solid waste also needs to be strengthened by the civic bodies. Behaviour change in the community will help to reduce the gap between practices and knowledge of dengue control, and decrease the burden of dengue. This could be helpful in realizing the government’s bigger initiatives such as Swachh Bharat and Swasth Bharat Missions.

Acknowledgements

I want to express my sincere thanks to Mr PK Singh, the then Additional Commissioner (H), North Delhi Municipal Corporation for his constant support and encouragement. I also want to extent my gratitude to the deputy health officers and entomologists of all the six zones of NDMC for helping me to carry out this study, especially Mrs Harleen Kaur, Entomologist, for helping me in data collection, and the entomological teams of the city zones for helping in data collection and compilation. My thanks also go to Mr Chitesh from PwC India for helping in data analysis.

150 Dengue Bulletin – Volume 40, 2018 KAP for prevention and control of dengue fever among community members in NDMC

References

[1] Comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. Revised and expanded edition. New Delhi: World Health Organization Regional Office for South-East Asia; 2011 (http://www.searo.who.int/entity/vector_borne_tropical_diseases/documents/SEAROTPS60/ en/, accessed 9 March 2019). [2] Gupta N, Srivastava S, Jain A, Chaturvedi UC. Dengue in India. Indian J Med Res. 2012;136:373–90. [3] Delhi Population Census data 2011 [website] (https://www.census2011.co.in/census/state/delhi.html, accessed 9 March 2019). [4] Singh RK, Mittal PK, Yadav NK, Gehlot OP, Dhiman RC. Aedes aegypti indices and KAP study in Sangam Vihar, south Delhi, during the XIX Commonwealth Games, New Delhi, 2010. Dengue Bulletin. 2011;35:131–40. [5] Matta S, Bhalla S, Singh D, Rasania SK, Singh S. Knowledge, attitude and practice (KAP) on dengue fever: a hospital based study. Indian J Community Med. 2006;31:185–6. [6] Taksande A, Lakhkar B. Knowledge, attitude and practice (KAP) of dengue fever in the rural area of central India. Shiraz E Med J. 2012;13:146–57. [7] Ahmed N, Taneepanichskul S. Knowledge, attitude and practices of dengue prevention among the people in Male, Maldives. J Health Res. 2008;22 (Suppl):33–37. [8] Winch PJ, Leontsini E, Rigau-Pérez JG, Ruiz-Pérez M, Clark GG, Gubler DJ. Community-based dengue prevention programs in Puerto Rico: impact on knowledge, behavior, and residential mosquito infestation. Am J Trop Med Hyg. 2002;67:363–70. [9] Hairi F, Ong CH, Suhaimi A, Tsung TW, bin Anis Ahmad MA, Sundaraj C et al. Knowledge, attitude and practices (KAP) study on dengue among selected rural communities in the Kuala Kangsar district, Malaysia. Asia Pac J Public Health. 2003;15:37–43. [10] Acharya A, Goswami K, Srinath S, Goswami A. Awareness about dengue syndrome and related preventive practices amongst residents of an urban resettlement colony of south Delhi. J Vector Borne Dis. 2005;42:122–7. [11] Swaddiwudhipong W, Lerdlukanavonge P, Khumklam P, Koonchote S, Nguntra P, Chaovakiratipong C. A survey of knowledge, attitude and practice of the prevention of dengue hemorrhagic fever in an urban community of Thailand. Southeast Asian J Trop Med Public Health. 1992;23:207–11. [12] Itrat A, Khan A, Javaid S, Kamal M, Khan H, Javed S et al. Knowledge, awareness and practices regarding dengue fever among the adult population of dengue hit cosmopolitan. PLoS One. 2008;3:e2620. [13] Dégallier N, Vilarinhos PT, De Carvalho MS, Knox MB, Caetano J Jr. People’s knowledge and practice about dengue, its vectors, and control means in Brasilia (DF), Brazil: its relevance with entomological factors. J Am Mosq Control Assoc. 2000;16:114–23. [14] Pérez-Guerra CL, Seda H, García-Rivera EJ, Clark GG. Knowledge and attitudes in Puerto Rico concerning dengue prevention. Rev Panam Salud Publica. 2005;17:243–53. [15] Snehalatha KS, Ramaiah KD, Vijay Kumar KN, Das PK. The mosquito problem and type and costs of personal protection measures used in rural and urban communities in Pondicherry region, South India. Acta Trop. 2003;88:3–9.

Dengue Bulletin – Volume 40, 2018 151 KAP for prevention and control of dengue fever among community members in NDMC

[16] Renganathan E, Parks W, Lloyd L, Nathan MB, Hosein E, Odugleh A et al. Towards sustaining behavioural impact in dengue prevention and control. Dengue Bulletin. 2003;27:6–12. [17] Tikoo D, Sharma G, Gupta M. Assessment of knowledge, attitude and practice of dengue in factory workers of Amritsar, Punjab. Int J Basic Clin Pharmacol. 2016;5:38–44. [18] Malhotra G, Yadav A, Dudeja P. Knowledge, awareness and practices regarding dengue among rural and slum communities in North Indian City, India. Int J Med Sci Public Health. 2014;3:295–9. [19] Swachh Bharat Mission campaign catching up. Press Information Bureau, Government of India (sbm. gov.in/sbmreport/home.aspx, accessed 9 March 2019). [20] Elder J, Lloyd LS. Achieving behaviour change for dengue control: methods, scaling-up, and sustainability. Working paper for the Scientific Working Group on Dengue Research, convened by the Special Programme for Research and Training in Tropical Diseases, Geneva, 1–5 October 2006 (http://www. who.int/tdr/publications/documents/swg_dengue_2.pdf, accessed 9 March 2019). [21] Daudé É, Mazumdar S, Solanki V. Widespread fear of dengue transmission but poor practices of dengue prevention: a study in the slums of Delhi, India. PLoS One. 2017;12:e0171543.

152 Dengue Bulletin – Volume 40, 2018 A geostatistical study to prioritize dengue-affected areas for implementation of effective control by municipal corporations of Delhi, India

Sanjeev Kumar Gupta,a Poonam Saroha,a Kumar Vikram,a NR Tuli,b Himmat Singh,a# Rekha Saxena,a# Aruna Srivastava,a BN Nagpal,a MC Joshic

aICMR-National Institute of Malaria Research, New Delhi bSouth Delhi Municipal Corporation of Delhi cKumaun University, Nainital, Uttarakhand

Abstract Background and objectives. To prioritize dengue-affected areas in Delhi, we conducted a geographic information system (GIS)-based study from 2009 to 2014 in 272 wards of the Municipal Corporation of Delhi (MCD), eight municipal wards of Delhi Cantonment (DCB) and nine subdivisions of New Delhi Municipal Council (NDMC). We noted spatial distribution patterns in the form of hotspots, high-risk areas and consistent high-risk wards for implementation of effective control by the MCDs, India. Methods. We collected epidemiological data from 2009 to 2014 from MCDs. Dengue cases were pooled and attached ward-wise. Further, the dengue rate (dR), described as dengue cases per thousand population (dR = dengue cases x 1000/ward population), was calculated for each year. Global Moran’s I index, Getis-Ord Gi* and spatial statistical tools were used in GIS for analysis. Results. We observed a significant clustered pattern of dengue cases in 2009, 2013 and 2014 (P<0.01), and in 2012 (P<0.10), and a random pattern in 2010 and 2011. One ward of the DCB was consistently identified for 3 years to be in the hotspot/high-risk category, whereas 26 wards from nine zones of Delhi were consistently identified for 2 years as being in the hotspot/high-risk category. We also observed that clustered distribution during one year led to an outbreak in the subsequent year. Priority control has been recommended in 27 wards that fall consistently in the hotspot and high-risk category. Interpretation and conclusion. Geostatistical-based hotspot and high-risk area analysis provides decision-makers with information to prioritize focused control of dengue. The study also revealed that clustered distribution during one year led to an outbreak during the subsequent year, which provides an opportunity for decision-makers to put in place vector control measures in advance. The study highlights the utility of GIS and spatial statistical tools for efficient processing of voluminous dengue epidemiological data at the micro level with a sound statistical base for focused and quick decision-making. Keywords: GIS; dengue; hotspot; high risk; geostatistical.

#E-mail: [email protected]

Dengue Bulletin – Volume 40, 2018 153 A geostatistical study to prioritize dengue-affected areas for implementation of effective control

Introduction

Dengue is a major public health problem globally. The World Health Organization states that dengue cases have increased 30-fold globally over the past five decades1. Dengue is endemic in 128 countries and 3.9 billion people are at risk of infection1. In India, the total number of dengue cases has significantly increased since 2001. In India, dengue is endemic in almost all states and is the leading cause of hospitalization2. In addition to the increased number of cases and disease severity, dengue is not only restricted to urban areas but has now spread to rural areas as well3. The frequency of dengue outbreaks has also increased in most of the states of India, with all four serotypes being prevalent4. The spatial distribution of dengue in India has been related to population growth, unplanned urbanization, changes in environmental factors, lack of sanitation, host–pathogen interactions and inadequate vector control measures3,5.

Dengue is driven by complex interactions among host, vector and virus, which are influenced by climatic factors. Both Aedes aegypti and Aedes albopictus are the main vectors for the dengue virus in India6. The National Capital Territory of Delhi is one of the dengue- endemic states of north India7. Severe dengue outbreaks were reported in Delhi during the past five decades, i.e. in 1967, 1970, 1982, 1988, 1996, 2003, 2006, 2010 and 20138–15. Delhi also experienced an outbreak of dengue in 2015, with 15 867 reported cases16.

The key challenges to the prevention and control of dengue are inadequate capacity of human resources, lack of funds and lack of a rapid response plan; knowledge gaps in diagnosis, case management and vector control; lack of evidence-based decision support and limited intersectoral collaboration. Factors that contribute to these challenges include (i) a rise in the number and size of densely populated urban cities, (ii) increased virus spread due to global movement, and (iii) extensive and indiscriminate use of insecticides resulting in insecticide resistance. In the absence of a dengue vaccine, vector control remains the key strategy for dengue prevention and control.

The ultimate goal is to reduce the disease burden by generating information that empowers the public to take protective action and helps public health agencies to allocate limited prevention, surveillance and control resources to the best effect. Timely information on the epidemiology of the disease and other causal factors is essential for public health practitioners.

The spatiotemporal dynamics of dengue or other vector-borne diseases related to the environment can be quantitatively analysed using geographical information systems (GIS) and other geospatial tools such as global positioning system (GPS). GIS is a tool that allows for the superimposition, analysis, manipulation, storage, retrieval and display of datasets from various sources. The statistical tool embedded in GIS can be used to understand the spatial variability of the disease. Therefore, we conducted a study to map dengue cases in Delhi on the GIS platform for identification of hotspots in order to support focused intervention.

154 Dengue Bulletin – Volume 40, 2018 A geostatistical study to prioritize dengue-affected areas for implementation of effective control

Material and methods

Data acquisition, thematic maps and tools

Software used: ESRI India’s ArcGIS Desktop 10.5

Thematic map and its source. The entire geographical territory of NCT of Delhi is divided into three statutory towns, i.e. Municipal Corporations of Delhi (MCD), New Delhi Municipal Council (NDMC) and Delhi Cantonment Board (DCB). As per the 2001 Census, there were 134 municipal wards in the MCD; however, after delimitation in 2007, the MCD area is presently divided into 272 wards17. The DCB presently has eight municipal wards whereas the NDMC has nine subdivisions17. We procured zone and ward maps of Delhi from a private GIS agency along with ward-wise Census data of 2011. The agency prepared digital maps by integrating location information from multiple sources, viz. secondary sources such as city maps, DDA publications, etc. In specific areas, we also undertook rapid reconnaissance field surveys.

Dengue epidemiological data. We collected dengue epidemiological data of Delhi for 6 years, i.e. from 2009 to 2014. These contained patient names, addresses, ages, sex, zones with dates of hospitalization and discharge from MCD. Dengue cases were pooled ward-wise and linked to the ward map of Delhi in the GIS. Further, the dengue rate (dR), described as dengue cases per thousand population, was calculated (dR = dengue cases x 1000/ward population) for each year.

GIS-based spatial statistical tools. The spatial autocorrelation tool Global Moran’s I was used to identify the spatial pattern, i.e. clustered, random or dispersed, based simultaneously on the features of the locations (wards of Delhi) and feature values, i.e. dR. The test compares the feature values of neighbouring units over the whole study area18. When neighbouring units over the whole study area have similar values, it indicates a strong positive autocorrelation, i.e. clustering. Otherwise, if neighbouring units have highly dissimilar values, then the statistics indicate a strong negative spatial autocorrelation, i.e. dispersion. The test returns Moran’s I index (between –1 and 1) and Z-score. A statistically significant Z score with a positive Moran’s I index indicates a tendency towards clustering while a negative value indicates dispersion. When the Moran’s I index is not significantly different from 0, there is no spatial autocorrelation, thus the spatial pattern is considered to be random. The null hypothesis (Ho) was set such that that there was no spatial clustering of dengue cases in the wards.

The hotspot analysis tool Getis-Ord Gi* identifies different spatial clustering patterns such as hotspots and high-risk areas over the entire study area with their statistical significance18. The test returns Z-scores and P-values. The larger the value of Z, the more intense the clustering, i.e. hotspot. High-risk areas have a lower significance level in comparison to hotspots.

Dengue Bulletin – Volume 40, 2018 155 A geostatistical study to prioritize dengue-affected areas for implementation of effective control

The fixed distance band method in spatial statistics has been used to ensure a consistent scale of analysis, as there is large variation in size of the wards in Delhi. Using the distance measuring tool of GIS, the average distance between the centroid to centroid of wards was found to be in the range of 1–8 km.

Spatial autocorrelation was measured on the basis of both ward locations and dRs for each year at difference bands ranging from 1 km to 8 km. The Z-score peaked at 4 km, i.e. a 4 km distance band reflects maximum spatial autocorrelation. Therefore, a 4 km distance band was considered for hotspot analysis.

Results and discussion

The results of Moran’s I index for dR for each year (2009–2014) is shown in Table 1. A significant clustered pattern was found in the years 2009, 2013 and 2014 (P<0.01) and in 2012 (P<0.10). A random pattern was found in the years 2010 and 2011, with Z-scores of –0.24 and 1.01, respectively, as shown in Figure 1. Therefore, the null hypothesis (H0) that dR is randomly distributed within the wards was accepted for the years 2010 and 2011 and rejected for the years 2009, 2012, 2013 and 2014.

Table 1: Global Moran’s I index, Z-values, P value and pattern of ward-wise dengue rate from 2009 to 2014

Moran’s Confidence Year Z-score P value (significance) Pattern index level 2009 0.151 8.865 <0.01 (significant) 99% Clustered 2010 –0.006 –0.241 0.809 (non-significant) - Random 2011 0.001 1.014 0.311 (non-significant) - Random 2012 0.029 1.761 0.078 (significant) 90% Clustered 2013 0.084 4.640 <0.01 (significant) 99% Clustered 2014 0.093 5.195 <0.01 (significant) 99% Clustered

156 Dengue Bulletin – Volume 40, 2018 A geostatistical study to prioritize dengue-affected areas for implementation of effective control

Figure 1: Spatial autocorrelation pattern using Moran’s I index showing (a) clustered pattern during 2009, 2013 and 2014 (P<0.01); (b) random during 2010 and 2011; and (c) clustered during 2012 (P<0.10)

Maps of hotspots and high-risk areas for the years 2009, 2012–2014 identified through Getis-Ord Gi* statistics are depicted in Figure 2. During the year 2009, a total of 36 wards identified under the hotspot and high-risk category were in the City, Central, South, Sadar- Paharganj, DCB and NDMC zones of Delhi. During 2012, 2013 and 2014, 17 wards of Karol Bagh, South, Nazafgarh and DCB zones; 34 wards of Karol Bagh, Narela, Nazafgarh, Rohini, Shahdara North, Shahdara South, West and DCB zones; and 28 wards of Central, City, Narela, Najafgarh, Sadar Paharganj, Shahdara South, West zones of Delhi, respectively, were identified under the hotspot and high-risk category.

It has been observed that clustered distribution during a year led to an outbreak during the subsequent year (Figure 4). A clustered distribution followed by a dengue outbreak was observed in 2009–2010, 2012–2013 and 2014–2015.

Out of four years, i.e. 2009, 2012–2014, the Cant-6 ward of DCB was identified for 3 years in the hotspot/high-risk category, whereas 26 wards from nine zones Delhi were identified for 2 years in the hotspot/high-risk category, as shown in Table 2 and Figure 5.

Dengue Bulletin – Volume 40, 2018 157 A geostatistical study to prioritize dengue-affected areas for implementation of effective control

Figure 2: Mapping of hotspot and high-risk areas using Getis-Ord G* statistics during 2009, 2012–2014 in wards of Delhi

Figure 3: Random pattern of dengue cases identified during 2010 and 2011

158 Dengue Bulletin – Volume 40, 2018 A geostatistical study to prioritize dengue-affected areas for implementation of effective control

Figure 4: Trends in cluster and random distribution identified using Global Moran’s I during 2009–2015

9 Clustered 0 Random 1 Random 2 Clustered 3 Clustered 4 Clustered 5 Outbreak 0 1 1 1 1 1 1

0 Distribution 0 Distribution 0 Distribution 0 0 0 0 2 2 2 2 2 Outbreak 2 2 (15867 Outbreak (5462 cases) cases) (6207 cases)

Priority control is recommended in 27 wards that fall consistently in the hotspot and high-risk category for 2–3 years.

Figure 5: Consistently occurring dengue cases (dR) in hotspot/high-risk areas of 27 wards of Delhi (1 ward consistent for 3 years, and 26 wards consistent for 2 years) during 2009, 2012–2014

Dengue Bulletin – Volume 40, 2018 159 A geostatistical study to prioritize dengue-affected areas for implementation of effective control

Table 2: Consistent hotspot/high-risk wards of Delhi

Consistent hotspot/high-risk Sr. no. Zone ward no. (ward name) 3 years 2 years 1 DCB Cant 6 Cant 3 Cant 5 2 Central – 155 (Lajpat Nagar) 156 (Bhogal) 3 City – 81 (Minto Road) 82 (Kucha Pandit) 84 (Turkman Gate)

4 Karol Bagh – 150 (Pusa) 151 (Inderpuri) 5 Najafgarh – 143 (Kapashera) 6 Sadar-Paharganj – 85 (Idgah Road) 89 (Paharganj) 7 Shahdara South – 222 (Laxmi Nagar) 8 South – 161 (Malviya Nagar) 162 (Village Hauz Rani) 163 (Safdarjang Enclave) 164 (Hauz Khas) 166 (Munirka) 167 (R.K. Puram) 169 (Lado Sarai) 184 (Pushp Vihar)

9 NDMC – NDMC 1 NDMC 2 NDMC 3 NDMC 4 TOTAL 1 26

In a study conducted in the district of Hulu Langar, Selangor, a mapping of spatial distribution of dengue cases was done via a combination of GIS and spatial statistic tools19. In another study, spatial mapping of temporal risk characteristics in Subang Jaya district of Selangor state, Malaysia was done based on dengue cases20. However, in our study, instead

160 Dengue Bulletin – Volume 40, 2018 A geostatistical study to prioritize dengue-affected areas for implementation of effective control of dengue cases, we attempted to identify the spatial distribution of dengue cases per thousand population using GIS tools, which removes area/population biases while analysing the patterns. We did not investigate the risk factors for occurrence of dengue cases, such as clustering leading to formation of hotspots/high-risk pockets at the micro level (locality-wise).

Hotspots detected through geostatistical-based studies enable the detection and targeting of vector-borne disease clusters and assist the health authorities in planning disease control activities. Bhunia et al. have shown the usefulness of hotspot analysis in the control of kala-azar21 whereas Handique et al. used spatial statistics analysis to delineate the malaria incidence hotspots at subcentre level in the north-eastern states of India22. Srividya et al. showed its importance in filariasis epidemiology and control23.

Conclusion

The results of our study show that epidemiological measures, if carefully applied, can delineate hotspots of disease incidence. Geostatistical tools can differentiate hotspots, high-risk and cold-spot areas on the basis of statistical significance, which provide information to decision- makers for priority control rather than focusing on the entire region. Our study also showed that clustered distribution during one year led to an outbreak during the subsequent year. This early information provides an opportunity for decision-makers to carry out vector control measures along with source reduction in advance. The study highlighted the utility of GIS and spatial statistical tools in efficient processing of voluminous dengue epidemiological data at the micro level with a sound statistical base for focused and quick decision-making.

Acknowledgements

The authors thank the Director, National Institute of Malaria Research, for supporting the study and acknowledge the Municipal Corporations of Delhi (SDMC, EDMC, NDMC) for providing epidemiological data of Delhi. The authors are also thankful to Mr Mritunjay Prasad Singh and Mr Pavan Kumar for managing the epidemiological database. This paper bears the NIMR publication screening committee approval no. 55/2019.

Declaration

The authors state that this article has not been published and will not be submitted for publication elsewhere if accepted for publication in the WHO Dengue Bulletin.

Dengue Bulletin – Volume 40, 2018 161 A geostatistical study to prioritize dengue-affected areas for implementation of effective control

References

[1] Dengue fact sheet. Neglected tropical diseases. New Delhi: World Health Organization; 2019 (http:// www.searo.who.int/entity/vector_borne_tropical_diseases/data/data_factsheet/en/, accessed 2 April 2019). [2] Ganeshkumar P, Murhekar MV, Poornima V, Saravanakumar V, Sukumaran K, Anandaselvasankar A et al. (2018) Dengue infection in India: a systematic review and meta-analysis. PLoS Negl Trop Dis. 2018;12(7):e0006618 (https://doi.org/10.1371/journal.pntd.0006618, accessed 2 April 2019). [3] Mutheneni SR, Morse AP, Caminade C, Upadhyayula SM. Dengue burden in India: recent trends and importance of climatic parameters. Emerg Microbes Infect. 2017;6(8):e70; doi:10.1038/emi.2017.57. [4] Gupta E, Ballani N. Current perspectives on the spread of dengue in India. Infect Drug Resist. 2014;7:337–342. [5] Deshkar ST, Raut SS, Khadse RK. Dengue infection in central India: a 5 years study at a tertiary care hospital. Int J Res Med Sci. 2017;5(6):2483–9. [6] Whitehorn J, Farrar J. Dengue. Br Med Bull. 2010;95:161–73. [7] Gupta E, Mohan S, Bajpai M, Choudhary A, Singh G. Circulation of dengue virus-1 (DENV-1) serotype in Delhi, during 2010–11 after dengue virus-3 (DENV-3) predominance: a single centre hospital-based study. J Vector Borne Dis. 2012;49(2):82–5. [8] Balaya S, Paul SD, D’Lima LV, Pavri KM. Investigations on an outbreak of dengue in Delhi in 1967. Indian J Med Res. 1969;57:767–74. [9] Diesh P, Pattanayak S, Singha P, Arora DD, Mathur PS, Ghosh TK et al. An outbreak of dengue fever in Delhi-1970. J Commun Dis. 1972;4:13–18. [10] Rao CVRM, Bagchi SK, Pinto BD, Ilkal MA, Bharadwaj M, Shaikh BH et al. The 1982 epidemic of dengue fever in Delhi. Indian J Med Res. 1985;82:271–5. [11] Kabra SK, Verma IC, Arora NK, Jain Y, Kalra V. Dengue haemorrhagic fever in children in Delhi. Bull World Health Organ. 1992;70:105–8. [12] Broor S, Dar L, Sengupta S, Chakraborty M, Wali JP, Biswas A et al. Recent dengue epidemic in Delhi, India. In: Saluzzo JE, Dodet B, editors. Factors in the emergence of arbovirus diseases. Paris: Elsevier; 1997:123–7. [13] Dar L, Gupta E, Narang P, Broor S. Co-circulation of dengue serotypes, Delhi, India. Emerg Infect Dis. 2006;12:352–3. [14] Gupta E, Dar L, Kapoor G, Broor S. The changing epidemiology of dengue in Delhi, India. Virol J. 2006;3:1–5. [15] Afreen N, Deeba F, Naqvi I, Shareef M, Ahmed A, Broor S et al. Molecular investigation of 2013 dengue fever outbreak from Delhi, India. PLoS Curr. 2014 September 2;6. [16] Savargaonkar D, Sinha S, Srivastava B, Nagpal BN, Sinha A, Shamim A et al. An epidemiological study of dengue and its coinfections in Delhi. Int J Infect Dis. 2018;74:41–6.

162 Dengue Bulletin – Volume 40, 2018 A geostatistical study to prioritize dengue-affected areas for implementation of effective control

[17] Census of India 2011 (http://censusindia.gov.in/2011-prov-results/data_files/delhi/1_PDFC-Paper-1- introductory_note_10-43.pdf, accessed 16 April 2019). [18] Saxena R, Nagpal BN, Das MK, Srivastava A, Gupta SK, Kumar A et al. A spatial statistical approach to analyze malaria situation at micro level for priority control in Ranchi district, Jharkhand. Indian J Med Res. 2012;136:124–30. [19] Zulkiflee AL, Mohamad MH. Mapping of dengue outbreak distribution using spatial statistics and geographical information system. In: 2nd International Conference on Information Science and Security, 14–16 December 2015, Seoul, South Korea. DOI: 10.1109/ICISSEC.2015.7371016 (https://ieeexplore. ieee.org/document/7371016/citations#citations, accessed 16 April 2019). [20] Dom NC, Ahmad AH, Adawiyah R, Ismail R. Spatial mapping of temporal risk characteristic of dengue cases in Subang Jaya. In: 2010 International conference on Sciences and Social Research (CSSR 2010), 5–7 December 2010, Kuala Lumpur, Malaysia (https://www.academia.edu/4017178/Spatial_mapping_ of_temporal_risk_characteristic_of_dengue_cases_in_Subang_Jaya, accessed 16 April 2019). [21] Bhunia GS, Kesari S, Chatterjee N, Kumar V, Das P. Spatial and temporal variation and hotspot detection of kala-azar disease in Vaishali district (Bihar), India. BMC Infect Dis. 2013;13:64. [22] Handique BK, Khan SA, Dutta P, Nath MJ, Qadir A, Raju PLN. Spatial Correlations of malaria incidence hotspots with environmental factors in Assam, North East India. In: ISPRS Annals of the Photogrammetry, Remote Sensing and Spatial Information Sciences, Volume III-8, 2016 XXIII ISPRS Congress, 12–19 July 2016, Prague, Czech Republic (https://www.researchgate.net/publication/303845627_SPATIAL_ CORRELATIONS_OF_MALARIA_INCIDENCE_HOTSPOTS_WITH_ENVIRONMENTAL_FACTORS_IN_ ASSAM_NORTH_EAST_INDIA, accessed 16 April 2019). [23] Srividya A, Michael E, Palaniyandi M, Pani SP, Das PK. A geostatistical analysis of the geographic distribution of lymphatic filariasis prevalence in Southern India. Am J Trop Med Hyg. 2002;67(5):480–48.

Dengue Bulletin – Volume 40, 2018 163 Reading levels of selected USA Federal Government dengue webpages

Jeffrey L. Lennon,a# Christopher M. Seitzb

aLiberty University, Department of Public and Community Health, Lynchburg, VA, USA bAppalachian State University, Department of Health and Exercise Science, Boone, NC, USA

Abstract A readability analysis was done of selected dengue webpages of the USA Federal Government Centers for Disease Control and Prevention. The study employed the readability analysis methods of SMOG, FOG, Fry and Flesch–Kincaid. Each readability analysis method had an average score that exceeded the recommended reading levels for health education reading materials. Recommendations were made on the basis of the analysis.

Introduction

Health education plays an important role in managing dengue fever. It provides content about dengue-related concerns as well as enhances awareness about the disease1,2. Written information forms an integral part of health education, especially programmes of communication for behavioural impact (COMBI) for dengue prevention and control3.

Web-based health education materials are becoming increasingly important as resources for health professionals as well as the general public. The website of the USA’s Centers for Disease Control and Prevention (CDC) has various webpages with dengue-related information4–7. A study of multiple websites with dengue-related content by Rao et al. used the DISCERN instruments for evaluation. Overall scores of website quality ranged from 2.24 to 3.76 on a 5-level Likert scale. Some sites were perceived to have unclear or limited information8. A missing component in the study of websites8 was that of readability analysis.

Health materials, whether print or electronic, need to be written at the reading level of the intended audience or population. Readability testing provides an estimate of the reading level appropriate for a given academic grade. Various readability formulas examine the complexity of words through multisyllabic word counts to average sentence lengths to assess reading levels9,10. For use by the general population of the USA, the reading level of materials should be no higher than that of eighth grade11.

#E-mail: : [email protected]

164 Dengue Bulletin – Volume 40, 2018 Reading levels of selected USA Federal Government dengue webpages

We assessed web-related readability for the following diverse health topics: common issues in internal medicine12, paediatric ophthalmology13 and smoking cessation14. The majority of these web-based health materials exceeded the recommended reading levels12–14. To date, no study has been published on readability levels of web-based dengue health materials. Therefore, we sought to determine reading levels of dengue-related health materials from the USA Federal Government’s website of the CDC.

Materials and methods

Webpages from the dengue website of the CDC were selected for this study4–7. This purposive sample was selected as the CDC is the premier government agency for disease control and prevention in the USA. Readability grade levels were assessed by the following readability formulas: SMOG9, Fry9, Flesch–Kincard9, and FOG15. The SMOG formula focused on the use of polysyllabic words9. The Fry formula focused on total syllables used in the written passage9. The Flesch–Kincaid formula centred on the average number of syllables and average sentence length9. The FOG formula employed a combination of average sentence length with the percentage of difficult words; for example, proper nouns are excluded in the FOG formula15. The Kruskal–Wallis H Test was also employed to examine possible significant differences between the samples of the various readability testing formulas16.

Results

The mean grade scores for the SMOG, FOG, Fry and Flesch–Kincaid tests were 12.75, 15.75, 13.5 and 10.25, respectively. The mean grade scores for Dengue (homepage), Frequently Asked Questions (FAQs), Travel and Dengue Outbreaks and Prevention were 13.5, 14.5, 12.5 and 11.75, respectively. The overall mean grade score for all webpages was 13.06. The Kruskal–Wallis test revealed a significant difference between readability formulas, x2(3)=10.92, P=0.0122. Table 1 gives the readability scores for webpages using the various readability formulas.

Table 1: Readability scores for webpages using various formulas

Flesch– Webpage Link SMOG FOG FRY Mean Kincaid Homepage cdc.gov/dengue/index.html 13 16 13 12 13.5 FAQs cdc.gov/dengue/faqfacts/index.html 14 19 15 10 14.5 Travel cdc.gov/dengue//traveloutbreaks/index.html 13 14 13 10 12.5 Prevention cdc.gov/dengue/prevention/index.html 11 14 13 9 11.75 Mean 12.75 15.75 13.5 10.25

Dengue Bulletin – Volume 40, 2018 165 Reading levels of selected USA Federal Government dengue webpages

Discussion this study aimed to assess the reading levels of dengue-related webpages of the CDC. The results of the reading levels of these webpages ranged from secondary school (high school) to the university level. They exceeded easy reading levels as well as the recommended reading levels11. This study on readability levels of dengue webpages compares with the high reading levels of other readability studies of health-related materials12–14. There were also differences in reading grade-level scores between the readability formulas. Even then, all scores exceeded the recommended reading level of less than eighth grade. This study highlights the challenge for health professionals to ensure that dengue-related health education materials be provided at a reading level appropriate for the population.

The study had the limitation that it examined webpages from only one website, the CDC. However, this government agency is considered to be USA’s premier disease prevention and control agency. To avoid a possible skewing of readability scores, multiple readability formulas were used. These results highlight the importance of assessing the readability of information related to health put out by state, local and private entities, and ensuring that it is provided according to the reading level of the intended population or subpopulation. Since the readability formulas were designed to correspond to the USA grade system, it would not be appropriate to use these formulas on the websites of other countries, without confirming the equivalence of grade systems. Also, the study had the limitation of using only English language webpages, as these readability formulas were tailored for the English language. However, readability formulas have been developed for many languages, such as Spanish and Japanese9. Though readability assessments are not the final criteria to ascertain the appropriateness of educational content, they should be integral to the process of developing health materials, especially dengue-related materials.

Recommendations

zz use readability analysis of health information materials before posting them on websites if such methods are available within a country and for specific languages.

zz When readability analysis is not available in a specific country, then use read-back analysis to ensure comprehension of materials at the recommended reading level. This approach was successfully used in the development of dengue-related health materials in the Philippines17.

zz When possible, replace technical words with simpler, colloquial words.

zz Ensure that health materials are available for readers with a low level of comprehension.

166 Dengue Bulletin – Volume 40, 2018 Reading levels of selected USA Federal Government dengue webpages

References

[1] Kusuma YS, Burman D, Kumari R, Lamkang AS, Babu BV. Impact of health education based intervention on community’s awareness of dengue and its prevention in Delhi, India. Glob Health Promot. 2017 Mar 1:1757975916686912. doi: 10.1177/1757975916686912 (accessed 20 March 2018). [2] Aziz AT, Al-Shami SA, Mahyoub JA, Hatabbi M, Ahmad AH, Md Rawi CS. Promoting health education and public awareness about dengue and its mosquito vector in . Parasit Vectors. 2014;7:487. [3] Azmawati MN, Aniza I, Ali M. Evaluation of Communication for Behavioral Impact (COMBI) program in dengue prevention: a qualitative and quantitative study in Selangor, Malaysia. Iran J Public Health. 2013;42(5):538–9. [4] Dengue. In: Centers for Disease Control and Prevention [website] (https://www. Cdc.gov/dengue/index. html, published 31 December 2013, accessed 3 March 2019). [5] Dengue: frequently asked questions. In: Centers for Disease Control and Prevention [website] (https:// www.cdc.gov/dengue/faqfacts/index.html, published 3 September 2009, accessed 10 March 2019). [6] Travel & dengue outbreaks. In: Centers for Disease Control and Prevention [website] (https://www. cdc.gov/dengue/traveloutbreaks/index.html, accessed 3 March 2019). [7] Prevention. In: Centers for Disease Control and Prevention [website] (https://.www.cdc.gov/dengue/ prevention/index.html, accessed 3 March 2019). [8] Rao NR, Mohapatra M, Mishra S, Joshi A. Evaluation of dengue-related health information on the internet. Perspect Health Inf Manag. 2012;9:1c. (http://www.ncbi.nlm.nih.gov/pmc/articles/ PMC3392950/pdf/phim0009-0001c.pdf, accessed 3 March 2019). [9] The Ohio State Medical Center. Clear Health Communication Program. Who’s reading your writing: how difficult is your text? (http://medicine.osu.edu/sitetool/sites/pdfs/ahecpublic/whos_reading_your_text. pdf, accessed 3 March 2019). [10] Strategic and Proactive Communication Branch. Simply put: a grade for creating easy-to-understand materials, 3rd ed. Atlanta, GA: Centers for Disease Control and Prevention; 2010:1–43 (https://www. cdc.gov/healthliteracy/pdf/simply_put.pdf, accessed 9 March 2019). [11] National Institutes of Health. How to write easy to read health materials. In: US National Library of Medicine [website] (http://www.nlm.nih.gov/medlineplus/etr.html, accessed 9 March 2019). [12] Hutchinson N, Baird GL, Garg M. Examining the reading level of internet medical information for common internal medicine diagnoses. Am J Med. 2016;129(6):637–9. doi: 10.1016/j.amjmed.2016.01.008. (accessed 21 March 2018). [13] John AM, John ES, Hansberry DR, Thomas PJ, Guo S. Analysis of online patient education included in pediatric ophthalmology. J AAPOS. 2015;19(5):430–4. [14] Seitz CM, Shiplo S, Filippini T, Kabir Z, Lennon JL, Fowler D. The reading level of government and voluntary health organization smoking cessation websites: a descriptive analysis. Am J Health Educ. 2017;48(6): 392–9. [15] The Gunning’s Fog Index (or FOG) Readability Formula. Readability formulas (http://www. readabilityformulas.com/, accessed 9 March 2019). [16] VassarStats: website for statistical computation (http://vassarstats.net/, accessed 9 March 2019). [17] Lennon JL, Coombs DW. The utility of a board game for dengue hemorrhagic fever health education. Health Educ. 2007;107(3):290–306

Dengue Bulletin – Volume 40, 2018 167 Short Note

Unusual complications of dengue fever

Rajeev Upreti,a# Monica Mahajan,b Ram Shankar Mishrab

aGeorge Eliot Hospital, Nuneaton, United Kingdom bMax Super Specialty Hospital, 1 Press Enclave Road, Saket, Delhi, India

Dengue fever (DF) is a mosquito-borne tropical viral haemorrhagic fever (VHF). A small proportion of patients may develop dengue haemorrhagic fever; however, the bleeding itself is rarely life-threatening. With the revision of the dengue classification scheme by the World Health Organization (WHO) in 2011, patients are now classified as having dengue fever (DF), dengue haemorrhagic fever without shock (DHF) or with shock (dengue shock syndrome [DSS]) and expanded dengue syndrome1,2. WHO coined the term “expanded dengue syndrome” to describe atypical cases that do not fall into either DSS or DHF. These unusual manifestations may be associated with coinfection, comorbidities or complications of prolonged shock. We discuss here two atypical manifestations of dengue. The first patient developed myositis, leading to compartment syndrome of the lower limb. The second patient developed congestive splenomegaly and splenic rupture.

Case reports

Case 1

A 24-year-old male presented with nausea and abdominal discomfort for the past 6 days. He had fever during the initial 4 days of the illness, with retro-orbital pain. He did not have any significant past medical history or history of dengue infection in past. On examination, he was afebrile, dehydrated with a pulse rate of 96/min and blood pressure (BP) of 100/70 mmHg. Detailed systemic examination was normal except for tenderness in the right hypochondrium. There was no rash, petechial haemorrhage, organomegaly or localized muscle tenderness. His initial investigations were haemoglobin (Hb) 14.7 g/dL, platelet count 45 000 x 109/L, total leukocyte count (TLC) 4.7 x 109/L. The total bilirubin was raised (2.1 mg/dL), as were transaminases (serum glutamic oxaloacetic transaminase [SGOT] 3280 IU/L, serum glutamic pyruvic transaminase [SGPT] 643 IU/L) and alkaline phosphatase (ALP 522 IU/L). The test for Dengue non-structural (NS)1 antigen was 3.1 (positive). The initial chest X-ray was normal. Ultrasonogram of the whole abdomen was suggestive of mild oedema of the gall bladder and

#E-mail: [email protected]

168 Dengue Bulletin – Volume 40, 2018 Unusual complications of dengue fever ascites. The patient was managed conservatively for DF with regular monitoring of platelets and administration of intravenous fluids and other supportive therapy.

He developed fever and severe myalgia 2 days later. He was empirically started on IV ceftriaxone and blood cultures were sent, which showed growth of Staphylococcus aureus. Antibiotics were accordingly upgraded to cefaperazone–sulbactum and linezolid. Urine culture was sterile. In spite of ongoing treatment, he was noted to be febrile, had tachycardia, and was tachypnoeic and hypoxic. The patient developed redness and swelling of the right leg and left thigh region. These were tense and tender on palpation, with pain on passive stretching of the leg muscles. He was shifted to the intensive care unit (ICU) for further management and monitoring, and was put on ventilator and inotropic support in view of the increasing breathlessness, hypotension and hypoxia. Cardiac markers were within normal limits, but the creatine phosphokinase (CPK) value was 1765 U/L. In view of the myositis and corpus syndrome, bedside fasciotomies of the right leg and left thigh were done by the plastic surgery team (Figure 1). The swab culture was also positive for Staphylococcus aureus.

Figure 1: Bulging muscles of the left thigh when the fasciotomy incision was given

Dengue Bulletin – Volume 40, 2018 169 Unusual complications of dengue fever

Postoperatively, regular dressings were done and continuous vaccum-assisted closure (VAC) was applied. CPK monitoring over several days revealed a rising trend with values ranging from 2445 U/L to 3367 U/L on day 3 and day 10 of surgery, respectively. Subsequently, he required several episodes of debridement of both the right as well as left thigh wounds. During his ICU stay, the patient continued to have fever and serial cultures were sent; antibiotics and antifungals were modified according to the reports. He also required multiple units of blood products. Gradually, his myositis settled after 1.5 months of treatment and then grafting was done. The patient was successfully weaned away from the ventilator after about 3 months of ventilator support. He continued physiotherapy for mobilization during this period and was discharged in a stable condition after a few more days of stay in a ward room.

Case 2

An 18-year-old female presented with complaints of high-grade fever for 5 days, vomiting and decreased oral intake since 1 day. She was conscious, oriented, with a pulse of 88 beats per minute and BP of 100/60 mmHg, and had no other remarkable physical findings. She was diagnosed to have dengue fever (dengue NS1 positive) and treatment was started accordingly.

Four days later, she developed diffuse abdominal pain with hypotension (BP decreased to 81/50 mmHg) and her Hb dropped from 10.9 g/dL to 7.3 g/dL. After an urgent ultrasound and CT scan of the abdomen, she was found to have splenic bleeding, which had developed into a haemoperitoneum. Immediate supportive measures were taken, along with endovascular splenic artery embolization by an interventional radiologist. However, she continued to remain haemodynamically unstable post-procedure and, the same day, she also underwent emergency diagnostic laparoscopy (to look for any other bleeding sites) with laparoscopic splenectomy, drainage of the haemoperitoneum and ligature of the bleeding splenic pedicle.

During the course of her illness, 9 units of packed red blood cells were transfused, 10 units of fresh frozen plasma, 2 bags of cryoprecipitate and 2 units of random donor platelet concentrate. She was given pneumococcal and meningococcal vaccines 14 days after surgery. She recovered well from the disease and surgery, and was discharged in a stable condition after 12 days of hospital stay.

Discussion

Dengue fever is a viral disease that results from one of four single-stranded, positive-sense RNA viruses (dengue virus types 1–4) of the genus Flavivirus (family Flaviviridae)3. It is spread by the mosquito vectors Aedes aegypti (primary vector) and Aedes albopictus. The infectious virus and virus-encoded NS1 antigen are present in the blood during the acute phase. After an incubation period of 3–7 days, symptoms start suddenly and follow three phases: an

170 Dengue Bulletin – Volume 40, 2018 Unusual complications of dengue fever initial febrile phase, a critical phase around the time of defervescence, and a spontaneous recovery phase. The usual complications of dengue infection include respiratory distress, ascites, vomiting, dehydration, bleeding, electrolyte and acid–base imbalances, blood glucose imbalance, coinfections and nosocomial infections.

Compartment syndrome is an unusual complication of dengue fever. There is polyserositis and third space collection of body fluid, which decreases the intravascular blood volume. This sets the stage for compartment syndrome by inducing ischaemic injury in the muscles (lower limbs in our case report 1). The increased pressure in the extravascular compartment is due to leaking capillaries. Bandopadhyay et al. have documented a similar case of compartment syndrome, which was successfully treated with timely surgical intervention4. In another case, Khoo et al. showed that a haematoma (due to arterial line insertion) and not the capillary leak may be the underlying pathophysiology of compartment syndrome in some cases5. The complication of compartment syndrome in DF is not restricted to the limbs; abdominal compartment syndrome has also been noticed, mainly in the paediatric population6. The treatment for compartment syndrome consists of relieving the pressure inside the compartment by surgical intervention, which needs to be done at the earliest to restrict ischaemic injury.

The spleen is frequently congested in cases of DF. Only anecdotal case reports have been documented to have splenic rupture in DF. Of the 11 reported dengue cases with splenic rupture (discussed case not included), 8 patients survived. Splenectomy is the treatment of choice for spontaneous splenic rupture and haemoperitoneum, though a few reports also advocate an initial trial of conservative management7,8. The choice of therapy between these two options depends on the haemodynamic status of the patient. Cases not responding to conservative therapy must undergo splenectomy.

In conclusion, patients with DF routinely require a detailed, thorough examination during the course of illness. Physicians should keep a high index of suspicion of complications on noticing any abnormal signs in a patient of DF. Secondary infections should be suspected in dengue cases in whom fever persists; superadded bacterial infections can be fatal. Internal bleeding should be suspected in patients with falling Hb levels and no signs of external blood loss. Survival among these cases is attributed to timely diagnosis and management rather than the natural course, which is expected to be poor if the diagnosis is missed.

References

[1] Comprehensive guidelines for prevention and control of dengue and dengue haemorrhagic fever. Revised and expanded edition. New Delhi: WHO Regional Office for South-East Asia; 2011 (http:// apps.who.int/iris/bitstream/10665/204894/1/B4751.pdf, accessed 13 March 2019). [2] Kadam DB, Salvi S, Chandanwale A. Expanded dengue. J Assoc Physicians India. 2016;64:59–63.

Dengue Bulletin – Volume 40, 2018 171 Unusual complications of dengue fever

[3] WHO, National Vector Borne Disease Control Programme. National guidelines for clinical management of dengue fever. New Delhi: WHO; 2015 (http://www.searo.who.int/india/publications/national_ guidelines_clinical_management_dengue1.pdf?ua=1, accessed 13 March 2019). [4] Bandyopadhyay D, Mondal P, Samui S, Bishnu S, Manna S. Acute compartment syndrome of upper limb as an unusual complication of dengue hemorrhagic fever. N Am J Med Sci. 2012;4:667–8. [5] Khoo CS, Chu GSE, Rosaida MS, Chidambaram SK. Dengue fever with compartment syndrome of the right arm. J R Coll Physicians Edinb. 2016;46:241–3. [6] Kamath SR, Ranjit S. Clinical features, complications and atypical manifestations of children with severe forms of dengue hemorrhagic fever in South India. Indian J Pediatr. 2006;73:889–95. [7] Seravali MR, Santos AH, Costa CE, Rangel DT, Valentim LF, Gonçalves RM. Spontaneous splenic rupture due to dengue fever: report of two cases. Braz J Infect Dis. 2008;12:538–40. [8] Sharma SK, Kadhiravan T. Spontaneous splenic rupture in dengue hemorrhagic fever. Am J Trop Med Hyg. 2008;78:7.

172 Dengue Bulletin – Volume 40, 2018 Instructions for contributors

Instructions for contributors

Dengue Bulletin welcomes all original research papers, short notes, review articles, letters to the Editor and book reviews which have a direct or indirect bearing on dengue fever/dengue haemorrhagic fever prevention and control, including case management. Papers should not contain any political statement or reference.

Manuscripts should be typewritten in English in double space on one side of white A4-size paper, with a margin of at least one inch on either side of the text and should not exceed 15 pages. The title should be as short as possible. The name of the author(s) should appear after the title, followed by the name of the institution and complete address. The e-mail address of the corresponding author should also be included and indicated accordingly.

References to published works should be listed on a separate page at the end of the paper. References to periodicals should include the following elements: name and initials of author(s); title of paper or book in its original language; complete name of the journal, publishing house or institution concerned; and volume and issue number, relevant pages and date of publication, and place of publication (city and country). References should appear in the text in the same numerical order (Arabic numbers in parentheses) as at the end of the article. For example:

(1) Kroeger A, Lenhart A, Ochoa M, Villegas E, Levy M, Alexander N, et al. Effective control of dengue vectors with curtains and water container covers treated with insecticide in Mexico and : cluster randomised trials. BMJ 2006;332:1247–52. (2) Dengue: guidelines for diagnosis, treatment, prevention and control. Geneva: World Health Organization; 2009. (3) Nathan MB, Dayal-Drager R. Recent epidemiological trends, the global strategy and public health advances in dengue: report of the Scientific Working Group on Dengue. Geneva: World Health Organization; 2006 (TDR/SWG/08). (4) DengueNet database and geographic information system. Geneva: World Health Organization; 2010. Available from: www.who.int/dengueNet [accessed 11 March 2010]. (5) Gubler DJ. Dengue and dengue haemorrhagic fever: Its history and resurgence as a global public health problem. In: Gubler DJ, Kuno G (ed.), Dengue and dengue haemorrhagic fever. CAB International, New York, NY,1997,1–22. Figures and tables (Arabic numerals), with appropriate captions and titles, should be included on separate pages, numbered consecutively, and included at the end of the text with instructions as to where they belong. Abbreviations should be avoided or explained at the first mention. Graphs or figures should be clearly drawn and properly labelled, preferably using MS Excel, and all data clearly identified.

Dengue Bulletin – Volume 40, 2018 173 Instructions for contributors

Articles should include a self-explanatory abstract at the beginning of the paper of not more than 300 words explaining the need/gap in knowledge and stating very briefly the area and period of study. The outcome of the research should be complete, concise and focused, conveying the conclusions in totality. Appropriate keywords and a running title should also be provided.

Articles submitted for publication should be accompanied by a statement that they have not already been published, and, if accepted for publication in the Bulletin, will not be submitted for publication elsewhere without the agreement of WHO, and that the right of republication in any form is reserved by the WHO Regional Offices for South-East Asia and the Western Pacific.

One hard copy of the manuscript with original and clear figures/tables and a computer diskette/CD-ROM indicating the name of the software should be submitted to:

The Editor Dengue Bulletin WHO Regional Office for South-East Asia Red Fort Capital Parsvnath Tower 1, Bhai Vir Singh Marg, Gole Market Sector 4, New Delhi 110 001, India Telephone: +91–11–4304 0200 Fax: +91–11–2336 8355 E-mail: [email protected]

Manuscripts received for publication are subjected to in-house review by professional experts and are peer-reviewed by experts in the respective disciplines. Papers are accepted on the understanding that they are subject to editorial revision, including, where necessary, condensation of the text and omission of tabular and illustrative material.

Original copies of articles submitted for publication will not be returned. The principal author will receive 10 reprints of the article published in the Bulletin. A pdf file can be supplied on request.

174 Dengue Bulletin – Volume 40, 2018 From the Editor’s Desk

There is about a 30-fold increase in dengue incidence over the past 50 years and it is now regarded as one of the most important arboviral infections in the world. There are many reasons for this increase, including faster spread of the virus through global travel, spread of the vectors to new geographical locations, rapid urbanization, global warming and climate change. About 52% of the population in the WHO South-East Asia Region is estimated to be at risk for dengue with 10 out of the 11 Member States (with the exception of the Democratic People's Republic of Korea) being endemic. All four serotypes of the virus are circulating in the Region. The Region has seen larger outbreaks of dengue in the past two years, with Sri Lanka experiencing the largest outbreak ever recorded in 2017. Many other countries, including India, Indonesia and Myanmar, have also seen focal outbreaks of increasing magnitude. All these resulted in researchers engaging in the clinical features, management, vector biology and control of dengue. In line with this priority, the Dengue Bulletin is published every year, encouraging researchers to explore different aspects of the disease and contribute to the knowledge gap and evidence base for combating the rapid spread of this deadly disease. The 40th volume of Dengue Bulletin is in your hands. It consists of papers on a new GIS surveillance tool, clinical management, vector behaviour, and the role of knowledge, attitudes and practices in dengue control. We now invite contributions for volume 41. The deadline for the receipt of the manuscripts is 31 October 2019. Contributors are requested to kindly follow the instructions given at the end of the Bulletin during the preparation of their manuscripts. Contributions should either be accompanied by flash drives and sent to the Editor, Dengue Bulletin, WHO Regional Office for South-East Asia, Red Fort Capital Parsvnath Tower 1, Bhai Vir Singh Marg, Gole Market Sector 4, New Delhi 110 001 India, or by email as a file attachment to the Editor at [email protected]. Readers who want copies of the Dengue Bulletin may write to the same address or the WHO Country Representative in their country of residence. The pdf version will be available on the WHO Regional Office website.

Dr Ahmed Jamsheed Mohamed Regional Adviser Neglected Tropical Diseases Control and Editor, Dengue Bulletin World Health Organization Regional Office for South-East Asia New Delhi, India 8 ISSN 0250-8362 ISSN 1 0 2 olume 40, December 2018 olume 40, December V Bulletin Dengue

Dengue Bulletin Volume 40, December 2018 O H W by . the the the and The also to 2 in on 6 are changing 3 review Bulletin 8 workers aspects. letters - management, management, 0 experts 5 and Dengue meetings 2 in-house information information infection, infection, 0 research related clinical to

N for annual S fever reviews other S updated the I international

all book strategies, strategies, subjected by and information are publish notes, control haemorrhagic haemorrhagic national/international national/international disseminate disseminate Asia strains provide of to short -reviewed o is T

publication peer DENV attempted attempted for South-East South-East articles, are fever/dengue fever/dengue Bulletin for new subjects. proceedings the and review received Office circulating of dengue -related patterns, of experts accepts managers, about Regional disciplines. objective DF/DHF status also manuscripts

on The All WHO The current epidemiological epidemiological information Bulletin editor programme published. professional respective