Kareem & Jarullah (2020): Investigating viruses in children May 2020 Vol. 23 Issue 9

Investigation of2Adenovirus, Infleunza A and B virus infections in children2under 5 years old in Thi-Qar Province

Sara kareem1*, Basim Abdul-Hussein Jarullah2

1Biology2department, collage of1science, Thi-Qar University, Thi-Qar, Iraq 2Collage of Veterinary2Medicine, Thi-Qar2University, Thi-Qar, Iraq

*Corresponding Author : Thi-Qar university , collage of science , Biology Department , Thi-Qar , Iraq , Tel : 07831084525 , E-Mail : [email protected] (Kareem)

Abstract

Background: Acute2respiratory1infection is a major killer of/children in developing3countries. Viruses are common causes of it among children and1Adenovirus, Influenza virus A and2B are some of them. Method: A2prospective cross sectional study was carried2out in children between 1-54years old with upper respiratory tract infections .Throat swabs were collected from 250 hospitalized children, and then the samples were transported by an ice bag from the hospital into the laboratory for storage in -20 ˚C. DNA –RNA extract PCR for diagnosis of viruses. Results: Adenovirus was detected in 42/250(16.8%) cases. Influenza A virus was detected in 38/250(15.2%), Influenza B virus was detected in 40/250(16%) cases. Co-infection between Influenza A and Adenovirus 8/250(3.2%) , Influenza B and Adenovirus 9/250(3.6%) ,Influenza A and B 10/250(4%), Influenza A and B and Adenovirus 4/250(1.6%). Unknown infections was14/250 (5.6%) cases. 38 of Influenza A virus-infected patients(60) were males and 22 were females, while 34 of Influenza B virus -infected children(63) were males and 29 were females , and 33 of Adenovirus –infected children (63) were males and 30 were females. Most the infections were detected in mainly less than one year old//children. Most the infections had occurred mainly//during winter and spring seasons. Conclusion: Adenovirus was more prevalent. Fever was the most common clinical signs followed by cough. The infections were prevalent in winter and early spring.

Keywords: Adenovirus, Influenza A virus, Influenza B virus, children

How to cite this article: Kareem S, Jarullah A-H (2020): Investigation of adenovirus, Influenza A and B virus infections in children under 5 years old in THi-Qar Province, Ann Trop Med & Public Health; 23(S9): SP23925. DOI: http://doi.org/10.36295/ASRO.2020.23925

1. Introduction

Viral/respiratory infections/ (VRIs) is a general term for//lung and airway (breathing passages)/infections./The disease burden from respiratory infection is/greater than that of any other cause of disease (1). In 2002, //18% of mortality for children younger than/5 years of age was caused

Annals of Tropical Medicine & Public Health : http://doi.org/10.36295/ASRO.2020.23925 Kareem & Jarullah (2020): Investigating viruses in children May 2020 Vol. 23 Issue 9 by respiratory infections; diarrheal disease/(15%) and malaria (11%) were the next greatest causes (2). In//2010, Nair and colleagues reported that 11.9/million and 3.0 million episodes of//severe and very severe VRTs respectively, contributed to//childhood hospitalization (3). Furthermore, 0.935lmillion deaths in 2013kwere attributed to RIs (4) .In developing/countries where pneumonia is responsible over 10-25 % of all deaths//among children under 5 years of ages the situation is more alarming (5). The respiratory2tract infection (RTI) in children is caused by several pathogenic3microorganisms such as bacteria,6viruses, parasites and fungi, but the major pathogenic viruses9particularly : Respiratory2syncytial virus (RSV) , human2rhinovirus (HRV) , human 2metapneumovirus (hMPV),2influenza viruses (Infv) , human parainfluenza virus(HPIV) ,2enteroviruses (EVs) ,2adenoviruses(Adv) , human3coronavirus (HCoV) and human3bocavirus (6) . Studies9focused on RSV, Infv, Adv, hMPV, have/revealed a high activity of viruses in children under 54years old, especially during the rainy. season (7). Indeed , previous studies have well/established that this viruses are leading cause of lower2respiratory tract infections (LRTIs) in young children with150% of children infected during their first1year of life, and 100 % having experienced at least one2infection by 3 years of age (8,7). Children are most susceptible to respiratory2tract infections which are attributed to several features that/attendant to physiological,4immunological and anatomical/characteristics (9).

2. Objectives: This study had two objectives. First, to determine the Adenovirus and Influenza virus A and B Virus , frequency and their co-infection in hospitalized ARI children, aged less than five years old, at one of the children’s referral different hospitals in Iraq in Thi-Qar province . Second, to describe the seasonal/prevalence of Adenovirus and 1Infleunza virus A and B Virus among this group during six months from December 2018 till May 2019.

3. Patients and Methods

3.1. Patients and samples

111This/descriptive study was based on the processing of throat swab from 250 children , aged under five years old, who were admitted to children hospitals of Thi-Qar/province with acute/respiratory//infections from December 2018 till May 2019; this was done for the detection of the frequency of Adenovirus , Influenza A and B/ infections and co-infections. Acute respiratory infections children aged less than five years, having fever and other symptoms of/ respiratory/infections were included in this study. Children with respiratory infections, aged above five years//old, were excluded. The specimens//were placed into a vial containing 1 mL of transport media/(VTM) and stored at /-20°C until use.

3.2. Nucleic Acid Extraction and cDNA Synthesis

11 Viral nucleic acids from specimens were extracted using Extraction Kit II (AcroGene-USA ) was designed specifically for efficient purification of viral DNA and RNA and done according to company instructions .//Reverse transcription was performed by using use TonkBio TM First Strand cDNA Synthesis kit for RT-PCR (Tonk Bioscience LLC –USA) and done according to the company instructions.

Annals of Tropical Medicine & Public Health : http://doi.org/10.36295/ASRO.2020.23925 Kareem & Jarullah (2020): Investigating viruses in children May 2020 Vol. 23 Issue 9

3.3. RT-PCR

RT-PCR technique was performed for detection of2Adenovirus , Infleunza2A and2B virus, RT- PCR master mix was prepared by using (AccuPower PCR PreMix Kit) and this master mix done according to company instructions. Oligonucleotide primers of Adenovirus and Influenza virus are shown in Table 1

Table (1): The primers that use for PCR

Name Forward Sequence Reverse Sequence location

Infleunza A AAG GGC TTT CAC CGA AGA GG CCC ATT CTC ATT ACT GCT TC NS Infleunza B ATGGCC ATC GGA TCC TCA AC TGT CAG CTA TTA TGG AGC TG NS Adenovirus GCC GAG AAG GGC GTG CGC ATG ACT TTT GAG GTG GAT Hexon AGG TA CCC

The PCR conditions were as follows: reverse1transcription at 50°C for 30 minutes , Initiatial1Denaturation 94°C for 5 minutes, followed by 45 cycles of1denaturation at 94°C for 30 seconds, annealing at 63°C for 30 seconds, Extension 72°C for 2 minutes, Final extension 72 ºC for 5 minutes .

3.4. Statistical Analysis

11Statistical analysis for comparison of relative value was performed by the SPSS//version 23 P < 0.01 were considered statistically//significant.

4- Results

4.1 Study population

From December 2018 to May 2019, 250 specimens were obtained from hospitalized children under five years old from different hospitals in Thi-Qar province for evolution of Adenovirus, Influenza A and B infections .

4.2. Adenovirus and Influenza virus Prevalence

Adenovirus was detected in 42(16.8%) cases, Influenza A virus was 38(15.2%) and Influenza B virus 40(16%) . Co-infection was detected as following: Influenza A and Adenovirus 8(3.2%) , Influenza B and Adenovirus 9(3.6%), Influenza A and B 10(4%), Influenza A and B and Adenovirus 4(1.6%) . Unknown infections was 14 (5.6%) cases, as in the table 2, 3,4,5,6

Table (2): Distribution of Influenza A and B , Adenovirus infections among group of children under 5 years old according to six

Annals of Tropical Medicine & Public Health : http://doi.org/10.36295/ASRO.2020.23925 Kareem & Jarullah (2020): Investigating viruses in children May 2020 Vol. 23 Issue 9

As shown in the table below, the percentage of male infection was higher than the female infection rate.

Six No. of +ve cases Flu A No. of +ve cases Flu B No. of +ve cases Adv Male 38(63%) 34(54%) 33(52%) Female 22(37%) 29(46%) 30(48%) Total 60 63 63 Table (2): Distribution of Influenza A and B, Adenovirus in patient group of children according to the age

As shown in the table below most the infections were in children less than one year

Age group (years) No. of +ve cases Flu A No. of +ve cases Flu B No. of +ve cases Adv ˂ ˂ 1 21 (35%) 20(32%) 25(39.6%) 1-2 9(32%) 25(39.6%) 15(23.8%) 2-3 10(17%) 10(15.8%) 15(23.8%) 3-4 4(7%) 5(7.9%) 3(4.7%) 4-5 6(10%) 3(4.7%) 5(8%) Total 60 63 63 Table (3) : Distribution of Influenza A and B , Adenoviru in patients group of children according to months of the year

As shown in the table below high rate of infection was in winter season (December and Jenuary)

The month No. of +ve cases Flu A No. of +ve cases Flu B No. of +ve cases Adv December 23(38%) 21(33.3%) 22(35%) Jenuary 17(28.3%) 21(33.3%) 20(32%) February 10(17%) 11(17.4%) 8(12.6%) March 6(10%) 7(11.1%) 8(12.6%) April 1(1.7%) 3(5%) 4(6.3%) May 3(5%) 0 1(1.5%) Total 60 63 63 Table (4): Distribution of patients with Influenza A and B , Adenovirus according to clinical symptoms

As it becomes clear in the table below , most of the infections were accompanied by high fever at the first level , then the other symptoms varied by appearance.

Annals of Tropical Medicine & Public Health : http://doi.org/10.36295/ASRO.2020.23925 Kareem & Jarullah (2020): Investigating viruses in children May 2020 Vol. 23 Issue 9

Clinical signs Frequency in Flu A Frequency in Flu B Frequency in Adv Fever 55(92%) 60(95%) 59(94%) Cough 48(80%) 52(82%) 45(71%) Wheezing 20(33%) 20(32%) 41(65%) Sneezing 40(67%) 39(62%) 12(19%) Rhinorrhoea 43(72%) 40(63%) 10(16%) Dyspnea 10(17%) 3(4.7%) 41(65%) Crepitation 1(1.6%) 1(1.5%) 5(8%) Croup 5(8%) 1(1.5%) 1(1.5%) Table (5): Distribution of Influenza A and B , Adenovirus according to the different cities in Thi-Qar province

As can be seen in the table below, the largest percentage of infection appeared in the city of Nasiriyah in the first level , and the infections varied in the rest of regions.

City No. of +ve cases Flu A No. of +ve cases Flu B No. of +ve cases Adv Al-Nasiriyah 30(50%) 30(47.6%) 22(35%) Sayed Dakhil 10(17%) 9(14%) 3(21%) Al-Shatrah 11(18%) 10(16%) 10(16%) Suq Alshyuokh 4(7%) 8(13%) 5(8%) Al-Rifai 2(3%) 1(1.4%) 5(8%) Al-Garraf 3(5%) 3(5%) 4(6%) Al-Batha 0 2(3%) 4(6%) Total 60 63 63

Figure (1): Agarose gel electrophoresis image that showed RT-PCR product analysis for of attachment Hexon for Adenovirus . M (Marker ladder 100-3000bp). Lane (1-10) showed some positive Adenovirus at 134 bp product size.

Annals of Tropical Medicine & Public Health : http://doi.org/10.36295/ASRO.2020.23925 Kareem & Jarullah (2020): Investigating viruses in children May 2020 Vol. 23 Issue 9

Figure (2): Agarose gel electrophoresis image that showed RT-PCR product analysis for of attachment nonstructural protien (NP) for Infleunza A virus. M (Marker ladder 100-3000bp). Lane (1-10) showed some positive Flu A at 190 bp product size.

Figure (3): Agarose gel electrophoresis image that showed RT-PCR product analysis for of attachment nonstructural protein (NP) for Influenza B virus. M (Marker ladder 100-3000bp). Lane (1-10) showed some positive Flu B at 249 bp product size.

5. Discussion

The study shows that 60, 63, 63 cases of infections of Influenza A, Influenza B /and Adenovirus, respectively, occurred among Iraqi children ˂ 5 years old, hospitalized with acute2respiratory tract infections. The proportion of Influenza A virus was (24%) and Influenza B virus was (25.2%) .This finding is in agreement with the findings of other studies that showed that2infants and young2children have an increased risk for hospitalization for influenza. Our result was higher that what found in study in2Kurdistan region in Iraq from1269 the overall incidence of influenza A and B viruses (9.9%) were 3.6% Flu A and 6.3% Flu B(10).In Iran study on 50 samples of RTI led to Flu A 12.5% and Flu B 50% (11).In Turkey 12.6% (12) , in Malaysia 11% (13). The proportion of Adenovirus was (25.2%) , which higher than those reported for study in1Kurdistan /Iraq from 269 cases were 18%(10) , In Kuwait 7.7% (14) , Iran 3.4% (15) . The di erence between the//prevalence rate of

Respiratory2viruses//infections and co-infections in severalff /2studies may be described by di erent groups of2patients, methods2usedllfor detection of2viruses, yearly variation in incidenceff and

Annals of Tropical Medicine & Public Health : http://doi.org/10.36295/ASRO.2020.23925 Kareem & Jarullah (2020): Investigating viruses in children May 2020 Vol. 23 Issue 9 other1variables (16). In table (2) the Prevalence of 2Flu A (63%male and 37%female) Flu B( 54%male and 46%female) . This result match with (17) who found that influenza was more frequent in males(52.6%) than females (47.4%) , also(18)found among the2cases influenza A-positive (53%) were male and (47%) were female. Prevalence of Adenovirus (52%male and 48%female), this result match with (19) found that among the adenovirus-positive cases 52% were male and 48%were female. In table (3) : Flu A highest ratio was in (˂1year)21(35%), followed by(1-2years)19(32%) ,while as Flu B highest ratio was in (˂1year) 20(32%), followed by(1-2years) 25(39.6%). This results1matched to (20) which found 78% of the children hospitalized with influenza A2infection were ˂4 years of age , also agreement2with the findings of other studies that showed that infants and young children have an increase risk for2hospitalization for influenza (21, 22).1Adenovirus high ratio was in (˂1) 25(39.6%) , followed by (1-2)(2-3 years)15(23.8%) for both .This results match with (23) that was mentioned2in his study hAdv most frequent in the age between (˂1-3) years old. Table (4) show the study was conducted1from December 2018 to May 2019 that mean in winter (Dec-Feb), spring (March-Apr), summer (May). Infections with respiratory viruses exhibit distinct seasonal patterns in most temperate2regions , viruses were detected throughout1the year , with a peak in winter (Dec –Jen –Feb) .That match with (24) in his study seasonality detection of respiratory1viruses when found viruses were detected throughout the year but it a peak in1winter .In table (5):Influenza A and B-positive patients suffered from : Fever (92%, 95%) , followed by2Cough (80%,82%), Rhinorrhoea (72% , 63%) , Sneezing (67%,62%) .This findings1are agree with (25) was found the patients with influenza were more likely1to have cough(93%), fever (68%) and nasal congestion1(91%) and show that cough and fever are good predictors1of influenza infection than either symptoms alone when Infleunza1is present within the community.1Adenovirus-positive patients suffered from1Fever (94%) , followed by Cough (71%) ,2Wheezing and Dyspnea (65%) for both .(26)indicate in his study in total 1300 patients with respiratory4infection among these patients2fever (66.5%) , cough (56.0%) in addition to another symptoms of (1pneumonia ,1bronchitis and1asthma) the diseases of lower1respiratory tract infection. In table (6): show that the study was conducted in Iraq / Thi-Qar2province in different cities ,but The biggest proportion of respiratory1infections were recorded in Al-Nasiriyah city more than the rest of Thi-Qar province(50% ,47.6% , 35%)for Flu A , Flu B and Adenovirus . Therefore, that’s due to , the large area of Al-Nasiriyah compared to other regions , secondly , the population density in Al- Nasiriyah is the highest compared to the rest of the regions .

Acknowledgment

This work was2supported by the Biology1Department , collage of sciences, Thi-Qar1university, Thi-Qar, Iraq. We would like to1thank the sta of all Thi-Qar2province hospitals for their helpful1assistance with this study. We also thankff Dr. Basim A. J. for his constant2advices for successful research.

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Annals of Tropical Medicine & Public Health : http://doi.org/10.36295/ASRO.2020.23925