FUO Cases Showing Prevalence of Scrub Typhus: a Comparative Study by ELISA and Rapid Test in a Tertiary Care Hospital in Andhra Pradesh, India

Int.J.Curr.Microbiol.App.Sci (2015) 4(2): 632-640

ISSN: 2319-7706 Volume 4 Number 2 (2015) pp. 632-640 http://www.ijcmas.com

Original Research Article FUO Cases Showing Prevalence of Scrub Typhus: A Comparative Study by ELISA and Rapid Test in a Tertiary Care Hospital in Andhra Pradesh, India

Ponugoti Munilakshmi1*, M.Venkata Krishna2, Maria Sindhura John1, T.Deepa1, G.Avinash1 and P.Sreenivasulu Reddy1

1Department of Microbiology, Narayana Medical College, Nellore-524003, A.P 2Department of Paediatrics, Narayana Medical College, Nellore-524003, A.P *Corresponding author

A B S T R A C T

Fever of unknown origin (FUO) has multiple causes. Scrub typhus is less known cause of FUO in India. There was high mortality in undiagnosed cases of FUO which lead to the diagnosis of scrub typhus. Scrub typhus is a rickettsial infection K e y w o r d s which is caused by Orientia tsutsugamushi and transmitted by the bite of the chigger of a mite. Present study done to know the prevalence of scrub typhus as a Fever of causative factor in FUO cases by detection of IgM antibodies by ELISA and rapid unknown test. This study was done over a period of six months in Narayana medical college origin, and hospital. 223 serum samples of FUO cases were analysed for IgM antibodies Orientia to Orientia tsutsugamushi along with dengue, malaria, typhoid, tuberculosis and tsutsugamushi, brucellosis. Scrub typhus IgM antibodies by ELISA were detected in 93 (41.7%) Elisa-enzyme patients. Scrub typhus positivity was significantly higher among female in linked immune comparison to males. Maximum positivity of scrub typhus was found in females of sorbent assay, 46-60 years age group. There was 98% correlation between ELISA and rapid Rapid test, method. The laboratory parameters were abnormal in most of the patients as Scrub typhus evident by thrombocytopenia, deranged liver functions and renal functions. The present study emphasizes the importance of scrub typhus among cases of FUO especially after rainy season and during early cooler months.

Introduction disease caused by Orientia tsutsugamushi, is Fever of unknown origin (FUO) is said 3 when the body temperature increases to a very less known cause of FUO . 38.3°C (101°F) or more several times a day Rickettsial infections are re-emerging with lasting longer than 3 weeks or failure to increased reports from different parts of the reach a diagnosis despite 1 week of inpatient world. Scrub typhus is an acute febrile evaluation 1Although there are multiple illness and delay in diagnosis is associated causes of FUO but infections such as enteric with considerable morbidity and mortality. fever, malaria, dengue, tuberculosis, Although it is a neglected disease in India, brucellosis are among most common but in recent years there are reports from causes. 2 Scrub typhus (ST), a rickettsial Maharashtra, Tamil Nadu, Karnataka,

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Kerala, Jammu and Kashmir, Uttaranchal, the cheapest and easily available, but this is Himachal Pradesh, Rajasthan, Assam and notoriously unreliable. Indirect West Bengal indicating the resurgence of the immunofluorescence test, the gold standard disease in this part of the world also. 4,5,6,7,8,9 is beyond affordability especially in poor countries and needs expertise for Scrub typhus or Bush typhus is a rickettsial interpretation as the choice of cut-off values infection which is caused by Orientia for positive diagnosis is influenced by tsutsugamushi and transmitted by some several factors such as antibody kinetics, species of trombiculid mites ("chiggers", geography, negative seroconversion and particularly a Leptotrombidium seasonality. 13,14 deliense).6 The name is derived from the prevalence of the mites in areas of heavy IgM ELISA has been evaluated and found to scrub vegetation. The disease is endemic in be quite satisfactory in comparison to the the geographical region known as gold standard, but samples need to be pooled "tsutsugamushi triangle" which extends for ELISA which can lead to delayed from northern Japan and far-eastern Russia diagnosis thus influencing the overall in the north, to the territories around the outcome. Rapid tests which are economic, Solomon Sea into northern Australia in the rapid and single tests can be carried out. south, and to Pakistan and Afghanistan in the west. The bite of this mite leaves a This study was carried out to know the characteristic black eschar which is useful to seroprevalence of scrub typhus in FUO the doctor for making the diagnosis. 5,10 cases and to compare a rapid test with IgM ELISA for the diagnosis of scrub typhus. The observation of the eschar is often missed and other signs and symptoms of the Materials and Methods disease are not characteristic leading to delayed diagnosis by the clinician. In view This is a cross-sectional study carried out on of low index of suspicion, non-specific signs serum samples from FUO cases received and symptoms, and absence of widely over a period of 10months extending from available sensitive and specific diagnostic March to December 2014. The samples were tests, these infections are difficult to processed for the detection of IgM diagnose. 11 antibodies for the diagnosis of scrub typhus by ELISA and Rapid test. Samples were also Failure of timely diagnosis leads to tested for dengue fever, typhoid fever and significant morbidity and mortality. With leptospirosis, tuberculosis and malaria. timely diagnosis treatment is easy, affordable and often successful with Detection IgM antibodies by ELISA- was dramatic response to antimicrobials. As done using In Bios International TM IgM antimicrobials effective for rickettsial ELISA. Detection of IgM antibodies by diseases are usually not included in Rapid method was done using SD Bioline empirical therapy of nonspecific febrile Tsutsugamushi, one-step scrub typhus illnesses, treatment of rickettsial diseases is antibody test. Clinical features of the not provided unless they are suspected. patients were retrieved from hospital Several tests are available with their own medical records. Statistical analysis was advantages and limitations. 12. Among all the done using SPSS 11.5 version. P-value was serological tests available Weil-Felix test is calculated using Chi-square test.

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Results and Discussion The present study reports 9.7% mortality in concurrence with previous studies.21,22,23,24 A total of 223 patients of FUO reported to The most common cause of death in our the Narayana hospital tested for ST IgM study was acute respiratory failure as most antibodies were detected in 93 (41.7%) of cases presented with fever, cough and cases. Among these 93 diagnosed cases 54 dyspnoea showing bilateral interstitial (58.06%) were females and 39 (41.94%) pneumonia in high-resolution computerized were males. Positivity for ST was higher tomogram. Fever, cough and vomitings have among female who were suffering from been reported as chief complaints in other FUO in comparison to males. The age of the studies also. 20,25Kammili et al., from patients ranged between 20 and 80 years. In Secunderabad, India tested for the females and males positivity for ST IgM prevalence of scrub typhus among patients antibody was highest in 40-60 years. who were provisionally diagnosed as dengue fever. Among the 100 patients studied, 19 There was 98% correlation between ELISA were found to be positive for antibodies for and rapid method. Of the 93 samples, only 2 scrub typhus by rapid immune- samples positive by ELISA were negative chromatography and Weil-Felix test. by rapid method. Seropositivity was equal among males and females with preponderance in old age Scrub typhus is a rickettsial disease caused group. Chief complaints included arthralgia by O. tsutsugamushi which is a Gram (2patients), haemorrhagic manifestations negative, intracellular bacterium. It is (3patients) and rash (3patients). 26 transmitted by the bite of mite belonging to Boorugu et al., reported a case from Andhra the genus Leptotrombidium Pradesh.The patient presented with fever (L.delienis)india.15 The prevalence of scrub which was associated with myalgia, loose typhus varies from 0-8% to 60% in different stools and dry cough. Patient was finally countries. 13,16In a study from Thailand, the diagnosed with scrub typhus associated with positivity for scrub typhus was 59.5% with thrombocytopenia, hepatitis, highest prevalence in 40-49 (77.7%) year hypoalbuminemia and bilateral pleural age group with no difference between the effusion. 27 A study from Chennai reported two sexes17. Gurung et al., tested 204 seizures, signs of consolidation, patients with fever of unknown origin of thrombocytopenia, elevated serum alkaline which 63 were confirmed positive of which phosphates and renal failure in 25%, 40%, 42 were males and 21 were females. 18 The 37.5%, 52% and 33% patients, respectively. study shows more positivity of ST in 6.25% of patients died of multi organ females particularly above 30 years of age. failure28 Vivekanandan M et al. also reported female preponderance in their study.19, In our study In the laboratory parameters, the most also there is female prepoderence and high important abnormality noticed was prevalence seen in 40-60 years age group. thrombocytopenia (63%). Other laboratory Fever with chills and rigors was the most findings include elevation of liver enzymes, common presentation in our study which is serum urea and serum creatinine. Similar similar to a hospital-based study in Taiwan abnormalities have been observed by and a study by Dass et al., from the state of Vivekanandan M et al. in their study19. Meghalaya, India. 20

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The diagnosis of scrub typhus poses a delayed diagnosis and treatment, which at problem due to low index of suspicion, non- times may be fatal. A rapid method which specific signs and symptoms, absence of the can provide the diagnosis on the same day. specific presentation of an eschar and the lack of diagnostic facilities in India. Therefore, we compared the rapid method with IgM ELISA for the diagnosis of scrub Tests are available like Weil-Felix, indirect typhus. Chinprasatsak et al.,30 evaluated a immunofluoresce, PCR, culture and ELISA rapid dot blot assay for rapid diagnosis and all of which have their own limitations. In reported the sensitivity and specificity of poor countries some of these tests like PCR, 87% and 94%, respectively. They concluded indirect immunofluorescence which provide that the dot blot immunoassay dipstick was accurate and specific diagnosis are either not accurate, rapid, easy to use and relatively available or are too expensive. Weil-Felix is inexpensive. No false positives were a commonly used inexpensive serological observed. Jang et al., evaluated IgM ELISA test which lacks both sensitivity and for the diagnosis of scrub typhus and specificity. 29 There is need for a rapid, reported sensitivity of 96.3% for IgG IFA- technically simple and economic test. positive samples and of 100% for IgM IFA- ELISA is an easy and comparatively positive samples. The specificity of the IgM economic test but the results of this test may capture ELISA was 99%, for IgM-positive not be available on the same day as samples samples. 31 need to be pooled for testing thus causing

Figure.1 Age wise distribution of scrub typhus

males females

20-40 40-60 60-80

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Figure.2 Sex wise distribution of scrub typhus

0 females males

Figure.3 Clinical presentation of seropositive patients

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Figure.4 laboratory parameters of scrub typhus patients

Figure.5 Comparison of efficacy ELISA and rapid test

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Gurung et al., used ELISA and rapid method unknown origin. Internist (Berl) for the diagnosis of scrub typhus. In their 2009;50:659-67. study one sample positive by rapid method 3. R Bithu, V Kanodia, RK Maheshwari was negative by ELISA. 18In our study using Possibility of scrub typhus in fever IgM ELISA and rapid test, there was 97% of unknown origin (FUO) cases: An correlation between the two tests. The experience from Rajasthan Indian difference was statistically significant Journal of Medical Microbiology, indicting the superiority of ELISA as 2014; 32( 4 ) 387-390 compared to rapid test. Therefore, it can be 4.Rathi N, Rathi A. Rickettsial infections: concluded that in centers with high Indian perspective. Indian Paediatric workload, ELISA should be used but with journal 2010;47:157-64. less number of samples rapid method may 5.Mahajan SK, Rolain JM, Kashyap R, be used for early diagnosis of scrub typhus. Bakshi D, Sharma V, Prasher BS, et al. Scrub typhus in Himalayas. Scrub typhus infections have been reported Emerg Infect Dis 2006;12:1590-2. from neighbouring states like Tamil Nadu, 6.Sundhindra BK, Vijaykumar S, Kutti AK, Kerala, Karnataka and Orissa. The climatic Tholpadi SR, Rajan AS, Mathai E, et conditions in Andhra Pradesh are more or al. Rickettsial spotted fever in less similar to these states therefore, it is Kerala. Natl Med J India highly likely that the various infections 2004;17:51-2. prevalent may be similar. Our study 7.Mathai E, Lloyd G, Cherian T, Abraham provides an evidence for the seropositivity OC, Cherian AM. Serological of scrub typhus in this state. evidence of the continued presence of human rickettsioses in southern Scrub typhus is prevalent but an India. Ann Trop Med Parasitol undiagnosed disease in India. It should be 2001;95:395-8. considered in the differential diagnosis of 8.Kamarasu K, Malathi M, Rajagopal V, patients suffering from acute febrile illness . Subramani K, Jagadeeshramasamy This is particularly important after the rainy D, Mathai E. Serological evidence season and early cooler months, i.e. between for wide distribution of spotted August and October months. Rapid and fevers and typhus fever in Tamil specific diagnostic methods using ELISA Nadu. Indian J Med Res can be carried out timely for early diagnosis 2007;126:128-30. of scrub typhus in patients with FUO. 9.Mittal V, Gupta N, Bhattacharya D, Kumar K, Ichhpujani RL, Singh S, et References al. Serological evidence of rickettsial infections in Delhi. Indian J Med Res 1.Seyed-Mohammad Alavi, Mohammad 2012;135:538-41. Nadimi, and Gholam Abbas 10. Chang WH. Current status of Zamani, Changing pattern of tsutsugamushi disease in Korea. J infectious etiology of fever of Korean Med Sci 1995;10:227-38. unknown origin (FUO) in adult 11.Vivekanandan M, Mani A, Priya YS, patients in Ahvaz, Iran Intern Med. Singh AP, Jayakumar S, Purty S. 2013 Summer; 4(3): 722 726. Outbreak of scrub typhus in 2.Schneidewind A, Ehrenstein B, Salzberger Pondicherry. J Assoc Physicians B. Infections as causes of fever of India 2010;58:24-8.

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