Journal of Infectious Disease and Pathology
Open Access

Scrub typhus; Antibodies; Liver function tests; Proteinuria; Immuno Chromatographic Test

Scrub typhus which is caused by Orientia tsutsugamushi previously called Ricketsia tsutsugamushi is endemic in North - East India. Clinically it mimics many other causes of acute febrile illness including malaria, typhoid, leptospirosis and viral exanthemata. Inability to identify the characteristic eschar in Indian population makes its diagnosis more difficult [1]. The aim of this retrospective case – control study was to analyse the infection pattern and adequacy of immune chromatographic test for total antibody detection (ICT) against Orientia tsutsugamushi for presumptive diagnosis and management of scrub typhus in a 132 bedded hospital in North -East India. All patients were administered Capsule Doxycycline 100 mg daily for a week.

In addition to ICT, serodiagnosis is possible by indirect immunofluorescence assay (IFA) which is regarded as the gold standard. The Weil-Felix test - a nonspecific agglutination test that utilizes antigenic cross-reactivity between rickettsiae and certain non-motile Proteus serotypes for detection of anti-rickettsial antibodies was used widely in the past. It requires paired sera and overnight incubation. It lacks both sensitivity and specificity and has fallen out of favour with pathologists and clinicians. Definite diagnosis of rickettsial infection is possible by culture in vitro on cell lines which requires biosafety level (BSL) 3 arrangements [2]. ELISA test for detection of immunoglobulin M is among the tests recommended by task force constituted by Indian Council of Medical Research [3]. Polymerase chain reaction-based test is the most accurate but requires infrastructure, expensive equipment, but can be affected by previous antibiotic therapy and has limitations in resource poor settings [4, 5]. In a study on 526 patients with scrub typhus found thrombocytopenia ≤ 20000/μl and serum creatinine ≥ 2mg/dl in 100 patients with severe disease [6].

Methods

A retrospective case - control study was carried out in a secondary care centre in North East India to examine the infection pattern and adequacy of serological diagnosis for scrub typhus in a limited resource setting. The authors had obtained clearance from the standing Ethical Committee Clearance of the hospital. This study included patients over one-year period from April 2017 to March 2018.

Inclusion criteria

Patients with history of fever exceeding one week and having a negative ICT result (n=67), while those with similar duration of fever but having a positive test result (n=56) were taken as controls and cases respectively for the purpose of this study.

Blood samples from 123 patients with history of acute febrile illness (AFI) were collected in red top Beckton Dickinson vacutainers and tested for presence of antibodies to Orientia tsutsugamushi using commercial SD Bioline Tsutsugamushi test kit (Standard Diagnostics, Inc, South Korea). The rapid test device contains a 56 Kilo Dalton’s major surface antigen O of Orientia tsutsugamushi. Inbuilt procedural control comprises of goat anti- mouse IgG. Briefly, 10 μl of serum was dispensed in the specimen well of the device after bringing it to room temperature. This was followed by addition of 3-4 drops of assay diluent. A positive result is indicated by the presence of both the control ‘C’ and test ‘T’ lines in the device and detects IgM, IgG and IgA antibodies together when interpreted after 10 -15 minutes of commencement. Thus, it is a solid phase ICT which provides only a preliminary diagnosis. Other additional diagnostic methods were not available anywhere in the city or state. A limitation of the test is that it is not possible to discern between the various iso types of immunoglobulin in the test. According to the manufacturer the sensitivity is 99% and specificity 96% with serological agreement of 97.5%.

Complete blood count (CBC) was done for all samples using a System XS 800 I hematology analyzer and peripheral blood smears (PBS) were examined. Routine urine examination was done for all 123 samples with Siemens Multistix® 10 SG on a Siemens Clinitek Status+semi-automated urine analyzer. Serum creatinine (n=108) and liver function tests (LFT n=95) including serum bilirubin, aspartate amino transferase (AST). Alanine amino transferase (ALT), alkaline phosphatase (ALKPO4), total proteins and serum albumin was done by dry chemistry on Vitros 250 Chemistry analyzers. Additional tests for excluding other causes of fever including blood culture, Widal test, immune chromatographic test for malarial antigen and cerebrospinal fluid examination were also carried out. C-Reactive proteins, other biochemical evaluations and radiological examinations were performed as indicated but shall not be discussed in this paper. Sixteen patients had received treatment in OPD of which two with additional illnesses were referred to a higher Centre. Remaining 14 patients had mild disease; while 10 of the admitted patients had severe disease. As it is a retrospective laboratory-based study, we classified those with any complications as severe disease, unlike Sriwongpan, et al. Who used an elaborate clinical risk algorithm to predict scrub typhus severity [7].

Statistical analysis of the accrued data was done by Excel version – using Microsoft office 10. Chi-square test was performed and p values were calculated: p ≤ 0.05 was considered as significant and p ≤ 0.01 was taken as highly significant.

In the patient cohort (n =56) which included seven children (age <18 years), the male to female distribution was 1:1with a mean age of 39.6 yrs. (3-85yrs). The control group (n =67) included 15 children and had a slight male preponderance with 38 males and 29 females. Mean age for the controls was 32.7 yrs. (4-84yrs). Follow up information was available for 33 patients (58.9%) who visited more than once and 31 controls (47%). Of 14 patients (25%) with severe complicated disease including septicemia, meningoencephalitis, thrombocytopenia acute respiratory distress syndrome and acute renal failure- ten recovered in the hospital. Of the remaining four patients, one succumbed to multi-organ failure, two required referrals, of which one had severe thrombocytopenia while the second had emphysematous pyelonephritis, gram negative septicemia with significantly raised serum creatinine. The fourth patient had staphylococcal septicemia (MRSA) with meningoencephalitis and raised intracranial tension who was improving, left on account of financial constraints. None of the patients had thrombocytopenia < 20,000/ μl -lowest being 30000/μl. ALT above five times the upper limit normal was observed in six patients (10.7%). Lymphocyte predominance was observed in four (7.14%) patients – all of whom were adults. Mean duration of hospitalization was 3.98 (1- 11 days) for the patients. Clinical signs are not documented due the laboratory-based nature of this study.

In the control group 52/67 patients (77.7%) required hospitalization the average period of hospitalization was three days (1 -11 days). Seven patients (10.4%) had severe disease of which four had meningoencephalitis, one each had renal failure, severe pneumonia and aseptic meningitis. Table 1 shows a comparison of laboratory parameters in the control and patient cohorts.

Table 1: Baseline investigations in 56 Patients cohort and 67 Controls.

Statistical analysis

In this study other than serum bilirubin all liver function tests and urinalysis for proteinuria showed statistical significance (Table 1). The p value for AST was lower than that for AST and equivalent to serum alkaline phosphatase. Other parameters in urinalysis, serum creatinine and CBC were not statistically significant.

A resurgence of scrub typhus had been reported in North-East India which mandates early diagnosis for effective management. Khan, et al. reported less than 33% positivity (108/314 samples) tested for IgM by the MAC ELISA kit Scrub Typhus DetectTM IgM (InBios International, Inc., USA) in a previous study [8]. In a study carried out by Pote, et al. to compare the sensitivity and specificity of three commonly available methods for serodiagnosis of acute scrub typhus infection, ICT was found to have specificity of 100 % and sensitivity of 38% [9]. Two studies - one from Thailand and another from Korea have reported a higher sensitivity of 66.7 and 72.6% respectively for the same kit [10, 11].

Only CBC and ICT for scrub typhus were done in all participants of this study. In resource poor settings where healthcare is not paid for by government or health insurance, many patients are unable to undergo all requisite testing which may translate into inadequate management. All subjects were not investigated even for all baseline tests as can be observed in Table 1 due to the poor socioeconomic background of the clientele. All commonly used LFT other than serum bilirubin were statistically highly significant (p <0.01) in scrub typhus patients as seen in Table 1.

In our centre mainstay of diagnosis is ICT for scrub typhus as the hospital doesn’t have an ELISA reader. At this centre ICT has been used for the last three years for the diagnosis and for guiding antibiotic therapy and the clinical correlation has been satisfactory considering the constraints of working in a remote secondary care centre. In this study 52/56 (93%) cases were managed satisfactorily including ten patients with severe disease. The reasons for low sensitivity in the previous study could be attributed to the Orientia tsutsugamushi antigens used for preparation of the kit. To best of our knowledge there is not much available data regarding circulating genotypes / serotypes of the organism in India. Although neither ICMR, nor Indian Academy of Paediatrics have recommended use of ICT for diagnosis and management of scrub typhus, we found it a very useful test which may to be used for presumptive diagnosis and management at primary health centres where other methods are not available, and can be duly supported with the most basic routine laboratory tests such as CBC, Urine routine examination and LFT.

Larger studies to compare the results of ICT with confirmatory tests will be of tremendous value in remote locations for presumptive diagnosis and management of scrub typhus in resource poor settings.

Conclusion

In limited resources settings ICT for combined antibody detection against Orientia tsutsugamushi along with routine urinalysis and liver function tests has been found to be adequate for presumptive diagnosis and management of scrub typhus.

Acknowledgment

The author would like to acknowledge his Department of Pathology from the Baptist Christian Hospital for their support during this work.

Conflicts of Interest

The author has no known conflicts of interested associated with this paper.

1. Pote K, Narang R, Deshmukh P. (2018) Diagnostic performance of serological tests to detect antibodies against acute scrub typhus infection in central India. Indian J Med Microbiol 36:108-112.

Indexed at, Google Scholar, Crossref

2. Luksameetanasan R, Blacksell SD, Kalambaheti T, Wuthiekanun V, Chierakul W et al. (2007)Patient and sample‑related factors that effect the success of In vitro isolation of Orientia tsutsugamushi. Southeast Asian J Trop Med Public Health 38: 91‑96.

Indexed at, Google Scholar

3. Rahi M, Gupte MD, Bhargava A, Varghese GM, Arora R. (2016)DHR‑ICMR guidelines for diagnosis and management of rickettsial diseases in India. Indian J Med Res 417-22

Indexed at, Google Scholar, Crossref

4. Paris DH, Blacksell SD, Nawtaisong P, Jenjaroen K, Teeraratkul A et al. (2011) Diagnostic accuracy of a loop‑mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute scrub typhus infection. PLoS Negl Trop Dis 5: e1307.

                 Indexed at, Google Scholar, Crossref

5. Lim C, Paris DH, Blacksell SD, Laongnualpanich A, Kantipong P,Chierakul W, et al. (2015)How to determine the accuracy of an alternative diagnostic test when it is actually better than the reference tests:A Re‑evaluation of diagnostic tests for scrub typhus using Bayesian LCMs. PLoS One10: e0114930.

Indexed at, Google Scholar , Crossref

6. Sriwongpan P, Krittigamas P, Kantipong P, Kunyanone N, Patumanond J, Namwongprom S. (2013) Clinical indicators for severe prognosis of scrub typhus. Risk Manag Healthy Policy 6:43-49.

  Google Scholar, Crossref

7. Sriwongpan P, Krittigamas P, Tantipong H, Patumanond J, Tawichasri C, Namwongprom S. (2014) Clinical risk-scoring algorithm to forecast scrub typhus severity. Risk Manag Healthc Policy 16: 7:11-7.

 Google Scholar, Crossref

8. Khan SA, Dutta P, Khan AM, Topno R, Borah J, Chowdhury P, Mahanta J. (2012) Re-emergence of scrub typhus in northeast India. Int J Infect Dis 16: e889-890.

 Indexed at, Google Scholar

9. Khandelwal S, Meena J K, Sharma B S. (2015) Scrub Typhus in Children: Clinical Profile and Complications. Pediatr on Call J 12: 95-98.

Indexed at, Google Scholar, Cross Ref

10. Silpasakorn S, Waywa D, Hoontrakul S, Suttinont C, Losuwanaluk K, Suputtamongkol Y, et al. (2012) Performance of SD bioline Tsutsugamushi assays for the diagnosis of scrub typhus in Thailand. J Med Assoc Thai 95 Suppl 2: S18‑22

Indexed at, Google Scholar  

11. Lee KD, Moon C, Oh WS, Sohn KM, Kim BN. (2014) Diagnosis of scrub typhus: Introduction of the immune chromatographic test in Korea. Korean J Intern Med 29:253‑255.

Indexed at, Google Scholar, Cross Ref

12. . Rathi N, Kulkarni A, Yewale V( 2017) IAP Guidelines on Rickettsial Diseases in Children. Indian Pediatr 54:223-229.  

 Indexed at, Google Scholar, Crossref