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Year : 2018  |  Volume : 61  |  Issue : 1  |  Page : 76-80
Prevalence of scrub typhus in pyrexia of unknown origin and assessment of interleukin-8, tumor necrosis factor-alpha, and interferon-gamma levels in scrub typhus-positive patients

Department of Microbiology, J. N. Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India

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Date of Web Publication22-Mar-2018


Background: Scrub typhus is lesser known cause of fever of unknown origin in India. Even if there have been reports documenting the prevalence of scrub typhus in different parts of India, it is still an unknown entity, and clinicians usually do not consider it as differential diagnosis. The present study was performed to document the prevalence of scrub typhus among febrile patients in western part of Uttar Pradesh and to assess the clinical profile of infected patients on the one hand and knowledge, attitude, and practices among clinicians on the other. Materials and Methods: A total of 357 adult patients with fever of more than 5-day duration were recruited. All patients underwent complete physical examination, and detailed clinical history was elicited as per predesigned pro forma. After primary screening to rule out malaria, enteric fever, and leptospirosis infection, secondary screening for scrub typhus was done by rapid screen test and IgM ELISA. Results: Scrub typhus infection was positive in 91 (25.5%) cases. The most common symptoms among the patients were fever (100%), pain in abdomen (79.1%), pedal edema 56 (61.5%), rash 44 (48.3%), headache 44 (48.3%), vomiting 42 (46.1%), constipation 33 (36.2%), cough 28 (30.7%), and lymphadenopathy 20 (21.9%). The median values of interleukin-8, interferon-gamma, and tumor necrosis factor-alpha in healthy controls were 15.54 pg/ml, 7.77 pg/ml, and 54.1 pg/ml, respectively, while the median values of these cytokines in scrub typhus-positive patients were 21.04 pg/ml, 8.74 pg/ml, and 73.8 pg/ml, respectively. Conclusion: Our results highlight that scrub typhus infection is an important cause of pyrexia of unknown origin, and active surveillance is necessary to assess the exact magnitude and distribution of the disease.

Keywords: ELISA, Orientia tsutsugamushi, pyrexia of unknown origin, scrub typhus

How to cite this article:
Rizvi M, Sultan A, Chowdhry M, Azam M, Khan F, Shukla I, Khan HM. Prevalence of scrub typhus in pyrexia of unknown origin and assessment of interleukin-8, tumor necrosis factor-alpha, and interferon-gamma levels in scrub typhus-positive patients. Indian J Pathol Microbiol 2018;61:76-80

How to cite this URL:
Rizvi M, Sultan A, Chowdhry M, Azam M, Khan F, Shukla I, Khan HM. Prevalence of scrub typhus in pyrexia of unknown origin and assessment of interleukin-8, tumor necrosis factor-alpha, and interferon-gamma levels in scrub typhus-positive patients. Indian J Pathol Microbiol [serial online] 2018 [cited 2023 Mar 25];61:76-80. Available from:

   Introduction Top

Scrub typhus is a zoonosis caused by Orientia tsutsugamushi which is transmitted through the bite of larval mites (chiggers) of the Trombiculidae family. It is a major pathogen in the Asia-Pacific region, where it accounts for up to 23% of all febrile episodes.[1] The nonspecific protean presentations of scrub typhus pose a great challenge to the clinicians in identifying these infections early in their clinical course when antibiotic therapy is most effective.[2] Common symptoms, such as fever, rash, headache, nausea, vomiting, and thrombocytopenia, can easily be confused with myriad of other diseases like malaria, chikungunya, dengue, enteric fever, leptospirosis, and  Brucellosis More Details.[3] Complications such as pneumonitis, meningoencephalitis, jaundice, renal failure, and myocarditis may also develop. The severity of infection ranges from mild features to multiorgan failure and death, which occurs in around 4% of the patients presenting to a hospital.[4],[5]

Scrub typhus in India has been documented from Jammu and Kashmir, Himachal Pradesh, Uttaranchal, Rajasthan, Assam, West Bengal, Maharashtra, Kerala, and Tamil Nadu.[6],[7],[8],[9] Hun and Troyoreported high magnitude of rickettsial diseases including scrub typhus, spotted fever, and Indian tick typhus.[3] Around 42.8% prevalence of scrub typhus has been documented in recent studies.[10]

However, the reported numbers in India are an underestimate due to the triad of lack of awareness of the disease, poor or absent community-based data, and nonavailability of confirmatory laboratory tests.[11] No report on scrub typhus has so far emanated from Uttar Pradesh. The present study was performed to document the prevalence of scrub typhus among febrile patients in western part of Uttar Pradesh and to assess the clinical profile of infected patients on the one hand and knowledge, attitude, and practices (KAP) among clinicians on the other. Interleukin (IL)-8, tumor necrosis factor (TNF)-alpha, and interferon (IFN)-gamma levels were analyzed in scrub typhus-positive patients.

   Materials and Methods Top

This study was conducted in the Department of Microbiology, J. N Medical College, A. M. U., Aligarh, over 2-month period from May to June 2014. A written informed consent was obtained from each patient. The study was cleared by the Institutional Ethical Committee of J. N Medical College. Adult patients attending outpatient department or admitted indoor with fever of more than 5-day duration associated with or without acute liver failure, respiratory distress, acute renal failure, and/or rash were recruited in the study. All patients underwent complete physical examination. Detailed clinical history was elicited as per predesigned pro forma.

Sample collection

Blood was collected aseptically from all patients with febrile illness of more than 5-day duration. Serum was separated by centrifugation, aliquoted, and stored at −20°C till further tests were performed.

Routine investigations

Complete hemogram, erythrocyte sedimentation rate, C-reactive protein (CRP), liver function tests, and renal function tests were also performed.

Primary screening

In all cases, peripheral smear and/or QBC test was performed to exclude malaria and Widal was put up to rule out enteric fever. In cases negative for malaria and enteric fever, the presence of leptospiral infection was investigated by rapid agglutination test (Leptocheck, Tulip) as well as by IgM ELISA (Panbio) as per manufacturer's instructions. All Leptospira-negative samples were processed for scrub typhus.

Screening for scrub typhus

All undifferentiated febrile cases were subjected to the rapid test for scrub typhus, pyrexia of unknown origin (PUO), screen (Tulip), and IgM ELISA (InBios) for scrub typhus as per the manufacturer's instructions and absorbance was read at 450 nm. IgM ELISA for scrub typhus was used here as confirmatory test for detection of scrub typhus infection. The results for scrub typhus IgM ELISA were interpreted according to the manufacturer's instructions, i.e., values, <0.2 optical density (OD) units were considered negative, 0.25–0.5 OD units were equivocal, and 0.5 OD units were positive. For samples showing equivocal results, another blood sample was drawn after 7 or more days, and the test was repeated. Negative and positive control sera were provided by the manufacturer and their absorbance was used for the calculation of the cutoff and for determining the validity of the test. As a part of assessing the specificity of IgM ELISA, ten Widal-positive and ten Leptospira-positive cases were also tested.

Cytokine assay

IL-8, TNF-alpha, and IFN-gamma levels in all scrub typhus-positive cases and thirty healthy control sera taken from blood bank were estimated by ELISA as per manufacturer's instructions.

Statistical analysis

Chi-square test and Student's t-test were used for scrub typhus prevalence.

Knowledge, attitude, and practices

KAP regarding Rickettsia among medical practitioners was assessed as per questionnaire to design an awareness program.

   Results Top

A total of 357 febrile patients were recruited in the study. Majority were females, 236 (66.11%) while 121 (33.8%) were males. While malaria was diagnosed in 26 (7.3%) patients, 162 (45.3%) patients were positive for enteric fever. Leptospirosis was identified in 67 (18.8%) patients. One hundred (28.01%) patients were positive for scrub typhus according to PUO rapid test screening of which 91 (25.4%) were positive by IgM ELISA. Thus, 11 cases were negative for the above etiologies and were classified under PUO.

Comparison of results of slide agglutination by pyrexia of unknown origin screen with IgM ELISA for scrub typhus pyrexia of unknown origin screen

Taking IgM ELISA as gold standard, nine cases identified by slide agglutination were false positive. Sensitivity and specificity of rapid PUO screen test was calculated against ELISA. Sensitivity was 97.85% (95% confidence interval [CI]: 92.43%–99.68%); specificity was 55% (95% CI: 31.55%–76.9%); positive predictive value was 91% (95% CI: 83.6%–95.79%); and negative predictive value was 84.62% (95% CI: 54.54%–93.63%). Thus, PUO rapid test had a high sensitivity but lower specificity than IgM ELISA. All ten Widal and Leptospira-positive cases were negative for scrub typhus.

All scrub typhus-positive patients were given appropriate treatment, all of which recovered thereafter.

Clinical profile of the patients with scrub typhus

The most common symptoms among the patients were fever (100%), pain in abdomen (79.1%), pedal edema 56 (61.5%), rash 44 (48.3%), headache 44 (48.3%), vomiting 42 (46.1%), constipation 33 (36.2%), cough 28 (30.7%), and lymphadenopathy 20 (21.9%). None of the patients diagnosed with scrub typhus had melena, convulsions, gum bleeding, clubbing, cyanosis, oliguria, epistaxis, or eschar in our study.

Gastrointestinal involvement was common. Majority of the patients, i.e., 72 (79%) had pain in abdomen at the time of admission. Diarrhea and vomiting were present in 24 (26.3%) and 42 (46.1%) patients, respectively. Hepatomegaly was present in 20 (21.9%) patients. Bilirubin levels were high in 11 (12%) patients while 47 (51.6%) had raised transaminase levels.

Respiratory complaints were present in very few patients. Cough was present in 28 (30.7%) patients, but tachypnea or acute respiratory distress syndrome was not observed in any of the patients.

There was no central nervous system involvement in any of the patient. However, 44 (48.3%) patients complained of headache.

Renal involvement was observed in the form of raised urine albumin levels, but none of the patients showed raised creatinine levels.

On comparing the clinical characteristics in scrub typhus-positive and negative individuals, it was found that rash, pallor, pedal edema, pain in abdomen, and raised transaminase levels were significantly associated with scrub typhus infection [Table 1]. On analyzing the hematological and biochemical parameters, it was found that CRP levels were significantly raised in scrub typhus patients [Table 2].
Table 1: Comparison of clinical characteristics of patients with and without scrub typhus infections

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Table 2: Laboratory findings in scrub typhus-positive and scrub typhus-negative patients

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Knowledge, attitude, and practices analysis

Analysis of the KAP regarding Rickettsia, especially scrub typhus, revealed that out of 20 doctors assessed, only 2 (10%) of doctors considered either of the two in their differential diagnosis for fever with given symptoms. Eighteen (90%) doctors thought it improbable that Rickettsia (scrub typhus) was circulating in this region.

Cytokine analysis

The median values of IL-8, IFN-gamma, and TNF-alpha in healthy controls were 15.54 pg/ml, 7.77 pg/ml, and 54.1 pg/ml, respectively, while the median values of these cytokines in scrub typhus-positive patients were 21.04 pg/ml, 8.74 pg/ml, and 73.8 pg/ml, respectively. Levels of IL-8 and TNF-alpha were raised in the study group whereas the levels of IFN-gamma were similar to that in the healthy controls.

   Discussion Top

Scrub typhus is an emerging public health problem and undoubtedly an important cause of PUO. These infections may pose a serious public health threat if not diagnosed on time or are misdiagnosed. Low index of suspicion, protean manifestations, and absence of cheap, sensitive, and specific diagnostic tests make these infections notoriously difficult to diagnose and treat. Moreover, failure of timely diagnosis and correct treatment leads to significant morbidity and mortality. A simple treatment with doxycycline is effective in treating the patient.

In developing countries like India where epidemiology and burden of rickettsial disease are largely unknown, the prohibitive cost of conducting high-end investigations such as immunofluorescence, western blot, or PCR-based tests further hampers accurate diagnosis and management of rickettsial diseases. However, simple, economical tests, such as Weil–Felix test and rapid agglutination tests, can guide a clinician in instituting appropriate treatment.[10] The test we assessed was a relatively cheap slide agglutination test – PUO screen (Tulip Diagnostics) which was compared with IgM ELISA.

In our study, 25.5% of the patients were diagnosed positive for scrub typhus fever by IgM ELISA. The positivity rate by slide agglutination was higher at 28.01%. In a study conducted by the National Centre for Disease Control, the prevalence of scrub typhus was much higher at 42.6%.[10] A study from Rajasthan reported around 49.1% prevalence of scrub typhus by IgM ELISA. One reason for lower rates of prevalence in our study may be due to the fact that it was conducted in the hot summer season. Earlier studies have reported higher rates of scrub typhus in rainy season.[12] This is because the mites are more active during or at the end of rainy season which coincides with the months of August to September in India.[13],[14] Majority of the patients belonged to 21–40 years of age (67/91; 73.6%) which is also comparable to the findings of Bithu et al.[12]

On analyzing the other etiologies, 7.3% of the patients were positive for malaria, 45.3% of the patients were positive for enteric fever, and 18.8% of the patients were positive for Leptospira. The prevalence of malaria was low (due to summer season) while that of enteric fever and leptospirosis is comparable to our previous reports.[15] IgM ELISA was highly specific as all the Widal and Leptospira-positive sera tested negative for scrub typhus. All patients recovered on institution of appropriate therapy.

Knowledge of geographical distribution, evidence of exposure to vector, and clinical features such as fever, rash, eschar, headache, and myalgia along with high index of suspicion are crucial factors for early diagnosis. In this study, clinical findings revealed that among the most common symptoms associated with scrub typhus-positive patients included fever, pain in abdomen, pedal edema, headache, pallor, vomiting, and rash. Among these, rash, pallor, pedal edema, and pain in abdomen were significantly associated (P< 0.001) with scrub typhus infection. High prevalence of rash in scrub typhus-positive patients was also found in studies conducted by Mittal et al.[10] and Rathi et al.[16] Eschar was not present in any of our patients. Varghese et al.[17] reported 55% incidence of eschar while there are authors who reported very low incidence of eschar (9.5% and 7.2%) in their study.[7],[18] Overall, an eschar may be a highly variable finding, which may be absent. It is clear from the above findings that clinical manifestations alone cannot shed light on the correct diagnosis. Correct diagnosis is essential for appropriate therapy to be instituted. As slide agglutination test had a high sensitivity but relatively lower specificity, it can serve as an initial but not only method to diagnose scrub typhus.

In our study, gastrointestinal involvement was more common as compared to respiratory involvement. Liver enzymes were deranged in 51.7% of the patients which is slightly lower than that reported by Varghese et al.[17] Among the respiratory symptoms, the presence of cough in 30.7% patients was comparable to Varghese et al.[17]

The laboratory findings revealed that the levels of total leukocyte count, alanine aminotransferase, CRP, and urine albumin were raised in the majority of scrub typhus-positive patients. These results are echoed in a similar study conducted by Rathi and Rathi [16] It is interesting to note that thrombocytopenia was not observed.

Levels of IL-8 in the study group were higher than in the control group. IL-8 is a potent neutrophil attractant. It thus plays a protective role in rickettsial infections. Elevated IL-8 levels were also observed in a similar study conducted on Leptospira-positive cases.[15] TNF-alpha production in O. tsutsugamushi infection is mediated by a heat-stable molecule not containing lipopolysaccharide or peptidoglycan.[19] TNF-alpha levels were significantly higher in scrub typhus cases in this study. Chierakul et al.[20] also reported the similar findings. TNF-alpha can have a dual role in rickettsial infections as it plays a protective role in rickettsial infections on the one hand, and on the other hand, it can work against the host by exacerbating the immunopathology. However, severe symptoms were not observed, maybe because treatment was instituted on time. IFN-gamma levels were not elevated in our study as against a study conducted by Chierakul et al.[20] who reported elevated levels. The pro-inflammatory cytokine IFN-gamma is a potent macrophage activator that plays a central role in cell-mediated immunity.[21] Its importance in host defense against intracellular pathogens has been demonstrated in several infectious disease models, including a mouse model in which IFN-gamma production was closely associated with the development of immunity against O. tsutsugamushi.[22] The low IFN-gamma levels in this study may point to low levels of IL-12 and IL-15 which are known to be potent inducers of IFN-gamma.

KAP was very poor among the clinicians as far as scrub typhus was concerned as only 10% clinicians considered rickettsial infections in the workup of PUO. Their awareness level was higher regarding Leptospira due to an ongoing project in our center.

Ours was a preliminary pilot study. A much wider, multicenter-based prevalence study will more accurately assess the prevalence of scrub typhus in Northern India.

   Conclusion Top

Scrub typhus infection is an important etiology in PUO in our area, and active surveillance is necessary to assess the exact magnitude and distribution of the disease. We recommend that all cases of undiagnosed fever should be screened for scrub typhus. Scrub typhus detection by slide agglutination is a relatively cheaper alternative and can be utilized as part of initial investigation to guide the clinicians in instituting appropriate treatment. The positive results can be confirmed by IgM antibody ELISA. In conclusion, it is possible for most microbiology laboratories to start screening for scrub typhus by slide agglutination test, assess the burden in their area, and later on add confirmatory tests such as IgM ELISA depending on the individual needs and facilities available.

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Conflicts of interest

There are no conflicts of interest.

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Correspondence Address:
Asfia Sultan
Department of Microbiology, J. N. Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/IJPM.IJPM_644_16

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