Study on rickettsial diseases evidenced by Weil-Felix test among febrile patients visiting a tertiary care hospital in Mymensingh

Background: Rickettsial diseases are neglected, re-emerging vector borne zoonosis & increasingly considered as one of the most important causes of pyrexia of unknown origin (PUO). They are grossly underdiagnosed due to wide range of non-specific symptoms, low index of clinical suspicion, lack of widely available specific diagnostic tools, leading to significant morbidity & mortality. Appropriate diagnosis in early stages is therefore necessary to prevent fatal complications associated with this disease. Objective: Present study was attempted to assess the burden of rickettsial illness by Weil-Felix agglutination test, among the suspected febrile patients, visiting a tertiary care hospital as well as to analyze the demographic profile & clinical manifestations of the seropositive cases. Methods: This was a cross-sectional study, conducted at department of microbiology, Mymensingh Medical College from March 2018 to February 2019. A total of 453 febrile patients of suspected rickettsial illness, irrespective of age and sex, were enrolled in this study. Serum sample from all the enrolled cases were then analyzed for rickettsial antibodies by Weil-Felix slide agglutination test. Results: Out of 453 cases, a total of 260 (57.39%) showed significant agglutination by Weil-Felix test, of which 101 (38.84%) were reactive to OX2 (spotted fever group rickettsiae), 65 (25%) were reactive to OXK (Scrub typhus) & 13 (5%) showed significant titers to OX19 (typhus fever). Remaining 81 (31.15%) sera were reactive to more than one antigens (mixed reactivity). Seropositivity was higher among female subjects (142; 54.61%) & age group >15-30 accounted for highest number of cases (95; 36.53%). Positive cases showed diverse clinical manifestations like headache (55.76%), myalgia (50.76%), skin rash (10.38%), eschar (9.23%), oliguria (7.3%), jaundice (10.76%), splenomegaly (6.81%), hepatomegaly (7.30%) etc. Conclusion: Rickettsial diseases should be considered as an important etiology of PUO & early diagnosis should be done to initiate proper treatment to prevent fatal complications. Though it lacks sensitivity & specificity, in a resource constraint area like Bangladesh, Weil-Felix test still serves as the cheapest initial diagnostic tool for rickettsial illness to guide the physician for further approaches.


Introduction
Rickettsiae are a group of small gram negative, non-motile, obligate intracellular organisms, those are transmitted directly to human hosts by the bite of arthropod vectors like-Louse, mite, fleas and ticks. 1 They are actually pleomorphic coccobacilli, not well stained with Gram's technique but can be seen under light microscope, when Giemsa or acridine orange staining are applied. 2 Rickettsial diseases are considered as a re-emerging threat to public health, if not early diagnosed or misdiagnosed. Various type of rickettsial infections are now enlisted as an important cause of pyrexia of unknown origin (PUO) and therefore it is required to be differentiated from many other common febrile illnesses like-dengue, malaria, enteric fever, leptospirosis, etc. 3 Rickettsial agents are categorized into 3 main biogroups. Typhus fever group comprising of R. prowazekii & R. typhi, which are responsible for classical epidemic typhus & endemic typhus respectively. Spotted fever group contain many species like-R. rickettsia, R. conorii, causing Rocky mountain spotted fever. Scrub typhus is another entity which is caused by a closely related rickettsial agent-Orientia tsutsugamushi, which is transmitted to human hosts by larval trombiculid mites. 4 Rickettsial diseases are regarded as one of the main causes of nonviral illnesses, that present with fever & rash. 5 Severities of this disease may vary from self-limiting febrile illness to a fulminant life threatening infection including multiorgan dysfunction with reported case fatality ranging from 1 % to 30%, if left untreated. 6 After an incubation period of 3-14 days, there is a sudden onset of non-specific symptoms like-fever, chill, headache, myalgia, dry cough, nausea, vomiting, lymphadenopathy and often a maculopapular rash on the extremeties. 7 An Eschar is a pathognomonic sign for scrub typhus, which is a cigarette-burn like cutaneous black necrotic lesion, appears on bite site of the larval mite, variably seen in 50% of cases. 8 However, occurrence of Escher is rare in south east Asian patients. 4 Recent studies in Asian countries have reported unusual complications of rickettsial diseases, those usually develop after first week of illness likegastrointestinal manifestation, renal failure, hepatitis, pneumonitis, acute respiratory distress syndrome (ARDS), septic shock, myocarditis, meningoencephalitis, tinnitus and hearing loss. 9,10 Rickettsial illnesses are frequently misdiagnosed by physicians due to lack of data regarding geographical distribution, low index of suspicion, non-specific clinical manifestations & unavailability of sensitive as well as specific diagnostic tests, leading to significant morbidity & mortality. 2,11 Serological methods like-Weil-Felix test (WFT), latex agglutination test, immunoperoxidase assay, Enzyme Linked Immunosorbent Assay (ELISA), and micro immunofluorescence ("gold standard"), can be used for diagnosing suspected rickettsial infections. The most widely used method is Weil-Felix test which is a non-specific heterophile agglutination test. It was developed from observation of Weil & Felix that, sera from patients with typhus fever agglutinated certain strains of Proteus like-Proteus vulgaris OX-19 and OX-2 as well as Proteus mirabilis OX-K. The test is usually done as tube agglutination though, rapid slide agglutination methods have been employed for screening. 12 Though this test lacks both sensitivity and specificity, in most of the laboratories in resource limiting area, this WF test is the only one available method. 1 Rickettsial infections prevail world-wide and they have been reported from all parts of India like Jammu, Kashmir, Himachal Pradesh, Rajasthan, Assam, Kerala, Uttar-Pradesh, Tamil-Nadu, West Bengal, Maharashtra. 13 Recently, rickettsial illnesses like-Scrub typhus and murine typhus also have been reported from Chittagong, Bangladesh.14 But the data regarding the burden of rickettsial infections in our country is very limited, which actually reflects only the tip of the iceberg.

Methodology
This was a cross-sectional study, conducted at department of microbiology, Mymensingh Medical College from the period of March 2018 to February 2019. A total of 453 Febrile patients (fever for more than 5 days) of suspected rickettsial illness, irrespective of age and sex, referred from outpatient and inpatient facilities of department of Medicine and department of Pediatrics, Mymensingh Medical College Hospital (MMCH) were included in the study. Febrile patients with already established cause of their illness other than rickettsial disease, were excluded from this study. Before collecting blood sample, informed consent was taken from all patients & socio-demographic data of all the subjects were collected by using pre-tested, printed questionnaire. Following all universal safety precautions, 2ml of venous blood was collected with a sterile disposable syringe & was transferred into a clean sterile test tube & blood was kept for 30 minutes for clot formation. Following centrifugation of blood at 800 rpm for 10 minutes, serum was separated from the clotted blood & then collected into a sterile microcentrifuge tube for Weil-Felix test. The serum samples were tested immediately. Whenever a delay in testing was anticipated, serum samples were stored in -20°C. According to the manufacturer's instructions, Weil-Felix slide agglutination assay were performed as a rapid screening test, on each serum sample, using all the three proteus strains-P. vulgaris OX-19, OX-2 and p. mirabilis OX-K (Cypress Diagnostics, Schotelveldstraat 3B-3012, Leuven, Belgium). First, the smooth stained Cypress antigen suspensions were mixed with patient's serum. Agglutination obtained within 1 minute was considered as positive reaction, indicating the presence of corresponding rickettsial antibodies in patient's serum. No agglutination indicated the absence of antibodies. This slide agglutination was done with doubling dilutions of each serum -1:80, 1:160 & 1: 320. According to the baseline titer for rickettsial diseases reported previously, a single Weil-Felix titer of 1:160 or more was considered as diagnostically significant. 15 Actually, this non-specific heterophil agglutination test is based on the principle that some strains of proteus share common somatic antigens with certain species of Rickettsiae. Therefore sera from patients of rickettsioses, containing rickettsial antibodies react with proteus antigen suspension & thus produce visible agglutination. Usually antigen suspension of P. vulgaris-OX2 react strongly with sera from patient infected with spotted fever group rickettsiae, except rocky mountain spotted fever (RMSF). Antigen suspension of P. vulgaris-OX19 reacts with sera of patient with typhus group as well as RMSF, while P. mirabilis-OXK antigen suspension strongly reacts with sera of patients with scrub typhus. 5

Results
Out of 453 serum samples, a total of 260 (57.39%) showed significant seroreactivity (titer ≥ 1: 160) by Weil-Felix agglutination test. Among these 260 positive samples, 179 (68.84%) sera showed reactivity only to one Weil-Felix antigen (single antigen reactivity), of which 101 (38.84%) were reactive to OX2 antigen, suggestive of spotted fever group rickettsiae, 65 (25%) were reactive to OXK antigen, suggestive of scrub typhus & 13 (5%) showed significant titers to OX19 antigen, indicating tick typhus or typhus fever group of rickettsiae. Remaining 81 (31.15%) Weil-Felix positive samples showed significant titers to more than one Proteus antigen (mixed antigen reactivity), making it difficult to interpret the result. Analyzing all the mixed titers, it was evident that 32 samples ( Figure 03]. All the seropositive cases were presented with fever. Apart from fever, headache (145; 55.76%) was the most common manifestation, followed by myalgia (132; 50.76%) and cough (108; 41.53%). Eschar was present only in 24 (9.23%) cases and 27 (10.38%) patients had rashes on their skin (Figure 04 showing eschar). 39 (15%) cases were presented with anemia, 28 (10.76%) cases had jaundice and only 11 (4.23%) patients were presented with neck rigidity [     29 We have documented oliguria in 19 (7.30%) cases indicating acute kidney injury (AKI). This finding is supported by a hospital based study in Chandigarh (India), which reported 26.53% (13/49) of scrub typhus cases having oliguria and they were all diagnosed to have AKI. 30 We have also documented 11 (4.23%) seropositive cases with neck rigidity, which is a definitive sign of meningitis. 26% (17/65) scrub typhus cases with evidence of neck rigidity were reported from a hospital based study in Pondicherry, India. 28 So, rickettsial illnesses, particularly scrub typhus should be regarded as an emerging cause of meningitis in clinical practices. This study also recorded that hepatomegaly & splenomegaly were found in 19 (7.30%) & 18 (6.81%) cases respectively. Two separate studies on rickettsial illness in Karnataka & Bengaluru (India), also documented a good number of seropositive cases with hepatomegaly & splenomegaly. 3,11 From the above discussion, it is very clear that rickettsial illness usually manifests as a non-specific febrile illness, but disease spectrum can be extended into fatal multi-organ involvement also. 31 So, this study clearly gives us an insight of the febrile patients with suspected rickettsiosis presenting to a tertiary care hospital & it is obvious that there are lots of genuine rickettsial cases, usually presenting with self-limiting non-specific manifestations as well as with multi-organ involvement like-hepatosplenomegaly, acute renal failure, hepatic failure, meningoencephalitis etc. So awareness among physicians & early diagnosis is very essential to prevent morbidity & mortality.

Limitation
Main limitation of the study was that the seropositive cases by Weil-Felix test, could not be compared with ELISA, immunofluorescence or other confirmatory tests in reference laboratories.

Conclusion
Our results show that, rickettsial infections are prevalent in Mymensingh region of Bangladesh & it should be considered as one of the most important causes of Fever of Unknown Origin. Nationwide active surveillance of rickettsial diseases is required to know the actual burden of the disease. In spite of all the drawbacks associated with Weil-Felix test, in a resource poor country like Bangladesh, it still serves as the available tool for diagnosis of the rickettsial illness to guide the clinicians for establishing early treatment.
With proper use of standardized antigens, inclusion of positive serum controls & proper clinical correlation with patient's sign-symptoms, Weil-Felix test can be considered as a cost effective option for initial screening of rickettsial illness.