Usefulness of Nested PCR Assay for the Molecular Diagnosis of Human Rickettsial Infection: A Study in Bangladesh

Background: Rickettsial infections are re-emerging arthropods born worldwide zoonotic disease caused by Rickettsia, which is responsible for spotted fever and typhus fever. The diagnosis of a rickettsial illness is important for appropriate antibiotic treatment. Aims: The study aimed to determine the diagnostic accuracy and clinical usefulness of using nested polymerase chain reaction (PCR) by comparing nested PCR, ELISA, and Weil-Felix (WF) tests. Methodology: This was a prospective type of cross-sectional study. A total of 135 clinically suspected rickettsial infection cases were enrolled. Peripheral blood was taken to detect gltA, 17 kDa lipoprotein antigen gene (17 kDa), ompA, and ompB gene of Rickettsia by nested PCR. ELISA and Weil-Felix tests were done to compare with nested PCR. Results: Out of 135 cases, we detected Rickettsia in 70(51.85%) cases by nested PCR assay (p<0.01), 33((24.4%) by Weil- Felix test, 34 (25.18%) by ELISA. Only 26.66% of cases were PCR positive, which were negative by both ELISA and Weil-Felix test. Fifteen (11.11%) cases were positive by all three tests. Among 70 PCR positive rickettsia cases most frequently detected gene was ompB 42(60%), followed by 17kDa 34(48.58%); gltA 21(30%), and ompA 3(4.28%). Multiple gene combinations (ompB, 17kDa and gltA) detected in 98.57 % cases. Conclusion: Nested PCR assays showed the highest rate of detection of rickettsia cases than ELISA and Weil-Felix test. Multiple gene combinations (ompB, 17kDa, and gltA) showed the highest positivity. Therefore, diagnosis of rickettsial infection can be confirmed by PCR assay, and clinicians can plan appropriate treatment for these patients.

Molecular Evidence for Flea-Borne Rickettsiosis in Febrile Patients from Madagascar

Rickettsiae may cause febrile infections in humans in tropical and subtropical regions. From Madagascar, no molecular data on the role of rickettsioses in febrile patients are available. Blood samples from patients presenting with fever in the area of the capital Antananarivo were screened for the presence of rickettsial DNA. EDTA (ethylenediaminetetraacetic acid) blood from 1020 patients presenting with pyrexia > 38.5 °C was analyzed by gltA-specific qPCR. Positive samples were confirmed by ompB-specific qPCR. From confirmed samples, the gltA amplicons were sequenced and subjected to phylogenetic analysis. From five gltA-reactive samples, two were confirmed by ompB-specific qPCR. The gltA sequence in the sample taken from a 38-year-old female showed 100% homology with R. typhi. The other sample taken from a 1.5-year-old infant was 100% homologous to R. felis. Tick-borne rickettsiae were not identified. The overall rate of febrile patients with molecular evidence for a rickettsial infection from the Madagascan study site was 0.2% (2/1020 patients). Flea-borne rickettsiosis is a rare but neglected cause of infection in Madagascar. Accurate diagnosis may prompt adequate antimicrobial treatment.

Prevalence and Molecular Characterization of Rickettsia spp. from Wild Small Mammals in Public Parks and Urban Areas of Bangkok Metropolitan, Thailand

Rural areas usually show a higher prevalence of rickettsial infection than urban areas. However, information on the rickettsial infection status in urban settings (e.g., built-up areas and city parks) is still limited, particularly in the Bangkok metropolitan area. In this study, we performed a molecular rickettsial survey of spleen samples of small mammals caught in public parks and built-up areas of Bangkok. Out of 198 samples, the Rattus rattus complex was found to be most prevalent. The amplification of rickettsial gltA fragment gene (338 bp) by nested PCR assay revealed positive results in four samples, yielding a low prevalence of infection of 2.02%. DNA sequencing results confirmed that three samples were matched with Rickettsia typhi, and one was identified as R. felis. It is noteworthy that this is the first report of the occurrence of R. felis DNA in rodents in Southeast Asia.

730. Ocular Involvement Associated with Rickettsial Infection

Background Rickettsiosis, a re-emerging disease, is characterized with a myriad clinical symptoms and various manifestations. Ocular involvement is often misdiagnosed since it’s rarely symptomatic. It especially involves the posterior segment. We aimed to study the clinical, laboratory and therapeutic features of ocular involvement associated with rickettsial infection. Methods We encountered a retrospective study including all patients hospitalized for rickettsial infection with ocular involvement in the infectious disease department between 2007 and 2020. The diagnosis was confirmed based on serology (seroconversion) and/or positive polymerase chain reaction for Rickettsia in skin biopsy. Results A total of 24 patients were included with a mean age of 40±12 years. There were 13 women (54.2%). Sixteen patients sought medical care during the warm months, from June to October (66.6%). The revealing clinical signs were febrile maculopapular skin rash (79.2%), cephalalgia (54.2%) and arthralgia (33.3%). Five patients had visual loss (20.8%). Physical examination revealed conjunctival hyperemia (37.5%) and pathognomonic eschar (29.1%). Laboratory investigations revealed elevated liver enzymes (79.1%), thrombocytopenia (75%) and cholestasis (58.3%). Ocular involvement was unilateral in 14 cases (58.3%). Retinitis was the most common manifestation (70.8%), followed by anterior uveitis (20.8%). Retinal fluorescein angiography, performed in ten cases (41.6%), confirmed retinitis in 8 cases (80%). Both retinal vasculitis and papillary hyperfluorescence were noted in two cases (20%). Patients received doxycycline in 21 cases (87.5%) and fluoroquinolones in three cases (12.5%). The median duration of treatment was 7[6-15] days. The disease evolution was favourable in all cases (100%). No ocular sequelae were noted. Complications were noted in two cases (8.2%) represented by thrombophlebitis (one case) and recurrent seizures (one case). Conclusion Systematic fundus examination should be performed in front of suspected rickettsiosis, even in the absence of ocular symptoms and signs. It provides clinical clues to promptly diagnose and treat rickettsiosis. Disclosures All Authors: No reported disclosures

Prevalence of Ticks and detection of Rickettsial Agents from blood of Tick-Infested animals in Lower Sindh: A MOLECULAR APPROACH

Ticks are important vectors of human and animal pathogens. They are considered as main vectors for transmission of rickettsial agents affecting animal and human health. The study was designed to investigate district wise pattern and detection of rickettsial agents by using molecular and conventional techniques in blood samples of infected cattles, buffalos, sheeps and goats. A survey study was carried out in lower Sindh (Tharparkar, Badin, Hyderabad, Karachi, Tando Muhammad khan, Thatta and Mirpurkhas). Blood samples were collected randomly from infected Cattles, buffalos, sheeps and goats and transported to the Molecular Parasitology laboratory, Sindh Agriculture University, Tandojam, followed by examinations under stereomicroscope and Polymerase Chain Reaction (PCR). The study showed that overall infection of Rickettsial agents among infected animals was recorded follwoing Microscopy/ Blood smear test in cattles, buffalos, sheeps and goats was 41.79, 49.09, 46 and 41.66% respectively, whereas overall infection through PCR in cattle, buffalo, sheep and goat was 39.55, 43.55, 46 and 55.55% respectively. Whereas animal-wise data through PCR indicates that in case of Goats (55.55%) were more susceptible to rickettsial infection as compared to sheep (46%), buffaloes (43.55%) and cattle (39.55%). The highest rate of rickettsial agents was found in district Tharparkar and lowest rate was found in district Karachi. Microscopy/Blood smear method indicates that Buffaloes were more susceptible for infection. Whereas PCR indicates Goats were more susceptible for infection.

Novel vector of scrub typhus in subantarctic Chile – evidence from human exposure

The exposure of a research team to chigger mites in southern Chile allowed the first identification of a trombiculid species as vector and reservoir of scrub typhus outside the tsutsugamushi triangle, providing unique insights into the ecology and transmission of this recently discovered rickettsial infection in South America.

Clinical Manifestations in a Fatal Case of Probable Rickettsia and Leptospira Coinfection in Yucatan, Mexico

Clinical or serological coinfections of Rickettsia and Leptospira are uncommon but should be included in differential diagnosis when poor sanitation and cohabitation with infected animals may converge. Rickettsial and leptospiral infections have been continuously increasing throughout the past decade in Yucatan, Mexico. Leptospirosis is a zoonotic disease caused by aerobic spirochetes, while rickettsiosis is an arthropod-borne disease. In 2020, 16% of all rickettsiosis cases and 10% of leptospirosis in the country originated in Yucatan. The objective of the present case report was to document an unusual case of probable coinfection with Rickettsia and Leptospira with emphasis on clinical manifestations and the epidemiological context that may orient future multidisciplinary measures. Here, we presented the case of a 12-year-old female whose mother had recently recovered from a rickettsial infection. The patient presented with fever and developed unspecific signs and symptoms of infection; however, her condition quickly deteriorated with gastrointestinal, hepatic, renal, and neurological dysfunction. After discounting rabies and identifying infection with Rickettsia and Leptospira, antibiotic treatment was indicated, but it was too late to prevent death. Simultaneous infections of Rickettsia and Leptospira may be considered in endemic regions when environmental, epidemiological, and clinical conditions converge.

Spotted Fever Rickettsial Infection in Pregnancy Causing Acute Hepatitis, Fatal Postpartum Hemorrhage and Possible Vertical Transmission

We present a rare case of a 31-year-old pregnant mother in 37 weeks of gestation who presented with a 3-day history of vomiting, epigastric pain, and icterus, subsequently diagnosed as a rickettsial disease in pregnancy (with positive IgG titer for SFG) complicated with hepatitis, coagulopathy, pulmonary hemorrhage, and post-partum hemorrhage, who expired despite intensive care management. The newborn baby also had high IgG titer for SFG suggesting a vertical transmission of the disease and recovered following treatment with chloramphenicol. The SFG rickettsial infection can cause diverse clinical manifestations in pregnancy including acute hepatitis and coagulopathy. Therefore, the possibility of SFG rickettsial infection needs to be considered in diagnostic workup in obscure clinical presentations. We postulate possible vertical transmission of SFG to newborns which needs further confirmation.

CLINICO-DEMOGRAPHIC PROFILE OF SCRUB TYPHUS IN UTTARAKHAND

BACKGROUND: Scrub typhus(ST) a rickettsial infection caused by Orientia tsutsugamushi and transmitted by trombiculid mites“chiggers” particularly Leptotrombidium deliense. It is one of the differential diagnoses of haemorrhagic fevers, especially if associated with acute respiratory distress syndrome and/or jaundice. A necrotic eschar at the inoculating site of the mite is pathognomic of ST.The western Himalayan regions of India, has been witnessing increased incidence of acute febrile illnesses over the years. AIM & OBJECTIVES : 1) To determine the frequency of ST in a tertiary health care hospital in Uttarakhand. 2) To understand the clinical spectrum and associated complications. 3) To analyze the demographic prole of ST cases. MATERIAL & METHODS: The retrospective study was carried out, over a period of 24 months (August 2015 to July 2017), which included cases of AFI, clinically suspected of ST. Serological testing was carried out by using commercial ELISA for specic IgM antibodies against O.tsutsugamushi. RESULTS: Out of the total 3854 cases of AFI attending the tertiary care hospital, the microbiology laboratory received a total of 760 clinically suspected cases of ST for serological testing, 494 cases (12.81%) were found positive for IgM antibodies against O.tsutsugamushi.The common symptoms noted were fever with headache, myalgias, gastrointestinal intolerance, followed by breathlessness, rash and jaundice. Eschar was seen only in 69 patients. The most common complication noticed was ARDS (8.29%) followed by hepatitis (6.88%), petechial heamorrhagic spots (38.3%) and sepsis (6.07%). Mortality rate was found to be 1.8%. The cases were mainly seen in the months between August to October which correlates the infection with the rainy season. CONCLUSION: ST should be considered as one of the differential diagnosis of AFI. Timely diagnosis and management becomes crucial to decrease/limit the diease burden, thus thwarting the onset of complications and mortality