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.

480. Alternate Diagnoses in Children Evaluated for Multisystem Inflammatory Syndrome in Children (MIS-C)

Background SARS-CoV-2 infection is typically a mild illness in children. Multisystem inflammatory syndrome in children (MIS-C) is a rare, post-infectious, hyperinflammatory condition associated with SARS-CoV-2 infection. The presentation of MIS-C is nonspecific and diagnostic criteria is broad. The Centers for Disease Control (CDC) defines MIS-C as a hospitalized patient &lt; 21 years presenting with fever, laboratory evidence of inflammation, no alternative plausible diagnosis, and with positive exposure history or testing for current or recent SARS-CoV-2 infection. Since there is no single diagnostic test for MIS-C, there are other disease processes that can mimic its presentation and delay prompt diagnosis and management. Methods Between March 2020 and February 2021, we reviewed 282 charts of patients admitted for evaluation of MIS-C at our institution. Results 101 were found to have MIS-C, 45 found to have Kawasaki Disease (KD), and 129 were ruled out. Of the ruled-out group, the most common final diagnoses were viral infection, urinary tract infection, and acute SARS-CoV-2 infection. Other diagnoses included rickettsial infections, pneumonia, rheumatologic conditions, and bloodstream infection. Rhinovirus/enterovirus, adenovirus, Epstein-Barr virus (EBV), and Herpes Simplex Virus (HSV) were the most common viruses other than SARS-CoV-2 identified. Conclusion These findings highlight the importance of maintaining a broad differential when evaluating a patient for MIS-C, especially as community seroprevalence rises, making antibody presence less predictive of MIS-C. Disclosures Susan Wu, MD, Eli Lilly (Shareholder)

Article Research Differentiation Between Typhoid and Typhus Fever Infection and Coinfection with Limited Resources in Iraq

Enteric fever is caused by the Gram-negative bacilli Salmonella typhi and paratyphi, which have similar signs and symptoms to typhus and rickettsial illness. Both typhoid fever and rickettsia typhus, whether endemic or epidemic, have similar presentations, making it difficult to distinguish between the two diseases. As a result, co-infection of typhoid and typhus fever can be ignored if not recognized clinically. Serum was collected from 182 patients from the Suweib and fourth shurta areas who had a temperature of more than 38°C and two or more signs and symptoms of typhoid and typhus, such as headache lasting more than a week, rash, myalgia, arthralgia, and bleeding. Started with widal test slide method All result results confirmed by tube method Same samples investigated for typhus infection by weil-felix tests. Data entered to analyzed by SPSS program. the results were appeared of the 182 patients tested, 58 (18%) yielded results suggested that rickettsial infections or typhoid were the most likely because of their illnesses. patients’ age ranged from 8 to 75 years with mean age 29.10 years. The majority were female and mainly student (41.4%) and housewife (25.9%) by age and sex for the 58 patients for whom data were recorded. Age groups did not differ significantly (p > 0.5). The frequency of disease was 69% in female patients and 31% in male patients (p < 0.005). Finally, can Concluded Both widal and weil-felix test should done specially if no responses to typhoid treatment and depend on trial therapy by adding doxycycline in case that more sensitive and accurate tests are not available.

A rare case of rickettsial fever with isolated third cranial nerve palsy causing hemophagocytic lymphohistiocytosis

Rickettsial infections are being increasingly diagnosed in the Indian subcontinent with the advent of affordable and sensitive diagnostic techniques. Rickettsial infections are sometimes complicated by the development of secondary hemophagocytic lymphohistiocytosis (HLH) which is life-threatening unless identified early and treated. Here, we describe a case of rickettsial fever in a 41-year-old male who presented with fever, skin rash, and neurological obtundation, complicated by both third cranial nerve palsy and HLH. The patient showed a good recovery in sensorium with intravenous doxycycline and dexamethasone, although the oculomotor nerve palsy persisted for about two months after discharge. This case highlights the need to initiate treatment for rickettsial fever and HLH on clinical grounds before waiting for confirmation by laboratory tests.

Pathogenic, but not non-pathogenic, Rickettsia manipulate inflammasome-dependent IL-1 responses to facilitate their replication and host dissemination

Rickettsia species (spp.) are strict obligate intracellular bacteria, symbiotic in their arthropod vector, and some being pathogenic in their mammalian host, including humans. One critical feature of these stealthy group of pathogens is their ability to manipulate hostile cytosolic environments to their benefits. Although our understanding of Rickettsia cell biology and pathogenesis are evolving, the mechanisms of host innate immune defense evasion by pathogenic Rickettsia spp. remains to be elucidated. Here, we showed that disease severity in wild-type (WT) C57BL/6J mice infected with R. typhi- (etiologic agent of murine typhus) and R. rickettsii (etiologic agent of Rocky Mountain Spotted Fever), but not with non-pathogenic R. montanensis, correlated with levels of bacterial burden as detected in the spleens, as well as the serum concentrations of pro-inflammatory cytokine IL-1α and to a lesser extent IL-1β. Antibody-mediated neutralization of IL-1α confirmed a key role in controlling mortality rates and bacterial burdens of rickettsiae-infected WT mice. As macrophages are a primary source of both IL-1α and IL-1β cytokines, we determined the mechanism of the anti-rickettsial activities using bone-marrow-derived macrophages. We found that pathogenic R. typhi and R. rickettsii, but not non-pathogenic R. montanensis, induced autophagy, and avoided autophagolysosomal destruction, while simultaneously eluded pro-IL-1α induction and benefited from the dampening of IL-1α secretion, via Caspase-11-Gsdmd-dependent mechanism, to facilitate intracytosolic replication. Adoptative transfer experiments identified that IL-1α secretion by macrophages was critical for controlling rickettsiosis in WT mice. In sum, we identified a previously unappreciated pathway by which pathogenic, unlike non-pathogenic, rickettsiae preferentially target the Caspase-11-Gsdmd-IL-1α signaling axis in macrophages, possibly via an autophagy-dependent mechanism, to support their replication and dissemination within the host.Significance StatementCurrently, no vaccines are available to prevent rickettsioses, and tick- and flea-borne rickettsial infections in humans are on the rise globally. In fact, the insufficient understanding of how pathogenic Rickettsia species circumvent host immune defense mechanisms has significantly hindered the development of more effective therapeutics. Here, we identified a previously unappreciated role for the Caspase-11-Gsdmd-IL-1α signaling axis, to limiting the survival and dissemination of pathogenic R. rickettsia and R. typhi species in macrophages and wild-type C57BL/6J mice. Adoptative transfer studies further identified IL-1α-secreting macrophages as critical mediators in controlling rickettsial infection in WT mice. Collectively, these findings provide insight into the potential mechanism of how pathogenic, but not non-pathogenic Rickettsia spp., become ubiquitinated, induce autophagy and benefit from the dampening of the Caspase-11-Gsdmd-mediated release of IL-1α to support host colonization.

Vaccine Design and Vaccination Strategies against Rickettsiae

Rickettsioses are febrile, potentially lethal infectious diseases that are a serious health threat, especially in poor income countries. The causative agents are small obligate intracellular bacteria, rickettsiae. Rickettsial infections are emerging worldwide with increasing incidence and geographic distribution. Nonetheless, these infections are clearly underdiagnosed because methods of diagnosis are still limited and often not available. Another problem is that the bacteria respond to only a few antibiotics, so delayed or wrong antibiotic treatment often leads to a more severe outcome of the disease. In addition to that, the development of antibiotic resistance is a serious threat because alternative antibiotics are missing. For these reasons, prophylactic vaccines against rickettsiae are urgently needed. In the past years, knowledge about protective immunity against rickettsiae and immunogenic determinants has been increasing and provides a basis for vaccine development against these bacterial pathogens. This review provides an overview of experimental vaccination approaches against rickettsial infections and perspectives on vaccination strategies.

The burden of scrub typhus in India: A systematic review

Background Scrub typhus, a vector-borne zoonotic infection caused by the bacteria Orientia tsutsugamushi, is one of the most common and clinically important rickettsial infections worldwide. An estimated one million cases occur annually with a high case fatality rate. Although scrub typhus is a major public health threat in India, the burden and distribution remains unclear. We aimed to estimate the burden of scrub typhus in India. Methodology We performed a systematic review of published literature on scrub typhus from India to extract information on epidemiology, morbidity, and mortality. Important databases were searched using keywords and appropriate combinations. We identified observational, interventional, and population-based studies and extracted the data to evaluate the number of cases diagnosed using serology or PCR and the number of deaths due to scrub typhus. We conducted a systematic narrative synthesis to summarize included studies. Principal findings In the last decade, there were 18,781 confirmed scrub typhus cases reported in 138 hospital-based studies and two community-based studies. IgM ELISA was used in 122 studies to confirm the cases in majority (89%). The proportion of scrub typhus among acute undifferentiated febrile illness (AUFI) studies was 25.3%, and community seroprevalence was 34.2%. Ninety studies had data published on multiple organ involvement out of which 17.4% of cases had multiple organ dysfunction syndromes, 20.4% patients required ICU admission, and 19.1% needed ventilation. The overall case-fatality rate was 6.3%, and the mortality among those with multi-organ dysfunction syndrome was as high as 38.9%. Conclusion/significance Scrub typhus, a common acute febrile illness in India causing severe morbidity, accounts for a large number of deaths. The burden of the disease has been underappreciated. Early diagnosis and prompt treatment can significantly reduce complications and mortality. Establishing good surveillance and instituting appropriate control measures are urgently needed.