Evidence of natural infection of Orientia tsutsugamushi in vectors and animal hosts � Risk of scrub typhus transmission to humans in Puducherry, South India

Background: Scrub typhus infection is endemic in India and reported to be the major cause for acute encephalitis syndrome (AES) in humans. Periodic occurrence of scrub typhus cases and presence of pathogen in rodents were also reported in areas with human cases of scrub typhus in Puducherry. Objectives: This study was carried out to screen Orientia tsutsugamushi in rodent/shrew reservoirs and vectors in villages of Puducherry with no reports of human scrub typhus cases. Methods: This study was conducted during October 2017 to January 2018 in ten randomly selected villages in Puducherry. Rodents/shrews in the peridomestic area were trapped using Sherman traps. Screening of O. tsutsugamushi in rodents/shrews and mite vectors was done by polymerase chain reaction (PCR). Weil-Felix test was done to screen antibodies against O. tsutsugamushi in rodent serum samples. Results: Among the 54 rodents trapped, Suncus murinus was the major small animal and Leptotrombidium deliense was the major mite species retrieved. PCR screening revealed pathogen positivity in 8 rodent blood and 3 pooled mite samples. Phylogenetic analysis has shown that Kato was the circulating serotype of O. tsutsugamushi. None of the rodent serum samples was tested positive for antibodies against O. tsutsugamushi by Weil-Felix test. Conclusions: The presence of pathogen in both vectors and reservoir animal hosts imposes a risk for scrub typhus transmission to the inhabitants; hence, initiation of vector control measures before the start of winter is recommended in the study area. It is also recommended to screen scrub typhus in patients with undifferentiated acute febrile illness and AES.

Isolation and Genetic Characterization of Orientia tsutsugamushi from Scrub Typhus Patients in Gyeongsangnam-do, Korea

Orientia tsutsugamushi (O. tsutsugamushi), which is endemic to an Asia-Pacific region, has increased its incidence and caused annually around 10 thousand patients infected with scrub typhus in Korea in the past several years. In the present study, we isolated 44 O. tsutsugamushi from the patients with febrile illness accompanied with or without an eschar in Gyeongsangnam-do, Korea. These isolates were characterized by genetic analysis of the major outer membrane protein, the 56-kDa type-specific antigen (tsa56), which is unique to O. tsutsugamushi. Two types of sequences of tsa56, designated by JJ1 and JJ2, were determined from 37 and 7 isolates of the 44 isolates, respectively. JJ1 and JJ2 showed 74.7~90.8% identity in nucleotide sequence and 66.1~90.5% identity in amino acid sequence with 33 reference strains except for Boryong and Kuroki. JJ1 and JJ2 had 100 and 99.9% nucleotide identity to Boryong strain, and 99.9 and 99.8% to Kuroki, which has been known to be similar to Boryong, respectively. In addition, they showed 77.9~ 81.4% nucleotide identity with the cluster of Gilliam-related genotypes, whereas they showed higher nucleotide identity (89.6~90.8%) with the cluster of Karp-related genotypes. To our knowledge, this is the first report to isolate O. tsutsugamushi and characterize their genotype as the Boryong in Jinju and West Gyeongsangnam-do, Korea, even though it has been reported that the Boryong was the predominant genotype in isolates from chiggers, domestic rodents, and patients in the southern part of Korea. Furthermore, our isolates could be useful source to study on the pathophysiology and epidemiology of scrub typhus in Korea.

Recent outbreak of scrub typhus in North Western part of India.

Background: Scrub typhus usually affects previously healthy active persons and if undiagnosed or diagnosed late, may prove to be life‑threatening. Diagnosis of scrub typhus should be largely based on a high index of suspicion and careful clinical, laboratory and epidemiological evaluation. Objective: To describe the diverse clinical and laboratory manifestations of scrub typhus diagnosed in Mahatma Gandhi Medical College and Hospital, Jaipur. Materials and Methods: All cases of febrile illness diagnosed as scrub typhus over a period of 3 months were analysed. Diagnosis was based on ELISA test for antibody detection against 56 kDa antigen. Results: Forty‑two cases of scrub typhus were seen over a period of 3 months (October, 2012‑December, 2012). Common symptoms were high grade fever of 4‑30 days duration, cough, haemoptysis and breathlessness. Eschar was not seen even in a single patient. Liver enzymes were elevated in nearly all cases (95.9%). Multiple organ dysfunction syndrome (MODS) was present in 16.66% of our patients (7 out of 42). Hypotension (6 patients, 14.2%), renal impairment (9 out of 15 patients, 60%), acute respiratory distress syndrome (4 patients, 9.52%) and meningitis (4 patients, 9.52%) were some of the important complications. There was a dramatic response to doxycycline in nearly all the patients, but initially when the disease was not diagnosed, seven patients had died. Conclusion: Scrub typhus has emerged as an important cause of febrile illness in Jaipur. Empirical treatment with doxycycline is justified in endemic areas.

Phylogenetic Analysis of the 56-kDa Type-Specific Protein Genes of Orientia tsutsugamushi in Central Korea

There are several antigenic variants of Orientia tsutsugamushi. The 56-kDa type-specific antigen (TSA) is responsible for the antigenic variation. Nucleotide sequences of the 56-kDa TSA obtained from 44 eschar samples of Korean scrub typhus patients and from 40 representative strains retrieved from the GenBank database were analyzed phylogenetically. Clinical patient data were assessed based on the genotyping results. Of the 44 nucleotide sequences, 32 (72.7%) clustered with the Boryong genotype, which is the major genotype in Korea. Eleven nucleotide sequences (25%) clustered with the Kawasaki genotype, not identified in Korea until 2010. One nucleotide sequence was consistent with the Karp genotype. The clinical course of the patients infected with each genotype showed no differences. Diagnostic performance of the immunofluorescence assay (IFA) using the 56-kDa TSA from Gilliam, Karp and Boryong as test antigens were not different for the Boryong and Kawasaki genotypes. Although Boryong is still the predominant genotype, the results suggest that Kawasaki genotype is quite prevalent in Chungbuk province of Korea.

Phylogenetic Analysis of the 56-kDa Type-Specific Protein Genes of Orientia tsutsugamushi in Central Korea

There are several antigenic variants of Orientia tsutsugamushi. The 56-kDa type-specific antigen (TSA) is responsible for the antigenic variation. Nucleotide sequences of the 56-kDa TSA obtained from 44 eschar samples of Korean scrub typhus patients and from 40 representative strains retrieved from the GenBank database were analyzed phylogenetically. Clinical patient data were assessed based on the genotyping results. Of the 44 nucleotide sequences, 32 (72.7%) clustered with the Boryong genotype, which is the major genotype in Korea. Eleven nucleotide sequences (25%) clustered with the Kawasaki genotype, not identified in Korea until 2010. One nucleotide sequence was consistent with the Karp genotype. The clinical course of the patients infected with each genotype showed no differences. Diagnostic performance of the immunofluorescence assay (IFA) using the 56-kDa TSA from Gilliam, Karp and Boryong as test antigens were not different for the Boryong and Kawasaki genotypes. Although Boryong is still the predominant genotype, the results suggest that Kawasaki genotype is quite prevalent in Chungbuk province of Korea.

Nested polymerase chain reaction on blood clots for gene encoding 56 kDa antigen and serology for the diagnosis of scrub typhus.

Purpose : Scrub typhus is a zoonotic illness endemic in the Asia-Pacific region. Early diagnosis and appropriate management contribute significantly to preventing adverse outcomes including mortality. Serology is widely used for diagnosing scrub typhus. Recent reports suggest that polymerase chain reaction (PCR) could be a rapid and reliable alternative. This study assessed the utility of these tests for scrub typhus diagnosis. Materials and Methods : Nested PCR to detect the 56 kDa antigen gene of O. tsutsugamushi was performed on blood clots from 87 individuals with clinically suspected scrub typhus. Weil-Felix test and scrub typhus IgM ELISA were performed on serum samples from the same patients. As a gold standard reference test was not available, latent class analysis (LCA) was used to assess the performance of the three tests. Results : The LCA analysis showed the sensitivity of Weil-Felix test, IgM ELISA and PCR to be 59%, 100% and 58% respectively. The specificity of ELISA was only 73%, whereas those of the Weil-Felix test and PCR were 94% and 100% respectively. Conclusion : Nested PCR using blood clots while specific, lacked sensitivity as compared to IgM ELISA. In resource-poor settings Weil-Felix test still remains valuable despite its moderate sensitivity.

The Attachment of Detergent-Extracted Outer Membrane Proteins of Orientia tsutsugamushi to the Host Cell Surface

Orientia tsutsugamushi, a causative agent of scrub typhus, is an obligate intracellular parasite. The mechanisms by which O. tsutsugamushi invade host cells are unknown. Given the importance of surface-exposed proteins in the pathogenesis of microbial pathogens, outer membrane proteins (OMP) of O. tsutsugamushi were extracted with detergents and their cellular binding was studied. Outer membrane fraction of O. tsutsugamushi was enriched by a sodium-lauryl sarcosinate (Sarkosyl) treatment of total membranes. Outer membrane proteins were extracted by the treatment with sodium dodecyl sulfate (SDS) and Sarkosyl. The resulting soluble fractions were examined for their cellular binding by the immunofluorescence microscopy. A fifty six kilodalton protein was found to bind to fixed ECV304 cells only when the outer membrane preparation was not treated by DTT or heat. These results suggest that the conformation the 56-kDa OMP is important for the attachment to the host cell surfaces.