Gilliam Strain Research Articles

Utility of next-generation sequencing for the etiological diagnosis of Orientia tsutsugamushi infection

BackgroundScrub typhus, an acute febrile disease caused by Orientia tsutsugamushi, is transmitted to humans through infected chigger mites. We present a case of scrub typhus in a previously healthy man from Shandong Province diagnosed using next-generation sequencing (NGS) and PCR and review recent literature on NGS for scrub typhus diagnosis. MethodsNGS was utilized for testing whole blood collected on admission. Confirmatory testing was done by detecting IgM and IgG antibodies to Orientia in acute and convalescent sera by ELISA. Orientia 47-kDa protein gene TaqMan and standard PCR of the 56-kDa protein gene and Sanger sequencing were performed on eschar scab DNA. ResultsThe NGS diagnosis was confirmed by 47-kDa protein gene TaqMan and sequencing of a fragment of the O. tsutsugamushi 56-kDa protein gene from the eschar scab. Analysis of this sequence and the NGS data indicated O. tsutsugamushi strain Cheeloo2020 is a novel genotype. Mapping of the NGS data against the O. tsutsugamushi Gilliam strain genome sequence identified 304 reads with high similarity. ConclusionsNGS is not only useful for multiplex diagnosis of scrub typhus, but also provides insight into the genetic diversity of O. tsutsugamushi. The common failure to submit sequences to databases makes it difficult to determine the minimal quantity and quality of NGS data being used for the positive identification of Orientia DNA in clinical specimens.

Pathological Responses in Asian House Shrews (Suncus murinus) to the Naturally Acquired Orientia tsutsugamushi Infection.

Scrub typhus is a re-emerging disease caused by Orientia tsutsugamushi, transmitted by mites belonging to the family Trombiculidae. Humans and rodents acquire the infection by the bite of larval mites/chiggers. Suncus murinus, the Asian house shrew, has been reported to harbor the vector mites and has been naturally infected with O. tsutsugamushi. The present study aimed to localize and record O. tsutsugamushi in the tissues and the host response in shrews naturally infected with O. tsutsugamushi. Sheehan's modified May-Grunwald Giemsa staining was carried out in 365 tissues from 87 animals, and rickettsiae were documented in 87 tissues from 20 animals. Immunohistochemical (IHC) staining, using polyclonal antibodies raised against selected epitopes of the 56-kDa antigen, was carried out, and 81/87 tissue sections were tested positive for O. tsutsugamushi. By IHC, in addition to the endothelium, the pathogen was also demonstrated by IHC in cardiomyocytes, the bronchiolar epithelium, stroma of the lungs, hepatocytes, the bile duct epithelium, the epithelium and goblet cells of intestine, the tubular epithelium of the kidney, and splenic macrophages. Furthermore, the pathogen was confirmed by real-time PCR using blood (n = 20) and tissues (n = 81) of the IHC-positive animals. None of the blood samples and only 22 out of 81 IHC-positive tissues were tested positive by PCR. By nucleotide sequencing of the 56-kDa gene, Gilliam and Karp strains were found circulating among these animals. Although these bacterial strains are highly virulent and cause a wide range of pathological alterations, hence exploring their adaptive mechanisms of survival in shrews will be of significance. Given that the pathogen localizes in various organs following a transient bacteremia, we recommend the inclusion of tissues from the heart, lung, intestine, and kidney of reservoir animals, in addition to blood samples, for future molecular surveillance of scrub typhus.

Differential cellular immune responses against Orientia tsutsugamushi Karp and Gilliam strains following acute infection in mice.

Scrub typhus is a leading cause of febrile illness in endemic countries due to infection with Orientia tsutsugamushi (Ot), a seriously understudied intracellular bacterium. Pulmonary involvement associated with vascular parasitism in patients is common and can develop into life threatening interstitial pneumonia. The diverse antigenicity of Ot genotypes and inter-strain differences in genome content are connected to varied virulence and clinical outcomes; however, detailed studies of strain-related pulmonary immune responses in human patients or small animal models of infection are lacking. In this study, we have used two clinically prevalent bacterial strains (Karp and Gilliam) to reveal cellular immune responses in inflamed lungs and potential biomarkers of disease severity. The results demonstrate that outbred CD-1 mice are highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice were susceptible to Karp, but resistant to Gilliam (with self-limiting infection), corresponding to their tissue bacterial burdens and lung pathological changes. Multicolor flow cytometric analyses of perfused B6 mouse lungs revealed robust and sustained influx and activation of innate immune cells (macrophages, neutrophils, and NK cells), followed by CD4+ and CD8+ T cells, during Karp infection, but such responses were greatly attenuated during Gilliam infection. The robust cellular responses in Karp-infected B6 mice positively correlated with significantly early and high levels of serum cytokine/chemokine protein levels (CXCL1, CCL2/3/5, and G-CSF), as well as pulmonary gene expression (Cxcl1/2, Ccl2/3/4, and Ifng). In vitro infection of B6 mouse-derived primary macrophages also revealed bacterial strain-dependent immune gene expression profiles. This study provided the lines of evidence that highlighted differential tissue cellular responses against Karp vs. Gilliam infection, offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis vs. infection control.

Molecular diagnosis and genotyping of Orientia tsutsugamushi in Maesot and Chiangrai, Thailand.

Scrub typhus is a neglected tropical disease with an estimated 1 million cases annually. The Asia-Pacific region is an endemic area for scrub typhus, especially in Thailand. Between June 2018 and December 2019, 31 patients with acute undifferentiated febrile illness (AUFI) were recruited for clinical trials and tested positive by a scrub typhus IgM RDT. Of the 17 buffy coat patient samples tested by 47kDa real-time PCR and 56kDa type-specific antigen (TSA) nested PCR, 94% (16/17) were positive, and of the 11 patients that presented with eschar lesions, 100% (11/11) of the eschar samples were confirmed positive. Genetic analysis of the 560 bp partial 56-kDa TSA gene demonstrated that most Orientia tsutsugamushi (Ot) infections were with Karp, Gilliam, Taiwan, P23, and CM606-like strains. This is the second occasion that the CM606-like and P23-like strains were reported in northern Thailand (first reported in 2011 and 2013, respectively). This study demonstrates that 1) the eschar remains the most reliable biological sample for PCR diagnosis of scrub typhus and 2) Northwestern Thailand has significant diversity of Ot strains, which underlines the requirement for ongoing surveillance to increase our understanding of Ot diversity to ensure accurate diagnostics and treatment.

Orientia tsutsugamushi Infection in Wild Small Mammals in Western Yunnan Province, China

Small mammals can transmit and serve as a reservoir for Orientia tsutsugamushi (Ot) in nature by carrying infected mites. In Yunnan, one of China's main foci of scrub typhus, etiological evidence and genetic diversity for Ot is limited. A total of 2538 small mammals were captured seasonally from 2015 to 2016 in the three counties of Yunnan, and the spleen or liver tissue was examined for Ot based on 56 kDa nPCR. The overall prevalence of Ot was 1.77%, ranging from 0.26 to 9.09% across different species. The Gilliam strain was found in 35.6% (16/45) of the wild small mammals, followed by the Karp 11.1% (5/45) and TA763 (1/45) strains, the last of which was discovered in western Yunnan for the first time. In Lianghe, Ot infection rates in wild small mammals were higher than in the other two counties. The infection rates of Eothenomys miletus with Ot were highest in the three dominant species. Ot infection rates in wild small mammals were higher in Lianghe (1200-1400 m) and Yulong (2800-3000 m). These findings could provide research clues for further confirmation of scrub typhus foci in western Yunnan or other similar natural environments.

Evaluation of an in-house PCR for diagnosing scrub typhus along with a preliminary study of genotypic characterization of Orientia tsutshugamushi circulating in Chhattisgarh: A prospective clinico-microbiological study

PurposeScrub typhus, caused by Orientia tsutsugamushi (O. tsutsugamushi) present nonspecific clinical features during manifestation of acute undifferentiated febrile illness (AUFI) to render its early diagnosis difficult. Accordingly, this study was undertaken to assess an in-house groEL PCR versus IgM ELISA for the diagnosis of scrub typhus and to genotypically characterise the randomly selected scrub typhus positive cases. MethodsBlood samples, collected from two hundred twenty one (221) AUFI cases were subjected to groEL PCR and IgM ELISA for diagnosis of scrub typhus. Eleven randomly selected PCR positive cases were processed for DNA sequencing to determine the genetic diversity of O. tsutsugamushi in Chhattisgarh. ResultsScrub typhus prevalence of 35.2% were detected among AUFI cases using both in-house groEL PCR and IgM ELISA. PCR alone showed sensitivity, specificity, positive and negative predictive values of 66.6% (CI: 55.08–76.94), 100% (CI: 90 to 100),100% (CI: 93.15 to 100) and 57.37% (CI: 44.05 to 69.96) while for IgM ELISA, these parameters were 62.8% (CI: 51.13–73.50), 100% (CI: 90 to 100), 100% (CI: 92.75 to 100) and 54.68% (CI: 41.75 to 67.18) respectively. PCR and ELISA could detect scrub typhus in 37.2% and 33.3% cases, when tested alone. groEL PCR detected the O. tsutsugamushi throughout the course of infection. Phylogenetic analysis depicted 5 of 11 positive cases belonged to Kuroki, Japan strain of O. tsutsugamushi, followed by Gilliam and Karp strain in 4 and 2 cases respectively. ConclusionScrub typhus should be considered in differential diagnosis of AUFI. groEL PCR may aid on to IgM ELISA test for optimum laboratory diagnosis of scrub typhus by its implementation especially in seronegative cases. Predominance of Kuroki-like strain followed by Gillian and Karp strains of O. tsutsugamushi in Chhattisgarh confirm variable geographical distribution of O. tsutsugamushi and provide the baseline epidemiological data which will eventually be used to help the researchers for developing better diagnostic tests and vaccine covering the predominant genotypes.

A time-course comparative clinical and immune response evaluation study between the human pathogenic Orientia tsutsugamushi strains: Karp and Gilliam in a rhesus macaque (Macaca mulatta) model.

BackgroundScrub typhus is a vector-borne febrile illness caused by Orientia tsutsugamushi transmitted by the bite of Trombiculid mites. O. tsutsugamushi has a high genetic diversity and is increasingly recognized to have a wider global distribution than previously assumed.Methodology/principle findingsWe evaluated the clinical outcomes and host immune responses of the two most relevant human pathogenic strains of O. tsutsugamushi; Karp (n = 4) and Gilliam (n = 4) in a time-course study over 80 days post infection (dpi) in a standardized scrub typhus non-human primate rhesus macaque model. We observed distinct features in clinical progression and immune response between the two strains; Gilliam-infected macaques developed more pronounced systemic infection characterized by an earlier onset of bacteremia, lymph node enlargement, eschar lesions and higher inflammatory markers during the acute phase of infection, when compared to the Karp strain. C-reactive protein (CRP) plasma levels, interferon gamma (IFN-γ, interleukin-1 receptor antagonist (IL-1ra), IL-15 serum concentrations, CRP/IL10- and IFN-γ/IL-10 ratios correlated positively with bacterial load in blood, implying activation of the innate immune response and preferential development of a T helper-type 1 immune response. The O. tsutsugamushi-specific immune memory responses in cells isolated from skin and lymph nodes at 80 dpi were more markedly elevated in the Gilliam-infected macaques than in the Karp-infected group. The comparative cytokine response dynamics of both strains revealed significant up-regulation of IFN-γ, tumor necrosis factor (TNF), IL-15, IL-6, IL-18, regulatory IL-1ra, IL-10, IL-8 and granulocyte-colony-stimulating factor (G-CSF). These data suggest that the clinical outcomes and host immune responses to scrub typhus could be associated with counter balancing effects of pro- and anti-inflammatory cytokine-mediated responses.Currently, no data on characterized time-course comparisons of O. tsutsugamushi strains regarding measures of disease severity and immune response is available. Our study provides evidence for the strain-specificity of host responses in scrub typhus, which supports our understanding of processes at the initial inoculation site (eschar), systemic disease progression, protective and/or pathogenic host immune mechanisms and cellular immune memory function.Conclusions/significanceThis study characterised an improved intradermal rhesus macaque challenge model for scrub typhus, whereby the Gilliam strain infection associated with higher disease severity in the rhesus macaque model than the previous Karp strain infection. Difficulties associated with inoculum quantitation for obligate-intracellular bacteria were overcome by using functional inoculum titrations in outbred mice. The Gilliam-based rhesus macaque model provides improved endpoint measurements and contributes towards the identification of correlates of protection for future vaccine development.

A Murine Model of Waning Scrub Typhus Cross-Protection between Heterologous Strains of Orientia tsutsugamushi.

Orientia tsutsugamushi, the etiologic agent of the life-threatening febrile disease scrub typhus, is an obligately intracellular small coccobacillary bacterium belonging to the family Rickettsiaceae and is transmitted by the parasitic larval stage of trombiculid mites. Progress towards a vaccine for protection against scrub typhus has been impeded by characteristics of the pathogen and the infection. There are numerous strains of O. tsutsugamushi in the Asia-Pacific region with geographical overlap. In human cases immunity has been described as poor against heterologous strains of the pathogen, as well as short-lived against the homologous strain, with a mean antibody reversion rate of less than one year. Animal models of cross-protection as well as of deterioration of this cross-protection are needed to enhance understanding of transient immunity to scrub typhus. To build upon current understanding of this ineffective protection we sought to utilize our recently developed models, sublethal intradermal infection followed by challenge via ordinarily lethal hematogenous dissemination. Mice that were initially infected sublethally with O. tsutsugamushi Gilliam strain and were challenged with an ordinarily lethal dose of heterologous Karp strain were protected from death by a robust immune response at one month after the primary infection as evidenced by an abundance of mononuclear cellular infiltrates in target organs such as lung, liver, and kidney; maintenance of body weight; and low bacterial loads in the organs. Waning protection from lethal Karp strain challenge indicated by weight loss mirroring that observed in naïve mice was observed as early as 9 months after primary Gilliam strain infection, and higher bacterial loads, severe disease, and eventual death in some mice was observed after challenge with Karp strain at 14 months post-initial heterologous infection.

Molecular Detection and Genetic Characterization of Orientia tsutsugamushi from Hospitalized Acute Encephalitis Syndrome Cases During Two Consecutive Outbreaks in Eastern Uttar Pradesh, India.

Background: Seasonal outbreaks of acute encephalitis syndrome (AES) have been reported especially in the pediatric population with a high case fatality rate in Eastern Uttar Pradesh, India. Orientia tsutsugamushi (OT) is a causative agent of scrub typhus that has been recently identified as a major cause of AES. However, the specific genotypes of OT responsible for AES cases of this region are not known. Therefore, the present study was undertaken to understand the molecular epidemiology of OT prevailing in the AES endemic Eastern Uttar Pradesh region of India. Methods: The study was conducted on 2529 hospitalized AES cases from August 2016 to December 2017. The presence of antibodies against OT from cerebrospinal fluid (CSF) and serum samples were tested using OT IgM enzyme-linked immunosorbent assay (ELISA), whereas OT DNA was tested from whole blood and CSF specimens targeting the partial gene of 56 kDa using nested PCR. Phylogenetic analysis was conducted with sequences (n = 241) generated in this study. Findings: Among the studied AES cases, 50% were found positive for antibodies against OT, whereas 37% of cases were positive for OT DNA. The genetic analysis study revealed that Gilliam (93.8%) is the prevailing genotype of OT followed by Karp (6.16%) genotype in AES cases. Furthermore, the Gilliam strains of this study showed they were >99% identical to earlier reported Gilliam strains from AES cases. Conclusion: We observed the presence of two main OT genotypes in AES cases, among which the majority of OT genotypes fall under the Gilliam clade. The understanding of predominant genotype will be beneficial for its future implications in vaccine development strategies and the development of rapid diagnostic tests.

A case of availability bias for COVID-19 causing scrub typhus diagnostic errors.

Although patients with scrub typhus develop a maculopapular rash all over the body, patients with COVID-19may also show a similar rash. At the first visit, we did not fully inspect his trunk and extremities under his clothes. Although scrub typhus and COVID-19have some similar symptoms, an eschar is a characteristic symptom of the former, and careful inspection is important to distinguish between the diseases.

Epidemiology of tsutsugamushi disease and its relationship with meteorological factors in Xiamen city, China.

Tsutsugamushi disease (TD) is an acute infectious disease caused by Orientia tsutsugamushi. This study aimed to analyze the epidemiological features of TD, investigate chigger mites and their hosts, and investigate the meteorological factors affecting TD incidence and the host of O. tsutsugamushi in Xiamen city, China. Data on reported TD cases were collected from 2006 to 2018. Spearman’s correlation test were used for identifying the relationship between meteorological factors and TD incidence and whether meteorological factors affect the host of O. tsutsugamushi. The incidence of reported TD increased gradually from 2006, reached a peak of 4.59 per 100,000 persons in 2014, and then decreased gradually. The TD incidence was seasonal, with epidemic periods occurred mainly in summer and autumn. Patients aged 40–60 years had the highest proportion of cases, accounting for 44.44% of the total cases. Farmers had the largest number of cases among all occupational groups. Rattus Norvegicus was the most common host, accounting for the largest proportion of rats (73.00%), and the highest rat density was observed in March and October every year. There were significant positive correlations between the number of reported cases and average temperature, sunshine duration, and rainfall as well as between rat density and average temperature. On phylogenetic analysis, 7 sequences of hosts and human TD cases obtained from health records demonstrated the highest similarities to the Kato, Karp, and Gilliam strains. No correlations were observed between rat density, and sunshine duration and rainfall. The transmission of TD in Xiamen city, China, was seasonal, and its incidence was affected by several meteorological factors including average temperature, sunshine duration, and rainfall. However, the host of O. tsutsugamushi was only affected by average temperature.

Outer Membrane Protein A Conservation among Orientia tsutsugamushi Isolates Suggests Its Potential as a Protective Antigen and Diagnostic Target.

Scrub typhus threatens one billion people in the Asia-Pacific area and cases have emerged outside this region. It is caused by infection with any of the multitude of strains of the bacterium Orientia tsutsugamushi. A vaccine that affords heterologous protection and a commercially-available molecular diagnostic assay are lacking. Herein, we determined that the nucleotide and translated amino acid sequences of outer membrane protein A (OmpA) are highly conserved among 51 O. tsutsugamushi isolates. Molecular modeling revealed the predicted tertiary structure of O. tsutsugamushi OmpA to be very similar to that of the phylogenetically-related pathogen, Anaplasma phagocytophilum, including the location of a helix that contains residues functionally essential for A. phagocytophilum infection. PCR primers were developed that amplified ompA DNA from all O. tsutsugamushi strains, but not from negative control bacteria. Using these primers in quantitative PCR enabled sensitive detection and quantitation of O. tsutsugamushi ompA DNA from organs and blood of mice that had been experimentally infected with the Karp or Gilliam strains. The high degree of OmpA conservation among O. tsutsugamushi strains evidences its potential to serve as a molecular diagnostic target and justifies its consideration as a candidate for developing a broadly-protective scrub typhus vaccine.

Rickettsial Illnesses as Important Causes of Febrile Illness in Chittagong, Bangladesh.

We conducted a yearlong prospective study of febrile patients admitted to a tertiary referral hospital in Chittagong, Bangladesh, to assess the proportion of patients with rickettsial illnesses and identify the causative pathogens, strain genotypes, and associated seasonality patterns. We diagnosed scrub typhus in 16.8% (70/416) and murine typhus in 5.8% (24/416) of patients; 2 patients had infections attributable to undifferentiated Rickettsia spp. and 2 had DNA sequence-confirmed R. felis infection. Orientia tsutsugamushi genotypes included Karp, Gilliam, Kato, and TA763-like strains, with a prominence of Karp-like strains. Scrub typhus admissions peaked in a biphasic pattern before and after the rainy season, whereas murine typhus more frequently occurred before the rainy season. Death occurred in 4% (18/416) of cases; case-fatality rates were 4% each for scrub typhus (3/70) and murine typhus (1/28). Overall, 23.1% (96/416) of patients had evidence of treatable rickettsial illnesses, providing important evidence toward optimizing empirical treatment strategies.

Orientia tsutsugamushi infection in rodents in Anhui Province of China

We conducted an investigation of Orientia tsutsugamushi infection among rodents in non-endemic areas in Anhui Province. Fifty-six (56) rodents including 44 Apodemus agrarius and 12 Niviventer niviventer were trapped and captured in autumn in the fields of three counties in Anhui Province. DNA samples were amplified and sequenced for the 56kDa protein gene of Orientia tsutsugamushi. The overall infection rate in the rodents was 23.2%(13/56). The rate of detection of O. tsutsugamushi in Apodemus agrarius and Niviventer niviventer were 27.3% and 8.3% respectively. Moreover, we identified two genotypes (Karp and Gilliam strains) of Orientia tsutsugamushi in rodents. Our study demonstrated that Apodemus agrarius is the main host for O. tsutsugamushi pathogen and this is the first report of Karp and Gilliam strains in these non-endemic areas in Anhui Province.

Murine models of scrub typhus associated with host control of Orientia tsutsugamushi infection.

BackgroundScrub typhus, a febrile illness of substantial incidence and mortality, is caused by infection with the obligately intracellular bacterium Orientia tsutsugamushi. It is estimated that there are more than one million cases annually transmitted by the parasitic larval stage of trombiculid mites in the Asia-Pacific region. The antigenic and genetic diversity of the multiple strains of O. tsutsugamushi hinders the advancement of laboratory diagnosis, development of long-lasting vaccine-induced protection, and interpretation of clinical infection. Despite the life-threatening severity of the illness in hundreds of thousands of cases annually, 85–93% of patients survive, often without anti-rickettsial treatment. To more completely understand the disease caused by Orientia infection, animal models which closely correlate with the clinical manifestations, target cells, organ involvement, and histopathologic lesions of human cases of scrub typhus should be employed. Previously, our laboratory has extensively characterized two relevant C57BL/6 mouse models using O. tsutsugamushi Karp strain: a route-specific intradermal model of infection and persistence and a hematogenously disseminated dose-dependent lethal model.Principal findingsTo complement the lethal model, here we illustrate a sublethal model in the same mouse strain using the O. tsutsugamushi Gilliam strain, which resulted in dose-dependent severity of illness, weight loss, and systemic dissemination to endothelial cells of the microcirculation and mononuclear phagocytic cells. Histopathologic lesions included expansion of the pulmonary interstitium by inflammatory cell infiltrates and multifocal hepatic lesions with mononuclear cellular infiltrates, renal interstitial lymphohistiocytic inflammation, mild meningoencephalitis, and characteristic typhus nodules.SignificanceThese models parallel characteristics of human cases of scrub typhus, and will be used in concert to understand differences in severity which lead to lethality or host control of the infection and to address the explanation for short duration of heterologous immunity in Orientia infection.

Genotypic characterization of Orientia tsutsugamushi from patients in two geographical locations in Sri Lanka

BackgroundTo date more than 20 antigenically distinct strains of Orientia tsutsugamushi (OT) reported within the tsutsugamushi triangle that cause an undifferentiated acute febrile illness in humans. Genotypic characterization of OT in different geographic regions or within the same country, is important in order to establish effective diagnostics, clinical management and to develop effective vaccines. Genetic and antigenic characterization of OT causing human disease in OT-endemic regions is not known for Sri Lanka.MethodsAdult patients and children who were admitted with an acute febrile illness and presumed to having acute scrub typhus based on presence of an eschar and other supporting clinical features were recruited. Eschar biopsies and buffy coat samples collected from patients who were confirmed having OT by IFA were further studied by real time PCR (Orientia 47 kD) and nested PCR (Orientia 56 kD) amplification. DNA sequences were obtained for 56 kD gene amplicons and phylogenetic comparisons were analyzed using currently available data in GenBank [Neucleotide substitution per 100 residues, 1000 Bootstrap Trials].ResultsTwenty eschar biopsies (Location1,19, Location 2,1) and eight buffy coat samples (Location1,6, Location2,2) examined by real time PCR revealed Orientia amplicons in 16 samples. DNA sequences were obtained for the 56 kD gene amplicons in 12 eschars and 4 buffy coat samples. The genotypes of the Location1 samples revealed that, 7 exhibiting close homology with JP1 [distantly related to UT177 Thai (Karp related)], five had close homology with Kato strain, two had close homology with JGv and JG AF [Distantly related to Kawasaki M63383] and one had close homology with Gilliam strain. The Location 2 strain was closely related to Kuroki-Boryong L04956, the genotype which is distributed in far eastern Asia. Similar to other patients in the cohort this patient also had never travelled out of Sri Lanka.ConclusionsWe observed all three main OT genotypes in Sri Lanka, and the majority fell into Thai Karp related clade. These results demonstrate great antigenic diversity of OT in the studied areas of Sri Lanka.

Determination of Scrub typhus Suggests a New Epidemic Focus in the Anhui Province of China.

In 2007, 19 cases of a scrub typhus epidemic occurred within a week at a sports school in Mingguang County, Anhui Province, where no previous incidence of this mite borne disease had been reported. Sero-surveillance in 2009 indicated that 10 of the 100 school students possessed anti-Orientia tsutsugamushi antibodies. From 2009 to 2013, 60 small animals and 2250 mites were collected in the vicinity of the school. 5 of the Apodemus agrarius samples and 1 group of Leptotrombidium linhuaikongense tested positive via PCR for O. tsutsugamushi. Two strains of O. tsutsugamushi were identified by injecting Kun Ming (KM) mice peritoneally with the organs of either Apodemus agrarius or Leptotrombidium linhuaikongense. Apart from sharing 98% homology with the O. tsutsugamushi Yongworl strain, genes encoding the membrane protein from the two O. tsutsugamushi isolates shared >99% sequence homology with each other, reflecting the consistency of the pathogen in both the vector and the host. In addition, we also characterized a chronic scrub typhus infection in a local patient. The membrane protein gene fragment from the patient’s blood shared 99% homology with O. tsutsugamushi Gilliam strain, suggesting that more than one O. tsutsugamushi strain is present at this location.

Prevalence and Phylogenetic Analysis of Orientia tsutsugamushi in Small Mammals in Hanoi, Vietnam.

Rodents are important reservoirs of many human pathogens transmitted via arthropod vectors. Arthropod-borne bacteria belonging to the family Rickettsiaceae cause acute febrile diseases in humans worldwide, but the real burdens of rickettsial diseases appear to be underestimated in Hanoi, Vietnam, because differential diagnosis on the basis of clinical signs and symptoms is confounded by the presence of other tropical infectious diseases with similar signs and symptoms. To know the prevalence of bacteria of the family Rickettsiaceae among small mammals in Hanoi, 519 animals thriving in the public places were captured and examined for the presence of bacterial sequences using duplex PCR. Nucleotide sequences specific for Orientia tsutsugamushi were detected in seven samples (1.3%). Out of seven animals, two were captured in a market, whereas five were in hospitals. None of the captured small mammals tested positive for the genus Rickettsia. The nucleotide sequence analysis of the genes encoding the 47-kDa high-temperature requirement A (47-kDa HtrA) and 56-kDa type-specific antigen (TSA) showed that these seven isolates were indistinguishable from each other. O. tsutsugamushi isolated in this study was closely related phylogenetically to the Gilliam strain, which was originally isolated at the border of Assam and Burma, rather than to those isolated in the central to southern part of Vietnam. It should be emphasized that Vietnamese hospitals were heavily infested by small rodents and some of them harbored O. tsutsugamushi. Strict hygienic control should be implemented to mitigate the potential risk posed by O. tsutsugamushi in hospital settings.

Is scrub typhus a cause of febrile illness among paediatric population of delhi?

Sir, Scrub typhus is a febrile illness caused by Orientia tsutsugamushi and transmitted through the bite of larval forms of trombiculid mites [1]. The signs and symptoms of the disease are relatively nonspecific and in areas like Delhi where outbreaks have not been reported so far, low index of suspicion makes the diagnosis of this infection difficult. Furthermore, there is a paucity of information regarding the role of scrub typhus as a cause of fever in paediatric population. In the present study, l00 paediatric patients, 1-12years of age and presenting with febrile illness to the paediatric outpatient department or ward of a tertiary care health centre from February 2013 to March 2014 were included. Common causes of fever (malaria, dengue, enteric fever) were ruled out by history, clinical examination and appropriate laboratory investigations. In 30 patients who remained febrile and no definitive etiological diagnosis could be made, blood samples (5 ml) were collected and the serum separated and preserved at -70°C till further analysis. These samples were tested for antibodies to O. tsutsugamushi employing the immunochromatographic test (ICT), SD Bioline Tsutsugamushi Assay (Standard Diagnostics, Yongin, Korea) that employs the 56-kDa major surface protein antigens from representative O. tsutsugamushi including Gilliam, Karp, and Kato strains and detects IgG, IgM, and IgA antibodies to O. tsutsugamushi. The test was performed as per manufacturer's instructions and any positive samples further tested by an IgM ELISA (InBiOS International Inc. USA) to diagnose current infection. Among these 30 cases, ICT was positive in three (10%) samples. Further analysis of the reactive samples by IgM ELISA revealed all three samples to be IgM positive. Outbreaks of scrub typhus have been reported from various parts of India in the recent past [2,3]. Thus, there is a strong possibility of presence of this disease in this region also. All three serologically confirmed cases in the present study were residents of Delhi with no history of recent travel to a scrub typhus endemic region, a strong evidence in favour of existence of the disease in the city. The 10% prevalence of scrub typhus reported in our study is in contrast to another report from Delhi where the prevalence of scrub typhus was reported to be quiet low [4]. A study conducted in paediatric population of Sri Lanka, a region endemic for scrub typhus, has reported a prevalence of 33.33% [5]. Scrub typhus is grossly under-diagnosed due to its non-specific clinical presentation, low index of suspicion among clinicians and lack of diagnostic facilities. Since antimicrobials effective for scrub typhus are usually not included in empirical therapy for febrile illnesses, early diagnosis is imperative to reduce the mortality and complications associated with the disease. Our study highlights the existence of scrub typhus in Delhi, a region non-endemic for this infection and the need to consider it as a possible cause of paediatric febrile illnesses. To the best of our knowledge this is the first study undertaken to estimate seroprevalence of scrub typhus in paediatric population of Delhi.

Diagnosis of scrub typhus: introduction of the immunochromatographic test in Korea

Diagnosis of scrub typhus: introduction of the immunochromatographic test in Korea