Evaluation of an immunoglobulin M capture enzyme-linked immunosorbent assay for diagnosis of Orientia tsutsugamushi infection

To differentiate scrub typhus from other acute febrile diseases, a rapid and reliable serological diagnosis is important. We developed an immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) for diagnosis of recent Orientia tsutsugamushi infections in humans. The 56-kDa major outer membrane protein of O. tsutsugamushi is well known as the most immunodominant antigen in scrub typhus. The test is based on the use of the biotinylated recombinant 56-kDa protein of O. tsutsugamushi Boryong, Bor56, which was expressed as a fusion protein with a maltose-binding protein in Escherichia coli. In the test, the serum IgM antibodies were captured by anti-human IgM antibodies coated onto a microtiter plate. The captured IgM antibodies were revealed through sequential addition of biotinylated Bor56 antigen and peroxidase-conjugated streptavidin to the plate. The IgM capture ELISA was compared with the immunofluorescence antibody assay (IFA) by testing 176 serum samples from patients with diagnosed cases of rickettsial disease and patients with other acute febrile diseases. Of the 81 IgG IFA-positive samples, 78 tested positive (sensitivity, 96.3%) and all 31 IgM IFA-positive samples tested positive (sensitivity, 100%) by the IgM capture ELISA. The specificity of the IgM capture ELISA was 99%, and 1 of the 95 IFA-negative samples was positive in the assay. These results strongly suggest that IgM capture ELISA using the recombinant Bor56 antigen is a reliable and detailed method for the detection of early O. tsutsugamushi infection.     

Expression and Refolding of Truncated Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi and Its Use in Enzyme-Linked Immunosorbent Assays

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. The gene encoding this protein from Karp strain was cloned into the expression vector pET11a. The recombinant protein (r56) was expressed as a truncated nonfusion protein (amino acids 80 to 456 of the open reading frame) which formed an inclusion body when expressed in Escherichia coli BL21. Refolded r56 was purified and compared to purified whole-cell lysate of the Karp strain of O. tsutsugamushi by immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for reactivity with rabbit sera prepared against eight antigenic prototypes of O. tsutsugamushi as well as several other species of Rickettsiales and nonrickettsial antigens. Refolded r56 exhibited broad reactivity with the rabbit antisera against the Orientia prototypes, and the ELISA reactions with the r56 and Karp whole-cell lysate antigens correlated well (r = 0.81, n = 22, sensitivity compared to that of standard ELISA of 91%). Refolded r56 did not react with most antisera against other rickettsial species or control antigens (specificity = 92%, n = 13) using a positive cutoff value determined with eight uninfected rabbit sera. Refolded r56 was evaluated further by ELISA, using 128 sera obtained from patients with suspected scrub typhus from Korat, Thailand, and 74 serum specimens from healthy Thai soldiers. By using the indirect immunoperoxidase assay as the reference assay, the recombinant antigen exhibited a sensitivity and specificity of 93% or greater for detection of both IgG and IgM in the ELISA at 1:400 serum dilution. These results strongly suggest that purified r56 is a suitable candidate for replacing the density gradient-purified, rickettsia-derived, whole-cell antigen currently used in the commercial dipstick assay available in the United States.     

Accuracy of AccessBio Immunoglobulin M and Total Antibody Rapid Immunochromatographic Assays for the Diagnosis of Acute Scrub Typhus Infection

Using archived samples, we assessed the diagnostic capacity of a rapid immunochromatographic test (ICT) for the detection of Orientia tsutsugamushi IgM and total antibodies to aid with the diagnosis of acute scrub typhus infection in febrile patients in Laos. The sensitivity and the specificity of the ICT for the detection of IgM were 96.8% (121/125 samples; 95% confidence interval [CI], 92.1 to 99.1%) and 93.3% (98/105 samples; 95% CI, 86.7 to 97.3%), respectively. For the detection of total antibodies, the sensitivity was 97.6% (122/125 samples; 95% CI, 93.1 to 99.5%), but the specificity was much lower, at 71.4% (75/105 samples; 95% CI, 61.8 to 79.8%).Scrub typhus, caused by Orientia tsutsugamushi, is an important acute febrile illness in the Asia-Pacific region. As very few health facilities have accessible accurate diagnostic tests, the diagnosis of scrub fever must be based on clinical features. However, this is difficult because the clinical symptoms and signs are similar to those of many other febrile diseases, such as murine typhus, leptospirosis, and dengue virus infection. The diagnosis of scrub typhus infection has relied on the detection of O. tsutsugamushi antibodies during the acute phase of the disease, and the “gold standard” assay is the indirect immunofluorescence antibody assay (IFA) (9). The development of rapid, diagnostic tests by the use of immunochromatographic test (ICT) technologies has provided a mechanism for point-of-care serological testing. The objective of the study described here was to assess the diagnostic capacities of two commercial rapid ICTs for the detection of O. tsutsugamushi IgM and whole antibodies to aid with the diagnosis of acute scrub typhus infection by the use of stored, characterized sera collected from febrile patients in the tropical environment of the Lao People''s Democratic Republic (Laos) and Thailand where scrub typhus is endemic.     

Early Diagnosis of Scrub Typhus with a Rapid Flow Assay Using Recombinant Major Outer Membrane Protein Antigen (r56) of Orientia tsutsugamushi

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. We developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to O. tsutsugamushi. The RFA employs a truncated recombinant 56-kDa protein from the Karp strain as the antigen. The performance of the RFA was evaluated with a panel of 321 sera (serial bleedings of 85 individuals suspected of scrub typhus) which were collected in the Pescadore Islands, Taiwan, from 1976 to 1977. Among these 85 individuals, IgM tests were negative for 7 cases by both RFA and indirect fluorescence assay (IFA) using Karp whole-cell antigen. In 29 cases specific responses were detected by the RFA earlier than by IFA, 44 cases had the same detection time, and 5 cases were detected earlier by IFA than by RFA. For IgG responses, 4 individuals were negative with both methods, 37 cases exhibited earlier detection by RFA than IFA, 42 cases were detected at the same time, and 2 cases were detected earlier by IFA than by RFA. The sensitivities of RFA detection of antibody in sera from confirmed cases were 74 and 86% for IgM and IgG, respectively. When IgM and IgG results were combined, the sensitivity was 89%. A panel of 78 individual sera collected from patients with no evidence of scrub typhus was used to evaluate the specificity of the RFA. The specificities of the RFA were 99% for IgM and 97% for IgG. The sensitivities of IFA were 53 and 73% for IgM and IgG, respectively, and were 78% when the results of IgM and IgG were combined. The RFA test was significantly better than the IFA test for the early detection of antibody to scrub typhus in primary infections, while both tests were equally sensitive with reinfected individuals.     

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.     

Diagnostic Performance of Serological Tests to Detect Antibodies Against Acute Scrub Typhus Infection in Central India

Background: Differentiating scrub typhus from other acute febrile illness is difficult due to non specificity of clinical symptoms and relative absence of eschar in Indian population. The diagnosis thus relies mainly on laboratory tests. Antibody based serological tests are mainstay of scrub typhus diagnosis. Here, we evaluated the diagnostic performance of IgM ELISA, IgM IFA and ICT to detect antibodies against O. tsutsugamushi in acute serum of febrile patients. Methodology: The serum samples from 600 randomly selected patients suffering from acute undifferentiated fever were tested by all the three tests mentioned above. We used latent class analysis to generate unbiased results as all the tests for scrub typhus diagnosis are imperfect and none of them can be considered as reference standard. Results: We found that IgM ELISA with cutoff titer 0.5 OD has high diagnostic accuracy (sensitivity 99.9% and specificity 99.15) than IgM IFA (sensitivity 96.8% and specificity 99.7%) for scrub typhus diagnosis. ICT used in our study had very high specificity 100% but low sensitivity (38%) which would limit its use for acute serum samples. ICT being a screening or point of care test, has to be more sensitive while some compromise with specificity is affordable. Hence, optimal cutoff for ICT should be evaluated under different settings. Conclusion: IgM ELISA being simple and affordable could be an alternative diagnostic test to IgM IFA which is subjective and costly.     

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.     

Possibility of scrub typhus in fever of unknown origin (FUO) cases: An experience from Rajasthan

Purpose: Fever of unknown origin (FUO) has multiple causes. Scrub typhus is less known cause of FUO in India. The present study reports a recent epidemic of scrub typhus amongst cases of FUO from different areas of Rajasthan, India. There was high mortality in undiagnosed cases of FUO which lead to the diagnosis of scrub typhus. Objective: To study the possibility of scrub typhus as a causative factor in FUO cases by qualitative detection of IgM antibodies with ELISA. Materials and Methods: From September 2012 to December 2012, 271 serum samples of FUO cases were analysed for IgM antibodies to Orientia tsutsugamushi along with dengue, malaria, typhoid, tuberculosis and brucellosis. Results: Scrub typhus IgM antibodies by ELISA were detected in 133 (49.1%) patients. Scrub typhus positivity was significantly higher among female in comparison to males (P < 0.05). Maximum positivity of scrub typhus was found in females of 46-60 years age group. The laboratory parameters were abnormal in most of the patients as evident by thrombocytopenia (63%), deranged liver functions (56%) and renal functions (25%). Conclusion: The present study emphasises the importance of scrub typhus among cases of FUO especially after rainy season and during early cooler months. The study also highlights the significance of ELISA method for rapid and early reporting and ruling out scrub typhus in FUO cases.     

Scrub Typhus in Previously Unrecognized Areas of Endemicity in China

Scrub typhus, caused by Orientia tsutsugamushi, has emerged recently in areas of northern China where the disease had not been known to exist. We analyzed epidemiological, clinical, and laboratory data for 104 patients who were admitted to a hospital in Fuyang City between 26 September and 1 November 2008. We showed that the major clinical manifestations of the patients were fever (100%), headache (82%), myalgias (77%), eschar (67%), rash (52%), and unusual facial flushing (62%). Among the 104 patients, the sera of 98% contained IgM antibodies to O. tsutsugamushi detected by indirect immunofluorescence assays (IFA), and DNA of the O. tsutsugamushi 56-kDa gene was amplified by PCR from the blood of 36 patients. We conclude that 104 patients were infected with scrub typhus in Fuyang City, Anhui Province. Our study indicates that physicians need to consider the diagnosis of scrub typhus for febrile patients living in northern China, where scrub typhus had not been considered to exist in the past.Scrub typhus, also known as tsutsugamushi disease, is an acute, febrile infectious illness. It is a zoonosis that is widespread in southern Asia, in a triangle from northern Japan and far-eastern Russia in the north to northern Australia in the south and to Pakistan and Afghanistan in the west, as well as in the islands of the western Pacific and Indian Oceans. More than half (55%) of the world''s population lives in areas where scrub typhus is endemic. The causative agent, Orientia tsutsugamushi (formerly Rickettsia tsutsugamushi), is an obligately intracellular bacterium and is transmitted to humans by the bite of a larval trombiculid mite, popularly known as a chigger. Scrub typhus has been known in southern China for thousands of years (5). However, the disease has emerged in northern China only in the last 2 decades (3). Here we report an outbreak of scrub typhus cases in the fall of 2008 in northwestern Anhui Province in central China, where the disease had not been known to occur previously.     

Comparison of Nested Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction Targeting 47kDa Gene for the Diagnosis of Scrub Typhus

Introduction: Scrub typhus is a zoonotic infection caused by Orientia tsutsugamushi which is transmitted by Leptotrombidium mites. The disease manifests as a mild-to-severe illness with non-specific clinical symptoms. Rapid diagnosis and prompt treatment are essential for patient management. Both serological and molecular methods are used for the diagnosis of scrub typhus. The present study assessed the usefulness of detection of the gene encoding the 47kDa outer-membrane protein (OMP) for the laboratory diagnosis of scrub typhus. Materials and Methods: Nested polymerase chain reaction (nPCR) and real-time PCR targeting 47 kDa OMP antigen gene of O. tsutsugamushi were performed on ethylenediaminetetraacetic acid blood samples. Results: Six of the 103 (5.8%) patients showed the presence of 47kDa gene by nPCR. Seventy of 103 (67.9%) cases showed the presence of 47kDa gene by qPCR. Among the 70 positive cases, the majority of them were females (40/70, 57.1%). The highest number of positive cases was observed during October–February. Conclusion: Real-time PCR targeting O. tsutsugamushi-specific 47-kDa gene is more sensitive than nPCR and may be the assay of choice for the detection of the organism in patients with suspected scrub typhus.     

A rapid, sensitive and reliable diagnostic test for scrub typhus in China

Purpose: To evaluate the performances for detection of IgM and IgG antibodies to Orientia. tsutsugamushi (Ot) using a gold conjugate-based rapid diagnostic test (RDT). Materials and Methods: The RDT employing mixture recombinant 56-kDa proteins of O. tsutsugamushi and the mIFA assay was performed on 33 patients from Fujian and Yunnan province respectively and 94 positive sera (36 from Hainan province and 58 from Jiangsu province) from convalescent stages of the patients with scrub typhus respectively and 82 negative sera from healthy farmers from Anhui province and Beijing City respectively in 2009. A comparison of the RDT and mIFA assay was performed by using the χ² test and the P level of ≤0.05 was considered to be significant. Results: Among these 94 positive sera from convalescent stages of the illness and 82 sera from control farmers, the specificity of RDT was 100% for both IgM and IgG tests. In 33 cases with scrub typhus, 5 cases were positively detected earlier by RDT than by mIFA for the IgM test, and 2 cases were positive for the IgG test. The sensitivities of RDT were 93.9% and 90.9% for IgM and IgG, respectively. Considering IgM and IgG together, the sensitivity was 100%. The geometric mean titre (GMT) of IFA and the RDT assay in diluted sera from confirmed cases were 1:37 versus 1:113 respectively (P<0.001) for IgM test and 1:99 versus 1:279 respectively (P<0.016) for IgG. Conclusions: The RDT was more sensitive than the traditional IFA for the early diagnosis of scrub typhus and was particularly suitable for use in rural areas.     

Use of Eschar for the Molecular Diagnosis and Genotypic Characterisation of Orientia tsutsugamushi Causing Scrub Typhus

Scrub typhus caused by Orientia tsutsugamushi presents as an acute febrile illness with a varied presentation from mild illness to fatal disease in the absence of appropriate antibiotic treatment. Performing polymerase chain reaction (PCR) on eschar sample acts a rapid diagnostic tool in the early stage of scrub typhus when blood is negative. A total of eight patients from whom both whole blood and eschar samples were collected and tested by nested PCR targeting 56 kDa trichostatin A (TSA) gene to detect O. tsutsugamushi DNA. All (100%) eschar samples and three whole blood samples tested positive. Genetic analysis of the 56 kDa TSA gene sequences showed that the majority were related to Karp reference strains, while one clustered with Kawasaki strain. When present, eschar should be favoured as a diagnostic sample over whole blood in the early phase of infection.     

Epidemiological,clinical and genetic characterization of scrub typhus in patients presenting with acute febrile illness in New Delhi

PurposeScrub typhus (ST) is a zoonotic disease, caused by O. tsutsugamushi is a major cause of acute febrile illness (AFI) in India. There is a need to study the prevalence and risk factors in various regions of India.MethodsA study to estimate the prevalence and study the risk factors of ST in patients presenting with acute febrile illness (AFI) was performed. All patients underwent serology for IgM antibodies to Orientia tsutsugamushi (In Bios International Inc, Seattle, WA) as per the manufacturers’ protocol. Following this, Polymerase Chain reaction (PCR) (real time SYBR green based targeting groEL gene and conventional PCR targeting 56 ?kDa type specific antigen gene) was performed from stored serum samples.ResultsDuring the study period, 473 patients were admitted. Of these 56 (11.8%) patients were ST positive by IgM serology. The conventional PCR targeting 56 ?kDa type specific antigen gene of O. tsutsugamushi was positive in six patients while Ot groEL SYBR green based PCR) was positive in five. PCR was positive in patients who had demonstrated a higher OD value in ELISA. Conventional PCR positive amplicons were sent for Sanger sequencing and confirmed to be O. tsutsugamushi. The mean age of the patients was 49 ?± ?18.3 years and males constituted a higher number of patients (67.9%, n ?= ?38). The pathognomonic eschar was present in 7 (12.5%) patients. Phylogenetic analysis revealed that sequences clustered close to Kato-like Hualein-20 strain and Karp-like Linh DT strains. All patients were administered doxycycline in our study. Mortality was recorded in 8.9% of the patients.ConclusionsIn patients presenting with acute febrile illness, ST should be considered as a differential diagnosis, especially in post-monsoon season. Along with serology, serum can also be used as sample for PCR in an intracellular bacterium like O. tsutsugamushi.     

Mapping of antigenic determinant regions of the Bor56 protein of Orientia tsutsugamushi.

The 56-kDa protein (Bor56) of Orientia tsutsugamushi is an immunoprotective antigen and is the target molecule of neutralizing antibodies. This antigen is recognized by almost all of the serum antibodies produced by patients in the convalescence phase of scrub typhus. We expressed the Bor56 open reading frame in Escherichia coli and generated from it a series of deletion constructs as MalE fusion proteins. Antibody-binding domains were characterized by using patient sera, mouse monoclonal antibodies (MAbs), and Bor56-immunized-mouse sera. None of the antibodies bound to a fusion protein containing the carboxy-terminal 140 amino acids (aa) of the Bor56 protein, suggesting that the carboxy-terminal domain of Bor56 is not exposed on the surface of the molecule. Human immunoglobulin M (IgM) antibodies predominantly bound to antigenic domain I (AD I; amino acids [aa] 19 to 113) and AD III (aa 243 to 328). Human IgG antibodies also showed preferential binding to AD I. The epitope recognized by strain-specific MAb (KI4) or group-specific MAb (KI57) was mapped to AD II (aa 142 to 203). Mouse serum antibodies, elicited by immunization with deletion mutants, consistently bound to AD III. Moreover, the carboxy-terminal 140 aa of the Bor56 protein did not elicit an antibody response in C3H/HeDub mice. A model of the antigenic structure of Bor56 is presented and discussed. These results suggest that antigenic fragments from AD I and AD III are useful in the induction of humoral immunity against O. tsutsugamushi. These antigenic analyses provide an important foundation for further analyses of the neutralizing-antibody responses generated during rickettsial infections. They also provide potential peptide substrates for diagnostic assays and vaccine strategies.     

Comparative Accuracy of the InBios Scrub Typhus Detect IgM Rapid Test for the Detection of IgM Antibodies by Using Conventional Serology

This study investigated the comparative accuracy of a recombinant 56-kDa type-specific antigen-based rapid diagnostic test (RDT) for scrub typhus for the detection of IgM antibodies by using conventional serology in well-characterized serum samples from undifferentiated febrile illness patients. The RDT showed high specificity and promising comparative accuracy, with 82% sensitivity and 98% specificity for samples defined positive at an IgM indirect immunofluorescence assay positivity cutoff titer of ≥1:1,600 versus 92% and 95% at ≥1:6,400, respectively.     

Kinetics and magnitude of antibody responses against the conserved 47-kilodalton antigen and the variable 56-kilodalton antigen in scrub typhus patients

Western blot analysis of Orientia tsutsugamushi whole-cell lysates with scrub typhus patient sera has identified at least five protein antigens of O. tsutsugamushi with molecular sizes of 22 kDa, 47 kDa, 56 kDa, 58 kDa, and 110 kDa. In this study, sera from serial bleedings of 108 patients were used to study the kinetics and the magnitude of specific antibody responses against the 47-kDa and 56-kDa antigens. Recombinant protein of the conserved 47-kDa antigen (r47b) or a mixture of truncated 56-kDa antigen (r56s) from three prototype strains was used as the antigen in an enzyme-linked immunosorbent assay (ELISA). Our results showed that 76% and 93% of these patients had elevated IgM and IgG against r47b, respectively, and 98% and 100% had elevated IgM and IgG against r56s, respectively. The kinetics of antibody responses against r47b and r56s can be grouped into three patterns. In the first type of response, IgM and IgG against r47b and r56s appeared about the same time. The IgM and IgG titers against r56s were much higher than those against r47b. In the second type of response, induction of IgM appeared to be similar to that in the first type. The major difference to the first type is that the IgG titers against r47b were induced at least 1 week later than those against the r56s. The third type showed strong IgG responses against both r47b and r56s, and low or no IgM responses indicated a secondary infection. This is the first systematic investigation of antibody response kinetics against the conserved 47-kDa antigen versus the variable 56-kDa antigen in scrub typhus patients.     

Identification of Outer Membrane Vesicles Derived from Orientia tsutsugamushi

Orientia tsutsugamushi, a causative pathogen of Scrub typhus, is a gram-negative intracellular bacterium. Outer membrane vesicles (OMVs) are produced from the membrane of bacteria and play many roles related to the survival of the pathogen. However, there have been no reports confirming whether O. tsutsugamushi indeed produce OMVs. O. tsutsugamushi boryong was cultured in ECV-304 cells for the purification of OMVs. Western blot analysis and immunoenrichment using anti-O. tsutsugamushi monoclonal antibody and electron microscopy were employed for identification and characterization of OMVs. We confirm the presence of OMVs derived from O. tsutsugamushi, and also found that those OMVs contain a major surface antigen of 56-kDa protein and variant immunogenic antigens.     

Early diagnosis of scrub typhus with a rapid flow assay using recombinant major outer membrane protein antigen (r56) of Orientia tsutsugamushi

The variable 56-kDa major outer membrane protein of Orientia tsutsugamushi is the immunodominant antigen in human scrub typhus infections. We developed a rapid immunochromatographic flow assay (RFA) for the detection of immunoglobulin M (IgM) and IgG antibodies to O. tsutsugamushi. The RFA employs a truncated recombinant 56-kDa protein from the Karp strain as the antigen. The performance of the RFA was evaluated with a panel of 321 sera (serial bleedings of 85 individuals suspected of scrub typhus) which were collected in the Pescadore Islands, Taiwan, from 1976 to 1977. Among these 85 individuals, IgM tests were negative for 7 cases by both RFA and indirect fluorescence assay (IFA) using Karp whole-cell antigen. In 29 cases specific responses were detected by the RFA earlier than by IFA, 44 cases had the same detection time, and 5 cases were detected earlier by IFA than by RFA. For IgG responses, 4 individuals were negative with both methods, 37 cases exhibited earlier detection by RFA than IFA, 42 cases were detected at the same time, and 2 cases were detected earlier by IFA than by RFA. The sensitivities of RFA detection of antibody in sera from confirmed cases were 74 and 86% for IgM and IgG, respectively. When IgM and IgG results were combined, the sensitivity was 89%. A panel of 78 individual sera collected from patients with no evidence of scrub typhus was used to evaluate the specificity of the RFA. The specificities of the RFA were 99% for IgM and 97% for IgG. The sensitivities of IFA were 53 and 73% for IgM and IgG, respectively, and were 78% when the results of IgM and IgG were combined. The RFA test was significantly better than the IFA test for the early detection of antibody to scrub typhus in primary infections, while both tests were equally sensitive with reinfected individuals.     

Seroprevalence of Scrub typhus at a tertiary care hospital in Andhra Pradesh

Introduction: Scrub typhus is a rickettsial infection which is caused by Orientia tsutsugamushi and transmitted by the bite of the chigger of a mite. Delay in diagnosis can be fatal otherwise the treatment is simple, doxycycline being the drug of choice. Indirect immunoflurescence is considered gold standard but it is not used in India as it is costly and also not available. There is need for rapid, economic and simple test for the diagnosis of scrub typhus. This study was taken up to study the seroprevalence of scrub typhus in Andhra Pradesh and to compare two commonly used serological methods; rapid test and IgM ELISA. Materials and methods: This is a prospective study in which 100 serum samples from clinically suspected cases collected over a period of 3 months were processed for the detection of IgM antibodies for scrub typhus by ELISA and Rapid test. Samples were also tested for leptospirosis and dengue fever which the other common causes of fever prevalent in this region. Results: Total number of samples processed was 100 of which 52 were males and 48 females. Among the hundred samples 39 were seropositive. Positivity was higher in the age group of patients between 16 and 30 yrs of age. There was 97% correlation between ELISA and rapid method. Of the 100 samples only three samples positive by ELISA were negative by rapid method. Fever was the most common manifestation and there was no eschar and no mortality reported. Conclusion: Scrub typhus should be included in the differential diagnosis of fever of unknown origin along with dengue, malaria and leptospirosis which are the other common endemic infections in this part of the country.     

Evaluation of nested PCR and loop mediated isothermal amplification assay (LAMP) targeting 47 ​kDa gene of Orientia tsutsugamushi for diagnosis of scrub typhus

PurposeDiagnostic testing, in particular early detection, is critical for scrub typhus, as most infected individuals have nonspecific symptoms that are easily confused with dengue and malaria. PCR and LAMP offer an alternative DNA amplification method for detection of Orientia tsutsugamushi. Detection of Orientia tsutsugamushi DNA by targeting the 47-kDa gene using nested PCR and LAMP for diagnosis of scrub typhus.MethodsA cross-sectional study in a tertiary care hospital in central India. The present study was done on a total of 274 patients with fever of five days or more and negative for other causes of fever viz. malaria, dengue and enteric fever. From each patient 5 ​ml of blood samples was collected in EDTA vial for molecular tests (PCR and LAMP) and in plain vial for serological tests (IgM IFA).The data was entered in Excel sheet and 2 ​× ​2 tables were created to find sensitivity, specificity, positive and negative likelihood ratios, disease prevalence, positive and negative predictive values and accuracy.ResultsPCR showed a sensitivity of 29.73% while the sensitivity of LAMP was 16.22%. The specificity of nested PCR and LAMP was very high, 99.58% and 99.16% respectively. The diagnostic accuracy of nested PCR (90.15%) was found to be marginally better than LAMP (87.96%).ConclusionsFor the treatment of scrub typhus, a gene-based diagnostic test would enable earlier and more accurate detection of the causative agents of the disease than serology in admission samples of patients with acute febrile illness in endemic areas.