Leptospirosis and Rickettsial Diseases Sero-Conversion Surveillance Among U.S. Military Personnel in Honduras.

INTRODUCTION: Leptospirosis and rickettsial diseases are global zoonotic diseases. In severe infection cases, mortality can range from 10% to 30%. Currently most epidemiological data available are based on outbreak investigations and hospital-based studies from endemic countries. The U.S. soldiers at military bases in these countries are highly vulnerable due to the fact that most of them are immunologically naïve to these pathogens. No risk assessment of leptospirosis and rickettsial diseases among U.S. military personnel in Honduras is currently available. This study was aimed at determining the prevalence of leptospirosis and rickettsial diseases in U.S. military personnel deployed to Honduras using serological assays. MATERIALS AND METHODS: A cohort of pre- and post-deployment sera from the most recent 1,000 military personnel stationed in Honduras for at least 6 months between 2000 and 2016 was identified for this study. Serum specimens from these eligible subjects were retrieved. All post-deployment serum specimens were screened at a dilution of 1:100 for the presence of IgG antibodies to Leptospira and Rickettsia pathogens. The pre-deployment sera from those individuals with post-deployment IgG antibodies above cutoff (i.e., seropositive) were tested to determine seroconversion. Seroconversion was defined as conversion of an optical density value from below the cutoff (i.e., negative) in a pre-deployed specimen to above the cutoff (i.e., positive) in a post-deployed specimen at a titer of 100. RESULTS: The seropositive post-deployment specimens for antibodies against Leptospira (causing leptospirosis), Rickettsia typhi (causing murine typhus [MT]), spotted fever group rickettsioses (SFGR, causing SFG Rickettsia), Orientia tsutsugamushi (causing scrub typhus [ST]), and Coxiella burnetii (causing Q fever [QF]) were 11.6%, 11.3%, 6%, 5.6%, and 8.0%, respectively. The seroconverted rate in those assigned to Honduras from 2000 to 2016 was 7.3%, 1.9%, 3.9%, 4.3%, and 2.7% for leptospirosis, MT, SFGR, ST, and QF, respectively. Among the seroconverted specimens, 27 showed seroconversion of at least two antibodies. These seroconverted individuals accounted for 8.8% (3 out of 34) of the personnel who looked for medical attention during their deployment. CONCLUSIONS: Our results suggest a leptospirosis seroconversion rate of 7.3%, which is higher than the 0.9% and 3.9% seroconversion in Korea and Japan, respectively. The higher rate of seroconversion indicates potential risk of Leptospira exposure. Additional testing of water samples in the pools and pits around the training sites to locate the infected areas is important to eliminate or reduce future exposure to Leptospira during trainings. The rates of seroconversion for ST, MT, spotted fever Rickettsia, and QF were 4.3%, 1.9%, 3.9%, and 2.7%, respectively, indicating the potential exposure to a variety of rickettsial-related pathogens. Testing of vectors for rickettsial pathogens in the areas could inform effective vector control countermeasures to prevent exposure. Proper precaution and protective measures are needed to better protect military personnel deployed to Honduras.

Investigation of the Sterility of Diluent in Prefilled Syringes Used for Vaccine Reconstitution at Department of Defense Recruit Training Sites.

INTRODUCTION: Measles, mumps, and rubella (MMR) and varicella (VAR) vaccines are the two vaccines administered in large recruit training sites (RTS) that require a single-use syringe to be prefilled with the diluent (ie water) before vaccine reconstitution. Since there are no preservatives in either MMR or VAR vaccines, it is critical to maintain the diluent sterile to ensure the sterility of the reconstituted vaccine. The Department of Defense/Defense Health Agency has instructions on reconstitution of lyophilized vaccines and guidelines for their storage. Vaccine manufacturers provide instructions on how to properly store the diluent. However, there is no clear guidance or standard operating procedures regarding the best practice for preparation and storage of the syringes prefilled with diluent. Various RTS across all four services have their respective routines to best fit their vaccination requirements. Currently, there are no available data on the sterility status of the diluent prepared using these various routines before they are used to reconstitute vaccines. MATERIALS AND METHODS: We investigated the sterility of the diluent (ie water) in prefilled syringes prepared using routines practiced at various RTS. Diluent was drawn up into single syringes and was kept under various conditions (4 °C or room temperature for overnight up to 24 hours) used by various RTS. At indicated time, diluent was injected into sterile vials and the sterility of the diluent was determined by monitoring the presence/growth of bacteria (including aerobic bacteria, mycoplasma, and an obligate intracellular bacterium, Coxiella burnetii), fungi, and viruses for up to 21 days after inoculation into proper and specific culture media. Both traditional cell culture and molecular assays were used to demonstrate the presence or absence of contamination that may compromise the sterility of the diluent. RESULTS: Our results demonstrate that the diluent, after being drawn up to fill the syringe, maintains sterility after storage for overnight up to 24 hours at room temperature or 4 °C with or without recapping the syringes, suggesting that current vaccine reconstitution routines practiced at large military RTS are safe. CONCLUSIONS: Our results demonstrate that in spite of variations in current practices used in various RTS, the diluent in the prefilled syringe tested from each site maintains its sterility and was determined to be safe for use in military health system-wide vaccine reconstitution.

Development of a Sensitive and Rapid Recombinase Polymerase Amplification Assay for Detection of Anaplasma phagocytophilum.

Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

Temporal analysis of mRNA expression profiles in Orientia infected C3HeB/FeJ mouse.

BACKGROUND: Scrub typhus causes up to 35% mortality if left untreated. One billion people living in the endemic regions are at risk. In spite of its heavy disease burden in some of the most populated areas in the world, there is no vaccine available. Although the disease can be effectively treated by proper antibiotics, timely and accurate diagnosis remains a challenge. Orientia tsutsugamushi infects a variety of mammalian cells in vitro and replicates in the cytoplasm of the infected cells. Microarray analysis has been used extensively to study host-pathogen interactions in in vitro models to understand pathogenesis. However there is a lack of in vivo studies. RESULTS: In this study, C3HeB/FeJ (C3H) mice were infected by O. tsutsugamushi via the intraperitoneal route and monitored gene expression at 10 different time points post infection. We observed two distinct types of expression profiles in the genes that we analyzed. There are two valleys (4-18 h and 2-4 days) with low number of differentially expressed genes (DEG) with three peaks with high number of DEG at 2 h, 1-day and 7-day post infection. Further analysis revealed that pathways like complement and coagulation cascade, and blood clotting cascade pathways showed significant global changes throughout entire time course. Real time quantitative Polymerase Chain Reaction (RT-qPCR) confirmed the change of expression for genes involved in complement and coagulation cascade. These results suggested dynamic regulation of the complement and coagulation cascades throughout most of the time post infection while some other specific pathways, such as fatty acid metabolism and tryptophan metabolism, are turned on or off at certain times post infection. CONCLUSIONS: The findings highlight the complex interconnection among all different biological pathways. It is conceivable that specific pathways such as cell growth control and cell development in the host are affected by Orientia in the initial phase of infection for Orientia to grow intracellularly. Once Orientia is replicating successfully inside the host as infection progresses, the infection could activate pathways involved in cellular immune responses to defend for host cell survival and try to eliminate the pathogen.

Leptospirosis Seroconversion Surveillance Among US Marines Assigned to Japan, 2011-2015.

INTRODUCTION: Leptospirosis is a global zoonotic disease spread through contact with contaminated water/soil. The US soldiers at the military bases in these countries are extremely vulnerable, as most of them are immunologically naïve to the responsible pathogen. No recent sero-epidemiological data of leptospirosis among US Marines stationed in Japan were available. MATERIALS AND METHODS: In this study, we analyzed the presence of leptospirosis in US Marines stationed in Japan. One thousand posttour sera samples were analyzed by enzyme-linked immunosorbent assay for Leptospira-specific Immunoglobulin G. RESULTS: Among these 1,000 posttour samples, 85 of them were positive and corresponding pretour samples were analyzed by enzyme-linked immunosorbent assay also. Seroconversion occurred for 35 (3.5%) Marines during their assignment to Japan. These results also indicate that 50 Marine personnels were exposed to leptospires before their assignment to Japan, perhaps because of previous exposure to leptospires at home. CONCLUSION: The 5% rate of seroconversion in 2013 and 2014 suggests that leptospirosis is a potential threat for Marines in the endemic region in Japan.

Regulation of Serum Exosomal MicroRNAs in Mice Infected with Orientia tsutsugamushi.

Exosomes are small extracellular vesicles that carry proteins, lipids, and nucleic acids. They are circulated in many body fluids and play an important role in intercellular communications. MicroRNAs (miRNAs), as major components of exosomes, are often regulated in many diseases including bacterial and viral infections. Functionally, exosome-carried miRNAs interact with various immune cells and affect their behavior. Little is known whether exosomal miRNAs are regulated during scrub typhus, a potentially lethal infection caused by intracellular bacteria, Orientiatsutsugamushi. In the present study, we utilized a scrub typhus mouse model and collected serum at various time points post infection. A custom quantitative PCR array covering 92 murine miRNAs was used to profile serum exosomal miRNAs. A total of 12 miRNAs were found to be significantly up- or down-regulated at least at one time point post infection when compared to uninfected animals. Further analysis identified multiple miRNAs in the let-7 family that were consistently down-regulated at early and late phase of infection. Functionally, serum exosomes isolated from infected mice displayed strong proinflammatory effect when incubated with bone marrow-derived macrophages. Our data revealed dynamic regulations of serum exosomal miRNA during scrub typhus infection, which could significantly influence host immune responses and disease outcome.

Serologic Evidence for Orientia Exposure in the Democratic Republic of Sao Tome and Principe.

Orientia tsutsugamushi is an obligate intracellular bacterium that causes scrub typhus in humans. Formerly thought to be confined to the "tsutsugamushi triangle" within the Asia-Pacific region, scrub typhus was recently identified in the Western Hemisphere. Moreover, a new species of Orientia bacterial genus was isolated from a patient in Dubai. This study investigated Orientia exposure in an African country, the Democratic Republic of Sao Tome and Principe. Two sets of samples were analyzed in the study: 240 dried blood spots (DBSs) collected in 2016 and 863 serum samples from 570 pregnant women in 2003. Antibodies against O. tsutsugamushi were examined by immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA). The positive samples were further confirmed by Western blot. The results of IFA showed that 5.8% (14/240) of DBSs and 20.4% (116/570) of the serum samples contained reactive antibodies, whereas IgG ELISA yielded a positive rate of 15.4% (88/570) for the serum samples. These findings provided serologic evidence of potential Orientia exposure even though case of scrub typhus has never been diagnosed in the nation. Further studies are needed to determine the epidemiology and the burden of this neglected tropical disease in Africa.

Assessment of a Sensitive qPCR Assay Targeting a Multiple-Copy Gene to Detect Orientia tsutsugamushi DNA.

Scrub typhus is caused by an obligated intracellular organism, Orientia tsutsugamushi (Orientia). The disease was traditionally thought to be limited in the tsutsugamushi triangle. Recently, scrub typhus has been confirmed in areas outside the triangle. Serological diagnosis of scrub typhus relies on indirect immunofluorescence assay (IFA). Molecular assays such as PCR, qPCR, loop-mediated isothermal amplification, and recombinase polymerase amplification are often targeting a single copy gene. These assays are sensitive and specific, yet they are not broadly used in clinical settings possibly due to low circulating Orientia in blood. In this study, we compared qPCR results using a multiple copy (traD) gene with those using a single copy (47 kDa) gene to assess the improvement of sensitivity and limit of detection. Our results demonstrate that the qPCR using the traD gene provides superior sensitivity in 15 Orientia strains. The limit of detection is below single Orientia genome equivalent and the assay retains specificity with excessive DNA from mouse, chiggers and human. The clinical utility was evaluated using confirmed scrub typhus positive and negative samples. The results show 100% sensitivity and specificity in these samples suggesting that the traD gene qPCR may be useful for clinical diagnosis of Orientia infection.

Simple Detection of Hepatitis B Virus in Using Loop-Mediated Isothermal Amplification Method.

INTRODUCTION: US Military and civilian personnel regularly deploy to regions that are endemic for the Hepatitis B virus (HBV), including the Western Pacific, Africa, Eastern Mediterranean, Southeast Asia, and Europe. When patients have life-threatening injuries that require any blood component that is not immediately available, they are typically transfused with locally collected fresh whole blood from a walking blood bank. Currently, there is no simple and easy method for sensitively screening fresh blood in deployed theaters of conflict. MATERIALS AND METHODS: In order to fill the gap, we have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of HBV in blood products. The primers were designed to target the gene of the pre-Surface/Surface antigen region of HBV. The amplification reaction mixture was incubated at 60°C for 60 min. The amplicon can be detected by a handheld fluorescence tube scanner or an immune-chromatography test strip. RESULTS: We were able to detect down to 10 copies of viral DNA by LAMP reaction for HBV DNA extracted from HBV-positive plasma. We also identified the optimal heat treatment condition (125°C for 10 min) for plasma specimens without requiring DNA extraction for the LAMP assay. The sensitivity of the assay was evaluated with polymerase chain reaction (PCR) confirmed HBV-positive samples. Using LAMP, we detected HBV in 107 out of 127 (84%) samples. CONCLUSION: This LAMP assay has the potential to be used in resource-limited settings to improve the safety of locally collected blood in endemic regions.

An Improved Enzyme-Linked Immunoassay for the Detection of Leptospira-Specific Antibodies.

Leptospirosis is a neglected zoonotic disease with worldwide endemicity and continues to be a significant public health burden on resource-limited populations. Previously, we produced three highly purified recombinant antigens (rLipL32, rLipL41, and rLigA-Rep) and evaluated their performance of detecting Leptospira-specific antibodies in enzyme-linked immunosorbent assay (ELISA) as compared with the microscopic agglutination test (MAT). The overall sensitivity of this assay approached 90%. Recently, another recombinant antigen (rLigB-Rep) was prepared. We tested each individual antigen and a 1:1:1:1 mixture of these four antigens for the detection of Leptospira-specific antibodies in ELISA. The performance of these recombinant antigens was evaluated with a much larger febrile patient panel (337 MAT-confirmed positive sera and 92 MAT-negative sera from febrile patients). Combining the detection results of immunoglobulin M and immunoglobulin G from these four individual antigens, the overall sensitivity was close to 90% but the specificity was only 66%, based on the MAT reference method. The overall sensitivity and specificity of the four-antigen mixture were 82% and 86%, respectively. The mixture of four antigens also exhibited a broader reactivity with MAT-positive samples of 18 serovars from six major pathogenic Leptospira species. Given the limitations of MAT, the data were further analyzed by Bayesian latent class model, showing that ELISA using a 1:1:1:1 mixture still maintained high sensitivity (79%) and specificity (88%) as compared with the sensitivity (90%) and specificity (83%) of MAT. Therefore, ELISA using a mixture of these four antigens could be a very useful test for seroprevalence studies.

Dissemination of Orientia tsutsugamushi, a Causative Agent of Scrub Typhus, and Immunological Responses in the Humanized DRAGA Mouse.

Scrub typhus is caused by Orientia tsutsugamushi, an obligated intracellular bacterium that affects over one million people per year. Several mouse models have been used to study its pathogenesis, disease immunology, and for testing vaccine candidates. However, due to the intrinsic differences between the immune systems in mouse and human, these mouse models could not faithfully mimic the pathology and immunological responses developed by human patients, limiting their value in both basic and translational studies. In this study, we have tested for the first time, a new humanized mouse model through footpad inoculation of O. tsutsugamushi in DRAGA (HLA-A2.HLA-DR4.Rag1KO.IL2RγcKO.NOD) mice with their human immune system reconstituted by infusion of HLA-matched human hematopoietic stem cells from umbilical cord blood. Upon infection, Orientia disseminated into various organs of DRAGA mice resulted in lethality in a dose-dependent manner, while all C3H/HeJ mice infected by the same route survived. Tissue-specific lesions associated with inflammation and/or necroses were observed in multiple organs of infected DRAGA mice. Consistent with the intracellular nature of Orientia, strong Th1, but subdued Th2 responses were elicited as reflected by the human cytokine profiles in sera from infected mice. Interestingly, the percentage of both activated and regulatory (CD4+FOXP3+) human T cells were elevated in spleen tissues of infected mice. After immunization with irradiated whole cell Orientia, humanized DRAGA mice showed a significant activation of human T cells as evidenced by increased number of human CD4+ and CD8+ T cells. Specific human IgM and IgG antibodies were developed after repetitive immunization. The humanized DRAGA mouse model represents a new pre-clinical model for studying Orientia-human interactions and also for testing vaccines and novel therapeutics for scrub typhus.

Evaluation of the stability of lyophilized loop-mediated isothermal amplification reagents for the detection of Coxiella burnetii.

Coxiella burnetii, the causative pathogen for Q fever, is an obligate intracellular bacterium and designated as a biosafety level 3 agent. Detection and quantification of the bacteria with conventional culturing methods is time-consuming and poses significant health risks. Polymerase chain reaction (PCR)-based assays have been developed for detecting C. burnetii and could provide rapid diagnosis. However, they require specialized equipment, including a cold chain for PCR reagents that maintains their stability during storage and transport. These requirements limit the advantage of PCR-based methods, especially in resource-limited areas. Previously, we had developed a lyophilized loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii. To simplify and improve this assay, the reagents for the LAMP assay and the detecting reagent, SYBR green, were lyophilized together. The stability of the lyophilized reagents was evaluated by measuring changes in detection limit for plasmid DNA encoding a C. burnetii gene upon storage at 4 °C, 25 °C, or 37 °C. Our data indicate that the lyophilized reagents remain stable for 24 months when stored at 4 °C, 28 days at 25 °C, and 2 days at 37 °C. This improved LAMP assay can be easily performed in a simple water bath or heating block. The stability at ambient temperature, the simplicity of assay procedure, and the availability of low cost equipment make this method ideal for use in resource-limited settings where Q fever is endemic.

Outer Membrane Protein OmpB Methylation May Mediate Bacterial Virulence.

Methylation of outer membrane proteins (OMPs) has been implicated in bacterial virulence. Lysine methylation in rickettsial OmpB is correlated with rickettsial virulence, and N- and O-methylations are also observed in virulence-relevant OMPs from several pathogenic bacteria that cause typhus, leptospirosis, tuberculosis, and anaplasmosis. We summarize recent findings on the structure of methylated OmpB, biochemical characterization, and crystal structures of OmpB methyltransferases. Native rickettsial OmpB purified from highly virulent strains contains multiple clusters of trimethyllysine, in contrast with mostly monomethyllysine, and no trimethyllysine is found in an avirulent strain. Crystal structure of the methyltransferases reveals mechanistic insights for catalysis, and a working model is discussed for this unusual post-translational modification.

Molecular Detection and Identification of Rickettsia Species in Ticks (Acari: Ixodidae) Collected From Belize, Central America.

Little is known about tick-borne rickettsial pathogens in Belize, Central America. We tested ixodid ticks for the presence of Rickettsia species in three of the six northern and western Belizean districts. Ticks were collected from domestic animals and tick drags over vegetation in 23 different villages in November 2014, February 2015, and May 2015. A total of 2,506 collected ticks were identified to the following species: Dermacentor nitens Neumann (46.69%), Rhipicephalus sanguineus (Latreille) (19.55%), Rhipicephalus microplus (Canestrini) (19.47%), Amblyomma cajennense complex (9.74%), Amblyomma maculatum Koch (3.47%), Amblyomma ovale Koch (0.68%), Ixodes nr affinis (0.16%), Amblyomma nr maculatum (0.12%), and Amblyomma nr oblongoguttatum (0.12%). Ticks were pooled according to species, life stage (larva, nymph, or adult), and location (n = 509) for DNA extraction and screened for genus Rickettsia by quantitative real-time polymerase chain reaction (qPCR). All 42 positive pools were found to be positive for spotted fever group (SFG) Rickettsia in pools of A. cajennense complex (n = 33), A. maculatum (n = 4), A. nr maculatum (n = 1), A. ovale (n = 1), R. sanguineus (n = 1), and I. nr affinis (n = 2). Rickettsia amblyommatis was identified from A. cajennense complex and A. nr maculatum. Rickettsia parkeri was found in A. maculatum, and Rickettsia sp. endosymbiont was detected in I. nr affinis. The presence of infected ticks suggests a risk of tick-borne rickettsioses to humans and animals in Belize. This knowledge can contribute to an effective tick management and disease control program benefiting residents and travelers.

An ELISA assay using a combination of recombinant proteins from multiple strains of Orientia tsutsugamushi offers an accurate diagnosis for scrub typhus.

BACKGROUND: Scrub typhus (ST) is a disease caused by an obligate intracellular bacterium, Orientia tsutsugamushi, an organism that requires a BSL3 laboratory for propagation. The disease is hallmarked by an eschar at the site of the chigger bite, followed by the development of fever, malaise, myalgia, anorexia, and papulomacular rash. Indirect immunofluorescent assay (IFA) is the gold standard for scrub typhus diagnosis, however, the subjectivity of the assay, the need for a specialized laboratory and instruments has limited the wide use of the test in resource limited areas. METHODS: A recombinant-protein based enzyme linked immunosorbent assay (ELISA) using the most abundant and immunodominant protein for the detection of Orientia specific antibodies in serum has been developed. The performance of the assay was evaluated using prospectively collected acute sera from 248 randomly selected patients in Thailand. The ELISA assay was evaluated using two different cutoff values. RESULTS: The receiver operating characteristic (ROC) curve generated cutoff values gave slightly better consistency with diagnosis of ST than those cutoff values established by averaging ELISA optical density of known negatives at 99% confidence interval. Both cutoff values provided similar statistical parameters when compared with the diagnosis of ST, indicating the validity of both calculations to derive cutoff values. These results suggest that both IgG and IgM ELISA performed well to accurately diagnose scrub typhus cases in endemic areas using only acute serum samples. CONCLUSIONS: We have successfully developed an ELISA assay for the detection of Orientia-specific antibodies in serum that could provide effective screening of acute sera under clinical setup and it is also a useful assay to estimate seroprevalence in various endemic areas.

Intraspecies comparative genomics of three strains of Orientia tsutsugamushi with different antibiotic sensitivity.

We recently reported the genome of Orientia tsutsugamushi (OT) strain Karp (GenBank Accession #: NZ_LYMA00000000.2, https://www.ncbi.nlm.nih.gov/nuccore/NZ_LYMA00000000.2) with > 2 Mb in size through clone-based sequencing and high throughput genomic shotgun sequencing (HTS). The genomes of OT strains AFSC4 and AFSC7 were similarly sequenced by HTS Since strains AFSC4 (GenBank Accession #: NZ_LYMT00000000.1, https://www.ncbi.nlm.nih.gov/nuccore/1035784408) and AFSC7 (GenBank Accession #: NZ_LYMB00000000.1, https://www.ncbi.nlm.nih.gov/nuccore/1035854767) were more resistant to antibiotics than strain Karp, we conducted comparative analysis of the three draft genomes annotated by RAST server aimed to identify possible genetic bases of difference in microbial antibiotic sensitivity. Intraspecies comparative genomics analysis of the three OT strains revealed that two ORFs encoding hypothetical proteins in both strains AFSC4 and AFSC7 are absent in strain Karp.

Utility of Loop-mediated Isothermal Amplification Assay, Polymerase Chain Reaction, and ELISA for Diagnosis of Leptospirosis in South Indian Patients.

BACKGROUND: Leptospirosis is a zoonotic disease which requires laboratory diagnosis for confirmation. MATERIALS AND METHODS: In this study serum samples from adults with acute undifferentiated fever (duration ≤15 days) were tested for IgM antibodies to Leptospira by ELISA, PCR for rrs gene and loop-mediated isothermal amplification (LAMP) assay for LipL32 and LipL41. RESULTS: Among the 150 sera tested, three were positive by PCR, LAMP and IgM ELISA/modified Faines' criteria, two by only PCR; seven only by LAMP assay and forty fulfilled modified Faine's criteria (illness clinically compatible and IgM ELISA positive for leptospirosis). Clinical correlation revealed renal compromise, low platelet count and severe jaundice were significantly related to leptospirosis (P < 0.05). CONCLUSION: This study suggests that LAMP assay could be useful for diagnosis of leptospirosis during the 1st week of illness whereas IgM ELISA forms the mainstay of diagnosis from the 2nd week onward. Further studies especially community based, comparing ELISA, PCR, LAMP, culture and microscopic agglutination test are required to evaluate the veracity of these findings.

Lateral Flow Rapid Test for Accurate and Early Diagnosis of Scrub Typhus: A Febrile Illness of Historically Military Importance in the Pacific Rim.

Scrub typhus (ST) is an infection caused by Orientia tsutsugamushi. Historically, ST was ranked as the second most important arthropod-borne medical problem only behind malaria during World War II and the Vietnam War. The disease occurs mainly in Southeast Asia and has been shown to emerge and reemerge in new areas, implying the increased risk for U.S. military and civilian personnel deployed to these regions. ST can effectively be treated by doxycycline provided the diagnosis is made early, before the development of severe complications. Scrub Typhus Detect is a lateral flow rapid test based on a mixture of recombinant 56-kDa antigens with broad reactivity. The performance of this prototype product was evaluated against indirect immunofluorescence assay, the serological gold standard. Using 249 prospectively collected samples from Thailand, the sensitivity and specificity for IgM was found to be 100% and 92%, respectively, suggesting a high potential of this product for clinical use. This product will provide a user friendly, rapid, and accurate diagnosis of ST for clinicians to provide timely and accurate treatments of deployed personnel.