Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO2 incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO2 for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC90 values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 μg/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research.

Melioidosis is a tropical infection caused by the Gram-negative soil saprophyte Burkholderia pseudomallei. Despite broad exposure of northeast Thais, disease develops in only a small proportion of individuals. Although diabetes is a risk factor, the mechanisms of host susceptibility to melioidosis are still poorly understood. We postulated that Toll-like receptors (TLRs) regulate host susceptibility to disease, and that genetic variation in TLRs is associated with melioidosis. We analyzed the frequency of eight previously described TLR pathway polymorphisms in 490 cases compared to 950 non-hospitalized controls or 458 hospitalized controls. Based on these results, we then analyzed the frequency of additional TLR4 or TLR6-1-10 region polymorphisms in cases and controls. We found that the TLR41196C>T variant was associated with protection from melioidosis when compared to non-hospitalized controls. The TLR1742A>G and TLR1−7202A>G variants were associated with melioidosis when compared to hospitalized controls. In further analyses, we found that two additional TLR4 region polymorphisms were associated with disease. In diabetics, three other TLR6-1-10 region polymorphisms were associated with disease when compared to hospitalized controls. We conclude that TLR genetic variants may modulate host susceptibility to melioidosis. Confirmation of these findings and further investigation of the mechanisms is required.

We hypothesized that the gold standard for diagnosing leptospirosis is imperfect. We used Bayesian latent class models and random-effects meta-analysis to test this hypothesis and to determine the true accuracy of a range of alternative tests for leptospirosis diagnosis.

Background. We observed that some patients with clinical leptospirosis supported by positive results of rapid tests were negative for leptospirosis on the basis of our diagnostic gold standard, which involves isolation of Leptospira species from blood culture and/or a positive result of a microscopic agglutination test (MAT). We hypothesized that our reference standard was imperfect and used statistical modeling to investigate this hypothesis.

Methods. Data for 1652 patients with suspected leptospirosis recruited during three observational studies and one randomized control trial that described the application of culture, MAT, immunofluorescence assay (IFA), lateral flow (LF) and/or PCR targeting the 16S rRNA gene were reevaluated using Bayesian latent class models and random-effects meta-analysis.

Results. The estimated sensitivities of culture alone, MAT alone, and culture plus MAT (for which the result was considered positive if one or both tests had a positive result) were 10.5% (95% credible interval [CrI], 2.7%–27.5%), 49.8% (95% CrI, 37.6%–60.8%), and 55.5% (95% CrI, 42.9%–67.7%), respectively. These low sensitivities were present across all 4 studies. The estimated specificity of MAT alone (and of culture plus MAT) was 98.8% (95% CrI, 92.8%–100.0%). The estimated sensitivities and specificities of PCR (52.7% [95% CrI, 45.2%–60.6%] and 97.2% [95% CrI, 92.0%–99.8%], respectively), lateral flow test (85.6% [95% CrI, 77.5%–93.2%] and 96.2% [95% CrI, 87.7%–99.8%], respectively), and immunofluorescence assay (45.5% [95% CrI, 33.3%–60.9%] and 96.8% [95% CrI, 92.8%–99.8%], respectively) were considerably different from estimates in which culture plus MAT was considered a perfect gold standard test.

Conclusions. Our findings show that culture plus MAT is an imperfect gold standard against which to compare alterative tests for the diagnosis of leptospirosis. Rapid point-of-care tests for this infection would bring an important improvement in patient care, but their future evaluation will require careful consideration of the reference test(s) used and the inclusion of appropriate statistical models.

Samples from 160 prospectively recruited febrile patients with typhus-like illness in an area of Thailand (Chiang Rai, northern Thailand) where scrub typhus is endemic were used to evaluate the diagnostic capabilities of four rapid immunochromatographic tests (ICTs) for the detection of Orientia tsutsugamushi IgM and total antibodies during acute scrub typhus infection. Of the 160 cases, 54 (34%) had been confirmed to have scrub typhus using the reference scrub typhus infection criteria (STIC), i.e., positive cell culture isolation, an admission IgM antibody titer of ≥1:12,800, a 4-fold rising IgM antibody titer, and/or positivity for ≥2 out of 3 PCR gene targets). The ICTs gave the following sensitivities and specificities: the Panbio IgM ICT, 46% (95% confidence interval [CI], 33 to 60) and 95% (95% CI, 89 to 98), respectively; the Standard Diagnostics IgM ICT, 68% (95% CI, 60 to 75) and 73% (95% CI, 68 to 78), respectively; the AccessBio IgM ICT, 56% (95% CI, 48 to 63) and 90% (95% CI, 87 to 94), respectively; and the AccessBio total antibody ABt ICT, 61% (95% CI, 53 to 68) and 68% (95% CI, 63 to 73), respectively. An isothermal loop amplification (LAMP) PCR assay for scrub typhus demonstrated a sensitivity of 52% (95% CI, 38 to 66) and a specificity of 94% (95% CI, 88 to 98). This study has revealed the diagnostic limitations of antibody-based assays in an acute care setting. However, the combination of ICTs with LAMP usually increased sensitivity with a minimal reduction in specificity. The best combination, the Panbio IgM ICT and LAMP, resulted in a sensitivity of 67% (95% CI, 53 to 79) and a specificity of 91% (95% CI, 83 to 95). The combination of antibody-based assays with DNA- or antigen-based tests shows promise for improved diagnostic sensitivity.

The Leptospira immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) has been recommended for the rapid diagnosis of leptospirosis in endemic areas. We conducted a retrospective case-control study of 218 patients (109 leptospirosis cases confirmed by Leptospira culture and/or microscopic agglutination test and 109 control patients with acute febrile illness) to evaluate the diagnostic accuracy of a commercial IgM ELISA (Panbio) in northeast Thailand. Paired serum samples taken on admission and at least 10 days after the onset of symptoms were tested. Using the cutoff value recommended by the manufacturer (11 Panbio units), sensitivity and specificity of IgM ELISA on paired sera were 90.8% and 55.1%. A receiver operating characteristic curve was used to determine the optimal cutoff value. This was 20 Panbio units, which gave a sensitivity and specificity of 76.1% and 82.6%, respectively, on paired sera. We conclude that using either cutoff value, the accuracy of IgM ELISA is limited in our setting.

Background

The Surviving Sepsis Campaign (SSC) guidelines describe best practice for the management of severe sepsis and septic shock in developed countries, but most deaths from sepsis occur where healthcare is not sufficiently resourced to implement them. Our objective was to define the feasibility and basis for modified guidelines in a resource-restricted setting.

Methods and Findings

We undertook a detailed assessment of sepsis management in a prospective cohort of patients with severe sepsis caused by a single pathogen in a 1,100-bed hospital in lower-middle income Thailand. We compared their management with the SSC guidelines to identify care bundles based on existing capabilities or additional activities that could be undertaken at zero or low cost. We identified 72 patients with severe sepsis or septic shock associated with S. aureus bacteraemia, 38 (53%) of who died within 28 days. One third of patients were treated in intensive care units (ICUs). Numerous interventions described by the SSC guidelines fell within existing capabilities, but their implementation was highly variable. Care available to patients on general wards covered the fundamental principles of sepsis management, including non-invasive patient monitoring, antimicrobial administration and intravenous fluid resuscitation. We described two additive care bundles, one for general wards and the second for ICUs, that if consistently performed would be predicted to improve outcome from severe sepsis.

Conclusion

It is feasible to implement modified sepsis guidelines that are scaled to resource availability, and that could save lives prior to the publication of international guidelines for developing countries.

Background

Pathogenic Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR. A proportion of patients who are positive by PCR are negative by culture. Leptospira spp. are fastidious bacteria, and we hypothesized that a false-negative culture result may represent infection with a distinct bacterial subset that fail to grow in standard culture medium.

Methods

We evaluated our hypothesis during a prospective study of 418 consecutive patients presenting to a hospital in northeast Thailand with an acute febrile illness. Admission blood samples were taken for Leptospira culture and PCR. A single tube nested PCR that amplified a region of the rrs gene was developed and applied, amplicons sequenced and a phylogenetic tree reconstructed.

Results

39/418 (9%) patients were culture-positive for Leptospira spp., and 81/418 (19%) patients were culture-negative but rrs PCR-positive. The species associated with culture-positive leptospirosis (37 L. interrogans and 2 L. borgpetersenii) were comparable to those associated with culture-negative, PCR-positive leptospirosis (76 L. interrogans, 4 L. borgpetersenii, 1 unidentified, possibly new species).

Conclusion

Molecular speciation failed to identify a unique bacterial subset in patients with culture-negative, PCR-positive leptospirosis. The rate of false-negative culture was high, and we speculate that antibiotic pre-treatment is the most likely explanation for this.

A 21-year survey conducted in northeast Thailand of antimicrobial resistance to parenteral antimicrobial drugs used to treat melioidosis identified 24/4,021 (0.6%) patients with one or more isolates resistant to ceftazidime (n = 8), amoxicillin-clavulanic acid (n = 4), or both drugs (n = 12). Two cases were identified at admission, and the remainder were detected a median of 15 days after starting antimicrobial therapy. Resistance to carbapenem drugs was not detected. These findings support the current prescribing recommendations for melioidosis.

Background

There is an urgent need to develop rapid and accurate point-of-care (POC) technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy.

Methodology/Principal Findings

In this study we present the clinical evaluation of loop-mediated isothermal PCR assay (LAMP) in the context of a prospective fever study, including 161 patients from scrub typhus-endemic Chiang Rai, northern Thailand.

A robust reference comparator set comprising following ‘scrub typhus infection criteria’ (STIC) was used: a) positive cell culture isolate and/or b) an admission IgM titer ≥1∶12,800 using the ‘gold standard’ indirect immunofluorescence assay (IFA) and/or c) a 4-fold rising IFA IgM titer and/or d) a positive result in at least two out of three PCR assays.

Compared to the STIC criteria, all PCR assays (including LAMP) demonstrated high specificity ranging from 96–99%, with sensitivities varying from 40% to 56%, similar to the antibody based rapid test, which had a sensitivity of 47% and a specificity of 95%.

Conclusions/Significance

The diagnostic accuracy of the LAMP assay was similar to realtime and nested conventional PCR assays, but superior to the antibody-based rapid test in the early disease course. The combination of DNA- and antibody-based detection methods increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute phase of scrub typhus.

Author Summary

There is an urgent need for alternative diagnostic methods for scrub typhus, but evaluation of these is hampered because the current serological gold standard (IFA) is imperfect. In a study from Thailand, 3 of 20 (15%) patients with fever had a positive Orientia tsutsugamushi PCR result despite negative serology. These findings could reflect potential benefits of the PCR assay in detecting rickettsaemia before antibody responses set in and/or a diagnostic advantage in endemic areas with high background levels of antibody in the population. Serology is complicated by the heterogeneity of strains present in Southeast Asia, but high resource costs and training make realtime PCR assays impractical for many areas where scrub typhus is endemic. This is where the new LAMP methodology has potential: it is inexpensive, simple to perform and requires only a waterbath or simple heating block instead of a thermocycler. In the context of a prospective fever study in a scrub typhus-endemic area in Thailand, the results support the validity of LAMP methodology for the diagnosis of scrub typhus, highlight the difficulties in comparing antibody- with DNA-based methods and also contribute towards understanding the dynamics of bacteraemia in this under recognised and under studied disease.

There is a lack of diagnostic tests for leptospirosis in technology-restricted settings. We developed loop-mediated isothermal amplification (LAMP) specific for the 16S ribosomal RNA gene (rrs) of pathogenic and intermediate group Leptospira species. The lower limit of detection was 10 genomic equivalents/reaction, and analytical specificity was high; we observed positive reactions for pathogenic/intermediate groups and negative reactions for non-pathogenic Leptospira species and other bacterial species. We evaluated this assay in Thailand by using a case–control study of 133 patients with laboratory-proven leptospirosis and 133 patients with other febrile illnesses. Using admission blood, we found that the rrs LAMP showed positive results in 58 of 133 cases (diagnostic sensitivity = 43.6, 95% confidence interval [CI] = 35.0–52.5) and in 22 of 133 controls (diagnostic specificity = 83.5, 95% CI = 76.0–89.3). Sensitivity was high for 39 patients who were culture positive for Leptospira spp. (84.6, 95% CI = 69.5–94.1). The rrs LAMP can provide an admission diagnosis in approximately half of patients with leptospirosis, but its clinical utility is reduced by a lower specificity.

Patients with diabetes have better survival from septic melioidosis than patients who without diabetes. This difference was seen only in patients taking glyburide prior to presentation and was associated with an anti-inflammatory effect of glyburide.

Background. Patients with diabetes mellitus are more prone to bacterial sepsis, but there are conflicting data on whether outcomes are worse in diabetics after presentation with sepsis. Glyburide is an oral hypoglycemic agent used to treat diabetes mellitus. This KATP-channel blocker and broad-spectrum ATP-binding cassette (ABC) transporter inhibitor has broad-ranging effects on the immune system, including inhibition of inflammasome assembly and would be predicted to influence the host response to infection.

Methods. We studied a cohort of 1160 patients with gram-negative sepsis caused by a single pathogen (Burkholderia pseudomallei), 410 (35%) of whom were known to have diabetes. We subsequently studied prospectively diabetics with B. pseudomallei infection (n = 20) to compare the gene expression profile of peripheral whole blood leukocytes in patients who were taking glyburide against those not taking any sulfonylurea.

Results. Survival was greater in diabetics than in nondiabetics (38% vs 45%, respectively, P = .04), but the survival benefit was confined to the patient group taking glyburide (adjusted odds ratio .47, 95% confidence interval .28–.74, P = .005). We identified differential expression of 63 immune-related genes (P = .001) in patients taking glyburide, the sum effect of which we predict to be antiinflammatory in the glyburide group.

Conclusions. We present observational evidence for a glyburide-associated benefit during human melioidosis and correlate this with an anti-inflammatory effect of glyburide on the immune system.

Melioidosis is an infectious disease endemic to northern Australia and Southeast Asia. In response to clinical confusion regarding the appropriate dose of amoxicillin-clavulanate, we have developed guidelines for the appropriate dosing of this second-line agent. For eradication therapy for melioidosis, we recommend 20/5 mg/kg orally, three times daily.

Background

Rapid PCR-based tests for the diagnosis of leptospirosis can provide information that contributes towards early patient management, but these have not been adopted in Thailand. Here, we compare the diagnostic sensitivity and specificity of two real-time PCR assays targeting rrs or lipL32 for the diagnosis of leptospirosis in northeast Thailand.

Methods/Principal Findings

A case-control study of 266 patients (133 cases of leptospirosis and 133 controls) was constructed to evaluate the diagnostic sensitivity and specificity (DSe & DSp) of both PCR assays. The median duration of illness prior to admission of cases was 4 days (IQR 2–5 days; range 1–12 days). DSe and DSp were determined using positive culture and/or microscopic agglutination test (MAT) as the gold standard. The DSe was higher for the rrs assay than the lipL32 assay (56%, (95% CI 47–64%) versus 43%, (95% CI 34–52%), p<0.001). No cases were positive for the lipL32 assay alone. There was borderline evidence to suggest that the DSp of the rrs assay was lower than the lipL32 assay (90% (95% CI 83–94%) versus 93%, (95%CI 88–97%), p = 0.06). Nine controls gave positive reactions for both assays and 5 controls gave a positive reaction for the rrs assay alone. The DSe of the rrs and lipL32 assays were high in the subgroup of 39 patients who were culture positive for Leptospira spp. (95% and 87%, respectively, p = 0.25).

Conclusions/Significance

Early detection of Leptospira using PCR is possible for more than half of patients presenting with leptospirosis and could contribute to individual patient care.

Background

Progress in therapy of cryptococcal meningitis has been slow because of the lack of a suitable marker of treatment response. Previously, we demonstrated the statistical power of a novel endpoint, the rate of clearance of infection, based on serial quantitative cultures of CSF, to differentiate the fungicidal activity of alternative antifungal drug regimens. We hypothesised that the rate of clearance of infection should also be a clinically meaningful endpoint.

Methods

We combined data from cohorts of patients with HIV-associated cryptococcal meningitis from Thailand, South Africa, and Uganda, for whom rate of clearance of infection was determined, and clinical and laboratory data prospectively collected, and explored the association between the rate of clearance of infection and mortality by Cox survival analyses.

Results

The combined cohort comprised 262 subjects. Altered mental status at presentation, a high baseline organism load, and a slow rate of clearance of infection were independently associated with increased mortality at 2 and 10 weeks. Rate of clearance of infection was associated with antifungal drug regimen and baseline CSF IFN-γ levels.

Conclusions

The results support use of rate of clearance, or early fungicidal activity, as a means to explore antifungal drug dosages and combinations in phase II studies. An increased understanding of how the factors determining outcome interrelate may help clarify opportunities for intervention.

Orientia tsutsugamushi is an intracellular α-proteobacterium which resides in trombiculid mites, and is the causative agent of scrub typhus in East Asia. The genome sequence of this species has revealed an unprecedented number of repeat sequences, most notably of the genes encoding the conjugative properties of a type IV secretion system (T4SS). Although this observation is consistent with frequent intragenomic recombination, the extent of homologous recombination (gene conversion) in this species is unknown. To address this question, and to provide a protocol for the epidemiological surveillance of this important pathogen, we have developed a multilocus sequence typing (MLST) scheme based on 7 housekeeping genes (gpsA, mdh, nrdB, nuoF, ppdK, sucD, sucB). We applied this scheme to the two published genomes, and to DNA extracted from blood taken from 84 Thai scrub typhus patients, from 20 cultured Thai patient isolates, 1 Australian patient sample, and from 3 cultured type strains. These data demonstrated that the O. tsutsugamushi population was both highly diverse [Simpson's index (95% CI) = 0.95 (0.92–0.98)], and highly recombinogenic. These results are surprising given the intracellular life-style of this species, but are broadly consistent with results obtained for Wolbachia, which is an α-proteobacterial reproductive parasite of arthropods. We also compared the MLST data with ompA sequence data and noted low levels of consistency and much higher discrimination by MLST. Finally, twenty-five percent of patients in this study were simultaneously infected with multiple sequence types, suggesting multiple infection caused by either multiple mite bites, or multiple strains co-existing within individual mites.

Author Summary

Scrub typhus, the rickettsial infectious disease caused by the obligate intracellular bacterium Orientia tsutsugamushi, is endemic across the Asia Pacific region. The bacterium is transmitted by the bite of larval stages of trombiculid mites (“chiggers”; Leptotrombidium spp.), which more typically feed on small rodents. Clinical features include fever, headache, myalgia, lymphadenopathy and an eschar at the site of the bite. Despite the importance of this pathogen, little is known of the population diversity or the role of homologous recombination in driving the microevolution of this species. Here, we describe the development and application of a multilocus sequence typing (MLST) scheme that can be applied directly to blood samples, and that was applied to 108 O. tsutsugamushi isolates. We found that this organism demonstrated a high rate of homologous recombination, a surprising finding given the intracellular life-style of this species. We also found that 25% of patients in our study were simultaneously infected with multiple sequence types, suggesting multiple infection caused by either multiple mite bites, or multiple strains co-existing within individual mites.

Melioidosis is an infectious disease with a propensity for relapse, despite prolonged antibiotic eradication therapy for 12 to 20 weeks. A pharmacokinetic (PK) simulation study was performed to determine the optimal dosing of cotrimoxazole (trimethoprim-sulfamethoxazole [TMP-SMX]) used in current eradication regimens in Thailand and Australia. Data for bioavailability, protein binding, and coefficients of absorption and elimination were taken from published literature. Apparent volumes of distribution were correlated with body mass and were estimated separately for Thai and Australian populations. In vitro experiments demonstrated concentration-dependent killing. In Australia, the currently used eradication regimen (320 [TMP]/1,600 [SMX] mg every 12 h [q12h]) was predicted to achieve the PK-pharmacodynamic (PD) target (an area under the concentration-time curve from 0 to 24 h/MIC ratio of >25 for both TMP and SMX) for strains with the MIC90 of Australian strains (≤1/19 mg/liter). In Thailand, the former regimen of 160/800 mg q12h would not be expected to attain the target for strains with an MIC of ≥1/19 mg/liter, but the recently implemented weight-based regimen (<40 kg [body weight], 160/800 mg q12h; 40 to 60 kg, 240/1,200 mg q12h; >60 kg, 320/1,600 mg q12h) would be expected to achieve adequate concentrations for strains with an MIC of ≤1/19 mg/liter. The results were sensitive to the variance of the PK parameters. Prospective PK-PD studies of Asian populations are needed to optimize TMP-SMX dosing in melioidosis.

Background

Symptoms and signs of leptospirosis are non-specific. Several diagnostic tests for leptospirosis are available and in some instances are being used prior to treatment of leptospirosis-suspected patients. There is therefore a need to evaluate the cost-effectiveness of the different treatment strategies in order to avoid misuse of scarce resources and ensure best possible health outcomes for patients.

Methods

The study population was adult patients, presented with uncomplicated acute febrile illness, without an obvious focus of infection or malaria or typical dengue infection. We compared the cost and effectiveness of 5 management strategies: 1) no patients tested or given antibiotic treatment; 2) all patients given empirical doxycycline treatment; patients given doxycycline when a patient is tested positive for leptospirosis using: 3) lateral flow; 4) MCAT; 5) latex test. The framework used is a cost-benefit analysis, accounting for all direct medical costs in diagnosing and treating patients suspected of leptospirosis. Outcomes are measured in length of fever after treatment which is then converted to productivity losses to capture the full economic costs.

Findings

Empirical doxycycline treatment was the most efficient strategy, being both the least costly alternative and the one that resulted in the shortest duration of fever. The limited sensitivity of all three diagnostic tests implied that their use to guide treatment was not cost-effective. The most influential parameter driving these results was the cost of treating patients with complications for patients who did not receive adequate treatment as a result of incorrect diagnosis or a strategy of no-antibiotic-treatment.

Conclusions

Clinicians should continue treating suspected cases of leptospirosis on an empirical basis. This conclusion holds true as long as policy makers are not prioritizing the reduction of use of antibiotics, in which case the use of the latex test would be the most efficient strategy.

Author Summary

Symptoms and signs of leptospirosis are non-specific. A number of diagnostic tests for leptospirosis are available. We compared the cost-benefit of 5 management strategies: 1) no patients tested or given antibiotic treatment; 2) all patients given empirical doxycycline treatment; patients given doxycycline when a patient is tested positive for leptospirosis using: 3) lateral flow; 4) MCAT; 5) latex test. Outcomes were measured in duration of fever which is then converted to productivity losses to capture the full economic costs. Empirical doxycycline treatment was found to be the most efficient strategy, being both the least costly alternative and the one that resulted in the lowest average duration of fever. The significantly higher relative cost of using a diagnostic test as compared with presumptive treatment, and the limited sensitivity of all the diagnostic tests implied that only the latex test could be considered cost-effective when compared with the no-antibiotic-treatment option, and that all three tests were still inferior to empirical treatment.

Orientia tsutsugamushi, the cause of scrub typhus, is a major pathogen in the Asia-Pacific region. The severity of infection ranges from mild features to multiorgan failure and death. The aim of this prospective study was to define the O. tsutsugamushi loads in the blood samples of patients with scrub typhus on the day of hospital admission and to determine whether this was associated with disease severity. Quantitation was performed using a real-time PCR assay targeting the 16S rRNA gene of O. tsutsugamushi. A total of 155 patients with a confirmed diagnosis of scrub typhus had a median (interquartile range [IQR], range) O. tsutsugamushi DNA load in blood of 13 (0 to 334, 0 to 310,253) copies/ml. This included 74 patients who had undetectable bacterial loads. An analysis of bacterial load versus clinical features for all 155 patents demonstrated that duration of illness (P < 0.001), presence of eschar (P = 0.004), and concentrations of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase (P < 0.001 for all three) were positively correlated with bacterial load. Patients who died had a significantly higher bacterial load than those who survived (mean [standard deviation] values: 17,154 [12.7] versus 281 [5.2] copies/ml; P < 0.001). This study has demonstrated a relationship between bacterial load and disease severity in adults with scrub typhus.

Background

Most information on invasive Staphylococcus aureus infections comes from temperate countries. There are considerable knowledge gaps in epidemiology, treatment, drug resistance and outcome of invasive S. aureus infection in the tropics.

Methods

A prospective, observational study of S. aureus bacteraemia was conducted in a 1000-bed regional hospital in northeast Thailand over 1 year. Detailed clinical data were collected and final outcomes determined at 12 weeks, and correlated with antimicrobial susceptibility profiles of infecting isolates.

Principal Findings

Ninety-eight patients with S. aureus bacteraemia were recruited. The range of clinical manifestations was similar to that reported from temperate countries. The prevalence of endocarditis was 14%. The disease burden was highest at both extremes of age, whilst mortality increased with age. The all-cause mortality rate was 52%, with a mortality attributable to S. aureus of 44%. Methicillin-resistant S. aureus (MRSA) was responsible for 28% of infections, all of which were healthcare-associated. Mortality rates for MRSA and methicillin-susceptible S. aureus (MSSA) were 67% (18/27) and 46% (33/71), respectively (p = 0.11). MRSA isolates were multidrug resistant. Only vancomycin or fusidic acid would be suitable as empirical treatment options for suspected MRSA infection.

Conclusions

S. aureus is a significant pathogen in northeast Thailand, with comparable clinical manifestations and a similar endocarditis prevalence but higher mortality than industrialised countries. S. aureus bacteraemia is frequently associated with exposure to healthcare settings with MRSA causing a considerable burden of disease. Further studies are required to define setting-specific strategies to reduce mortality from S. aureus bacteraemia, prevent MRSA transmission, and to define the burden of S. aureus disease and emergence of drug resistance throughout the developing world.

Background

Burkholderia pseudomallei is a soil-dwelling saprophyte and the cause of melioidosis. Horizontal gene transfer contributes to the genetic diversity of this pathogen and may be an important determinant of virulence potential. The genome contains genomic island (GI) regions that encode a broad array of functions. Although there is some evidence for the variable distribution of genomic islands in B. pseudomallei isolates, little is known about the extent of variation between related strains or their association with disease or environmental survival.

Results

Five islands from B. pseudomallei strain K96243 were chosen as representatives of different types of genomic islands present in this strain, and their presence investigated in other B. pseudomallei. In silico analysis of 10 B. pseudomallei genome sequences provided evidence for the variable presence of these regions, together with micro-evolutionary changes that generate GI diversity. The diversity of GIs in 186 isolates from NE Thailand (83 environmental and 103 clinical isolates) was investigated using multiplex PCR screening. The proportion of all isolates positive by PCR ranged from 12% for a prophage-like island (GI 9), to 76% for a metabolic island (GI 16). The presence of each of the five GIs did not differ between environmental and disease-associated isolates (p > 0.05 for all five islands). The cumulative number of GIs per isolate for the 186 isolates ranged from 0 to 5 (median 2, IQR 1 to 3). The distribution of cumulative GI number did not differ between environmental and disease-associated isolates (p = 0.27). The presence of GIs was defined for the three largest clones in this collection (each defined as a single sequence type, ST, by multilocus sequence typing); these were ST 70 (n = 15 isolates), ST 54 (n = 11), and ST 167 (n = 9). The rapid loss and/or acquisition of gene islands was observed within individual clones. Comparisons were drawn between isolates obtained from the environment and from patients with melioidosis in order to examine the role of genomic islands in virulence and clinical associations. There was no reproducible association between the individual or cumulative presence of five GIs and a range of clinical features in 103 patients with melioidosis.

Conclusion

Horizontal gene transfer of mobile genetic elements can rapidly alter the gene repertoire of B. pseudomallei. This study confirms the utility of a range of approaches in defining the presence and significance of genomic variation in natural populations of B. pseudomallei.

In an evaluation of the API 20NE for the identification of Burkholderia spp., 792/800 (99%) Burkholderia pseudomallei and 17/19 (89%) B. cepacia isolates were correctly identified but 10 B. mallei and 98 B. thailandensis isolates were not correctly identified. A latex agglutination test was positive for 796/800 (99.5%) B. pseudomallei isolates and negative for 120 other oxidase-positive gram-negative bacilli.

Leptospirosis and scrub typhus are important causes of acute fever in Southeast Asia. Options for empirical therapy include doxycycline and azithromycin, but it is unclear whether their efficacies are equivalent. We conducted a multicenter, open, randomized controlled trial with adult patients presenting with acute fever (<15 days), without an obvious focus of infection, at four hospitals in Thailand between July 2003 and January 2005. Patients were randomly allocated to receive either a 7-day course of doxycycline or a 3-day course of azithromycin. The cure rate, fever clearance time, and adverse drug events were compared between the two study groups. A total of 296 patients were enrolled in the study. The cause of acute fever was determined for 151 patients (51%): 69 patients (23.3%) had leptospirosis; 57 patients (19.3%) had scrub typhus; 14 patients (4.7%) had murine typhus; and 11 patients (3.7%) had evidence of both leptospirosis and a rickettsial infection. The efficacy of azithromycin was not inferior to that of doxycycline for the treatment of both leptospirosis and scrub typhus, with comparable fever clearance times in the two treatment arms. Adverse events occurred more frequently in the doxycycline group than in the azithromycin group (27.6% and 10.6%, respectively; P = 0.02). In conclusion, doxycycline is an affordable and effective choice for the treatment of both leptospirosis and scrub typhus. Azithromycin was better tolerated than doxycycline but is more expensive and less readily available.