Direk Limmathurotsakul

Increasing Incidence of Human Melioidosis in Northeast Thailand

Melioidosis is a serious community-acquired infectious disease caused by the Gram-negative environmental bacterium Burkholderia pseudomallei. A prospective cohort study identified 2,243 patients admitted to Sappasithiprasong Hospital in northeast Thailand with culture-confirmed melioidosis between 1997 and 2006. These data were used to calculate an average incidence rate for the province of 12.7 cases of melioidosis per 100,000 people per year. Incidence increased incrementally from 8.0 (95% confidence interval [CI] = 7.2–10.0) in 2000 to 21.3 (95% CI = 19.2–23.6) in 2006 (P < 0.001; χ2 test for trend). Male sex, age ≥ 45 years, and either known or undiagnosed diabetes were independent risk factors for melioidosis. The average mortality rate from melioidosis over the study period was 42.6%. The minimum estimated population mortality rate from melioidosis in 2006 was 8.63 per 100,000 people (95% CI = 7.33–10.11), the third most common cause of death from infectious diseases in northeast Thailand after human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and tuberculosis.

Direct In Vivo Assessment of Microcirculatory Dysfunction in Severe Falciparum Malaria

BACKGROUND This study sought to describe and quantify microcirculatory changes in the mucosal surfaces of patients with severe malaria, by direct in vivo observation using orthogonal polarization spectral (OPS) imaging. METHODS The microcirculation in the rectal mucosa of adult patients with severe malaria was assessed by use of OPS imaging, at admission and then daily. Comparison groups comprised patients with uncomplicated falciparum malaria, patients with bacterial sepsis, and healthy individuals. RESULTS Erythrocyte velocities were measured directly in 43 adult patients with severe falciparum malaria, of whom 20 died. Microcirculatory blood flow was markedly disturbed, with heterogeneous obstruction that was proportional to severity of disease. Blocked capillaries were found in 29 patients (67%) and were associated with concurrent hyperdynamic blood flow (erythrocyte velocity, >750 mm/s) in adjacent vessels in 27 patients (93%). The proportion of blocked capillaries correlated with the base deficit in plasma and with the concentration of lactate. Abnormalities disappeared when the patients recovered. In healthy individuals and in patients with uncomplicated malaria or sepsis, no stagnant erythrocytes were detected, and, in patients with sepsis, hyperdynamic blood flow was prominent. CONCLUSION Patients with severe falciparum malaria show extensive microvascular obstruction that is proportional to the severity of the disease. This finding underscores the prominent role that microvascular obstruction plays in the pathophysiology of severe malaria and illustrates the fundamental difference between the microvascular pathophysiology of malaria and that of bacterial sepsis.

Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis

Burkholderia pseudomallei, a highly pathogenic bacterium that causes melioidosis, is commonly found in soil in Southeast Asia and Northern Australia1,2. Melioidosis can be difficult to diagnose due to its diverse clinical manifestations and the inadequacy of conventional bacterial identification methods3. The bacterium is intrinsically resistant to a wide range of antimicrobials, and treatment with ineffective antimicrobials may result in case fatality rates (CFRs) exceeding 70%4,5. The importation of infected animals has, in the past, spread melioidosis to non-endemic areas6,7. The global distribution of B. pseudomallei and the burden of melioidosis, however, remain poorly understood. Here, we map documented human and animal cases and the presence of environmental B. pseudomallei and combine this in a formal modelling framework8–10 to estimate the global burden of melioidosis. We estimate there to be 165,000 (95% credible interval 68,000–412,000) human melioidosis cases per year worldwide, from which 89,000 (36,000–227,000) people die. Our estimates suggest that melioidosis is severely underreported in the 45 countries in which it is known to be endemic and that melioidosis is probably endemic in a further 34 countries that have never reported the disease. The large numbers of estimated cases and fatalities emphasize that the disease warrants renewed attention from public health officials and policy makers.

Melioidosis: a clinical overview

INTRODUCTION Melioidosis, an infection caused by the environmental Gram-negative bacillus Burkholderia pseudomallei, has emerged as an important cause of morbidity and mortality in Southeast Asia and northern Australia. SOURCES OF DATA a review of the literature using PubMed. AREAS OF AGREEMENT Approaches to diagnosis and antimicrobial therapy. AREAS OF CONTROVERSY Whether seroconversion signals the presence of a quiescent bacterial focus and an increase in long-term risk of melioidosis. AREAS TIMELY FOR DEVELOPING RESEARCH Melioidosis is potentially preventable, but there is a striking lack of evidence on which to base an effective prevention programme. An accurate map defining the global distribution of B. pseudomallei is needed, together with studies on the relative importance of different routes of infection. There is a marked difference in mortality from melioidosis in high-income versus lower income countries, and affordable strategies that reduce death from severe sepsis (from any cause) in resource-restricted settings are needed.

Determinants of Mortality in a Combined Cohort of 501 Patients With HIV-Associated Cryptococcal Meningitis: Implications for Improving Outcomes.

Cerebrospinal fluid fungal burden, altered mental status, and rate of clearance of infection predict acute mortality in HIV-associated cryptococcal meningitis. The identification of factors associated with mortality informs strategies to improve outcomes.

Independent association between rate of clearance of infection and clinical outcome of HIV-associated cryptococcal meningitis: analysis of a combined cohort of 262 patients.

BACKGROUND Progress in therapy for 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 cerebrospinal fluid, to differentiate the fungicidal activity of alternative antifungal drug regimens. We hypothesized that the rate of clearance of infection should also be a clinically meaningful endpoint. METHODS We combined data from cohorts of patients with human immunodeficiency virus-associated cryptococcal meningitis from Thailand, South Africa, and Uganda, for whom the 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 cerebrospinal fluid interferon-gamma levels. CONCLUSIONS The results support the use of the rate of clearance of infection 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.

Toll-Like Receptor 2 Impairs Host Defense in Gram-Negative Sepsis Caused by Burkholderia pseudomallei (Melioidosis)

Background Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis. Methods and Findings Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury. Conclusions Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.

Relationship of cerebrospinal fluid pressure, fungal burden and outcome in patients with cryptococcal meningitis undergoing serial lumbar punctures

Objectives:To assess impact of serial lumbar punctures on association between cerebrospinal fluid (CSF) opening pressure and prognosis in HIV-associated cryptococcal meningitis; to explore time course and relationship of opening pressure with neurological findings, CSF fungal burden, immune response, and CD4 cell count. Design:Evaluation of 163 HIV-positive ART-naive patients enrolled in three trials of amphotericin B-based therapy for cryptococcal meningitis in Thailand and South Africa. Methods:Study protocols required four lumbar punctures with measurements of opening pressure over the first 2 weeks of treatment and additional lumbar punctures if opening pressure raised. Fungal burden and clearance, CSF immune parameters, CD4 cell count, neurological symptoms and signs, and outcome at 2 and 10 weeks were compared between groups categorized by opening pressure at cryptococcal meningitis diagnosis. Results:Patients with higher baseline fungal burden had higher baseline opening pressure. High fungal burden appeared necessary but not sufficient for development of high pressure. Baseline opening pressure was not associated with CD4 cell count, CSF pro-inflammatory cytokines, or altered mental status. Day 14 opening pressure was associated with day 14 fungal burden. Overall mortality was 12% (20/162) at 2 weeks and 26% (42/160) at 10 weeks, with no significant differences between opening pressure groups. Conclusion:Studies are needed to define factors, in addition to fungal burden, associated with raised opening pressure. Aggressive management of raised opening pressure through repeated CSF drainage appeared to prevent any adverse impact of raised opening pressure on outcome in patients with cryptococcal meningitis. The results support increasing access to manometers in resource-poor settings and routine management of opening pressure in patients with cryptococcal meningitis.

Fool's Gold: Why Imperfect Reference Tests Are Undermining the Evaluation of Novel Diagnostics: A Reevaluation of 5 Diagnostic Tests for Leptospirosis

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.

Biological Relevance of Colony Morphology and Phenotypic Switching by Burkholderia pseudomallei

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42 degrees C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.