Three phylogenetic groups have driven the recent population expansion of Cryptococcus neoformans.

Cryptococcus neoformans (C. neoformans var. grubii) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype.

Screen of whole blood responses to flagellin identifies TLR5 variation associated with outcome in melioidosis.

Melioidosis is a severe infection caused by the flagellated bacterium Burkholderia pseudomallei. The nonsense polymorphism TLR51174C>T is associated with improved outcome in Thais with melioidosis. We hypothesized that other TLR5 variants may modulate the host response and determine outcome in melioidosis. We genotyped 12 TLR5 variants selected de novo from the HapMap database and examined the association of each with cytokines induced by flagellin stimulation of whole blood from healthy Thai subjects. We found a blunted cytokine response for three related markers that were in linkage disequilibrium (LD) with a non-synonymous variant, TLR51846T>C. Carriers of TLR51846T>C had broadly impaired cytokine responses induced by flagellin. TLR51846T>C was associated with protection against death in melioidosis patients (odds ratio: 0.62, 95% confidence interval: 0.42-0.93, P=0.021). We observed no impairment in TLR51846C-dependent nuclear factor κB activation, however, suggesting an alternative mechanism for the effect. We found that TLR51846T>C was in strong LD with TLR51174C>T. Many of the blunted cytokine responses observed and the association of TLR51846T>C with survival in melioidosis patients may be attributable to TLR51174C>T, but we could not exclude an independent effect of TLR51846T>C. These data identify novel associations for TLR51846T>C, enhance our understanding of TLR5 genetic architecture in Thais and highlight the role of TLR5 in melioidosis.

Toll-like receptor 4 region genetic variants are associated with susceptibility to melioidosis.

Melioidosis is a tropical infection caused by the Gram-negative soil saprophyte Burkholderia pseudomallei. Despite broad exposure of northeastern 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 with 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 TLR4(1196C>T) variant was associated with protection from melioidosis when compared with non-hospitalized controls. The TLR1(742A>G) and TLR1(-7202A>G) variants were associated with melioidosis when compared with 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 with hospitalized controls. We conclude that TLR genetic variants may modulate host susceptibility to melioidosis. Confirmation of these findings and further investigation of the mechanisms are required.

An open randomized controlled trial of desmopressin and pulse dexamethasone as adjunct therapy in patients with pulmonary involvement associated with severe leptospirosis.

Pulmonary involvement in leptospirosis is emerging as a common complication of severe leptospirosis. A prospective randomized controlled trial of desmopressin or high-dose (pulse) dexamethasone as adjunctive therapy in 68 patients with pulmonary involvement associated with severe leptospirosis was conducted between July 2003 and October 2006 at five hospitals in Thailand. There were 23 patients in the desmopressin group, 22 in the pulse dexamethasone group, and 23 in a control group who received standard critical care alone. The diagnosis of leptospirosis was confirmed in 52 patients (77%). There were 15 deaths (22%), of which eight patients received desmopressin, four patients received pulse dexamethasone, and three patients received critical care alone (p 0.19). Eight patients with confirmed leptospirosis died (five patients in the desmopressin group, one in the pulse dexamethasone group and two in the control group). The mortality was not significantly different in the desmopressin group or pulse dexamethasone group compared to the control group in both intention-to-treat patients, and in patients with confirmed leptospirosis. There were no serious events associated with desmopressin treatment, although pulse dexamethasone treatment was associated with a significant increase in nosocomial infection. The results of logistic regression analysis revealed that serum bilirubin level was the only significant risk factor associated with mortality (OR 0.759, 95% CI 0.598-0.965, p 0.024). The results obtained in the present study do not support the use of either pulse dexamethasone or desmopressin as adjunct therapy for pulmonary involvement associated with severe leptospirosis.

Epidemiology and clinical aspects of rickettsioses in Thailand.

Scrub typhus and murine typhus are widespread in Thailand. Clinical manifestations of both diseases are nonspecific and vary widely. Acute undifferentiated fever (AUF), with or without organ dysfunction, is a major clinical presentation of these two diseases. The epidemiology and clinical manifestations including severe complications of scrub typhus and murine typhus in Thailand are summarized. Sixteen hundred and sixty-three patients with AUF were studied in six hospitals in Thailand between 2000 and 2003. Scrub typhus and murine typhus were diagnosed in 16.1% and 1.7% of them, respectively. Clinical spectrum of murine typhus was similar to scrub typhus. Hepatic dysfunction and pulmonary involvement were common complications. Multi-organ dysfunction mimicking sepsis syndrome occurred in 11.9% of patients with scrub typhus. The mortality of severe scrub typhus varied from 2.6% to 16.7%. Awareness that scrub typhus and murine typhus are prominent causes of AUF in adults in Thailand improves the probability of an accurate clinical diagnosis. Early recognition and appropriate treatment reduces morbidity and mortality. Results from recent clinical studies from Thailand indicated that rational antimicrobial therapy would be doxycycline in mild cases and a combination of either cefotaxime or ceftriaxone and doxycycline in severe cases. Azithromycin could be considered as an alternative treatment when doxycycline allergy is suspected. This would be either curative, or have no ill effect, in the majority of instances. Failure to improve or defervesce within 48 hours would indicate the need to perform a thorough re-evaluation of clinical findings and initial laboratory investigation results, as well as a need to change antibiotic.

Rheumatological manifestations in patients with melioidosis.

Melioidosis, an infection caused by the bacterium Burkholderia pseudomallei, has a wide range of clinical manifestations. Here, we describe rheumatological melioidosis (involving one or more of joint, bone or muscle), and compare features and outcome with patients without rheumatological involvement. A retrospective study of patients with culture-confirmed melioidosis admitted to Sappasithiprasong Hospital, Ubon Ratchathani during 2002 and 2005 identified 679 patients with melioidosis, of whom 98 (14.4%) had rheumatological melioidosis involving joint (n=52), bone (n = 5), or muscle (n = 12), or a combination of these (n=29). Females were over-represented in the rheumatological group, and diabetes and thalassemia were independent risk factors for rheumatological involvement (OR; 2.49 and 9.56, respectively). Patients with rheumatological involvement had a more chronic course, as reflected by a longer fever clearance time (13 vs 7 days, p = 0.06) and hospitalization (22 vs 14 days, p < 0.001), but lower mortality (28% vs 44%, p = 0.005). Patients with signs and symptoms of septic arthritis for longer than 2 weeks were more likely to have extensive infection of adjacent bone and muscle, particularly in diabetic patients. Surgical intervention was associated with a survival benefit, bur not a shortening of the course of infection.

Release of granzymes and chemokines in Thai patients with leptospirosis.

The plasma concentrations of granzymes are considered to reflect the involvement of cytotoxic T-cells and natural killer cells in various disease states. Interferon (IFN)-gamma-inducible protein-10 (IP-10) and monokine induced by IFN-gamma (Mig) are members of the non-ELR CXC chemokine family that act on T-cells and natural killer cells. This study revealed that the plasma concentrations of granzyme B (but not granzyme A), IP-10 and Mig were higher in 44 Thai patients with definite or possible leptospirosis than in healthy blood donors. These data suggest that activation of cell-mediated immunity is part of the early host response to leptospirosis.

In vitro-clinical correlations for amphotericin B susceptibility in AIDS-associated cryptococcal meningitis.

Reliable measures of antifungal drug susceptibility are needed. We tested the susceptibility of Cryptococcus neoformans from patients treated with amphotericin B. In vitro susceptibility employed a modified broth macrodilution method. We demonstrate a strong correlation between the quantitative measures of in vitro amphotericin B susceptibility and the quantitative response observed in patients.

The changing pattern of bloodstream infections associated with the rise in HIV prevalence in northeastern Thailand.

A survey of bloodstream infections was conducted in the large regional hospital in Ubon Ratchatani, northeastern Thailand between 1989 and 1998, during the onset of the HIV epidemic. The incidence of Staphylococcus aureus, Escherichia coli, Klebsiella/Enterobacter and Pseudomonas aeruginosa bacteraemias remained constant whereas infections caused by Burkholderia pseudomallei, non-typhoid Salmonellae, Cryptococcus neoformans, Penicillum marneffei and to a lesser extent Streptococcus pneumoniae all rose. Burkholderia pseudomallei infections were unrelated to HIV, whereas the other infections were associated directly with HIV. Group D non-typhoid Salmonellae bloodstream infections (mainly Salmonella enteritidis) rose coincident with the increase in HIV seroprevalence, and preceded the increase in the other HIV-associated infections. Other non-typhoid Salmonella bacteraemias increased two years after the rise in group D infections, and invasive yeast infections increased four years later, coincident with the increase in AIDS. Increasing Group D non-typhoid Salmonella bloodstream infections are an early warning signal of an impending rise in AIDS.

A comparison of artesunate alone with combined artesunate and quinine in the parenteral treatment of acute falciparum malaria.

In some areas clinicians have combined parenteral artesunate and quinine in the belief that the 2 drugs would be additive or synergistic in severe malaria. A randomized comparison of the effectiveness of intravenous (i.v.) artesunate versus i.v. artesunate and i.v. quinine together on parasite clearance was conducted in 1998/99 amongst 69 patients with uncomplicated and severe Plasmodium falciparum malaria in western Thailand. The parasite clearance time did not differ significantly between the 2 treatment groups (P = 0.12), but adverse events were significantly more frequent in the artesunate plus quinine group (P = 0.05). Quinine did not have a significant antipyretic effect and artesunate did not affect the electrocardiographic QTc interval. There is no benefit evident from combining parenteral administration of these 2 antimalarial drugs in the acute phase of treatment.

A comparison of the in vivo kinetics of Plasmodium falciparum ring-infected erythrocyte surface antigen-positive and -negative erythrocytes.

Ring-infected erythrocyte surface antigen (RESA)-positive, Plasmodium falciparum-negative red blood cells (RBCs) are cells from which the malaria parasite has been removed by the host without the destruction of the erythrocyte ("pitting"). The survival of RESA-RBCs in vivo was assessed in 14 severe and 6 uncomplicated falciparum malaria patients. The mean RESA-RBC life of 183 hours (95% confidence interval [CI], 136-246) was longer than the median parasite clearance time of 66 hours (range, 30-108 hours) but shorter than the mean red cell life of 1027 hours (95% CI, 840-1213) (P =.0004), with a median ratio of 0.2:1.0 (range, 0.1-0.7). The estimated median percentage of parasites pitted/body transit was 0.003% (range, 0.001%-0.05%). The rate of rise of the RESA-RBC count during the first 24 hours after antimalarial treatment was significantly faster (P =.036) and the subsequent RESA-RBC survival significantly shorter (P =.017) after treatment with an artemisinin derivative than after treatment with quinine. Parasitization of red cells leads to changes in the erythrocyte that shorten their survival even if the parasite is removed subsequently.