Field evaluation of a simple fluorescence method for detection of viable Mycobacterium tuberculosis in sputum specimens during treatment follow-up.

Simple tuberculosis (TB) treatment monitoring tools are needed. We assessed the performance of fluorescein-diacetate (FDA) smear microscopy for detection of viable Mycobacterium tuberculosis in sputum specimens (n = 288) of TB cases under treatment compared to culture (17.4% culture positivity). FDA sensitivity was moderate (83.7% [95% confidence interval {CI}, 70.3 to 92.6]), and specificity was low (66.1% [59.5 to 72.2]). The good negative predictive value (94.8% [90.1 to 97.8]) and negative likelihood ratio (0.2) suggest using this method to rule out treatment failure in settings without access to culture.

Factors associated with acute renal failure in adults with severe falciparum malaria.

We conducted a retrospective study of patients with severe falciparum malaria to determine factors associated with malarial acute renal failure (MARF). We reviewed 262 medical records of adults hospitalized with severe falciparum malaria in Thailand from 2004 to 2008. The incidence of MARF in our study population was 44% (115/262); 75% (86/115) of these had MARF on admission and 25% (29/115) developed MARF during hospitalization. The majority of MARF patients presented in a hypercatabolic state (62%, 68/109) and were non-oliguric (48%, 55/115) or oliguric (44%, 51/115). Forty-six percent of MARF patients (53/115) required renal replacement therapy for a median duration of 4.5 days. Patients with MARF had significantly higher complication rates (p < 0.001), longer duration of hospitalization (p < 0.001) and a higher case fatality rate (p = 0.001). Using stepwise multiple logistic regression analysis by backward selection method, factors associated with MARF were advanced age [odds ratios (OR); 95% confidence intervals (CI) 1.037 (1.011-1.063), p = 0.005], being, referred from another hospital [2.876 (1.447-5.714), p = 0.003], an elevated total bilirubin level [(1.168 (1.101-1.241), p < 0.001], requiring inotropic drugs [4.879 (2.255-10.557), p < 0.001] and developing a hospital acquired infection [3.425; 1.406-8.343, p = 0.007]. Clinicians should be aware of these factors associated with MARF.

Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand.

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99%). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5%), followed by the wild-type A383/A553 (17.5%) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99 percent). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7 percent), 57I/58R/117T/N (1 percent), 57L/58R/117T (5.8 percent) and 58R/61M/117N (14.5 percent). The most common Pvdhfr alleles were 57I/58R/117T (77.7 percent), 57I/58R/117T/N (1 percent), 57L/58R/117T (5.8 percent) and 58R/61M/117N (14.5 percent). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5 percent), followed by the wild-type A383/A553 (17.5 percent) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

Evaluation of rapid diagnostics for Plasmodium falciparum and P. vivax in Mae Sot Malaria endemic area, Thailand.

Prompt and accurate diagnosis of malaria is the key to prevent disease morbidity and mortality. This study was carried out to evaluate diagnostic performance of 3 commercial rapid detection tests (RDTs), i.e., Malaria Antigen Pf/Pan™, Malaria Ag-Pf™, and Malaria Ag-Pv™ tests, in comparison with the microscopic and PCR methods. A total of 460 blood samples microscopically positive for Plasmodium falciparum (211 samples), P. vivax (218), mixed with P. falciparum and P. vivax (30), or P. ovale (1), and 124 samples of healthy subjects or patients with other fever-related infections, were collected. The sensitivities of Malaria Ag-Pf™ and Malaria Antigen Pf/Pan™ compared with the microscopic method for P. falciparum or P. vivax detection were 97.6% and 99.0%, or 98.6% and 99.0%, respectively. The specificities of Malaria Ag-Pf™, Malaria Ag-Pv™, and Malaria Antigen Pf/Pan™ were 93.3%, 98.8%, and 94.4%, respectively. The sensitivities of Malaria Ag-Pf™, Malaria Antigen Pf/Pan™, and microscopic method, when PCR was used as a reference method for P. falciparum or P. vivax detection were 91.8%, 100%, and 96.7%, or 91.9%, 92.6%, and 97.3%, respectively. The specificities of Malaria Ag-Pf™, Malaria Ag-Pv™, Malaria Antigen Pf/Pan™, and microscopic method were 66.2%, 92.7%, 73.9%, and 78.2%, respectively. Results indicated that the diagnostic performances of all the commercial RDTs are satisfactory for application to malaria diagnosis.

Study on association between genetic polymorphisms of haem oxygenase-1, tumour necrosis factor, cadmium exposure and malaria pathogenicity and severity.

BACKGROUND: Malaria is the most important public health problems in tropical and sub-tropical countries. Haem oxygenase (HO) enzyme and the pro-inflammatory cytokine tumour necrosis factor (TNF) have been proposed as one of the factors that may play significant role in pathogenicity/severity of malaria infection. HO is the enzyme of the microsomal haem degradation pathway that yields biliverdin, carbon monoxide, and iron. In this study, the association between malaria disease pathogenicity/severity and (GT)n repeat polymorphism in the promoter region of the inducible HO-1 including the effect of cadmium exposure (potent inducer of HO-1 transcription) as well as polymorphism of TNF were investigated. METHODS: Blood samples were collected from 329 cases non-severe malaria with acute uncomplicated Plasmodium falciparum malaria (UM) and 80 cases with Plasmodium vivax malaria (VM), and 77 cases with severe or cerebral malaria (SM) for analysis of genetic polymorphisms of HO-1 and TNF and cadmium levels. These patients consisted of 123 (25.3%) Thai, 243 (50.0%) Burmese and 120 (24.7%) Karen who were present at Mae Sot General Hospital, Mae Sot, Tak Province, Thailand. RESULTS: The number of (GT)n repeats of the HO-1 gene in all patients varied between 16 and 39 and categorized to short (S), medium (M) and long (L) GTn repeats. The genotype of (GT)n repeat of HO-1 was found to be significantly different among the three ethnic groups of patients. Significantly higher frequency of S/L genotype was found in Burmese compared with Thai patients, while significantly lower frequencies of S/S and M/L but higher frequency of M/M genotype was observed in Burmese compared with Karen patients. No significant association between HO-1 and TNF polymorphisms including the inducing effect of cadmium and malaria pathogenicity/severity was observed. CONCLUSIONS: Difference in the expression of HO-1 genotype in different ethnic groups may contribute to different severity of malaria disease. With this limited sample size, the finding of the lack of association between malaria disease pathogenicity/severity genetic polymorphisms of HO-1 (GT)n repeat as well as TNF observed in this study may not entirely exclude their possible link with malaria disease pathogenicity/severity. Further study in larger sample size is required.

Assessment of the pharmacokinetics and dynamics of two combination regimens of fosmidomycin-clindamycin in patients with acute uncomplicated falciparum malaria.

BACKGROUND: This study investigated the pharmacokinetics of fosmidomycin when given in combination with clindamycin at two dosage regimens in patients with acute uncomplicated falciparum malaria. METHODS: A total of 70 patients with acute uncomplicated Plasmodium falciparum malaria who fulfilled the enrolment criteria were recruited in the pharmacokinetic study. Patients were treated with two different dosage regimens of fosmidomycin in combination with clindamycin as follows:Group I: fosmidomycin (900 mg) and clindamycin (300 mg) every 6 hours for 3 days (n = 25); and Group II: fosmidomycin (1,800 mg) and clindamycin (600 mg) every 12 hours for 3 days (n = 54). RESULTS: Both regimens were well tolerated with no serious adverse events. The 28-day cure rates for Group I and Group II were 91.3 and 89.7%, respectively. Steady-state plasma concentrations of fosmidomycin and clindamycin were attained at about 24 hr after the first dose. The pharmacokinetics of both fosmidomycin and clindamycin analysed by model-independent and model-dependent approaches were generally in broad agreement. There were marked differences in the pharmacokinetic profiles of fosmidomycin and clindamycin when given as two different combination regimens. In general, most of the dose-dependent pharmacokinetic parameters (model-independent C(max): 3.74 vs 2.41 microg/ml; C(max-ss): 2.80 vs 2.08 microg/ml; C(max-min-ss): 2.03 vs 0.71 microg/ml; AUC: 23.31 vs 10.63 microg.hr/ml (median values) were significantly higher in patients who received the high dose regimen (Group II). However, C(min-ss) was lower in this group (0.80 vs 1.37 microg/ml), resulting in significantly higher fluctuations in the plasma concentrations of both fosmidomycin and clindamycin following multiple dosing (110.0 vs 41.9%). Other pharmacokinetic parameters, notably total clearance (CL/F), apparent volume of distribution (V/F, V(z)/F) and elimination half-life (t(1/2z), t(1/2e)) were also significantly different between the two dosage regimens. In addition, the dose-dependent pharmacokinetics of both fosmidomycin and clindamycin tended to be lower in patients with recrudescence responses in both groups. CONCLUSION: The findings may suggest that dosing frequency and duration have a significant impact on outcome. The combination of fosmidomycin (900 mg) and clindamycin (300-600 mg) administered every six hours for a minimum of five days would constitute the lowest dose regimen with the shortest duration of treatment and which could result in a cure rate greater than 95%.

Microsatellite polymorphism in the heme oxygenase-1 gene promoter is associated with susceptibility to cerebral malaria in Myanmar.

Cerebral malaria (CM) is a serious complication of Plasmodium falciparum malaria, and its pathogenesis leading to coma remains unknown. Heme oxygenase-1 (HO-1) catalyzes heme breakdown, eventually generating bilirubin, iron and carbon monoxide. The HO-1 gene promoter contains a polymorphic (GT)n repeat which may influence the expression level of HO-1. To explore the correlation between this (GT)n polymorphism and susceptibility to CM, we analyzed the frequencies of the (GT)n alleles in 120 Myanmarese patients with uncomplicated malaria (UM) and 30 patients with CM. The frequency of homozygotes for the short (GT)n alleles (<28 repeats) in CM patients was significantly higher than those in UM patients (P < 0.008, OR = 3.14). Thus, short (GT)n alleles represent a genetic risk factor for CM.

Fosmidomycin, a novel chemotherapeutic agent for malaria.

In previous studies, fosmidomycin has been shown to possess activity against Plasmodium falciparum in vitro and in the mouse model. It has a novel mode of action through inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase, an enzyme of the nonmevalonate pathway of isoprenoid biosynthesis, which is absent in humans. In this open-label, uncontrolled trial, the efficacy and safety of fosmidomycin, in an oral dose of 1,200 mg every 8 h for 7 days, were evaluated in the treatment of acute uncomplicated Plasmodium falciparum malaria in 20 adult subjects in Gabon and Thailand. Clinical assessments were performed and thick blood smears were evaluated every 8 h until parasite clearance and resolution of symptoms were achieved; assessments continued at weekly intervals thereafter for the duration of the 28-day followup period. All subjects were clinically and parasitologically cured on day 7 (primary end point). Parasite and fever clearance were rapid, with means of 44 and 41 h, respectively. On day 28, seven out of nine subjects (78%) were cured in Gabon and two out of nine subjects (22%) were cured in Thailand. The drug was well tolerated, although mild gastrointestinal side effects were recorded for five subjects. Analysis of hematological and biochemical parameters showed no clinically significant changes throughout the study. Fosmidomycin is an effective and safe antimalarial drug, although its use as a single agent is restricted by the occurrence of recrudescent infections. However, its role in combination therapy should be explored.