Genetic analysis of the dihydrofolate reductase-thymidylate synthase gene from geographically diverse isolates of Plasmodium malariae

Plasmodium malariae, the parasite responsible for quartan malaria, is transmitted in most areas of malaria endemicity and is associated with significant morbidity. The sequence of the gene coding for the enzyme dihydrofolate reductase-thymidylate synthase (DHFR-TS) was obtained from field isolates of P. malariae and from the closely related simian parasite Plasmodium brasilianum. The two sequences were nearly 100% homologous, adding weight to the notion that they represent genetically distinct lines of the same species. A survey of polymorphisms of the dhfr sequences in 35 isolates of P. malariae collected from five countries in Asia and Africa revealed a low number of nonsynonymous mutations in five codons. In five of the isolates collected from southeast Asia, a nonsynonymous mutation was found at one of the three positions known to be associated with antifolate resistance in other Plasmodium species. Five isolates with the wild-type DHFR could be assayed for drug susceptibility in vitro and were found to be sensitive to pyrimethamine (mean 50% inhibitory concentration, 2.24 ng/ml [95% confidence interval, 0.4 to 3.1]).     

Thiamin deficiency and uncomplicated falciparum malaria in Laos

OBJECTIVE: Thiamin deficiency complicates severe Plasmodium falciparum malaria in Thailand and may contribute to acidosis. We therefore estimated the frequency of biochemical thiamin deficiency in patients presenting with uncomplicated falciparum malaria in southern Laos. METHODS: Red cell transketolase activation coefficients (alpha) were measured in 310 patients presenting with uncomplicated falciparum malaria and 42 days after starting treatment. RESULTS: Twelve per cent of patients had biochemical evidence of severe deficiency (alpha values >1.31) at presentation, declining to 3% 42 days later. CONCLUSION: Thiamin deficiency was common in Lao patients admitted with uncomplicated P. falciparum infection and was reduced following treatment of malaria and multivitamin supplementation. The role of this preventable and treatable disorder in malaria and other acute infections, and the incidence of beriberi in rural Laos, needs further investigation.     

Factors associated with a measles outbreak in children admitted at Mahosot Hospital,Vientiane, Laos

Background  

In 2002 and 2003 there were large outbreaks of measles in many provinces of Laos, including in Vientiane. We therefore conducted a study to determine risk factors associated with measles amongst children admitted at Mahosot Hospital, Vientiane.     

Causes of community-acquired bacteremia and patterns of antimicrobial resistance in Vientiane, Laos

There is no published information on the causes of bacteremia in the Lao PDR (Laos). Between 2000 and 2004, 4512 blood culture pairs were taken from patients admitted to Mahosot Hospital, Vientiane, Laos, with suspected community-acquired bacteremia; 483 (10.7%) cultures grew a clinically significant community-acquired organism, most commonly Salmonella enterica serovar typhi (50.9%), Staphylococcus aureus (19.0%), and Escherichia coli (12.4%). S. aureus bacteremia was common among infants (69.2%), while children 1-5 years had a high frequency of typhoid (44%). Multi-drug-resistant S. Typhi was rare (6%). On multiple logistic regression analysis, typhoid was associated with younger age, longer illness, diarrhea, higher admission temperature, and lower peripheral white blood cell count than non-typhoidal bacteremia. Empirical parenteral ampicillin and gentamicin would have some activity against approximately 88% of clinically significant isolates at a cost of US $1.4/day, an important exception being B. pseudomallei. Bacteremic infants in this setting require an anti-staphylococcal antibiotic.     

Fake antimalarials in Southeast Asia are a major impediment to malaria control: multinational cross-sectional survey on the prevalence of fake antimalarials

OBJECTIVE: To assess the prevalence of counterfeit antimalarial drugs in Southeast (SE) Asia. DESIGN: Cross-sectional survey. SETTING: Pharmacies and shops selling antimalarial drugs in Myanmar (Burma), Lao PDR, Vietnam, Cambodia and Thailand. MAIN OUTCOME MEASURES: Proportion of artemisinin derivatives or mefloquine containing drugs of substandard quality. RESULTS: Of the 188 tablet packs purchased which were labelled as 'artesunate' 53% did not contain any artesunate. All counterfeit artesunate tablets were labelled as manufactured by 'Guilin Pharma', and refinements of the fake blisterpacks made them often hard to distinguish from their genuine counterparts. No other artemisinin derivatives were found to be counterfeited. Of the 44 mefloquine samples, 9% contained <10% of the expected amount of active ingredient. CONCLUSIONS: An alarmingly high proportion of antimalarial drugs bought in pharmacies and shops in mainland SE Asia are counterfeit, and the problem has increased significantly compared with our previous survey in 1999-2000. This is a serious threat to public health in the region.     

Combined molecular and clinical assessment of Plasmodium falciparum antimalarial drug resistance in the Lao People's Democratic Republic (Laos)

Molecular markers provide a rapid and relatively inexpensive approach for assessing antimalarial drug susceptibility. We collected 884 Plasmodium falciparum-infected blood samples from 17 Lao provinces. Each sample was genotyped for 11 codons in the chloroquine resistance transporter (pfcrt), dihydrofolate reductase (pfdhfr), and dihydropteroate synthase (pfdhps) genes. The samples included 227 collected from patients recruited to clinical trials. The pfcrt K76T mutation was an excellent predictor of treatment failure for both chloroquine and chloroquine plus sulfadoxine-pyrimethamine, and mutations in both pfdhfr and pfdhps were predictive of sulfadoxine-pyrimethamine treatment failure. In multivariate analysis, the presence of the pfdhfr triple mutation (51 + 59 + 108) was strongly and independently correlated with sulfadoxine-pyrimethamine failure (odds ratio = 9.1, 95% confidence interval = 1.4-60.2, P = 0.017). Considerable geographic heterogeneity in allele frequencies occurred at all three loci with lower frequencies of mutant alleles in southern than in northern Laos. These findings suggest that chloroquine and sulfadoxine-pyrimethamine are no longer viable therapy in this country.     

Rickettsial infections and fever, Vientiane, Laos

Rickettsial diseases have not been described previously from Laos, but in a prospective study, acute rickettsial infection was identified as the cause of fever in 115 (27%) of 427 adults with negative blood cultures admitted to Mahosot Hospital in Vientiane, Laos. The organisms identified by serologic analysis were Orientia tsutsugamushi (14.8%), Rickettsia typhi (9.6%), and spotted fever group rickettsia (2.6% [8 R. helvetica, 1 R. felis, 1 R. conorii subsp. indica, and 1 Rickettsia "AT1"]). Patients with murine typhus had a lower frequency of peripheral lymphadenopathy than those with scrub typhus (3% vs. 46%, p<0.001). Rickettsioses are an underrecognized cause of undifferentiated febrile illnesses among adults in Laos. This finding has implications for the local empiric treatment of fever.     

A prospective assessment of the accuracy of commercial IgM ELISAs in diagnosis of Japanese encephalitis virus infections in patients with suspected central nervous system infections in Laos

Japanese encephalitis virus (JEV) is a major cause of encephalitis in Asia. We estimated the diagnostic accuracy of two anti-JEV immunoglobulin M (IgM) enzyme-linked immunosorbent assays (ELISAs) (Panbio and XCyton JEVCheX) compared with a reference standard (AFRIMS JEV MAC ELISA) in a prospective study of the causes of central nervous system infections in Laos. Cerebrospinal fluid (CSF; 515 patients) and serum samples (182 patients) from those admitted to Mahosot Hospital, Vientiane, were tested. The CSF from 14.5% of acute encephalitis syndrome (AES) patients and 10.1% from those with AES and meningitis were positive for anti-JEV IgM in the reference ELISA. The sensitivities for CSF were 65.4% (95% confidence interval [CI] = 51-78) (Xcyton), 69.2% (95% CI = 55-81) (Panbio), however 96.2% (95% CI = 87-100) with Panbio Ravi criteria. Specificities were 89-100%. For admission sera from AES patients, sensitivities and specificities of the Panbio ELISA were 85.7% (95% CI = 42-100%) and 92.9% (95% CI = 83-98%), respectively.