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We assessed the knowledge of malaria diagnosis and management by community health providers in rural Vientiane and Savannakhet Provinces, Lao PDR. Sixty health providers (17 pharmacy owners/drug sellers and 43 village health volunteers) were interviewed. All diagnosed malaria using symptoms and signs only; 14% were aware of >2 criteria for the diagnosis of severe malaria. Although chloroquine and quinine, the then recommended Lao national policy for uncomplicated malaria treatment, were the most common antimalarials prescribed - 65% gave incorrect doses and 70% did not know the side effects. Although not recommended by the then national policy, 27% of the health providers used combinations of antimalarials as they considered monotherapy ineffective. This study strongly suggests that further training of Lao rural health providers in malaria diagnosis and management is needed to improve the quality of health services in areas remote from district hospitals.

Summary 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 (α) 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 (α 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. Objectif La deficience en thiamine complique la malaria severe aPlasmodium falciparum en Thailande et peut contribuer a de l'acidose. Nous avons estime la frequence de la deficience biochimique en thiamine chez des patients presentant une malaria falciparum non compliquee dans le sud du Laos. Methode Les coefficients ‘α‘ de l'activation de la transcetolase dans les globules rouges ont ete mesures chez 310 patients avec une malaria falciparum non compliquee a leur presentation et a 42 jours apres instauration du traitement. Resultats 12% des patients avaient une evidence biochimique de deficience severe (valeurs de α > 1,31) a la presentation. Ce taux diminuait a 3% lorsque mesure 42 jours plus tard. Conclusion La deficience en thiamine etait commune chez les patients Laotiens admis avec une infection non compliquee aP. falciparum. Cette deficience a ete reduite apres traitement de la malaria et supplementation en multivitamines. Le role de cette deficience evitable et traitable dans la malaria et dans d'autres infections aigues, ainsi que l'incidence du beriberi chez les Laotiens ruraux, necessite des recherches supplementaires. Objetivo La deficiencia de tiamina complica la malaria severa por Plasmodium falciparum en Tailandia y puede contribuir a la acidosis. El objetivo de este estudio fue estimar la frecuencia de deficiencia bioquimica de tiamina en pacientes que presentan malaria no complicada por falciparum en el sur de Laos. Metodo Los coeficientes (α) de activacion de la transquetolasa de los eritrocitos fueron medidos en 310 pacientes al momento de presentarse con malaria no complicada por falciparum, asi como 42 dias despues de comenzar tratamiento. Resultados Un doce por ciento de los pacientes presentaban evidencia bioquimica de deficiencia severa (valores α > 1.31) al momento de presentarse con malaria. Este valor disminuia a un 3% 42 dias despues. Conclusion La deficiencia de tiamina era comun en pacientes de Laos admitidos con malaria no complicada por P.falciparum, y se redujo despues del tratamiento de la malaria y de la suplementacion con un complejo multivitaminico. Seria conveniente investigar mas a fondo el papel de este desorden prevenible y tratable, en malaria y otras infecciones agudas, y la incidencia de beriberi en Laos rural,.

Recent drug trials in Laos have shown high levels of Plasmodium falciparum resistance to chloroquine, but there are no published data on in vitro antimalarial drug susceptibility. We used the double-site enzyme-linked pLDH immunodetection (DELI) assay to estimate the in vitro antimalarial drug susceptibility of 108 fresh P. falciparum isolates from southern Laos. The geometric mean (95% confidence interval) 50% inhibitory concentration values (nmol/L) were 152.4 (123.8-187.6) for chloroquine, 679.8 (533.8-863.0) for quinine, 45.9 (37.9-55.7) for mefloquine, 5.0 (4.4-6.4) for artesunate, 6.3 (4.5-8.9) for dihydroartemisinin, and 59.1 (46.4-75.3) for lumefantrine. The proportion of isolates defined as resistant were 65%, 40%, and 8% for chloroquine, quinine, and mefloquine, respectively. Of 53 isolates genotyped for the pfcrt T76K chloroquine-resistance mutation, 48 (91%) were mutants. P. falciparum in Laos is multi-drug resistant; antimalarial immunity resulting from the use of ineffective chloroquine before 2005 probably contributes significantly to the therapeutic responses in clinical trials.

If copy number variants (CNVs) are predominantly deleterious, we would expect them to be more efficiently purged from populations with a large effective population size (Ne) than from populations with a small Ne. Malaria parasites (Plasmodium falciparum) provide an excellent organism to examine this prediction, because this protozoan shows a broad spectrum of population structures within a single species, with large, stable, outbred populations in Africa, small unstable inbred populations in South America and with intermediate population characteristics in South East Asia. We characterized 122 single-clone parasites, without prior laboratory culture, from malaria-infected patients in seven countries in Africa, South East Asia and South America using a high-density single-nucleotide polymorphism/CNV microarray. We scored 134 high-confidence CNVs across the parasite exome, including 33 deletions and 102 amplifications, which ranged in size from

Background Tropheryma whipplei is a bacterium commonly found in feces of young children in Africa, but with no data from Asia. We estimated the prevalence of T. whipplei carriage in feces of children in Lao PDR (Laos). Methods/Principal Findings Using specific quantitative real-time PCR, followed by genotyping for each positive specimen, we estimated the prevalence of T. whipplei in 113 feces from 106 children in Vientiane, the Lao PDR (Laos). T. whipplei was detected in 48% (51/106) of children. Those aged ≤4 years were significantly less frequently positive (17/52, 33%) than older children (34/54, 63%; p< 0.001). Positive samples were genotyped. Eight genotypes were detected including 7 specific to Laos. Genotype 2, previously detected in Europe, was circulating (21% of positive children) in 2 kindergartens (Chompet and Akad). Genotypes 136 and 138 were specific to Chompet (21% and 15.8%, respectively) whereas genotype 139 was specific to Akad (10.55%). Conclusions/Significance T. whipplei is a widely distributed bacterium, highly prevalent in feces of healthy children in Laos. Further research is needed to identify the public health significance of this finding., Author Summary Tropheryma whipplei is a common bacterium carried in feces of young children. Here, using specific PCR, we estimated the prevalence of T. whipplei in 113 feces from 106 children in Vientiane, the Lao PDR (Laos). T. whipplei was detected in 48% (51/106) of children. Eight genotypes were detected, including 7 specific to Laos. Genotype 2, previously detected in Europe, was circulating (21% of positive children) in 2 kindergartens (Chompet and Akad). Genotypes 136 and 138 were specific to Chompet (21% and 15.8%, respectively), whereas genotype 139 was specific to Akad (10.55%). Long regarded as a rare bacterium, now we can affirm that T. whipplei is a widely distributed bacterium, highly prevalent in feces including those from children in Vientiane.

In southeast Asia, the widespread high prevalence of counterfeits tablets of the vital antimalarial artesunate is of great public health concern. To assess the seriousness of this problem, we quantified the amount of active ingredient present in artesunate tablets by liquid chromatography coupled to mass spectrometry. This method, in conjunction with analysis of the packaging, classified tablets as genuine, substandard, or fake and validated results of the colorimetric Fast Red TR test. Eight (35%) of 23 fake artesunate samples contained the wrong active ingredients, which were identified as different erythromycins and paracetamol. Raman spectroscopy identified calcium carbonate as an excipient in 9 (39%) of 23 fake samples. Multivariate unsupervised pattern recognition results indicated two major clusters of artesunate counterfeits, those with counterfeit foil stickers and containing calcium carbonate, erythromycin, and paracetamol, and those with counterfeit holograms and containing starch but without evidence of erythromycin or paracetamol.

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.

Rickettsia spp. are an underrecognized cause of undifferentiated febrile illness., 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

We conducted a randomized open trial of oral chloramphenicol (50mg/kg/day in four divided doses for 14 days) versus ofloxacin (15 mg/kg/day in two divided doses for 3 days) in 50 adults with culture-confirmed uncomplicated typhoid fever in Vientiane, Laos. Patients had been ill for a median (range) of 8 (2-30) days. All Salmonella enterica serotype typhi isolates were nalidixic acid-sensitive, four (8%) were chloramphenicol-resistant and three (6%) were multidrug-resistant. Median (range) fever clearance times were 90 (24-224) hours in the chloramphenicol group and 54 (6-93) hours in the ofloxacin group (P

In 2003, a stratified random sample survey was conducted in the Lao People's Democratic Republic (Laos) to study the availability and quality of antimalarials in the private sector. In 2012, this survey was repeated to allow a statistically valid analysis of change through time. The counterfeit detection device 3 (CD-3) was used to assess packaging quality in the field and HPLC and mass spectroscopy analysis chemical analysis performed. The availability of oral artesunate monotherapies had significantly decreased from 22.9% (22) of 96 outlets in southern Laos in 2003 to 4.8% (7) of 144 outlets in 2012 (P < 0.0001). All the samples collected in the 2012 survey contained the correct active pharmaceutical ingredients (APIs) in contrast to the 21 (84%) falsified artesunate samples found in the 2003 survey. Although none of the medicines found in 2012 survey had evidence for falsification, 25.4% (37) of the samples were outside the 90-110% pharmacopeial limits of the label claim, suggesting that they were substandard or degraded. Results obtained from this survey show that patients are still exposed to poorly manufactured drugs or to ineffective medicines such as chloroquine. The quality of artemisinin-based combination therapies (ACTs) used in Laos needs to be monitored, since falsified ACTs would have devastating consequences in public health. Fil: Tabernero, Patricia. Lao Oxford Mahosot Hospital; China Fil: Mayxay, Mayfong. University of Oxford; Reino Unido Fil: Culzoni, Maria Julia. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Dwivedi, Prabha. University Of Heath Sciences; China Fil: Swamidoss, Isabel. U.S. Centers for Disease Control and Prevention; Estados Unidos Fil: Allan, Elizabeth Louise. Georgia Institute of Techology; Estados Unidos Fil: Khanthavong, Maniphone. U.S. Centers for Disease Control and Prevention; Estados Unidos Fil: Phonlavong, Chindaphone. U.S. Centers for Disease Control and Prevention; Estados Unidos Fil: Vilayhong, Chantala. Centre of Malariology, Parasitology and Entomology; China Fil: Yeuchaixiong, Sengchanh. Centre of Malariology, Parasitology and Entomology; China Fil: Sichanh, Chanvilay. Bureau of Food and Drug Inspection; China Fil: Sengaloundeth, Sivong. Food and Drug Department; China Fil: Kaur, Harparkash. University of London; Reino Unido Fil: Fernandez, Facundo M.. Georgia Institute of Techology; Estados Unidos Fil: Green, Michael D.. National Center for Infectious Diseases; Estados Unidos Fil: Newton, Paul N.. University of Oxford; Reino Unido

Mechanisms propelling drug resistance If it were to spread, resistance to the drug artemisinin would seriously derail the recent gains of global malaria control programs (see the Perspective by Sibley). Mutations in a region called the K13-propeller are predictive for artemisinin resistance in Southeast Asia. Mok et al. looked at the patterns of gene expression in parasites isolated from more than 1000 patients sampled in Africa, Bangladesh, and the Mekong region. A range of mutations that alter protein repair pathways and the timing of the parasite's developmental cycle were only found in parasites from the Mekong region. Straimer et al. genetically engineered the K13 region of parasites obtained from recent clinical isolates. Mutations in this region were indeed responsible for the resistance phenotypes. Science , this issue p. 431 , p. 428 ; see also p. 373

The etiology of fever in rural Lao People's Democratic Republic (Laos) has remained obscure until recently owing to the lack of laboratory facilities. We conducted a study to determine the causes of fever among 229 patients without malaria in Savannakhet Province, southern Laos; 52% had evidence of at least one diagnosis (45% with single and 7% with apparent multiple infections). Among patients with only one diagnosis, dengue (30.1%) was the most common, followed by leptospirosis (7.0%), Japanese encephalitis virus infection (3.5%), scrub typhus (2.6%), spotted fever group infection (0.9%), unspecified flavivirus infection (0.9%), and murine typhus (0.4%). We discuss the empirical treatment of fever in relation to these findings.

We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population.

Indirect clinical measures assessing anti-malarial drug transmission-blocking activity in falciparum malaria include measurement of the duration of gametocytaemia, the rate of gametocyte clearance or the area under the gametocytaemia-time curve (AUC). These may provide useful comparative information, but they underestimate dose-response relationships for transmission-blocking activity. Following 8-aminoquinoline administration P. falciparum gametocytes are sterilized within hours, whereas clearance from blood takes days. Gametocytaemia AUC and clearance times are determined predominantly by the more numerous female gametocytes, which are generally less drug sensitive than the minority male gametocytes, whereas transmission-blocking activity and thus infectivity is determined by the more sensitive male forms. In choosing doses of transmission-blocking drugs there is no substitute yet for mosquito-feeding studies.

Background Both endocarditis and Bartonella infections are neglected public health problems, especially in rural Asia. Bartonella endocarditis has been described from wealthier countries in Asia, Japan, Korea, Thailand and India but there are no reports from poorer countries, such as the Lao PDR (Laos), probably because people have neglected to look. Methodology/Principal Findings We conducted a retrospective (2006–2012), and subsequent prospective study (2012–2013), at Mahosot Hospital, Vientiane, Laos, through liaison between the microbiology laboratory and the wards. Patients aged >1 year admitted with definite or possible endocarditis according to modified Duke criteria were included. In view of the strong suspicion of infective endocarditis, acute and convalescent sera from 30 patients with culture negative endocarditis were tested for antibodies to Brucella melitensis, Mycoplasma pneumoniae, Bartonella quintana, B. henselae, Coxiella burnetii and Legionella pneumophila. Western blot analysis using Bartonella species antigens enabled us to describe the first two Lao patients with known Bartonella henselae endocarditis. Conclusions/Significance We argue that it is likely that Bartonella endocarditis is neglected and more widespread than appreciated, as there are few laboratories in Asia able to make the diagnosis. Considering the high prevalence of rheumatic heart disease in Asia, there is remarkably little evidence on the bacterial etiology of endocarditis. Most evidence is derived from wealthy countries and investigation of the aetiology and optimal management of endocarditis in low income countries has been neglected. Interest in Bartonella as neglected pathogens is emerging, and improved methods for the rapid diagnosis of Bartonella endocarditis are needed, as it is likely that proven Bartonella endocarditis can be treated with simpler and less expensive regimens than “conventional” endocarditis and multicenter trials to optimize treatment are required. More understanding is needed on the risk factors for Bartonella endocarditis and the importance of vectors and vector control., Author Summary Infection of heart valves (endocarditis) with bacteria is an important condition, especially afflicting those with rheumatic heart disease, and has a high mortality if untreated. Most of the evidence for optimal antibiotic and surgical management comes from wealthy countries. There are no published data from poorer countries in SE Asia despite a high burden of rheumatic heart disease. We investigated the bacterial infections of heart valves in the Lao PDR (Laos) through heart ultrasound scans and analysis of patients' blood. We provide evidence of infection with the poorly understood bacteria Bartonella henselae (the cause of cat scratch disease) in two patients from Laos. We argue that it is likely that Bartonella endocarditis is more widespread than appreciated, as there are few laboratories in Asia able to make the diagnosis. This is important as it is likely that proven Bartonella endocarditis can be treated with simpler and less expensive regimens than “conventional” endocarditis. There have been great advances in the wealthy world in the diagnosis and treatment of endocarditis but these have not been assessed or implemented in poorer countries. More evidence on the causes and optimal management of endocarditis in low income countries is needed.

The dihydropteroate synthase (dhps) genes of 44 P. malariae strains from four Asian countries were isolated. Only a limited number of polymorphisms were observed. Comparison with homologous mutations in other Plasmodium species showed that these polymorphisms are unlikely to be associated with sulfadoxine resistance.

The contribution of humoral immunity to the therapeutic response in acute falciparum malaria was assessed in a case-control study. Forty adult Thai patients with acute falciparum malaria who had subsequent recrudescent infections and 40 patients matched for age, therapeutic regimen, and disease severity who were cured by Day 28 were studied. All cured patients had positive immunoglobulin (Ig) G to ring-infected erythrocyte surface antigen (RESA) in their admission plasma, compared with only 60% of patients who failed to respond to treatment (P < 0.001). The proportion of IgM-positive cases at admission was also higher in the successfully treated group than in the group with failure (70% versus 30%) (P < 0.001). The geometric mean (95% confidence interval) reciprocal IgG titer at admission was significantly higher in cured patients (187.0 [83.5-418.3]) compared with those who experienced treatment failure (11.6 [5.1-26.5]) (P < 0.001). The patients with uncomplicated malaria who were both IgG and IgM positive at admission had significantly shorter fever clearance times and lower admission parasitemia levels compared with those who were negative (P = 0.01 and P = 0.02, respectively). The median (range) in vitro parasite multiplication rate was significantly lower in cultures containing positive anti-RESA antibody plasma compared with those containing normal plasma (0.7 [0.1-3.5] versus 2.6 [0.1-12.1]; P < 0.001). These results suggest that antimalarial antibodies may play an important supportive role in the therapeutic response to antimalarial drugs during acute falciparum malaria.

In Thailand, approximately 8% of patients treated for vivax malaria are found subsequently to have coinfection with Plasmodium falciparum. A P. falciparum histidine rich protein 2 (PfHRP-2) dipstick test was evaluated as a predictor of mixed infections with subpatent P. falciparum in a prospective study of 238 patients admitted to the hospital with acute vivax malaria. Of these, 23 (10%) had subsequent development of falciparum malaria without reexposure. Patients with cryptic P. falciparum infection had a significantly lower mean (standard deviation) hematocrit than those with P. vivax alone: 29.6 (7.6%) versus 37.2 (6.4%) (P < 0.0001). Using microscopic appearance of P. falciparum after the start of treatment as the reference standard, the PfHRP-2 test was 74% sensitive and 99% specific in predicting mixed infections with subpatent P. falciparum parasitemia at presentation. The PfHRP-2 dipstick test may be a useful adjunct to microscopy in areas where mixed infections are common.

The potential for Plasmodium falciparum histidine-rich protein-2 (PfHRP-2) dipstick tests to predict antimalarial treatment failure was investigated in a prospective study in Thailand of 38 patients admitted with severe malaria and 54 hospitalized with uncomplicated P. falciparum infections. Of these, 40 had subsequent recrudescence of their infections. Overall, 89% of patients with severe malaria and 61% of patients with uncomplicated malaria had positive PfHRP-2 dipstick tests for > 2 weeks following the start of treatment. Persistence was correlated positively with admission parasite counts, PfHRP-2 intensity scores and disease severity. PfHRP-2 tests which remained positive for > 2 weeks and PfHRP-2 reactive intensity scores on admission, at day 7 and day 14 did not predict treatment failure independent of admission parasitaemia. Freezing and thawing the blood samples did not significantly affect PfHRP-2 results tested by the dipstick technique. The PfHRP-2 dipstick test provides a useful indicator of recent severe malaria, but does not predict the therapeutic response.

BACKGROUND: In a recent study one third of Lao patients presenting with uncomplicated Plasmodium falciparum malaria had biochemical evidence of thiamin deficiency, which was associated with a higher incidence of adverse events. Thiamin supplementation might, therefore, reduce adverse events in this population. METHODS: An exploratory, double-blind, parallel group, placebo-controlled, superiority trial of thiamin supplementation in patients of all ages with uncomplicated and severe falciparum malaria was conducted in Xepon District, Savannakhet Province, southern Laos. Patients were randomly assigned to either oral thiamin 10 mg/day for 7 days immediately after standard anti-malarial treatment then 5 mg daily until day 42, or identical oral placebo. RESULTS: After interim analyses when 630 patients (314 in thiamin and 316 in placebo groups) had been recruited, the trial was discontinued on the grounds of futility. On admission biochemical thiamin deficiency (alpha ≥ 25%) was present in 27% of patients and 9% had severe deficiency (alpha > 31%). After 42 days of treatment, the frequency of thiamin deficiency was lower in the thiamin (2%, 1% severe) compared to the placebo (11%, 3% severe) groups (p 0.05). Clinical, haematological, and parasitological responses to treatment did not differ significantly between the two groups. CONCLUSION: Thiamin supplementation reduced biochemical thiamin deficiency among Lao malaria patients following anti-malarial drug treatment, but it did not reduce the frequency of adverse events after anti-malarial therapy or have any detected clinical or parasitological impact. TRIAL REGISTRATION: ISRCTN 85411059.

BACKGROUND: The emergence of Plasmodium falciparum resistance to artemisinins in Southeast Asia threatens the control of malaria worldwide. The pharmacodynamic hallmark of artemisinin derivatives is rapid parasite clearance (a short parasite half-life), therefore, the in vivo phenotype of slow clearance defines the reduced susceptibility to the drug. Measurement of parasite counts every six hours during the first three days after treatment have been recommended to measure the parasite clearance half-life, but it remains unclear whether simpler sampling intervals and frequencies might also be sufficient to reliably estimate this parameter. METHODS: A total of 2,746 parasite density-time profiles were selected from 13 clinical trials in Thailand, Cambodia, Mali, Vietnam, and Kenya. In these studies, parasite densities were measured every six hours until negative after treatment with an artemisinin derivative (alone or in combination with a partner drug). The WWARN Parasite Clearance Estimator (PCE) tool was used to estimate "reference" half-lives from these six-hourly measurements. The effect of four alternative sampling schedules on half-life estimation was investigated, and compared to the reference half-life (time zero, 6, 12, 24 (A1); zero, 6, 18, 24 (A2); zero, 12, 18, 24 (A3) or zero, 12, 24 (A4) hours and then every 12 hours). Statistical bootstrap methods were used to estimate the sampling distribution of half-lives for parasite populations with different geometric mean half-lives. A simulation study was performed to investigate a suite of 16 potential alternative schedules and half-life estimates generated by each of the schedules were compared to the "true" half-life. The candidate schedules in the simulation study included (among others) six-hourly sampling, schedule A1, schedule A4, and a convenience sampling schedule at six, seven, 24, 25, 48 and 49 hours. RESULTS: The median (range) parasite half-life for all clinical studies combined was 3.1 (0.7-12.9) hours. Schedule A1 consistently performed the best, and schedule A4 the worst, both for the individual patient estimates and for the populations generated with the bootstrapping algorithm. In both cases, the differences between the reference and alternative schedules decreased as half-life increased. In the simulation study, 24-hourly sampling performed the worst, and six-hourly sampling the best. The simulation study confirmed that more dense parasite sampling schedules are required to accurately estimate half-life for profiles with short half-life (≤ three hours) and/or low initial parasite density (≤ 10,000 per μL). Among schedules in the simulation study with six or fewer measurements in the first 48 hours, a schedule with measurements at times (time windows) of 0 (0-2), 6 (4-8), 12 (10-14), 24 (22-26), 36 (34-36) and 48 (46-50) hours, or at times 6, 7 (two samples in time window 5-8), 24, 25 (two samples during time 23-26), and 48, 49 (two samples during time 47-50) hours, until negative most accurately estimated the "true" half-life. For a given schedule, continuing sampling after two days had little effect on the estimation of half-life, provided that adequate sampling was performed in the first two days and the half-life was less than three hours. If the measured parasitaemia at two days exceeded 1,000 per μL, continued sampling for at least once a day was needed for accurate half-life estimates. CONCLUSIONS: This study has revealed important insights on sampling schedules for accurate and reliable estimation of Plasmodium falciparum half-life following treatment with an artemisinin derivative (alone or in combination with a partner drug). Accurate measurement of short half-lives (rapid clearance) requires more dense sampling schedules (with more than twice daily sampling). A more intensive sampling schedule is, therefore, recommended in locations where P. falciparum susceptibility to artemisinins is not known and the necessary resources are available. Counting parasite density at six hours is important, and less frequent sampling is satisfactory for estimating long parasite half-lives in areas where artemisinin resistance is present.

Scrub typhus, murine typhus, and spotted fever group rickettsia all occur in the Lao PDR (Laos) [1], [2]. Scrub typhus and murine typhus account for ∼16% and 10%, respectively, of acute undifferentiated fever in blood culture–negative adults admitted to hospital in the capital city, Vientiane [1]. However, typhus-like illnesses are significant diagnostic challenges; patients with leptospirosis, dengue, typhoid, and malaria are also common and can present with similar symptoms and signs. Although these pathogens are common and mixed (or concurrent) infections are expected, the laboratory diagnosis of mixed infection is a vexed subject. Reports of mixed infections often use only serological criteria. The problems of antibody persistence and interspecies cross-reaction raise uncertainty as to whether these results represent true mixed infections, sequential infections, or cross-reactions. We report a patient with concurrent scrub typhus and murine typhus, demonstrated by dual PCR positivity, and discuss evidence for identifying mixed infections.

Background Respiratory illness (RI) remains a public health problem in Laos, but little is known about the overall burden and people’s healthcare-seeking behavior for RI. Understanding the burden of RI and community patterns of healthcare-seeking behavior would provide better guidance for Lao public health program and policy planners to improve RI public health practice, surveillance systems, and prevention strategies. Methods A quantitative and qualitative survey was conducted in 14 randomly selected villages of two purposively selected peri-urban and two rural provinces in Laos. A pre-designed and pre-tested questionnaire was used to collect information on RI in household members (defined as new fever with cough and/or sore-throat in the absence of other diagnoses during the preceding 30 days) from all heads of household in each village. Sixteen focus group discussions were conducted to obtain more information to support the quantitative survey. Results Among 1,751 households (9,114 people) studied, 3.5% (317/9,114) had experienced RI (fever, cough, and/or sore-throat) in the 30 days before the survey [6.2% in rural and 2.4% in peri-urban areas (p

BACKGROUND: Malaria morbidity and mortality have been significantly reduced through the proper use of insecticide-treated mosquito nets, but the extra protection afforded by the insecticide diminishes over time. The insecticide depletion rates vary according to location where wash frequency and wear are influenced by cultural habits as well as the availability of water. Monitoring of available insecticides on the net surface is essential for determining the effective life of the net. Therefore, a rapid and inexpensive colorimetric field test for cyanopyrethroids (Cyanopyrethroid Field Test or CFT) was used to measure surface levels of deltamethrin on insecticide-coated polyester nets (PowerNets™) in rural Lao PDR over a two-year period. METHODS: Net surface levels of deltamethrin were measured by wiping the net with filter paper and measuring the adsorbed deltamethrin using the CFT. A relationship between surface levels of deltamethrin and whole net levels was established by comparing results of the CFT with whole levels assayed by high-performance liquid chromatography (HPLC). An effective deltamethrin surface concentration (EC80) was determined by comparing mosquito mortality (WHO Cone Test) with CFT and HPLC results. Five positions (roof to bottom) on each of 23 matched nets were assayed for deltamethrin surface levels at 6, 12, and 24 months. Mosquito mortality assays (WHO Cone Tests) were performed on a subset of eleven 24-month old nets and compared with the proportion of failed nets as predicted by the CFT. RESULTS: At six months, the nets retained about 80% of the baseline (new net) levels of deltamethrin with no significant differences between net positions. At 12 months, ~15-40%, and at 24 months CONCLUSION: The CFT is a useful and accurate indicator of net efficacy and may be substituted for mosquito bioassays.

Background Mass drug (antimalarial) administration (MDA) is currently under study in Southeast Asia as part of a package of interventions referred to as targeted malaria elimination (TME). This intervention relies on effective community engagement that promotes uptake and adherence in target communities (above 80%). Objective Based on the experienced of designing and implementing the community engagement for TME in Laos, in this article we aim to present the elements of effective community engagement for mass antimalarial administration. Methods The design and implementation of community engagement, which took place from September 2015 to August 2016 was recorded as field notes, meeting minutes and photographs. These data underwent qualitative content analysis. Results The community engagement strategy that accompanied TME in Laos was successful in terms of contributing to high levels of participation in mass anti-malarial administration (above 85%). Based on the experience of designing and implementing the community engagement, five key elements were identified: (1) stakeholder and authority engagement, which proceeded from national level, to regional/district and local level; (2) local human resources, particularly the recruitment of local volunteers who were integral to the design and implementation of activities in the study villages; (3) formative research, to rapidly gain insight into the local social and economic context; (4) responsiveness whereby the approach was adapted according to the needs of the community and their responses to the various study components; and (5) sharing control/leadership with the community in terms of decisions on the organization of TME activities. Conclusions The community engagement that accompanied TME in Laos had to deal with challenges of implementing a complex study in remote and linguistically isolated villages. Despite these challenges, the study recorded high population coverage. Lessons learnt from this experience are useful for studies and intervention programs in diverse contexts.

In Vientiane, Laos, on July 21, 2016, the University of Health Sciences, Ministry of Health, in collaboration with the Lao–Oxford–Mahosot Hospital–Wellcome Trust Research Unit, organised the first Science Café event in Laos. More than 50 students and staff of the Faculties of Medicine, Pharmacy, Dentistry, Nursing Sciences, Medical Technology, Basic Sciences, and Public Health attended the 2 h long event on Medical and Research Ethics. Lively discussions included what makes a research study ethical, what makes consent valid, and whether children should be involved as participants in clinical research.

Background Mycetoma is a chronic, localized, slowly progressing infection of the cutaneous and subcutaneous tissues caused either by fungi (eumycetoma or implantation mycosis) or by aerobic actinomycetes (actinomycetoma). It is acquired by traumatic implantation, most commonly in the tropics and subtropics, especially in rural agricultural communities. Although well recognized elsewhere in Asia, it has not been reported from the Lao People’s Democratic Republic (Laos). Case presentation A 30 year-old female elementary school teacher and rice farmer from northeast Laos was admitted to Mahosot Hospital, Vientiane, with a massive growth on her left foot, without a history of trauma. The swelling had progressed slowly but painlessly over 5 years and multiple draining sinuses had developed. Ten days before admission the foot had increased considerably in size and became very painful, with multiple sinuses and discharge, preventing her from walking. Gram stain and bacterial culture of tissue biopsies revealed a branching filamentous Gram-positive bacterium that was subsequently identified as Actinomadura madurae by 16S rRNA gene amplification and sequencing. She was treated with long-term co-trimoxazole and multiple 3-week cycles of amikacin with a good therapeutic response. Conclusion We report the first patient with actinomycetoma from Laos. The disease should be considered in the differential diagnosis of chronic skin and bone infections in patients from rural SE Asia.

Background The emergence of artemisinin resistance in South East Asia threatens the efficacy of artemisinin derivatives (AD). Since the pharmacodynamic hallmark of AD is rapid parasite clearance, the clinical phenotype of slow clearance characterises resistance. This indicator remains critically important to monitor the extent of the problem in the absence of molecular marker(s) associated with artemisinin resistance and lack of sensitivity of current in vitro tests. Frequent parasite counts are needed to define clearance rate but it is uncertain what sampling frequency is required to ensure reliable estimates.

Background In 2007, a regulation on smoke-free health facilities and institutions was adopted by the Lao government. Little is known about health policymakers’ knowledge and opinions regarding tobacco policy control, including physicians’ behaviour. This paper aims to describe the knowledge of Lao health policymakers and their opinions regarding physicians tobacco use and national smoking policy control. Methods In 2007, we made a qualitative explorative study with data from a purposive sample of 18 key informants through semi-structured, face-to-face interviews. The key informants, who were heads of departments, directors of hospitals and directors of centres, mainly worked at the national level, and some provincial levels. Content analysis was used. Results Policymakers perceived the inadequate implementation of a smoke-free regulation and policy as being a barrier and that the general public may not accept physicians smoking, since they are regarded as role models. Most of the respondents mentioned that regulations or laws related to control of smoking in health institutions are available in Laos, but they lacked detailed knowledge of them probably because regulations as well as the smoke-free policy documents were not widely disseminated. The respondents agreed that anti-smoking education should be integrated in the training curricula, especially in the medical schools, and that the provision of counselling on health consequences from smoking and methods of smoking cessation was important. Conclusion This study contributes to tobacco policy evidence and to knowledge regarding factors related to the uptake of evidence into policymaking. Dissemination and implementation of a tobacco control policy nationally, and integration of tobacco cessation training programs in the curricula were found to be productive approaches for improvement.

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.

Narrowing Down Artemisinin Resistance Knowing that antimalarial drug resistance is characterized by selective sweeps and reduced diversity around resistance mutations, Cheeseman et al. (p. 79 ) looked for signatures of selection in a modified genome-wide association study in parasite populations from Cambodia, Laos, and Thailand. Thirty-three regions showed evidence of selection and enrichment of known antimalarial resistance genes. Fine-mapping of parasite samples taken during the past decade narrowed the association down to a 35-kb region of seven genes on chromosome 13 that seemed to explain at least 35% of the observed reduction in parasite clearance rate. However, the absence of strong candidate mutations suggests the involvement of noncoding regulatory mutations.

Background The Lao Government changed the national policy for uncomplicated Plasmodium falciparum malaria from chloroquine to artemether-lumefantrine (AL) in 2005. Since then, no information on AL efficacy has been reported. With evidence of resistance to artemisinin derivatives in adjacent Cambodia, there has been a concern as to AL efficacy. Monitoring of AL efficacy would help the Lao Government to make decisions on appropriate malaria treatment. Methods The efficacy of a three-day, twice daily oral artemether-lumefantrine for the treatment of uncomplicated falciparum malaria in Xepon District, Savannakhet Province, southern Laos was studied over 42 days follow-up. This was part of a trial of thiamin supplementation in falciparum malaria. Results Of 630 patients with P. falciparum enrolled in the trial of thiamin treatment, 549 (87%, 357 children ≤15 years and 192 adults) were included in this study. The per protocol 42-day cure rates were 97% (524/541) [96% (337/352) for children and 99% (187/189) for adults, p = 0.042]. By conventional intention-to-treat analysis, the 42-day cure rates adjusted for re-infection, were 97% (532/549) [96% (342/357) in children and 99% (190/192) in adults, p = 0.042]. The proportion of patients who remained parasitaemic at day 1 after treatment was significantly higher in children [33% (116/356)] compared to adults [15% (28/192)] (p Conclusions The current nationally-recommended anti-malarial treatment (artemether-lumefantrine) remains highly efficacious for the treatment of uncomplicated falciparum malaria five years after introduction in Laos. Regular monitoring is required in case artemisinin-resistant P. falciparum parasites should appear. Trial registration ISRCTN85411059.

The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence—archeological and genetic—suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade.

Widespread use of antibiotics may be important in the spread of antimicrobial resistance. We estimated the proportion of Lao in- and outpatients who had taken antibiotics before medical consultation by detecting antibiotic activity in their urine added to lawns of Bacillus stearothermophilus, Escherichia coli, and Streptococcus pyogenes. In the retrospective (N = 2,058) and prospective studies (N = 1,153), 49.7% (95% confidence interval [CI] = 47.4-52.0) and 36.2% (95% CI = 33.4-38.9), respectively, of Vientiane patients had urinary antibiotic activity detected. The highest frequency of estimated antibiotic pre-treatment was found in patients recruited with suspected central nervous system infections and community-acquired septicemia (both 56.8%). In Vientiane, children had a higher frequency of estimated antibiotic pre-treatment than adults (60.0% versus 46.5%; P < 0.001). Antibiotic use based on patients histories was significantly less frequent than when estimated from urinary antibiotic activity (P < 0.0001).

We investigated the significance and risk factors of bowel obstruction caused by the consumption of wild bananas (BOWB) in Laos. Of six patients with BOWB in Luang Namtha, North Laos, five required enterotomy for phytobezoars. All had eaten wild banana (WB) seeds. Of 227 other patients/relatives: 91.2% had eaten WB; 46.3% had also eaten the seeds and 45.4% knew of complications resulting from eating WB; 42.3% were aware of the complications of ingesting the seeds (constipation [37.9%], appendicitis/abdominal pain/vomiting [2.6% each] and bloated stomach/death [1.3% each]). Middle/highland Lao ethnicity was associated with WB and seed consumption (odds ratio [OR] 9.91 and 2.33), male sex with WB consumption and unawareness (OR 4.31 and 1.78). At all surgically-equipped hospitals in Laos, 33/44 doctors knew of BOWB, describing patients as young adults (16/30), male (24/30) and from middleland Lao (18/30). Countrywide, 46/48 patients with BOWB required laparotomy in 2009 (incidence 0.8/100,000). All consumed WB seeds. BOWB is widespread in Laos, especially among young middleland Lao men consuming WB seeds on an empty stomach.

Background Beriberi occurs in Vientiane, Lao PDR, among breastfed infants. Clinical disease may be the tip of an iceberg with subclinical thiamin deficiency contributing to other illnesses. Thiamin treatment could improve outcome. Methodology/Principal Findings A cohort of 778 sick infants admitted during one year without clinical evidence of beriberi were studied prospectively and erythrocyte transketolase assays (ETK) performed. Biochemical thiamin deficiency was defined both in terms of the activation coefficient (α>31%) and basal ETK activity 31% and 13.4 % basal ETK, Author Summary Infantile beriberi, or clinical thiamin (vitamin B1) deficiency in infants, is a forgotten disease in Asia, where 100 years ago it was a major public health problem. Infants with this deficiency, commonly aged ∼ 2–3 months, present in cardiac failure but usually rapidly improve if given thiamin injections. It remains relatively common in Vientiane, Lao PDR (Laos), probably because of prolonged intra- and post-partum food avoidance behaviours. Clinical disease may be the tip of an iceberg with subclinical thiamin deficiency contributing to sickness in infants without overt clinical beriberi. We therefore recruited 778 sick infants admitted during one year at Mahosot Hospital, Vientiane, without clinical evidence of beriberi, and performed erythrocyte transketolase (ETK) assays. 13.4 % of infants had basal ETK

We conducted an open, randomized clinical trial of oral dihydroartemisinin-piperaquine (DP) versus artesunate-mefloquine (AM) in 300 patients in Laos with uncomplicated Plasmodium falciparum malaria as part of a multicentre study in Asia. Survival analysis and adjustment for re-infection showed that the 63-day cure rates (95% confidence interval [CI]) were 100% for AM and 99.5% (96.4-99.8%) for DP. The 63-day cure rates per protocol were 99% (97 of 98) for AM and 99.5% (196 of 197) for DP (P = 0.55). The difference (AM minus DP) in cure rates (95% CI) was -0.5% (-5.1 to 2.0%), which is within the 5% non-inferiority margin. The median fever and parasite clearance times were also similar for AM and DP. The proportion of patients with at least one recorded potential adverse event was significantly higher in the AM group (38 of 87, 44%) than in the DP group (57 of 182, 31%) (relative risk = 0.6, 95% CI = 0.4-0.9; P = 0.04). Dihydroartemisinin-piperaquine is not inferior to AM in the treatment of uncomplicated P. falciparum malaria in Laos and is associated with fewer adverse effects. The results of this study were similar to those of the larger multicentre study.

Comparative genomic hybridization studies have revealed elevated copy number (CN) at the reticulocyte-binding protein 1 gene (PfRh1) in fast growing lab-adapted parasites, while genetic manipulation demonstrates a causal link between cell invasion and PfRh1 CN. We therefore examined PfRh1 copy number variation (CNV) in 202 single clone parasite isolates from four countries to quantify the extent of CNV within natural populations. Surprisingly, we found that no natural parasite infections showed elevated CN. In contrast, 4/28 independent laboratory reference strains show elevated CN. One possibility is that amplification of PfRh1 (or neighboring loci) is selected during laboratory culture. In the case of FCR3 group of parasites, clone trees show that PfRh1 amplification arose in laboratory lines following establishment in culture. These data show that CNV at PfRh1 is rare or non-existent in natural populations, but can arise during laboratory propagation. We conclude that PfRh1 CNV is not an important determinant of gene expression, cell invasion or growth rate in natural parasite populations.

Neorickettsia sennetsu has been described from Japan and Malaysia, causing a largely forgotten infectious mononucleosis-like disease. Because it is believed to be contracted from eating raw fish, frequently consumed in the Lao PDR, we looked for evidence of N. sennetsu among Lao patients and fish. A buffy coat from 1 of 91 patients with undifferentiated fever was positive by 16S rRNA amplification and sequencing and real-time polymerase chain reactions (PCR) targeting two N. sennetsu genes. Lao blood donors and patients with fever, hepatitis, or jaundice (N = 1,132) had a high prevalence (17%) of immunofluorescence assay IgG anti-N. sennetsu antibodies compared with 4% and 0% from febrile patients (N = 848) in Thailand and Malaysia, respectively. We found N. sennetsu DNA by PCR, for the first time, in a fish (Anabas testudineus). These data suggest that sennetsu may be an under-recognized cause of fever and are consistent with the hypothesis that it may be contracted from eating raw fish.

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]).

We describe an adult with uncomplicated Plasmodium falciparum malaria who did not improve clinically despite 5 days of intramuscular artemether therapy. He was prescribed a lower dose (kg body weight) than that recommended, and a vial from the packet contained only 74% of the artemether dose as stated by the manufacturer. The combination of underdosing, poor-quality drug, and the intrinsic low bioavailability of artemether may have contributed to his poor clinical response. Analysis of the packaging and chemical "fingerprinting" of the artemether suggested that the drug was genuine but was either substandard or had deteriorated after manufacture.

BACKGROUND: Malaria is a very important cause of anaemia in tropical countries. Anaemia is assessed either by measurement of the haematocrit or the haemoglobin concentration. For comparisons across studies, it is often necessary to derive one measure from the other. METHODS: Data on patients with slide-confirmed uncomplicated falciparum malaria were pooled from 85 antimalarial drug trials conducted in 25 different countries, to assess the haemoglobin/haematocrit relationship at different time points in malaria. Using a linear random effects model, a conversion equation for haematocrit was derived based on 3,254 measurements from various time points (ranging from day 0 to day 63) from 1,810 patients with simultaneous measurements of both parameters. Haemoglobin was also estimated from haematocrit with the commonly used threefold conversion. RESULTS: A good fit was obtained using Haematocrit = 5.62 + 2.60 * Haemoglobin. On average, haematocrit/3 levels were slightly higher than haemoglobin measurements with a mean difference (+/- SD) of -0.69 (+/- 1.3) for children under the age of 5 (n = 1,440 measurements from 449 patients). CONCLUSION: Based on this large data set, an accurate and robust conversion factor both in acute malaria and in convalescence was obtained. The commonly used threefold conversion is also valid., Epicentre

We describe two patients who developed severe illness after eating the skin and eggs of a toad, probably Bufo melanostictus Schneider, in southeastern Laos. One boy died, and one developed a digoxin toxicity-like syndrome with bradycardia and heart failure but survived. A telephone survey of 16 Lao provincial hospitals suggested that toad poisoning occurs in at least six provinces. That 93% of villagers in three villages in southeastern Laos were aware that toads are poisonous but that 51% had encountered patients with toad toxicity suggests that the potential gravity is not appreciated. These data indicate that toad poisoning may be underestimated and that education on the seriousness of toad toxins could be a useful public health measure.

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.

Populations of Plasmodium falciparum show striking differences in linkage disequilibrium, population differentiation and diversity, but only fragmentary data exists on the genetic structure of Plasmodium vivax. We genotyped nine tandem repeat loci bearing 2-8 bp motifs from 345 P. vivax infections collected from three Asian countries and from five locations in Colombia. We observed 9-37 alleles per locus and high diversity (He=0.72-0.79, mean=0.75) in all countries. Numbers of multiple clone infections varied considerably: these were rare in Colombia and India, but > 60% of isolates carried multiple alleles in at least one locus in Thailand and Laos. However, only one or two of the nine loci show >1 allele in many samples, suggesting that mutation within infections may result in overestimation of true multiple carriage rates. Identical nine-locus genotypes were frequently found in Colombian populations, contributing to strong linkage disequilibrium. These identical genotypes were strongly clustered in time, consistent with epidemic transmission of clones and subsequent breakdown of allelic associations, suggesting high rates of inbreeding and low effective recombination rates in this country. In contrast, identical genotypes were rare and loci were randomly associated in all three Asian populations, consistent with higher rates of outcrossing and recombination. We observed low but significant differentiation between different Asian countries (standardized FST = 0.13-0.45). In comparison, we see greater differentiation between collection locations within Colombia (standardized FST = 0.4-0.7), and strong differentiation between continents (standardized FST = 0.48-0.79). The observed heterogeneity in multiple clone carriage rates, linkage disequilibrium and population differentiation are similar in some, but not all, respects to those observed in P. falciparum, and have important implications for the design of association mapping studies, and interpretation of P. vivax epidemiology.

We assessed the knowledge of malaria diagnosis and management by community health providers in rural Vientiane and Savannakhet Provinces, Lao PDR. Sixty health providers (17 pharmacy owners/drug sellers and 43 village health volunteers) were interviewed. All diagnosed malaria using symptoms and signs only; 14% were aware of2 criteria for the diagnosis of severe malaria. Although chloroquine and quinine, the then recommended Lao national policy for uncomplicated malaria treatment, were the most common antimalarials prescribed - 65% gave incorrect doses and 70% did not know the side effects. Although not recommended by the then national policy, 27% of the health providers used combinations of antimalarials as they considered monotherapy ineffective. This study strongly suggests that further training of Lao rural health providers in malaria diagnosis and management is needed to improve the quality of health services in areas remote from district hospitals.