Kanungnit Congpuong

Selection and spread of artemisinin-resistant alleles in Thailand prior to the global artemisinin resistance containment campaign.

The recent emergence of artemisinin resistance in the Greater Mekong Subregion poses a major threat to the global effort to control malaria. Tracking the spread and evolution of artemisinin-resistant parasites is critical in aiding efforts to contain the spread of resistance. A total of 417 patient samples from the year 2007, collected during malaria surveillance studies across ten provinces in Thailand, were genotyped for the candidate Plasmodium falciparum molecular marker of artemisinin resistance K13. Parasite genotypes were examined for K13 propeller mutations associated with artemisinin resistance, signatures of positive selection, and for evidence of whether artemisinin-resistant alleles arose independently across Thailand. A total of seven K13 mutant alleles were found (N458Y, R539T, E556D, P574L, R575K, C580Y, S621F). Notably, the R575K and S621F mutations have previously not been reported in Thailand. The most prevalent artemisinin resistance-associated K13 mutation, C580Y, carried two distinct haplotype profiles that were separated based on geography, along the Thai-Cambodia and Thai-Myanmar borders. It appears these two haplotypes may have independent evolutionary origins. In summary, parasites with K13 propeller mutations associated with artemisinin resistance were widely present along the Thai-Cambodia and Thai-Myanmar borders prior to the implementation of the artemisinin resistance containment project in the region.

Field Evaluation of a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Assay, RealAmp, for the Diagnosis of Malaria in Thailand and India

BACKGROUNDnTo eliminate malaria, surveillance for submicroscopic infections is needed. Molecular methods can detect submicroscopic infections but have not hitherto been amenable to implementation in surveillance programs. A portable loop-mediated isothermal amplification assay called RealAmp was assessed in 2 areas of low malaria transmission.nnnMETHODSnRealAmp was evaluated in 141 patients from health clinics in India (passive surveillance) and in 127 asymptomatic persons in Thailand (active surveillance). The diagnostic validity, precision, and predictive value of RealAmp were determined using polymerase chain reaction (PCR) as the reference method. A pilot study of RealAmp was also performed on samples from patients presenting at a Thai health center.nnnRESULTSnA total of 96 and 7 positive cases were detected in India and Thailand, respectively, via PCR. In comparison with nested PCR, the sensitivity and specificity of RealAmp in India were 94.8% (95% confidence interval [CI], 88.3%-98.3%) and 100% (95% CI, 92.1%-100%), respectively, with correct identification of all 5 Plasmodium vivax cases. In Thailand, compared with pooled real-time PCR, RealAmp demonstrated 100% sensitivity (95% CI, 59.0%-100%) and 96.7% specificity (95% CI, 91.7%-99.1%). Testing at the health center demonstrated RealAmps potential to serve as a point-of-care test with results available in 30-75 minutes.nnnCONCLUSIONnRealAmp was comparable to PCR in detecting malaria parasites and shows promise as a tool to detect submicroscopic infections in malaria control and elimination programs worldwide.

Tracking Origins and Spread of Sulfadoxine-Resistant Plasmodium falciparum dhps Alleles in Thailand

ABSTRACT The emergence and spread of drug-resistant Plasmodium falciparum have been a major impediment for the control of malaria worldwide. Earlier studies have shown that similar to chloroquine (CQ) resistance, high levels of pyrimethamine resistance in P. falciparum originated independently 4 to 5 times globally, including one origin at the Thailand-Cambodia border. In this study we describe the origins and spread of sulfadoxine-resistance-conferring dihydropteroate synthase (dhps) alleles in Thailand. The dhps mutations and flanking microsatellite loci were genotyped for P. falciparum isolates collected from 11 Thai provinces along the Burma, Cambodia, and Malaysia borders. Results indicated that resistant dhps alleles were fixed in Thailand, predominantly being the SGEGA, AGEAA, and SGNGA triple mutants and the AGKAA double mutant (mutated codons are underlined). These alleles had different geographical distributions. The SGEGA alleles were found mostly at the Burma border, while the SGNGA alleles occurred mainly at the Cambodia border and nearby provinces. Microsatellite data suggested that there were two major genetic lineages of the triple mutants in Thailand, one common for SGEGA/SGNGA alleles and another one independent for AGEAA. Importantly, the newly reported SGNGA alleles possibly originated at the Thailand-Cambodia border. All parasites in the Yala province (Malaysia border) had AGKAA alleles with almost identical flanking microsatellites haplotypes. They were also identical at putatively neutral loci on chromosomes 2 and 3, suggesting a clonal nature of the parasite population in Yala. In summary, this study suggests multiple and independent origins of resistant dhps alleles in Thailand.

Compliance with a three-day course of artesunate-mefloquine combination and baseline anti-malarial treatment in an area of Thailand with highly multidrug resistant falciparum malaria

BackgroundArtemisinin-based combination therapy (ACT) is presently recommended by the World Health Organization as first-line treatment for uncomplicated Plasmodium falciparum malaria in several countries, as a mean of prolonging the effectiveness of first-line malaria treatment regimens. A three-day course of artesunate-mefloquine (4 mg/kg body weight once daily for three consecutive days, plus 15 and 10 mg/kg body weight mefloquine on the first and second days) has been adopted by Malaria Control Programme of Thailand as first-line treatment for uncomplicated falciparum malaria all over the country since 2008. The gametocytocydal anti-malarial drug primaquine is administered at the dose of 30 mg (0.6 mg/kg) on the last day. The aim of the present study was to assess patient compliance of this combination regimen when applied to field condition.MethodsA total of 240 patients (196 males and 44 females) who were attending the malaria clinics in Mae-Sot, Tak Province and presenting with symptomatic acute uncomplicated falciparum malaria, with no reappearance of Plasmodium vivax parasitaemia during follow-up were included into the study. The first dose of the treatment was given to the patients under direct supervision. All patients were given the medication for self-treatment at home and were requested to come back for follow-up on day 3 of the initial treatment. Baseline (day 0) and day 3 whole blood mefloquine and plasma primaquine concentrations were determined by high performance liquid chromatography.ResultsTwo patients had recrudescence on days 28 and 35. The Kaplan-Meier estimate of the 42-day efficacy rate of this combination regimen was 99.2% (238/240). Based on whole blood mefloquine and plasma primaquine concentrations on day 3 of the initial treatment, compliance with mefloquine and primaquine in this three-day artesunate-mefloquine combination regimen were 96.3% (207/215), and 98.5% (197/200), respectively. Baseline mefloquine and primaquine levels were observed in 24 and 16% of the patients.ConclusionThe current first-line treatment and a three-day combination regimen of artesunate-mefloquine provides excellent patient compliance with good efficacy and tolerability in the treatment of highly multidrug resistance falciparum malaria. Previous treatment with mefloquine and primaquine were common in this area.

Active Case Detection with Pooled Real-Time PCR to Eliminate Malaria in Trat Province, Thailand

We conducted contact tracing and high-risk group screening using pooled real-time polymerase chain reaction (PCR) to support malaria elimination in Thailand. PCR detected more Plasmodium infections than the local and expert microscopists. High-throughput pooling technique reduced costs and allowed prompt reporting of results.

SYBR Green I and Taqman Quantitative Real-Time Polymerase Chain Reaction Methods for the Determination of Amplification of Plasmodium falciparum Multidrug Resistance-1 Gene (PFMDR1)

abstract:u2003 The pfmdr1 gene, which encodes P-glycoprotein homolog 1, has been shown to be a reliable marker of resistance for Plasmodium falciparum related to artesunate and mefloquine combination therapy. The aims of this study are to investigate the copy number of pfmdr1 in P. falciparum isolates collected from the 4 malaria-endemic areas of Thailand (Kanchanaburi, Mae Hongson, Ranong, and Tak) along the Thailand–Myanmar (Burma) border (Thai–Myanmar border) by using SYBR Green I and the standard method TaqMan real-time polymerase chain reaction (RT-PCR) and to compare the efficiency (sensitivity and specificity) of SYBR Green I with TaqMan RT-quantitative (q)PCR methods in determining pfmdr1 gene copy number. Ninety-six blood samples were collected onto filter paper from patients with uncomplicated falciparum malaria who attended malaria clinics in the Kanchanaburi (n u200a=u200a 45), Mae Hongson (n u200a=u200a 18), Ranong (n u200a=u200a 11), and Tak (n u200a=u200a 22) provinces in Thailand. Parasite genomic DNA was extracted from dried blood spots by using QIAcube™ automated sample preparation. Pfmdr1 gene copy number was determined by TaqMan (63 samples) and SYBR Green I (96 samples) real-time PCR. Seventy-one (74.0%), 14 (14.6%), 10 (10.4%), and 1 (1%) isolates carried 1, 2, 3, and 4 pfmdr1 gene copies, respectively. Forty-three of 48 (89.6%), 6 of 11 (54.5%), and 3 of 4 (75.0%) samples, respectively, showed agreement with results of 1, 2, and 3 pfmdr1 gene copies as determined by both methods. The efficiency of SYBR Green I in identifying pfmdr1 gene copy number was found to be significantly correlated with that of TaqMan. Considering its simplicity and relatively low cost, SYBR Green I RT-qPCR is therefore a promising alternative technique for the determination of pfmdr1 copy number.

Pharmacodynamic interaction between mefloquine and retinol in Plasmodium falciparum in vitro.

ZusammenfassungMefloquin, ein 4-Chinolinmethanol, wurde in Thailand zur Behandlung der Malaria Tropica eingeführt nachdem Plasmodium falciparum fast universelle Resistenz gegen die 4-Aminochinoline und Folsäurehemmer entwickelt hatte. Obwohl auch Resistenz gegen Mefloquin zu einem Problem wurde, war Artemisinin-basierende Kombinationstherapie (ACT) mittels Mefloquine und Artesunat bis vor kurzem ausreichend wirksam. Da die Wirkung von Chinin, einem anderen 4-Chinolinmethanol, durch Retinol erheblich verstärkt wird, war eine Erweiterung der Untersuchungen auf Mefloquin naheliegend. Die Interaktion zwischen Mefloquin und Retinol, letzteres bei Konzentrationen entsprechend der 50., 65. und 80. Perzentile der physiologischen Serumkonzentrationen bei gesunden Erwachsenen, wurde bei 37 frischen Isolaten von P. falciparum untersucht. Die mittleren IC50, IC90 und IC99 Werte für Mefloquin lagen bei 1,76, 9,81 und 39,78 μM, jene für Mefloquin + Retinol niedrig bei 0,33, 1,37 und 4,33 μM, jene für Mefloquin + Retinol mittel bei 0,29, 1,15 und 3,48 μM, und jene für Mefloquin + Retinol hoch bei 0,20, 0,85 und 2,70 μM. Evidenz für beträchtlichen Synergismus zwischen Mefloquin und Retinol bei P. falciparum war hoch signifikant.SummaryMefloquine, a 4-quinolinemethanol derivative, was introduced in Thailand after Plasmodium falciparum had acquired almost universal resistance to the 4-aminoquinolines and antifols. However, also resistance to mefloquine has become an increasing problem, but artemisinin-based combination therapy (ACT) with mefloquine and artesunate remained until recently sufficiently effective. Since synergistic interaction between quinine, another 4-quinolinemethanol, with retinol was observed earlier, the investigations were expanded to mefloquine. The interaction between mefloquine and retinol at concentrations equal to the 50th, 65th and 80th percentile of the physiological retinol levels in healthy adults was determined in 37 fresh isolates of P. falciparum. The mean IC50, IC90 and IC99 values for mefloquine were 1.76, 9.81 and 39.78 μM, those for mefloquine + retinol low 0.33, 1.37 and 4.33 μM, those for mefloquine + retinol medium 0.29, 1.15 and 3.48 μM, and those for mefloquine + retinol high 0.20, 0.85 and 2.70 μM. Evidence for strong synergism between mefloquine and retinol in P. falciparum was highly significant.

Efficacy of a 3-day artesunate-mefloquine combination in the treatment of uncomplicated falciparum malaria in Kanchanaburi province of Thailand

Abstract Background: In Kanchanaburi province located on the Thai-Myanmar border, Plasmodium falciparum parasites have developed significant resistance to commonly-used anti-malarials. For use against falciparum malaria, 2-day artesunate-mefloquine combination (MAS2) has recently been replaced by a 3-day artesunate-mefloquine combination (MAS3) that is an artemisinin-based combination therapy regimen recommended by the WHO. Objective: Investigate the efficacy and safety of MAS3 in the treatment of uncomplicated falciparum malaria in patients of Kanchaburi province. Methods: The study was conducted at Bongtee sub-district, Sai Yok district, Kanchanaburi province between June and November 2009. Fifty-one uncomplicated falciparum malaria patients were enrolled. Inclusion, exclusion and study method followed the WHO protocol for assessment and monitoring of antimalarial drug efficacy for the treatment of uncomplicated falciparum malaria. Patients received a MAS3 and were followed for 42 days. Results: All patients clinically recovered, but four patients were again parasitaemic on day 21, (1 patient) 28 (2 patients) and 42 (1 patient), respectively. Molecular analyses suggested that all recurrences were caused by recrudescence. There were no severe adverse events, but complaints of headache, gastrointestinal upset, nausea, and vomiting. Delay in parasite clearance was found. Proportion of parasite clearance on day 1, 2, 3 and 7 were 17.7%, 62.7%, 80.4%, and 100%, respectively. Conclusion: MAS3 is comparable to MAS2, and meet the WHO efficacy criteria for use against falciparum malaria, but the effect on parasite clearance was inferior to that of MAS2. Close monitoring evaluation is required.

Synergism between quinine and retinol in fresh isolates of Plasmodium falciparum.

ZusammenfassungDie pharmakodynamische Interaktion zwischen Retinol und Chinin wurde an 38 frischen Isolaten von Plasmodium falciparum in Thailand, in einem Gebiet mit Chininresistenz, untersucht. Die Kombination von Chinin mit Retinol in Konzentrationen wie sie der 50., 65. und 80. Perzentile der physiologischen Bandbreite bei Gesunden entsprechen, führte im Vergleich zu Chinin allein zu einer signifikanten Herabsetzung der EC50, EC90, EC99 und der GMCOC für Chinin. Die FIC-Werte für EC90 und EC99 zeigen steigenden Synergismus mit steigendem EC-Wert und Retinolanteil, und bis auf 0,2420 sinkende mittlere SFIC-Werte, typisch für ausgeprägten Synergismus.SummaryFollowing earlier reports of synergism between retinol and various antimalarial compounds, the pharmacodynamic interaction between retinol and quinine was investigated in 38 fresh isolates of Plasmodium falciparum. The study was carried out in western Thailand, an area with quinine-resistant P. falciparum. The combination of quinine with retinol in concentrations corresponding to the 50th, 65th and 80th percentile of the physiological values in healthy subjects, significantly reduced the EC50, EC90, EC99 and GMCOC for quinine. The FIC values at EC90 and EC99 indicate increasing synergism with rising EC and retinol concentration. The mean SFIC value dropped to a level as low as 0.2420, indicating strong synergism

Pharmacodynamic interaction between monodesbutyl-benflumetol and artemisinin as well as proguanil in Plasmodium falciparum in vitro

ZusammenfassungDie Sensibilität von Plasmodium falciparum gegenüber Artemisinin, Monodebutyl-Benflumetol und einer 1:3 Kombination beider Stoffe wurde bei 51 frischen Parasitenisolaten untersucht. Obwohl der Vergleich zwischen den Werten für volle Hemmung durch Artemisinin allein (63,33 nM), DBB allein (50,15 nM) und die Kombination (23,92 nM) klar synergistisches Verhalten zwischen Artemisinin und DBB anzeigte, war dies weniger ersichtlich beim Vergleich der Log-Probit Regressionen. Ferner stiegen die geometrischen Mittelwerte der Summe der fraktionellen Hemmkonzentrationen (SFIC) mit Zunahme der effektiven Hemmkonzentrationen (EC). Die Interaktion zwischen DBB und Proguanil wurde mittels einer 3:1 m/m Kombination beider Stoffe an 24 frischen Isolaten von P. falciparum untersucht. Proguanil allein hatte schwache blutschizontozide Wirkung. Die Log-Probit Regressionen zeigten für die Kombination höhere Aktivität im Vergleich zu DBB allein. Die SFIC Werte waren im Bereich von mäßigem Synergismus. Durch den Zusatz von Proguanil konnte die Wirksamkeit von DBB nahezu verdoppelt werden. Dies könnte bei möglicher künftiger therapeutischer oder prophylaktischer Anwendung von DBB vorteilhaft sein.SummaryThe sensitivity of Plasmodium falciparum against artemisinin, monodebutyl-benflumetol (DBB) and a 1:3 m/m combination of both compounds was assessed in 51 fresh parasite isolates. Although a comparison between fully inhibitory concentrations (GMCOC) of artemisinin alone (63.33 nM), DBB alone (50.15 nM) and the combination (23.92 nM) indicated significant synergism between artemisinin and DBB, this was less evident when comparing the log-probit regressions. Moreover, the geometric mean values of the fractional inhibitory concentrations (SFIC) showed a rising tendency with increasing EC level. In a study comprising 24 fresh isolates of P. falciparum, the interaction between DBB and proguanil was explored with a 3:1 m/m combination of both compounds. Proguanil alone showed weak blood schizontocidal activity. The log-probit regressions indicated higher activity of the combination as compared to DBB alone. The SFIC values indicated moderate synergism between DBB and proguanil that could be an advantage in an eventual therapeutic and prophylactic use of DBB.