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Molecular characterisation of drug-resistant Plasmodium falciparum from Thailand
Dinora Lopes, Kanchana Rungsihirunrat, Fátima Nogueira, Aree Seugorn, José Gil, Virgilio E do Rosário, Pedro Cravo
Malaria Journal , 2002, DOI: 10.1186/1475-2875-1-12
Abstract: In the present study, we have established the in vitro sensitivity to CQ, mefloquine (MF), quinine (QUIN) and amodiaquine (AMQ) of 52 P. falciparum isolates collected in Thailand, and assessed the prevalence of four putative genetic polymorphisms of drug resistance, pfcrt K76T, pfmdr1 N86Y, pfmdr1 D1042N and pfmdr1 Y1246D, by PCR-RFLP.The percentage of isolates resistant to CQ, MF, and AMQ was 96% (50/52), 62% (32/52), and 58% (18/31), respectively, while all parasites were found to be sensitive to QUIN. In addition, 41 (79%) of the isolates assayed were resistant simultaneously to more than one drug; 25 to CQ and MF, 9 to CQ and AMQ, and 7 to all three drugs, CQ, MF and AMQ. There were two significant associations between drug sensitivity and presence of particular molecular markers, i) CQ resistance / pfcrt 76T (P = 0.001), and ii) MF resistance / pfmdr1 86N (P < 0.001)i) In Thailand, the high levels of CQ pressure have led to strong selection of the pfcrt 76T polymorphism and ii) pfmdr1 86N appears to be a good predictor of in vitro MF resistance.Malaria due to Plasmodium falciparum affects 300 million people and claims an estimated 1.5 million lives every year. Our present inability to synthesise a fully protective vaccine means that chemotherapy stands as the only effective measure in the control of the disease. However, in many parts of the world the parasite P. falciparum has become resistant to most drugs presently used [1], seriously undermining efforts for controlling malaria.Chloroquine (CQ) has long been the drug of choice for the treatment of malaria; however, CQ-resistant parasites are now present in most areas where malaria is endemic [2]. Chloroquine resistance is especially well established in Thailand, after having been first described in that country in the late 1950's [3]. The decline in the efficacy of chloroquine has led to the use of alternative antimalarials, such as antifolates, mefloquine and artemisinin derivatives, but parasite resistance
Longitudinal study of Plasmodium falciparum and Plasmodium vivax in a Karen population in Thailand
Waraphon Phimpraphi, Richard E Paul, Surapon Yimsamran, Supalarp Puangsa-art, Nipon Thanyavanich, Wanchai Maneeboonyang, Sutthiporn Prommongkol, Samarn Sornklom, Wutthichai Chaimungkun, Irwin F Chavez, Herve Blanc, Sornchai Looareesuwan, Anavaj Sakuntabhai, Pratap Singhasivanon
Malaria Journal , 2008, DOI: 10.1186/1475-2875-7-99
Abstract: A clinical malaria case treatment programme was carried out over a decade in a Karen community composed of seven hamlets on the Thai-Myanmar border.From 1994 to 2004, prevalence rates of both P. falciparum and P. vivax decreased by 70–90% in six of the seven study hamlets, but were unchanged in one hamlet. Overall, incidence rates decreased by 72% and 76% for P. falciparum and P. vivax respectively over the period 1999–2004. The age-incidence and prevalence curves suggested that P. vivax was more transmissible than P. falciparum despite a greater overall burden of infection with P. falciparum. Male gender was associated with increased risk of clinical presentation with either parasite species. Children (< 15 years old) had an increased risk of presenting with P. vivax but not P. falciparum.There was a considerable reduction in incidence rates of both P. vivax and P. falciparum over a decade following implementation of a case treatment programme. The concern that intervention methods would inadvertently favour one species over another, or even lead to an increase in one parasite species, does not appear to be fulfilled in this case.Over the last decade in Thailand, a concerted programme at the national level of clinical case treatment coupled with vector control programmes has led to a greatly reduced burden of malaria [1], restricting malaria transmission to the border provinces. The Tak Malaria Initiative (TMI) sought to address the malaria problem in one of these border provinces, Tak, implementing a system of early diagnosis and treatment with mefloquine-artesunate combination therapy [2]. This initiative proved most successful in reducing morbidity and mortality of P. falciparum but had seemingly little impact on P. vivax. Plasmodium vivax accounts for over half of all malaria infections outside Africa and in 2002 the Multilateral Initiative on Malaria convened a special conference, "Vivax Malaria Research: 2002 and Beyond" [3]. The conference highlighted the di
Clinical Factors for Severity of Plasmodium falciparum Malaria in Hospitalized Adults in Thailand  [PDF]
Patrick Sagaki, Vipa Thanachartwet, Varunee Desakorn, Duangjai Sahassananda, Supat Chamnanchanunt, Wirongrong Chierakul, Punnee Pitisuttithum, Prajej Ruangkanchanasetr
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0071503
Abstract: Plasmodium falciparum is a major cause of severe malaria in Southeast Asia, however, there is limited information regarding clinical factors associated with the severity of falciparum malaria from this region. We performed a retrospective case-control study to compare clinical factors and outcomes between patients with severe and non-severe malaria, and to identify clinical factors associated with the requirement for intensive care unit (ICU) admission of patients with severe falciparum malaria among hospitalized adults in Southeast Asia. A total of 255 patients with falciparum malaria in the Hospital for Tropical Diseases in Bangkok, Thailand between 2006 and 2012 were included. We identified 104 patients with severe malaria (cases) and 151 patients with non-severe malaria (controls). Patients with falciparum malaria with following clinical and laboratory characteristics on admission (1) referrals, (2) no prior history of malaria, (3) body temperature of >38.5°C, (4) white blood cell counts >10×109/μL, (5) presence of schizonts in peripheral blood smears, and (6) albumin concentrations of <3.5 g/dL, were more likely to develop severe malaria (P<0.05). Among patients with severe malaria, patients who met ≥3 of the 2010 WHO criteria had sensitivity of 79.2% and specificity of 81.8% for requiring ICU admission. Multivariate analysis identified the following as independent associated factors for severe malaria requiring ICU admission; (1) ethnicity of Thai [odds ratio (OR) = 3.601, 95% confidence interval (CI) = 1.011–12.822] or Myanmar [OR = 3.610, 95% CI = 1.138–11.445]; (2) referrals [OR = 3.571, 95% CI = 1.306–9.762]; (3) no prior history of malaria [OR = 5.887, 95% CI = 1.354–25.594]; and (4) albumin concentrations of <3.5 g/dL [OR = 7.200, 95% CI = 1.802–28.759]. Our findings are important for the clinical management of patients with malaria because it can help early identification of patients that could develop severe malaria and require ICU admission. Early identification and the timely initiation of appropriate treatments may well improve the outcomes and reduce the mortality of these patients.
Diversifying Selection on the Thrombospondin-Related Adhesive Protein (TRAP) Gene of Plasmodium falciparum in Thailand  [PDF]
Jun Ohashi, Yuji Suzuki, Izumi Naka, Hathairad Hananantachai, Jintana Patarapotikul
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0090522
Abstract: Sporozoites of Plasmodium falciparum are transmitted to human hosts by Anopheles mosquitoes. Thrombospondin-related adhesive protein (TRAP) is expressed in sporozoites and plays a crucial role in sporozoite gliding and invasion of human hepatocytes. A previous study showed that the TRAP gene has been subjected to balancing selection in the Gambian P. falciparum population. To further study the molecular evolution of the TRAP gene in Plasmodium falciparum, we investigated TRAP polymorphisms in P. falciparum isolates from Suan Phueng District in Ratchaburi Province, Thailand. The analysis of the entire TRAP coding sequences in 32 isolates identified a total of 39 single nucleotide polymorphisms (SNPs), which comprised 37 nonsynonymous and two synonymous SNPs. McDonald–Kreitman test showed that the ratio of the number of nonsynonymous to synonymous polymorphic sites within P. falciparum was significantly higher than that of the number of nonsynonymous to synonymous fixed sites between P. falciparum and P. reichenowi. Furthermore, the rate of nonsynonymous substitution was significantly higher than that of synonymous substitution within Thai P. falciparum. These results indicate that the TRAP gene has been subject to diversifying selection in the Thai P. falciparum population as well as the Gambian P. falciparum population. Comparison of our P. falciparum isolates with those from another region of Thailand (Tak province, Thailand) revealed that TRAP was highly differentiated between geographically close regions. This rapid diversification seems to reflect strong recent positive selection on TRAP. Our results suggest that the TRAP molecule is a major target of the human immune response to pre-erythrocytic stages of P. falciparum.
Comparison of PCR and microscopy for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand
Russell E Coleman, Jetsumon Sattabongkot, Sommai Promstaporm, Nongnuj Maneechai, Bousaraporn Tippayachai, Ampornpan Kengluecha, Nattawan Rachapaew, Gabriela Zollner, Robert Miller, Jefferson A Vaughan, Krongtong Thimasarn, Benjawan Khuntirat
Malaria Journal , 2006, DOI: 10.1186/1475-2875-5-121
Abstract: The study was performed from May 2000 to April 2002 in the village of Kong Mong Tha, located in western Thailand. Plasmodium vivax (PV) and Plasmodium falciparum (PF) are the predominant parasite species in this village, followed by Plasmodium malariae (PM) and Plasmodium ovale (PO). Each month, fingerprick blood samples were taken from each participating individual and used to prepare thick and thin blood films and for PCR analysis.PCR was sensitive (96%) and specific (98%) for malaria at parasite densities ≥ 500/μl; however, only 18% (47/269) of P. falciparum- and 5% (20/390) of P. vivax-positive films had parasite densities this high. Performance of PCR decreased markedly at parasite densities <500/μl, with sensitivity of only 20% for P. falciparum and 24% for P. vivax at densities <100 parasites/μl.Although PCR performance appeared poor when compared to microscopy, data indicated that the discrepancy between the two methods resulted from poor performance of microscopy at low parasite densities rather than poor performance of PCR. These data are not unusual when the diagnostic method being evaluated is more sensitive than the reference method. PCR appears to be a useful method for detecting Plasmodium parasites during active malaria surveillance in Thailand.The detection of asexual parasites by light microscopy of Giemsa-stained thick and thin films remains the standard laboratory method for the diagnosis of malaria [1,2]. Although detection of parasites in symptomatic patients reporting to local malaria clinics is the primary means used for malaria diagnosis in Thailand, use of active (cross-sectional) surveillance provides a tool for detection of patients with asymptomatic malaria and relatively low parasite rates. In Thailand, active surveillance is used in remote areas where individuals may have difficulty in reaching a malaria clinic – in this situation malaria clinic personnel make periodic visits to a given village and examine blood smears from all individ
Country-wide assessment of the genetic polymorphism in Plasmodium falciparum and Plasmodium vivax antigens detected with rapid diagnostic tests for malaria
Natacha Mariette, Céline Barnadas, Christiane Bouchier, Magali Tichit, Didier Ménard
Malaria Journal , 2008, DOI: 10.1186/1475-2875-7-219
Abstract: A country-wide assessment of polymorphism of the PfHRP2, PfHRP3, pLDH and aldolase antigens was carried out in 260 Plasmodium falciparum and 127 Plasmodium vivax isolates, by sequencing the genes encoding these antigens in parasites originating from the various epidemiological strata for malaria in Madagascar.Higher levels of polymorphism were observed for the pfhrp2 and pfhrp3 genes than for the P. falciparum and P. vivax aldolase and pldh genes. Pfhrp2 sequence analysis predicted that 9% of Malagasy isolates would not be detected at parasite densities ≤ 250 parasites/μl (ranging from 6% in the north to 14% in the south), although RDTs based on PfHRP2 detection are now recommended in Madagascar.These findings highlight the importance of training of health workers and the end users of RDTs in the provision of information about the possibility of false-negative results for patients with clinical symptoms of malaria, particularly in the south of Madagascar.Since the emergence and spread of Plasmodium falciparum parasites resistant to inexpensive anti-malarial drugs, such as chloroquine (CQ) and sulphadoxine-pyrimethamine (SP), routine malaria case management has changed in endemic countries, such as Madagascar. Malaria diagnosis in these areas – particularly in zones not well covered by healthcare facilities – was entirely based on clinical examination, with CQ widely administered for any fever with no obvious alternative cause [1-3]. Since the introduction of more effective, more expensive anti-malarial drug combinations, such as artemisinin combination therapy (ACT), the WHO recommends the establishment of an accurate biological diagnosis before treatment and the withdrawal of presumptive anti-malarial treatment for all patients other than children under the age of five years in hyperendemic areas. This change in medical practice is now a public health priority in Africa, ensuring that effective anti-malarial drugs are administered only to the patients who need them
Genetic diversity of Plasmodium vivax and Plasmodium falciparum in Honduras
Lopez Ana,Ortiz Andres,Coello Jorge,Sosa-Ochoa Wilfredo
Malaria Journal , 2012, DOI: 10.1186/1475-2875-11-391
Abstract: Background Understanding the population structure of Plasmodium species through genetic diversity studies can assist in the design of more effective malaria control strategies, particularly in vaccine development. Central America is an area where malaria is a public health problem, but little is known about the genetic diversity of the parasite’s circulating species. This study aimed to investigate the allelic frequency and molecular diversity of five surface antigens in field isolates from Honduras. Methods Five molecular markers were analysed to determine the genotypes of Plasmodium vivax and Plasmodium falciparum from endemic areas in Honduras. Genetic diversity of ama-1, msp-1 and csp was investigated for P. vivax, and msp-1 and msp-2 for P. falciparum. Allelic frequencies were calculated and sequence analysis performed. Results and conclusion A high genetic diversity was observed within Plasmodium isolates from Honduras. A different number of genotypes were elucidated: 41 (n = 77) for pvama-1; 23 (n = 84) for pvcsp; and 23 (n = 35) for pfmsp-1. Pvcsp sequences showed VK210 as the only subtype present in Honduran isolates. Pvmsp-1 (F2) was the most polymorphic marker for P. vivax isolates while pvama-1 was least variable. All three allelic families described for pfmsp-1 (n = 30) block 2 (K1, MAD20, and RO33), and both allelic families described for the central domain of pfmsp-2 (n = 11) (3D7 and FC27) were detected. However, K1 and 3D7 allelic families were predominant. All markers were randomly distributed across the country and no geographic correlation was found. To date, this is the most complete report on molecular characterization of P. vivax and P. falciparum field isolates in Honduras with regards to genetic diversity. These results indicate that P. vivax and P. falciparum parasite populations are highly diverse in Honduras despite the low level of transmission.
Transmission of malaria and genotypic variability of Plasmodium falciparum on the Island of Annobon (Equatorial Guinea)
Jorge Cano, Pedro Berzosa, Aida de Lucio, Miguel Descalzo, Leonardo Bobuakasi, Sisinio Nzambo, Melchor Ondo, Jesus N Buatiche, Gloria Nseng, Agustin Benito
Malaria Journal , 2007, DOI: 10.1186/1475-2875-6-141
Abstract: A blood sample was taken from the selected children in order to determine Plasmodium infection by microscopical examination and by semi-nested multiplex PCR. The diversity of P. falciparum circulating alleles was studied on the basis of the genes encoding for the merozoite surface proteins, MSP-1 and MSP-2 of P. falciparum.The crude parasite rate was 17% during the dry season and 60% during the rainy season. The percentage of children sleeping under a bed net was over 80% in the two surveys. During the rainy season, 33.3% of the children surveyed were anaemic at the time of the study. No association was found between the crude parasite rate, the use of bed nets and gender, and anaemia. However, children between five and nine years of age were five times less at risk of being anaemic than those aged less than one year. A total of 28 populations of the three allelic families of the msp-1 gene were identified and 39 of the msp-2 gene. The variability of circulating allelic populations is significantly higher in the rainy than in the dry season, although the multiplicity of infections is similar in both, 2.2 and 1.9 respectively.Based on the high degree of geographical isolation of the Annobon population and the apparent marked seasonality of the transmission, it is feasible to believe that malaria can be well controlled from this small African island.Plasmodium falciparum is a highly polymorphic parasite with a high antigens heterogeneity [1]. This heterogeneity may represent a major obstacle to the development of an effective vaccine [2].In general, the P. falciparum infections include a complex mixture of biologically and genetically different populations, as has been demonstrated by different techniques, including the Restriction Fragment Length Polymorphism (RFLP) [3] and the Polymerase Chain Reaction (PCR) [4]. PCR has been used to study the existing polymorphisms in various markers, such as the Merozoite Surface Proteins 1 (MSP-1) and 2 (MSP-2), the circumsporozo
Longitudinal in vitro surveillance of Plasmodium falciparum sensitivity to common anti-malarials in Thailand between 1994 and 2010
Parker Daniel,Lerdprom Rujira,Srisatjarak Wanna,Yan Guiyun
Malaria Journal , 2012, DOI: 10.1186/1475-2875-11-290
Abstract: Background Drug and multidrug-resistant Plasmodium falciparum malaria has existed in Thailand for several decades. Furthermore, Thailand serves as a sentinel for drug-resistant malaria within the Greater Mekong sub-region. However, the drug resistance situation is highly dynamic, changing quickly over time. Here parasite in vitro drug sensitivity is reported for artemisinin derivatives, mefloquine, chloroquine and quinine, across Thailand. Methods Blood was drawn from patients infected with P. falciparum in seven sentinel provinces along Thai international borders with Cambodia, Myanmar, Laos, and Malaysia. In vitro parasite sensitivity was tested using the World Health Organization’s microtest (mark III) (between 1994 and 2002) and the histidine-rich protein-2 (HRP2)-based enzyme-linked immunosorbent assay (in 2010). Following World Health Organization protocol, at least 30 isolates were collected for each province and year represented in this study. Where possible, t-tests were used to test for significant differences. Results There appears to be little variation across study sites with regard to parasite sensitivity to chloroquine. Quinine resistance appears to have been rising prior to 1997, but has subsequently decreased. Mefloquine sensitivity appears high across the provinces, especially along the north-western border with Myanmar and the eastern border with Cambodia. Finally, the data suggest that parasite sensitivity to artemisinin and its derivatives is significantly higher in provinces along the north-western border with Myanmar. Conclusions Parasite sensitivity to anti-malarials in Thailand is highly variable over time and largely mirrors official drug use policy. The findings with regard to reduced sensitivity to artemisinin derivatives are supported by recent reports of reduced parasite clearance associated with artemisinin. This trend is alarming since artemisinin is considered the last defence against malaria. Continued surveillance in Thailand, along with increased collaboration and surveillance across the entire Greater Mekong sub-region, is clearly warranted.
The Limits and Intensity of Plasmodium falciparum Transmission: Implications for Malaria Control and Elimination Worldwide  [PDF]
Carlos A Guerra,Priscilla W Gikandi,Andrew J Tatem,Abdisalan M Noor,Dave L Smith,Simon I Hay ,Robert W Snow
PLOS Medicine , 2008, DOI: 10.1371/journal.pmed.0050038
Abstract: Background The efficient allocation of financial resources for malaria control using appropriate combinations of interventions requires accurate information on the geographic distribution of malaria risk. An evidence-based description of the global range of Plasmodium falciparum malaria and its endemicity has not been assembled in almost 40 y. This paper aims to define the global geographic distribution of P. falciparum malaria in 2007 and to provide a preliminary description of its transmission intensity within this range. Methods and Findings The global spatial distribution of P. falciparum malaria was generated using nationally reported case-incidence data, medical intelligence, and biological rules of transmission exclusion, using temperature and aridity limits informed by the bionomics of dominant Anopheles vector species. A total of 4,278 spatially unique cross-sectional survey estimates of P. falciparum parasite rates were assembled. Extractions from a population surface showed that 2.37 billion people lived in areas at any risk of P. falciparum transmission in 2007. Globally, almost 1 billion people lived under unstable, or extremely low, malaria risk. Almost all P. falciparum parasite rates above 50% were reported in Africa in a latitude band consistent with the distribution of Anopheles gambiae s.s. Conditions of low parasite prevalence were also common in Africa, however. Outside of Africa, P. falciparum malaria prevalence is largely hypoendemic (less than 10%), with the median below 5% in the areas surveyed. Conclusions This new map is a plausible representation of the current extent of P. falciparum risk and the most contemporary summary of the population at risk of P. falciparum malaria within these limits. For 1 billion people at risk of unstable malaria transmission, elimination is epidemiologically feasible, and large areas of Africa are more amenable to control than appreciated previously. The release of this information in the public domain will help focus future resources for P. falciparum malaria control and elimination.
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