Benjawan Khuntirat

Bio: Benjawan Khuntirat is an academic researcher from United States Department of the Army. The author has contributed to research in topics: Plasmodium vivax & Plasmodium falciparum. The author has an hindex of 14, co-authored 18 publications receiving 1018 citations.

Detection of Four Plasmodium Species by Genus- and Species-Specific Loop-Mediated Isothermal Amplification for Clinical Diagnosis

Abstract: Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method, was developed for the clinical detection of four species of human malaria parasites: Plasmodium falciparum, P. vivax, P. malariae, and P. ovale. We evaluated the sensitivity and specificity of LAMP in comparison with the results of microscopic examination and nested PCR. LAMP showed a detection limit (analytical sensitivity) of 10 copies of the target 18S rRNA genes for P. malariae and P. ovale and 100 copies for the genus Plasmodium, P. falciparum, and P. vivax. LAMP detected malaria parasites in 67 of 68 microscopically positive blood samples (sensitivity, 98.5%) and 3 of 53 microscopically negative samples (specificity, 94.3%), in good agreement with the results of nested PCR. The LAMP reactions yielded results within about 26 min, on average, for detection of the genus Plasmodium, 32 min for P. falciparum, 31 min for P. vivax, 35 min for P. malariae, and 36 min for P. ovale. Accordingly, in comparison to the results obtained by microscopy, LAMP had a similar sensitivity and a greater specificity and LAMP yielded results similar to those of nested PCR in a shorter turnaround time. Because it can be performed with a simple technology, i.e., with heat-treated blood as the template, reaction in a water bath, and inspection of the results by the naked eye because of the use of a fluorescent dye, LAMP may provide a simple and reliable test for routine screening for malaria parasites in both clinical laboratories and malaria clinics in areas where malaria is endemic.

Comparison of PCR and microscopy for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand

Abstract: The main objective of this study was to compare the performance of nested PCR with expert microscopy as a means of detecting Plasmodium parasites during active malaria surveillance in western Thailand. 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.

Genetic diversity and multiple infections of Plasmodium vivax malaria in Western Thailand.

Abstract: Using two polymorphic genetic markers, the merozoite surface protein-3alpha (MSP-3alpha) and the circumsporozoite protein (CSP), we investigated the population diversity of Plasmodium vivax in Mae Sod, Thailand from April 2000 through June 2001. Genotyping the parasites isolated from 90 malaria patients attending two local clinics for the dimorphic CSP gene revealed that the majority of the parasites (77%) were the VK210 type. Genotyping the MSP3-alpha gene indicated that P. vivax populations exhibited an equally high level of polymorphism as those from Papua New Guinea, a hyperendemic region. Based on the length of polymerase chain reaction products, three major types of the MSP-3alpha locus were distinguished, with frequencies of 74.8%, 18.7%, and 6.5%, respectively. The 13 alleles distinguished by restriction fragment length polymorphism analysis did not show a significant seasonal variation in frequency. Genotyping the MSP-3alpha and CSP genes showed that 19.3% and 25.6% of the patients had multiple infections, respectively, and the combined rate was 35.6%. Comparisons of MSP-3alpha sequences from nine clones further confirmed the high level of genetic diversity of the parasite and also suggested that geographic isolation may exist. These results strongly indicate that P. vivax populations are highly diverse and multiple clonal infections are common in this malaria-hypoendemic region of Thailand.

Evidence for Subclinical Avian Influenza Virus Infections Among Rural Thai Villagers

Abstract: Background Regions of Thailand reported sporadic outbreaks of A/H5N1 highly pathogenic avian influenza (HPAI) among poultry between 2004 and 2008 Kamphaeng Phet Province, in north-central Thailand had over 50 HPAI poultry outbreaks in 2004 alone, and 1 confirmed and 2 likely other human HPAI infections between 2004 and 2006 Methods In 2008, we enrolled a cohort of 800 rural Thai adults living in 8 sites within Kamphaeng Phet Province in a prospective study of zoonotic influenza transmission We studied participants’ sera with serologic assays against 16 avian, 2 swine, and 8 human influenza viruses Results Among participants (mean age 496 years and 58% female) 65% reported lifetime poultry exposure of at least 30 consecutive minutes Enrollees had elevated antibodies by microneutralization assay against 3 avian viruses: A/Hong Kong/1073/1999(H9N2), A/Thailand/676/2005(H5N1), and A/Thailand/384/2006(H5N1) Bivariate risk factor modeling demonstrated that male gender, lack of an indoor water source, and tobacco use were associated with elevated titers against avian H9N2 virus Multivariate modeling suggested that increasing age, lack of an indoor water source, and chronic breathing problems were associated with infection with 1 or both HPAI H5N1 strains Poultry exposure was not associated with positive serologic findings Conclusions These data suggest that people in rural central Thailand may have experienced subclinical avian influenza infections as a result of yet unidentified environmental exposures Lack of an indoor water source may play a role in transmission

Comparison of artificial membrane feeding with direct skin feeding to estimate the infectiousness of Plasmodium vivax gametocyte carriers to mosquitoes.

Abstract: The efficacy of a membrane-feeding apparatus as a means of infecting Anopheles dirus mosquitoes with Plasmodium vivax was compared with direct feeding of mosquitoes on gametocyte carriers. Volunteers participating in the study were symptomatic patients reporting to malaria clinics in western Thailand. Direct mosquito feeds were conducted on 285 P. vivax-infected individuals. Four methods of preparing blood for the membrane-feeding apparatus were evaluated. They included 1) replacement of patient plasma with sera from a P. vivax-naive donor (n = 276), 2) replacement of patient plasma with plasma from a P. vivax-naive donor (n = 83), 3) replacement of patient plasma with that individual’s own plasma (n = 80), and 4) whole blood added directly to the feeder (n = 221). Criteria used to compare the different methods included 1) number of feeds infecting mosquitoes, 2) percent of mosquitoes with oocysts, and 3) mean number of oocysts per positive mosquito. For most parameters, the direct- feeding method was not significantly different from methods that replaced patient plasma with sera/plasma from a P. vivax-naive donor. However, direct feeding was more effective than use of whole blood or blood that was reconstituted with the patient’s own plasma. These data suggest a possible role of transmission-blocking antibody. The implications towards development of a membrane-feeding assay for the evaluation of candidate transmission-blocking malaria vaccines is discussed.

Rodent-borne diseases and their risks for public health

Abstract: Rodents are the most abundant and diversified order of living mammals in the world. Already since the Middle Ages we know that they can contribute to human disease, as black rats were associated with distribution of plague. However, also in modern times rodents form a threat for public health. In this review article a large number of pathogens that are directly or indirectly transmitted by rodents are described. Moreover, a simplified rodent disease model is discussed.

Antibody-dependent enhancement of severe dengue disease in humans

Abstract: For dengue viruses 1 to 4 (DENV1-4), a specific range of antibody titer has been shown to enhance viral replication in vitro and severe disease in animal models. Although suspected, such antibody-dependent enhancement of severe disease has not been shown to occur in humans. Using multiple statistical approaches to study a long-term pediatric cohort in Nicaragua, we show that risk of severe dengue disease is highest within a narrow range of preexisting anti-DENV antibody titers. By contrast, we observe protection from all symptomatic dengue disease at high antibody titers. Thus, immune correlates of severe dengue must be evaluated separately from correlates of protection against symptomatic disease. These results have implications for studies of dengue pathogenesis and for vaccine development, because enhancement, not just lack of protection, is of concern.

Epidemiology and Infectivity of Plasmodium falciparum and Plasmodium vivax Gametocytes in Relation to Malaria Control and Elimination

Abstract: Malaria remains a major cause of morbidity and mortality in the tropics, with Plasmodium falciparum responsible for the majority of the disease burden and P. vivax being the geographically most widely distributed cause of malaria. Gametocytes are the sexual-stage parasites that infect Anopheles mosquitoes and mediate the onward transmission of the disease. Gametocytes are poorly studied despite this crucial role, but with a recent resurgence of interest in malaria elimination, the study of gametocytes is in vogue. This review highlights the current state of knowledge with regard to the development and longevity of P. falciparum and P. vivax gametocytes in the human host and the factors influencing their distribution within endemic populations. The evidence for immune responses, antimalarial drugs, and drug resistance influencing infectiousness to mosquitoes is reviewed. We discuss how the application of molecular techniques has led to the identification of submicroscopic gametocyte carriage and to a reassessment of the human infectious reservoir. These components are drawn together to show how control measures that aim to reduce malaria transmission, such as mass drug administration and a transmission-blocking vaccine, might better be deployed.