The infectivity of Plasmodium-infected humans in western Thailand was estimated by feeding laboratory-reared Anopheles dirus Peyton and Harrison mosquitoes on venous blood placed in a membrane-feeding apparatus. Between May 2000 and November 2001, a total of 6,494 blood films collected during an active malaria surveillance program were checked by microscopy for the presence of Plasmodium parasites: 3.3, 4.5, and 0.1% of slides were P. falciparum- (Pf), P. vivax- (Pv), and P. malariae (Pm)-positive. Venous blood was collected from 70, 52, 6, and 4 individuals infected with Pf, Pv, Pm, and mixed Pf/Pv, respectively, with 167 uninfected individuals serving as negative controls. Only 10% (7/70), 13% (7/52), and 0% (0/6) of membrane feeds conducted on Pf-, Pv-, and Pm-infected blood yielded infected mosquitoes. One percent (2/167) of microscope-negative samples infected mosquitoes; however, both samples were subsequently determined to be Pf-positive by polymerase chain reaction. Gametocytes were observed in only 29% (4/14) of the infectious samples. All infections resulted in low oocyst loads (average of 1.2 oocysts per positive mosquito). Only 4.5% (10/222) of mosquitoes fed on the seven infectious Pf samples developed oocysts, whereas 2.9% (9/311) of mosquitoes fed on the seven infectious Pv samples developed oocysts. The probability of a mosquito becoming infected with Pf or Pv after a blood meal on a member of the human population in Kong Mong Tha was estimated to be 1 in 6,700 and 1 in 5,700, respectively. The implications toward malaria transmission in western Thailand are discussed.

Objective: 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. Methods: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.Results: 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. Conclusion: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.

Abstract. Microscopy of Giemsa-stained thick and thin films by a skilled microscopist has remained the standard laboratory method for the diagnosis of malaria. However, diagnosis of malaria with this method is problematic since interpretation of results requires considerable expertise, particularly at low parasite levels. We compared the efficacy of "field" and "expert laboratory" microscopy for active surveillance of Plasmodium falciparum and P. vivax in western Thailand. Field microscopy consisted of an approximately five-minute read (50−100 fields) of a thick film at ×700 using a natural light source, whereas expert laboratory microscopy consisted of a 20-minute read (number of parasites per 500 leukocytes) at ×1,000 using a high-quality, well-maintained microscope with an artificial light source. All discordant and 20% of concordant results were cross-checked blindly. A total of 3,004 blood films collected between May and November 2000 were included in the study, of which 156 (5.2%) were positive for P. falciparum, 177 (5.9%) for P. vivax, and 4 (0.1%) for both P. falciparum and P. vivax by expert microscopy. A total of 84.4% (135 of 160) of the P. falciparumpositive slides and 93.9% of the P. vivax-positive slides had a parasitemia of less than 500/L. Field microscopy was specific (99.3%) but not sensitive (10.0%) for the diagnosis of P. falciparum malaria, with a positive predictive value (PPV) of 43.2% and a negative predictive value (NPV) of 95.1%. The corresponding specificity and sensitivity for the diagnosis of P. vivax malaria were 99.2% and 7.1%, respectively, with a PPV of 38.7% and an NPV of 93.9%. Field microscopy, as defined in this study, is not an effective method for active malaria surveillance in western Thailand, where prevalence and parasitemia rates are low.

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 ‫ס‬ 276), 2) replacement of patient plasma with plasma from a P. vivax-naive donor (n ‫ס‬ 83), 3) replacement of patient plasma with that individual's own plasma (n ‫ס‬ 80), and 4) whole blood added directly to the feeder (n ‫ס‬ 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.

Controlled human malaria infection (CHMI) provides a highly informative means to investigate hostpathogen interactions and enable in vivo proof-of-concept efficacy testing of new drugs and vaccines. However, unlike Plasmodium falciparum, well-characterized P. vivax parasites that are safe and suitable for use in modern CHMI models are limited. Here, two healthy malaria-naïve UK adults with universal donor blood group were safely infected with a clone of P. vivax from Thailand by mosquito-bite CHMI.Parasitemia developed in both volunteers and, prior to treatment, each volunteer donated blood to produce a cryopreserved stabilate of infected red blood cells. Following stringent safety screening, the parasite stabilate from one of these donors ("PvW1") was thawed and used to inoculate six healthy malaria-naïve UK adults by blood-stage CHMI, at three different dilutions. Parasitemia developed in all volunteers, who were then successfully drug treated. PvW1 parasite DNA was isolated and sequenced to produce a high quality genome assembly by using a hybrid assembly method. We analysed leading vaccine candidate antigens and multigene families, including the Vivax interspersed repeat (VIR) genes of which we identified 1145 in the PvW1 genome. Our genomic analysis will guide future assessment of candidate vaccines and drugs, as well as experimental medicine studies.