Transmission efficiency of Plasmodium vivax at low parasitaemia.

BACKGROUND: Plasmodium vivax is responsible for much of malaria outside Africa. Although most P. vivax infections in endemic areas are asymptomatic and have low parasite densities, they are considered a potentially important source of transmission. Several studies have demonstrated that asymptomatic P. vivax carriers can transmit the parasite to mosquitoes, but the efficiency has not been well quantified. The aim of this study is to determine the relationship between parasite density and mosquito infectivity, particularly at low parasitaemia. METHODS: Membrane feeding assays were performed using serial dilutions of P. vivax-infected blood to define the relationship between parasitaemia and mosquito infectivity. RESULTS: The infection rate (oocyst prevalence) and intensity (oocyst load) were positively correlated with the parasite density in the blood. There was a broad case-to-case variation in parasite infectivity. The geometric mean parasite density yielding a 10% mosquito infection rate was 33 (CI 95 9-120) parasites/µl or 4 (CI 95 1-17) gametocytes/µl. The geometric mean parasite density yielding a 50% mosquito infection rate was 146 (CI 95 36-586) parasites/µl or 13 (CI 95 3-49) gametocytes/µl. CONCLUSION: This study quantified the ability of P. vivax to infect Anopheles dirus at over a broad range of parasite densities. It provides important information about parasite infectivity at low parasitaemia common among asymptomatic P. vivax carriers.

Controlled human malaria infection with a clone of Plasmodium vivax with high-quality genome assembly.

Controlled human malaria infection (CHMI) provides a highly informative means to investigate host-pathogen 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, 2 healthy malaria-naive United Kingdom 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 RBCs. Following stringent safety screening, the parasite stabilate from one of these donors (PvW1) was thawed and used to inoculate 6 healthy malaria-naive United Kingdom adults by blood-stage CHMI, at 3 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 analyzed 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.

Sensitive detection of Plasmodium vivax malaria by the rotating-crystal magneto-optical method in Thailand.

The rotating-crystal magneto-optical detection (RMOD) method has been developed for the rapid and quantitative diagnosis of malaria and tested systematically on various malaria infection models. Very recently, an extended field trial in a high-transmission region of Papua New Guinea demonstrated its great potential for detecting malaria infections, in particular Plasmodium vivax. In the present small-scale field test, carried out in a low-transmission area of Thailand, RMOD confirmed malaria in all samples found to be infected with Plasmodium vivax by microscopy, our reference method. Moreover, the magneto-optical signal for this sample set was typically 1-3 orders of magnitude higher than the cut-off value of RMOD determined on uninfected samples. Based on the serial dilution of the original patient samples, we expect that the method can detect Plasmodium vivax malaria in blood samples with parasite densities as low as [Formula: see text]5-10 parasites per microliter, a limit around the pyrogenic threshold of the infection. In addition, by investigating the correlation between the magnitude of the magneto-optical signal, the parasite density and the erythrocytic stage distribution, we estimate the relative hemozoin production rates of the ring and the trophozoite stages of in vivo Plasmodium vivax infections.

Malaria cross-sectional surveys identified asymptomatic infections of Plasmodium falciparum, Plasmodium vivax and Plasmodium knowlesi in Surat Thani, a southern province of Thailand.

OBJECTIVES: Malaria cross-sectional surveys are rarely conducted in very low transmission settings. This study aimed to determine the prevalence and risk factors of Plasmodium infection in a near-elimination setting in southern Thailand. METHODS: Two cross-sectional surveys were conducted in areas of active transmission in the Surat Thani province of Thailand in January and May 2019. PCR was used to detect Plasmodium infection. RESULTS: The prevalence of Plasmodium blood infection was 0.45% and 0.61% in January and May 2019, respectively. The major parasite species was Plasmodium falciparum in January and Plasmodium vivax in May. Unexpectedly, Plasmodium knowlesi infections were also detected. Most infections, including those of Plasmodium knowlesi, were asymptomatic. Being male and staying outdoors at night-time were the only significant identified risk factors. Of people infected in January 28.0% were positive in May for the same parasite species, suggesting persistent asymptomatic infections. CONCLUSIONS: Despite the very low incidence rate in Surat Thani, most malaria infections were asymptomatic. Outdoor mosquito biting at night-time is likely an important mode of malaria transmission. Unexpectedly, asymptomatic Plasmodium knowlesi infection was found, confirming previous reports of such infection in mainland Southeast Asia.

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

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 > or = 500/microl; 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/microl, with sensitivity of only 20% for P. falciparum and 24% for P. vivax at densities <100 parasites/microl. 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.

Infectivity of asymptomatic Plasmodium-infected human populations to Anopheles dirus mosquitoes in western Thailand.

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.

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

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.

Comparison of field and expert laboratory microscopy for active surveillance for asymptomatic Plasmodium falciparum and Plasmodium vivax in western Thailand.

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 x700 using a natural light source, whereas expert laboratory microscopy consisted of a 20-minute read (number of parasites per 500 leukocytes) at x1,000 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. falciparum-positive slides and 93.9% of the P. vivax-positive slides had a parasitemia of less than 500/microL. 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.

Field evaluation of the ICT Malaria Pf/Pv immunochromatographic test for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand.

Rapid antigen assays provide an effective tool for the detection of malaria in symptomatic patients. However, the efficacy of these devices for detecting asymptomatic malaria, where parasite levels are normally significantly lower than in symptomatic patients, is less well established. We evaluated the efficacy of a new combined Plasmodium falciparum-Plasmodim vivax immunochromatographic test (ICT Malaria Pf/Pv) in a cross-sectional malaria survey of the village of Ban Kong Mong Tha, Kanchanaburi Provice, Thailand, from August to December 2000. A total of 1,976 bleeds were made from 559 individuals over the course of the study. Blinded microscopy of thick and thin blood films was used as the gold standard; all discordant and 10% of concordant results were cross-checked. Of 1,976 ICT Malaria Pf/Pv dipsticks tested, 98.3% (n = 1,943) performed as expected, as evidenced by the appearance of the control line. The ICT Malaria Pf/Pv test was both sensitive (100.0%) and specific (99.7 %) for the diagnosis of falciparum malaria with parasitemias of > or = 500 trophozoites/microL; however, only 15.9% (13/82) of infected individuals had parasitemia rates this high. When P. falciparum parasitemia rates were < 500/microL, the sensitivity of the diagnosis was only 23.3%, with a positive predictive value (PPV) and a negative predictive value (NPV) of 76.2 and 97.2%, respectively. The ICT Malaria Pf/Pv test was specific, but not sensitive, for the diagnosis of vivax malaria with parasite rates of > or = 500 trophozoites/microl, with sensitivity, specificity, PPV, and NPV of 66.7%, 99.9%, 66.7%, and 99.9%, respectively. At parasite rates of < 500/microL, corresponding values were 0.0%, 99.9%, 0%, and 95.1%. Because of the relatively high cost of these assays, low parasite rates found in the majority of asymptomatic individuals, and low sensitivity of this assay with rates of < 500/microl, use of this assay as a tool for active case detection is of limited value in western Thailand.

Short report: Failure of the OptiMAL rapid malaria test as a tool for the detection of asymptomatic malaria in an area of Thailand endemic for Plasmodium falciparum and P. vivax.

We evaluated the efficacy of the OptiMAL assay in a cross-sectional malaria survey in western Thailand from April to August 2001. Expert microscopy of Giemsa-stained thick and thin blood films was used as the gold standard. Positive control lines were evident in 99% (1,128 of 1,137) of the assays tested. However, 34% (384 of 1,128) of assays produced an aberrant result (a positive P. falciparum-specific line and a negative panmalarial line). False-positive panmalarial and Plasmodium falciparum-specific lines occurred in 25.9% (270 of 1,042) and 60.3% (628 of 1,042) of microscopy-negative samples, respectively. Due to the preponderance of false-positive test results, it was necessary to develop subjective criteria for test positivity based on line intensity. For determination of assay performance during this study, we therefore considered all test lines that were scored as intermediate or strong as positive and lines that were faint as negative. Using these criteria, we determined that the sensitivity of the OptiMAL assay for P. falciparum was 25% with > 500 parasites/microl and 10.5% with > 100 parasites/microl, while for P. vivax, the sensitivity at the same parasite rates was 100% and 41.7%, respectively. Further studies are required to determine whether the problems we identified are limited to this particular lot of OptiMAL assays.