Vivax Gametocytes Research Articles

Long-lasting infectivity of Plasmodium vivax present in malarial patient blood to Anopheles aquasalis

Experimental studies for understanding the relationship between Plasmodium vivax and its vector hosts are difficult, because of to the lack of a long-term, in vitro continuous culture system unavailability of infected blood samples, seasonality of the disease, and the concentration of most cases in remote areas. This study evaluates the duration of the infectivity of P. vivax to Anopheles aquasalis after collecting blood from malaria-infected patients. Blood was collected from patients and stored at 4 °C and 37 °C. Every day, for 4 days, the blood was fed to An. aquasalis adult females, and a Giemsa-stained thick blood smear was mounted to account for sexual (gametocytes) and asexual (trophozoites and schizonts) stages and calculate parasitemia. Oocysts in the midgut of the mosquitoes were counted on the seventh day after feeding. Kruskal-Wallis test was used to compare the mean number of oocysts (MO) and the parasite density (PD) in each storage condition and post-infection time-points. The Mann-Whitney test was used to compare the number of oocysts for each day between temperatures. The results show that P. vivax stored at 4 °C and at 37 °C has its infectivity to An. aquasalis preserved for 2 days and 3 days, respectively. Infection rate (IR), PD and MO were higher on the day of blood collection and decreased gradually over time. The parasite density (number of parasites/μL) diminished faster at 4 °C than at 37 °C. In this study, a preservation protocol is shown for long-lasting infectivity of P. vivax in a blood sample taken from malaria-infected patients. These results show that infectivity of P. vivax stored at 4 °C and at 37 °C to An. aquasalis persist until 3 days after blood collection, but parasite density, infection rate, and mean of oocysts decreased 24h after blood collection. Since the malaria cases are increasingly far from the urban areas these results indicate that is possible, losing some infectivity, to realize experimental infections several dozen hours after the blood collection. However, it is necessary to improve the procedures for preserving P. vivax gametocytes for mosquito infection in the laboratory.

Delayed diagnosis of Plasmodium vivax malaria in an elderly Sri Lankan pilgrim in India

Malaria was rampant in Sri Lanka two decades ago but we have since been declared free of malaria transmission by the WHO in 2016. However, neighboring India still has a high incidence of malaria, and visitors to India carry a high risk of contracting this disease. Despite the elimination of indigenous cases of malaria in Sri Lanka, a fair number of cases are detected from travelers coming from endemic regions of the globe. Delay in diagnosis occurs due to a lack of awareness among the medical community and a missed travel history as observed in this case scenario. We report a 71-year-old previously healthy Sri Lankan male who developed a febrile illness after sixteen days of traveling in India on pilgrimage. He presented with a six day history of of illness and it took a further seven days to consider malaria as a possible diagnosis. Malaria antigen was positive on day thirteen of the illness with Plasmodium vivax trophozoites and gametocytes seen on the thick and thin films. He was treated with chloroquine and recovered slowly with clearing of parasitaemia. A correct diagnosis and close liaison with the anti-malaria campaign helped in the successful management of our patient. This report is an eye opener to consider malaria as a diagnostic possibility and a clinical dilemma and to take a detailed travel history in patients presenting fever. Raising awareness of travelers about prevention against malaria and the need for malaria prophylaxis is also necessary.

Human Plasmodium vivax mosquito experimental transmission

Plasmodium vivax bench research greatly lags behind Plasmodium falciparum because of an inability to culture in vitro. A century ago, intentionally inducing a malaria infection was a strategy commonly used to cure late-stage syphilis. These controlled human malaria infections were used with expertise and persisted to the end of World War II. While controlled malaria liver-stage infection has been achieved for both P. vivax and P. falciparum, controlled human transmission to mosquitoes falls short for both species. In this issue of the JCI, Collins et al. present groundbreaking work that establishes a system to transmit P. vivax gametocytes from humans to mosquitoes. The authors injected a unique human isolate of P. vivax that reached high gametocyte density within weeks. This study provides a technical advance that will facilitate the study and eradication of the human parasite P. vivax.

Hematological Profile and Gametocyte Carriage in Malaria Patients from Southern Pakistan.

BackgroundMalarial infection is a major cause of concern, both worldwide and in Pakistan. Gametocytes are the sexual forms of the parasite that are essential for transmission. They fuse inside the mosquito to develop sporozoites. Gametocytes of the plasmodium parasites, which cause the infection, differentiate into male and female gametocytes. These gametocytes constitute the sexual stage of the malaria parasite and are essential in transmission of the disease from human to vector Anopheles. Gametocytes are affected by factors such as host immunity, drug treatment, reticulocytemia, anemia, low levels of asexual parasitemia and stress to the parasite. The aim of this study was to observe the hematological parameters, age and gametocyte carriage in an area of seasonal malaria transmission.MethodsThe study was conducted at Aga Khan University Hospital (AKUH) Laboratory over the period of one year and 294 patients with uncomplicated malaria were recruited. Patients infected with Plasmodium falciparum (P. falciparum) or Plasmodium vivax (P. vivax) malaria and no co-morbidities were included in the study.ResultsGametocytemia was highest during the period of July to November, with P. vivax, 267 (90.8%), predominating compared to P. falciparum, 27 (9.2%). P. vivax gametocytes were observed from May to October and P. falciparum gametocytes were observed from July to December. Low hemoglobin in females and low platelet levels were observed. The mean platelet count was significantly lower in cases of P. vivax having gametocytes compared to P. falciparum with gametocytes. Higher parasitic index was associated with lower platelet count. The most significantly altered parameters were hemoglobin, hematocrit, white blood cell (WBC), and platelet count. Hemoglobin and platelets were significantly lower during the malaria season in study participants, both male and female.ConclusionIn conclusion, infection with P. falciparum and P. vivax modulates significant changes in hematological parameters in populations living in malaria endemic regions. In the malaria season males were more frequently affected by malaria with thrombocytopenia. Gametocyte carriage remains unaffected by seasonal changes thus ensuring parasite transmission during the dry season.

Assessment of ultra-sensitive malaria diagnosis versus standard molecular diagnostics for malaria elimination: an in-depth molecular community cross-sectional study

Submicroscopic malaria infections contribute to transmission in exposed populations but their extent is underestimated even by standard molecular diagnostics. Sophisticated sampling and ultra-sensitive molecular methods can maximise test sensitivity but are not feasible in routine surveillance. Here we investigate the gains achievable by using increasingly sensitive methods with the aim to understand what diagnostic sensitivity is necessary to guide malaria interventions. Venous blood samples were collected from participants in a cross-sectional survey in two coastal medium-endemic villages in Madang province, Papua New Guinea. Using ultra-sensitive quantitative PCR (us-qPCR) on concentrated high-volume blood samples (2 mL) as reference, we quantified the proportion of Plasmodium falciparum and Plasmodium vivax infections and gametocyte carriers detectable in fingerprick blood volumes (200 μL) by standard 18S rRNA qPCR, us-qPCR, rapid diagnostic test (RDT), and ultra-sensitive P falciparum RDT. We further compared the epidemiological patterns observed with each diagnostic approach in the study population. Venous blood samples were collected from 300 participants between Dec 5, 2016, and Feb 24, 2017 (ie, during peak rainy season). Standard qPCR identified 87 (54%) of 161 P falciparum infections and 73 (52%) of 141 P vivax infections detected by the reference method. us-qPCR identified an additional 11 (7%) P falciparum infections and 14 (10%) P vivax infections. 80 (86%) of 93 P falciparum gametocyte carriers and 75 (91%) of 82 P vivax gametocyte carriers were found among infections detectable by us-qPCR. Ultra-sensitive RDT missed half of P falciparum infections detected by standard qPCR, including high gametocytaemic infections. Epidemiological patterns corresponded well between standard qPCR and the reference method. As the prevalence of P vivax decreased with increasing age, the proportion of P vivax infections undetectable by standard qPCR increased. Almost all potentially transmitting parasite carriers were identified with us-qPCR on fingerprick blood volumes. Analysing larger blood volumes revealed a large pool of ultra-low-density P falciparum and P vivax infections, which are unlikely to be transmitted. Therefore, current RDTs cannot replace molecular diagnostics for identifying potential P falciparum transmitters. Swiss National Science Foundation.

Genetic diversity and transmissibility of imported Plasmodium vivax in Qatar and three countries of origin

Malaria control program in the Arabian Peninsula, backed by adequate logistical support, has interrupted transmission with exception of limited sites in Saudi Arabia and sporadic outbreaks in Oman. However, sustained influx of imported malaria represents a direct threat to the above success. Here we examined the extent of genetic diversity among imported P. vivax in Qatar, and its ability to produce gametocytes, compared to parasites in main sites of imported cases, the Indian subcontinent (india) and East Africa (Sudan and Ethiopia). High diversity was seen among imported P. vivax in Qatar, comparable to parasites in the Indian subcontinent and East Africa. Limited genetic differentiation was seen among imported P. vivax, which overlapped with parasites in India, but differentiated from that in Sudan and Ethiopia. Parasite density among imported cases, ranged widely between 26.25–7985934.1 Pv18S rRNA copies/µl blood, with a high prevalence of infections carried gametocytes detectable by qRT-PCR. Parasitaemia was a stronger predictor for P. vivax gametocytes density (r = 0.211, P = 0.04). The extensive diversity of imported P. vivax and its ability to produce gametocytes represent a major threat for re-introduction of malaria in Qatar. The genetic relatedness between P. vivax reported in Qatar and those in India suggest that elimination strategy should target flow and dispersal of imported malaria into the region.

Infection of Anopheles aquasalis from symptomatic and asymptomatic Plasmodium vivax infections in Manaus, western Brazilian Amazon

BackgroundAsymptomatic individuals are one of the major challenges for malaria elimination programs in endemic areas. In the absence of clinical symptoms and with a lower parasite density they constitute silent reservoirs considered important for maintaining transmission of human malaria. Studies from Brazil have shown that infected individuals may carry these parasites for long periods.ResultsPatients were selected from three periurban endemic areas of the city of Manaus, in the western Brazilian Amazon. Symptomatic and asymptomatic patients with positive thick blood smear and quantitative real-time PCR (qPCR) positive for Plasmodium vivax were invited to participate in the study. A standardised pvs25 gene amplification by qPCR was used for P. vivax gametocytes detection. Anopheles aquasalis were fed using membrane feeding assays (MFA) containing blood from malaria patients. Parasitemia of 42 symptomatic and 25 asymptomatic individuals was determined by microscopic examination of blood smears and qPCR. Parasitemia density and gametocyte density were assessed as determinants of infection rates and oocysts densities. A strong correlation between gametocyte densities (microscopy and molecular techniques) and mosquito infectivity (P < 0.001) and oocysts median numbers (P < 0.05) was found in both groups. The ability to infect mosquitoes was higher in the symptomatic group (41%), but infectivity in the asymptomatic group was also seen (1.42%).ConclusionsAlthough their infectivity to mosquitoes is relatively low, given the high prevalence of P. vivax asymptomatic carriers they are likely to play and important role in malaria transmission in the city of Manaus. The role of asymptomatic infections therefore needs to be considered in future malaria elimination programs in Brazil.

High proportions of asymptomatic and submicroscopic Plasmodium vivax infections in a peri-urban area of low transmission in the Brazilian Amazon

BackgroundPopulation-based studies conducted in Latin America have shown a high proportion of asymptomatic and submicroscopic malarial infections. Considering efforts aiming at regional elimination, it is important to investigate the role of this asymptomatic reservoir in malaria transmission in peri-urban areas. This study aimed to estimate the prevalence of Plasmodium spp. and gametocyte burden on symptomatic and asymptomatic infections in the Brazilian Amazon.ResultsTwo cross-sectional household surveys (CS) were conducted including all inhabitants in a peri-urban area of Manaus, western Amazonas State, Brazil. Malaria parasites were detected by light microscopy (LM) and qPCR. Sexual stages of Plasmodium spp. were detected by LM and RT-qPCR. A total of 4083 participants were enrolled during the two surveys. In CS1, the prevalence of Plasmodium vivax infections was 4.3% (86/2010) by qPCR and 1.6% (32/2010) by LM. Fifty percent (43/86) of P. vivax infected individuals (qPCR) carried P. vivax gametocytes. In CS2, 3.4% (70/2073) of participants had qPCR-detectable P. vivax infections, of which 42.9% (30/70) of infections were gametocyte positive. The P. vivax parasite density was associated with gametocyte carriage (P < 0.001). Sixty-seven percent of P. vivax infected individuals and 53.4% of P. vivax gametocyte carriers were asymptomatic.ConclusionsThis study confirms a substantial proportion of asymptomatic and submicroscopic P. vivax infections in the study area. Most asymptomatic individuals carried gametocytes and presented low asexual parasitemia. This reservoir actively contributes to malaria transmission in the Brazilian Amazon, underscoring a need to implement more efficient control and elimination strategies.

Infection of laboratory colonies of Anopheles mosquitoes with Plasmodium vivax from cryopreserved clinical isolates

Plasmodium vivax is the most geographically widespread malaria parasite. Unique features of transmission biology complicate P. vivax control. Interventions targeting transmission are required for malaria eradication. In the absence of an in vitro culture, transmission studies rely on live isolates from non-human primates or endemic regions. Here, we demonstrate P. vivax gametocytes from both India and Brazil are stable during cryopreservation. Importantly, cryopreserved gametocytes from Brazil were capable of infecting three anopheline mosquito species in feedings done in the United States. These findings create new opportunities for transmission studies in diverse locales.

Risk factors for Plasmodium falciparum and Plasmodium vivax gametocyte carriage in Papua New Guinean children with uncomplicated malaria

There are limited data on gametocytaemia risk factors before/after treatment with artemisinin combination therapy in children from areas with transmission of multiple Plasmodium species. We utilised data from a randomised trial comparing artemether-lumefantrine (AL) and artemisinin-naphthoquine (AN) in 230 Papua New Guinean children aged 0.5–5 years with uncomplicated malaria in whom determinants of gametocytaemia by light microscopy were assessed at baseline using logistic regression and during follow-up using multilevel mixed effects modelling. Seventy-four (32%) and 18 (8%) children presented with P. falciparum and P. vivax gametocytaemia, respectively. Baseline P. falciparum gametocytaemia was associated with Hackett spleen grade 1 (odds ratio (95% CI) 4.01 (1.60–10.05) vs grade 0; P<0.001) and haemoglobin (0.95 (0.92–0.97) per 1g/L increase; P<0.001), and P. falciparum asexual parasitaemia in slide-positive cases (0.36 (0.19–0.68) for a 10-fold increase; P=0.002). Baseline P. vivax gametocytaemia was associated with Hackett grade 2 (12.66 (1.31–122.56); P=0.028), mixed P. falciparum/vivax infection (0.16 (0.03–1.00); P=0.050), P. vivax asexual parasitaemia (5.68 (0.98–33.04); P=0.053) and haemoglobin (0.94 (0.88–1.00); P=0.056). For post-treatment P. falciparum gametocytaemia, independent predictors were AN vs AL treatment (4.09 (1.43–11.65)), haemoglobin (0.95 (0.93–0.97)), presence/absence of P. falciparum asexual forms (3.40 (1.66–0.68)) and day post-treatment (0.086 (0.82–0.90)) (P<0.001). Post-treatment P. vivax gametocytaemia was predicted by presence of P. vivax asexual forms (596 (12–28,433); P<0.001). Consistent with slow P. falciparum gametocyte maturation, low haemoglobin, low asexual parasite density and higher spleen grading, markers of increased prior infection exposure/immunity, were strong associates of pre-treatment gametocyte positivity. The persistent inverse association between P. falciparum gametocytaemia and haemoglobin during follow-up suggests an important role for bone marrow modulation of gametocytogenesis. In P. vivax infections, baseline and post-treatment gametocyte carriage was positively related to the acute parasite burden, reflecting the close association between the development of asexual and sexual forms.

Plasmodium vivax gametocyte infectivity in sub-microscopic infections.

BackgroundThe use of molecular techniques has put in the spotlight the existence of a large mass of malaria sub-microscopic infections among apparently healthy populations. These sub-microscopic infections are considered an important pool for maintained malaria transmission.MethodsIn order to assess the appearance of Plasmodium vivax gametocytes in circulation, gametocyte density and the parasite infectivity to Anopheles mosquitoes, a study was designed to compare three groups of volunteers either experimentally infected with P. vivax sporozoites (early infections; n = 16) or naturally infected patients (acute malaria, n = 16 and asymptomatic, n = 14). In order to determine gametocyte stage, a quantitative reverse transcriptase PCR (RT-qPCR) assay targeting two sexual stage-specific molecular markers was used. Parasite infectivity was assessed by membrane feeding assays (MFA).ResultsIn early infections P. vivax gametocytes could be detected starting at day 7 without giving rise to infected mosquitoes during 13 days of follow-up. Asymptomatic carriers, with presumably long-lasting infections, presented the highest proportion of mature gametocytes and were as infective as acute patients.ConclusionsThis study shows the potential role of P. vivax asymptomatic carriers in malaria transmission should be considered when new policies are envisioned to redirect malaria control strategies towards targeting asymptomatic infections as a tool for malaria elimination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1104-1) contains supplementary material, which is available to authorized users.

Strategies for Understanding and Reducing the Plasmodium vivax and Plasmodium ovale Hypnozoite Reservoir in Papua New Guinean Children: A Randomised Placebo-Controlled Trial and Mathematical Model

BackgroundThe undetectable hypnozoite reservoir for relapsing Plasmodium vivax and P. ovale malarias presents a major challenge for malaria control and elimination in endemic countries. This study aims to directly determine the contribution of relapses to the burden of P. vivax and P. ovale infection, illness, and transmission in Papua New Guinean children.Methods and FindingsFrom 17 August 2009 to 20 May 2010, 524 children aged 5–10 y from East Sepik Province in Papua New Guinea (PNG) participated in a randomised double-blind placebo-controlled trial of blood- plus liver-stage drugs (chloroquine [CQ], 3 d; artemether-lumefantrine [AL], 3 d; and primaquine [PQ], 20 d, 10 mg/kg total dose) (261 children) or blood-stage drugs only (CQ, 3 d; AL, 3 d; and placebo [PL], 20 d) (263 children). Participants, study staff, and investigators were blinded to the treatment allocation. Twenty children were excluded during the treatment phase (PQ arm: 14, PL arm: 6), and 504 were followed actively for 9 mo. During the follow-up time, 18 children (PQ arm: 7, PL arm: 11) were lost to follow-up. Main primary and secondary outcome measures were time to first P. vivax infection (by qPCR), time to first clinical episode, force of infection, gametocyte positivity, and time to first P. ovale infection (by PCR). A basic stochastic transmission model was developed to estimate the potential effect of mass drug administration (MDA) for the prevention of recurrent P. vivax infections. Targeting hypnozoites through PQ treatment reduced the risk of having at least one qPCR-detectable P. vivax or P. ovale infection during 8 mo of follow-up (P. vivax: PQ arm 0.63/y versus PL arm 2.62/y, HR = 0.18 [95% CI 0.14, 0.25], p < 0.001; P. ovale: 0.06 versus 0.14, HR = 0.31 [95% CI 0.13, 0.77], p = 0.011) and the risk of having at least one clinical P. vivax episode (HR = 0.25 [95% CI 0.11, 0.61], p = 0.002). PQ also reduced the molecular force of P. vivax blood-stage infection in the first 3 mo of follow-up (PQ arm 1.90/y versus PL arm 7.75/y, incidence rate ratio [IRR] = 0.21 [95% CI 0.15, 0.28], p < 0.001). Children who received PQ were less likely to carry P. vivax gametocytes (IRR = 0.27 [95% CI 0.19, 0.38], p < 0.001). PQ had a comparable effect irrespective of the presence of P. vivax blood-stage infection at the time of treatment (p = 0.14). Modelling revealed that mass screening and treatment with highly sensitive quantitative real-time PCR, or MDA with blood-stage treatment alone, would have only a transient effect on P. vivax transmission levels, while MDA that includes liver-stage treatment is predicted to be a highly effective strategy for P. vivax elimination. The inclusion of a directly observed 20-d treatment regime maximises the efficiency of hypnozoite clearance but limits the generalisability of results to real-world MDA programmes.ConclusionsThese results suggest that relapses cause approximately four of every five P. vivax infections and at least three of every five P. ovale infections in PNG children and are important in sustaining transmission. MDA campaigns combining blood- and liver-stage treatment are predicted to be a highly efficacious intervention for reducing P. vivax and P. ovale transmission.Trial registrationClinicalTrials.gov NCT02143934

Purification Methodology for Viable and Infective Plasmodium vivax Gametocytes That Is Compatible with Transmission-Blocking Assays.

Significant progress toward the control of malaria has been achieved, especially regarding Plasmodium falciparum infections. However, the unique biology of Plasmodium vivax hampers current control strategies. The early appearance of P. vivax gametocytes in the peripheral blood and the impossibility of culturing this parasite are major drawbacks. Using blood samples from 40 P. vivax-infected patients, we describe here a methodology to purify viable gametocytes and further infect anophelines. This method opens new avenues to validate transmission-blocking strategies.

Blood-Stage Parasitaemia and Age Determine Plasmodium falciparum and P. vivax Gametocytaemia in Papua New Guinea

A better understanding of human-to-mosquito transmission is crucial to control malaria. In order to assess factors associated with gametocyte carriage, 2083 samples were collected in a cross-sectional survey in Papua New Guinea. Plasmodium species were detected by light microscopy and qPCR and gametocytes by detection of pfs25 and pvs25 mRNA transcripts by reverse-transcriptase PCR (qRT-PCR). The parasite prevalence by PCR was 18.5% for Plasmodium falciparum and 13.0% for P. vivax. 52.5% of all infections were submicroscopic. Gametocytes were detected in 60% of P. falciparum-positive and 51% of P. vivax-positive samples. Each 10-fold increase in parasite density led to a 1.8-fold and 3.3-fold increase in the odds of carrying P. falciparum and P. vivax gametocytes. Thus the proportion of gametocyte positive and gametocyte densities was highest in young children carrying high asexual parasite densities and in symptomatic individuals. Dilution series of gametocytes allowed absolute quantification of gametocyte densities by qRT-PCR and showed that pvs25 expression is 10-20 fold lower than pfs25 expression. Between 2006 and 2010 parasite prevalence in the study site has decreased by half. 90% of the remaining infections were asymptomatic and likely constitute an important reservoir of transmission. However, mean gametocyte densities were low (approx. 1-2 gametocyte/μL) and it remains to be determined to what extent low-density gametocyte positive individuals are infective to mosquitos.

Recombinant Pvs48/45 antigen expressed in E. coli generates antibodies that block malaria transmission in Anopheles albimanus mosquitoes.

Transmission of malaria parasites from humans to Anopheles mosquitoes can be inhibited by specific antibodies elicited during malaria infection, which target surface Plasmodium gametocyte/gamete proteins. Some of these proteins may have potential for vaccine development. Pvs48/45 is a P. vivax gametocyte surface antigen orthologous to Pfs48/45, which may play a role during parasite fertilization and thus has potential for transmission blocking (TB) activity. Here we describe the expression of a recombinant Pvs48/45 protein expressed in Escherichia coli as a ∼60kDa construct which we tested for antigenicity using human sera and for its immunogenicity and transmission blocking activity of specific anti-mouse and anti-monkey Pvs48/45 antibodies. The protein reacted with sera of individuals from malaria-endemic areas and in addition induced specific IgG antibody responses in BALB/c mice and Aotus l. griseimembra monkeys. Sera from both immunized animal species recognized native P. vivax protein in Western blot (WB) and immunofluorescence assays. Moreover, sera from immunized mice and monkeys produced significant inhibition of parasite transmission to An. Albimanus mosquitoes as shown by membrane feeding assays. Results indicate the presence of reactive epitopes in the Pvs48/45 recombinant product that induce antibodies with TB activity. Further testing of this protein is ongoing to determine its vaccine potential.

Plasmodium vivax gametocyte proteins, Pvs48/45 and Pvs47, induce transmission-reducing antibodies by DNA immunization

Malaria transmission-blocking vaccines (TBV) aim to interfere with the development of the malaria parasite in the mosquito vector, and thus prevent spread of transmission in a community. To date three TBV candidates have been identified in Plasmodium vivax; namely, the gametocyte/gamete protein Pvs230, and the ookinete surface proteins Pvs25 and Pvs28. The Plasmodium falciparum gametocyte/gamete stage proteins Pfs48/45 and Pfs47 have been studied as TBV candidates, and Pfs48/45 shown to induce transmission-blocking antibodies, but the candidacy of their orthologs in P. vivax, Pvs48/45 (PVX_083235) and Pvs47 (PVX_083240), for vivax TBV have not been tested. Herein we investigated whether targeting Pvs48/45 and Pvs47 can inhibit parasite transmission to mosquitoes, using P. vivax isolates obtained in Thailand. Mouse antisera directed against the products from plasmids expressing Pvs48/45 and Pvs47 detected proteins of approximately 45- and 40-kDa, respectively, in the P. vivax gametocyte lysate, by Western blot analysis under non-reducing conditions. In immunofluorescence assays Pvs48/45 was detected predominantly on the surface and Pvs47 was detected in the cytoplasm of gametocytes. Membrane feeding transmission assays demonstrated that anti-Pvs48/45 and -Pvs47 mouse sera significantly reduced the number of P. vivax oocysts developing in the mosquito midgut. Limited amino acid polymorphism of these proteins was observed among 27 P. vivax isolates obtained from Thailand, Vanuatu, and Colombia; suggesting that polymorphism may not be an impediment for the utilization of Pvs48/45 and Pvs47 as TBV antigens. In one Thai isolate we found that the fourth cysteine residue in the Pvs47 cysteine-rich domain (CRD) III (amino acid position 337) is substituted to phenylalanine. However, antibodies targeting Pvs47 CRDI-III showed a significant transmission-reducing activity against this isolate, suggesting that this substitution in Pvs47 was not critical for recognition by the generated antibodies. In conclusion, our results indicate that Pvs48/45 and Pvs47 are potential transmission-blocking vaccine candidates of P. vivax.

Membrane Feeding Assay to Determine the Infectiousness of Plasmodium vivax Gametocytes.

The evaluation of Plasmodium vivax gametocyte infectiousness by the membrane feeding assay is herein described. While P. vivax cannot be cultured and different parasite isolates may infect mosquitoes at different rates, the protocol described in this chapter identifies critical parameters to be considered when performing the assay such as methods for the preparation of the mosquitoes, the size of the blood cup, and the blood volume used. In previous studies the data have shown that the membrane feeding assay is useful for studies of parasite biology, and the effects of transmission blocking drugs and vaccines.

Anopheles darlingi (Diptera: Culicidae) displays increased attractiveness to infected individuals with Plasmodium vivax gametocytes.

BackgroundMost hematophagous insects use host odours as chemical cues. The odour components, some physiological parameters and host attractiveness are affected by several conditions, including infection by parasites, e.g., plasmodia and, therefore, change the epidemiological scenario. This study evaluated the attractiveness of individuals with vivax malaria before, during (7 days) and after treatment (14 days) with specific antimalarial drugs.FindingsMosquito attractiveness to vivax-infected patients was assessed using a vertical olfactometer using the foot as a source of body odour. The ratio of Anopheles darlingi mosquitoes in the lower chamber of the olfactometer was used to calculate the attractiveness, and patient temperature was measured using a digital thermometer. An increased attractiveness was found only in patients bearing vivax gametocytes during the first experiment (early infection) (P < 0.001). Patients in the first experiment tended to have a higher body temperature, but grouping patients into fever and non-fever resulted in a higher attractiveness only in the fever group of gametocyte carriers, suggesting a synergistic effect of temperature and gametocytes in the host attractiveness to A. darlingi.ConclusionsGametocyte presence and fever in vivax malaria patients increased short distance host attractiveness to An. darlingi.

Extradural hematoma in Plasmodium vivax malaria: Are we alert to detect?

Sir, Malaria continues to be an important parasitic disease in tropics and contributes to morbidity and mortality.[1] We report a case of Plasmodium vivax infection who presented with features of spontaneous extradural bleed in order to create an awareness of this unusual entity among practitioners. A 22-years-old male lorry driver was referred from a private hospital to our emergency department (ED) for altered sensorium, while he was on treatment for 4 days for smear positive vivax malaria. He recently had a trip to northern India in the lorry. There was no history of trauma, fall from height, seizure, bleeding diathesis, substance abuse, or intake of any herbal preparations. Prior to referral he was given chloroquine, paracetamol, domperidone orally, and intravenous fluids for 3 days. However, fever persisted even after a course of oral chloroquine as per WHO recommendation. On examination in ED, he was drowsy, disoriented with Glasgow Coma Scale of 9 (E2, M5, and V2), febrile (102.5°F), dehydrated, pale, and icteric. There were no other bleeding manifestations. He was hemodynamically stable and maintaining adequate saturation in room air. He had diffuse abdominal tenderness with hepatosplenomegaly. His pupils were unequal with sluggish reaction to light and planters were bilaterally extensor. There was no neck rigidity. His investigations revealed hemoglobin of 6.0 gm/dl (normal: 12.5-14.5), platelet count of 45,000/cumm (140,000-400,000), serum creatinine of 1.8 mg/dl, total bilirubin 9.6 mg/dl (0.4-1), International normalized ratio (INR) of 1.12, and activated partial thromboplastin time (APTT) of 30 s (24-32). His remaining parameters were within normal limits. His HIV status was negative. Peripheral blood smear was positive for Plasmodium vivax gametocytes and ring stages with marked parasitaemia and rapid card test was done to rule out undetected mixed infection, which is based on detecting specific Plasmodium LDH antigen by using monoclonal antibody directed against iso-forms of the enzyme. Noncontrast computed tomography of the cranium demonstrated left side extradural hematoma [Figure 1]. He was started on intravenous artesunate 2.4 mg/kg at the time of admission, then at 12 and 24 h, and then, once a day for 5 days. In view of his deteriorating neurological status, he underwent prompt surgical decompression and 50 ml of dark red blood clots were evacuated. As he had high parasitic index, partial exchange transfusion (1350 ml) was carried out which reduced the parasitaemia. He had a favorable outcome, with remarkable clinical improvement in the immediate postoperative period. We did not start oral artesunate tablets because it also contains mefloquine, which can precipitate neuropsychiatric complications. The repeat peripheral smear at the time of discharge was free of malarial parasite and he was discharged on primaquine 15 mg/day for 14 days after screening for G6PD deficiency. Figure 1 Noncontrast axial cranial CT revealing left temporoparietal acute extradural hematoma Plasmodium vivax infection though considered to be benign and self-limiting disease, in recent past it has produced severe and complicated malaria.[2] The altered sensorium in a malarial patient is often mistaken for cerebral malaria.[3] The occurrence of intracranial hematoma is rare in malaria. Earlier extradural bleed and subdural hematoma were demonstrated in falciparum malaria.[4,5,6,7] It appears that this patient developed spontaneous extradural bleed in the absence of other demonstrable causes for bleeding. Extradural bleed in this patient may be due to the rupture of a small vessel and/or cerebral venules plugged by parasitized erythrocytes coupled with thrombocytopenia. The microrheologic research had clearly demonstrated that P. vivax infection enhances the aggregation, erythrocyte clumping, and reduces deformability affecting the microcirculation.[8] When infected erythrocyte sequestrates, there is local production of lactate due to impaired tissue perfusion. Thus, the cellular hypoxia increases the production of serum tumor necrosis factor-α (TNF-α), a potential immunogenetic marker, and vary with severity of disease. TNF induces profound alterations in endothelial integrity. Also, complex interaction occurs between platelet and parasitized red blood cells leading to vascular injury. All these might have contributed to extradural hematoma in this case. Polymerase chain reaction could not be done due technical limitations. The lesson learnt is that development of an altered sensorium in an established case of malaria while on specific therapy should not be mistaken only as cerebral malaria since it is a diagnosis of exclusion and treated without reconsideration or confirmation by additional entities and investigations will increase the mortality and morbidity. The failure to clear parasitaemia is an indication that the treatment is no longer useful and should be changed.

A novel reverse transcription polymerase chain reaction reveals a high prevalence of Plasmodium vivax gametocyte carriage in an endemic area of Thailand

Background: Gametocytes are precursors of malaria sexual stages that are infective to mosquito vectors and play crucial roles in maintaining cycle of infection. Microscopy cannot determine the status of gametocyte carriage in those who had submicroscopic gametocytemia that may serve as infectious reservoirs in endemic areas. Meanwhile, gametocytes possess stage-specific mRNA that can be detected by molecular methods. Objective: To develop a sensitive method for detection of Plasmodium vivax gametocytes using reverse transcription polymerase chain reaction (RT-PCR) and determine its diagnostic performance in clinical samples. Materials and Methods: A nested RT-PCR was devised using primers targeting Pvs25, a mature gametocytespecific mRNA transcript of P. vivax (nested Pvs25 RT-PCR). Performance of the assay was evaluated using mRNA extracted from blood samples of 180 febrile patients attending a malaria clinic in Tak Province. Total RNA was extracted from blood samples that were preserved in RNAlater???? and from dried blood on filter papers. Malaria species was determined by microscopy from Giemsa stained blood smears and reaffirmed by nested PCR targeting mitochondrial cytochrome b (nested mtCytb PCR). Results: Of 180 blood samples, malaria was diagnosed in 120 patients (69 P. vivax and 51 P. falciparum) by microscopy and 125 patients by nested mtCytb PCR (69 P. vivax, 51 P. falciparum and 5 coinfections with both these species). Microscopy detected gametocytes in 30 of all 74 (40.5%) P. vivax positives by nested mtCytb PCR. Meanwhile, 67 of 74 (90.5%) P. vivax-positive isolates that were preserved in RNAlater???? gave positive results by nested Pvs25 RT-PCR. Therefore, nested Pvs25 RT-PCR identified mature P. vivax gametocytes more than twice as frequently as microscopy. The minimum detection threshold for nested Pvs25 RT-PCR was 10 copies of template DNA whereas no cross-reactivity with other human malaria species was observed. Dried blood collected on filter papers offered comparable results for Pvs25 mRNA detection with blood stored in RNA preservative with only 2.7% difference in positive rates. Conclusion: The nested RT-PCR targeting Pvs25 developed in this study is sensitive and specific for diagnosing mature P. vivax gametocytes and can be efficiently applied to both blood samples kept in RNA preservative and dried blood on filter paper. Keywords: Plasmodium vivax, Gametocyte, Pvs25, reverse transcription polymerase chain reaction, Thailand