Implementation of a novel PCR based method for detecting malaria parasites from naturally infected mosquitoes in Papua New Guinea

<p>Abstract</p> <p>Background</p> <p>Detection of <it>Plasmodium species </it>in mosquitoes is important for designing vector control studies. However, most of the PCR-based detection methods show some potential limitations. The objective of this study was to introduce an effective PCR-based method for detecting <it>Plasmodium vivax </it>and <it>Plasmodium falciparum </it>from the field-caught mosquitoes of Papua New Guinea.</p> <p>Methods</p> <p>A method has been developed to concurrently detect mitochondrial cytochrome b (<it>Cyt b</it>) of four human <it>Plasmodium </it>species using PCR (<it>Cytb</it>-PCR). To particularly discriminate <it>P. falciparum </it>from <it>P. vivax</it>, <it>Plasmodium ovale </it>and <it>Plasmodium malariae</it>, a polymerase chain reaction-repeated fragment length polymorphism (PCR-RFLP) has further been developed to use with this method. However, due to limited samples number of <it>P. ovale </it>and <it>P. malariae</it>; this study was mainly confined to <it>P. vivax </it>and <it>P. falciparum</it>. The efficiency of <it>Cytb</it>-PCR was evaluated by comparing it with two 'gold standards' enzyme linked immunosorbent assay specific for circumsporozoite protein (CS-ELISA) using artificially infected mosquitoes; and nested PCR specific for small subunit ribosomal RNA (<it>SSUrRNA</it>) using field caught mosquitoes collected from three areas (Kaboibus, Wingei, and Jawia) of the East Sepic Province of Papua New Guinea.</p> <p>Results</p> <p>A total of 90 mosquitoes were artificially infected with three strains of <it>Plasmodium</it>: <it>P. vivax-</it>210 (<it>n </it>= 30), <it>P. vivax</it>-247 (<it>n </it>= 30) and <it>P. falciparum </it>(<it>n </it>= 30). These infected mosquitoes along with another 32 unfed mosquitoes were first checked for the presence of <it>Plasmodium </it>infection by CS-ELISA, and later the same samples were compared with the <it>Cytb</it>-PCR. CS-ELISA for <it>P. vivax</it>-210, <it>P. vivax</it>-247 and <it>P. falciparum </it>detected positive infection in 30, 19 and 18 mosquitoes respectively; whereas <it>Cytb</it>-PCR detected 27, 16 and 16 infections, respectively. The comparison revealed a close agreement between the two assays (κ = 0.862, 0.842 and 0.894, respectively for Pv-210, Pv-247 and <it>P. falciparum </it>groups). It was found that the eight CS-ELISA-positive mosquitoes detected negative by <it>Cytb</it>-PCR were false-positive results. The lowest detection limit of this <it>Cytb</it>-PCR was 10 sporozoites. A highly concordance result was also found between nested PCR and <it>Cytb</it>-PCR using 107 field caught mosquitoes, and both tests concordantly detected <it>P. falciparum </it>in an <it>Anopheles punctulatus </it>mosquito collected from Kaboibus. Both tests thus suggested an overall sporozoite rate of 0.9% (1/107) in the study areas. Subsequently, PCR-RFLP efficiently discriminated <it>P. falciparum </it>from <it>P. vivax </it>for all of the <it>Cytb</it>-PCR positive samples.</p> <p>Conclusion</p> <p>A single step PCR based method has been introduced here that is highly sensitive, efficient and reliable for identifying <it>P. vivax </it>and <it>P. falciparum </it>from mosquitoes. The reliability of the technique was confirmed by its ability to detect <it>Plasmodium </it>as efficiently as those of CS-ELISA and nested PCR. Application of the assay offers the opportunity to detect vector species of Papua New Guinea and may contribute for designing further vector control programmes.</p

Transmission-blocking activity induced by malaria vaccine candidates Pfs25/Pvs25 is a direct and predictable function of antibody titer

<p>Abstract</p> <p>Background</p> <p>Mosquito stage malaria vaccines are designed to induce an immune response in the human host that will block the parasite's growth in the mosquito and consequently block transmission of the parasite. A mosquito membrane-feeding assay (MFA) is used to test transmission-blocking activity (TBA), but in this technique cannot accommodate many samples. A clear understanding of the relationship between antibody levels and TBA may allow ELISA determinations to be used to predict TBA and assist in planning vaccine development.</p> <p>Methods</p> <p>Rabbit anti-Pfs25 sera and monkey anti-Pvs25 sera were generated and the antibody titers were determined by a standardized ELISA. The biological activity of the same sera was tested by MFA using <it>Plasmodium </it>gametocytes (cultured <it>Plasmodium falciparum </it>or <it>Plasmodium vivax </it>from malaria patients) and <it>Anopheles </it>mosquitoes.</p> <p>Results</p> <p>Anti-Pfs25 and anti-Pvs25 sera showed that ELISA antibody units correlate with the percent reduction in the oocyst density per mosquito (Spearman Rank correlations: 0.934 and 0.616, respectively), and fit a hyperbolic curve when percent reduction in oocyst density is plotted against antibody units of the tested sample. Antibody levels also correlated with the number of mosquitoes that failed to become infected, and this proportion can be calculated from the reduction in oocyst numbers and the distribution of oocysts per infected mosquito in control group.</p> <p>Conclusion</p> <p>ELISA data may be used as a surrogate for the MFA to evaluate transmission-blocking vaccine efficacy. This will facilitate the evaluation of transmission-blocking vaccines and implementation of this malaria control strategy.</p

Highly heterogeneous residual malaria risk in western Thailand

Over the past decades, the malaria burden in Thailand has substantially declined. Most infections now originate from the national border regions. In these areas, the prevalence of asymptomatic infections is still substantial and poses a challenge for the national malaria elimination program. To determine epidemiological parameters as well as risk factors for malaria infection in western Thailand, we carried out a cohort study in Kanchanaburi and Ratchaburi provinces on the Thailand-Myanmar border. Blood samples from 999 local participants were examined for malaria infection every 4 weeks between May 2013 and Jun 2014. Prevalence of Plasmodium falciparum and Plasmodium vivax was determined by quantitative PCR (qPCR) and showed a seasonal variation with values fluctuating from 1.7% to 4.2% for P. vivax and 0% to 1.3% for P. falciparum. Ninety percent of infections were asymptomatic. The annual molecular force of blood-stage infection (molFOB) was estimated by microsatellite genotyping to be 0.24 new infections per person-year for P. vivax and 0.02 new infections per person-year for P. falciparum. The distribution of infections was heterogenous, that is, the vast majority of infections (>80%) were found in a small number of individuals (<8% of the study population) who tested positive at multiple timepoints. Significant risk factors were detected for P. vivax infections, including previous clinical malaria, occupation in agriculture and travel to Myanmar. In contrast, indoor residual spraying was associated with a protection from infection. These findings provide a recent landscape of malaria epidemiology and emphasize the importance of novel strategies to target asymptomatic and imported infections

Malaria Elimination in the Greater Mekong Subregion: Challenges and Prospects

Malaria is a significant public health problem and impediment to socioeconomic development in countries of the Greater Mekong Subregion (GMS), which comprises Cambodia, China’s Yunnan Province, Lao People’s Democratic Republic, Myanmar, Thailand, and Vietnam. Over the past decade, intensified malaria control has greatly reduced the regional malaria burden. Driven by increasing political commitment, motivated by recent achievements in malaria control, and urged by the imminent threat of emerging artemisinin resistance, the GMS countries have endorsed a regional malaria elimination plan with a goal of eliminating malaria by 2030. However, this ambitious, but laudable, goal faces a daunting array of challenges and requires integrated strategies tailored to the region, which should be based on a mechanistic understanding of the human, parasite, and vector factors sustaining continued malaria transmission along international borders. Malaria epidemiology in the GMS is complex and rapidly evolving. Spatial heterogeneity requires targeted use of the limited resources. Border malaria accounts for continued malaria transmission and represents sources of parasite introduction through porous borders by highly mobile human populations. Asymptomatic infections constitute huge parasite reservoir requiring interventions in time and place to pave the way for malaria elimination. Of the two most predominant malaria parasites, Plasmodium falciparum and P. vivax, the prevalence of the latter is increasing in most member GMS countries. This parasite requires the use of 8-aminoquinoline drugs to prevent relapses from liver hypnozoites, but high prevalence of glucose-6-phosphate dehydrogenase deficiency in the endemic human populations makes it difficult to adopt this treatment regimen. The recent emergence of resistance to artemisinins and partner drugs in P. falciparum has raised both regional and global concerns, and elimination efforts are invariably prioritized against this parasite to avert spread. Moreover, the effectiveness of the two core vector control interventions—insecticide-treated nets and indoor residual spraying—has been declining due to insecticide resistance and increased outdoor biting activity of mosquito vectors. These technical challenges, though varying from country to country, require integrated approaches and better understanding of the malaria epidemiology enabling targeted control of the parasites and vectors. Understanding the mechanism and distribution of drug-resistant parasites will allow effective drug treatment and prevent, or slow down, the spread of drug resistance. Coordination among the GMS countries is essential to prevent parasite reintroduction across the international borders to achieve regional malaria elimination

Improvement of culture conditions for long-term in vitro culture of Plasmodium vivax

Background: The study of the biology, transmission and pathogenesis of Plasmodium vivax is hindered due to the lack of a robustly propagating, continuous culture of this parasite. The current culture system for P. vivax parasites still suffered from consistency and difficulties in long-term maintenance of parasites in culture and for providing sufficient biological materials for studying parasite biology. Therefore, further improvement of culture conditions for P. vivax is needed. Methods: Clinical samples were collected from patients diagnosed with P. vivax in western Thailand. Leukocyte-depleted P. vivax infected blood samples were cultured in a modified McCoy's 5A medium at 5% haematocrit under hypoxic condition (5% O 2 , 5% CO 2 , and 90% N 2 ). Reticulocytes purified from adult peripheral blood were added daily to maintain 4% reticulocytes. Parasites were detected by microscopic examination of Giemsa-stained smears and molecular methods. Results: The effects of culture variables were first analysed in order to improve the culture conditions for P. vivax. Through analysis of the sources of host reticulocytes and nutrients of culture medium, the culture conditions better supporting in vitro growth and maturation of the parasites were identified. Using this system, three of 30 isolates could be maintained in vitro for over 26 months albeit parasite density is low. Conclusions: Based on the analysis of different culture variables, an improved and feasible protocol for continuous culture of P. vivax was developed

Natural infection of Plasmodium falciparum induces inhibitory antibodies against gametocyte development in human hosts.

SUMMARY: We identified naturally induced antibodies from malaria patients in Thailand and clarified the effect of the antibodies on gametocyte development. Fifty-nine percent of the Plasmodium falciparum-infected blood samples (17 of 29) fed to female Anopheles mosquitoes showed no oocyst infection. Seventeen percent of the samples (5 of 29) distorted the morphology and hampered the maturity of the gametocytes. A possible mechanism for the gametocyte inhibitory activity was shown by the binding of the plasma antibodies to live, immature, intraerythrocytic gametocytes during the incubation period. One hundred fifty-seven proteins specific to different gametocyte stages were explored to find the targets of the antisera that bound to the live gametocytes. However, no additional gametocyte transmission-blocking vaccine candidate was detected. Therefore, the development of alternative transmission-blocking vaccines in high-transmission areas should focus on the identification of more gametocyte antigens-inducing inhibitory antibodies that reduce gametocytemia

Population dynamics of sporogony for Plasmodium vivax parasites from western Thailand developing within three species of colonized Anopheles mosquitoes

BACKGROUND: The population dynamics of Plasmodium sporogony within mosquitoes consists of an early phase where parasite abundance decreases during the transition from gametocyte to oocyst, an intermediate phase where parasite abundance remains static as oocysts, and a later phase where parasite abundance increases during the release of progeny sporozoites from oocysts. Sporogonic development is complete when sporozoites invade the mosquito salivary glands. The dynamics and efficiency of this developmental sequence were determined in laboratory strains of Anopheles dirus, Anopheles minimus and Anopheles sawadwongporni mosquitoes for Plasmodium vivax parasites circulating naturally in western Thailand. METHODS: Mosquitoes were fed blood from 20 symptomatic Thai adults via membrane feeders. Absolute densities were estimated for macrogametocytes, round stages (= female gametes/zygotes), ookinetes, oocysts, haemolymph sporozoites and salivary gland sporozoites. From these census data, five aspects of population dynamics were analysed; 1) changes in life-stage prevalence during early sporogony, 2) kinetics of life-stage formation, 3) efficiency of life-stage transitions, 4) density relationships between successive life-stages, and 5) parasite aggregation patterns. RESULTS: There was no difference among the three mosquito species tested in total losses incurred by P. vivax populations during early sporogony. Averaged across all infections, parasite populations incurred a 68-fold loss in abundance, with losses of ca. 19-fold, 2-fold and 2-fold at the first (= gametogenesis/fertilization), second (= round stage transformation), and third (= ookinete migration) life-stage transitions, respectively. However, total losses varied widely among infections, ranging from 6-fold to over 2,000-fold loss. Losses during gametogenesis/fertilization accounted for most of this variability, indicating that gametocytes originating from some volunteers were more fertile than those from other volunteers. Although reasons for such variability were not determined, gametocyte fertility was not correlated with blood haematocrit, asexual parasitaemia, gametocyte density or gametocyte sex ratio. Round stages and ookinetes were present in mosquito midguts for up to 48 hours and development was asynchronous. Parasite losses during fertilization and round stage differentiation were more influenced by factors intrinsic to the parasite and/or factors in the blood, whereas ookinete losses were more strongly influenced by mosquito factors. Oocysts released sporozoites on days 12 to 14, but even by day 22 many oocysts were still present on the midgut. The per capita production was estimated to be approximately 500 sporozoites per oocyst and approximately 75% of the sporozoites released into the haemocoel successfully invaded the salivary glands. CONCLUSION: The major developmental bottleneck in early sporogony occurred during the transition from macrogametocyte to round stage. Sporozoite invasion into the salivary glands was very efficient. Information on the natural population dynamics of sporogony within malaria-endemic areas may benefit intervention strategies that target early sporogony (e.g., transmission blocking vaccines, transgenic mosquitoes)

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 ∼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

Radical curative efficacy of tafenoquine combination regimens in Plasmodium cynomolgi-infected Rhesus monkeys (Macaca mulatta)

<p>Abstract</p> <p>Background</p> <p>Tafenoquine is an 8-aminoquinoline being developed for radical cure (blood and liver stage elimination) of <it>Plasmodium vivax</it>. During monotherapy treatment, the compound exhibits slow parasite and fever clearance times, and toxicity in glucose-6-phosphate dehydrogenase (G6PD) deficiency is a concern. Combination with other antimalarials may mitigate these concerns.</p> <p>Methods</p> <p>In 2005, the radical curative efficacy of tafenoquine combinations was investigated in <it>Plasmodium cynomolgi</it>-infected naïve Indian-origin Rhesus monkeys. In the first cohort, groups of two monkeys were treated with a three-day regimen of tafenoquine at different doses alone and in combination with a three-day chloroquine regimen to determine the minimum curative dose (MCD). In the second cohort, the radical curative efficacy of a single-day regimen of tafenoquine-mefloquine was compared to that of two three-day regimens comprising tafenoquine at its MCD with chloroquine or artemether-lumefantrine in groups of six monkeys. In a final cohort, the efficacy of the MCD of tafenoquine against hypnozoites alone and in combination with chloroquine was investigated in groups of six monkeys after quinine pre-treatment to eliminate asexual parasites. Plasma tafenoquine, chloroquine and desethylchloroquine concentrations were determined by LC-MS in order to compare doses of the drugs to those used clinically in humans.</p> <p>Results</p> <p>The total MCD of tafenoquine required in combination regimens for radical cure was ten-fold lower (1.8 mg/kg <it>versus </it>18 mg/kg) than for monotherapy. This regimen (1.8 mg/kg) was equally efficacious as monotherapy or in combination with chloroquine after quinine pre-treatment to eliminate asexual stages. The same dose of (1.8 mg/kg) was radically curative in combination with artemether-lumefantrine. Tafenoquine was also radically curative when combined with mefloquine. The MCD of tafenoquine monotherapy for radical cure (18 mg/kg) appears to be biologically equivalent to a 600-1200 mg dose in humans. At its MCD in combination with blood schizonticidal drugs (1.8 mg/kg), the maximum observed plasma concentrations were substantially lower than (20-84 <it>versus </it>550-1,100 ng/ml) after administration of 1, 200 mg in clinical studies.</p> <p>Conclusions</p> <p>Ten-fold lower clinical doses of tafenoquine than used in prior studies may be effective against <it>P. vivax </it>hypnozoites if the drug is deployed in combination with effective blood-schizonticidal drugs.</p