Insecticidal bed nets and filariasis transmission in Papua New Guinea.

BACKGROUND: Global efforts to eliminate lymphatic filariasis are based on the annual mass administration of antifilarial drugs to reduce the microfilaria reservoir available to the mosquito vector. Insecticide-treated bed nets are being widely used in areas in which filariasis and malaria are coendemic. METHODS: We studied five villages in which five annual mass administrations of antifilarial drugs, which were completed in 1998, reduced the transmission of Wuchereria bancrofti, one of the nematodes that cause lymphatic filariasis. A total of 21,899 anopheles mosquitoes were collected for 26 months before and 11 to 36 months after bed nets treated with long-lasting insecticide were distributed in 2009. We evaluated the status of filarial infection and the presence of W. bancrofti DNA in anopheline mosquitoes before and after the introduction of insecticide-treated bed nets. We then used a model of population dynamics to estimate the probabilities of transmission cessation. RESULTS: Village-specific rates of bites from anopheline mosquitoes ranged from 6.4 to 61.3 bites per person per day before the bed-net distribution and from 1.1 to 9.4 bites for 11 months after distribution (P<0.001). During the same period, the rate of detection of W. bancrofti in anopheline mosquitoes decreased from 1.8% to 0.4% (P=0.005), and the rate of detection of filarial DNA decreased from 19.4% to 14.9% (P=0.13). The annual transmission potential was 5 to 325 infective larvae inoculated per person per year before the bed-net distribution and 0 after the distribution. Among all five villages with a prevalence of microfilariae of 2 to 38%, the probability of transmission cessation increased from less than 1.0% before the bed-net distribution to a range of 4.9 to 95% in the 11 months after distribution. CONCLUSIONS: Vector control with insecticide-treated bed nets is a valuable tool for W. bancrofti elimination in areas in which anopheline mosquitoes transmit the parasite. (Funded by the U.S. Public Health Service and the National Institutes of Health.).

Multiplex assay for species identification and monitoring of insecticide resistance in Anopheles punctulatus group populations of Papua New Guinea.

Anopheles punctulatus sibling species (An. punctulatus s.s., Anopheles koliensis, and Anopheles farauti species complex [eight cryptic species]) are principal vectors of malaria and filariasis in the Southwest Pacific. Given significant effort to reduce malaria and filariasis transmission through insecticide-treated net distribution in the region, effective strategies to monitor evolution of insecticide resistance among An. punctulatus sibling species is essential. Mutations in the voltage-gated sodium channel (VGSC) gene have been associated with knock-down resistance (kdr) to pyrethroids and DDT in malarious regions. By examining VGSC sequence polymorphism we developed a multiplex assay to differentiate wild-type versus kdr alleles and query intron-based polymorphisms that enable simultaneous species identification. A survey including mosquitoes from seven Papua New Guinea Provinces detected no kdr alleles in any An. punctulatus species. Absence of VGSC sequence introgression between species and evidence of geographic separation within species suggests that kdr must be monitored in each An. punctulatus species independently.

High throughput multiplex assay for species identification of Papua New Guinea malaria vectors: members of the Anopheles punctulatus (Diptera: Culicidae) species group.

Malaria and filariasis are transmitted in the Southwest Pacific region by Anopheles punctulatus sibling species including An. punctulatus, An. koliensis, the An. farauti complex 1-8 (includes An. hinesorum [An. farauti 2], An. torresiensis [An. farauti 3]). Distinguishing these species from each other requires molecular diagnostic methods. We developed a multiplex polymerase chain reaction (PCR)-based assay specific for known species-specific nucleotide differences in the internal transcribed spacer 2 region and identified the five species most frequently implicated in transmitting disease (An. punctulatus, An. koliensis, An. farauti 1, An. hinesorum, and An. farauti 4). A set of 340 individual mosquitoes obtained from seven Papua New Guinea provinces representing a variety of habitats were analyzed by using this multiplex assay. Concordance between molecular and morphological diagnosis was 56.4% for An. punctulatus, 85.3% for An. koliensis, and 88.9% for An. farauti. Among 158 mosquitoes morphologically designated as An. farauti, 33 were re-classified by PCR as An. punctulatus, 4 as An. koliensis, 26 as An. farauti 1, 49 as An. hinesorum, and 46 as An. farauti 4. Misclassification results from variable coloration of the proboscis and overlap of An. punctulatus, An. koliensis, the An. farauti 4. This multiplex technology enables further mosquito strain identification and simultaneous detection of microbial pathogens.

High-throughput molecular diagnosis of circumsporozoite variants VK210 and VK247 detects complex Plasmodium vivax infections in malaria endemic populations in Papua New Guinea.

Malaria is endemic in lowland and coastal regions of Papua New Guinea (PNG), and is caused by Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. Infection by P. vivax is attributed to distinct strains, VK210 and VK247, which differ in the sequence of the circumsporozoite protein (pvcsp). Here, based upon sequence polymorphisms in pvcsp, we developed a post-PCR ligation detection reaction-fluorescent microsphere assay (LDR-FMA) to distinguish these P. vivax strains. This diagnostic assay was designed to detect the presence of both VK210 and VK247 P. vivax strains simultaneously in a high-throughput 96-well format. Using this assay, we analyzed human blood samples from the Wosera (n=703) and Mugil (n=986) regions to evaluate the prevalence of these P. vivax strains. VK210 and VK247 strains were found in both study sites. In the Wosera, single infections with VK210 strain were observed to be most common (41.7%), followed by mixed-strain (36.8%) and VK247 single-strain infections (21.5%). Similarly, in Mugil, VK210 single-strain infections were most common (51.6%), followed by mixed-strain (34.4%) and VK247 single-strain infections (14%). These results suggest that the distribution of P. vivax infections was similar between the two study sites. Interestingly, we observed a non-random distribution of these two P. vivax strains, as mixed-strain infections were significantly more prevalent than expected in both study sites (Wosera and Mugil χ(2)p-value<0.001). Additionally, DNA sequence analysis of a subset of P. vivax infections showed that no individual pvcsp alleles were shared between the two study sites. Overall, our results illustrate that PNG malaria-endemic regions harbor a complex mixture of P. vivax strains, and emphasize the importance of malaria control strategies that would be effective against a highly diverse parasite population.

Pyrethroid susceptibility in natural populations of the Anopheles punctulatus group (Diptera: Culicidae) in Papua New Guinea.

The development of insecticide resistance has compromised mosquito control efforts in many parts of the world. Papua New Guinea (PNG) has a long history of dichlorodiphenyltrichloroethane (DDT) use and currently distributes pyrethroid-treated nets for malaria control. This study is the first to investigate the status of pyrethroid resistance in the Anopheles punctulatus group, the major malaria and filariasis vectors of PNG. The study used World Health Organization standard susceptibility bioassays to detect knockdown phenotypes and a novel nested polymerase chain reaction to detect the knockdown resistant (kdr) allele in these vectors. Our results show 100% susceptibility to pyrethroids in all populations surveyed and an absence of the kdr allele.

Molecular-based assay for simultaneous detection of four Plasmodium spp. and Wuchereria bancrofti infections.

Four major malaria-causing Plasmodium spp. and lymphatic filariasis-causing Wuchereria bancrofti are co-endemic in many tropical and sub-tropical regions. Among molecular diagnostic assays, multiplex polymerase chain reaction (PCR)-based assays for the simultaneous detection of DNAs from these parasite species are currently available only for P. falciparum and W. bancrofti or P. vivax and W. bancrofti. Using a post-PCR oligonucleotide ligation detection reaction-fluorescent microsphere assay (LDR-FMA), we developed a multiplex assay that has the capability to simultaneously detect all four Plasmodium spp. and W. bancrofti infections in blood samples. Compared with microfilarial positivity in the blood, the LDR-FMA assay is highly concordant (91%), sensitive (86%), and specific (94%), and has high reproducibility for Plasmodium spp. (85-93%) and W. bancrofti (90%) diagnoses. The development of this assay for the simultaneous diagnosis of multiple parasitic infections enables efficient screening of large numbers of human blood and mosquito samples from co-endemic areas.

Application of pharmacogenomics to malaria: a holistic approach for successful chemotherapy.

Drug resistance in malaria jeopardizes the most elementary objectives of malaria control--reducing suffering and eliminating mortality. An important, and so far the only known, mechanism of drug resistance appears to be polymorphisms in the malaria parasite genes. Efforts to circumvent antimalarial drug resistance now range from the use of combination therapies with existing agents to genomics-based studies directed toward discovering novel targets and agents. However, the potential contribution of host genetic/molecular factors, particularly those associated with antimalarial drug metabolism, remains largely unexplored. Our knowledge concerning the basic mechanisms involved in the pharmacokinetics of antimalarial drugs is fragmentary. In addition, the link between antimalarial drug pharmacokinetics and treatment outcomes is generally unclear. The purpose of this article is to provide general background information on antimalarial drug resistance and associated parasite genetic factors, and subsequently highlight the aforementioned unexplored and unclear areas, with a view to stimulate much needed further research.