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

BACKGROUND: Detection of Plasmodium species 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 Plasmodium vivax and Plasmodium falciparum from the field-caught mosquitoes of Papua New Guinea. METHODS: A method has been developed to concurrently detect mitochondrial cytochrome b (Cyt b) of four human Plasmodium species using PCR (Cytb-PCR). To particularly discriminate P. falciparum from P. vivax, Plasmodium ovale and Plasmodium malariae, 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 P. ovale and P. malariae; this study was mainly confined to P. vivax and P. falciparum. The efficiency of Cytb-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 (SSUrRNA) using field caught mosquitoes collected from three areas (Kaboibus, Wingei, and Jawia) of the East Sepic Province of Papua New Guinea. RESULTS: A total of 90 mosquitoes were artificially infected with three strains of Plasmodium: P. vivax-210 (n = 30), P. vivax-247 (n = 30) and P. falciparum (n = 30). These infected mosquitoes along with another 32 unfed mosquitoes were first checked for the presence of Plasmodium infection by CS-ELISA, and later the same samples were compared with the Cytb-PCR. CS-ELISA for P. vivax-210, P. vivax-247 and P. falciparum detected positive infection in 30, 19 and 18 mosquitoes respectively; whereas Cytb-PCR detected 27, 16 and 16 infections, respectively. The comparison revealed a close agreement between the two assays (kappa = 0.862, 0.842 and 0.894, respectively for Pv-210, Pv-247 and P. falciparum groups). It was found that the eight CS-ELISA-positive mosquitoes detected negative by Cytb-PCR were false-positive results. The lowest detection limit of this Cytb-PCR was 10 sporozoites. A highly concordance result was also found between nested PCR and Cytb-PCR using 107 field caught mosquitoes, and both tests concordantly detected P. falciparum in an Anopheles punctulatus 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 P. falciparum from P. vivax for all of the Cytb-PCR positive samples. CONCLUSION: A single step PCR based method has been introduced here that is highly sensitive, efficient and reliable for identifying P. vivax and P. falciparum from mosquitoes. The reliability of the technique was confirmed by its ability to detect Plasmodium 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.

Molecular phylogeography of Culex quinquefasciatus mosquitoes in central Bangladesh.

Mosquitoes in the Culex pipiens complex are a major vector of numerous parasitic and arboviral diseases. Here we report the phylogeography of a prevalent Culex mosquito, Cx. quinquefasciatus, from three locations in Bangladesh: Dhaka, Savar and Mymensingh. Sequence analysis of the genes encoding mitochondrial cytochrome oxidase subunit II, nuclear elongation factor-1 alpha, and acetylcholinesterase-2 revealed the lack of a population genetic structure among the three locations. Moreover, the highly divergent ribosomal internal transcribed spacer 2 suggests that this locus has not evolved in concert. The results further show evidence of historical introgression of internal transcribed spacer 2 from Cx. pipiens to Cx. quinquefasciatus of Bangladesh, and that the introgression occurred before Cx. quinquefasciatus had dispersed within this region. The study also reveals historical population expansion in this region, followed by a post-expansion Wolbachia sweep.

Genetic diversity in two sibling species of the Anopheles punctulatus group of mosquitoes on Guadalcanal in the Solomon Islands.

BACKGROUND: The mosquito Anopheles irenicus, a member of the Anopheles punctulatus group, is geographically restricted to Guadalcanal in the Solomon Islands. It shows remarkable morphological similarities to one of its sibling species, An. farauti sensu stricto (An. farauti s.s.), but is dissimilar in host and habitat preferences. To infer the genetic variations between these two species, we have analyzed mitochondrial cytochrome oxidase subunit II (COII) and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences from Guadalcanal and from one of its nearest neighbours, Malaita, in the Solomon Islands. RESULTS: An. farauti s.s. was collected mostly from brackish water and by the human bait method on both islands, whereas An. irenicus was only collected from fresh water bodies on Guadalcanal Island. An. irenicus is distributed evenly with An. farauti s.s. (Phi SC = 0.033, 0.38%) and its range overlaps in three of the seven sampling sites. However, there is a significant population genetic structure between the species (Phi CT = 0.863, P < 0.01; Phi ST = 0.865, P < 0.01 and FST = 0.878, P < 0.01). Phylogenetic analyses suggest that An. irenicus is a monophyletic species, not a hybrid, and is closely related to the An. farauti s.s. on Guadalcanal. The time estimator suggests that An. irenicus diverged from the ancestral An. farauti s.s. on Guadalcanal within 29,000 years before present (BP). An. farauti s.s. expanded much earlier on Malaita (texp = 24,600 BP) than the populations on Guadalcanal (texp = 16,800 BP for An. farauti s.s. and 14,000 BP for An. irenicus). CONCLUSION: These findings suggest that An. irenicus and An. farauti s.s. are monophyletic sister species living in sympatry, and their populations on Guadalcanal have recently expanded. Consequently, the findings further suggest that An. irenicus diverged from the ancestral An. farauti s.s. on Guadalcanal.

Intertidal snail-trematode communities on the southern Thailand before and after the South Asia tsunami.

Intertidal snail-trematode communities in southern Thailand were examined before and after the South Asia tsunami. Infection rates and species diversity of cercaria in the host snail Cerithidea in tidal zones did not change significantly from one year before to one month after the tsunami. However, the host snails C. quadrata, C. alata and C. obtusa disappeared from greatly damaged sites. It is important to follow up on the intertidal snail-trematode community recovery process after destruction of the intertidal ecosystem.