Kwang Shik Choi

Background: Recent studies presented two clades (clades I and II) within the major malaria vector, Anopheles funestus s.s. on the mitochondrial DNA. We describe a hydrolysis probe analysis (Taqman assay) method for the rapid identification of these two clades. Findings: A total of 53 An. funestus s.s. from Malawi and Mozambique were tested for detection of clade types using the hydrolysis probe analysis. Results were compared to DNA sequence analysis to verify the accuracy of the probes Taqman assay for this vector species. Analysis using the hydrolysis probe revealed that there were 21 individuals from Malawi and 13 individuals from Mozambique for clade I, and 19 individuals from Mozambique for clade II. The results were consistent with the results of DNA sequences. A field sample from northern Zambia revealed the presence of both clade types. Conclusion: A diagnostic method using the hydrolysis probe analysis was developed to identify clade types within An. funestus s.s. This assay will be useful for screening clade types of field-collected An. funestus specimens accurately and efficiently in malaria vector research and control studies.

Anopheles funestus s.s., one of the major malaria vectors in sub-Saharan Africa, belongs to a group of eleven African species that are morphologically similar at the adult stage, most of which do not transmit malaria. The population structure of An. funestus based on mitochondrial DNA data led to the description of two cryptic subdivisions, clade I widespread throughout Africa and clade II known only from Mozambique and Madagascar. In this study, we investigated five common members of the Anopheles funestus group in southern Africa in order to determine relationships within and between species.

Background: Malaria vector control programmes that rely on insecticide-based interventions such as indoor house spraying with residual insecticides or insecticide treated bed nets, need to base their decision-making process on sound baseline data. More and more commercial entities in Africa, such as mining companies, are realising the value to staff productivity of controlling malaria transmission in their areas of operation. This paper presents baseline entomological data obtained during surveys conducted for four mining operations in Ghana, West Africa. Results: The vast majority of the samples were identified as Anopheles gambiae S form with only a few M form specimens being identified from Tarkwa. Plasmodium falciparum infection rates ranged from 4.5 to 8.6% in An. gambiae and 1.81 to 8.06% in An. funestus. High survival rates on standard WHO bioassay tests were recorded for all insecticide classes except the organophosphates that showed reasonable mortality at all locations (i.e. > 90%). The West African kdr mutation was detected and showed high frequencies in all populations. Conclusions: The data highlight the complexity of the situation prevailing in southern Ghana and the challenges facing the malaria vector control programmes in this region. Vector control programmes in Ghana need to carefully consider the resistance profiles of the local mosquito populations in order to base their resistance management strategies on sound scientific data.

Anopheles longipalpis is morphologically similar to the major African malaria vector Anopheles funestus at the adult stage although it is very different at the larval stage. Despite the development of the species-specific multiplex PCR assay for the An. funestus group, the genomic DNA of Anopheles longipalpis type C specimens can be amplified with the Anopheles vaneedeni and Anopheles parensis primers from this assay. The standard, species-specific An. funestus group PCR, results in the amplification of two fragments when An. longipalpis type C specimens are included in the analysis. This result can easily be misinterpreted as being a hybrid between An. vaneedeni and An. parensis. Anopheles longipalpis type C can be identified using a species-specific PCR assay but this assay is not reliable if other members of the An. funestus group, such as An. funestus, An. funestus-like and An. parensis, are included. The present study provides a multiplex assay that will identify An. longipalpis along with other common members of the African An. funestus group, including Anopheles leesoni.

Background: Knockdown resistance (kdr) caused by a single base pair mutation in the sodium channel gene is strongly associated with pyrethroid insecticide resistance in Anopheles gambiae in West-Central Africa. Recently, various molecular techniques have been developed to screen for the presence of the kdr mutations in vector populations with varying levels of accuracy. In this study, the results of the hydrolysis probe analysis for detecting the kdr mutations in An. gambiae s.s. from the Republic of the Congo were compared with DNA sequence analysis. Methods: A total of 52 pyrethroid and DDT resistant An. gambiae from Pointe-Noire (Congo-Brazzaville) were tested for detection of the two kdr mutations (kdr-e and kdr-w) that are known to occur in this species. Results from the hydrolysis probe analysis were compared to DNA sequencing to verify the accuracy of the probe analysis for this vector population. Results: Fifty-one specimens were found to be An. gambiae S-form and one was a M/S hybrid. DNA sequencing revealed that more than half of the specimens (55.8%) carried both the kdr-e and kdr-w resistance mutations, seven specimens (13.5%) were homozygous for the kdr-e mutation, and 14 specimens (26.9%) were homozygous for the kdr-w mutation. A single individual was genotyped as heterozygous kdr-e mutation (1.9%) only and another as heterozygous kdr-w mutation (1.9%) only. Analysis using hydrolysis probe analysis, without adjustment of the allelic discrimination axes on the scatter plots, revealed six specimens (11.5%) carrying both mutations, 30 specimens (57.8%) as homozygous kdr-w, six specimens (11.5%) homozygous for the kdr-e mutation, one specimen (1.9%) heterozygous for the kdr-w mutation and one specimen (1.9%) present in wild type form. Eight of the specimens (15.4%) could not be identified using unadjusted hydrolysis probe analysis values. No heterozygous kdr-e mutations were scored when adjustment for the allelic discrimination axes was omitted. However, when the axes on the scatter plots were adjusted the results were consistent with those of the DNA sequence analysis, barring two individuals that were mis-scored in the hydrolysis probe analysis. Conclusion: Both the kdr-e and kdr-w mutations were abundant in An. gambiae S-form from Pointe-Noire. The hydrolysis probe analysis can lead to misleading results if adjustment to allelic discrimination axes is not investigated. This is mainly relevant when both kdr-e and kdr-w are present in a population in a high frequency. This report highlights the importance of concurrent screening for both mutations. Therefore, performing routine assay protocols blindly can result in the misinterpretation of results. Although hydrolysis probe analysis of kdr is still held as the gold standard assay, this paper highlights the importance of kdr mutation confirmation via sequencing especially in regions where kdr frequency has never been reported before or where both the kdr-e and kdr-w mutations are present simultaneously.