Simultaneous identification of the Anopheles funestus group and Anopheles longipalpis type C by PCR-RFLP

<p>Abstract</p> <p>Background</p> <p><it>Anopheles longipalpis </it>is morphologically similar to the major African malaria vector <it>Anopheles funestus </it>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 <it>An. funestus </it>group, the genomic DNA of <it>Anopheles longipalpis </it>type C specimens can be amplified with the <it>Anopheles vaneedeni </it>and <it>Anopheles parensis </it>primers from this assay. The standard, species-specific <it>An. funestus </it>group PCR, results in the amplification of two fragments when <it>An. longipalpis </it>type C specimens are included in the analysis. This result can easily be misinterpreted as being a hybrid between <it>An. vaneedeni </it>and <it>An. parensis</it>. <it>Anopheles longipalpis </it>type C can be identified using a species-specific PCR assay but this assay is not reliable if other members of the <it>An. funestus </it>group, such as <it>An. funestus</it>, <it>An. funestus</it>-like and <it>An. parensis</it>, are included. The present study provides a multiplex assay that will identify <it>An. longipalpis </it>along with other common members of the African <it>An. funestus </it>group, including <it>Anopheles leesoni</it>.</p> <p>Methods</p> <p>A total of 70 specimens from six species (<it>An. funestus</it>, <it>An. funestus</it>-like, <it>An. parensis</it>, <it>Anopheles rivulorum</it>, <it>An. vaneedeni </it>and <it>An. leesoni</it>) in the <it>An. funestus </it>group and <it>An. longipalpis </it>type C from Malawi, Mozambique, South Africa and Zambia were used for the study. A restriction fragment length polymorphism (RFLP) assay was designed based on the DNA sequence information in the GenBank database.</p> <p>Results</p> <p>The enzyme, <it>EcoRI </it>digested only <it>An. longipalpis </it>type C and <it>An. funestus</it>-like after the species-specific <it>An. funestus </it>group PCR assay. The <it>An. longipalpis </it>and <it>An. funestus</it>-like digestion profiles were characterized by three fragments, 376 bp, 252 bp and 211 bp for <it>An. longipalpis </it>type C and two fragments, 375 bp and 15 bp for <it>An. funestus</it>-like.</p> <p>Conclusions</p> <p>An RFLP method for the group was developed that is more accurate and efficient than those used before. Hence, this assay would be useful for field-collected adult specimens to be identified routinely in malaria vector research and control studies.</p

Challenges for Malaria Elimination in Zanzibar: Pyrethroid Resistance in Malaria Vectors and Poor Performance of Long-Lasting Insecticide Nets.

Long-lasting insecticide treated nets (LLINs) and indoor residual house spraying (IRS) are the main interventions for the control of malaria vectors in Zanzibar. The aim of the present study was to assess the susceptibility status of malaria vectors against the insecticides used for LLINs and IRS and to determine the durability and efficacy of LLINs on the island. Mosquitoes were sampled from Pemba and Unguja islands in 2010--2011 for use in WHO susceptibility tests. One hundred and fifty LLINs were collected from households on Unguja, their physical state was recorded and then tested for efficacy as well as total insecticide content. Species identification revealed that over 90% of the Anopheles gambiae complex was An. arabiensis with a small number of An. gambiae s.s. and An. merus being present. Susceptibility tests showed that An. arabiensis on Pemba was resistant to the pyrethroids used for LLINs and IRS. Mosquitoes from Unguja Island, however, were fully susceptible to all pyrethroids tested. A physical examination of 150 LLINs showed that two thirds were damaged after only three years in use. All used nets had a significantly lower (p < 0.001) mean permethrin concentration of 791.6 mg/m2 compared with 944.2 mg/m2 for new ones. Their efficacy decreased significantly against both susceptible An. gambiae s.s. colony mosquitoes and wild-type mosquitoes from Pemba after just six washes (p < 0.001). The sustainability of the gains achieved in malaria control in Zanzibar is seriously threatened by the resistance of malaria vectors to pyrethroids and the short-lived efficacy of LLINs. This study has revealed that even in relatively well-resourced and logistically manageable places like Zanzibar, malaria elimination is going to be difficult to achieve with the current control measures

Vectorial status and insecticide resistance of Anopheles funestus from a sugar estate in southern Mozambique

<p>Abstract</p> <p>Background</p> <p>The dual problems of rising insecticide resistance in the malaria vectors and increasing human malaria cases since 2001 in southern Mozambique are cause for serious concern. The selection of insecticides for use in indoor residual spraying (IRS) programmes is highly dependent on the extent to which local mosquitoes are susceptible to the approved classes of insecticides. The insecticide resistance status and role in malaria transmission of <it>Anopheles funestus </it>was evaluated at the Maragra Sugar Estate in southern Mozambique where an IRS vector control programme has been in operation for seven years using the carbamate insecticide bendiocarb.</p> <p>Results</p> <p>No <it>Anopheles </it>species were captured inside the sugar estate control area. <it>Anopheles funestus </it>group captured outside of the estate represented 90% (n = 475) of the total collections. Of the specimens identified to species by PCR (n = 167), 95% were <it>An. funestus s.s. </it>One <it>An. rivulorum </it>was identified and seven specimens did not amplify. The <it>Anopheles gambiae </it>complex was less abundant (n = 53) and of those identified (n = 33) 76% were <it>An. arabiensis </it>and 24% <it>An. merus</it>. Insecticide susceptibility tests showed that wild-caught and F-1 family <it>An. funestus </it>were resistant to deltamethrin (32.5% mortality) and lambda-cyhalothrin (14.6% mortality), less so to bendiocarb (71.5% mortality) and fully susceptible to both malathion and DDT (100%). Bendiocarb and pyrethroid resistance was nullified using 4% piperonyl butoxide (Pbo), strongly suggesting that both are mediated by P450 monooxygenase detoxification. ELISA tests <it>of An. funestus </it>for <it>Plasmodium falciparum</it>, gave a sporozoite rate of 6.02% (n = 166). One unidentified member of the <it>An. gambiae </it>complex tested positive for <it>P. falciparum </it>sporozoites.</p> <p>Conclusion</p> <p><it>Anopheles funestus </it>was found to be the most abundant and principle vector of malaria in this area, with members of the <it>An. gambiae </it>complex being secondary vectors. Despite the continual use of bendiocarb within the estate for seven years and the level of <it>An. funestus </it>resistance to this insecticide, the IVC programme is still effective against this and other <it>Anopheles </it>in that no vectors were found inside the control area. However, the Mozambique National Malaria Control Programme ceased the use of DDT and bendiocarb in this area of its operations in 2009, and replaced these insecticides with a pyrethroid which will increase insecticide resistance selection pressure and impact on control programmes such as the Maragra IVC.</p

The effect of a single blood meal on the phenotypic expression of insecticide resistance in the major malaria vector Anopheles funestus

<p>Abstract</p> <p>Background</p> <p><it>Anopheles funestus </it>is a major malaria vector in southern Africa. Vector control relies on the use of insecticide chemicals to significantly reduce the number of malaria vectors by targeting that portion of the female population that takes blood meals and subsequently rests indoors. It has been suggested that the intake of a blood meal may assist female mosquitoes to tolerate higher doses of insecticide through vigour tolerance. It is hypothesized that during the process of blood digestion, detoxification mechanisms required for the neutralizing of harmful components in the blood meal may also confer an increased ability to tolerate insecticide intoxication through increased enzyme regulation.</p> <p>Methods</p> <p>Bottle bioassays using a range of concentrations of the pyrethroid insecticide permethrin were performed on pyrethroid susceptible and resistant laboratory strains of <it>An. funestus </it>in order to detect differences in insecticide susceptibility following a single blood meal. Based on these results, a discriminating dosage was identified (double the lowest dosage that resulted in 100% mortality of the susceptible strain). Blood-fed and unfed females drawn from the resistant strain of <it>An. funestus </it>were then assayed against this discriminating dose, and the percentage mortality for each sample was scored and compared.</p> <p>Results</p> <p>In the insecticide dose response assays neither the fully susceptible nor the resistant strain of <it>An. funestus </it>showed any significant difference in insecticide susceptibility following a blood meal, regardless of the stage of blood meal digestion. A significant increase in the level of resistance was however detected in the resistant <it>An. funestus </it>strain following a single blood meal, based on exposure to a discriminating dose of permethrin.</p> <p>Conclusion</p> <p>The fully susceptible <it>An. funestus </it>strain did not show any significant alteration in susceptibility to insecticide following a blood meal suggesting that vigour tolerance through increased body mass (and increased dilution of internalized insecticide) does not play a significant role in tolerance to insecticide intoxication. The increase in insecticide tolerance in the pyrethroid resistant strain of <it>An. funestus </it>following a blood meal suggests that insecticide detoxification mechanisms involved in insecticide resistance are stimulated by the presence of a blood meal prior to insecticide exposure, leading to enhanced expression of the resistance phenotype. This finding may be significant in terms of the methods used to control indoor resting populations of <it>An. funestus </it>if the mass killing effect of insecticide application proves increasingly inadequate against blood-feeding females already carrying the insecticide resistance phenotype.</p

Malaria vectors and transmission dynamics in coastal south-western Cameroon

BACKGROUND: Malaria is a major public health problem in Cameroon. Unlike in the southern forested areas where the epidemiology of malaria has been better studied prior to the implementation of control activities, little is known about the distribution and role of anophelines in malaria transmission in the coastal areas. METHODS: A 12-month longitudinal entomological survey was conducted in Tiko, Limbe and Idenau from August 2001 to July 2002. Mosquitoes captured indoors on human volunteers were identified morphologically. Species of the Anopheles gambiae complex were identified using the polymerase chain reaction (PCR). Mosquito infectivity was detected by the enzyme-linked immunosorbent assay and PCR. Malariometric indices (plasmodic index, gametocytic index, parasite species prevalence) were determined in three age groups (<5 yrs, 5–15 yrs, >15 yrs) and followed-up once every three months. RESULTS: In all, 2,773 malaria vectors comprising Anopheles gambiae (78.2%), Anopheles funestus (17.4%) and Anopheles nili (7.4%) were captured. Anopheles melas was not anthropophagic. Anopheles gambiae had the highest infection rates. There were 287, 160 and 149 infective bites/person/year in Tiko, Limbe and Idenau, respectively. Anopheles gambiae accounted for 72.7%, An. funestus for 23% and An. nili for 4.3% of the transmission. The prevalence of malaria parasitaemia was 41.5% in children <5 years of age, 31.5% in those 5–15 years and 10.5% in those >15 years, and Plasmodium falciparum was the predominant parasite species. CONCLUSION: Malaria transmission is perennial, rainfall dependent and An. melas does not contribute to transmission. These findings are important in the planning and implementation of malaria control activities in coastal Cameroon and West Africa

Detoxification enzymes associated with insecticide resistance in laboratory strains of Anopheles arabiensis of different geographic origin

Background The use of insecticides to control malaria vectors is essential to reduce the prevalence of malaria and as a result, the development of insecticide resistance in vector populations is of major concern. Anopheles arabiensis is one of the main African malaria vectors and insecticide resistance in this species has been reported in a number of countries. The aim of this study was to investigate the detoxification enzymes that are involved in An. arabiensis resistance to DDT and pyrethroids. Methods The detoxification enzyme profiles were compared between two DDT selected, insecticide resistant strains of An. arabiensis, one from South Africa and one from Sudan, using the An. gambiae detoxification chip, a boutique microarray based on the major classes of enzymes associated with metabolism and detoxification of insecticides. Synergist assays were performed in order to clarify the roles of over-transcribed detoxification genes in the observed resistance phenotypes. In addition, the presence of kdr mutations in the colonies under investigation was determined. Results The microarray data identifies several genes over-transcribed in the insecticide selected South African strain, while in the Sudanese population, only one gene, CYP9L1, was found to be over-transcribed. The outcome of the synergist experiments indicate that the over-transcription of detoxification enzymes is linked to deltamethrin resistance, while DDT and permethrin resistance are mainly associated with the presence of the L1014F kdr mutation. Conclusions These data emphasise the complexity associated with resistance phenotypes and suggest that specific insecticide resistance mechanisms cannot be extrapolated to different vector populations of the same species

A comparison of DNA sequencing and the hydrolysis probe analysis (TaqMan assay) for knockdown resistance (kdr) mutations in Anopheles gambiae from the Republic of the Congo

<p>Abstract</p> <p>Background</p> <p>Knockdown resistance (<it>kdr</it>) caused by a single base pair mutation in the sodium channel gene is strongly associated with pyrethroid insecticide resistance in <it>Anopheles gambiae </it>in West-Central Africa. Recently, various molecular techniques have been developed to screen for the presence of the <it>kdr </it>mutations in vector populations with varying levels of accuracy. In this study, the results of the hydrolysis probe analysis for detecting the <it>kdr </it>mutations in <it>An. gambiae </it>s.s. from the Republic of the Congo were compared with DNA sequence analysis.</p> <p>Methods</p> <p>A total of 52 pyrethroid and DDT resistant <it>An. gambiae </it>from Pointe-Noire (Congo-Brazzaville) were tested for detection of the two <it>kdr </it>mutations (<it>kdr</it>-e and <it>kdr</it>-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.</p> <p>Results</p> <p>Fifty-one specimens were found to be <it>An. gambiae </it>S-form and one was a M/S hybrid. DNA sequencing revealed that more than half of the specimens (55.8%) carried both the <it>kdr</it>-e and <it>kdr</it>-w resistance mutations, seven specimens (13.5%) were homozygous for the <it>kdr</it>-e mutation, and 14 specimens (26.9%) were homozygous for the <it>kdr</it>-w mutation. A single individual was genotyped as heterozygous <it>kdr</it>-e mutation (1.9%) only and another as heterozygous <it>kdr</it>-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 <it>kdr</it>-w, six specimens (11.5%) homozygous for the <it>kdr</it>-e mutation, one specimen (1.9%) heterozygous for the <it>kdr</it>-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 <it>kdr</it>-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.</p> <p>Conclusion</p> <p>Both the <it>kdr</it>-e and <it>kdr</it>-w mutations were abundant in <it>An. gambiae </it>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 <it>kdr</it>-e and <it>kdr</it>-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 <it>kdr </it>is still held as the gold standard assay, this paper highlights the importance of <it>kdr </it>mutation confirmation via sequencing especially in regions where <it>kdr </it>frequency has never been reported before or where both the <it>kdr</it>-e and <it>kdr</it>-w mutations are present simultaneously.</p