The impact of indoor residual spraying on Plasmodium falciparum microsatellite variation in an area of high seasonal malaria transmission in Ghana, West Africa.

Here, we report the first population genetic study to examine the impact of indoor residual spraying (IRS) on Plasmodium falciparum in humans. This study was conducted in an area of high seasonal malaria transmission in Bongo District, Ghana. IRS was implemented during the dry season (November-May) in three consecutive years between 2013 and 2015 to reduce transmission and attempt to bottleneck the parasite population in humans towards lower diversity with greater linkage disequilibrium. The study was done against a background of widespread use of long-lasting insecticidal nets, typical for contemporary malaria control in West Africa. Microsatellite genotyping with 10 loci was used to construct 392 P. falciparum multilocus infection haplotypes collected from two age-stratified cross-sectional surveys at the end of the wet seasons pre- and post-IRS. Three-rounds of IRS, under operational conditions, led to a >90% reduction in transmission intensity and a 35.7% reduction in the P. falciparum prevalence (p < .001). Despite these declines, population genetic analysis of the infection haplotypes revealed no dramatic changes with only a slight, but significant increase in genetic diversity (He : pre-IRS = 0.79 vs. post-IRS = 0.81, p = .048). Reduced relatedness of the parasite population (p < .001) was observed post-IRS, probably due to decreased opportunities for outcrossing. Spatiotemporal genetic differentiation between the pre- and post-IRS surveys (D = 0.0329 [95% CI: 0.0209 - 0.0473], p = .034) was identified. These data provide a genetic explanation for the resilience of P. falciparum to short-term IRS programmes in high-transmission settings in sub-Saharan Africa.

Evaluation of piperonyl butoxide in enhancing the efficacy of pyrethroid insecticides against resistant Anopheles gambiae s.l. in Ghana.

BACKGROUND: Malaria vector control methods involving the use of pyrethroids remain the strategies being used against malaria vectors in Ghana. These methods include the use of long-lasting insecticidal nets and indoor residual spraying in many areas in Ghana. However, there is evidence that pyrethroid resistance is widespread in many areas in Ghana. Synergists have been shown to be useful in inhibiting the enzymes that are responsible for the development of resistance and hence enhance the insecticide susceptibility of Anopheles gambiae sensu lato (s.l.) in many areas. The present study investigated the effect of piperonyl butoxide (PBO) on the susceptibility status of An. gambiae s.l. across some sentinel sites in Ghana. METHODS: Three to five day old An. gambiae s.l. reared from larvae were used in WHO susceptibility tube assays. Batches of 20-25 female adult An. gambiae s.l. were exposed simultaneously to the insecticide alone and to the PBO + insecticide. The knock down rate after 60 min and mortality at 24 h were recorded. RESULTS: Deltamethrin and permethrin resistance of An. gambiae s.l. was observed in all the sites in 2015 and 2016. The mortality after 24 h post exposure for deltamethrin ranged from 16.3% in Weija to 82.3% in Kade, whereas that for permethrin ranged from 3.8% in Gomoa Obuasi to 91.3% in Prestea. A significant increase in susceptibility to deltamethrin and less to permethrin was observed during both 2015 and 2016 years in most of the sites when An. gambiae s.l. mosquitoes were pre-exposed to PBO. CONCLUSION: Findings from this study showed that the use of PBO significantly enhanced the susceptibility of An. gambiae s.l. mosquitoes in most of the sentinel sites. It is recommended that vector control strategies incorporating PBO as a synergist can be effective in killing mosquitoes in the presence of deltamethrin and permethrin resistance.

A reduction in malaria transmission intensity in Northern Ghana after 7 years of indoor residual spraying.

BACKGROUND: Indoor residual spraying (IRS) is being implemented as one of the malaria prevention methods in the Northern Region of Ghana. Changes in longevity, sporozoite and entomological inoculation rates (EIRs) of major malaria vectors were monitored to assess the impact of IRS in selected districts. METHODS: Monthly human landing catches (HLCs) were used to collect mosquitoes from sentinel sites in three adjacent districts between July 2009 and December 2014: Savelugu Nanton (SND) where IRS had been implemented from 2008 to 2014; Tolon Kumbungu (TKD) where IRS had been implemented between 2008 and 2012 and Tamale Metropolis (TML) with no history of IRS. Mosquitoes were morphologically identified to species level and into sibling species, using PCR. Samples of Anopheles gambiae sensu lato (s.l.) were examined for parity and infectivity. EIR was calculated from biting and infectivity rates of malaria vectors. RESULTS: Parity rates of An. gambiae s.l. decreased significantly (p < 0.0001) in SND from 44.8% in 2011 to 28.1% by 2014, and in TKD from 53.3% in 2011 to 46.6% in 2012 (p = 0.001). However 2 years after IRS was discontinued in TKD, the proportion of parous An. gambiae s.l. increased significantly to 68.5% in 2014 (p < 0.0001). Parity rates in the unsprayed district remained high throughout the study period, ranging between 68.6% in 2011 and 72.3% in 2014. The sum of monthly EIRs post-IRS season (July-December) in SND ranged between 2.1 and 6.3 infective bites/person/season (ib/p/s) during the 3 years that the district was sprayed with alphacypermethrin. EIR in SND was reduced to undetectable levels when the insecticide was switched to pirimiphos methyl CS in 2013 and 2014. Two years after IRS was withdrawn from TKD the sum of monthly EIRs (July-December) increased by about fourfold from 41.8 ib/p/s in 2012 to 154.4 ib/p/s in 2014. The EIR in the control area, TML, ranged between 35 ib/p/s in 2009 to 104.71 ib/p/s by 2014. CONCLUSIONS: This study demonstrates that IRS application did have a significant impact on entomological indicators of malaria transmission in the IRS project districts of Northern Ghana. Transmission indicators increased following the withdrawal of IRS from Tolon Kumbungu District.

Indoor residual spraying with a non-pyrethroid insecticide reduces the reservoir of Plasmodium falciparum in a high-transmission area in northern Ghana.

High-malaria burden countries in sub-Saharan Africa are shifting from malaria control towards elimination. Hence, there is need to gain a contemporary understanding of how indoor residual spraying (IRS) with non-pyrethroid insecticides when combined with long-lasting insecticidal nets (LLINs) impregnated with pyrethroid insecticides, contribute to the efforts of National Malaria Control Programmes to interrupt transmission and reduce the reservoir of Plasmodium falciparum infections across all ages. Using an interrupted time-series study design, four age-stratified malariometric surveys, each of ~2,000 participants, were undertaken pre- and post-IRS in Bongo District, Ghana. Following the application of three-rounds of IRS, P. falciparum transmission intensity declined, as measured by a >90% reduction in the monthly entomological inoculation rate. This decline was accompanied by reductions in parasitological parameters, with participants of all ages being significantly less likely to harbor P. falciparum infections at the end of the wet season post-IRS (aOR = 0.22 [95% CI: 0.19-0.26], p-value < 0.001). In addition, multiplicity of infection (MOI var ) was measured using a parasite fingerprinting tool, designed to capture within-host genome diversity. At the end of the wet season post-IRS, the prevalence of multi-genome infections declined from 75.6% to 54.1%. This study demonstrates that in areas characterized by high seasonal malaria transmission, IRS in combination with LLINs can significantly reduce the reservoir of P. falciparum infection. Nonetheless despite this success, 41.6% of the population, especially older children and adolescents, still harboured multi-genome infections. Given the persistence of this diverse reservoir across all ages, these data highlight the importance of sustaining vector control in combination with targeted chemotherapy to move high-transmission settings towards pre-elimination. This study also points to the benefits of molecular surveillance to ensure that incremental achievements are not lost and that the goals advocated for in the WHO's High Burden to High Impact strategy are realized.

Assessing the Presence of Wuchereria bancrofti Infections in Vectors Using Xenomonitoring in Lymphatic Filariasis Endemic Districts in Ghana.

Mass drug administration (MDA) is the current mainstay to interrupt the transmission of lymphatic filariasis. To monitor whether MDA is effective and transmission of lymphatic filariasis indeed has been interrupted, rigorous surveillance is required. Assessment of transmission by programme managers is usually done via serology. New research suggests that xenomonitoring holds promise for determining the success of lymphatic filariasis interventions. The objective of this study was to assess Wuchereria bancrofti infection in mosquitoes as a post-MDA surveillance tool using xenomonitoring. The study was carried out in four districts of Ghana; Ahanta West, Mpohor, Kassena Nankana West and Bongo. A suite of mosquito sampling methods was employed, including human landing collections, pyrethrum spray catches and window exit traps. Infection of W. bancrofti in mosquitoes was determined using dissection, conventional and real-time polymerase chain reaction and loop mediated isothermal amplification assays. Aedes, Anopheles coustani, An. gambiae, An. pharoensis, Culex and Mansonia mosquitoes were sampled in each of the four study districts. The dissected mosquitoes were positive for filarial infection using molecular assays. Dissected An. melas mosquitoes from Ahanta West district were the only species found positive for filarial parasites. We conclude that whilst samples extracted with Trizol reagent did not show any positives, molecular methods should still be considered for monitoring and surveillance of lymphatic filariasis transmission.

Species composition and insecticide resistance status of Anopheles gambiae (s.l.) (Culicidae) in Kome, southern Chad and the implications for malaria control.

BACKGROUND: The development and spread of insecticide resistance among malaria vectors, is a threat to the continued effectiveness of interventions to control and eliminate the disease. The status of insecticide resistance among malaria vector populations at two sites in Kome, southern Chad, was evaluated to inform decisions on vector control. METHODS: Mosquito larvae were collected from temporary rain-filled and semi-permanent breeding places at two sites and reared in a laboratory. Emerging Anopheles gambiae (senso lato) (s.l.) adults were morphologically identified, sorted and evaluated for susceptibility to WHOPES recommended insecticides. Standardized biomolecular and biochemical methods were used to determine sibling species and molecular forms: knockdown resistant alleles (kdr-w) for pyrethroids and DDT; acetylcholinesterase-1 resistant alleles for organophosphate and carbamates; biochemical resistance through measurement of the levels of non-specific esterase (α and ß), oxidase and glutathione-s-transferases activities. RESULTS: Anopheles gambiae (s.l.) was the main vector group in the two study sites and comprised of Anopheles gambiae (senso stricto) (s.s.) and An. arabiensis, respectively, at 71 and 29 % in Site A, and 60 and 40 % at Site B. Anopheles gambiae (s.s.) was composed of M (Anopheles coluzzii) and S [nominotypical An. gambiae (s.s.)] molecular forms. Anopheles coluzzii accounted for over 98 % of the sub-group. There was extensive phenotypic resistance to pyrethroids, DDT and carbamates, but full susceptibility to organophosphates. Population-wide frequency of knockdown resistant allele in An. gambiae (s.l.) was 43 homozygous (RR), 19 heterozygous (RS) and 38 % homozygous susceptible (SS). When segregated by species and molecular forms, An. coluzzii had the highest kdr-w frequency of 37.4 homozygous resistant alleles, and 17.5 % heterozygous, with 8.3 % homozygote susceptible alleles. An. gambiae (s.s.) had 1 % homozygous resistant allele. Levels of esterase, oxidase and glutathione-s-transferases were not significantly different compared to fully susceptible laboratory raised An. gambiae (s.s.) Kisumu reference, although few individuals showed significant elevation of esterases (> 0.04 µg/protein), indicating a likely start of biochemical enzyme resistance. CONCLUSIONS: There is an urgent need for action to stop and reverse significant insecticide resistance in the area. A comprehensive entomological surveillance and monitoring program is needed to understand the full extent of resistance to enable realistic insecticide resistance management strategy, and also to track future changes in the vector populations.

Transmission indices and microfilariae prevalence in human population prior to mass drug administration with ivermectin and albendazole in the Gomoa District of Ghana.

BACKGROUND: The Lymphatic Filariasis Elimination Programme in Ghana involves annual mass drug administration (MDA) of ivermectin and albendazole to persons living in endemic areas. This is repeated annually for 4-6 years to span across the reproductive lifespan of adult worms. In order to stimulate participation of community members in the MDA programme, this study was carried out to understand local views on transmission, management and prevention of the disease. The study also presents baseline transmission indices and microfilariae prevalence in the human population in eight endemic communities of coastal Ghana prior to the MDA. METHODS: A descriptive survey was carried out to explore perceptions on causes, treatment and prevention of lymphatic filariasis. Perceptions on community participation in disease control programmes were also assessed. After participants were selected by cluster sampling and 100 µl of blood sampled from each individual and examined for mf microfilariae. A similar volume of blood was used to determine the presence of circulating filarial antigen. Mosquitoes were collected simultaneously at all sites by human landing catches for 4 days per month over a six-month period. All Anopheles mosquitoes were dissected and examined for the larval stages of the parasite following which molecular identification of both vector and parasite was done. RESULTS: Eight hundred and four persons were interviewed, of which 284 (32.9%; CI 31.1-34.5) acknowledged elephantiasis and hydrocoele as health related issues in the communities. Thirty-three people (3.8%; CI 2.1-5.5) thought sleeping under bed net could help prevent elephantiasis. Microfilariae prevalence was 4.6% (43/941) whiles 8.7% (75/861) were positive for circulating filarial antigen. A total of 17,784 mosquitoes were collected, majority (55.8%) of which were Anopheles followed by Culex species (40%). Monthly biting rates ranged between 311 and 6116 bites/person for all the eight communities together. Annual transmission potential values for An. gambiae s.s. and An. funestus were 311.35 and 153.50 respectively. CONCLUSION: Even though the highest mf density among inhabitants was recorded in a community that had the lowest Anopheles density with Culex species constituting 95% of all mosquitoes collected, Anopheles gambiae s.s. and An. funestus remained the main vectors.

Role of species composition in malaria transmission by the Anopheles funestus group (Diptera: Culicidae) in Ghana.

Malaria remains a public health problem in Ghana, with Anopheles gambiae and Anopheles funestus as the predominant vectors. While much information exists on the species composition of An. gambiae, very little exists for An. funestus. This study was carried out to determine the species composition of An. funestus Giles populations from three ecological areas in Ghana and investigate their role in malaria transmission. Mosquitoes were collected using human landing and pyrethrum spray methods. A total of 10,254 Anopheles individuals were collected, out of which An. funestus constituted 53.6% (5,496). An. funestus sensu stricto (s.s.) and Anopheles lessoni were identified as the only members of the An. funestus group in all three ecological areas. All 62 sporozoite positive specimens that were identified as An. funestus s.s. were highly anthropophilic with a human blood index in the range of 80-96%, whereas more than 83% of the An. leesoni had fed on either bovine, goat, or sheep. Malaria transmission was higher in the Sahel savannah area than the rest of the ecological zones, with An. funestus s.s. being implicated as a vector of malaria in all ecological zones. Anopheles leesoni occurred in all the ecological areas but played no role in malaria transmission. The study established the importance of An. funestus s.s. in malaria transmission in Ghana.

Characterization of malaria transmission by vector populations for improved interventions during the dry season in the Kpone-on-Sea area of coastal Ghana.

BACKGROUND: Malaria is a major public health problem in Ghana. We present a site-specific entomological study of malaria vectors and transmission indices as part of an effort to develop a site for the testing of improved control strategies including possible vaccine trials. METHODS: Pyrethrum spray catches (PSC), and indoor and outdoor human landing collections of adult female anopheline mosquitoes were carried out over a six-month period (November 2005 - April 2006) at Kpone-on-Sea, a fishing village in southern Ghana. These were morphologically identified to species level and sibling species of the Anopheles gambiae complex further characterized by the polymerase chain reaction (PCR). Enzyme-linked immunosorbent assay was used to detect Plasmodium falciparum mosquito infectivity and host blood meal sources. Parity rate was examined based on dilatation of ovarian tracheoles following dissection. RESULTS: Of the 1233 Anopheles mosquitoes collected, An. gambiae s.l. was predominant (99.5%), followed by An. funestus (0.4%) and An. pharoensis (0.1%). All An. gambiae s.l. examined (480) were identified as An. gambiae s.s. with a majority of M molecular form (98.2%) and only 1.8% S form with no record of M/S hybrid. A significantly higher proportion of anophelines were observed outdoors relative to indoors (χ2 = 159.34, df = 1, p < 0.0000). Only An. gambiae M molecular form contributed to transmission with a high degree of anthropophily, parity rate and an estimated entomological inoculation rate (EIR) of 62.1 infective bites/person/year. The Majority of the infective bites occurred outdoors after 09.00 pm reaching peaks between 12.00-01.00 am and 03.00-04.00 am. CONCLUSION: Anopheles gambiae M molecular form is responsible for maintaining the status quo of malaria in the surveyed site during the study period. The findings provide a baseline for evidence-based planning and implementation of improved malaria interventions. The plasticity observed in biting patterns especially the combined outdoor and early biting behavior of the vector may undermine the success of insecticide-based strategies using insecticide treated nets (ITN) and indoor residual spray (IRS). As such, novel or improved vector interventions should be informed by the local malaria epidemiology data as it relates to vector behavior.

Lymphatic filariasis in Ghana: establishing the potential for an urban cycle of transmission.

Lymphatic filariasis is a significant public health and economic problem in many tropical and sub-tropical regions. Unplanned urbanization leading to a lack of proper sanitary conditions has resulted in an increase in the urban-based transmission of a number of vector-borne diseases, including lymphatic filariasis. It has been well established that lymphatic filariasis is endemic in rural areas of Ghana. The goal of this study was to determine if there is a potential of establishing urban transmission cycles in Ghana's major cities. We clinically and immunologically assessed 625 individuals from the three major urban areas (Bawku, Bolgatanga and Secondi/Takoradi), finding that the prevalence of infection with Wuchereria bancrofti ranged from 0 to 12.5%. The results of a polymerase chain reaction based analysis of mosquitoes collected from these areas suggested that there is a low but detectable prevalence of mosquitoes infected with W. bancrofti. We conclude that there may be a potential for an established urban transmission of lymphatic filariasis in Ghana.

Detection of malaria sporozoites by standard ELISA and VecTestTM dipstick assay in field-collected anopheline mosquitoes from a malaria endemic site in Ghana.

We compared the VecTestTM dipstick assay for detection of Plasmodium sporozoites in Anopheles vectors of malaria with standard circumsporozoite (CS) microplate ELISA for detection of Plasmodium falciparum circumsporozoite protein (PfCSP) in Anopheles mosquitoes. Mosquitoes were collected from a malaria endemic site (Kassena Nankana district) in northern Ghana. Of 2620 randomly sampled mosquitoes tested, the standard CS-ELISA gave a sporozoite rate of 10.8% compared with 11.2% by VecTestTM, which was not statistically different (P = 0.66). Visual reading of the CS-ELISA results gave a sporozoite rate of 13.4%, which was higher than the other tests (P > 0.05). To allow a more objective evaluation of the sensitivity of the dipstick, an additional 136 known CS-ELISA-positive specimens were analysed. The prevalence of the test (including the additional samples) was 14.6% and 14.7% for CS-ELISA and dipstick, respectively (P > 0.05). The estimated prevalence by visual assessment of the CS-ELISA results was 17.5%. The relative specificity and sensitivity of the VecTestTM dipstick and visually read ELISA were estimated based on the CS-ELISA as a gold standard. The specificities of the dipstick and visual ELISA were high, 98.0% and 96.6%, respectively. However, the sensitivities of the two assays were 88.8% for VecTest and 100% for visual ELISA (P < 0.01). Concordance between VecTest and CS-ELISA was good (kappa = 0.86). Similarly, there was a good concordance between the dipstick and the visually read ELISA (kappa = 0.88). Extrapolating from PfCSP controls (titrated quantities of P. falciparum sporozoites), mean sporozoite loads of CS-ELISA-positive An. gambiae (286 +/- 28.05) and An. funestus (236 +/- 19.32) were determined (P = 0.146). The visual dipstick grades showed high correlation with sporozoite load. The more intense the dipstick colour, the higher the mean sporozoite load (+ = 108, ++ = 207, +++ = 290, r = 0.99, r2 = 1). The VecTest dipstick offers practical advantages for field workers needing rapid and accurate means of detection of sporozoites in mosquitoes.