Nine LF endemic communities in the Gomoa district of Ghana (between Latitude 5° 24’ - 35’N and Longitude 0° 25’ - 36’W) were selected based on available data on the disease epidemiology in the population and vector species distribution 10 11 12 . These are Amanful, Ayesuano, Dago, Fawomanye, Hwida, Kyiren, Mampong, Obiri and Okyereko. The district lies in the coastal savannah zone of Ghana and is located 50 km west of Accra, the capital city of Ghana. Average annual rainfall ranges between 760 and 1000 mm, whilst mean annual temperature ranges between 26 and 30°C. The main occupations of the inhabitants are farming and fishing for those living near the shores of the Atlantic Ocean.

This study was conducted from April to June 2004, the fourth year of MDA with ivermectin and albendazole in these communities. The areas also form part of an on-going annual longitudinal community-based intervention study. Human participation and the mosquito collection were done by cluster sampling method. Mass screening of the study population for mf was done by collecting 100 μl finger-pricked blood from each individual into heparinised capillary tubes and immediately mixing with 900 μl 3% acetic acid. Quantification of parasitaemia used the Sedgwick-Rafter counting chamber method with the compound microscope set at ×100 magnification 13 .

After consenting to participate, twelve adult volunteers with varying mf levels slept under partially opened mosquito nets hung over beds in their rooms. At the mid-point of each collection hour, finger-pricked blood was taken and mf density estimated using the same procedure described above. Mosquitoes trapped in the nets were collected each hour from 21.00 hours to 06.00 hours on the next day using an aspirator. About half the number of mosquitoes collected were killed immediately and dissected for ingested mf. The remaining mosquitoes were fed on 10% sugar solution and maintained for up to 13 days in paper-cups at 26-28×C, relative humidity 70-80% and 12-hour photoperiod in the insectary 14 . Mosquitoes that died before day 13 were dissected for developing stages of W. bancrofti, whilst those that survived until the last day were dissected for the presence of infective stage L₃ larvae of the parasite.

Molecular identifications of An. gambiae, An. funestus and W. bancrofti were conducted using already established methods 15 16 17 . For the vector species identification, genomic DNA was extracted from the carcasses of mosquitoes after homogenisation with sterile Konte’s plastic pestles in 100 μl bender buffer. The homogenate was then incubated at 65°C for 30 min, followed by the addition of 125 μl of phenol. The Centrifuge 5415 C (Eppendorf) was used in all spinning of samples, unless otherwise stated. The mixture was vortexed and spun at 14,000 rpm for 10 min. The supernatant was transferred into a fresh tube and 250 μl of pre-chilled absolute ethanol and 10 μl of 8 M potassium acetate were added. This was incubated at -40°C for an hour, spun at 10,000 rpm for 10 min and supernatant poured off. The pellet was then rinsed with 200 μl of 70% ethanol, spun at 10,000 rpm for 5 min, and supernatant poured off. The pellet was dried and re-dissolved in 50 μl TE + RNAse and then kept at 4°C until ready for PCR (Table 1). Each PCR reaction mixture of 25 μl contained 1× PCR buffer (Sigma, USA), 200 μM each of the four deoxyribonucleotide triphosphates, 10 μM each of the oligonucleotide primers (Table 1), and 0.125 units of Taq Polymerase enzyme (Sigma, USA). A microliter of the genomic DNA was used as template for the amplification reaction. Anopheles gambiae s.s. were further identified and differentiated into the M and S molecular forms by enzymatic restriction of the PCR product as described by Fanello et al. 18 . This was done by amplification of 1.3 kb rDNA followed by restriction fragment length polymorphism (RFLP) with restriction enzyme Hha I (Sigma-Aldrich, USA).

After gene amplification and digestion, the PCR products were electrophoresed separately in 2% agarose gel. The gel was prepared by adding TAE buffer to the powder, which was placed in a microwave oven (230 V, 50 Hz, 2660 W, 12.0A) for 1 minute to dissolve the solute, and then stained with 0.5 μg/ml Ethidium Bromide. For the electrophoresis, 8 μl of each sample was added to 1 μl of orange G (5X) gel loading dye after placing the solidified gel in 1X TAE buffer in a mini gel system (BIORAD USA). One hundred volts of electric current was passed through it for an hour and the gel photographed over a UV trans-illuminator (UPC, USA) at short wavelength using a Polaroid camera and film type 667 (Polaroid, USA). The sizes of the PCR products were estimated by comparison with the mobility of a 100 base pair molecular weight size marker (Sigma).

After the carcass of infected mosquito was scrapped into 1.5 ml eppendorf tubes, DNeasy Tissue Kit (QIAGEN Inc., USA) was used in the extraction of the parasite’s genomic DNA from animal tissues following the manufacturer’s recommended protocol. After the DNA extraction, aliquots of 5 μl of the filarial DNA extract from the mosquitoes were used as templates for the amplification reaction. The PCR assay was performed using two published specific oligonucleotide primers, NV-1 and NV-2 17 . The PCR products were electrophoresed in 2% agarose gel as described in the previous section.

Microfilariae (mf) in human blood samples that were preserved in 3% acetic acid were also characterised after extracting the genomic DNA using the same kit described above. Infected blood samples were amplified and identified using the same procedure described in previous sections.

For the yearly MDA and mass screening for mf prevalence in the communities, oral informed consent was sought from all participants. Subsequently, written consent was obtained from each volunteer who slept under bed nets after the study purpose, procedures, entry and exit criteria were explained to them. All volunteers and the entire community members received that year’s round of MDA immediately after the blood sample collection. The Institutional Review Board of Noguchi Memorial Institute for Medical Research approved the study.

Data were entered into Microsoft Access and analysed for the vector competency of Anopheles spp. in supporting the development of mf to the infective stage larvae. The same software was used to calculate the geometric mean intensity on mf in the human population. One-way analysis of variance (ANOVA) was used to test for the significance of age- and gender-specific variations between the human population and mf, with p value set at 0.05.

The overall prevalence of mf in the study communities (N = 1083) was 1.6%; mf prevalence among males and females (2.05% and 1.10% respectively) was not significantly different (p = 0.39). The mf levels ranged from 0 to 59/100 μl blood with geometric mean intensity of 1.1 mf/ml of blood (Table 2). There was no significant variation in mf intensity and age-group (p = 0.40); likewise no significant difference between mf intensity and gender of participants (p = 0.91) (Table 2). Four out of the nine communities namely Ayesuano, Dago, Hwida and Okyereko recorded positive cases, with Okyereko having the highest number of cases (Table 3). Among the positive cases, Okyereko recorded 155.6 mf/ml of blood whilst Dago recorded 15.3 mf/ml of blood.

*Antilog [Σ log (x + 1)/ n], where x is the number of mf per ml of blood in mf individuals and n is the number of people examined 9 .

The 564 mosquitoes collected consisted of 350 (62.1%) Anopheles, 182 (32.3%) Mansonia, 28 (5%) Aedes and 4 (0.7%) Culex species, (Figure 1). The Anopheles species comprised of 310 (88.6%) An. funestus, 32 (9.1%) An. gambiae and 8 (2.3%) An. Pharoensis, (Figure 1). The hourly biting rates of An. gambiae and An. funestus were 6.4 and 62 bites/person/night respectively (Table 4). Of the mosquito species collected, 192 (34%) were engorged with blood-meals (Table 5). Whereas 6/350 (1.7%) of the Anopheles and 6/182 (3.3%) of the Mansonia species were found with the mf (L₁ stage) of W. bancrofti, there was no recovery of L₃ or L₂ stage larvae after 12 days of maintenance. While each of the infected An. gambiae had an average of one mf, each An. funestus had an average of eight mf when killed immediately after collection (Table 5). The mf load in the peripheral blood and biting rates of Anopheles mosquitoes peaked concurrently between 0.30 and 2.30 hours (Figure 2).

^aNumber of mosquitoes caught/ number of collectors x number of captures (bites/ person/ night); ^bNumber of mosquitoes infected/ number of mosquitoes dissected; ^cL₃ in the head and proboscis/ number of surviving mosquitoes; ^dNumber of mosquitoes with L₃ in the head and proboscis/ number of mosquitoes with L₃; ^eNumber of surviving mosquitoes at the end of study/ number of engorged mosquitoes. ^fNumber of L₃ x 100/ number of mf ingested among dissected mosquitoes 19 20 21 .

All mf found in mosquitoes were of L₁ stage, neither L₂ nor L₃ stages were found. All mosquitoes caught were dissected latest by end of the maintenance (13 days).

Of the 32 An. gambiae s.l. collected, 30 were identified as An. gambiae s.s. as they showed the expected diagnostic band size of 390 base pairs. After restriction enzyme treatment with Hha I, M forms remained a single band of 390 bp since there was no digestion. S forms on the other hand resulted in two bands of 110 and 280 bp. Of the An. gambiae s.s digested, 21 (70%) were M forms with 9 (30%) S molecular forms. Among the 310 An. funestus s.l. collected, 286 were identified by PCR; of which 267 (86%) were An. funestus s.s. with diagnostic band sizes of 460 base pairs, and 19 (6%) were An. Leesoni with band sizes of 146 base pairs. The presence of W. bancrofti in 20 infected mosquitoes and 10 human blood samples were confirmed at 188 bp.