الفهرس 
Transmission of Wuchereria bancroftiOnly 14 pages are availabe for public view
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Abstract
VI- Summary
The Global Program for Elimination of Lymphatic Filariasis (GPELF) aimed to reduce blood microfilariae (MF) below the threshold necessary for mosquitoes to transmit the filarial parasite. The strategy is 5-year mass drug administration (MDA) of an annual dose of a combined regimen to residents of endemic populations.
In 2000, the Egyptian Ministry of Health and Population (MOHP) in collaboration with the WHO has implemented a 5-year national programme for elimination of filariasis that was based on repeated annual mass treatment of eligible people with a combined regimen of a single dose of 6 mg/kg body weight DEC and 400 mg/subject Alb (DEC/Alb).
1- Comparison of indoor-resting and outdoor-gravid mosquito collection methods
After five years of MDA with DEC/Alb, blood MF is expected to have vanished or at least to be extremely low. Therefore, for PCR detection of potentially residual W. bancrofti MF DNA (molecular xenomonitoring), a large number of female mosquitoes (estimated 3,000 per locality) that would have ingested blood is needed. In this perspective, two mosquito sampling methods were compared in Kafr El Tarayna village (Menofiya governorate) and El Aziziya village (Giza governorate): Aspiration of indoor-resting females (morning and night aspiration) and the use of outdoor-gravid traps.
- In Kafr El Tarayna, Cx. pipiens females collected by gravid traps were significantly more numerous (110.1 ± 89.5/trap) than females collected by aspiration either in the morning (3.6 ± 3.7/house-morning) or at night (2.4 ± 1.9/house-night).
- Similarly, in El Aziziya, Cx. pipiens females collected by gravid traps were significantly more numerous (70.5 ± 101.9/trap) than females collected by aspiration either in the morning (3.6 ± 3.5/house-morning) or at night (3.4 ± 3.2/house-night). Accordingly, the gravid trap collection method was adopted for further collection of Cx. pipiens in selected study sites.
2. Molecular xenomonitoring the effects of five rounds of MDA in selected villages
Mosquito catches were initiated in Kafr Hamza village (Qaliubiya Governorate) at the onset of the mosquito breeding season in May 2007. For this, 54 traps were distributed (on the periphery and also in the core) so as to cover the whole village. Each trap contained four liters of a fermented mixture of 500 g straw and 114 L water and operated. Only Cx. pipiens were collected and counted and females sorted according to their abdominal appearance (Sella stages) into empty (non-blood-fed or non-gravid), blood-fed, half-gravid, and gravid.
- Unexpectedly, 46.1% of collected mosquitoes were males (N = 2,401), with 44.5 ± 42.7 males per trap.
- Of all collected females (N= 2,811), large numbers of empty females were attracted (77.6%), as well as half-gravid and gravid females (20.3%), in addition to some blood fed ones.
Such disparity in the collection was presumed to be the fact of either the oviposition medium used, or the time of year of mosquito collections. Therefore, different oviposition media were tested in order to select the infusion yielding the larger number of gravid or half-gravid females.
2.1. Selection of an oviposition medium
An experiment was designed to compare relative attraction of half-gravid and gravid mosquitoes to three oviposition media in Eastern Kafr Hamza village.
- All three media attracted as much males as females. The proportion of female mosquitoes in the collections ranged from 48.2 to 56.2%.
- Again, most females were empty (66.9 to 72.5%), some were blood fed (9.4 to 10.2%) and only 18.1 to 22.9% were gravid or half-gravid. The mean number of gravid and half-gravid females by tested medium did not vary significantly.
This indicated that there was no significant difference among media in the number of collected females per individual trap; and that any oviposition infusion could be indifferently used for capturing half-gravid and gravid Cx. pipiens.
Therefore, it was presumed that the time of year of mosquito collection could be an important parameter for collecting older mosquitoes. For this, mosquito collections were postponed from May to July or later.
2.2. Comparison of mosquito collections in early and mid-summer season
Mosquito collections carried out in Kafr Hamza in May 19 and August 1st were compared. In May, only Cx. pipiens were collected, whereas in August three female Anopheles pharoensis were captured in addition to Cx. pipiens.
- Both collections yielded males. More importantly, the proportion of females collected in August approached 70% of collected Cx. pipiens, compared to 54% females collected in May. In August, the mean number of females per trap exceeded by far (166.3 ± 111.0) that of females collected in May (52.1 ± 49.9).
- Both May and August collections yielded empty and also blood fed females.
- In August, the proportion of half-gravid and gravid females exceed by 2.7 times that of such females collected in May. Indeed, in August, the mean number of half-gravid and gravid females per trap (88.3 ± 74.3) significantly exceeded that number in May (10.6 ± 14.2).
These data indicated that the time of year of mosquito collection is an important factor and that mid- or late summer mosquito collection would yield a majority of older, half-gravid and gravid mosquitoes necessary for xenomonitoring, compared to younger, empty females.
2.3. Mosquitoes collected in mid and late summer
Gravid trap collections of Cx. pipiens were carried out in six villages: Kafr Hamza and Sanden-hour (Qaliubiya), El Roda and Ibrash (Sharqiya), Beqsa and Kafr Nafra (Menofiya). In each village, 50 traps were placed all over from sunset to early morning the next day. This was repeated a second night, so that 100 traps were used to capture mosquitoes in each village.
- Density of Sharqiya mosquitoes (61.1± 51.2 mosquitoes/trap) was remarkably low compared to that of Menofiya (172.9 ± 172.9 mosquitoes/trap) and Qaliubiya (162.9 ± 164.5 mosquitoes/trap) governorates.
- The proportion of collected Cx. pipiens females ranged from 51.6 to 94.8%, with 48.1 ± 42.7 to 223.7 ± 212.2 females per trap. In addition to Cx. pipiens, few anophelines were captured. Those were Anopheles pharoensis in Kafr Hamza (N = 3), Sanden-hour (N = 4), El Roda (N = 3), and Kafr Nafra (N = 2). One female An. multicolor was captured in Sanden-hour and one An. tenebrosus in El Roda.
- For PCR detection of potentially low vector infection rates after MDA, a large number of female mosquitoes (N = 3,000) that have taken at least one blood meal (half-gravid or gravid and sometimes blood fed ones) was needed. This number was obtained by a single night collection in Kafr Hamza (N = 4,415), Kafr Nafra (N = 4,320) and Beqsa (N = 9,800); and by repeating mosquito sampling a second night in Sanden-Hour (N1 = 1,846; N2 = 1,985); El Roda (N1 = 1,707; N2 = 2,695) and Ibrash (N1 = 1,556; N2 = 1,073).
- Collected Cx. pipiens varied in their abdominal appearance. In addition to half-gravid and gravid mosquitoes, the collections contained empty females (5.9 to 42.5%) and blood fed ones (0.4 to 4.4%) as well.
2.4. Detection of W. bancrofti DNA in mosquitoes
Molecular xenomonitoring (MX) is the use of molecular techniques for detection of filarial DNA in mosquitoes for evaluating the effect of MDA on mosquito filarial infection.
The impact of five rounds of MDA with DEC/Alb on the potential transmission of W. bancrofti by Cx pipiens in selected villages was evaluated in pools of 15 mosquitoes each (200 pools per village). The polymerase chain reaction (PCR) assay was performed in a DNA thermal cycler. DNA was extracted by the silica/guanidinium thiocyanate method or the QIAGEN DNeasy kit and silica glass beads. Extracted DNA was amplified using two oligonucleotide primers, NV-1 and NV-2 for detection of W. bancrofti DNA in Cx. pipiens. The PCR-generated fragment sizes that were visualized by gel electrophoresis under ultraviolet light.
- PoolScreen point estimates of mosquito infection measures the probability that the mosquito infection parameter in these villages falls between two set values with a 95% confidence interval.
- After five rounds of MDA, no infected mosquito was detected in Kafr Hamza, Ibrash and Beqsa. However, PoolScreen point estimates did not completely discard mosquito infection in these villages, but in no case infection exceeded 0.07%. This value is well below the provisional 0.25% threshold necessary to sustain filarial transmission by Cx. pipiens.
- In contrast, pools of infected mosquitoes were recognized in Sanden-hour, El Roda and Kafr Nafra (N= 1, N= 1 and N= 4 pools, respectively).
- Assuming that at least one infected mosquito was included in an infected pool, the minimum infection rate in these villages was 0.03, 0.03 and 0.13%, respectively. The upper limit of PoolScreen point estimate was 0.17% in Sanden-hour, and 0.2% in El Roda. But in Kafr Nafra, however, this limit amounted to 0.35%, suggesting a probable persisting transmission in this village.
- An attempt was made to investigate whether villages (Sanden-hour, El Roda, Kafr Nafra) from which infected mosquito pools were captured had a higher density of Cx. pipiens compared to villages (Kafr Hamza, Ibrash, Beqsa) from which no infected mosquito was caught.
- Unexpectedly, density of mosquitoes collected in villages from where no infected mosquito was captured (123.5± 139.9) was significantly higher than that in villages recording infected mosquito pools (63.0± 57.1).
- Moreover, in Qaliubiya governorate, although Sanden-hour recorded infected females, mosquito density in this village was significantly lower than that of Kafr Hamza where no infected mosquito was detected.
- Similarly, in Menofiya governorate, Kafr Nafra had the highest mosquito infection rate (four positive pools recorded), although its mosquitoes were less abundant than in Beqsa which showed no infected mosquito.
- Also, in Sharqiya governorate, mosquito density did not vary significantly between EL-Roda (one positive pool recorded) and Ibrach (all pools negative).
These results indicated that mosquito density of Cx. pipiens females in study villages may not be the reason for the persistence of mosquito infection after MDA.
- By examining pre-treatment MF prevalence rates, it was observed, and as expected, that two of three mosquito negative villages (Kafr Hamza and Ibrash) recorded low pre-treatment MF prevalence (1.9% and 3.0%, respectively). However, the third village (Beqsa) had a relatively high pre-treatment MF prevalence rate (6.6%).
- As for the mosquito positive villages, two of such villages (Sanden-hour, El Roda) had relatively high pre-treatment MF prevalence rates (11.3% and 8.0%, respectively). However, the third of such villages (Kafr Nafra) had an extremely low pre-treatment MF prevalence rate (1.0%).
Accordingly, pre-treatment MF prevalence rates may reflect to a certain extent why villages with high pre-treatment MF have infected mosquitoes around after five rounds of MDA.
3. Screening of mosquito breeding sites in selected study sites
Study villages were scanned for potential mosquito breeding habitats during the day made to each village. Potential larval habitats were geo-referenced using a Global Positioning System (GPS) and inspected for the presence of mosquito immature stages using a standard 350 ml dipper. For each village, location of breeding sites was represented on a map.
- Screening of potential Cx. pipiens breeding sites in study villages categorized six types of larval habitats. These habitats included drainage canals and drains, irrigation canals and channels, cesspits, cesspools, seepage water and abandoned wells.
- In Kafr Hamza village, a total of 137 sites representing six types of potential mosquito breeding habitats were detected. Of such breeding habitats, seepage water (47 sites) represented the most common potential larval habitat (34.3%). Twenty four out of 137 (17.5%) sites contained mosquitoes.
- In Sanden-hour village, a total of 84 sites representing six types of potential breeding habitats were detected. Of such breeding habitats, irrigation canals and ditches (22 sites) and cesspits (21 sites) represented the most common potential larval habitat (26.2% and 25.0%, respectively). Twenty four out of 84 (28.6%) sites contained mosquitoes.
- In El Roda, a total of 54 sites representing five types of potential breeding habitats were detected. Of these, cesspits (28 sites) represented the most common potential larval habitat (51.9%). Seven out of 54 (12.9%) sites contained mosquitoes.
- In Ibrash, a total of 101 sites representing five types of potential breeding habitats were detected. Of these, irrigation canals and ditches (32 sites) represented the most common potential larval habitat (31.7%). Thirty three out of 101 (32.7%) sites contained mosquitoes.
- In Kafr Nafra, a total of 108 sites representing six types of potential breeding habitats were detected. Of such breeding habitats, irrigation canals and ditches (44 sites) represented the most common potential larval habitat (40.7%). Twenty one out of 108 (19.4%) sites contained mosquitoes.
- In Beqsa, a total of 65 sites representing six types of potential breeding habitats were detected. Of such habitats, seepage water (20 sites) represented the most common potential larval habitat (30.8%). Nineteen out of 65 (29.2%) sites contained mosquitoes.
- Location of potential mosquito breeding sites containing immature stages of Cx. pipiens in selected study sites were more prevalent on the periphery (17.2 to 40.3%) than in the core of the village (8.0 to 34.6%).
- In conclusion. xenomonitoring filaria elimination is best implemented by outdoor mid- or late summer collection of 3,000 gravid Cx. pipiens per study site. PoolScreen estimates indicated that filaria transmission has probably been eliminated from five of the study villages. In Kafr Nafra however, a provisional 0.25% mosquito infection threshold has been exceeded. These data were not related to mosquito density, but rather to pre-treatment blood MF, except for Kafr Nafra