Application of hydrolysis probe analysis to identify clade types of the malaria vector mosquito Anopheles funestus sensu stricto from Muheza,northeastern Tanzania

A hydrolysis probe analysis (TaqMan assay) was used to study clade types in Anopheles funestus sensu stricto Giles, a major malaria vector in sub‐Saharan Africa, with specimens collected from Muheza in Tanga, northeastern Tanzania. A total of 186 An. funestus specimens were analysed, revealing that 176 (94.6%) were of clade I and 10 (5.4%) of clade II. These findings extend the distribution of clade type II from southern Mozambique and northern Zambia to northeastern Tanzania. The technique used can also be of great value in assessing the role and contribution of these clade types in malaria transmission and insecticide resistance frequencies for An. funestus s.s.     

Cryptic species within Anopheles longipalpis from southern Africa and phylogenetic comparison with members of the An. funestus group

House-resting Anopheles mosquitoes are targeted for vector control interventions; however, without proper species identification, the importance of these Anopheles to malaria transmission is unknown. Anopheles longipalpis, a non-vector species, has been found in significant numbers resting indoors in houses in southern Zambia, potentially impacting on the utilization of scarce resources for vector control. The identification of An. longipalpis is currently based on classical morphology using minor characteristics in the adult stage and major ones in the larval stage. The close similarity to the major malaria vector An. funestus led to investigations into the development of a molecular assay for identification of An. longipalpis. Molecular analysis of An. longipalpis from South Africa and Zambia revealed marked differences in size and nucleotide sequence in the second internal transcribed spacer (ITS2) region of ribosomal DNA between these two populations, leading to the conclusion that more than one species was being analysed. Phylogenetic analysis showed the Zambian samples aligned with An. funestus, An. vaneedeni and An. parensis, whereas the South African sample aligned with An. leesoni, a species that is considered to be more closely related to the Asian An. minimus subgroup than to the African An. funestus subgroup. Species-specific primers were designed to be used in a multiplex PCR assay to distinguish between these two cryptic species and members of the An. funestus subgroup for which there is already a multiplex PCR assay.     

Anopheles arabiensis and An. funestus are equally important vectors of malaria in Matola coastal suburb of Maputo, southern Mozambique

Transmission characteristics of malaria were studied in Matola, a coastal suburb of Maputo, the capital City, in southern Mozambique, from November 1994 to April 1996. The local climate alternates between cool dry season (May-October) and hot rainy season (November-April) with mean annual rainfall 650-850 mm. Saltmarsh and freshwater pools provide mosquito breeding sites in Matola. Malaria prevalence reached approximately 60% among people living nearest to the main breeding sites of the vectors. Plasmodium falciparum caused 97% of malaria cases, others being P. malariae and P. ovale. Potential malaria vector mosquitoes (Diptera: Culicidae) collected at Matola during daytime indoor-resting (n = 1021) and on human bait at night (n = 5893) comprised 12% Anopheles coustani Laveran (93% biting outdoors), 46% An. funestus Giles (68% biting indoors) and 42% An. gambiae Giles sensu lato (60% biting outdoors). All 215 specimens of An. gambiae s.l. identified genetically were An. arabiensis Patton. Anopheles funestus populations remained stable throughout the year, whereas densities of the An. gambiae complex fluctuated considerably, with An. arabiensis peaking during the rainy season. No concomitant rise in malaria incidence was observed. Human landing indices of An. funestus and An. arabiensis averaged 1.8 and 3.8 per man-night, respectively. Overall Plasmodium sporozoite rates were 2.42+/-1.24% in 2181 An. funestus and 1.11+/-1.25% in 1689 An. arabiensis dissected and examined microscopically. Mean daily survival rates were 0.79 for both vector species. Estimated infective bites/person/year were 15 An. funestus and 12 An. arabiensis. Biting rates were greatest at 2100-24.00 hours for An. funestus (68% endophagic) and 21.00-03.00 hours for An. arabiensis (40% endophagic). The entomological inoculation rate (EIR) declined sharply over very short distances (50% per 90m) away from breeding-sites of the vectors. Consequently, P. falciparum prevalence among Matola residents was halved 350 m within the town. Implications for the protective effectiveness of a 'cordon sanitaire' by residual house-spraying and/or the use of insecticide-treated bednets are discussed.     

Malaria transmission potential of Anopheles mosquitoes in the Mwea-Tebere irrigation scheme, Kenya

1. Anopheles arabiensis Patton and An. funestus Giles were identified as vectors of Plasmodium falciparum malaria in the Mwea-Tebere irrigation scheme, Kenya. An. arabiensis was the only member of the An. gambiae complex identified from chromosome characteristics. Other Anopheles species found included An. pharoensis Theobald, An. rufipes Gough and An. coustani Laveran. Survival rates per gonotrophic cycle for An. arabiensis averaged 0.37 during the short rains (October-November), 0.49 during the dry season (February) and 0.78 during the long rains (May-June). Vectorial capacities were correspondingly low due to low survival rates and a high degree of zoophily. The average duration of infective life for P. falciparum was 0.2 days for both An. arabiensis and An. funestus. In contrast, entomological inoculation rates were comparatively high: 6-8 infective bites/man/month. An. pharoensis averaged 110 bites/man/night during the short rains; 1/999 (0.1%) was positive by ELISA for P. falciparum circumsporozoite antigen, but the ELISA evidence is not conclusive for vector incrimination. In correspondence with clinical observations, the transmission of P. malariae and P. ovale is unlikely due to the low vector survival rates. The observed anomaly between low vectorial capacities and high entomological inoculation rates demonstrates the importance of accurately estimating vector sporozoite rates to monitor unstable malaria transmission in irrigated areas.     

Does moonlight influence the biting behaviour of Anopheles funestus?

The possible effect of moonlight on the biting behaviour of mosquitoes in southern Mozambique, in particular that of Anopheles funestus (Diptera: Culicidae), a primary vector of malaria, was investigated by comparing catches indoors and outdoors using CDC light traps and 'Furvela' tent traps, respectively, for 35 consecutive nights, from 9 September to 15 October 2008. Collections were separated into three 4-hourly samples each night. A total of 17 591 mosquitoes belonging to nine species were collected, 6747 in light traps and 10 844 in tent traps. Anopheles funestus (n = 7634) and Mansonia africana (n = 4859) were the most abundant species. Fluctuations in temperature and humidity were the two environmental variables associated with changes in relative abundance of mosquitoes. Most An. funestus were collected indoors, with the majority collected in the first 4 h of the night. This was most evident on nights when moonlight was present in the early part of the night. A total of 3488 An. funestus were dissected for gonotrophic age determination. Parous rates did not change with lunar phase, but estimated oviposition cycle length was significantly shorter on nights when moonlight was present at the time of oviposition. Moonlight at dusk did not, however, affect the proportion of newly emerged insects with mating plugs collected. Outdoor transmission of malaria, especially on moonlit nights, remains a problem for control programmes.     

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.     

Studies on the bionomics of male Anopheles gambiae Giles and male Anopheles funestus Giles from southern Mozambique

Little is known about the fitness of wild male mosquitoes, the females of which are vectors of malaria. The problem of studying male biology has been exacerbated by difficulties associated with catching them. In southern Mozambique, however, almost the entire adult population of An. funestus and An. gambiae s.l. rest inside houses. They leave in a dusk exodus, which makes them easy to collect. In 8,348 exit collections from a village from 2003 to 2009, 567,195 male An. funestus and 34,591 male An. gambiae s.l. were collected. During the study, numbers of An. funestus increased but numbers of An. gambiae s.l. declined to the point of extinction. Overall numbers of An. gambiae s.l. were positively correlated with temperature, whilst the relationship between temperature and numbers of An. funestus changed from an initially positive one in the first three years of the study to a negative one in the last three years. Marked males were recaptured up to 300 m from the release site, with most recaptures occurring within 150 m. Estimated mean daily survival of male An. funestus was 0.86 (95% C.I. 0.869-0.850). For the years 2003-2007, estimated mean daily survival of male An gambiae s.l. was 0.660 (95% C.I. 0.682-0.638). For either species, there was no relationship between mean weekly temperature and estimated daily survival. These results imply that males of An. funestus live as long as females but have a relatively short flight range. They are discussed in the light of possible release strategies of sterile or genetically modified mosquitoes.     

Anopheles funestus resistant to pyrethroid insecticides in South Africa

Northern Kwazulu/Natal (KZN) Province of South Africa borders on southern Mozambique, between Swaziland and the Indian Ocean. To control malaria vectors in KZN, houses were sprayed annually with residual DDT 2 g/ m2 until 1996 when the treatment changed to deltamethrin 20-25 mg/m2. At Ndumu (27 degrees 02'S, 32 degrees 19'E) the recorded malaria incidence increased more than six-fold between 1995 and 1999. Entomological surveys during late 1999 found mosquitoes of the Anopheles funestus group (Diptera: Culicidae) resting in sprayed houses in some sectors of Ndumu area. This very endophilic-vector of malaria had been eliminated from South Africa by DDT spraying in the 1950s, leaving the less endophilic An. arabiensis Patton as the only vector of known importance in KZN. Deltamethrin-sprayed houses at Ndumu were checked for insecticide efficacy by bioassay using susceptible An. arabiensis (laboratory-reared) that demonstrated 100% mortality. Members of the An. funestus group from Ndumu houses (29 males, 116 females) were identified by the rDNA PCR method and four species were found: 74 An. funestus Giles sensu stricto, 34 An. parensis Gillies, seven An. rivulorum Leeson and one An. leesoni Evans. Among An. funestus s.s. females, 5.4% (4/74) were positive for Plasmodium falciparum by ELISA and PCR tests. To test for pyrethroid resistance, mosquito adults were exposed to permethrin discriminating dosage and mortality scored 24h post-exposure: survival rates of wild-caught healthy males were 5/10 An. funestus, 1/9 An. rivulorum and 0/2 An. parensis; survival rates of laboratory-reared adult progeny from 19 An. funestus females averaged 14% (after 1h exposure to 1% permethrin 25:75cis:trans on papers in WHO test kits) and 27% (after 30 min in a bottle with 25 microg permethrin 40:60cis:trans). Anopheles funestus families showing >20% survival in these two resistance test procedures numbered 5/19 and 12/19, respectively. Progeny from 15 of the families were tested on 4% DDT impregnated papers and gave 100% mortality. Finding these proportions of pyrethroid-resistant An. funestus, associated with a malaria upsurge at Ndumu, has serious implications for malaria vector control operations in southern Africa.     

Efficacy of permethrin-impregnated curtains for malaria vector control

Preliminary results obtained by the use of permethrin-impregnated curtains against the Afrotropical malaria vectors Anopheles gambiae Giles s.l. and An.funestus Giles are reported and discussed. Field trials were carried out in villages near Ouagadougou, Burkina Faso. Houses were provided with curtains made from 100% cotton netting, impregnated with permethrin at the dose of 1 g a.i./m2, to cover the doorway, the window(s) and the space under the eaves. Entomological data collected during the period 1985-86 showed residual permethrin activity for about a year, and almost complete prevention of indoor-resting mosquitoes. Increased exit-rate and mortality-rate of house-entering malaria vectors were also obtained. Utilization of this malaria vector control method in primary health care programmes is advocated.     

ELISA study of oocyst-sporozoite transition in malaria vectors

Intrinsic vector characteristics and environmental factors affect the sporogonic development of P. falciparum in Anopheles mosquitoes. We tested for the presence of the circumsporozoite protein, as a marker of the oocyst to sporozoite transition in naturally infected Anopheles gambiae s.l. and Anopheles funestus. Malaria vectors were collected in a village in the Sahel of Niger during the rainy and dry seasons. ELISA-CSP was carried out on abdomen and head/thorax portions from more than 2000 samples. No significant difference was found in the overall rates of infection of An. gambiae s.l. (4.13%) and An. funestus (3.58%). Given the differences in duration of the two parasite stages, P. falciparum CSP antigen prevalence was nearly as high in the abdomen as in the head/thorax, and did not differ significantly between An. gambiae s.l. and An. funestus. These preliminary results suggest that development from oocysts to salivary gland sporozoites is similar in the two vectors. However, these developmental indices varied as a function of the season in which samples were collected, particularly for An. gambiae s.l. This simple method may be useful for field studies assessing the effect of environmental and genetic factors on parasite survival.     

Quantitation of malaria sporozoites in the salivary glands of wild Afrotropical Anopheles

The number of malaria sporozoites in the salivary glands was determined microscopically for 1137 wild, naturally infected Anopheles from western Kenya. Infective Anopheles gambiae Giles sensu lato (n = 874) contained a geometric mean (GM) of 962 sporozoites and An.funestus Giles (n = 263) contained 812. No significant differences were detected in geometric mean numbers of sporozoites between species, collection techniques or sites. Of the infective An.gambiae, 1.7% (15/874) contained more than 41,830 sporozoites, the maximum observed for An.funestus. Microscopic techniques were found to be more sensitive than enzyme-linked immunosorbent assays (ELISA) for detecting low-grade sporozoite infections in salivary glands. Salivary gland sporozoites from 83.6% of the 1137 gland infections were identified by ELISA as either Plasmodium falciparum Welch (n = 910), P.ovale Stephens (n = 7), P.malariae Grassi & Feletti (n = 3) or mixed (n = 30). The 187 gland infections which could not be identified by ELISA contained significantly fewer sporozoites (GM = 242) than those which could be identified (GM = 1200).     

Year to year and seasonal variations in vector bionomics and malaria transmission in a humid savannah village in west Burkina Faso

A longitudinal entomological study was carried out from 1999 to 2001 in Lena, a humid savannah village in the western region of Burkina Faso in order to establish malaria vector bionomics and the dynamics of malaria transmission. In the first year, malaria transmission was mainly due to An. gambiae s.s., but during the two later years was due to An. funestus, which were observed in high frequency towards the end of the rainy season. PCR identification of samples of An. gambiae s.1. showed 93% to be An. gambiae s.s. and 7% An. arabiensis. An. funestus constituting more than 60% of the vectors were identified in PCR as An. funestus s.s. The persistence of intense vectorial activity in this village was probably due to the road building in a swampy area creating a semi-permanent swamp that provided large sites for larval mosquitoes. These swampy sites seemed to be more favorable for An. funestus than for An. gambiae s.s. Thus, land development must be monitored and subjected to planning to minimize vector proliferation. Such a system of planning could lead to the restriction or even elimination of the swamp that is the source of larvae developing in the heart of the village.     

Moderate transmission but high prevalence of malaria in Madagascar

Malaria transmission remains poorly documented in areas of low transmission. A study has been carried out over two consecutive years in Analamiranga, a village located at an altitude of 885m on the western edge of the Malagasy highlands, with the aim of generating and updating malariometric indexes for both mosquitoes and schoolchildren. In this village, no vector control measures were performed during the study period nor during previous decades. Mosquitoes were collected monthly when landing on human volunteers and in various resting-places. Blood samples were taken every 3 months from schoolchildren aged 6-12 years and microscopically examined. Of 7,480 mosquitoes collected on human subjects, 5,790 were anophelines. Ten anopheline species were represented and three of these, Anopheles funestus, Anopheles arabiensis and Anopheles mascarensis, accounted for 59.2% of the collection. Of these three species 4,640 were also collected in resting places. The proportion of mosquitoes fed on bovids was high; conversely, the anthropophilic rate (mosquitoes fed on human beings) was especially low: 31%, 7% and 1%, respectively, for A. funestus, A. arabiensis and A. mascarensis. The only confirmed malaria vector was A. funestus with a low sporozoite index (of 6,830 A. funestus, five were positive for Plasmodium falciparum and four for Plasmodium vivax). The annual entomological inoculation rate (number of bites of infected anophelines per adult person) was estimated at 2.49 with low variation over the 2 years. Overall, 909 thick blood smears were tested from blood samples taken from schoolchildren with 30.3% being malaria-positive. The four Plasmodium species infecting human subjects were detected in the following proportions: P. falciparum 78.9%, P. vivax 19.4%, Plasmodium malariae 1.0% and Plasmodium ovale 0.7%. The proportions of children who were infected with any Plasmodium ranged from 10.7% in February to 51.0% in September. Parasitemic children with fever (axillary temperature >37.5 degrees C) accounted for 16.4% of the children sampled. This study demonstrates that there are substantial parasitological consequences of even a relatively low entomological transmission and also recommends including exterior resting-places of mosquitoes in future spraying campaigns in the highlands of Madagascar.     

Anopheles and malaria transmission in Ambohimena, a village in the Occidental fringe of Madagascar Highlands

The anopheline vectors and malaria transmission were investigated in the Middle West of Madagascar, in the village of Ambohimena (at the altitude of 940 meters) during two years (August 1996 to July 1998). This village is located outside the vector control area, where yearly DDT house spraying campaigns have been conducted between 1993 and 1998. Collection of mosquitoes was mainly based on all night man billing collections (650 man-nights), pyrethrum spray catches (224 bedrooms) and direct collections in outdoor resting places (140 toilets, 61 pigsties, 33 holes, 19 sheds, 79 sisal hedges, 70 cart shelters). Blood fed anophelines allowed analysis of the origin of blood with an ELISA method. Presence of circum-sporozoite protein was assessed with another ELISA method. The total number of collected anophelines was 14,280. Two malaria vectors were identified: Anopheles funestus Giles, 1900 and An. arabiensis Patton, 1902. An. funestus was the most abundant mosquito, especially during the hot rainy season. Two peaks of abundance were observed (in December and April). Endophagic rate (for mosquitoes aggressive for man) of 35.3%, an endophilic rate (for resting mosquitoes) of 78.0% and an anthropophilic rate (for indoor resting mosquitoes) of 64.0% were calculated. The average parity rate was relatively low (61.2%). The Plasmodium falciparum immunological sporozoite rate was 0.20%. An. funestus presented a higher vectorial capacity during the first round of rice cultivation (January) than during the second round (April-May). An. arabiensis was mostly abundant in December and January at the beginning of the rainy season. This species was exophagic (endophagic rate = 27.5%) and zoophilic (anthropophilic rate = 7.8%). The sporozoitic index was determined as zero (number of examined mosquitoes = 871). In this village, An. arabiensis presented only marginal importance for malaria transmission. Malaria transmission occurred from December to April. Annual entomological inoculation rate, only due to An. funestus, was 8.96 during the first year, and 3.17 during the second year. In this area where transmission is moderately stable, we suggest an extension of vector imagocidal control activities up to the western fringes of the Highlands.     

The impact of permethrin-impregnated bednets on malaria vectors of the Kenyan coast

Abstract. The effects of introducing permethrin-impregnated bednets on local populations of the malaria vector mosquitoes Anopheles funestus and the An.gambiae complex was monitored during a randomized controlled trial at Kilifi on the Kenyan coast. Pyrethrum spray collections inside 762 households were conducted between May 1994 and April 1995 after the introduction of bednets in half of the study area. All-night human bait collections were performed in two zones (one control and one intervention) for two nights each month during the same period. PCR identifications of An.gambiae sensu lato showed that proportions of sibling species were An.gambiae sensu stricto > An.merus > An.arabiensis.
Indoor-resting densities of An.gambiae s.l. and the proportion of engorged females decreased significantly in intervention zones as compared to control zones. However, the human blood index and Plasmodium falciparum sporozoite rate remained unaffected. Also vector parous rates were unaltered by the intervention, implying that survival rates of malaria vectors were not affected. The human-biting density of An.gambiae s.l. , the predominant vector, was consistently higher in the intervention zone compared to the control zone, but showed 8% reduction compared to pre-intervention biting rates - versus 94% increase in the control zone.
Bioassay, susceptibility and high-performance liquid chromatography results all indicated that the permethrin content applied to the nets was sufficient to maintain high mortality of susceptible vectors throughout the trial. Increased rates of early outdoor-biting, as opposed to indoor-biting later during the night, were behavioural or vector composition changes associated with this intervention, which would require further monitoring during control programmes employing insecticide-treated bednets.     

Chromosomal and bionomic heterogeneities suggest incipient speciation in Anopheles funestus from Burkina Faso

Sampling of day-resting Anopheles funestus was carried out in September-November 1991, October-December 1992, and November 1994 at two sites near Ouagadougou, Burkina Faso: the small village of Noungou where humans outnumber cattle, and the nearby Fulani settlement of Loumbila where cattle outnumber humans. Collections made inside human dwellings were supplemented in 1992 by outdoor-resting samples from artificial pit-shelters. Indoor-resting An. funestus were also collected in November 1992 and November 1994 in four villages of the Banfora area (southern Burkina Faso) and in a sudanese-sahelian village in northern Burkina Faso (Tougouri). Half-gravid female sub-samples were preserved in carnoy's fixative and processed for polytene chromosome analysis. The material from the two villages near Ouagadougou was analysed by ELISA to know (i) the human/animal origin of the blood meal; (ii) the infectivity for Plasmodium falciparum malaria; and (iii) the possible correlation of these parameters with chromosomal variants. A total of 1416 An. funestus could be scored for the whole polytenic complement, while the origin of the blood meal and circumsporozoite protein (CSP) positivity were asserted from 1076 and 1154 specimens, respectively. With a few exceptions, four polymorphic paracentric inversions (3a, 3b, 2a and 5a in decreasing order of frequency) were observed in all populations. Inversion 2s, whose breaking points include those of inversion 2a, was found only as the heterokaryotype 2s/+ floating at an overall frequency of 3.7% in two villages of the Banfora area and in the two sites near Ouagadougou. Two heterokaryotypes 2a/t out of 186 scored specimens were observed in different years from one village of the Banfora area. Wide variations in inversion frequencies were observed among the samples without consistent geographical or temporal clines. Highly significant departures from Hardy-Weinberg equilibrium were recorded for inversions 3a and 3b in most samples, with the alternative homokaryotypes (standard and inverted) significantly more frequent than expected. Conversely, inversion 5a was in Hardy-Weinberg equilibrium in most samples, whereas the 2a-s inversion system was intermediate between these extremes. However, a deficit of heterokaryotypes was apparent practically in all samples. Significantly higher frequencies of the standard homokaryotypes were recorded (i) in the exophilic samples collected in Loumbila for arrangement 3a; (ii) in the animal-fed sub-sample collected outdoors in Noungou vs. the parallel human-fed sub-sample for arrangements 2a-s, 3a, and 3b, or vs. the samples obtained from indoor catches in both Loumbila and Noungou in the case of inversion 3a; (iii) in the December CSP-negative sub-sample from Loumbila vs. the parallel CSP-positive sub-sample for arrangement 2a. A plausible working hypothesis is that An. funestus in Burkina Faso includes two taxonomic units, one of which is mainly monomorphic standard with most inverted arrangements floating at very low frequencies, and probably uniquely characterised by arrangement 2s, while the other taxon is nearly fixed for arrangement 3ab and polymorphic for all the other inversions at intermediate to high frequencies. The latter would be characterised by a higher vectorial capacity and would probably correspond to An. funestus s.s. from East Africa. About the former hypothetical taxon, its endophily and anthropophily appear less marked and its relationship with other members of the An. funestus subgroup will require specific investigations.     

ELISA absorbance cut-off method affects malaria sporozoite rate determination in wild Afrotropical Anopheles

ABSTRACT. Malaria sporozoite infection rates in a mixed species group of 244 Anopheles gambiae Giles sensu lato and 115 An. funestus Giles wild female mosquitoes were compared using three methods to determine cutoff absorbance values for positivity of a Plasmodium falciparum Welch enzyme-linked immunosorbent assay (ELISA). Positive controls were based on P. falciparum circumsporozoite protein. As negative controls, four wild male Anopheles were included on each microtitre plate; tests were repeated on four consecutive days for each plate.
Infection rates were estimated at 13.1–22.8% using the mean absorbance value of negative controls plus three standard deviations, 11.7–12.8% using double the mean and 12.5–13.6% using the fixed cut-off value of 0.20 (allowing for 20% variation in negative control absorbance values).
Observed agreement for positivity or negativity among samples tested four times was 98.6% for the 2× mean method, 97.2% for the fixed cut-off 0.20 value, but only 82.7% for the mean +3 SD method. It was concluded that the 2× mean cut-off method is most reliable for field studies. P. falciparum sporozoite rates of 12.2% in An. funestus and 11.9% in An. gambiae s. l . were thus determined on the basis of the 2× mean cut-off method.
This comparative evaluation demonstrates that vector infectivity rates can be seriously over-estimated from sporozoite ELISA tests, by as much as 87% in one case considered here, depending on the absorbance cut-off method applied for negative controls.     

Adult anopheline ecology and malaria transmission in irrigated areas of South Punjab, Pakistan

Surface irrigation in the Punjab province of Pakistan has been carried out on a large scale since the development of the Indus Basin Irrigation System in the late 19th century. The objective of our study was to understand how the population dynamics of adult anopheline mosquitoes (Diptera: Culicidae) could be related to malaria transmission in rural areas with intensive irrigation and a history of malaria epidemics. In this paper we present our observations from three villages located along an irrigation canal in South Punjab. The study was carried out from 1 April 1999 to 31 March 2000. Mosquitoes were collected from bedrooms using the pyrethroid spraycatch method and from vegetation and animal sheds using backpack aspirators. Overall, Anopheles subpictus Grassi sensu lato predominated (55.6%), followed by An. stephensi Liston s.l. (41.4%), An. culicifacies Giles s.l. (2.0%), An. pulcherrimus Theobald (1.0%) and An. peditaeniatus Leicester (0.1%). Most mosquitoes (98.8%) were collected from indoor resting-sites whereas collections from potential resting-sites outdoors accounted for only 1.2% of total anopheline densities, confirming the endophilic behaviour of anophelines in Pakistan. Anopheles stephensi, An. culicifacies and An. subpictus populations peaked in August, September and October, respectively. High temperatures and low rainfall negatively affected seasonal abundance in our area. There were interesting differences in anopheline fauna between villages, with An. culicifacies occurring almost exclusively in the village at the head of the irrigation canal, where waterlogged and irrigated fields prevailed. Monthly house-to-house fever surveys showed that malaria transmission remained low with an overall slide positivity rate of 2.4% and all cases were due to infection with Plasmodium vivax. The most plausible explanation for low transmission in our study area seems to be the low density of Pakistan's primary malaria vector, An. culicifacies. The role of other species such as An. stephensi is not clear. Our observations indicate that, in South Punjab, irrigation-related sites support the breeding of anopheline mosquitoes, including the vectors of malaria. As our study was carried out during a year with exceptionally hot and dry climatic conditions, densities and longevity of mosquitoes would probably be higher in other years and could result in more significant malaria transmission than we observed. To assess the overall importance of irrigation-related sites in the epidemiology of malaria in the Punjab, more studies are needed to compare irrigated and non-irrigated areas.