Extensive multiple test centre evaluation of the VecTest malaria antigen panel assay

To determine which species and populations of Anopheles transmit malaria in any given situation, immunological assays for malaria sporozoite antigen can replace traditional microscopical examination of freshly dissected Anopheles. We developed a wicking assay for use with mosquitoes that identifies the presence or absence of specific peptide epitopes of circumsporozoite (CS) protein of Plasmodium falciparum and two strains of Plasmodium vivax (variants 210 and 247). The resulting assay (VecTest Malaria) is a rapid, one-step procedure using a 'dipstick' test strip capable of detecting and distinguishing between P. falciparum and P. vivax infections in mosquitoes. The objective of the present study was to test the efficacy, sensitivity, stability and field-user acceptability of this wicking dipstick assay. In collaboration with 16 test centres world-wide, we evaluated more than 40 000 units of this assay, comparing it to the standard CS ELISA. The 'VecTest Malaria' was found to show 92% sensitivity and 98.1% specificity, with 97.8% accuracy overall. In accelerated storage tests, the dipsticks remained stable for > 15 weeks in dry conditions up to 45 degrees C and in humid conditions up to 37 degrees C. Evidently, this quick and easy dipstick test performs at an acceptable level of reliability and offers practical advantages for field workers needing to make rapid surveys of malaria vectors.     

Evaluation of monoclonal antibodies against Plasmodium vivax sporozoites for ELISA development

Nine monoclonal antibodies (MAbs) developed against Plasmodium vivax (Grassi & Feletti) salivary gland sporozoites were evaluated for use in an enzyme-linked immunosorbent assay (ELISA), using sporozoites developed in Anopheles dirus Peyton & Harrison An. gambiae Giles and An.maculatus Theobald. Four of the antibodies were unsuitable due to the low sensitivity of the resulting assays or the requirement for high concentrations of capture antibody. An additional two MAbs were rejected because they resulted in assays with high background absorbance, attributed to self-binding. Of the three remaining MAbs, the use of Navy vivax sporozoite (NVS) 3 resulted in an ELISA with the highest sensitivity and the lowest concentration requirement for capture antibody. Assay sensitivity varied with sporozoite strain indicating possible quantitative epitope heterogeneity. None of the MAbs cross-reacted with the heterologous sporozoites tested by immunofluorescence antibody assay (IFA). The IFA activity was not an indicator of ELISA sensitivity. The use of MAb NVS 3 in a standardized ELISA method resulted in an assay 10 times more sensitive than reported previously for P. vivax sporozoites, with a detection limit of fewer than 100 sporozoites per mosquito.     

Interactions of human malaria parasites, Plasmodium wVaxand P.falciparum, with the midgut of Anopheles mosquitoes

Abstract Present understanding of the development of sexual stages of the human malaria parasites Plasmodium vivax and P.falciparum in the Anopheles vector is reviewed, with particular reference to the role of the mosquito midgut in establishing an infection. The sexual stages of the parasite, the gametocytes, are formed in human erythrocytes. The changes in temperature and pH encountered by the gametocyte induce gametogenesis in the lumen of the midgut. Macromolecules derived from mosquito tissue and second messenger pathways regulate events leading to fertilization. In An.tessellatus the movement of the ookinete from the lumen to the midgut epithelium is linked to the release of trypsin in the midgut and the peritrophic matrix is not a firm barrier to this movement. The passage of the P. vivax ookinete through the peritrophic matrix may take place before the latter is fully formed. The late ookinete development in P.falciparum requires chitinase to facilitate penetration of the peritrophic matrix. Recognition sites for the ookinetes are present on the midgut epithelial cells. N-acetyl glucosamine residues in the oligosaccharide side chains of An.tessellatus midgut glycoproteins and peritrophic matrix proteoglycan may function as recognition sites for P.vivax and P.falciparum ookinetes. It is possible that ookinetes penetrating epithelial cells produce stress in the vector. Mosquito molecules may be involved in oocyst development in the basal lamina, and encapsulation of the parasite occurs in vectors that are refractory to the parasite. Detailed knowledge of vector-parasite interactions, particularly in the midgut and the identification of critical mosquito molecules offers prospects for manipulating the vector for the control of malaria.     

Selection of Anopheles dirus for refractoriness and susceptibility to Plasmodium yoelii nigeriensis

Two lines of the Oriental malaria vector mosquito Anopheles dirus species A (Diptera: Culicidae), one fully refractory and one fully susceptible to Plasmodium yoelii nigeriensis (an African rodent malaria parasite), were established after 17 generations of mass selection, followed by single female selection for one or two generations. Prior to selection, the stock colony of An. dirus was 17% refractory. Both lines of An. dirus produced abundant ookinetes that started to invade the midgut within 24h post-infection, as seen in histological sections. In most of the refractory mosquitoes, oocysts stopped development <12 h post-invasion, indicating a rapid defence mechanism. Dead P. y. nigeriensis parasites were apparently localized as small melanized spots (2-5 microm) seen in wet preparations of mosquito midguts dissected 5-7 days post infective bloodmeal. In some refractory An. dirus females, apart from the spots, a small number of totally encapsulated oocysts (c. 10 microm) were also present. These larger melanized parasites predominated in a few females: they appeared 2-3 days post-infection as a secondary delayed defence mechanism. The progeny of reciprocal matings between susceptible and refractory lines had approximately 50% susceptibility. Backcrosses of F1 hybrids with susceptible or refractory lines increased or decreased the susceptibility of backcross progeny accordingly. Overall, these results suggest polygenic control of susceptibility to P. y. nigeriensis infection. The refractory line of An. dirus showed normal susceptibility to natural infections of the human malarias P. falciparum and P. vivax from local patients.     

Selection of Anopheles stephensi for refractoriness and susceptibility to Plasmodium falciparum

Variation in susceptibility of the vector Anopheles stephensi Liston to the human malaria parasite Plasmodium falciparum (Welch) was demonstrated using twelve strains of mosquitoes and one strain of parasites cultured in vitro. The Beech strain of An. stephensi exhibited greatest natural refractoriness, but with high intrapopulation variability. By selection for the required characteristic, two refractory lines of the Punjab strain and one highly susceptible line of the Sind strain were obtained. The median number of oocysts in the two refractory lines was less than 4% of that in the unselected line, whilst the highly susceptible line yielded about twice as many oocysts as the unselected line. Selection progressed more by keeping the descendants of individual females separate and selecting between them (individual selection) rather than pooling the progeny of all selected mosquitoes (mass selection). Using the former procedure many lines were lost due to inbreeding depression, but the outcome was more successful.     

Seroprevalence of Plasmodium vivax Circumsporozoite Protein Antibody in High-Risk Malaria Areas in Korea

The circumsporozoite protein (CSP) of Plasmodium spp. is a diagnostic antigen and useful biomarker for monitoring short-term/seasonal changes to malaria transmission. Using P. vivax CSP antibody ELISA, epidemiological characteristics were analyzed in the residents of Ganghwa, Cheorwon, Paju, and Goseong from 2017 to 2018. In Ganghwa and Cheorwon, 1.6% and 1.2% of residents, respectively, were PvCSP-antibody-positive in 2018, which indicates a decrease of 0.4% in the positive rate compared to 2017. The annual parasite incidence (API) in Ganghwa and Cheorwon was 24.9 and 10.5 in 2017 and 20.3 and 10.7 in 2018, respectively. Although the changes were not significant, the API in Ganghwa decreased slightly by 4.5 in 2018 compared to the previous year. In Paju and Goseong, 3.9% and 2.0% of residents were positive for the PvCSP antibody. The API in Paju was 13.1 in 2017 and 16.0 in 2018, although no malaria patients were reported for the 2 years. Therefore, the results suggest that PvCSP is a useful antigen for confirming initial malaria infection. Additionally, considering that the antibody is relatively transient, it can be employed for sero-epidemiological studies to determine the extent of malaria transmission in the current year.     

A novel nested polymerase chain reaction assay targeting Plasmodium mitochondrial DNA in field‐collected Anopheles mosquitoes

Sensitive techniques for the detection of Plasmodium (Aconoidasida: Plasmodiidae) sporozoites in field‐collected malaria vectors are essential for the correct assessment of risk for malaria transmission. A real‐time polymerase chain reaction (RT‐PCR) protocol targeting Plasmodium mtDNA proved to be much more sensitive in detecting sporozoites in mosquitoes than the widely used enzyme‐linked immunosorbent assay targeting Plasmodium circumsporozoite protein (CSP‐ELISA). However, because of the relatively high costs associated with equipment and reagents, RT‐PCRs are mostly used to assess the outcomes of experimental infections in the frame of research experiments, rather than in routine monitoring of mosquito infection in the field. The present authors developed a novel mtDNA‐based nested PCR protocol, modified from a loop‐mediated isothermal amplification (LAMP) assay for Plasmodium recognition in human blood samples, and compared its performance with that of routinely used CSP‐ELISAs in field‐collected Anopheles coluzzii (Diptera: Culicidae) samples. The nested PCR showed 1.4‐fold higher sensitivity than the CSP‐ELISA. However, nested PCR results obtained in two laboratories and in different replicates within the same laboratory were not 100% consistent, probably because the copy number of amplifiable Plasmodium mtDNA was close in some specimens to the threshold of nested PCR sensitivity. This implies that Plasmodium‐positive specimens should be confirmed by a second nested PCR to avoid false positives. Overall, the results emphasize the need to use molecular approaches to obtain accurate estimates of the actual level of Plasmodium circulation within malaria vector populations.     

Detection of Plasmodium sporozoites in mosquitoes by polymerase chain reaction and oligonucleotide rDNA probe, without dissection of the salivary glands

Dried Anopheles gambiae mosquito head + thorax portions, infected with Plasmodium falciparum sporozoites, were processed by the polymerase chain reaction. The PCR product was hybridized to an oligonucleotide probe (known as 114R or AW34) diagnostic for Plasmodium . The detection level by autoradiography was ten sporozoites per mosquito. Head + thorax of mosquitoes that contained mature P.falciparum oocysts, without sporozoites, gave no positive signal, indicating that the test detects only infective mosquitoes. This test can be applied to wild mosquito specimens collected, prepared and processed at different time intervals. The technique is convenient, highly sensitive, and could be used with a non-radioactive detection system and specific probes to differentiate Plasmodium spp.     

Antibodies to Anopheles midgut reduce vector competence for Plasmodium vivax malaria

Anopheles tessellatus mosquitoes ingested Plasmodium vivax gametocytes in human erythrocytes suspended in rabbit sera with and without anti-mosquito midgut antibodies. When the mosquito bloodmeal contained anti-midgut antibodies, fewer oocysts of P. vivax developed on the mosquito midgut and the proportion of mosquitoes becoming infected was significantly reduced. Complement inactivated serum also reduced the infection rate and load. A second bloodmeal containing anti-midgut antibodies, given 48 or 72 h later, did not enhance the transmission-blocking effect. IgG purified from antimidgut sera was shown to mediate the transmission-blocking effect.     

Anopheles pharoensis and transmission of Plasmodium falciparum in the Senegal River delta, West Africa

Abstract. 1. Anopheles pharoensis Theobald was found to be the prevalent man-biting anopheline mosquito in the central area of the Senegal River delta.
2. Blood-fed females of An.pharoensis were obtained during September-December 1987 from mosquito bednets in the village of Souhlloul, near the Boundoum dam, 70 km NE of St Louis.
3. Dried mosquito specimens were identified morphologically and each thorax processed using monoclonal antibody against the circum-sporozoite protein of Plasmodium falciparum.
4. Five An.pharoensis out of 912 examined were sporozoite positive, while ninety-eight An.gambiae Giles sensu lato were all negative. This finding strongly supports the local importance of An.pharoensis as a malaria vector.
5. Successful use of pyrethroid-impregnated bednets against malaria transmission in this situation has helped to achieve more than 90% reduction of malaria prevalence.     

The influence of permethrin-impregnated bednets and mass drug administration on the incidence of Plasmodium falciparum malaria in children in Sabah, Malaysia

A small-scale trial was carried out in the Upper Kinabatangan district of Sabah, Malaysia, to determine the effect of using permethrin-impregnated bednets on malaria transmission. A total of 306 nylon bednets with cotton borders, impregnated at a dose estimated to have been 0.062 g permethrin/m2 of nylon netting, were distributed to 139 households in five villages. At the time of distributing bednets, mass drug administration with Fansidar plus primaquine was also administered to the human population to clear all parasitaemias due to Plasmodium falciparum Welch. In another village, for comparison, mass drug administration was the only intervention. After intervention measures in December 1984 and January 1985, the parasite rates in children declined in all villages during the first month, significantly more in the villages with impregnated bednets than in the control, thus proving that the nets had an impact on malaria. However, after about 2 months, parasite rates started to increase again. After 4-6 months, parasite rates in the villages with bednets approached the rate in the control village without nets. The increase in parasite rates was paralleled by a significant deterioration in the quality, physical condition and the degree of non-utilization of bednets. Entomological evaluation proved the efficacy of permethrin-impregnated nets for controlling Anopheles balabacensis Baisas and other anophelines. Bioassays (1 h exposure) of permethrin-impregnated bednets gave 100% mortality initially and 44-61% mortality after 85-106 days. Mosquito collections in treated bednets were significantly reduced for at least 217 days. The project failed to achieve prolonged suppression of malaria transmission for a combination of entomological, sociological and practical reasons which are discussed in relation to the objectives and implementation of future bednet studies.     

DNA probes for species identification of mosquitoes in the Anopheles gambiae complex

Abstract. Identification of species within the Anopheles gambiae Giles species complex is essential for the correct evaluation of malaria vector ecology studies and control programmes. The development of DNA probes to distinguish species of the An.gambiae complex is described. Genomic libraries were prepared for four members of the An.gambiae complex. These were screened using radiolabeled DNA from different species of An. gambiae sensu lato and a number of clones selected on the basis of their species specificity. These clones could be divided into two groups, each containing homologous sequences. Sequences homologous to group 1 inserts are highly reiterated in the genomes of Anopheles arabiensis Patton and Anopheles merus Dönitz, present in low copy number in Anopheles melas Theobald, but were not detected in Anopheles gambiae sensu stricto. Studies on the organization of this sequence in the genome of An.arabiensis show that homologous sequences are male specific and interspersed within the chromatin. Sequences homologous to group 2 inserts are highly repeated in the genomes of An.merus and An.melas, but present in low copy number in An.gambiae s.s. and An.arabiensis. Group 2 homologous sequences are not sex-specific in the species tested and appear to be tandemly repeated. When used as hybridization probes, these sequences provide a sensitive means for the identification of species within the Anopheles gambiae complex.     

Host choice of anopheline mosquitoes in a malaria endemic area of western Venezuela

Abstract. Bloodmeals of exophilic anopheline mosquitoes collected resting on vegetation in a malaria endemic area in western Venezuela were identified by ELISA. Using a TMB peroxidase substrate in the ELISA, human bloodmeals were readily identified up to 40 h after ingestion in all laboratory-fed mosquitoes tested. Assay sensitivity declined to 75% identifiable 44 h post-feeding.
The Human Blood Index and the Feeding Index of each species differed between the three villages studied. An.triannulatus was generally more anthropophilic than An.nuneztovari and An.oswaldoi. These contrasting results emphasize the difficulties of interpreting host choice data.     

Impact of untreated bednets on prevalence of Wuchereria bancrofti transmitted by Anopheles farauti in Papua New Guinea

Abstract Despite the growing evidence that insecticide‐treated mosquito nets reduce malaria morbidity and mortality in a variety of epidemiological conditions, their value against lymphatic filariasis infection and disease is yet to be established. The impact of untreated bednets on the prevalence of Wuchereria bancrofti (Cobbold) (Nematoda: Filarioidea) infection and disease was investigated on Bagabag island in Papua New Guinea, where both malaria and filariasis are transmitted by the same vector mosquitoes of the Anopheles punctulatus Dönitz group (Diptera: Culicidae). Community‐wide surveys were conducted recording demographic characteristics including bednet usage. Physical examinations for hydrocoele and lymphoedema were performed and blood samples assessed for filarial and malaria parasites. Mosquitoes were sampled using the all‐night landing catch method and individually dissected to determine W. bancrofti infection and infective rates. Bednet usage among residents was 61% and the mean age of users (25.6 years) was similar to non‐users (22.5 years). Anopheles farauti Laveran was the only species were found to contain filarial larvae: 2.7% infected (all stages), 0.5% infective (L3). The overall W. bancrofti microfilaraemia and antigenaemia rates were 28.5% and 53.1%, respectively. Bednet users had lower prevalence of W. bancrofti microfilaraemia, antigenaemia and hydrocoele rates than non‐users. In comparison, untreated bednets had no effect on the prevalence and intensity of Plasmodium falciparum and P. vivax infections. The impact of bednet usage on rates of microfilaraemia and antigenaemia remained significant even when confounding factors such as age, location and sex were taken into account, suggesting that untreated bednets protect against W. bancrofti infection.     

A generalized approach to detection of organophosphate resistance in mosquitoes

Insecticide bioassays and biochemical microtitre assays were compared for detection of resistance to the organophosphate insecticides malathion and fenitrothion, using inbred laboratory strains of malaria vectors Anopheles albimanus Wiedemann, An.arabiensis Patton and An.stephensi Liston. With susceptible mosquitoes, the LT100 values determined from bioassays corresponded closely with times taken to abolish the activity of acetylcholinesterase activity in biochemical assays: approximately 2 h for malathion and 3 h for fenitrothion. Resistant strains of all three anophelines showed longer survival correlated with prolonged acetylcholinesterase activity. An.albimanus strains with insensitive acetylcholinesterase survived bioassays with discriminating doses of 1 h exposure to 5% malathion or 1% fenitrothion and were judged as resistant. It is concluded that enzyme-specific microassays provide a reliable means of detecting resistant individuals, with practical advantages over bioassays which do not reveal the resistance mechanism and require large numbers of healthy mosquitoes.     

Performance Evaluation of Biozentech Malaria Scanner in Plasmodium knowlesi and P. falciparum as a New Diagnostic Tool

The computer vision diagnostic approach currently generates several malaria diagnostic tools. It enhances the accessible and straightforward diagnostics that necessary for clinics and health centers in malaria-endemic areas. A new computer malaria diagnostics tool called the malaria scanner was used to investigate living malaria parasites with easy sample preparation, fast and user-friendly. The cultured Plasmodium parasites were used to confirm the sensitivity of this technique then compared to fluorescence-activated cell sorting (FACS) analysis and light microscopic examination. The measured percentage of parasitemia by the malaria scanner revealed higher precision than microscopy and was similar to FACS. The coefficients of variation of this technique were 1.2–6.7% for Plasmodium knowlesi and 0.3–4.8% for P. falciparum. It allowed determining parasitemia levels of 0.1% or higher, with coefficient of variation smaller than 10%. In terms of the precision range of parasitemia, both high and low ranges showed similar precision results. Pearson’s correlation test was used to evaluate the correlation data coming from all methods. A strong correlation of measured parasitemia (r²=0.99, P<0.05) was observed between each method. The parasitemia analysis using this new diagnostic tool needs technical improvement, particularly in the differentiation of malaria species.     

Six new species of the Anopheles leucosphyrus group, reinterpretation of An. elegans and vector implications

Abstract. Among Oriental anopheline mosquitoes (Diptera: Culicidae), several major vectors of forest malaria belong to the group of Anopheles (Cellia) leucosphyrus Dönitz. We have morphologically examined representative material (> 8000 specimens from seven countries) for taxonomic revision of the Leucosphyrus Group. Six new species are here described from adult, pupal and larval stages (with illustrations of immature stages) and formally named as follows: An. latens n. sp. (= An. leucosphyrus species A of Baimai et al., 1988b), An. cracens n. sp., An. scanloni n. sp., An. baimaii n. sp. (formerly An. dirus species B, C, D, respectively), An. mirans n. sp. and An. recens n. sp. Additionally, An. elegans (James) is redescribed and placed in the complex of An. dirus Peyton & Harrison (comprising An. baimaii, An. cracens, An. dirus, An. elegans, An. nemophilous Peyton & Ramalingam, An. scanloni and An. takasagoensis Morishita) of the Leucosphyrus Subgroup, together with An. baisasi Colless and the An. leucosphyrus complex (comprising An. balabacensis Baisas, An. introlatus Baisas, An. latens and An. leucosphyrus). Hence, the former Elegans Subgroup is renamed the Hackeri Subgroup (comprising An. hackeri Edwards, An. pujutensis Colless, An. recens and An. sulawesi Waktoedi). Distribution data and bionomics of the newly defined species are given, based on new material and published records, with discussion of morphological characters for species distinction and implications for ecology and vector roles of such species. Now these and other members of the Leucosphyrus Group are identifiable, it should be possible to clarify the medical importance and distribution of each species. Those already regarded as vectors of human malaria are: An. baimaii[Bangladesh, China (Yunnan), India (Andamans, Assam, Meghalaya, West Bengal), Myanmar, Thailand]; An. latens[Borneo (where it also transmits Bancroftian filariasis), peninsular Malaysia, Thailand]; probably An. cracens (Sumatra, peninsular Malaysia, Thailand); presumably An. scanloni (Thailand); perhaps An. elegans (the Western Ghat form of An. dirus, restricted to peninsular India); but apparently not An. recens (Sumatra) nor An. mirans[Sri Lanka and south-west India (Karnataka, Kerala, Tamil Nadu)], which is a natural vector of simian malarias. Together with typical An. balabacensis, An. dirus and An. leucosphyrus, therefore, the Leucosphyrus Group includes about seven important vectors of forest malaria, plus at least a dozen species of no known medical importance, with differential specific distributions collectively spanning > 5000 km from India to the Philippines.     

Location of genes on chromosome arms in the Anopheles gambiae group of species and their correlation to linkage data for other anopheline mosquitoes

The use of paracentric inversions as genetic markers in the Anopheles gambiae group of mosquitoes is described. The gene for dieldrin resistance is assigned to chromosome 2 which in turn is correlated to the previous assignment of the gene to linkage group II. The locus of the enzyme phosphoglucomutase 2 (Pgm 2) is similarly assigned to chromosome 2 and evidence is presented for possible linkage between Pgm 2 and dieldrin resistance. There was no linkage or correlation of chromosome 2 and loci of the enzymes superoxide dismutase (Sod) and octanol dehydrogenase (Odh). These genes are therefore assumed to be on chromosome 3 (linkage group III). Evidence that such gene linkage group/chromosome correlations may extend to other species for which chromosome maps and homologies have been worked out is discussed.