Age-grading the biting midge Culicoides sonorensis using near-infrared spectroscopy.

Age-grading of insects is important in the control and monitoring of both insect populations and vector-borne diseases. Microscopy and morphological techniques exist to age-grade most blood-feeding flies, but these techniques are laborious, often destructive to the insects, and slow. Near-infrared spectroscopy (NIRS) can be automated and is a non-destructive technique for age-grading. We applied NIRS techniques to age-grade females of the biting midge, Culicoides sonorensis Wirth & Jones (Diptera: Ceratopogonidae), the vector of bluetongue and other arboviruses in North America. Female flies of five known age cohorts (1, 3, 6, 9 and 12 days post-emergence) from three laboratory colonies were used. The data indicate that NIRS can be used to differentiate age groups of C. sonorensis.

Sex separation of tsetse fly pupae using near-infrared spectroscopy.

Implementation of the sterile insect technique for tsetse (Glossina spp.) requires that only sterile male insects be released; thus, at some stage of the fly production process the females have to be removed. A further constraint in the use of the sterile insect technique for tsetse is that the females are needed for colony production and hence, a non-destructive method of sex separation is required. In most tsetse sterile insect technique programmes thus far, females have been eliminated from the released material by hand-separation of chilled adults. Using near-infrared (NIR) spectroscopy, significant differences have been found between the spectra for the pupae of male and female G. pallidipes Austen. Significantly, the differences appear to be maximized 4-5 days before emergence of the adults. Tsetse fly pupae up to five days before emergence can be sexed with accuracies that generally range from 80 to 100%. This system, when refined, will enable effective separation of male and female pupae to be carried out, with emerged females being returned to the colony and males being irradiated and released. If separation can be achieved five days before emergence, this will also enable irradiated male pupae to be shipped to other destinations as required. Other Diptera were evaluated using this system but had lower classification accuracies of 50-74%. This may be due to the difference in reproductive physiology between these different fly groups.

Repellents: past, present, and future.

The use of repellents in protecting people against vector-borne diseases is predicated on the assertion that reducing human/vector contact will reduce the incidence of disease. The methods that have been used in developing countries have been simple to apply and relatively cheap. This article will discuss the use of repellents for protection against vector-borne disease in Southeast Asia and the Southwest Pacific region.

In vitro and in vivo evaluation of albumin-encapsulated primaquine diphosphate prepared by nebulization into heated oil.

Nebulization of an aqueous mixture of primaquine diphosphate and albumin into heated vegetable oil produces spherical particles with an average size of 6 microm. The microparticles are relatively stabile in buffers of pH 7.2 and 4.5 and completely degrade when exposed to proteolytic enzymes such as trypsin. Pharmacokinetic evaluation of the albumin-encapsulated primaquine diphosphate shows significantly higher levels in mouse liver tissue relative to free drug 2-48 h post-IP administration. Higher AUC (2.8x), lower steady-state volume of distribution (10x) and slower half-life (2.5x) relative to an equivalent dose of free primaquine diphosphate suggest liver targeting and sustained release of the drug from the microparticles.

Quantification of Plasmodium malariae infection in mosquito vectors.

Plasmodium malariae occurs in various tropical regions throughout the world and causes low, yet significant, levels of morbidity in human populations. One means of studying the ecology and frequency of this parasite is by measuring sporozoite loads in the salivary glands of infected mosquitoes. An effective, species-specific test that can be used to detect the presence of sporozoites in mosquitoes is the circumsporozoite ELISA. The aim of the present study was to standardize the circumsporozoite ELISA for P.malariae, by setting quantification parameters using, as antigen, either a synthetic peptide or extracts of whole sporozoites. The standard quantification curves produced indicated that the assay had a lower threshold of sensitivity of 250 sporozoites in a 50-microl sample, equivalent to about 1250 sporozoites in a mosquito.

Quantitative determination of sporozoites and circumsporozoite antigen in mosquitoes infected with Plasmodium falciparum or P. vivax.

It is essential for malariologists and researchers to have simple and accurate means of assessing the threat of Plasmodium parasites. An attempt was therefore made to re-standardize one of the circumsporozoite (CS) ELISA that can be used to detect and quantify the circumsporozoite antigens of P. falciparum and P. vivax. A two-site, 'sandwich' ELISA based on a monoclonal antibody was used to test for the CS antigen and sporozoites of each Plasmodium species simultaneously. Using the resultant optical-density values, standard curves, that permit the number of sporozoites in an infected mosquito to be estimated from the quantification of the CS antigen, were constructed. Using these plots and the CS ELISA, the presence of just 12.5 sporozoites (i.e. 0.8 pg CS antigen) of P. falciparum, four sporozoites (3.2 pg antigen) of P. vivax-210 or 12.5 sporozoites (32.0 pg antigen) of P. vivax-247 could be demonstrated.

Field evaluation of a lethal ovitrap against dengue vectors in Brazil.

Field evaluation of a "lethal ovitrap" (LO) to control dengue vector Aedes mosquitoes (Diptera: Culicidae), was undertaken in two Brazilian municipalities, Areia Branca and Nilopolis, in the State of Rio de Janeiro. The LO is designed to kill Aedes via an insecticide-treated ovistrip (impregnated with deltamethrin). In each municipality, the intervention was applied to a group of 30 houses (10 LOs/house) and compared to 30 houses without LOs in the same neighbourhood. Five LOs were put outside and five LOs inside each treated house. Three methods of monitoring Aedes density were employed: (i) percentage of containers positive for larvae and/or pupae; (ii) total pupae/house; (iii) total adult females/house collected by aspirator indoors. Weekly mosquito surveys began during the month before LO placement, by sampling from different groups of 10 houses/week for 3 weeks pre-intervention (i.e. 30 houses/month) and for 3 months post-intervention in both treated and untreated areas. Prior to LO placement at the end of February 2001, Aedes aegypti (L) densities were similar among houses scheduled for LO treatment and comparison (untreated control) at each municipality. Very few Ae. albopictus (Skuse) were found and this species was excluded from the assessment. Post-intervention densities of Ae. aegypti were significantly reduced for most comparators (P < 0.01), as shown by fewer positive containers (4-5 vs. 10-18) and pupae/house (0.3-0.7 vs. 8-10) at LO-treated vs. untreated houses, 3 months post-treatment at both municipalities. Numbers of adult Ae. aegypti females indoors were consistently reduced in LO-treated houses at Areia Branca (3.6 vs. 6.8/house 3 months post-intervention) but not at Niloplis (approximately 3/house, attributed to immigration). These results demonstrate sustained impact of LOs on dengue vector population densities in housing conditions of Brazilian municipalities.

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.

Authentication of artemether, artesunate and dihydroartemisinin antimalarial tablets using a simple colorimetric method.

The recent and widespread appearance of counterfeit antimalarial tablets in South-east Asia prompted the search for simple field assays to identify genuine drugs. In a recently described colorimetric assay for artesunate, Fast red TR salt reacted with an alkali-decomposition product of artesunate to produce a distinct yellow colour. However, that assay is specific for artesunate and it cannot be used to test for artemether. Because of potential concerns over artemether tablet counterfeiting, the colorimetric assay was modified to detect artemether, dihydroartemisinin and artesunate tablets. Other common antimalarials (artemisinin, chloroquine diphosphate, mefloquine HCl, sulphadoxine and pyrimethamine), as well as aspirin and acetaminophen, were negative in the assay, indicating its specificity for artemether, dihydroartemisinin and artesunate. The colorimetric method can be used to obtain a rapid visual assessment of tablet authenticity. The method can also be used to quantify the drug content of tablets, when used in conjunction with a spectrophotometer.

Dipsticks for rapid detection of plasmodium in vectoring anopheles mosquitoes.

Malaria remains the most serious vector-borne disease, affecting some 300-500 million people annually, transmitted by many species of Anopheles mosquitoes (Diptera: Culicidae). Monoclonal antibodies developed against specific circumsporozoite (CS) proteins of the main malaria parasites Plasmodium falciparum and P. vivax have been used previously for enzyme-linked immunosorbent assays (ELISA), widely employed for detection of malaria sporozoites in vector Anopheles for local risk assessment, epidemiological studies and targeting vector control. However, ELISA procedures are relatively slow and impractical for field use. To circumvent this, we developed rapid wicking assays that identify the presence or absence of specific peptide epitopes of CS protein of the most important P. falciparum and two strains (variants 210 and 247) of the more widespread P. vivax. The resulting assay is a rapid, one-step procedure using a 'dipstick' wicking test strip. In laboratory assessment, dipsticks identified 1 ng/ mL of any of these three CS protein antigens, with sensitivity nearly equal to the CS standard ELISA. We have developed and are evaluating a combined panel assay that will be both qualitative and quantitative. This quick and easy dipstick test (VecTest Malaria) offers practical advantages for field workers needing to make rapid surveys of malaria vectors.

Anti-mosquito midgut antibodies block development of Plasmodium falciparum and Plasmodium vivax in multiple species of Anopheles mosquitoes and reduce vector fecundity and survivorship.

The mosquito midgut plays a central role in the sporogonic development of malaria parasites. We have found that polyclonal sera, produced against mosquito midguts, blocked the passage of Plasmodium falciparum ookinetes across the midgut, leading to a significant reduction of infections in mosquitoes. Anti-midgut mAbs were produced that display broad-spectrum activity, blocking parasite development of both P. falciparum and Plasmodium vivax parasites in five different species of mosquitoes. In addition to their parasite transmission-blocking activity, these mAbs also reduced mosquito survivorship and fecundity. These results reveal that mosquito midgut-based antibodies have the potential to reduce malaria transmission in a synergistic manner by lowering both vector competence, through transmission-blocking effects on parasite development, and vector abundance, by decreasing mosquito survivorship and egg laying capacity. Because the intervention can block transmission of different malaria parasite species in various species of mosquitoes, vaccines against such midgut receptors may block malaria transmission worldwide.

Geographic subdivision of the range of the malaria parasite Plasmodium vivax.

We examined geographically distinct isolates of Plasmodium vivax and categorized them according to developmental success in Anopheles albimanus. We found that parasites from Central America and Colombia form a group distinct from those of Asia. New World isolates have a distinct chromosomal translocation and an episomal variation in the open reading frame (ORF) 470 DNA sequence that distinguishes them from the other isolates tested. Old World types of P. vivax were introduced into the Americas, and a remnant of this lineage remains in P. simium. It is indistinguishable from Old World P. vivax to the extent determinable by using our encoded markers and the examination of its developmental pattern in mosquitoes. The cohesive characteristics that separate types of P. vivax are predictors of range and potential for transmission and hence require taxonomic distinction.

Malaria vectors in the municipality of Serra do Navio, State of Amapá, Amazon Region, Brazil

We conducted a survey to determine the vectors of malaria in six localities of Serra do Navio municipality, State of Amapá, from 1990 to 1991. Malaria infection rates of 29.3 percent, 6.2 percent and 20.4 percent were detected by human blood smears in Colônia ægua Branca, Porto Terezinha and Arrependido, respectively. There was no malaria infection detected in Serra do Navio. Fifteen species were identified among 3,053 anopheline mosquitoes collected by human bait and 64.4 percent were identified as Anopheles albitarsis s.l., 16.7 percent An. braziliensis, 9.5 percent An. nuneztovari and 5.8 percent An. triannulatus. An. darlingi, the main vector of malaria in the Amazon region of Brazil, was scare. Using enzyme-linked immunosorbent assay (ELISA), a total positive rate of 0.8 percent (23/2876) was found for six species: fifteen An. albitarsis s.l., four An. nuneztovari, and one of each: An. braziliensis, An. triannulatus, An. oswaldoi and An. rangeli. Nine of 23 positive mosquitoes were infected with Plasmodium malariae, eight with P. vivax VK210, three with P. vivax VK247 and three with P. falciparum. Since An. albitarsis s.l. was collected feeding on humans, was present in the highest density and was positive by ELISA for malaria sporozoites, it probably plays an important role in malaria transmission in this area

Evaluation of the efficacy of lambda-cyhalothrin applied as ultra-low volume and thermal fog for emergency control of Aedes aegypti in Honduras.

An extended-duration formulation of lambda-cyhalothrin (Demand CS) applied as either an ultra-low volume (ULV) or thermal fog spray from a new handheld sprayer (Terrier) against Aedes aegypti was evaluated in Honduras. Spray applications were made at the front door for 1 min or to each room for 15 sec, both for the ULV and thermal fog applications to houses in separate blocks for each treatment. The efficacy and duration of effectiveness of the spray was determined from sentinel caged mosquito mortality and collection of mosquitoes within houses with a backpack power aspirator. Sentinel caged mosquito mortality in both open and sequestered locations was 97-100% for all spray treatments, with control mortality less than 2%. Both ULV applications (front door and each room) provided 4 wk of significant control (P < 0.01) based on adult Ae. aegypti house collections.

Light, carbon dioxide, and octenol-baited mosquito trap and host-seeking activity evaluations for mosquitoes in a malarious area of the Republic of Korea.

Two field trials for commercially available and experimental mosquito traps variously baited with light, carbon dioxide, octenol, or combinations of these were evaluated in a malarious area at Paekyeon-Ri near Tongil-Chon (village) and Camp Greaves, Paju County, Kyonggi Province, Republic of Korea. The host-seeking activity for common mosquito species was determined using hourly aspirator collections from a human- and propane lantern-baited Shannon trap. The total number of mosquitoes and number of each species captured during the test were compared using 8 x 8 and 5 x 5 Latin square designs based on trap location. Significant differences were observed for the total number of mosquitoes collected in the 8 x 8 test, such that counterflow geometry (CFG) with CO2 > or = CFG with CO2 and octenol > or = Shannon trap > or = Mosquito Magnet with octenol > American Biophysics Corporation (ABC) light trap with light, CO2 (500 ml/min), and octenol > or = ABC light trap with light and dry ice > or = ABC light trap with light and CO2 > ABC light trap with light only. A concurrent 5 x 5 test found significant differences in trap catch, where Mosquito Magnet with octenol > New Jersey light trap > or = EPAR Mosquito Killer with CO2 > or = ABC light trap with light and dry ice > Centers for Disease Control (CDC) light trap (manufactured by John W. Hock) with light and octenol. Significant differences in trap catch were noted for several species including: Aedes vexans, Anopheles sinensis, An. yatsushiroensis, An. lesteri, Culex pipiens, and Cx. orientalis. Traps baited with octenol captured significantly fewer Cx. pipiens than those not baited with octenol. Likewise, no Cx. orientalis were captured in octenol-baited traps. Host-seeking activity showed a similar bimodal pattern for all species captured. Results from these field trap evaluations can significantly enhance surveillance efforts. Significantly greater numbers of mosquitoes were captured with mosquito traps using counterflow technology (e.g., Mosquito Magnet and CFG traps) when compared to standard light and carbon dioxide-baited traps. Additionally, field evaluations demonstrate that various traps can be utilized for isolation and detection of arboviruses and other pathogens.

A colorimetric field method to assess the authenticity of drugs sold as the antimalarial artesunate.

Artesunate is the most widely used of the artemisinin derivatives. These drugs are being used increasingly throughout the tropical world, and are an essential component of the treatment of multi-drug resistant malaria. The recent and widespread appearance of counterfeit artesunate tablets in several countries in Southeast Asia poses a serious threat to health in this region. We have developed a simple, inexpensive colorimetric test to determine artesunate authenticity in tablets. The test is based on a reaction between an alkali decomposition product of artesunate and a diazonium salt, fast red TR (FRTR). The appearance of a yellow color indicates the presence of artesunate. The specificity of the test is dependent on the pH of the reaction. Among other antimalarials tested, (i.e. artemisinin, artemether, chloroquine, quinine, primaquine, sulfadoxine, and pyrimethamine) only artesunate produced a positive color reaction at pH 4. The assay requires only 1% of the total weight of a standard tablet containing 50 mg of artesunate and can be completed within 10 min. The method was tested on six genuine artesunate tablets and six counterfeit artesunate tablets obtained in Southeast Asia. The average amount of artesunate in the genuine tablets was determined to be 50.8 +/- 2.9 mg while the counterfeit tablets were found to contain no artesunate.

Field evaluation of deet and a piperidine repellent against Aedes communis (Diptera: Culicidae) and Simulium venustum (Diptera: Simuliidae) in the Adirondack Mountains of New York.

Repellent efficacy of N,N-diethyl-3-methyl-benzamide (deet), the piperidine, 1-[3-cyclohexen-1-ylcarbonyl]-2-methylpiperidine (AI3-37220), and a 1:1 ratio of deet + AI3-37220 were evaluated topically (0.25 mg/cm2 applied in ethanol solution) on human volunteers against the mosquito Aedes communis (DeGeer) and the black fly Simulium venustum Say. The average repellency of all three formulations was > 95% at 4 h. For both mosquitoes and black flies, deet alone provided < 90% protection at 6 h, whereas AI3-37220 provided > 95% protection. Although repellent treatments were not significantly different overall, the contrasts between AI3-3720 versus deet were significant at 6 and 8 h. The 95% confidence interval on percent repellency at 6 h ranged from 90.1 to 98.9% for AI3-37220 versus 64.3 to 82.2% for deet, and at 8 h ranged 76.1 to 88.5% for AI3-37220 versus 47.8 to 64.0% for deet. Similarly, the confidence interval for protection against black flies at 6 h by (AI3-37220 ranged from 86.3 to 99.5% and did not overlap with the confidence interval provided by deet alone (51.2 to 78.8%). There was no evidence of synergistic repellency from a combination of the two compounds; i.e., protection from combined compounds was no better than either repellent used alone.

Mosquito collections following local transmission of Plasmodium falciparum malaria in Westmoreland County, Virginia.

A 63-year-old woman from Colonial Beach, Westmoreland County, VA, was diagnosed with Plasmodium falciparum malaria on July 19, 1998. The woman had no history of international travel, intravenous drug use, blood transfusion, or other risk factor for contracting the disease. She seldom left the county and generally spent her evenings indoors, leading to the conclusion that she had been bitten locally by an infected mosquito. Colonial Beach is host to a population of migrant agricultural laborers from areas in which malaria occurs, but a blood survey of 89 Haitians and Mexicans failed to find Plasmodium parasites, specific antibodies, or clinical cases of malaria. Mosquito surveys were conducted during 2 days (July 22 and 28, 1998) with carbon-dioxide-baited light traps, larval and pupal collections, and landing collections. Thirteen species of mosquitoes were identified morphologically, including 4 potential vectors: Anopheles crucians, An. punctipennis, An. smaragdinus (new state record), and An. quadrimaculatus s.s. (new state record). Identifications of the latter 2 species were confirmed by sequencing of the ITS2 DNA region from adults reared from locally collected larvae. Anopheles smaragdinus was the most common biting species among the potential vectors, although An. crucians was the most abundant in other kinds of collections. In addition, Ae. albopictus was collected in Westmoreland County for the 1st time.

Development and evaluation of a dipstick assay for detection of Plasmodium falciparum and P. vivax sporozoites in mosquitoes (Diptera: Culicidae).

We developed a nitrocellulose-based, dipstick circumsporozoite (CS)-enzyme immunoassay [ELISA] for the simultaneous detection of Plasmodium falciparum and P. vivax-210 CS protein. The assay had a detection threshold of < 250 P. falciparum or 400 P. vivax sporozoites per sample, gave results concordant with dissection of salivary glands and CS-ELISA, but was slightly less sensitive than the CS-ELISA in microtiter plates. The assay consistently detected one infected mosquito in a pool of 10 or 20 mosquitoes, and was 100% specific in discriminating between species of Plasmodium when mosquito suspensions were spiked with sporozoites. The assay could be completed in 1 h, required no specialized equipment, and therefore was useful for field applications.

Different prevalences of Plasmodium vivax phenotypes VK210 and VK247 associated with the distribution of Anopheles albimanus and Anopheles pseudopunctipennis in Mexico.

The geographic distribution of Plasmodium vivax circumsporozoite protein phenotypes from patient blood used to infect colonized Anopheles albimanus and An. pseudopunctipennis was investigated in southern Mexico. Parasite phenotype types were determined in blood samples by a polymerase chain reaction and oligoprobe hybridization or by immunofluorescent assay of sporozoites. The proportion of infected mosquitoes and the number of oocysts per mosquito confirmed previous in vitro observations indicating that An. albimanus is more susceptible to VK210 and that An. pseudopunctipennis is more susceptible to VK247. All patients living on the coast were infected with VK210 and most patients living above 170 meters above sea level had VK247. Both phenotypes infected patients from intermediate altitudes. These results concur with the distribution of the anophelines, indicating that An. albimanus is the main vector of the phenotype VK210, but that An. pseudopunctipennis transmits both phenotypes. These conditions have direct implications on parasite transmission rates and malaria epidemiology in Mexico.