Attrition, physical integrity and insecticidal activity of long-lasting insecticidal nets in sub-Saharan Africa and modelling of their impact on vectorial capacity.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the primary malaria prevention and control intervention in many parts of sub-Saharan Africa. While LLINs are expected to last at least 3 years under normal use conditions, they can lose effectiveness because they fall out of use, are discarded, repurposed, physically damaged, or lose insecticidal activity. The contributions of these different interrelated factors to durability of nets and their protection against malaria have been unclear. METHODS: Starting in 2009, LLIN durability studies were conducted in seven countries in Africa over 5 years. WHO-recommended measures of attrition, LLIN use, insecticidal activity, and physical integrity were recorded for eight different net brands. These data were combined with analyses of experimental hut data on feeding inhibition and killing effects of LLINs on both susceptible and pyrethroid resistant malaria vectors to estimate the protection against malaria transmission-in terms of vectorial capacity (VC)-provided by each net cohort over time. Impact on VC was then compared in hypothetical scenarios where one durability outcome measure was set at the best possible level while keeping the others at the observed levels. RESULTS: There was more variability in decay of protection over time by country than by net brand for three measures of durability (ratios of variance components 4.6, 4.4, and 1.8 times for LLIN survival, use, and integrity, respectively). In some countries, LLIN attrition was slow, but use declined rapidly. Non-use of LLINs generally had more effect on LLIN impact on VC than did attrition, hole formation, or insecticide loss. CONCLUSIONS: There is much more variation in LLIN durability among countries than among net brands. Low levels of use may have a larger impact on effectiveness than does variation in attrition or LLIN degradation. The estimated entomological effects of chemical decay are relatively small, with physical decay probably more important as a driver of attrition and non-use than as a direct cause of loss of effect. Efforts to maximize LLIN impact in operational settings should focus on increasing LLIN usage, including through improvements in LLIN physical integrity. Further research is needed to understand household decisions related to LLIN use, including the influence of net durability and the presence of other nets in the household.

Evaluation of the long-term efficacy of K-Othrine® PolyZone on three surfaces against laboratory reared Anopheles gambiae in semi-field conditions.

BACKGROUND: In this semi-field study, a new polymer-enhanced deltamethrin formulation, K-Othrine® PolyZone, was compared to a standard deltamethrin product for residual activity against a susceptible strain of laboratory-reared Anopheles gambiae using standard WHO cone bioassays. METHODS: Residual insecticide efficacy was recorded after exposure to metal, cement and wood panels maintained in experimental huts in sub-tropical environmental conditions in north central Florida, USA, and panels stored in a climate controlled chamber located at the Centers for Disease Control and Prevention, Georgia, USA. CONCLUSIONS: K-Othrine® PolyZone demonstrated 100% control on metal and cement panels 1 year post application and > 80% control on wood panels up to 6 mo. The new formulation should be considered for use in indoor residual spray programmes requiring long-term control of malaria vectors.

Insecticide resistance in Anopheles arabiensis from Ethiopia (2012-2016): a nationwide study for insecticide resistance monitoring.

BACKGROUND: Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) remain the cornerstones of malaria vector control. However, the development of insecticide resistance and its implications for operational failure of preventative strategies are of concern. The aim of this study was to characterize insecticide resistance among Anopheles arabiensis populations in Ethiopia and describe temporal and spatial patterns of resistance between 2012 and 2016. METHODS: Between 2012 and 2016, resistance status of An. arabiensis was assessed annually during the long rainy seasons in study sites from seven of the nine regions in Ethiopia. Insecticide resistance levels were measured with WHO susceptibility tests and CDC bottle bioassays using insecticides from four chemical classes (organochlorines, pyrethroids, organophosphates and carbamates), with minor variations in insecticides tested and assays conducted between years. In selected sites, CDC synergist assays were performed by pre-exposing mosquitoes to piperonyl butoxide (PBO). In 2015 and 2016, mosquitoes from DDT and deltamethrin bioassays were randomly selected, identified to species-level and screened for knockdown resistance (kdr) by PCR. RESULTS: Intense resistance to DDT and pyrethroids was pervasive across Ethiopia, consistent with historic use of DDT for IRS and concomitant increases in insecticide-treated net coverage over the last 15 years. Longitudinal resistance trends to malathion, bendiocarb, propoxur and pirimiphos-methyl corresponded to shifts in the national insecticide policy. By 2016, resistance to the latter two insecticides had emerged, with the potential to jeopardize future long-term effectiveness of vector control activities in these areas. Between 2015 and 2016, the West African (L1014F) kdr allele was detected in 74.1% (n = 686/926) of specimens, with frequencies ranging from 31 to 100% and 33 to 100% in survivors from DDT and deltamethrin bioassays, respectively. Restoration of mosquito susceptibility, following pre-exposure to PBO, along with a lack of association between kdr allele frequency and An. arabiensis mortality rate, both indicate metabolic and target-site mutation mechanisms are contributing to insecticide resistance. CONCLUSIONS: Data generated by this study will strengthen the National Malaria Control Programme's insecticide resistance management strategy to safeguard continued efficacy of IRS and other malaria control methods in Ethiopia.

A Comparison of Carbon Dioxide Sources for Mosquito Capture in Centers for Disease Control and Prevention Light Traps on the Florida Gulf Coast (1).

Traditional sources of carbon dioxide (CO2), dry ice, and compressed gas, were tested against 3 combinations of food-grade reagents known to generate CO2using a compact, lightweight generator delivery system with Centers for Disease Control and Prevention light traps. Three 6 × 6 Latin square trials were completed near the Florida Gulf Coast in the Lower Suwannee Wildlife Refuge during the summer of 2013, collecting a total of 31,632 female mosquitoes. Treatments included dry ice, compressed CO2gas, a control trap (no CO2), citric acid + sodium bicarbonate, vinegar + sodium bicarbonate, and yeast + sugar. Decreasing order of trap collections (treatment mean number of mosquitoes per trap night ± standard error) were dry ice 773.5 (± 110.1) > compressed gas 440.7 (± 42.3) > citric acid + sodium bicarbonate 197.6 (± 30.4), yeast + sugar 153.6 (± 27.4) > vinegar + sodium bicarbonate 109.6 (± 16.2) > control 82.4 (± 14.0). A 2-way Kruskal-Wallis analysis by treatment, site, and treatment × site interaction identified significant differences between all treatments. Although dry ice and compressed CO2gas collected significantly more mosquitoes than other combinations (P < 0.05), use of citric acid and sodium bicarbonate or yeast and sugar greatly outperformed unbaited traps and offer a good alternative to dry ice and compressed gas in areas where these agents are not readily available or are difficult to obtain due to logistical constraints. An inexpensive, portable CO2generator for use with food-grade reagents is described.

Optimizing Collection of Anopheles gambiae s.s. (Diptera: Culicidae) in Biogents Sentinel Traps.

Surveillance of malaria vectors in Africa is most often accomplished using CDC-type light traps or human landing catches (HLCs). Over the past 30 yr, a variety of commercial and experimental mosquito traps have been developed for residential mosquito control or for improved surveillance of disease vector species, including the BG Sentinel (BGS) trap. To optimize collection of Anopheles gambiae Giles using this trap, BGS traps were modified with an opening (vent) added to the trap base to decrease exhaust airflow. Four traps configurations were tested with colony-reared host-seeking female An. gambiae in free-flying laboratory enclosures. Six attractant treatments (three attractants: BG-Lure, Limburger cheese, and a blank, with and without CO2) were tested concurrently. Across all trap-attractant combinations, significantly more mosquitoes (P < 0.05) were collected in standard, unmodified traps set upright (mean: 10.0) or upside down (9.8) than vented traps, whether upright (5.9) or upside down (7.0). The BG-Lure + CO2 and Limburger Cheese + CO2 bait combinations were more attractive than the other attractants tested alone. All attractant combinations collected significantly more mosquitoes than unbaited traps. Field studies are needed to determine if BG-Lure + CO2- or Limburger cheese + CO2-baited BGS traps are comparable with HLCs in collecting host-seeking An. gambiae.

SR450 And Superhawk XP Applications Of Bacillus thuringiensis israelensis Against Culex quinquefasciatus.

Sprayer comparisons and larval morality assays were conducted following SR450 backpack mist blower and Superhawk XP thermal fogger applications of Vectobac® WDG Bacillus thuringiensis israelensis (Bti) against Culex quinquefasciatus. Bacillus thuringiensis israelensis was applied at maximum label rate in a 232.26-m(2) field plot located in north-central Florida with containers placed at 2 heights (ground level and 1.52 m above ground) on stakes positioned 3.04, 6.09, 9.14, 12.19, and 15.24 m from the spray line. Results indicated that there was no significant (P > 0.05) difference in 24- and 48-h larval mortality between the 2 sprayers or between the 2 heights. There was significant difference (P < 0.05) among the 5 rows, with mortality continuously decreasing with increasing distance from sprayer. Both sprayers provided on average >70% larval mortality 3.04-9.14 m from the spray line, and <60% mortality at 12.19 and 15.24 m. The data suggest that the SR450 and Superhawk XP may be comparable sprayers for use with Bti to control mosquito larvae.

Evaluating RNAlater® as a preservative for using near-infrared spectroscopy to predict Anopheles gambiae age and species.

BACKGROUND: Mosquito age and species identification is a crucial determinant of the efficacy of vector control programmes. Near-infrared spectroscopy (NIRS) has previously been applied successfully to rapidly, non-destructively, and simultaneously determine the age and species of freshly anesthetized African malaria vectors from the Anopheles gambiae s.l. species complex: An. gambiae s. s. and Anopheles arabiensis. However, this has only been achieved on freshly-collected specimens and future applications will require samples to be preserved between field collections and scanning by NIRS. In this study, a sample preservation method (RNAlater(®)) was evaluated for mosquito age and species identification by NIRS against scans of fresh samples. METHODS: Two strains of An. gambiae s.s. (CDC and G3) and two strains of An. arabiensis (Dongola, KGB) were reared in the laboratory while the third strain of An. arabiensis (Ifakara) was reared in a semi-field system. All mosquitoes were scanned when fresh and rescanned after preservation in RNAlater(®) for several weeks. Age and species identification was determined using a cross-validation. RESULTS: The mean accuracy obtained for predicting the age of young (<7 days) or old (≥ 7 days) of all fresh (n = 633) and all preserved (n = 691) mosquito samples using the cross-validation technique was 83% and 90%, respectively. For species identification, accuracies were 82% for fresh against 80% for RNAlater(®) preserved. For both analyses, preserving mosquitoes in RNAlater(®) was associated with a highly significant reduction in the likelihood of a misclassification of mosquitoes as young or old using NIRS. Important to note is that the costs for preserving mosquito specimens with RNAlater(®) ranges from 3-13 cents per insect depending on the size of the tube used and the number of specimens pooled in one tube. CONCLUSION: RNAlater(®) can be used to preserve mosquitoes for subsequent scanning and analysis by NIRS to determine their age and species with minimal costs and with accuracy similar to that achieved from fresh insects. Cold storage availability allows samples to be stored longer than a week after field collection. Further study to develop robust calibrations applicable to other strains from diverse ecological settings is recommended.

Detection of broadly distributed sodium channel alleles characteristic of insect pyrethroid resistance in West Nile virus vector Culex pipiens complex mosquitoes in the United States.

West Nile virus (WNV) has emerged as a health threat to the North American population since its initial outbreak in New York City in 1999. Culex (Culex) pipiens complex mosquitoes have been considered to play the primary role in the enzootic maintenance and transmission of WNV in North America. The voltage-gated sodium channel (NaCh) gene contains pyrethroid resistance-associated mutations in the coding region in many insect species. However, the knowledge of potential NaCh mutations was minimal in Culex. Seeking pyrethroid resistance alleles in Culex, we evaluated a transect along the east coast of the United States with an NaCh-based genotyping tool that amplified a portion of the transcribed sequence containing kdr mutations and the intron immediately downstream of the mutation site. Three genotypes that are typically associated with pyrethroid resistance in insects have been identified in Culex pipiens complex mosquitoes in this study: susceptible wild type kds, the classical knock-down resistance Leu --> Phe mutation (Phe/kdr), and a second resistance mechanism, a Leu --> Ser mutation (Ser/kdr). Moreover, we observed heterozygotic individual mosquitoes possessing both kdr alleles. Results of this study advance our knowledge of the potential for pyrethroid insecticide resistance among the populations of Cx. pipiens complex in the United States.

Use of vector diagnostics during military deployments: recent experience in Iraq and Afghanistan.

Vector-borne diseases such as malaria, dengue, and leishmaniasis are a threat to military forces deployed outside of the United States. The availability of specific information on the vector-borne disease threat (e.g., presence or absence of a specific disease agent, temporal and geographic distribution of competent vectors, and vector infection rates) allows for effective implementation of appropriate measures to protect our deployed military forces. Vector diagnostics can provide critical, real-time information crucial to establishing effective vector prevention/control programs. In this article we provide an overview of current vector diagnostic capabilities, evaluate the use of vector diagnostics in Operation Enduring Freedom and Operation Iraqi Freedom, and discuss the concept of operations under which vector diagnostics are employed.

Determination of oseltamivir quality by colorimetric and liquid chromatographic methods.

We developed a colorimetric and chromatographic assay for oseltamivir to assess the authenticity of Tamiflu (F. Hoffmann-La Roche Ltd., Basel, Switzerland) because of a growing concern about counterfeit oseltamivir. The colorimetric assay is quantitative and relies on an extractable colored ion-pair complex of oseltamivir with Congo red or bromochlorophenol blue. The reverse-phase chromatographic assay uses an alkaline mobile phase with UV detection. Both methods were evaluated for variability and selectivity and subsequently applied to batches of oseltamivir products acquired through the Internet. The Congo red test showed greater assay sensitivity, linearity, and accuracy. Colorimetric and chromatographic analysis showed all batches of oseltamivir product were within +/-15% of the stated amount of active ingredient.

Identification of blood meal source and infection with Trypanosoma cruzi of Chagas disease vectors using a multiplex cytochrome b polymerase chain reaction assay.

Long-term control of triatomine bugs in Chagas endemic regions will depend on a full understanding of vector-parasite-host interactions. Herein we describe a cytochrome b multiplex polymerase chain reaction (PCR)-based strategy for blood meal source identification in bug foregut contents. This technique discriminates human from animal blood, and has been tested in five Triatoma species from México. Host identification has been validated for human, four rodent species, two bat species, dog, rabbit, sheep, and opossum. In addition, Trypanosoma cruzi can be identified simultaneously using S34/S67-specific kinetoplast DNA primers. Both host and parasite identification were possible as long as 10 weeks after bug feeding, and in samples stored up to 6 years. The blood meal identification procedure described here represents a powerful tool for large-scale studies identifying the biological, ecological, and environmental variables associated with Chagas disease transmission.

Evidence for transmission of Plasmodium vivax among a duffy antigen negative population in Western Kenya.

We present evidence that a parasite with characteristics of Plasmodium vivax is being transmitted among Duffy blood group-negative inhabitants of Kenya. Thirty-two of 4,901 Anopheles gambiae and An. funestus (0.65%) collected in Nyanza Province were ELISA positive for the P. vivax circumsporozoite protein VK 247. All positives were found late in the rainy season, when An. funestus predominated, and disproportionately many were found at a single village. A P. vivax specific sequence of the SSU rRNA gene was amplified from three of six ELISA-positive mosquitoes. Erythrocytes from 31 children, including 9 microscopically diagnosed as infected with P. vivax, were negative by flow cytometry for the Fy3 or Fy6 epitopes, which indicate Duffy blood group expression. A DNA fragment specific for the C terminus of the gene for P. vivax merozoite surface protein 1 (MSP-1) was amplified from the blood of four of these children and subsequently sequenced from two.

Biting behavior and Plasmodium infection rates of Anopheles arabiensis from Sille, Ethiopia.

The man-biting behavior and Plasmodium infection rates of anopheline mosquitoes were investigated in Sille, a hyperendemic malarious area in southern Ethiopia. Seven Anopheles species were identified from all night landing collections, conducted from 18:00 to 06:00h between October 2001 and August 2002. The predominant species was Anopheles arabiensis (55.8%), followed by Anopheles coustani (31.5%), Anopheles pharoensis (9.5%), Anopheles funestus (2.2%), Anopheles nili (0.5%), Anopheles marshallii (0.4%) and Anopheles demeilloni (0.2%). Dissection of A. arabiensis showed an average parous rate of 73.2%. A large proportion of the parous mosquitoes were caught biting in the latter part of the night. Malaria sporozoite rates were determined by ELISA for A. arabiensis, with 0.5% (4/796) infective with Plasmodium falciparum and 1.76% (14/796) with Plasmodium vivax; there were no mixed infections. From our small sample of sporozoite positives we found no association between biting behavior and sporozoite infection status.

Determination of Insecticidal Effect (LC50 and LC90) of Organic Fatty Acids Mixture (C8910+Silicone) Against Aedes aegypti and Aedes albopictus (Diptera: Culicidae).

Emerging and re-emerging vector-borne diseases such as chikungunya and dengue and associated Aedes vectors are expanding their historical ranges; thus, there is a need for the development of novel insecticides for use in vector control programs. The mosquito toxicity of a novel insecticide and repellent consisting of medium-chain carbon fatty acids (C8910) was examined. Determination of LC50 and LC90 was made against colony-reared Aedes aegypti (L.) and Aedes albopictus (Skuse) using probit analysis on mortality data generated by Centers for Disease Control and Prevention bottle bioassays. Six different concentrations of C8910 + silicone oil yielded an LC50 of 160.3 µg a.i/bottle (147.6-182.7) and LC90 of 282.8 (233.2-394.2) in Ae. aegypti; five concentrations yielded an LC50 of 125.4 (116.1-137.6) and LC90 of 192.5 (165.0-278.9) in Ae. albopictus. Further development of C8910 and similar compounds could provide vector control specialists novel insecticides for controlling insect disease vectors.

Near-Infrared Spectroscopy, a Rapid Method for Predicting the Age of Male and Female Wild-Type and Wolbachia Infected Aedes aegypti.

Estimating the age distribution of mosquito populations is crucial for assessing their capacity to transmit disease and for evaluating the efficacy of available vector control programs. This study reports on the capacity of the near-infrared spectroscopy (NIRS) technique to rapidly predict the ages of the principal dengue and Zika vector, Aedes aegypti. The age of wild-type males and females, and males and females infected with wMel and wMelPop strains of Wolbachia pipientis were characterized using this method. Calibrations were developed using spectra collected from their heads and thoraces using partial least squares (PLS) regression. A highly significant correlation was found between the true and predicted ages of mosquitoes. The coefficients of determination for wild-type females and males across all age groups were R2 = 0.84 and 0.78, respectively. The coefficients of determination for the age of wMel and wMelPop infected females were 0.71 and 0.80, respectively (P< 0.001 in both instances). The age of wild-type female Ae. aegypti could be identified as < or ≥ 8 days old with an accuracy of 91% (N = 501), whereas female Ae. aegypti infected with wMel and wMelPop were differentiated into the two age groups with an accuracy of 83% (N = 284) and 78% (N = 229), respectively. Our results also indicate NIRS can distinguish between young and old male wild-type, wMel and wMelPop infected Ae. aegypti with accuracies of 87% (N = 253), 83% (N = 277) and 78% (N = 234), respectively. We have demonstrated the potential of NIRS as a predictor of the age of female and male wild-type and Wolbachia infected Ae. aegypti mosquitoes under laboratory conditions. After field validation, the tool has the potential to offer a cheap and rapid alternative for surveillance of dengue and Zika vector control programs.

Rapid and Non-destructive Detection and Identification of Two Strains of Wolbachia in Aedes aegypti by Near-Infrared Spectroscopy.

The release of Wolbachia infected mosquitoes is likely to form a key component of disease control strategies in the near future. We investigated the potential of using near-infrared spectroscopy (NIRS) to simultaneously detect and identify two strains of Wolbachia pipientis (wMelPop and wMel) in male and female laboratory-reared Aedes aegypti mosquitoes. Our aim is to find faster, cheaper alternatives for monitoring those releases than the molecular diagnostic techniques that are currently in use. Our findings indicate that NIRS can differentiate females and males infected with wMelPop from uninfected wild type samples with an accuracy of 96% (N = 299) and 87.5% (N = 377), respectively. Similarly, females and males infected with wMel were differentiated from uninfected wild type samples with accuracies of 92% (N = 352) and 89% (N = 444). NIRS could differentiate wMelPop and wMel transinfected females with an accuracy of 96.6% (N = 442) and males with an accuracy of 84.5% (N = 443). This non-destructive technique is faster than the standard polymerase chain reaction diagnostic techniques. After the purchase of a NIRS spectrometer, the technique requires little sample processing and does not consume any reagents.

Plasmodium-infected Anopheles mosquitoes collected in Virginia and Maryland following local transmission of Plasmodium vivax malaria in Loudoun County, Virginia.

Two recent outbreaks of locally acquired, mosquito-transmitted malaria in Virginia in 1998 and 2002 demonstrate the continued risk of endemic mosquito-transmitted malaria in heavily populated areas of the eastern United States. Increasing immigration, growth in global travel, and the presence of competent anopheline vectors throughout the eastern United States contribute to the increasing risk of malaria importation and transmission. On August 23 and 25, 2002, Plasmodium vivax malaria was diagnosed in 2 teenagers in Loudoun County, Virginia. The Centers for Disease Control and Prevention (CDC) deemed these cases to be locally acquired because of the lack of risk factors for malaria, such as international travel, blood transfusion, organ transplantation, or needle sharing. The patients lived approximately 0.5 mi apart; however, 1 patient reported numerous visits to friends who lived directly across the street from the other patient. Two Anopheles quadrimaculatus s.l. female pools collected in Loudoun County, Virginia, and 1 An. punctipennis female pool collected in Fairfax County, Virginia, tested positive for P. vivax 210 with the VecTest panel assay and enzyme-linked immunosorbent assay (ELISA). In addition, 2 An. quadrimaculatus s.l. female pools collected in Montgomery, Maryland, tested positive for P. vivax 210. The CDC confirmed these initial results with the circumsporozoite ELISA. The authors believe that this is the 1st demonstration of Plasmodium-infected mosquitoes collected in association with locally acquired human malaria in the United States since the current national malaria surveillance system began in 1957.

Optimizing malarial epidemiological studies in areas of low transmission.

Malaria risk factor studies have traditionally used microscopy readings of blood slides as the measure of malaria infection in humans, although alternatives are available. There is the need for an assessment of how the use of these alternative diagnostic approaches will influence the efficiency and significance of epidemiological studies. In an area of Sri Lanka with known risk factors for malaria, two cross-sectional surveys were done at the start and at the peak of transmission season. Microscopy was compared with enzyme-linked immunosorbent assays (ELISA) and polymerase chain reaction (PCR). The major risk factor in this area was the location of houses relative to confirmed vector breeding sites. At the peak of the transmission season, the results pointed in the same direction, irrespective of the diagnostic method used. However, the importance of distance from the breeding site was not statistically significant when microscopy was used, which can be explained by the lower prevalence of microscopy positivity in comparison to the prevalence of ELISA- and PCR-positivity. This study suggests that in low-transmission areas, such as Sri Lanka, smaller sample sizes can be used for epidemiological research studies using PCR instead of microscopy to estimate parasite prevalence. This efficiency gain has to be weighed against the higher cost and complexity of the PCR. PCR cannot replace microscopy as the standard diagnostic procedure at the field level. ELISA is not directly comparable with microscopy and PCR but it can also be a useful tool in malaria epidemiological studies. This study indicates that cross-sectional surveys are only efficient if they take place during peak transmission season. Cross sectional surveys currently implemented by the Sri Lankan government in response to local malaria outbreaks can form the basis for valid epidemiological studies and be used for the generation of malaria risk maps if samples were also analyzed using PCR.

Polymerase chain reaction-based identification and genotyping of Anopheles mosquitoes with a 96-pin bacterial replicator.

A simple method for rapid identification of large numbers of Anopheles mosquitoes was developed based on polymerase chain reaction (PCR) amplification of the rDNA intergenic spacer and internal transcribed spacer 2. By means of previously described primers for the Anopheles gambiae and An. quadrimaculatus species complexes, rDNA was amplified simultaneously from 96 whole mosquitoes or parts. No homogenization or individual DNA preparation was necessary, and transfer of 96 samples to PCR reactions was performed simultaneously with a bacterial replicator. Control reactions indicate that the level of cross-contamination is negligible, and false-negative findings are rare. The method was tested on larvae, pupae, adult heads, whole adult males and females, and single tarsi. All parts except tarsi provided satisfactory template. Fresh, ethanol-preserved, dried, and frozen adults were also tested with similar results. The method was also tested for amplification of a single-copy gene, white. Results were generally positive, although some false-negative findings were observed. This method allows rapid analysis of large numbers of mosquitoes without robotic equipment and should enable rapid and extensive PCR analysis of field-collected samples and laboratory specimens.

Emergence of a new neotropical malaria vector facilitated by human migration and changes in land use.

In a region of northeastern Amazonia, we find a species previously of minor importance, Anopheles marajoara, to be the principal malaria vector. In a total of five collections during 1996-97 in three replicated sites near the city of Macapá, Amapá state, this species occurs in much greater abundance compared with the presumed vector Anopheles darlingi. Also, a significantly higher proportion of An. marajoara is infected with malaria parasites, determined by the ELISA technique. This appears to be the result of increased abundance of An. marajoara due to alterations in land use, invasion of its primary breeding sites by human immigrants, and its anthropophilic behavior. This discovery highlights one of the challenges of Neotropical malaria control, namely that the targeting of specific vectors may be complicated by a changing mosaic of different locally important vectors and their interactions with human populations.