Response to: Bouwman, H. et al. Hallogenated pollutants in terrestrial and aquatic bird eggs: converging patterns of pollutant profiles, and impacts and risks from higher levels Environ. Res. (2013) http://dx.doi.org/10.1016/j.envres.2013.06.003.

Bouwman and coauthors present data and analyses of DDT and other halogenated pollutants in environmental samples and based on their data and analyses thereof, argue against the use of DDT for malaria control. Regrettably, the analyses, presentations, and interpretations of data presented by Bouwman and coauthors are biased and erroneous.

Malaria diagnosis and hospitalization trends, Brazil.

We focused on rates of malaria in the state of Amazonas and city of Manaus, Brazil. Plasmodium vivax accounted for an increased number and rate of hospital admissions, while P. falciparum cases decreased. Our observations on malaria epidemiology suggest that the increased hospitalization rate could be due to increased severity of P. vivax infections.

Molecular epidemiology of Plasmodium vivax in the State of Amazonas, Brazil.

Over the past 2 decades, the Amazon region of Brazil has experienced reemergence of Plasmodium vivax malaria, with reported occurrence of severe disease. The frequency and manifestations of this severe disease are unlike previous clinical experience. The hypothesis has been raised that the occurrence of severe disease may relate to the emergence of a variant form of the parasite. To test this hypothesis, we conducted a retrospective cohort study of P. vivax strains in the State of Amazonas. We determined nucleic acid sequences of segments of three genes, the 18S SSUrRNA Type A gene, the circumsporozoite surface protein (CSP) gene and the MSP-1 gene. Sequences were determined for parasites infecting 11 hospitalized (Inpatients) and 21 non-hospitalized (Outpatients) patients. We observed two common polymorphisms in the 18S SSUrRNA Type A gene; a thymidine (T)/adenine (A) polymorphism at residue 117 was significantly more common in the Inpatient group (p<0.05). Types of variation in the CSP gene included the numbers of repeat nonapeptide segments, alanine/aspartic acid polymorphism at position 5 of the nonapeptide repeat, and sporadic mutations. Alanine was more common as the fifth residue of the nonapeptide repeat in Inpatients and in strains causing second infections (both, p<0.05). Synonymous substitutions of the common repeat sequence occurred frequently in codons 1, 2, and 7, while the mutations at codon 5 were always non-synonymous, indicating that variation at codon 5 reflected selective pressure. Among MSP-1 gene sequences, recombination among progenitor strains, related to the Salvador I and Belém strains, was the main source of diversity. Phylogenetic analyses that incorporated sequence data for all three genes tested did not reveal clustering of sequences from inpatients. Our data do not affirm that the hypothesis that severe P. vivax disease in Amazonas is related to emergence of a new variant, but do suggest that variation in the fifth position of the CSP gene nonapeptide repeat may relate to disease manifestations.

A mark release-recapture study to define the flight behaviors of Anopheles vestitipennis and Anopheles albimanus in Belize, Central America.

The recapture rates of wild-caught, unengorged Anopheles vestitipennis and Anopheles albimanus females were determined at 0, 400, and 800 m from a fixed release point in Belize, Central America. Three sampling trials, each consisting of two 12-hour collections, were performed at each distance during September-October 2003. A total of 1,621 An. vestitipennis and 1,326 An. albimanus were marked and released during the course of the study. The recapture rate of An. vestitipennis was greatest at 0 m (7.9%; 44/ 556) and declined from 3.0% (16/531) at 400 m to 0.2% (1/534) at 800 m. Anopheles albimanus females were recaptured only at the 0-m distance and in extremely low numbers (1.1%; 5/446). Biting patterns for the unmarked natural populations were similar to those previously described for Belize, and recaptures for both species occurred during these normal biting times. The overall recapture rates for An. vestitipennis (3.76%; 61/ 1,621) and An. albimanus (0.38%; 5/1,326) indicate that An. vestitipennis has a higher probability of being attracted to a human habitation.

Inhibition of kinesin motor proteins by adociasulfate-2.

Kinesin motor proteins are involved in cell division and intracellular transport of vesicles and organelles, and as such, they play a role in neurological disease, cancer, and developmental disorders. Inhibitors of kinesin would be valuable as probes of cell physiology and as potential therapeutics. Adociasulfate-2 (AS-2) is the only known natural product inhibitor of kinesins, but its mechanism of action is unknown. We utilized kinetic studies, dynamic light scattering, and transmission electron microscopy to investigate the inhibitory action of AS-2. Our data suggest that AS-2 is not a classical 1:1 inhibitor. Instead, a rodlike aggregate that mimics microtubules is complexed with kinesin and inhibits its ATPase activity. An intriguing implication of this hypothesis is that aggregates of a chiral natural product can have interesting and biologically relevant properties. This mode of action might represent one way in which a small molecule can disrupt a protein-protein interaction.

Use of remote sensing and geographic information systems to predict locations of Anopheles darlingi-positive breeding sites within the Sibun River in Belize, Central America.

Previous studies have identified several anopheline species integral to the transmission of malaria in Belize. The highly efficient vector, Anopheles darlingi Root, is currently considered the most important. The preferred larval habitat of An. darlingi has been described as floating detritus patches, which are commonly associated with overhanging spiny bamboo, Guadua longifolia (E. Fourn.), along river margins. The objectives of this study were to use remote sensing and geographic information system (GIS) tools to 1) define the landscape features (i.e., river curvature, land cover, and house locations) associated with An. darlingi-positive breeding habitats and 2) determine the association between cleared land cover and the growth of spiny bamboo. A systematic survey was conducted in which all detritus patches of at least 1 m2 were sampled, mapped using GPS, and characterized by cause of habitat lodging. Bamboo stretches growing along the river margins also were mapped. Spatial analyses of satellite imagery found no associations between river characteristics or land cover with positive An. darlingi habitats. In addition, there was no significant difference in cleared versus forested land cover in relation to the presence or absence of bamboo. Results indicate that the average distance from homes to negative habitats was significantly greater than from positive detritus mats. Based on the land cover and river characteristics used, our results do not support the use of remote sensing as a predictive tool to locate specific areas within rivers positive for An. darlingi habitats.

A delayed release mechanism for mark-release-recapture studies.

A delayed release mechanism was designed for a mark-recapture study that evaluated the flight behavior of anopheline mosquitoes in Belize, Central America. The design prevents marked mosquitoes from being released until after a set time. The time lag allows researchers to return to collecting posts before release and reduces the potential of marked females tracking an odor plume generated during the release procedure. This is especially important in study designs evaluating temporal host-seeking behavior patterns. Detailed descriptions of the design are presented.

Experimental evaluation of overhanging bamboo in Anopheles darlingi larval habitat selection in Belize, Central America.

Previous studies in Belize have shown the preferred breeding habitats of the malaria vector Anopheles darlingi were floating detritus patches within riverine systems that were associated with overhanging bamboo. The present study focused on an experimental evaluation of overhanging bamboo in An. darlingi habitat selection using larval counts as an indicator of attractiveness. Four sets of 1-m2 floating screened enclosures were placed at a location with a documented presence of both larval and adult An. darlingi populations. Each enclosure set comprised a control (i.e., open water) and three other experimental treatments consisting of: 1) detritus, 2) detritus with overhanging bamboo, and 3) overhanging bamboo alone. Larvae were sampled from all treatments on Day 5, Day 11, and Day 17 post-setup. A total of 2,461 An. darlingi larvae were collected and identified from three trials conducted from March-May 2002. Of these, 1,997 larvae were sampled from detritus treatments, 256 from enclosures with bamboo and detritus, 139 from bamboo treatments, and 69 from control enclosures. The detritus treatment had a significantly higher average count of An. darlingi larvae than the other treatments (P<0.01), and no difference existed between the control treatment and the treatment containing overhanging bamboo alone (P=0.423). More importantly, enclosures containing the overhanging bamboo with detritus treatments did not have greater larval populations than the enclosures with only detritus. These data suggest that bamboo does not contribute to An. darlingi larval habitat attractiveness but may function as a barrier to surface water flow causing the lodging of debris, the preferred habitat, that will then attract gravid females for oviposition.

Biting patterns and seasonal densities of Anopheles mosquitoes in the Cayo District, Belize, Central America with emphasis on Anopheles darlingi.

The present study utilized an experimental hut to conduct human-baited landing collections for characterizing the all-night biting patterns and seasonal densities of adult Anopheles darlingi in the centrally located Cayo District of Belize, Central America. A total of 25 all-night collections (i.e., sunset to sunrise) were conducted from January 2002 to May 2003, capturing a total of 18,878 An. darlingi females. Anopheles darlingi exhibited a bimodal nightly biting pattern with one predominate peak occurring three h after sunset and a smaller peak occurring one h prior to sunrise. Biting females were collected throughout the night in higher densities indoors (9,611) than outside (9,267) the experimental hut (O:I=1.00:1.04). Seasonal adult collections show An. darlingi densities were highest during the transitional months between the end of the wet and beginning of the dry season (January) and the end of the dry season and beginning of the wet season (May). A total of 2,010 An. darlingi females was captured in 31 two-h, human-baited landing collections performed from January to October 2002. Anopheles darlingi monthly population densities were found to have no significant associations with high or low temperatures, precipitation, or river level. However, qualitative data examination indicates an inverse relationship between river level and An. darlingi adult collections suggesting a disturbance of larval habitats. All-night biting and seasonal distribution patterns for other anopheline species are also described. None of the adult specimens collected throughout the entire study tested positive for Plasmodium spp. infection using the VecTest rapid diagnostic kit.

Comparative susceptibility of three species of Anopheles from Belize, Central America, to Plasmodium falciparum (NF-54).

In August of 2000, a comparative susceptibility study was conducted using 3 species of Anopheles mosquitoes from Belize, Central America, and a standard species used in laboratory infection studies, Anopheles stephensi. Test populations were fed human blood infected with cultured Plasmodium falciparum (NF-54 strain) parasites via a membrane feeder. The control species, An. stephensi, exhibited the highest infections, with 73.8% of dissected specimens positive for sporozoites in the salivary glands. The control species also showed heavier sporozoite loads; 74.0% of positive glands having greater than 200 sporozoites. Of species from Belize, Anopheles darlingi was the most susceptibile, e.g., 41.0% of salivary glands were positive, with more than 200 sporozoites per gland. Anopheles vestitipennis had a low salivary gland infection rate (9.3%) and a moderate number of sporozoites in glands (i.e., 85.7% containing 50-250 sporozoites). Anopheles albimanus was the least susceptible species to infection. No specimens of An. albimanus from the Golden Stream population developed sporozoites in the salivary glands, yet 20.7% of dissected specimens had positive midgut infections. The An. albimanus Buena Vista population showed similar results with only a 2.2% salivary gland infection rate and a 21.5% midgut infection rate. Oocysts in An. stephensi increased in size by 20% after day 10. Development peaked at day 12, with a mean oocyst diameter of 58 microm at onset of oocyst differentiation. Oocysts developed more slowly in An. vestitipennis until day 10. After day 10, there was a 53% increase in oocyst development over the previous 10 days. Oocyst differentiation was not observed until day 13 postfeed. As with An. vestitipennis, both populations of An. albimanus showed similar slow rates of oocyst development; however, no dramatic growth increase occurred after day 10. The oocysts in the Golden Stream population exhibited a cessation of growth after day 10, peaking at a mean of 30 microm. The Buena Vista population did not exhibit the same level of reduced oocyst development. A gradual increase in growth continued until days 13 and 14 (36.7 and 35.7 microm, respectively).

A mark-release-recapture study using a novel portable hut design to define the flight behavior of Anopheles darlingi in Belize, Central America.

Knowledge of the flight behavior of local vectors is of paramount importance in mosquito control programs. The following study defined the recapture rate of wild-caught, unengorged Anopheles darlingi females at 0, 400, and 800 m from a fixed release point in Belize, Central America, using a newly designed portable experimental hut. Three sampling trials, each consisting of 2 12-h collections, were performed at all distances from July 2002 to June 2003. A total of 1,185 An. darlingi were marked and released during the course of the study. The recapture rate was greatest at 0 m (29.0%; 124/428) and declined from 11.6% (37/318) at 400 m to 5.8% (21/361) at the 800-m site. There was no difference between the average number of marked mosquitoes recaptured inside the experimental hut versus outside the hut at any distance location. Recapture rates of each trial were highest during the first night's collection at all locations. Further examination of the first night data revealed a variation in the peak time of recapture by distances from the release point. The peak in nightly recapture at both the 0- and 400-m sites occurred within the first 2 h after sunset, and the peak recapture at the 800-m site occurred during the 7th h after sunset. Information from the present study is the first to describe the flight behavior of An. darlingi in Belize and will benefit in the development of adult-density risk assessments at the house level based on distances from potential vector breeding sites.

A novel high-throughput screening system to evaluate the behavioral response of adult mosquitoes to chemicals.

A modular and novel assay system for rapid mass screening of chemical compounds for contact irritant and spatial repellent actions against adult mosquitoes is described. The responses of Aedes aegypti to various concentrations of 3 topical repellents, deet, Bayrepel, and SS220, were evaluated. At treatment concentrations > or = 25 nmol/cm2 of SS220, mosquitoes exhibited significant contact irritant (escape) and spatial repellent (movement away from the chemical source) responses, whereas, a 10-fold increase in the treatment concentration of deet and Bayrepel was required to produce similar responses. The novel bioassay system detected contact irritancy and spatial repellency activity with reproducible results and provided baseline data for determining minimum effective concentrations for other chemicals. The system is compact in size, easy to decontaminate, and requires only a minute quantity of chemical compound.

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.

Evaluation of habitat management strategies for the reduction of malaria vectors in northern Belize.

Mowing and burning of emergent vegetation were evaluated as potential management strategies for the control of the malaria vector, Anopheles vestitipennis, in northern Belize, Central America. The primary aim was reduction of tall dense macrophytes (dominated by Typha domingensis) as preferred larval habitat for An. vestitipennis. Nine experimental plots were established in a Typha marsh in Orange Walk District, Belize. Three plots were burned, three were treated by subaquatic mowing, and three were unaltered controls. After treatment, Typha height was most dramatically affected by the mow treatment. Plant heights at 21 and 95 days post-treatment reflected an 89% and 48% decrease, respectively, compared to pretreatment conditions. The Typha height in the burn plots was not as severely affected. Heights at 21 days post-treatment were 39% lower than those of pre-treatment vegetation, with a return to near pre-test heights by 95 days post-treatment. Both treatments resulted in a significant reduction in the number of An. vestitipennis larvae collected as compared to control plots. Conversely, the treatments resulted in increased larval densities of several other vector and pest mosquito species. Larval population densities ofAn. albimanus, Ochlerotatus taeniorhynchus, and Culex coronator were significantly higher in burn plots. In mow plots, there were significant increases in An. albimanus and Oc. taeniorhynchus larval populations. Non-target invertebrate species affected by the treatments were adult Tropisternus collaris, larval Corythrella, and adult Parapleapuella.

Spatial correlations of mapped malaria rates with environmental factors in Belize, Central America.

BACKGROUND: The purposes of this study were to map overall malaria incidence rates from 1989 through 1999 for villages in Belize; to assess the seasonal distribution of malaria incidence by region; and to correlate malaria incidence rates with vegetation cover and rivers in villages, using geographic information system technology.Malaria information on 156 villages was obtained from an electronic database maintained by the Belize National Malaria Control Program. Average annual malaria incidence rates per 1000 population over 10 years were calculated for villages using the 1991 population census as a denominator. Malaria incidence rates were integrated with vegetation cover from a 1995 vegetation map, and with river data from a digital data set. RESULTS: Mapping malaria incidence over the 10-year period in the study villages indicated the existence of a spatial pattern: the southern and western areas of Belize had consistently higher rates of malaria than northern areas. Examination of the seasonal distribution of malaria incidence by month over 10 years indicated that a statistically significant difference existed among districts and among months (p < 0.05). Spatial analysis of malaria incidence rates and of vegetation in Belize showed villages with high malaria rates having more broadleaf hill forests, agricultural land, and wetland vegetation types (i.e. SWF-seasonally waterlogged fire-induced shrubland of the plains). Statistical and spatial analyses of malaria incidence and of river distributions in Belize determined the high 10 percentile malaria incidence villages in western and southern Belize to have more rivers within two kilometers of the center of a village and a statistically significant correlation between proximity to rivers and villages (Spearman's gamma = -0.23; p < 0.05), especially in Stann Creek District (Spearman's gamma = -0.82; p < 0.05). CONCLUSIONS: Examination of the distribution of malaria during 10 years indicated transmission varied among geographic areas and among seasons. Additional studies are needed to examine, in more detail, the association between environmental and meteorological factors and malaria transmission. Furthermore, the role of An. darlingi in malaria transmission in Stann Creek needs further study since, of the three main vectors in Belize, An. darlingi has been found strongly associated with rivers.

Use of polymerase chain reaction technique to confirm VecTest screening results in Plasmodium falciparum and Plasmodium vivax VK 210 laboratory-infected Anopheles stephensi mosquitoes.

We evaluated polymerase chain reaction (PCR) to confirm immunoassays for malaria parasites in mosquito pools after a failure to detect malaria with PCR during an outbreak in which pools tested positive using VecTest and enzyme-linked immunosorbent assay (ELISA). We combined VecTest, ELISA, and PCR to detect Plasmodium falciparum and Plasmodium vivax VK 210. Each mosquito pool, prepared in triplicate, consisted of 1 exposed Anopheles stephensi and up to 9 unfed mosquitoes. The results of VecTest and ELISA were concordant. DNA from a subset of the pools, 1 representative of each ratio of infected to uninfected mosquitoes, was extracted and used as template in PCR. All P. vivax pools were PCR positive but some needed additional processing for removal of apparent inhibitors before positive results were obtained. One of the pools selected for P. falciparum was negative by PCR, probably because of losses or contamination during DNA extraction; 2 remaining pools at this ratio were PCR positive. Testing pools by VecTest, ELISA, and PCR is feasible, and PCR is useful for confirmation of immunoassays. An additional step might be needed to remove potential inhibitors from pools prior to PCR.

Vector competence of three North American strains of Aedes albopictus for West Nile virus.

To evaluate the potential for North American (NA) Aedes albopictus to transmit West Nile virus (WN), mosquito strains derived from 3 NA sources (Frederick County, Maryland, FRED strain; Cheverly, MD, CHEV strain; Chambers and Liberty counties, Texas, TAMU strain) were tested. These strains were tested along with a previously tested strain from a Hawaiian source (OAHU strain). Mosquitoes were fed on 2- to 3-day-old chickens previously inoculated with a New York strain (Crow 397-99) of WN. All of the NA strains were competent laboratory vectors of WN, with transmission rates of 36, 50, 83, and 92% for the FRED, CHEV, OAHU, and TAMU strains, respectively. The extrinsic incubation period for WN in Ae. albopictus held at 26 degrees C was estimated to be 10 days. Based on efficiency of viral transmission, evidence of natural infection, bionomics, and distribution, Ae. albopictus could be an important bridge vector of WN in the southeastern USA.

Host feeding preferences of Anopheles species collected by manual aspiration, mechanical aspiration, and from a vehicle-mounted trap in the Toledo District, Belize, Central America.

The host-feeding patterns of Anopheles albimanus, Anopheles vestitipennis, and Anopheles punctimacula from the Toledo District in southern Belize were studied with blood-fed females that were collected by manual aspiration, a backpack aspirator, and a vehicle-mounted trap for sampling in-flight mosquito populations. Female An. vestitipennis collected from both inside and outside house walls by manual aspiration tested positive for human blood meals (88 and 67%, respectively). At increasing distances from the houses, specimens of An. vestitipennis collected from vegetation with the backpack aspirator were equally positive for human and cattle blood (44 and 43%, respectively). In contrast, 68% of the An. albimanus specimens (148) collected by backpack aspiration tested positive for cattle blood. Engorged An. vestitipennis from vehicle-mounted trap collections tested positive for cattle (108) and human (52) blood. Almost all specimens of An. albimanus from these collections were positive for cow (95%). After analyzing the data from the An. vestitipennis samples using the feeding index, the ratio of human blood to all other bloodmeal sources showed indices greater than 1. Both An. albimanus and An. punctimacula fed mostly on cattle and rarely fed on humans. Foraging ratios for the 3 Anopheles species were very similar to the feeding indexes. Ratios based on data from all collection methods showed that An. vestitipennis feeds predominately on humans. The foraging ratios for An. albimanus demonstrated consistent preferences for nonhuman hosts. As with previous studies. An. albimanus seemed to prefer cattle and pigs to almost all other host species.

Environmental factors associated with larval habitats of malaria vectors in northern Kyunggi Province, Republic of Korea.

The larval habitats of malaria vectors near the Demilitarized Zone of the Republic of Korea (ROK) were sampled from June through September 2000 to determine larval abundance and to identify environmental factors associated with high larval density. Six primary habitats were identified: rice fields, irrigation ditches, drainage ditches, stream pools, irrigation pools, and marshes. Most habitats harbored similar densities of larvae until August and September, when population densities in rice fields declined and those in irrigation pools increased. The primary vector in the ROK, Anopheles sinensis, occurred in water with a wide range of values for environmental factors, including pH, total dissolved solids, percent of surface covered with floating vegetation, and nitrate and phosphate concentrations. No environmental factor or combination of factors were found that were predictive of high larval densities. This study suggests that larval Anopheles are capable of developing in a wide range of stagnant, freshwater habitats in northern Kyunggi Province, ROK.

Mosquito studies in Belize, Central America: records, taxonomic notes, and a checklist of species.

Data from mosquito collections made in Belize, Central America, between September 1990 and April 1993 are presented. A total of 537 collections yielding 15,139 specimens are summarized. One genus, 4 subgenera, and 31 species are recorded from Belize for the 1st time. A checklist of the 111 mosquito species now known to occur in Belize is presented.