The mosquito Anopheles (Cellia) oreios sp. n., formerly species 6 of the Australasian Anopheles farauti complex, and a critical review of its biology and relation to disease.

Species 6 of the Australasian Anopheles farauti sibling species complex (Diptera: Culicidae) is described and formally named Anopheles oreios Bangs & Harbach, sp. n. Adult, pupal and fourth-instar larval specimens collected in the Baliem Valley, Papua Province, Indonesia, are characterized and compared with those of Anopheles farauti, Anopheles hinesorum, Anopheles irenicus and Anopheles torresiensis (formerly informally denoted as species 1, 2, 7 and 3, respectively). The variable wings of adult females, the male genitalia, the pupa and the fourth-instar larva of An. oreios are illustrated and DNA sequence data are included for regions coding for sections of the mitochondrial COI and COII genes. The biology of An. oreios and its relation to malaria transmission are discussed in detail and contrasted with the biology and disease relations of some members of the An. farauti and Anopheles punctulatus sibling species complexes.

Field evaluations of disposable sticky lures for surveillance of Aedes aegypti (Stegomyia aegypti) and Culex quinquefasciatus in Jakarta.

From December 1997 to April 1998, disposable sticky lures (1608 lure days) were trialled in homes in north Jakarta, Indonesia as surveillance tools for Aedes aegypti (Stegomyia aegypti) (Diptera: Culicidae) and Culex quinquefasciatus (Diptera: Culicidae), referenced to indoor resting adult collections (92 × 10 min). The lures collected 89.4% of the total of 1339 Ae. aegypti and 92.1% of the total of 1272 Cx. quinquefasciatus collected by all methods. Because there were no significant differences with respect to numbers collected in bedrooms, living rooms and kitchens, bedrooms were selected for subsequent trials for reasons of convenience. The main trials involved a replicated complete block design with L-lysine and sodium carbonate. Lures without attractant or with four different dilutions of L-lysine collected 3.4-8.5 times more Ae. aegypti and 4.2-8.1 times more Cx. quinquefasciatus than were collected by mouth aspirator. Lures with or without dilutions of sodium carbonate collected 2.7-5.0 times more Ae. aegypti and 1.8-4.2 times more Cx. quinquefasciatus than aspirator collections. The precision associated with catches of sticky lures was better than that for aspirator collections. Although olfactants generally improved the numbers of mosquitoes collected, the differences in catch between lures with and without attractants were usually non-significant. Any deficit in catch may be offset by increasing the surveillance period to ≥30 days to detect all four dengue serotypes from infected mosquitoes.

Randomized, parallel placebo-controlled trial of primaquine for malaria prophylaxis in Papua, Indonesia.

Malaria causes illness or death in unprotected travelers. Primaquine prevents malaria by attacking liver-stage parasites, a property distinguishing it from most chemoprophylactics and obviating 4-week postexposure dosing. A daily adult regimen of 30 mg primaquine prevented malaria caused by Plasmodium falciparum and P. vivax for 20 weeks in 95 of 97 glucose-6-phosphate dehydrogenase (G6PD)-normal Javanese transmigrants in Papua, Indonesia. In comparison, 37 of 149 subjects taking placebo in a parallel trial became parasitemic. The protective efficacy of primaquine against malaria was 93% (95% confidence interval [CI] 71%-98%); against P. falciparum it was 88% (95% CI 48%-97%), and >92% for P. vivax (95% CI >37%-99%). Primaquine was as well tolerated as placebo. Mild methemoglobinemia (mean of 3.4%) returned to normal within 2 weeks. Blood chemistry and hematological parameters revealed no evidence of toxicity. Good safety, tolerance, and efficacy, along with key advantages in dosing requirements, make primaquine an excellent drug for preventing malaria in nonpregnant, G6PD-normal travelers.

Allelic variants of the Plasmodium falciparum merozoite surface antigen 2 (MSA-2) in a geographically restricted area of Irian Jaya.

Blood samples were collected from 12 residents of 4 villages in the Oksibil area of Irian Jaya. Eleven patients were positive for Plasmodium falciparum infection as evidenced by successful amplification of the MSA-2 gene by the polymerase chain reaction. Two patients showed evidence of infection by 2 strains of Plasmodium falciparum. All MSA-2 genes were completely sequenced and all could be assigned to one of the two major allelic families of MSA-2, however all MSA-2 gene sequences differed from previously described alleles. Five new allelic forms were identified, one of which was present in 8 of the 11 patients. Within small natural populations of P. falciparum, it appears that variation in MSA-2 approximates that seen world-wide. All samples were also analysed by hybridisation of amplified DNA to family specific probes and all samples hybridised to known probes. Our results demonstrate that there is a degree of microheterogeneity of MSA-2 that is undetectable by hybridisation studies alone.

Prevalence of serum antibodies to human T lymphotropic virus-1 in an isolated tribe in the highlands of Irian Jaya, Indonesia.

There have been several recent reports on the high prevalence of serum antibodies to human T lymphotropic virus-1 (HTLV-1) in isolated populations residing in the coastal areas and highlands of Papua New Guinea. In the absence of significant cases of clinical disease, it has been surmised that this reactivity might be the consequence of serologic recognition of yet undefined human retroviruses or parasite antigens. These observations prompted an investigation of the prevalence of anti-HTLV-1 antibodies among members of the Ngalum tribe that dwells in a secluded highland valley in the eastern Jayawijaya Mountains of Irian Jaya, Indonesian New Guinea. Of 165 tribespeople, 85 (52%) were positive for IgG antibodies to HTLV-1 in an indirect enzyme-linked immunosorbent assay. Eighty-two were more than 10 years of age. On the Western blot, all positive sera reacted strongly with the p19 core antigen, but recognition of the envelope antigens, gp46 and gp21, was conspicuously absent. Thirty-four of the 85 villagers with these indeterminant blots had active Plasmodium falciparum infections, but antibody absorption studies with HTLV-1 and P. falciparum erythrocytic stage antigens failed to confirm suspected serologic cross-reactivities. Thirty-three others had acute malaria and/or high titers of anti-malaria antibodies but were seronegative for HTLV-1. We suspect that indeterminant Western blots for HTLV-1 reflect antibody responses to related latent retroviruses that are activated as a consequence of immunosuppression following malaria infection and chloroquine therapy.

Resistance to antimalarials by Plasmodium falciparum in Arso PIR, Irian Jaya, Indonesia.

Between 1987 and 1990, susceptibility of Plasmodium falciparum to chloroquine and to Fansidar was measured in vivo in 151 volunteers using the standard 7-day test. All volunteers lived in Arso PIR, Irian Jaya. A 25 mg/kg dose of chloroquine base was administered over a three-day period to 92 volunteers positive for P. falciparum rings (greater than 10 rings/200 white blood cells). Fifty volunteers (54%) showed results consistent with resistance. Twenty-nine were classified RII, and 21 RIII. In November 1989, a single curative dose of Fansidar was administered to 59 volunteers divided among three groups with 18 months, four years, and life-long exposure to endemic malaria. The proportion of volunteers in each group still positive for P. falciparum on day 7 of followup was 54%, 0%, and 14%, respectively. Thus, immune status profoundly effected clinically response to Fansidar. Standard in vitro microtests were also performed on parasites from 11 volunteers against chloroquine, amodiaquine, quinine, pyrimethamine/sulfadoxine, and and mefloquine. Nine of ten isolates showed in vitro growth consistent with resistance to chloroquine. Tests with other drugs showed few isolates with results considered indicative of susceptibility. Arso PIR has a severe drug resistance problem.

Heightened transmission of stable malaria in an isolated population in the highlands of Irian Jaya, Indonesia.

Malaria at an elevation of 1,500 meters is uncommon and is usually unstable when it occurs. To confirm reports of a recent increase in transmission of stable malaria in the Oksibil Valley, which is at an elevation of 1,250-1,500 meters in the Jayawijaya Mountains of Irian Jaya, Indonesia, five malariometric surveys were conducted in four villages between May 1990 and July 1991. A total of 3,380 blood smears, representative of 1,949 persons, was examined. Prevalence rates over the survey period were consistent in each of the four villages, averaging 10% for infants, 50% for children 1-4 years of age, 35% for those 5-9 years old, 28% for those 10-14 years old, and 16% for adults (greater than 15 years old). The spleen rate for the those less than five years old was 96%, with an average enlarged spleen score of 2.32. Plasmodium falciparum accounted for 55% of the infections in the valley, but P. vivax was the predominant species in those less than 10 years old. In the village of Kutdol at an elevation of 1,500 meters, P. malariae was identified in 43% of the positive smears. Four cases were diagnosed as P. ovale. Infection with P. falciparum without obvious clinical symptoms was common in both adults and children. Entomologic and epidemiologic data suggested that the recent upsurge in transmission coincided with the replacement of traditional village huts with the more modern social housing. This replacement required the extensive construction of drainage ditches, which inadvertently also served as additional vector breeding sites. We suspect that this manipulation of the environment, in an effort to improve the quality of life, created conditions conductive for heightened transmission of stable malaria.

PIP: Malaria at an elevation of 1500 meters is uncommon and is usually unstable when it occurs. To confirm reports of a recent increase in the transmission of stable malaria in the Oksibil Valley, at an elevation of 1250-1500 meters in the Jayawijaya Mountains of Irian Jaya, Indonesia, 5 malariometric surveys were conducted in 4 villages between May 1990 and July 1991. A total of 3380 blood smears from 1949 people was examined. Prevalence rates over the survey period were consistent in each of the 4 villages,with averages of 10% for infants, 50% for children 1-4 years old, 35% for those 5-9 years old, 28% for those 10-14 years old, and 16% for adults (over 15 years of age). The spleen rate for those less than 5 years old was 96%, with an average enlarged spleen score of 2.32. Plasmodium falciparum accounted for 55% of the infections in the valley, but P. vivax was the predominant species in those less than 10 years old. In the village of Kutdol, at an elevation of 1500 meters, P. Malariae was identified in 43% of the positive smears. 4 cases were diagnosed as P. ovale. Infection with P. falciparum without obvious clinical symptoms was common in both adults and children. Entomologic and epidemiologic data suggested that the recent upsurge in transmission coincided with the replacement of traditional village huts with more modern social housing. This replacement required the extensive construction of drainage ditches, which inadvertently also served as additional vector breeding sites. The authors suspect that this manipulation of the environment, while attempting to improve the quality of life, created conditions which were conducive for the increased transmission of stable malaria.

Resistance to chloroquine by Plasmodium vivax in Irian Jaya, Indonesia.

Evidence of emerging resistance to chloroquine by Plasmodium vivax is described from Irian Jaya (Indonesian New Guinea). Sixteen of 24 residents in the village of Arso PIR II taking supervised weekly chloroquine prophylaxis (5 mg base/kg) had asexual parasitemia with P. vivax at least once during eight weeks of surveillance. An American working in the same village developed symptomatic P. vivax parasitemia despite chloroquine prophylaxis. Five days after therapy with 600 mg chloroquine base, the asexual parasitemia in the American increased 40-fold, but cleared after treatment with 1,500 mg chloroquine base. Serum samples were not available from many of the cases, but six local residents and the American had serum levels of chloroquine in excess of the ordinarily suppressive 15 ng/ml at the time of their asexual parasitemias (16-70 ng/ml). The weekly 300 mg base tablet of chloroquine, which has been the standard for prophylaxis against malaria for more than 40 years, was not effective against P. vivax in Arso PIR, Irian Jaya.

Cultivation of sexually mature Brugia malayi in vitro.

Sexually mature male and female Brugia malayi were developed from third stage larvae after 60 days in the in vitro culture system described by Franke and others in 1987 (Am J Trop Med Hyg 37: 370-375). Between 75 and 100 days in culture, many worms produced living microfilariae. Each gravid female produced 200-1,500 microfilariae/day.

Malaria vaccine study site in Irian Jaya, Indonesia: Plasmodium falciparum incidence measurements and epidemiologic considerations in sample size estimation.

Malaria epidemiologic and entomologic studies were performed during both the high transmission and low transmission seasons to characterize the Plasmodium falciparum malaria transmission at a proposed malaria vaccine trial site in Irian Jaya, Indonesia. The study population consisted of two subsets: native Irianese men with lifelong exposure to malaria and transmigrants who arrived from a nonmalarious area 2.5 years before the start of the study. All subjects received a radical cure for malaria and were then monitored weekly by blood film. Both P. falciparum malaria attack rates and incidence densities were calculated; transmigrants had a significantly higher rate (P = 0.003) than the Irianese during the low transmission season study (20-weeks long) but not during the high transmission season study (12-weeks long). Lack of exposure-induced immunity left the transmigrants at a minimum 17-25% greater relative risk of becoming parasitemic compared with the Irianese during the low transmission season study. During the high transmission season study, 50% of the transmigrants were P. falciparum positive by week 6 and 50% of the Irianese by week 9. During the low transmission season, 50% of the transmigrants were positive by week 10 and 43% of the Irianese were positive by week 17. Entomologic studies showed that Anopheles koliensis was the predominant vector (> 98% of anopheline catch). Entomologic inoculation rates for P. falciparum were 0.018 and 0.39 infective bites/person/night for the low and high transmission seasons, respectively. New P. vivax cases represented between 16% and 42% of all initial malaria cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-specific prevalence of Plasmodium falciparum among six populations with limited histories of exposure to endemic malaria.

The age-specific prevalence of Plasmodium falciparum parasitemia among residents of six villages in northeastern Irian Jaya, Indonesia, has been measured for a period of five years. All study subjects were transmigrants from Java living in Irian Jaya for three weeks to 72 months, depending upon the village and point of measurement. Fifteen separate estimates of prevalence were obtained from 4,554 Giemsa-stained thick blood films from 91 to 701 people (mean sample size = 304) among the six villages. The prevalence of parasitemia among people who had lived in Irian Jaya for less than one year did not decrease as a function of age, except in one village at eight months. In contrast, after 16 months to two years or more of residence, the prevalence of parasitemia decreased markedly with increasing age beyond 6-10 or 11-15 years. Social, behavioral, or entomologic characteristics of these populations did not explain the decreasing prevalence of parasitemia with age. An age-dependent naturally acquired protective immunity appeared to develop in all of these villages after 1-2 years of exposure to hyperendemic malaria.

Age-dependent acquired protection against Plasmodium falciparum in people having two years exposure to hyperendemic malaria.

An epidemiologic study of susceptibility to frequent and high-grade parasitemia by Plasmodium falciparum revealed that age-dependent acquired protection developed within a two-year period of exposure to hyperendemic infection pressure. The study was conducted in a single village in northeastern Irian Jaya, Indonesia, where half the residents were native to the province and the other half were transmigrants from areas of Java, where there is little or no malaria transmission. Five separate measures of susceptibility to the asexual parasitemia of falciparum malaria were derived from results of four months of biweekly surveillance of 240 volunteers. Increasing protection as a function of age among the Javanese was a consistent pattern among the five estimates of susceptibility. These age-dependent functions of protection were quantitatively parallel to those among life-long residents of Irian Jaya. When humoral immune responsiveness to ring-infected erythrocyte surface antigen (RESA) was measured by ELISA, a similar pattern emerged; the relative level of antibody to RESA increased as parallel functions of age among the two subpopulations. Acquired protective immunity against P. falciparum was not the cumulative product of many years of heavy exposure to antigen. Instead, the full benefit of protection appeared to develop quickly. The degree of protection was governed by recent exposure and age, independent of history of chronic heavy exposure.

Primaquine for prophylaxis against malaria among nonimmune transmigrants in Irian Jaya, Indonesia.

A comparison of primaquine versus chloroquine for prophylaxis among nonimmune transmigrants from Java and Bali in the hyperendemic Arso region of Irian Jaya, Indonesia was conducted. Forty-five subjects received 0.5 mg of primaquine base/kg of body weight every other day, and 54 people in the same village received weekly 5 mg of chloroquine base/kg for 16-19 weeks beginning in December 1992. Plasmodium falciparum accounted for 18 of 30 infections with chloroquine, and four of five infections among subjects receiving primaquine. Plasmodium vivax was found in 12 people taking chloroquine but in just one person taking primaquine. The risk of malaria among people taking chloroquine relative to that among subjects taking primaquine was 3.96 (P = 0.014) for P. falciparum and 10.56 (P = 0.012) for P. vivax. Primaquine was better tolerated than chloroquine. The minimal protective efficacy for primaquine prophylaxis was 74% against P. falciparum and 90% against P. vivax among nonimmune children and adults living in Irian Jaya. These findings require confirmation with randomized, double-blinded, and placebo-controlled trials.

Assessment of age-dependent immunity to malaria in transmigrants.

Sixty-six Javanese transmigrants moving from Java, an area of very low malaria transmission, to Irian Jaya, an area of high malaria transmission, were monitored to evaluate the effects of exposure to malaria transmission and age on resistance to infection and the induction of humoral immunity. The risk of acquiring Plasmodium falciparum parasitemia was not statistically greater in children (5-15 years of age) than in adults (> 15 years of age) during the first 14 months of exposure. However, during the cross-sectional survey at 14 months of exposure. children did have significantly higher P. falciparum asexual blood-stage parasite densities. Serum antibody titers to R32LR, a peptide containing sequences from the P. falciparum circumsporozoite repeat region, and MSP19, a proteolytic fragment of merozoite surface protein-1 (MSP-1) from P. falciparum, were measured by enzyme-linked immunosorbent assay. Exposure for both six and 14 months produced statistically significant increased antibody titers to both R32LR and MSP-1; no age-dependent difference in antibody titers was observed. In this population, exposure to malaria transmission induced antibodies to antigens associated with immunity to malaria. In addition, we noted an age-dependent difference in the parasitemia density of P. falciparum.

In vivo responses to antimalarials by Plasmodium falciparum and Plasmodium vivax from isolated Gag Island off northwest Irian Jaya, Indonesia.

There is renewed interest in the rich nickel and cobalt deposits of Pulau Gag, an isolated but malarious island off the northwest coast of Irian Jaya. In preparation for an expanded workforce, an environmental assessment of malaria risk was made, focusing upon malaria prevalence in the small indigenous population, and the in vivo sensitivity of Plasmodium falciparum and P. vivax to chloroquine (CQ) and sulfadoxine/pyrimethamine (S/P), the respective first- and second-line drugs for uncomplicated malaria in Indonesia. During April-June 1997, mildly symptomatic or asymptomatic malaria infections were found in 24% of 456 native residents. Infections by P. falciparum accounted for 60% of the cases. Respective day 28 cure rates for CQ (10 mg base/kg on days 0 and 1; 5 mg/kg on day 2) in children and adults were 14% and 55% (P < 0.005). Type RII and RIII resistance characterized only 5% of the CQ failures. Re-treatment of 36 P. falciparum CQ treatment failures with S/P (25 mg/kg and 1.25 mg/kg, respectively) demonstrated rapid clearance and complete sensitivity during the 28-day follow-up period. More than 97% of the P. vivax malaria cases treated with CQ cleared parasitemia within 48 hr. Three cases of P. vivax malaria recurred between days 21 and 28, but against low drug levels in the blood. The low frequency of RII and RIII P. falciparum resistance to CQ, the complete sensitivity of this species to S/P, and the absence of CQ resistance by P. vivax are in contrast to in vivo and in vitro test results from sites on mainland Irian Jaya.

Chloroquine-resistant Plasmodium vivax in transmigration settlements of West Kalimantan, Indonesia.

Malariometric surveys were conducted during July 1996 in native Dayak villages and predominantly Javanese transmigration settlements in Ketapang district of West Kalimantan, Indonesia. Malaria prevalence ranged from 0.9% to 2.7% in Dayak villages and from 1% to 20% in the transmigration settlements. Plasmodium falciparum accounted for 67% of the cases among Dayaks but P. vivax was dominant among transmigrants, accounting for more than 72% of the infections. Chloroquine sensitivity/resistance was assessed by 28-day in vivo testing of uncomplicated malaria infections and measurement of chloroquine blood levels in cases where parasitemias reappeared within the 28-day test period. Resistance was based on the appearance of asexual parasites against chloroquine plus desethylchloroquine levels exceeding the minimally effective whole blood concentrations proposed for sensitive parasite strains (P. vivax, 100 ng/ml; P. falciparum, 200 ng/ml). All parasitemias cleared initially within four days of beginning supervised chloroquine therapy (25 mg base/kg over a 48-hr period), but asexual parasites reappeared within 28 days in 27 of 52 P. vivax and three of 12 P. falciparum cases. Chloroquine blood levels at the time of recurrent parasitemias revealed resistance in 12 of the 27 P. vivax cases and in one of the three P. falciparum cases. Genotypes of nine of the 12 recurrent P. vivax isolates matched with their primary isolates and ruled out reinfection. These findings establish the presence of chloroquine-resistant P. vivax on the island of Borneo. The pattern of malaria and the high frequency of chloroquine resistance by P. vivax at the West Kalimantan location may relate to demographic, ecologic, agricultural, and socioeconomic changes associated with transmigration.

Epidemic dengue transmission in southern Sumatra, Indonesia.

An outbreak of dengue fever (DF), dengue haemorrhagic fever (DHF), and dengue shock syndrome (DSS) in the city of Palembang, south Sumatra, Indonesia was investigated to (i) validate epidemic occurrence, (ii) confirm dengue virus aetiology and associated serotype(s), (iii) provide a demonstrable measure of community impact, and (iv) identify causative relationship (if any) with climatic El Niño Southern Oscillation (ENSO) influences. Trend analysis based on a 6-year retrospective review of hospital records demonstrates a 3-fold increase in clinical cases for the outbreak period (January-April 1998), relative to historical records. In the 2 hospitals surveyed, the monthly mean number of outbreak-related dengue cases over 4 months was 833 (range 650-995 cases/month); the mean monthly value for the previous 72 months was 107 (range 14-779 cases/month). An apparent trend in epidemic transmission was observed, evolving from a 5-year cyclic phenomenon to an annual occurrence, often indistinguishable from one year to the next. The proportional distribution of clinical outbreak cases into DF, DHF and DSS diagnostic categories was 24%, 66%, and 10%, respectively. The population aged 10-19 years accounted for the largest (35%) proportion of hospitalized DHF cases, followed by children aged 5-9 years (25%) and children aged 4 years (16%). Serum samples obtained during acute illness from 221 hospitalized patients were examined using serology, RT-PCR, and virus isolation in cell culture: 59% of samples had laboratory evidence of a dengue infection. All 4 dengue virus serotypes (DEN 1-4) were identified in epidemic circulation, with DEN 3 predominating (43%). DEN 1 was the principal serotype associated with less severe dengue illness, suggesting that virulence may be, in part, a function of infecting serotype. The climatic influence of ENSO on rainfall and temperature in the months leading up to and during the outbreak was dramatic, and is likely to contribute to favourable outbreak conditions.

Susceptibility of various mosquitoes of California to subperiodic Brugia malayi.

Laboratory experiments were conducted to determine the susceptibility of six species of mosquitoes, representing three genera, to subperiodic Brugia malayi. The black-eye, Liverpool strain of Aedes aegypti was the susceptible control. Mosquitoes were fed on microfilaremic jirds (Meriones unguiculatus). All mosquitoes, except wild caught Culex erythrothorax, were laboratory-reared and allowed to feed when 8 to 10 days old. Anopheles freeborni, Anopheles hermsi, and Culiseta inornata proved refractory. Both Anopheles species allowed invasion of flight muscle and development to the late first stage, after which larval growth ceased and melanization occurred. Culiseta inornata prevented any larval development. Culex tarsalis and Cx. erythrothorax proved highly susceptible to B. malayi infection. In all, 95.6% and 88.7% of the Cx. tarsalis harbored third-stage larvae after infective feedings of 15.7 and 81.8 mf/microliters of blood, respectively, while only 11.5% were found susceptible when microfilaremia was low (1.1 mf/microliters). Culex erythrothorax demonstrated a susceptibility rate of 82.3% with 17.0 mf/microliters. Both Culex species appear to be excellent experimental hosts for subperiodic B. malayi. This is the first conclusive evidence that mosquitoes of the genus Culex can naturally support the complete development of a stain of subperiodic B. malayi.

Rediscovery of Anopheles (Cellia) clowi (Diptera: Culicidae), a rarely recorded member of the Anopheles punctulatus group.

Anopheline specimens collected in Papua New Guinea were morphologically identified as the rarely recorded Anopheles clowi Rozeboom & Knight. Amplification of the rDNA ITS2 region of this material revealed a fragment of 750 bp confirming its placement in the Anopheles punctulatus group. This group contains 12 species and includes the major malaria vectors in the islands of the southwest Pacific. Digestion of the ITS2 with the restriction enzyme MspI produced restriction fragment-length polymorphism with bands at 380, 300, and 150 bp, a pattern shared by no other members of this group. Phylogenetic analysis involving the sequencing of a 2 kb region of the rDNA 18S gene indicated that An. clowi was monophyletic and basal to the rest of the group and showed considerable independent evolution from the other members. This is the first record of An. clowi in Papua New Guinea and only the third collection of this species since its discovery in 1945.

Detection of dengue viral RNA in Aedes aegypti (Diptera: Culicidae) exposed to sticky lures using reverse-transcriptase polymerase chain reaction.

Active surveillance for dengue (DEN) virus infected mosquitoes can be an effective way to predict the risk of dengue infection in a given area. However, doing so may pose logistical problems if mosquitoes must be kept alive or frozen fresh to detect DEN virus. In an attempt to simplify mosquito processing, we evaluated the usefulness of a sticky lure and a seminested reverse-transcriptase polymerase chain reaction assay (RT-PCR) for detecting DEN virus RNA under laboratory conditions using experimentally infected Aedes aegypti (L.) mosquitoes. In the first experiment, 40 male mosquitoes were inoculated with 0.13 microl of a 10(4) pfu/ml DEN-2 stock solution. After a 7-d incubation period, the mosquitoes were applied to the sticky lure and kept at room temperatures of 23-30 degrees C. Following 7, 10, 14, and 28 d application, 10 mosquitoes each were removed from the lure, pooled, and assayed for virus. DEN virus nucleic acid was clearly detectable in all pools up to 28 d after death. A second study evaluated sensitivity and specificity using one, two, and five DEN-infected mosquitoes removed after 7,10, 14, 21, and 30 d application and tested by RT-PCR. All four DEN serotypes were individually inoculated in mosquitoes and evaluated using the same procedures as experiment 1. The four serotypes were detectable in as few as one mosquito 30 d after applications to the lure with no evidence of cross-reactivity. The combination of sticky lures and RT-PCR show promise for mosquito and dengue virus surveillance and warrant further evaluation.