Field detection of Tembusu virus in western Thailand by rt-PCR and vector competence determination of select culex mosquitoes for transmission of the virus.

Tembusu virus (TMUV; Ntaya serocomplex) was detected in two pools of mosquitoes captured near Sangkhlaburi, Thailand, as well as from sera from sentinel ducks from the same area. Although TMUV has been isolated from several mosquito species in Asia, no studies have ever shown competent vectors for this virus. Therefore, we allowed mosquitoes captured near Sangkhlaburi to feed on young chickens that had been infected with TMUV. These mosquitoes were tested approximately 2 weeks later to determine infection, dissemination, and transmission rates. Culex vishnui developed high viral titers after feeding on TMUV-infected chicks and readily transmitted virus to naïve chickens. In contrast, Cx. fuscocephala seemed less susceptible to infection, and more importantly, zero of five fuscocephala with a disseminated infection transmitted virus by bite, indicating a salivary gland barrier. These results provide evidence for the involvement of Culex mosquitoes in the transmission of TMUV in the environment.

Evaluation of a field-portable DNA microarray platform and nucleic acid amplification strategies for the detection of arboviruses, arthropods, and bloodmeals.

Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors.

A field-expedient method for detection of leptospirosis causative agents in rodents.

We have developed a thermal-stable, pathogenic Leptospira TaqMan PCR assay intended to support pathogen surveillance in reservoir populations. The assay is packaged specifically for use with a portable, ruggedized, real-time PCR thermocycler. Limit of detection was established at ≤ 100 fg (20 organisms). Sensitivity and specificity were 100% concordant with conventional PCR results using a broad test panel of human pathogenic and nonpathogenic Leptospira, genetic near neighbors, and clinically significant organisms. In blind testing using a panel (n=50) of pathogenic Leptospira infected and noninfected Rattus species samples, assay sensitivity results were 100% concordant with conventional PCR. Tests performed under field conditions using wild-collected rodent kidney extracts demonstrated the mobility of the system. During field evaluation, samples were processed and analyzed in 3 hours. Thermal stabilized reagents allowed transportation, storage, and analyses under ambient temperatures. The system provides a promising aid in leptospirosis control programs.

Emergence of Japanese encephalitis virus genotype V in the Republic of Korea.

BACKGROUND: Japanese encephalitis virus (JEV) genotype V reemerged in Asia (China) in 2009 after a 57-year hiatus from the continent, thereby emphasizing a need to increase regional surveillance efforts. Genotypic characterization was performed on 19 JEV-positive mosquito pools (18 pools of Culex tritaeniorhynchus and 1 pool of Cx. bitaeniorhynchus) from a total of 64 positive pools collected from geographically different locations throughout the Republic of Korea (ROK) during 2008 and 2010. FINDINGS: Two regions of the JEV genome were sequenced from 19 pools; the envelope gene and the nonstructural protein 5 (NS5)/3'-untranslated region (UTR). Eighteen pools of Culex tritaeniorhynchus and one pool of Cx. bitaeniorhynchus were positive for genotype I and genotype V, respectively. Sequence alignment of the complete E gene from Cx. bitaeniorhynchus showed high amino acid similarity (98.8%) to the Muar strain, characterized as the first report of genotype V, isolated from an encephalitis patient in Malaysia in 1952. CONCLUSION: This study represents the first report of JEV genotype V in the ROK. The reemergence of genotype V in Asia (China and ROK) after more than a half-century and its discovery in Cx. bitaeniorhynchus, a mosquito species previously unknown to carry JEV in the ROK, emphasizes the need for enhanced JE surveillance to monitor the dynamics of JEV strains within the region. Future findings may have implications with regard to JEV vaccination/prevention strategies.

Japanese encephalitis virus in culicine mosquitoes (Diptera: Culicidae) collected at Daeseongdong, a village in the demilitarized zone of the Republic of Korea.

In total, 22,846 (17,793 culicines and 5,053 Anopheles spp.) female mosquitoes were captured by a Mosquito Magnet trap at Daeseongdong, a small village adjacent to the military demarcation line (center of the demilitarized zone) in northern Gyeonggi Province, Republic of Korea (ROK). Culicine mosquitoes were identified to species, placed in pools of up to 30 mosquitoes each, and screened for flavivirus using a SYBR Green I-based real-time polymerase chain reaction. In total, 51/660 pools positive for flaviviruses and confirmed by DNA sequencing of the NS5 region, were positive for Japanese encephalitis virus (family Flaviviridae, genus Flavivirus, JEV) (50 Culex tritaeniorhynchus Giles and one Culex bitaeniorhynchus Giles). The JEV maximum likelihood estimations (MLEs) (estimated number of viral RNA-positive mosquitoes per 1,000) for Cx. tritaeniorhynchus and Cx. bitaeniorhynchus were 9.7 and 0.9, respectively. This is the first report of a Cx. bitaeniorhynchus positive for JEV in the ROK. JEV is a local civilian and military health threat and a significant concern for nonimmune (unvaccinated) U.S. soldiers, civilians, and family members deployed to the ROK.

Comparison of PCR and microscopy for the detection of asymptomatic malaria in a Plasmodium falciparum/vivax endemic area in Thailand.

OBJECTIVE: The main objective of this study was to compare the performance of nested PCR with expert microscopy as a means of detecting Plasmodium parasites during active malaria surveillance in western Thailand. METHODS: The study was performed from May 2000 to April 2002 in the village of Kong Mong Tha, located in western Thailand. Plasmodium vivax (PV) and Plasmodium falciparum (PF) are the predominant parasite species in this village, followed by Plasmodium malariae (PM) and Plasmodium ovale (PO). Each month, fingerprick blood samples were taken from each participating individual and used to prepare thick and thin blood films and for PCR analysis. RESULTS: PCR was sensitive (96%) and specific (98%) for malaria at parasite densities > or = 500/microl; however, only 18% (47/269) of P. falciparum- and 5% (20/390) of P. vivax-positive films had parasite densities this high. Performance of PCR decreased markedly at parasite densities <500/microl, with sensitivity of only 20% for P. falciparum and 24% for P. vivax at densities <100 parasites/microl. CONCLUSION: Although PCR performance appeared poor when compared to microscopy, data indicated that the discrepancy between the two methods resulted from poor performance of microscopy at low parasite densities rather than poor performance of PCR. These data are not unusual when the diagnostic method being evaluated is more sensitive than the reference method. PCR appears to be a useful method for detecting Plasmodium parasites during active malaria surveillance in Thailand.

Morphological variations of Anopheles minimus A in Tak Province, Thailand.

Anopheles minimus Theobald is one of the major vectors of malaria throughout the Oriental Region, and it's complex consists of at least 2 sibling species (A and C) in Thailand. This study aimed to determine the morphological variations of wings of An. minimus A and to clarify the specific status of An. minimus in Ban Khun Huay, Ban Pa Dae, and Ban Tham Seau, Mae Sot district, Tak Province, Thailand. Anopheline larvae were collected from the fields between October 2002 and September 2003, allowed to emerge into adults in the laboratory and identified by morphological and molecular characterization. About 1,715 of female An. minimus A were separated into 8 groups based on their wing scale patterns. Polymerase Chain Reaction Restriction Fragment Length Polymorphism (PCR-RFLP) assay (ribosomal DNA ITS2) confirmed the identification of An. minimus A in all 8 groups.

Water quality and breeding habitats of anopheline mosquito in northwestern Thailand.

Malaria transmission is dependent upon many hydrology-driven ecological factors that directly affect the vectorial competence, including the presence of suitable habitats for the development of anopheline larvae. Larval habitats were identified and characterized at three malaria endemic villages (Ban Khun Huay, Ban Pa Dae, and Ban Tham Seau) in Mae Sot district, Tak Province, in northwestern Thailand between July 2002 and June 2003. The Global Positioning System (GPS) was used to provide precise locational data for the spatial distribution of anopheline mosquito larvae and their habitats. Ten habitat categories were identified. Eighteen adult Anopheles species were identified from larvae in all the surveyed habitats. An. minimus was the most common species throughout the year. The relationship between eight abiotic variables (temperature, hardness, carbon dioxide, dissolved oxygen, nitrate, phosphate, silica and pH) and the abundance of four major species of malaria vectors (An. (Cel.) dirus, An. (Cel.) minimus, An. (Cel.) maculatus, and An. (Cel.) sawadwongporni), and six species of non-vectors (An. (Cel.) kochi, An. (Cel.) jamesii, An. (Ano.) peditaeniatus, An. (Ano.) barbirostris, An. (Ano.) campestris, and An (Cel.) vagus) larvae was investigated. The results from the multiple regression models suggest that hardness, water temperature and carbon dioxide are the best predictor variables associated with the abundance of An. minimus larvae (p < 0.001); water pH for An. dirus larvae (p < 0.001); temperature and pH for An. kochi larvae (p < 0.01); temperature and silica concentration for An. jamesii larvae (p < 0.001); dissolved oxygen and silica concentration for An. campestris larvae (p < 0.001); and pH and silica concentration for An. vagus larvae (p < 0.001). We could not identify key environmental variables for An. maculatus, An. sawadwongporni, An. peditaeniatus, and An. barbirostris.

Geographical distribution of Anopheles minimus species A and C in western Thailand.

Elucidating vector distribution based on an accurate species identification is important to understanding the nature of the species complex in order to achieve vector control. Morphologically, An. minimus s.l. is difficult to distinguish from both its species complex and its closely related species. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and a single multiplex-allele specific PCR developed for species identification were applied in this study in comparison with morphological identification. Both methods were used, combining with geographical information systems to determine the distribution of An. minimus species A and C. The investigation on the breeding habitats was performed in the malarious area of western Thailand. Anopheles larvae were collected from 36 bodies of water among five districts (Sangkhaburi, Thong Pha Phum, Si Sawat, Muang, and Sai Yok) of Kanchanaburi Province, Thailand. In this study, An. minimus A larvae were present in all study districts but the association differed when focusing on study sites within each district. Although there were many reports of An. minimus A in Ban Phu Rat and Ban Phu Toei villages in Sai Yok District, we did not find the breeding sites of species A in those two areas. An. minimus A and C were found in Ban Phu Ong Ka village in Sai Yok District. The breeding habitats of An. minimus C were present covering 30-40 km of distance in northern part of Sai Yok and this species was also found in the central and southern parts of Si Sawat District.