Development and application of an AllGlo probe-based qPCR assay for detecting knockdown resistance (kdr) mutations in Anopheles sinensis.

BACKGROUND: Anopheles sinensis is one of the most important malaria vectors in China and other Southeast Asian countries. High levels of resistance have been reported in this species due to the long-term use of insecticides, especially pyrethroids, for public health and agricultural purposes. Knockdown resistance (kdr) caused by a single base pair mutation in the gene encoding the sodium channel is strongly associated with pyrethroid insecticide resistance in many Anopheles mosquitoes. There are few methods currently available for detecting kdr mutations in An. sinensis. METHODS: A novel AllGlo probe-based qPCR (AllGlo-qPCR) method was developed to screen for the predominant kdr mutations in An. sinensis mosquitoes from the Jiangsu Province. The results from AllGlo-qPCR, allele-specific PCR (AS-PCR), and TaqMan-MGB probe-based qPCR (TaqMan-qPCR) were compared. A comparative analysis of the equipment required, ease of use and cost of the available methods was also performed. Finally, the AllGlo-qPCR method was used to detect the frequencies of kdr mutations from the other four provinces in central China. RESULTS: Six kdr genotypes were detected in An. sinensis from the Jiangsu Province by DNA sequencing. The AllGlo-qPCR method detected all of the kdr genotypes with a high level of accuracy (97% sensitivity and 98% specificity). AllGlo-qPCR correctly determined the kdr genotypes of 98.73% of 158 An. sinensis samples, whereas TaqMan-qPCR and AS-PCR correctly identified 96.84% and 88.61% of mutations, respectively. Furthermore, the AllGlo-qPCR method is simpler to perform, requires less equipment, and exhibits a moderate expense cost comparing with the other tested methods of kdr mutation detection. Samples collected from four of the other provinces in central China showed a high frequency of kdr mutation in An. sinensis, as detected by the established AllGlo-qPCR method. CONCLUSION: The novel AllGlo-qPCR method developed for kdr mutation detection in An. sinensis exhibits greater specificity and sensitivity than currently available methods and is more cost-effective; therefore, it represents a useful tool for entomological surveillance.

An aquaporin and an aquaglyceroporin have roles in low temperature adaptation of mosquitoes (Anopheles sinensis).

Mosquitoes (Anopheles sinensis), widely geographically distributed in Asia including China, are the primary vector of the malaria parasite Plasmodium vivax and other parasitic diseases such as Malayan filariasis. An. sinensis can survive through low winter temperatures. Aquaporin channels are found in all life forms, where they facilitate environmental adaptation by allowing rapid trans-cellular movement of water (classical aquaporins) or water and solutes such as glycerol (aquaglyceroporins). Here, we identified and characterized 2 aquaporin (AQP) homologs in An. sinensis: AsAQP2 (An. sinensis aquaglyceroporin) and AsAQP4 (An. sinensis aquaporin). When expressed in frog (Xenopus laevis) oocytes, AsAQP2 transported water, glycerol, and urea; AsAQP4 transported only water. Water permeation through AsAQP2 and AsAQP4 was inhibited by mercuric chloride. AsAQP2 expression was slightly higher in adult female mosquitoes than in males, and AsAQP4 expression was significantly higher in adult males. The 2 AsAQPs were highly expressed in Malpighian tubules and midgut. AsAQP2 and AsAQP4 expression was up-regulated by blood feeding compared with sugar feeding. At freezing point (0 °C), the AsAQP4 expression level increased and An. sinensis survival time reduced compared with those at normal temperature (26 °C). At low temperature (8 °C), the AsAQP2 and AsAQP4 expression levels decreased and survival time was significantly longer compared with those at 26 °C. These results suggest that AsAQP2 and AsAQP4 have roles in water homeostasis during blood digestion and in low temperature adaptation of A. sinensis. Together, our results show that the 2 AQPs are important for mosquito diuresis after blood feeding and when exposed to low temperatures.

The complete mitochondrial genome of major malaria vector Anopheles anthropophagus (Diptera: Culicidae) in China.

Anopheles anthropophagus (Xu and Feng 1975) is the major vector of malaria in Eastern and Southern China. The species An. anthropophagus is considered a synonym of An. lesteri (Baisas & Hu, 1936), although they differ in several key biological characteristics. Here, we report the complete mitochondrial genome of An. anthropophagus for the first time. The mitogenome of An. anthropophagus is a typical circular, double-stranded molecule with a total length of 15,413 base pairs, and contains 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and an AT-rich control region. A phylogenetic analysis of the complete mitogenomes of 16 species of Anopheles (Culicidae) revealed that An. anthropophagus is closely related to An. sinensis (Wiedemann 1828), in the family Culicidae. The An. anthropophagus mitogenome provides new data for further taxonomic and phylogenetic studies of the genus Anopheles.

Hepatitis B virus nucleic acid testing in Chinese blood donors with normal and elevated alanine aminotransferase.

BACKGROUND: Nucleic acid testing (NAT) is currently not a routine donor test in China. The aim of this study was to evaluate the current residual risk of hepatitis B virus (HBV) transmission and the value of ALT testing in preventing HBV infection. STUDY DESIGN AND METHODS: From January 2008 to September 2009, a total of 5521 qualified donations by routine screening and 5034 deferred donations due to elevated ALT alone were collected from five blood centers. Samples were tested for HBV DNA by triplex individual-donation (ID)-NAT (ULTRIO assay, on the TIGRIS system, Novartis Diagnostics). HBV NAT-reactive samples were further analyzed by HBV serology, alternative NAT, and viral load and were diluted to simulate if they could be detected in a minipool-NAT. RESULTS: There was no significant difference in the HBV NAT-yield rate between the qualified donations group (5/5521) and the deferred donations group (4/5034). Of these nine potential HBV-yield cases, one donor (11%) was a possible HBV window-period donor, one (11%) was a chronic HBV carrier, and seven (78%) had probable or confirmed occult HBV infections. Of seven potential HBV-yield cases quantified, the viral loads were less than or equal to 70.0 IU/mL. Minipool testing (minipools of 4, 8, and 16 donations) would miss 43% to 79% of the nine HBV-yield donations. CONCLUSIONS: Based on our findings in qualified donations, we estimate that the nationwide implementation of ID-NAT testing for HBV DNA in China would detect an additional 9964 viremic donations per year. ALT testing seems to have no significant value in preventing transfusion-transmitted HBV infection. ID-NAT versus simulated minipool-NAT using the ULTRIO test demonstrates the benefit to implement a more sensitive NAT strategy in regions of high HBV endemicity.

[Analysis of Causes and Condition of Blood Donation Reaction Under Collective Emergency Blood Donation].

OBJECTIVE: To explore the causes and specific conditions of blood donation reaction under the collective emergency unpaid blood donation, and to provide theoretical basis and decision-making reference for drafting the collective emergency unpaid blood donation and blood donation safety. METHODS: Through a combination of prospective and retrospective models, and statistical methods were used to analyze the causes and conditions of the blood donation response of 10401 people participating in collective emergency unpaid blood donation during 2016.1-2018.8. RESULTS: A total of 10401 person-times donated blood in a sitting manner, and a total of 293 blood donation reactions occurred. By improving the blood donation services year by year, the moderate blood donation reaction during the year 2017 and 2018 was significantly lower than that in 2016 (P＜0.05). In the actual blood donation group of≤100, 200, 300 and 400 ml, the incidence of blood donation reaction was statistically significant (P＜0.05); the incidence of blood donation reaction in the blood donors for 1,2,3 and ＞3 drnations was also statistically significant (P＜0.05); the blood donation reactions rate of B antigen containers was significantly different from the donors without B antigen (P＜0.05); the incidence of blood donation reaction with related to the weight of the donor. CONCLUSION: The blood donation reaction of collective emergency unpaid blood donation closely relates with mental factors, blood donation service, blood donation frequency and body weight of the blood donor. The first blood donation is more likely to produce blood donation reaction. The blood donation volum≤ 100 ml from blood donors is resulted mostly from blood donation reactions.

[Research Progress in Acute Hemolysis and Safe Blood Transfusion of Glucose-6-Phosphate Dehydrogenase Deficiency-Review].

Glucose-6-phosphate dehydrogenase deficiency (G6PD) is one of hereditary diseases sariously influencing the human health. G6PD is characterized by wide distribution, high incidence, inducing the hemolysis, complex mechanism of hemolysis and common occurence in children and so on. The blood transfusion is most effective method for acute ouset of hemolysis, but the risk is more high, thereby it is necessary to guarante the safety of blood transfusion. In addition of the routine managementin blood transfusion and standard procedures, the basic rules and indicators of blood transfusion must be grasped, the blood preparations should be known well, the blood protection must be strengthened, and the measures of personalized psychologic interference must be drawn up carnestly and performed strictily so as to improve the therapeutic efficacy and safe of blood transfusion. Through the promotion and development of social pubbicity, elucation activities, preventive and control measures and medical levels, many patients have been freed from healthy problems as soon as possible. In this review, the research progress on acute hemolysis and safe blood transfusion in G6PD are summarized.

Monitoring of malaria vectors at the China-Myanmar border while approaching malaria elimination.

BACKGROUND: Tengchong County was one of the counties located at the China-Myanmar border with high malaria incidence in the previous decades. As the pilot county for malaria elimination at the border area, Tengchong County is aiming to be the first county to achieve malaria elimination goal. A cross-sectional entomological survey was carried out to evaluate the feasibility of elimination approach and assess the receptivity of malaria reintroduction. METHODS: Light traps associated with live baits were used to investigate the abundance of adult mosquitoes in nine villages in Tengchong County. Light traps were set to collect adult mosquitoes in both human houses and cowsheds from dusk till dawn in each site. RESULTS: A total of 4948 adult Anopheles mosquitoes were collected from May to December in two villages. Of the mosquitoes were captured, 24.2% were in human houses and 75.8% in cowsheds. The peak of abundance occurred in July for An. sinensis and in September-October for An. minimus (s.l.) Ten Anopheles species were collected, the most prevalent being An. sinensis (50.3%), An. peditaeniatus (31.6%) and An. minimus (s.l.) (15.8%), contributing to 97.6% of the sample. Potential breeding sites were also investigated and a total of 407 larvae were collected, with An. sinensis (50.1%) and An. minimus (s.l.) (46.2%) as predominant species. Ponds and rice fields were the two preferred breeding sites for Anopheles mosquitoes; however, the difference between the number of adults and larvae captured suggest other breeding sites might exist. Both An. sinensis and An. minimus (s.l.) were found zoophilic with human blood index as 0.21 and 0.26, respectively. No Plasmodium positive Anopheles specimens were found by PCR among 4,000 trapped mosquitoes. CONCLUSIONS: Although no indigenous malaria cases have been reported in Tengchong County since 2013, there is still a risk from the presence of vectors in the context of human population movements from neighboring malaria endemic areas. The presence of An. sinensis, associated to rice fields, is particularly worrying. Sustained entomological surveillance is strongly suggested even after malaria elimination certification.

[Effect of different temperatures on development of Aedes albopictus].

OBJECTIVE: To explore the effect of different temperatures on the different development stages of Aedes albopictus. METHODS: The changes at different development stages of mosquitoes (egg, larva, pupae) and gonotrophic cycle were observed at different temperature conditions of 10, 15, 20, 25, 30, 35 °C and 40 °C. The full developmental cycles were compared during different temperatures. RESULTS: All the stages of the mosquitoes could not develop at 10 °C. Under the different temperatures of 15, 20, 25, 30, 35 °C and 40 °C, the hatchabilities of the mosquitoes were 0, 32%, 82%, 83%, 82% and 59% respectively; the pupation rates of the mosquitoes were 38%, 53%, 84%, 88%, 72% and 42% respectively; and the emergence rates of the mosquitoes were 92%, 95%, 97%, 97%, 83% and 17% respectively. The mosquitoes could well develop at 20, 25, 30 °C and 35 °C, the development time was 37.73, 18.50, 16.92 and 13.66 days respectively. CONCLUSION: The development time of Aedes albopictus is shorter at the higher temperature. The optimum temperature for the mosquitoes to develop is between 25-30 °C, and higher or lower the temperatures will suppress the development of the mosquitoes.

[Analysis of the sequences of the ribosomal DNA ITS2 region for differentiating anopheline mosquitoes within hyrcanus group].

OBJECTIVE: To analyze the genetic characteristics of ribosomal DNA ITS2 region for differentiating anopheline mosquitoes within Hyrcanus group. METHODS: The ribosomal DNA ITS2 region of both laboratory line and filed collected An. anthropophagus and An. sinensis as well as the field collected An. yatsushiroensis and An. lesteri were amplified and sequenced. The sequencing data were then analyzed for the restriction mapping using Omega Sequencing analysis program. RESULTS AND CONCLUSION: The length of the sequences of An. sinensis, An. anthropophagus, An. yatsushiroensis and An. lesteri are 472, 452, 456 and 456 bp respectively. The restriction mapping showed that there were different restriction digesting sites among the ribosomal DNA ITS2 region sequences from An. sinensis, An. anthropophagus, An. yatsushiroensis and An. lesteri. On the basis of the sequence differences among the anopheline species within Hyrcanus group, it is possible to develop new technique for genetic identification of anopheline mosquitoes.

[Experimental observation of toxic effect of Bacillus thuringiensis var. israelensis against Aedes, Culex and Anopheles larvae].

OBJECTIVE: To evaluate the toxic effect of Bacillus thuringiensis var. israelensis (Bti) wettable powder against Aedes, Culex and Anopheles larvae. METHODS: The biological assay was applied to test the lethal concentration of 50% (LC50) of Bti wettable powder against Aedes, Culex and Anopheles larvae. RESULTS: The LC50(s) of Bti wettable powder against Aedes albopictus, Culex pipiens pallens and Anopheles sinensis larvae were 0.104, 0.160 microg/ml and 0.324 microg/ml, respectively; its biological potencies against them were 0.125, 0.192 IU/ml and 0.389 IU/ml, respectively. The LC50(s) of continuous contact of Bti wettable powder with An. sinensis stage III larvae for 1, 2 d and 3 d were 0.324, 0.092 microg/ml and 0.032 microg/ml, respectively, and its biological potencies were 0.389, 0.110 IU/ml and 0.038 IU/ml, respectively. The LC50(s) of the bacteria against An. sinensis stage I , II, III, IV were 0.024, 0.137, 0.324 microg/ml and 0.450 microg/ml, respectively, and the biological potencies were 0.029, 0.164, 0.389 IU/ml and 0.540 IU/ml, respectively. CONCLUSION: Bti wettable powder has a good toxicity to Aedes, Culex and Anopheles larvae, especially for the latter two. It is better to apply the bacteria at the early stage of mosquito larvae.

[Surveillance of malaria in provincial surveillance sites in Jiangsu Province, 2013].

OBJECTIVE: To understand the epidemic situation and influencing factors of malaria in Jiangsu Province and grasp its epidemic regularity and trend. METHODS: According to the malaria prevalence in Jiangsu Province, 6 counties (city, district) including Yixing, Suining, Wujin, Hai'an, Ganyu and Xuyi were selected as provincial surveillance sites to survey malaria epidemic conditions. The basic information, blood test results of fever patients, case investigation, information of malaria patients, monitoring data of investigation and disposition of the malaria focus were collected and analyzed. RESULTS: In 2013, the blood tests of 66 723 fever patients were performed, the average blood smear checking rate was 1.10%, and the average positive rate was 0.08% (52 plasmodium positive individuals) in the 6 areas. For these 52 plasmodium positive individuals, the blood retests and case investigations were completed within 3 days after these cases were reported by the network system, and the investigation confirmed that they were foreign imported malaria cases. The malaria focus investigation and disposition were finished within 1 week and the data were reported by the Parasitic Diseases Information System. Four of 52 cases were recrudescence during the follow-up. Among the 52 cases, 20 people went abroad themselves and 4 were labors of private enterprises, 21 people came back without the accompanied. CONCLUSIONS: With the development of the malaria elimination program in Jiangsu Province, the eliminating malaria "targeted 1-3-7" working pattern has been comprehensively implemented. The personnel monitoring for labors who returned from overseas working will be a key in the future.

[Study on TaqMan-MGB real-time fluorescence quantitative PCR to detect gene mutation of kdr from Anopheles sinensis].

OBJECTIVE: To establish a Real-time Fluorescence Quantitative PCR to detect the kdr gene mutation in Anopheles sinensis. METHODS: One pair of primers and three TaqMan-MGB probes were designed based on kdr gene and its L1014 locus mutations of A. sinensis. After optimization, the Real-time Fluorescence Quantitative PCR was verified by using 6 types of A. sinensis samples with different kdr gene types. Additionally, 50 laboratory samples and 113 field samples were tested by this method. RESULTS: The established Real-time Fluorescence Quantitative PCR could identify 6 different kdr gene types in A. sinensis. The mutation could be detected by single-tube Fluorescence Quantitative PCR, and the detail mutation type could be further identified by double-tube Fluorescence Quantitative PCR. By using this method, 50 laboratory samples were confirmed as wild type homozygotes. Among 113 field samples, 12 were wild type homozygotes, others were L1014F or L1014C mutations, and the total mutation frequency was 87.61%. CONCLUSION: The new established TaqMan-MGB Real-time Fluorescence Quantitative PCR can be used to detect the kdr gene L1014 mutations of A. sinensis.

[Establishment of malaria early warning system in Jiangsu Province II application of digital earth system in malaria epidemic management and surveillance].

OBJECTIVES: To explore a new mode of malaria elimination through the application of digital earth system in malaria epidemic management and surveillance. METHODS: While we investigated the malaria cases and deal with the epidemic areas in Jiangsu Province in 2011, we used JISIBAO UniStrong G330 GIS data acquisition unit (GPS) to collect the latitude and longitude of the cases located, and then established a landmark library about early-warning areas and an image management system by using Google Earth Free 6.2 and its image processing software. RESULTS: A total of 374 malaria cases were reported in Jiangsu Province in 2011. Among them, there were 13 local vivax malaria cases, 11 imported vivax malaria cases from other provinces, 20 abroad imported vivax malaria cases, 309 abroad imported falciparum malaria cases, 7 abroad imported quartan malaria cases (Plasmodium malaria infection), and 14 abroad imported ovale malaria cases (P. ovale infection). Through the analysis of Google Earth Mapping system, these malaria cases showed a certain degree of aggregation except the abroad imported quartan malaria cases which were highly sporadic. The local vivax malaria cases mainly concentrated in Sihong County, the imported vivax malaria cases from other provinces mainly concentrated in Suzhou City and Wuxi City, the abroad imported vivax malaria cases concentrated in Nanjing City, the abroad imported falciparum malaria cases clustered in the middle parts of Jiangsu Province, and the abroad imported ovale malaria cases clustered in Liyang City. CONCLUSION: The operation of Google Earth Free 6.2 is simple, convenient and quick, which could help the public health authority to make the decision of malaria prevention and control, including the use of funds and other health resources.

[Cloning and sequence analysis of cDNA encoding aquaporin (AQP) gene from Anopheles sinensis].

OBJECTIVE: To clone and analyze the full-length sequence of aquaporin gene of Anopheles sinensis (AsAQP), so as to provide an insight into its biology functions. METHODS: The degenerate primers were used to amplify conserved region of AQP from An. sinensis cDNA. After then, the full-length cDNA of AsAQP was obtained by rapid amplification of cDNA ends (RACE). Concurrently, the bioinformatics methods were applied to analyze the obtained sequence. RESULTS: The obtained full-length cDNA of AsAQP consisted of 762 bp and 253 deduced amino acids with a predicted molecular mass of 63.2 kD. Bioinformatics analysis demonstrated that AsAQP had a typical structure with six membrane-spanning domains and an internal symmetry showing two highly conserved Asn-Pro-Ala (NPA) motif and possessing the consensus sequence of major intrinsic protein (MIP) superfamily. The AsAQP shared the identities of 76% and 78% with those of Culex quinquefasciatus and Aedes aegypti AQPs, respectively. Phylogenetic analysis indicated that AsAQP was clustered with Aedes and Culex AQPs. CONCLUSIONS: The full-length AsAQP is cloned by degenerate primers and RACE from An. sinensis. The AsAQP gene is a member of MIP protein family, and has the typical function region. The study lays the foundation for further research on the function of AsAQP.

[Molecular identification of Anopheles hyrcanus complex by using single enzyme digestion PCR-RFLP method].

OBJECTIVE: To establish a novel molecular identification method for discrimination of members within Anopheles hyrcanus complex. METHODS: The sequences of the ribosomal DNA second internal transcribed spacer (rDNA ITS2) region of An. hyrcanus complex, including An. anthropophagus, An. lesteri, An. sinesis and An. yatsushiroensisi were analyzed by using molecular biology software Vector NTI 9.0, and a specificity restriction enzyme was selected based on the restriction fragment length polymorphism. Thus the single enzyme digestion PCR-RFLP method was established for genetic identification of An. hyrcanus complex, and 452 anopheline mosquitoes captured in the field were tested, comparing with the results of the previously established double enzyme digestion PCR-RFLP method and traditional morphological classification. RESULTS: The molecular software analysis revealed that the restriction enzyme Dde I could digest rDNA ITS2 region of An. hyrcanus complex into different fragments, thus it could be used for single enzyme PCR-RFLP for An. hyrcanus complex identification, and the result was further confirmed by laboratory experiment. Furthermore, a total of 452 anopheline mosquitoes captured from 4 malaria endemic areas were tested by this single enzyme digestion PCR-RFLP method, and 20 of them were identified as An. anthropophagus, 6 as An. lesteri, 391 as An. sinesis, and 35 as An. yatsushiroensisi. The results were 100% accordant to the double enzyme digestion PCR-RFLP method, and 93.4% accordant to the traditional morphological classification. CONCLUSIONS: The newly established single enzyme digestion PCR-RFLP method can be used for An. hyrcanus complex identification, and is more simple and reliable than the traditional morphological classification, and it is a suitable tool for field entomology surveillance.

[Detection of Plasmodium sporozoites in mosquitoes by using fluorescent quantitative PCR].

OBJECTIVE: To establish a fluorescent quantitative PCR (FQ-PCR) method for quantitative detection and species identification of Plasmodium sporozoites in Anopheles mosquitoes. METHODS: One pair of human Plasmodium genus-specific primers based on 18S rRNA genes were used and the reaction system and reaction condition of FQ-PCR were optimized by using the mixture of Plasmodium 18S rRNA gene recombinant plasmids and Anopheles DNA as a template. The specificity was verified by using four Plasmodium spp. 18S rRNA gene plasmid DNA as well as mosquito DNA and the Plasmodium species was identified according to the value of melting temperature (Tm). The standard curve was made by using P. vivax 18S rRNA gene recombinant plasmids which were serially diluted by negative Anopheles DNA as a template. The sensitivity was analysed by using plasmid DNA and laboratory infected sporozoite positive mosquito DNA, respectively. The different parts and different amounts of Anopheles DNA were added into the reaction system to investigate the influence of Anopheles DNA on the assessment. RESULTS: There was no specific amplification for mosquito DNA and human blood DNA. There was specific amplification for Plasmodium 18S RNA gene recombinant plasmids and the Tm(s) of P. malariae, P. falciparum, P. ovale and P. vivax were 71.0, 72.7, 73.9 degrees C and 75.9 degrees C, respectively, which were easy to be identified. The standard curve indicated a good linear relationship between the cycle threshold (Ct) and template concentration (r = -0.99). The sensitivity was 50 copies of plasmid DNA or one sporozoite positive mosquito DNA diluted by 32 times of mosquito DNA. Anopheles DNA could inhibite the FQ-PCR reaction. The Ct value of amplification showed a good reproducibility both within the same experiment and among different experiments. CONCLUSION: The novel SYBR Green I based FQ-PCR method developed in this study shows a high sensitivity and specificity and it can be used for quantitative detection and species identification of sporozoites in mosquitoes.

[Establishment of early warning system of malaria epidemic situation in Jiangsu Province I impact of temperature on development of Plasmodium vivax in Anopheles sinensis in Jiangsu Province].

OBJECTIVE: To observe the developmental threshold temperature and the effective accumulated temperature of Plasmodium vivax sporozoites in Anopheles sinensis in Jiangsu Province, and to analyze the impact of temperature on the development of Plasmodium vivax. METHODS: The Anopheles sinensis mosquitoes were maintained under different temperatures of 16 +/- 0.5, 19 +/- 0.5, 22 +/- 0.5, 25 +/- 0.5, 28 +/- 0.5 degrees C and 31 +/- 0.5 degrees C in incubators after membrane feeding with Plasmodium vivax infected blood at laboratory. The salivary glands of Anopheles sinensis were dissected to confirm the development of sporozoite under different temperatures, and the developmental threshold temperature and the effective accumulated temperature of Plasmodium vivax sporozoites in Anopheles sinensis were calculated. The theoretical number of generations of Plasmodium vivax in Anopheles sinensis per year was calculated based on the average monthly temperatures of 13 municipalities in Jiangsu Province. RESULTS: The average developmental threshold temperature of Plasmodium vivax in Anopheles sinensis in Jiangsu Province was 15.31 degrees C, the average effective accumulated temperature was 109.81 day-degrees, and the optimal temperature for the proliferation of Plasmodium vivax was 25-28 degrees C. The theoretical generation number of Plasmodium vivax in sinensis in south Jiangsu was 1-3 generations being more than that in the north of the province. CONCLUSIONS: Temperature is one of the important influencing factors for the development of Plasmodium. The chance for Anopheles sinensis to transmit malaria increases under the temperature of 25-28 degrees C. However, other factors should be considered in the establishment of early warning system of malaria.

[Survey on malaria vectors in Jiangsu Province, 2005-2009].

OBJECTIVE: To understand the species, density and seasonal variation of malaria vectors in Jiangsu Province, so as to provide scientific evidence for malaria control. METHODS: From 2005 to 2009, 5 towns in Jiangsu Province were selected as surveillance sites, and the species of malaria vectors and their density and seasonal variation were studied by the outdoor and indoor trapping methods. The data of malaria cases were analyzed by the circular distribution method. RESULTS: Only Anopheles sinensis was captured in the 5 surveillance sites from 2005 to 2009, and its density peak was mainly appeared in the first half of July. The peak incidence of malaria was on 16th August, the distribution of cases was accordant with the seasonal variation of vectors. CONCLUSION: The surveillance and control of vectors should still be strengthened in the malaria control, so as to prevent the epidemic from rebounding as the increase of the density of Anopheles sinensis.

[Sensitivity of Anopheles sinensis to insecticides in Jiangsu Province].

OBJECTIVE: To understand the sensitivity of Anopheles sinensis to deltamethrin, DDT and malathion in Jiangsu province. METHODS: The adult mosquitoes were captured from the fields of Sihong, Yangzhong and Yixing counties (cities) and cultured, and by using the method recommended by WHO, their first filial generations knocked down at 10, 15, 20, 30, 40, 50, and 60 min after exposure with insecticides aforementioned were counted, and the mortality of mosquitoes tested after 24 h was recorded. RESULTS: The knock down rates of mosquitoes, which were the first generation (F0) of Anopheles sinensis captured from the field of Sihong, Yangzhong, Yixing counties (cities) to 0.05% deltamethrin were 28.57%, 57.14%, 52.38%, respectively 60 min after the exposure; and the mortality rates 24 h-post-exposure were 35.71%, 57.14%, 61.90%, respectively. The resistance degree to deltamethrin was assessed as "R" level. The knock down rates of mosquitoes 60 min after the exposure to 4% DDT were 9.52%, 2.38%, 4.76%, respectively, and the mortality rates 24 h-post-exposure were 47.62%, 50.00%, 40.48%, respectively. The resistance degree to DDT was assessed as "R" level. The knock down rates of mosquitoes 60 min after the exposure to 5% malathion were 11.90%, 28.57%, 28.13%, respectively, and the mortality rates 24 h after the exposure were 80.95%, 85.71%, 93.75%, respectively. The resistance degree to malathion was assessed as level "M". The knock down rates of Anopheles sinensis captured in day 1, day 7, day 15 and the F1 from Yixing 60 min after the exposure to 0.05% deltamethrin were 54.76%, 76.19%, 92.86%, and 52.38%, respectively, and the mortality rates post-24 h were 54.76%, 76.19%, 95.23% and 61.90%, respectively. The difference of mortality post-24 h between the mosquitoes of 1 day post-captured and F1 was not statistically significant (P > 0.05). The knock down rates 60 min after the exposure to 0.05% deltamethrin to the female and male F1 of Anopheles sinensis from Sihong were 28.57% and 40.48%, and the mortality rates post-24 h were 35.71%, 42.86% respectively, and the difference was not statistically significant (P > 0.05). CONCLUSIONS: Anopheles sinensis in the field of Jiangsu Province has developed severe resistance to deltamethrin and DDT, initial resistance to malathion. In order to prevent the development of resistance to the insecticides, the integrated management measures should be adopted in the future.

Adaptation of a visualized loop-mediated isothermal amplification technique for field detection of Plasmodium vivax infection.

BACKGROUND: Loop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions. METHODS: A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection. RESULTS: The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method. CONCLUSIONS: This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.