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.

[Evaluation on malaria situation in areas along Yellow River and Huaihe River by indirect fluorescent antibody test].

OBJECTIVE: To evaluate malaria situation by indirect fluorescent antibody test (IFAT) in 12 villages of six counties along Yellow River and Huihe River in Anhui, Henan and Hubei Provinces. METHODS: Cross sectional seroepidemiologic survey was conducted through IFAT in Anopheles sinensis area and A. anthropophagus plus A. sinensis area respectively in 2006 and 2007. The blood-filter papers collected from residents were tested by IFAT to calculate malaria antibody positive rate (APR) and positive GMRT in different age groups. The predictive infection probability was evaluated by reversible catalytic model based on antibody positive rate in the group of less than 20 year-old and compared to the annual parasite incidence. RESULTS: In 2006 and 2007, the average antibody positive rate in the investigated villages was 6.1% and 12.0% respectively. In An. sinensis areas, the APR in 2007 (12.0%) was significantly higher than that in 2006 (4.1%) (chi2 = 69.9, P < 0.01) while in An. anthropophagus plus An. sinensis areas there was no significant difference in 2006 (10.3%) and 2007 (12.1%) (chi2 = 0.17, P > 0.05). The average positive GMRT of the 12 villages was 26.2 in 2007, slightly higher than that in 2006. In both An. sinensis area and An. anthropophagus plus An. sinensis area, the predictive infection probabilities were higher than the annual parasite incidence with an average 117.3 and 17.20 times higher than the latter respectively. 87.8% of the antibody positive cases were asymptomatic, indicating that there were potential infection sources with symptomatic parasitaemia in the areas. CONCLUSION: Malaria is still prevalent in areas along Yellow River and Huihe River and the actual number of malaria cases are underestimated by the case report system. Malaria reemergence may occur in An. sinensis area but relatively stable in An. anthropophagus plus An. sinensis area.

[Study on the thresholds of malaria transmission by Anopheles anthropophagus in Hubei Province].

OBJECTIVE: To study the thresholds and potential of malaria transmission by Anopheles anthropophagus in Hubei Province and provide indicators for the disease surveillance, early warning, prevention and control in the locality. METHODS: From July to August 2001, field investigations on vectors and malaria situation were carried out in the village of Yanjiafan, Suizhou City, where the malaria incidence was high. The entomological investigations included the man-biting rate, the proportion of parous anophelines, the human blood index and the blood preference. The others included malaria incidence and parasite rate in human population, the intervals from episode to treatment of the cases, and collection of data on the mean temperature in the area. Based on the formula of basic reproductive rate, the critical man-biting rate was estimated. RESULTS: 92.6% (63/68) of An. anthropophagus were found to have human blood meals, it occupied 91.5% (97/106) of the mosquitoes in human dwellings, its human blood index and vectorial capacity were 12.5 times (0.50/0.04) and 6.5 times (0.9448/0.1449) higher than those of An. sinensis. The critical man-biting rate was 0.2823 and the adjusted man-biting rate was 3.5 times of its critical man-biting rate (0.9892/0.2823). The malaria incidence was 0.65% (12/1844) and the parasite rate in pupils was 0.51%(1/198). CONCLUSION: A reduction of the adjusted man-biting rate of An. anthropophagus by 71.5% is needed for interrupting malaria transmission by this vector in the study area.

[Epidemic situation and control strategy of imported malaria in Hubei Province from 2006 to 2011].

OBJECTIVE: To understand the characteristics of imported malaria in Hubei Province so as to provide the evidence for formulating the prevention and control strategy. METHODS: The data of all imported malaria cases from 2006-01 to 2011-07 were retrospectively analyzed, and at the same time, the imported malaria cases since 2010 were interviewed through telephone and the cases since 2011-05 were interviewed face to face. All data were analyzed with EpiInfo. RESULTS: There were 195 imported malaria cases from 2006-01-01 to 2011-07-07, and there was a rising trend but there was no obvious difference among seasons. The interval between the disease onset and diagnosis was shorter in high educated people than low educated people (chi2 = 10.93, P < 0.01) and in the severe cases than the slight cases (chi2 = 4.58, P < 0.05). The severe rates of malaria cases were 70.4% and 82.9% in the low educated group and the high educated group, respectively (chi2 = 7.02, P < 0.01). Non-condition regression analysis showed that there were three influence factors which affected severe malaria, including whether or not self-treatment instead of seeing doctor when being fever; whether or not having health education before going abroad; whether or not considering malaria of the initial diagnosis doctor. CONCLUSION: It is very important to strengthen the personnel abroad malaria health education, the malaria blood smear microscopic inspection in medical institutions, and the initial diagnosis doctor's awareness on malaria.

[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.