Development of a real-time PCR assay for Trypanosoma cruzi detection in blood samples

The aim of this study was to develop a real-time PCR technique to detect Trypanosoma cruzi DNA in blood of chagasic patients. Analytical sensitivity of the real-time PCR was assessed by two-fold serial dilutions of T. cruzi epimastigotes in seronegative blood (7.8 down to 0.06 epimastigotes/mL). Clinical sensitivity was tested in 38 blood samples from adult chronic chagasic patients and 1 blood sample from a child with an acute congenital infection. Specificity was assessed with 100 seronegative subjects from endemic areas, 24 seronegative subjects from non-endemic area and 20 patients with Leishmania infantum-visceral leishmaniosis. Real-time PCR was designed to amplify a fragment of 166 bp in the satellite DNA of T. cruzi. As internal control of amplification human RNase P gene was coamplified, and uracil-N-glycosylase (UNG) was added to the reaction to avoid false positives due to PCR contamination. Samples were also analysed by a previously described nested PCR (N-PCR) that amplifies the same DNA region as the real-time PCR. Sensitivity of the real-time PCR was 0.8 parasites/mL (50% positive hit rate) and 2 parasites/mL (95% positive hit rate). None of the seronegative samples was positive by real-time PCR, resulting in 100% specificity. Sixteen out of 39 patients were positive by real-time PCR (41%). Concordance of results with the N-PCR was 90%. In conclusion, real-time PCR provides an optimal alternative to N-PCR, with similar sensitivity and higher throughput, and could help determine ongoing parasitaemia in chagasic patients.     

Ovipositional Behavior of Anopheles gambiae Mosquitoes

Mosquito eggs laid within two hours are necessary for transgenic (injection) studies, because mosquito eggs become hard after that period. Thus, in order to have eggs available within this two-hour window, it is important to understand the ovipositional behavior of Anopheles gambiae s.s.. In the present study, the ovipositional behavior of An. gambiae s.s. (Kisumu) was investigated in several different conditions: age of mosquitoes, time post blood meal to access oviposition substrate, and light conditions. Two groups of mosquitoes, 3–5 days old and 9–11 days old were blood-fed. For those mosquito groups, an oviposition dish was set either at 48 hours or 72 hours after the blood meal either in a light condition or in an artificial dark condition. The number of laid eggs was compared among the different conditions. The 3–5 day-old mosquitoes apparently produced a higher number of eggs than 9–11 day-old mosquitoes, while there was no significant difference between the two groups. The number of laid eggs per one surviving blood-fed mosquito in the dark condition was significantly higher than that in the light condition (p = 0.03). Providing an oviposition dish at 72 hours after blood meal resulted in a significantly higher number of laid eggs per one surviving blood-fed mosquito than at 48 hours after blood meal (p = 0.03). In conclusion, the optimal condition to have readily available egg supply for transgenic analysis was as follows: 3–5 day-old mosquitoes with an oviposition dish placed at 72 hours after the blood meal in a dark environment.     

Chlorfenapyr: a pyrrole insecticide for the control of pyrethroid or DDT resistant Anopheles gambiae (Diptera: Culicidae) mosquitoes

Owing to the development and spread of pyrethroid resistance in Anopheles gambiae in Africa there is an urgent need to develop alternative insecticides to supplement the pyrethroids. Chlorfenapyr is a pyrrole insecticide first commercialized for the control of agricultural pests and termites. Performance against An. gambiae bearing kdr (pyrethroid and DDT resistance) or Ace-1(R) insensitive acetylcholinesterase (organophosphate and carbamate resistance) mechanisms was studied using a variety of adult bioassay tests including a simulated-experimental hut system (tunnel tests) that allows uninhibited mosquito behaviour/insecticide interactions. Strains resistant to pyrethroids and organophosphates showed no cross resistance to chlorfenapyr. In cone bioassays on treated netting the mortality of adult mosquitoes showed an unexpected curvilinear response, with highest mortality occurring at intermediate dosages. Adults expressed irritability to chlorfenapyr at higher dosages, which might explain the dosage-mortality trend. Toxic activity of chlorfenapyr was slow compared to conventional neurotoxic insecticides and additional mortality occurred between 24h and 72 h. In tunnel tests, the dosage-mortality trend showed a more typical sigmoid response and most mortality occurred during the first 24h. Mosquito penetration through the holed, treated netting showed only limited inhibition and blood-feeding was not inhibited. Mortality rates in the kdr strain exposed to chlorfenapyr treated netting in tunnel tests were much higher than with permethrin treated netting over the same 100-500 mg/m(2) dosage range. Chlorfenapyr has potential for malaria control in treated-net or residual spraying applications in areas where mosquitoes are pyrethroid resistant. For treated-net applications chlorfenapyr might be combined with pyrethroid as a mixture to provide personal protection as well as to give control of resistant mosquitoes.     

Distribution of the molecular forms of Anopheles gambiae and pyrethroid knock down resistance gene in Nigeria

We investigated the distribution of the molecular M and S forms of Anopheles gambiae and the knock down resistance (kdr) gene associated with pyrethroid and DDT resistance in A. gambiae s.s. at 13 localities across Nigeria. Two-three days old adult female mosquito reared from larval collections were tested using standard WHO procedures, diagnostic test kits and impregnated papers to assess their pyrethroid resistance status. Specimens were identified by PCR assays and characterized for the kdr gene. DNA from adult A. gambiae s.s. collected from human dwellings were also tested for the presence of the kdr gene. The overall collection was a mix of the molecular M and S forms across the mangrove (63:37%), forest (56:44%), and transitional (36:64%) ecotypes, but almost a pure collection of the S form in the Guinea and Sudan-savanna. Results of insecticide susceptibility tests showed that mosquitoes sampled at seven localities were susceptible to permethrin, deltamethrin, and DDT, but populations of A. gambiae resistant to these insecticides were recorded at six other localities mainly in the transitional and Guinea-savanna ecotypes. The kdr gene was found only in the molecular S forms, including areas where both forms were sympatric. The overall kdr frequency was low: <47% in forest, 37-48% in the transitional, and 45-53% in Guinea-savanna. The data suggest that pyrethroid resistance in A. gambiae in Nigeria is not as widespread when compared to neighbouring West African countries.     

Repellency of essential oils of some plants from the Kenyan coast against Anopheles gambiae

Volatile oils extracted by hydrodistillation from six plant species growing in the Kenyan coast, Croton pseudopulchellus Pax, Mkilua fragrans Verdc. (Annonaceae), Endostemon tereticaulis (poir.) Ashby, Ocimum forskolei Benth., Ocimum fischeri Guerke and Plectranthus longipes Baker (Labiateae), were evaluated for repellency on forearms of human volunteers against Anopheles gambiae sensu stricto. All oils were found to be more repellent (RC50 range = 0.67-9.21 x 10(-5) mg cm(-2)) than DEET (RC50 = 33 x 10(-5) mg cm(-2)). The individual components of the oils were identified by GC-MS and GC co-injections with authentic standards. The repellency of 15 of the main constituents of the different oils (which had not been previously assayed) was evaluated. Although some of these showed relatively high individual repellencies, none was comparable to the parent essential oils. Partial synthetic blends of selected constituents with moderate or relatively high individual repellency against the vector were also assayed. Four of these exhibited activities comparable to or higher than those of the corresponding parent oils, indicating interesting blend effects in the repellent action of the oils against the mosquito. The implication of these results in the utilization of the plants is discussed.     

Development of a real-time PCR assay for the diagnosis of scrub typhus cases in India and evidence of the prevalence of new genotype of O. tsutsugamushi

A qualitative syber green real-time PCR with primers designed for a truncated portion of the 56kDa major outer membrane antigen gene of Orientia tsutsugamushi was used to diagnose scrub typhus from the blood or serum of suspected patients. Sixty-six blood and/or sera samples from fever cases, either with high index of suspicion for scrub typhus and/or positive by Weil-Felix test (> or = 1:160), were tested with the PCR. Specificity of the PCR was confirmed by end point melt curve analysis and sequencing of the amplicons. A nested PCR for determination of the serotypes of O. tsutsugamushi was performed on to the samples. In real-time PCR strong positive fluorescence was obtained in 73% of the suspected samples. Serotype-specific PCR amplification of some of the positive samples was indicative of the Kuroki type whereas the rest were non-responsive to this test. Sequence analyses of PCR amplicons indicated the presence of new, previously undescribed type of O. tsutsugamushi in this region. This one-step real-time PCR can be used for the detection and confirmation of scrub typhus, when used independently or in conjunction with, the Weil-Felix test, which is still the only available detection test for scrub typhus in most parts of the developing world. Elaborate studies need to be taken up to further evaluate its suitability as specific molecular tool for the diagnosis of scrub typhus and to delineate the prevalent strain types in these regions for a clear epidemiological understanding of this emerging infectious disease.     

From the Cover: Transmission potential of Rickettsia felis infection by Anopheles gambiae mosquitoes

A growing number of recent reports have implicated Rickettsia felis as a human pathogen, paralleling the increasing detection of R. felis in arthropod hosts across the globe, primarily in fleas. Here Anopheles gambiae mosquitoes, the primary malarial vectors in sub-Saharan Africa, were fed with either blood meal infected with R. felis or infected cellular media administered in membrane feeding systems. In addition, a group of mosquitoes was fed on R. felis-infected BALB/c mice. The acquisition and persistence of R. felis in mosquitoes was demonstrated by quantitative PCR detection of the bacteria up to day 15 postinfection. R. felis was detected in mosquito feces up to day 14. Furthermore, R. felis was visualized by immunofluorescence in salivary glands, in and around the gut, and in the ovaries, although no vertical transmission was observed. R. felis was also found in the cotton used for sucrose feeding after the mosquitoes were fed infected blood. Natural bites from R. felis-infected An. gambiae were able to cause transient rickettsemias in mice, indicating that this mosquito species has the potential to be a vector of R. felis infection. This is particularly important given the recent report of high prevalence of R. felis infection in patients with “fever of unknown origin” in malaria-endemic areas.In 2002, Rickettsia felis, an obligate intracellular bacterium that belongs to the spotted fever group of Rickettsia, was definitively described (1, 2). Over the past 2 decades, an increasing number of reports have implicated R. felis as a human pathogen, paralleling an increase in reports of the detection of R. felis in arthropod hosts throughout the world (1, 3).By 2011, more than 70 human cases of R. felis had been reported worldwide, including in Central and South America, Asia, northern Africa, and Europe (1). More cases have been published since then, including the first probable human cases in Australia (4). In sub-Saharan Africa, recent studies have challenged the importance of R. felis infection in patients with “fever of unknown origin,” with this bacterium detected in up to 15% of such patients (5–7). In 2011, a potential R. felis primary infection, called “yaaf,” was suspected in the case of an 8-mo-old girl in Senegal with polymorphous skin lesions similar to those seen in patients from Mexico (8). The epidemiologic and clinical picture of this emerging infection in Africa, including its potential vectors, is poorly understood, however.Various arthropods, but primarily fleas, have been associated with R. felis (1, 3). More specifically, the cat flea Ctenocephalides felis is the arthropod in which R. felis has been most frequently detected. To date, it is the sole confirmed biological vector of R. felis, with both horizontal and vertical transmission making this flea a potential reservoir for this bacterium (9–11). However, in some countries where R. felis appears to be highly prevalent, such as Senegal, neither cat fleas nor other arthropods have been implicated in its transmission (12).Mosquitoes are the most important vectors of infectious diseases in humans, with more than one-half of the global population at risk for exposure to mosquito-borne infections (13, 14). Anopheles gambiae is known to be the primary vector of malaria in Africa, whereas Aedes albopictus is a vector of dengue and chikungunya (15, 16). Interestingly, Ae. albopictus and An. gambiae mosquito cells support R. felis growth (1, 17). In 2012, Ae. albopictus from Gabon and An. gambiae molecular form S (the primary African malarial vector) from Ivory Coast tested positive for R. felis by species-specific real-time quantitative PCR (qPCR) (17, 18). More recently, several mosquito species from Senegal were found to harbor R. felis, including Ae. luteocephalus, An. arabiensis, An. ziemanni, An. pharoensis, An. funestus, and Mansonia uniformis (5). These data raise new issues with respect to the epidemiology of R. felis in Africa, including the degree of vector competence of mosquitoes. The objective of this work was to study the acquisition and transmission of R. felis by An. gambiae mosquitoes in an experimental model of infection.     

Multiple insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus from Benin, West Africa

Because free-insecticide treated net distribution is planned in Benin (West Africa) during the next few years, we investigated the type, frequency and distribution of insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus mosquitoes in four localities selected on the basis of contrasting agricultural practices, use of insecticides and environment. Bioassays with WHO diagnostic test kits were carried out using pyrethroid, carbamate, organophosphate and organochlorine insecticides. An. gambiae mosquitoes were identified to species and to M or S molecular forms using PCR techniques. Molecular and biochemical assays were carried out to identify kdr and Ace.1 mutations in individual mosquitoes and to detect any increase in the activity of enzymes typically involved in insecticide metabolism (oxidase, esterase and glutathion-S-transférases). WHO diagnostic tests showed high frequency of resistance in An. gambiae and Cx. quinquefasciatus to permethrin and DDT in three areas. This was consistent with the presence of target site insensitivity due to kdr mutation and to increased metabolism through enzymatic activity. Kdr was expressed in both M and S forms. However, less than 1% of An. gambiae or Cx. quiqnuefasciatus showed the presence of the Ace.1^R mutation. Carbamate/OP resistance was present at higher frequency in Culex than in An. gambiae. Dieldrin resistance was present in both species at all four localities. A higher frequency of pyrethroid-resistance was found in An. gambiae mosquitoes collected in urban areas compared to those collected in rice growing areas. The expansion of vegetable growing within urban areas probably contributed to selection pressure on mosquitoes. The detection of multiple resistance mechanisms in both An. gambiae and Cx. quinquefasciatus in Benin may represent a threat for the efficacy of ITNs and other forms of vector control such as indoor residual spraying in the future.     

The effects of rainfall and evapotranspiration on the temporal dynamics of Anopheles gambiae s.s. and Anopheles arabiensis in a Kenyan village

The population dynamics of the larval and adult life stages of the malaria vector Anopheles gambiae Giles were studied in Miwani, western Kenya, in relation to meteorological conditions. Larval density within a habitat, the number of larval habitats and sibling species composition were investigated as determinants of larval population dynamics. Female vector densities inside local houses and sibling species composition were investigated as determinants of adult population dynamics. Larval densities were estimated using a modified area-sampling method. Within the habitats, all instars showed a highly aggregated distribution, with the exception of second instars. A longitudinal study on the larval populations of A. gambiae s.l. in two different types of habitat (dirt track and ditch) was carried out, using a novel sampling procedure. A. gambiae s.s. and Anopheles arabiensis, the two sibling species occurring sympatrically in the study area, showed some spatial segregation between the two types of habitat. Rainfall was significantly correlated with the number of A. gambiae s.l. larval habitats during the first 6 weeks of study taking 1 week time lag into account, while over the entire 5-month study period correlations were less clear. With 1 week time lag, rainfall was also significantly correlated with the number of female A. gambiae s.l. collected from CDC-light traps in the study houses. Both larval and adult populations showed a significant increase in the proportion of A. gambiae s.s. within the mixed population of A. gambiae s.s. and A. arabiensis over time. Although not significantly correlated, the ratio of rainfall over precipitation/potential evapotranspiration (P/PE), indicative of the humidity conditions in the area, was probably the driving force of this increase.     

rDNA-ITS2-PCR assay for grouping the cryptic species of Anopheles culicifacies complex (Diptera: Culicidae)

Anopheles culicifacies, a predominant vector of malaria in India exists as a complex of five sibling species A, B, C, D and E, of which, except species B, all the rest are vectors with varying vectorial capacities. With a combination of PCR assays, it is possible to identify all the five members of this species complex. These assays include amplification of the rDNA-ITS2 region followed by digestion of the ITS2 amplicon using restriction enzyme, Rsa I which groups the five members of the An. culicifacies complex into two categories: species A and D forming one category and species B, C and E forming another. The samples grouped thus are then subjected to two allele-specific PCR assays (AD-PCR and BCE-PCR), which has been designed using sequence differences in the mitochondrial cytochrome oxidase II (CO II) subunit. The AD-PCR assay distinguishes species A and D, whereas the BCE-PCR assay distinguishes species B, C and E. In the present study, the differences in the ITS2 region of the five species was used to design a PCR assay which groups the five members into the same two categories as obtained after digestion of the ITS2-PCR product. This assay uses a common forward primer based on the 5.8S region and two reverse primers, which is specific for the two categories. Amplification of a PCR product of size 253bp indicates the presence of species A/D, while a product of size 409bp indicates the presence of species B/C/E. By using this ITS2 PCR assay, the three-step procedure is reduced to two cutting down the time and cost involved. The ITS2 PCR assay has been validated on specimens collected from different regions of India and the results confirm to the earlier reports on the distribution of the members of the An. culicifacies complex.     

Identification of four members of the Anopheles funestus (Diptera: Culicidae) group and their role in Plasmodium falciparum transmission in Bagamoyo coastal Tanzania

The role of Anopheles funestus group in malaria transmission was investigated in Bagamoyo coastal Tanzania, in the process of characterizing the area as a malaria vaccine testing site. Mosquitoes were sampled inside houses and multiplex PCR was used to identify 649 specimens. The following species were found: A. funestus s.s. (84.3%), A. leesoni (13.6%), A. rivulorum (1.5%) and A. parensis (0.6%). Multiplex PCR of 147 blood-fed specimens showed that over half (57.1%) of the identifiable blood meals were taken from human hosts, and human blood index in A. funestus and A. leesoni was 55% and 82% respectively. Plasmodium falciparum infection rate determined by nested PCR was 11% in A. funestus s.s. Although the abundance was low, 26 specimens of A. leesoni, two of A. rivolurum and one of A. parensis were found positive for P. falciparum. The presence of four A. funestus species in Tanzania emphasizes the relevance to define precisely their spatial and temporal distribution, specific behaviour, ecology and exact role in malaria transmission.     

Development and evaluation of a polymerase chain reaction (PCR) assay for the detection of Opisthorchis viverrini in fish

Human opisthorchiais caused by the liver fluke Opisthorchis viverrini is a major fish-borne trematode infection endemic in the Southeast Asian countries. The infection is acquired through consumption of raw fish harboring metacercariae of O. viverrini. Owing to potential risk of transmission of opisthorchiasis through fish trade, rapid and reliable detection methods have gained importance to ensure food safety. In the study described here, we report successful development and evaluation of a polymerase chain reaction (PCR) assay for the detection of O. viverrini, based on the nucleotide sequence derived in this study. The assay is specific with no cross-reaction with other trematodes commonly found in fish including the closely related species, Clonorchis sinensis. The sensitivity of the assay was determined to be 10(-12)ng of O. viverrini DNA while in artificially spiked fish meat, 3 metacercariae could be detected. The results suggest that the PCR method described here is specific to O. viverrini with potential application in fish quarantining.     

Real-time PCR assay for the identification of cutaneous Leishmania parasite species in Constantine region of Algeria

We evaluated the use of real-time PCR for the identification of cutaneous Leishmania species. The assay, based on the melting curve analysis of fluorescent products, allows the discrimination of four culture adapted strains (L. infantum MHOM/TN/80/IPT1, L. major MHOM/SU/73/5-ASKH, L. donovani MHOM/IN/80/DD8 and L. tropica MHOM/SU/74/K27). One hundred and twenty-nine skin lesions, spotted on filter paper, were collected from patients consulting for suspicion of cutaneous leishmaniasis (CL) at the parasitology laboratory of Constantine Hospital (Algeria). Ninety-seven (75.2%) of the samples analyzed were positive. Sixty-one (5%) were related to L. major strain. These results indicate that PCR assay provides pleasant results with filter paper and represents a tool for the identification of old world CL.     

Loss of heterozygosity testing using real-time PCR analysis of single nucleotide polymorphisms

Purpose: Colon cancer is a genetic disease, caused by mutations in different oncogenes and tumor-suppressor genes. The aim of this study is to evaluate the usefulness of real-time PCR SNP analysis as a new technique in the loss of heterozygosity (LOH) analysis at the E-cadherin gene locus in sporadic colon cancer. Methods: One-hundred cases of human sporadic colon cancer and corresponding normal tissue samples were analyzed using two flanking polymorphic markers commonly used in the LOH analysis at the E-cadherin gene locus by conventional VNTR–LOH analysis. Two intragenic E-cadherin SNP markers were analyzed using real-time PCR SNP analysis. Results: LOH (17.6%) was detected using flanking markers, however, no LOH was detected when the intragenic E-cadherin SNP markers were introduced into our study. Since these markers are intragenic they more accurately represent the status of the E-cadherin gene than the previously used flanking markers. Conclusion: In conclusion, real-time PCR SNP analysis was found to be more accurate, faster, simpler, and a more high-throughput method than the conventional VNTR–LOH analysis.     

Malaria vectors in the Republic of Benin: Distribution of species and molecular forms of the Anopheles gambiae complex

Members of the Anopheles gambiae complex are among the best malaria vectors in the world, but their vectorial capacities vary between species and populations. A large-scale sampling of An. gambiae sensu lato was carried out in 2006 and 2007 in various bioclimatic areas of Benin (West Africa). The objective of this study was to collate data on the relative frequencies of species and forms within the An. gambiae complex and to produce a map of their spatial distribution. Sampling took place at 30 sites and 2122 females were analyzed. Two species were identified through molecular methods. The overall collection showed a preponderance of An. gambiae s.s., but unexpectedly, An. arabiensis was reported in the coastal-Guinean bioclimatic area characterized by a mean annual rainfall of >1500 mm where only An. gambiae s.s. was reported previously. Our study of Benin indicates that An. arabiensis would be adapted not only to the urban areas but also to the rural humid regions. Among 1717 An. gambiae s.s., 26.5% were of the M form and 73.3% were S form. Few hybrid specimens between the M and S forms were observed (0.2%). Only the spatial distribution of the M form appears to be mainly a function of bioclimatic area.Factors that influence the distribution of these malaria vectors are discussed. This study underlines the need of further investigations of biological, ecological, and behavioral traits of these species and forms to better appreciate their vectorial capacities. Acquisition of entomological field data appears essential to better estimate the stratification of malaria risk and help improve malaria vector control interventions.     

利用荧光定量RT-PCR和PCR方法检测结核分枝杆菌复合群

目的建立一种新的检测结核分枝杆菌复合群的荧光定量试验方法(R/P分析),探讨R/P分析检测临床标本中结核分枝杆菌复合群的应用价值。方法根据结核分枝杆菌复合群基因保守序列设计引物和探针构建质粒标准品。运用R/P分析检测54例确诊结核病人临床样本,检测的结果同时与培养法、荧光定量PCR法进行比较。结果不同临床标本用R/P分析诊断结核病,其敏感性高于荧光定量PCR法和培养法,阳性检出率分别为96.3%、83.3%和55.6%。结论 R/P方法是一种快速、特异的直接检测方法,它可以区分结核分枝杆菌复合群的死菌与活菌,因此可以更好的指导医生进行治疗,更准确地做出公共卫生决策。     

SYBR GreenⅠ染料法定量PCR用于人体疟原虫定量检测及虫种鉴别的研究

目的建立一种可用于疟原虫检测并同时鉴别虫种的荧光定量PCR方法。方法根据人体疟原虫18SrRNA基因序列特征,在疟原虫种特异性区域两侧的属特异性保守区设计引物,对4种人体疟原虫进行PCR扩增,将得到的扩增产物采用TA克隆法插入pGEMT载体制备标准质粒,用于制备标准曲线进行定量分析,并进行熔解曲线分析,测定各虫种扩增产物的熔解温度。结果 4种人体疟原虫均能扩增得到特异性片断,荧光定量PCR方法中建立的标准曲线相关关系较好（相关系数r=-1.00）,熔解曲线分析结果显示,4种人体疟原虫的熔解温度相差较大,分别为三日疟原虫71.3℃、恶性疟原虫72.8℃、卵形疟原虫74.6℃和间日疟原虫75.8℃。结论建立的SYBR GreenI染料法荧光定量PCR技术可同时进行人体疟原虫的定量检测和虫种鉴别。     

Evaluation of a PCR-RFLP-ITS2 assay for discrimination of Anopheles species in northern and western Colombia

Anopheles mosquitoes are routinely identified using morphological characters of the female that often lead to misidentification due to interspecies similarity and intraspecies variability. The aim of this work was to evaluate the applicability of a previously developed PCR–RFLP–ITS2 assay for accurate discrimination of anophelines in twelve localities spanning three Colombian malaria epidemiological regions: Atlantic Coast, Pacific Coast, and Uraba-Bajo Cauca-Alto Sinu region. The evaluation of the stability of the PCR–RFLP patterns is required since variability of the ITS2 has been documented and may produce discrepancies in the patterns previously reported. The assay was used to evaluate species assignation of 939 mosquitoes identified by morphology. Strong agreement between the morphological and molecular identification was found for species Anopheles albimanus, Anopheles aquasalis, Anopheles darlingi and Anopheles triannulatus s.l. (p ≥ 0.05, kappa = 1). However, disagreement was found for species Anopheles nuneztovari s.l., Anopheles neomaculipalpus, Anopheles apicimacula and Anopheles punctimacula (p ≤ 0.05; kappa ranging from 0.33 to 0.80). The ITS2–PCR–RFLP assay proved valuable for discriminating anopheline species of northern and western Colombia, especially those with overlapping morphology in the Oswaldoi Group.     

First report on Anopheles fluviatilis U in southeastern Iran

Anopheles fluviatilis James, one of the malaria vectors in Iran, is a complex of at least three cryptic species provisionally designated as species S, T and U. These species are morphologically indistinguishable at any stage of their life cycle and can be identified only by the examination of species-specific fixed inversions in the polytene chromosomes. Recently, sequence analysis of 28S D3 and second internal transcribed spacer (ITS2) regions of ribosomal DNA has revealed 7 haplotypes of S, U, T1, T2, Y, X and V within the complex. Identification of the cryptic species of the complex is of paramount importance in a disease control program due to contrasting differences in their vectorial efficiency, preference for feeding on humans and resting behavior. In this study we analyzed the sequence of 28S D3- and ITS2-rDNA loci to identify the species composition of the An. fluviatilis complex in Jiroft and Chabahar districts, two of the most important endemic malaria foci in southeastern corner of Iran. The ITS2 sequence analysis revealed that all of the An. fluviatilis specimens were identical to the Y/T2 haplotype of An. fluviatilis T, whereas D3 sequence analysis revealed presence of species T in Jiroft and species U in Chabahar district. It is the first report of species U in Iran.