Biomphalaria molluscs (Gastropoda: Planorbidae) in Rio Grande do Sul, Brazil

The present study was aimed at characterising Biomphalaria species using both morphological and molecular (PCR-RFLP) approaches. The specimens were collected in 15 localities in 12 municipalities of the southern region of the state of Rio Grande do Sul, Brazil. The following species were found and identified: Biomphalaria tenagophila guaibensis, Biomphalaria oligoza and Biomphalaria peregrina. Specimens of the latter species were experimentally challenged with the LE Schistosoma mansoni strain, which showed to be refractory to infection.

Preliminary studies investigating the occurrence of Biomphalaria cousini in Brazil

Specific genetic profiles of Brazilian Biomphalaria species were previously standardized by molecular taxonomy through the analysis of restriction fragments, which were generated by digesting the internal transcribed spacer region of rDNA with the DdeI endonuclease. Biomphalaria amazonica displayed three distinct profiles. To investigate these distinct profiles, the same molecular technique, polymerase chain reaction and restriction fragment length polymorphism, was used with different endonucleases. In addition, morphological data were also used to compare B. amazonica specimens that were collected from Brazil, Colombia and Bolivia. The morphological characters of Bolivian molluscs were similar to B. amazonica, displayed a molecular profile of five restriction fragments and morphological data, whereas the Colombian mollusc population showed morphological characters similar to Biomphalaria cousini and a molecular profile of three restriction fragments, similar to B. cousini. The Brazilian specimens showed the B. amazonica and B. cousini molecular profiles as well as a third profile, which resembled a combination of the Colombian and Bolivian molecular profiles.

Prospective universal application of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat genotyping to characterize Mycobacterium tuberculosis isolates for fast identification of clustered and orphan cases.

The use of molecular tools for genotyping Mycobacterium tuberculosis isolates in epidemiological surveys in order to identify clustered and orphan strains requires faster response times than those offered by the reference method, IS6110 restriction fragment length polymorphism (RFLP) genotyping. A method based on PCR, the mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping technique, is an option for fast fingerprinting of M. tuberculosis, although precise evaluations of correlation between MIRU-VNTR and RFLP findings in population-based studies in different contexts are required before the methods are switched. In this study, we evaluated MIRU-VNTR genotyping (with a set of 15 loci [MIRU-15]) in parallel to RFLP genotyping in a 39-month universal population-based study in a challenging setting with a high proportion of immigrants. For 81.9% (281/343) of the M. tuberculosis isolates, both RFLP and MIRU-VNTR types were obtained. The percentages of clustered cases were 39.9% (112/281) and 43.1% (121/281) for RFLP and MIRU-15 analyses, and the numbers of clusters identified were 42 and 45, respectively. For 85.4% of the cases, the RFLP and MIRU-15 results were concordant, identifying the same cases as clustered and orphan (kappa, 0.7). However, for the remaining 14.6% of the cases, discrepancies were observed: 16 of the cases clustered by RFLP analysis were identified as orphan by MIRU-15 analysis, and 25 cases identified as orphan by RFLP analysis were clustered by MIRU-15 analysis. When discrepant cases showing subtle genotypic differences were tolerated, the discrepancies fell from 14.6% to 8.6%. Epidemiological links were found for 83.8% of the cases clustered by both RFLP and MIRU-15 analyses, whereas for the cases clustered by RFLP or MIRU-VNTR analysis alone, links were identified for only 30.8% or 38.9% of the cases, respectively. The latter group of cases mainly comprised isolates that could also have been clustered, if subtle genotypic differences had been tolerated. MIRU-15 genotyping seems to be a good alternative to RFLP genotyping for real-time interventional schemes. The correlation between MIRU-15 and IS6110 RFLP findings was reasonable, although some uncertainties as to the assignation of clusters by MIRU-15 analysis were identified.

Optimized molecular resolution of cross-contamination alerts in clinical mycobacteriology laboratories

BACKGROUND. The phenomenon of misdiagnosing tuberculosis (TB) by laboratory cross-contamination when culturing Mycobacterium tuberculosis (MTB) has been widely reported and it has an obvious clinical, therapeutic and social impact. The final confirmation of a cross-contamination event requires the molecular identification of the same MTB strain cultured from both the potential source of the contamination and from the false-positive candidate. The molecular tool usually applied in this context is IS6110-RFLP which takes a long time to provide an answer, usually longer than is acceptable for microbiologists and clinicians to make decisions. Our purpose in this study is to evaluate a novel PCR-based method, MIRU-VNTR as an alternative to assure a rapid and optimized analysis of cross-contamination alerts. RESULTS. MIRU-VNTR was prospectively compared with IS6110-RFLP for clarifying 19 alerts of false positivity from other laboratories. MIRU-VNTR highly correlated with IS6110-RFLP, reduced the response time by 27 days and clarified six alerts unresolved by RFLP. Additionally, MIRU-VNTR revealed complex situations such as contamination events involving polyclonal isolates and a false-positive case due to the simultaneous cross-contamination from two independent sources. CONCLUSION. Unlike standard RFLP-based genotyping, MIRU-VNTR i) could help reduce the impact of a false positive diagnosis of TB, ii) increased the number of events that could be solved and iii) revealed the complexity of some cross-contamination events that could not be dissected by IS6110-RFLP.

Analysis of the genetic variability of PvMSP-3α among Plasmodium vivax in Brazilian field isolates

Reliable molecular markers are essential for a better understanding of the molecular epidemiology of Plasmodium vivax, which is a neglected human malaria parasite. The aim of this study was to analyze the genetic diversity of P. vivax isolates from the Brazilian Amazon using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the highly polymorphic merozoite surface protein-3alpha (PvMSP-3α) gene. To accomplish this, 60 isolates of P. vivax from different endemic areas in the Brazilian Amazon were collected. The PvMSP-3α gene was amplified by nested-PCR. Three major types of the PvMSP-3α locus were detected at different frequencies: type A (68%), B (15%) and C (17%). A single sample showed two PCR fragments, which corresponded to infection with types A and C. PCR-RFLP analysis using the HhaI restriction enzyme for 52 isolates clearly identified 11 haplotypes, eight of which were from type A, two from type B and only one from type C. Seven other isolates did not show a clear pattern using PCR-RFLP. This result might be due to multiple clone infections. This study showed a high diversity of the PvMSP-3α gene among P. vivax isolates from the Brazilian Amazon, but also indicated that the detection performance of PCR-RFLP of the PvMSP-3α gene may not be sufficient to detect multiple clone infections.

Natural Leishmania sp. reservoirs and phlebotomine sandfly food source identification in Ibitipoca State Park, Minas Gerais, Brazil

Leishmania spp are distributed throughout the world and different species are associated with varying degrees of disease severity. However, leishmaniasis is thought to be confined to areas of the world where its insect vectors, sandflies, are present. Phlebotomine sandflies obtain blood meals from a variety of wild and domestic animals and sometimes from humans. These vectors transmit Leishmania spp, the aetiological agent of leishmaniasis. Identification of sandfly blood meals has generally been performed using serological methods, although a few studies have used molecular procedures in artificially fed insects. In this study, cytochrome b gene (cytB) polymerase chain reaction (PCR) was performed in DNA samples isolated from 38 engorged Psychodopygus lloydi and the expected 359 bp fragment was identified from all of the samples. The amplified product was digested using restriction enzymes and analysed for restriction fragment length polymorphisms (RFLPs). We identified food sources for 23 females; 34.8% yielded a primate-specific banding profile and 26.1% and 39.1% showed banding patterns specific to birds or mixed restriction profiles (rodent/marsupial, human/bird, rodent/marsupial/human), respectively. The food sources of 15 flies could not be identified. Two female P. lloydi were determined to be infected by Leishmania using internal transcribed spacer 1 and heat shock protein 70 kDa PCR-RFLP. The two female sandflies, both of which fed on rodents/marsupials, were further characterised as infected with Leishmania (Viannia) braziliensis. These results constitute an important step towards applying methodologies based on cytB amplification as a tool for identifying the food sources of female sandflies.

Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

Novel polymerase chain reaction-restriction fragment length polymorphism assay to determine internal transcribed spacer-2 group in the Chagas disease vector, Triatoma dimidiata (Latreille, 1811)

Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2) is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR) product and polymerase slippage near the 5' end. To overcome these challenges we have designed new primers that amplify only the 3'-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment length polymorphism (RFLP) approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.

Incidence and transmission patterns of tuberculosis among indigenous populations in Brazil

Approximately 10% of the Brazilian indigenous population lives in the state of Mato Grosso do Sul (MS), where a large number of new cases of tuberculosis (TB) are reported. This study was conducted to assess TB occurrence, transmission and the utility of TB diagnosis based on the Ogawa-Kudoh (O-K) culture method in this remote population. The incidence of TB was estimated by a retrospective review of the surveillance data maintained by the Notifiable Diseases Surveillance System for the study region. The TB transmission pattern among indigenous people was assessed by genotyping Mycobacterium tuberculosis isolates using the IS 6110restriction fragment length polymorphism (RFLP) technique. Of the 3,093 cases identified from 1999-2001, 610 (~20%) were indigenous patients (average incidence: 377/100,000/year). The use of the O-K culture method increased the number of diagnosed cases by 34.1%. Of the genotyped isolates from 52 indigenous patients, 33 (63.5%) belonged to cluster RFLP patterns, indicating recently transmitted TB. These results demonstrate high, on-going TB transmission rates among the indigenous people of MS and indicate that new efforts are needed to disrupt these current transmissions.

Seasonal fluctuations of bacterial community diversity in agricultural soil and experimental validation by laboratory disturbance experiments.

Natural fluctuations in soil microbial communities are poorly documented because of the inherent difficulty to perform a simultaneous analysis of the relative abundances of multiple populations over a long time period. Yet, it is important to understand the magnitudes of community composition variability as a function of natural influences (e.g., temperature, plant growth, or rainfall) because this forms the reference or baseline against which external disturbances (e.g., anthropogenic emissions) can be judged. Second, definition of baseline fluctuations in complex microbial communities may help to understand at which point the systems become unbalanced and cannot return to their original composition. In this paper, we examined the seasonal fluctuations in the bacterial community of an agricultural soil used for regular plant crop production by using terminal restriction fragment length polymorphism profiling (T-RFLP) of the amplified 16S ribosomal ribonucleic acid (rRNA) gene diversity. Cluster and statistical analysis of T-RFLP data showed that soil bacterial communities fluctuated very little during the seasons (similarity indices between 0.835 and 0.997) with insignificant variations in 16S rRNA gene richness and diversity indices. Despite overall insignificant fluctuations, between 8 and 30% of all terminal restriction fragments changed their relative intensity in a significant manner among consecutive time samples. To determine the magnitude of community variations induced by external factors, soil samples were subjected to either inoculation with a pure bacterial culture, addition of the herbicide mecoprop, or addition of nutrients. All treatments resulted in statistically measurable changes of T-RFLP profiles of the communities. Addition of nutrients or bacteria plus mecoprop resulted in bacteria composition, which did not return to the original profile within 14 days. We propose that at less than 70% similarity in T-RFLP, the bacterial communities risk to drift apart to inherently different states.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Intestinal parasites and genotyping of Giardia duodenalis in children: first report of genotype B in isolates from human clinical samples in Mexico

Giardia duodenalis is one of the most prevalent enteroparasites in children. This parasite produces several clinical manifestations. The aim of this study was to determine the prevalence of genotypes of G. duodenalis causing infection in a region of southeastern Mexico. G. duodenalis cysts were isolated (33/429) from stool samples of children and molecular genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, targeting the triosephosphate isomerase ( tpi ) and glutamate dehydrogenase ( gdh ) genes. The tpi gene was amplified in all of the cyst samples, either for assemblage A (27 samples) or assemblage B (6 samples). RFLP analysis classified the 27 tpi -A amplicons in assemblage A, subgenotype I. Samples classified as assemblage B were further analysed using PCR-RFLP of the gdh gene and identified as assemblage B, subgenotype III. To our knowledge, this is the first report of assemblage B of G. duodenalis in human clinical samples from Mexico.

Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

Polymerase chain reaction detection of LeishmaniaDNA in skin biopsy samples in Sri Lanka where the causative agent of cutaneous leishmaniasis is Leishmania donovani

Leishmania donovani is the known causative agent of both cutaneous (CL) and visceral leishmaniasis in Sri Lanka. CL is considered to be under-reported partly due to relatively poor sensitivity and specificity of microscopic diagnosis. We compared robustness of three previously described polymerase chain reaction (PCR) based methods to detectLeishmania DNA in 38 punch biopsy samples from patients presented with suspected lesions in 2010. Both, Leishmaniagenus-specific JW11/JW12 KDNA and LITSR/L5.8S internal transcribed spacer (ITS)1 PCR assays detected 92% (35/38) of the samples whereas a KDNA assay specific forL. donovani (LdF/LdR) detected only 71% (27/38) of samples. All positive samples showed a L. donovani banding pattern upon HaeIII ITS1 PCR-restriction fragment length polymorphism analysis. PCR assay specificity was evaluated in samples containing Mycobacterium tuberculosis, Mycobacterium leprae, and human DNA, and there was no cross-amplification in JW11/JW12 and LITSR/L5.8S PCR assays. The LdF/LdR PCR assay did not amplify M. leprae or human DNA although 500 bp and 700 bp bands were observed in M. tuberculosis samples. In conclusion, it was successfully shown in this study that it is possible to diagnose Sri Lankan CL with high accuracy, to genus and species identification, using Leishmania DNA PCR assays.