First genotyping of Giardia lamblia from human and animal feces in Argentina, South America

The purpose of this study was to investigate the genotypes of Giardia lamblia from human and animal feces and their epidemiological and clinical characteristics in Argentina, South America. Seventy isolates, 60 from humans (adults and children), eight from dogs and two from cows were processed by polymerase chain reaction-restriction fragment length polymorphism. Data corresponding to demographic, socio-cultural and environmental variables and presence/absence of signs/symptoms were collected. The triosephosphate isomerase gene was amplified from 43 (71.66%) of the 60 human fecal samples. Among these, 3/43 (6.98%) were genotype AII and 40/43 (93.02%) were genotype B. Assemblage AII was detected in three children who lived together in a shantytown and they were oligosymptomatic and none had diarrhea. This genotype was not found in animals. Genotype B showed a high prevalence in both adults and children. It was also found in polysymptomatic people, many of whom presented diarrhea. It was also found only in one dog. The present study represents the first contribution to the knowledge of G. lamblia genotypes in Argentina.

Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64%), followed by VGII (21%), VNII (5%), VGIII (4%), VGI and VNIV (3% each), and VNIII (< 1%). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil.

Onychomycosis insensitive to systemic terbinafine and azole treatments reveals non-dermatophyte moulds as infectious agents.

BACKGROUND: Dermatophytes are the main cause of onychomycoses, but various non-dermatophyte filamentous fungi are often isolated from abnormal nails. OBJECTIVE: Our aim was the in situ identification of the fungal infectious agent in 8 cases of onychomycoses which could not be cured after systemic terbinafine and itraconazole treatment. METHODS: Fungal DNA was extracted from nail samples, and infectious fungi were identified by restriction fragment length polymorphism (RFLP) of amplified fungal ribosomal DNA using a previously described PCR/RFLP assay. RESULTS: PCR/RFLP identification of fungi in nails allows the identification of the infectious agent: Fusarium sp., Acremonium sp. and Aspergillus sp. were found as a sole infectious agent in 5, 2 and 1 cases, respectively. CONCLUSIONS: Fusarium spp. and other non-dermatophyte filamentous fungi are especially difficult to cure in onychomycoses utilising standard treatment with terbinafine and itraconazole. PCR fungal identification helps demonstrate the presence of moulds in order to prescribe alternative antifungal treatments.

Evaluation of the new advanced 15-loci MIRU-VNTR genotyping tool in Mycobacterium tuberculosis molecular epidemiology studies

Background. During the last few years, PCR-based methods have been developed to simplify and reduce the time required for genotyping Mycobacterium tuberculosis (MTB) by standard approaches based on IS6110-Restriction Fragment Length Polymorphism (RFLP). Of these, MIRU-12-VNTR (Mycobacterial interspersed repetitive units- variable number of tandem repeats) (MIRU-12) has been considered a good alternative. Nevertheless, some limitations and discrepancies with RFLP, which are minimized if the technique is complemented with spoligotyping, have been found. Recently, a new version of MIRU-VNTR targeting 15 loci (MIRU-15) has been proposed to improve the MIRU-12 format. Results. We evaluated the new MIRU-15 tool in two different samples. First, we analyzed the same convenience sample that had been used to evaluate MIRU-12 in a previous study, and the new 15-loci version offered higher discriminatory power (Hunter-Gaston discriminatory index [HGDI]: 0.995 vs 0.978; 34.4% of clustered cases vs 57.5%) and better correlation (full or high correlation with RFLP for 82% of the clusters vs 47%). Second, we evaluated MIRU-15 on a population-based sample and, once again, good correlation with the RFLP clustering data was observed (for 83% of the RFLP clusters). To understand the meaning of the discrepancies still found between MIRU-15 and RFLP, we analyzed the epidemiological data for the clustered patients. In most cases, splitting of RFLP-clustered patients by MIRU-15 occurred for those without epidemiological links, and RFLP-clustered patients with epidemiological links were also clustered by MIRU-15, suggesting a good epidemiological background for clustering defined by MIRU-15. Conclusion. The data obtained by MIRU-15 suggest that the new design is very efficient at assigning clusters confirmed by epidemiological data. If we add this to the speed with which it provides results, MIRU-15 could be considered a suitable tool for real-time genotyping.

Systematic survey of clonal complexity in tuberculosis at a populational level and detailed characterization of the isolates involved.

Clonally complex infections by Mycobacterium tuberculosis are progressively more accepted. Studies of their dimension in epidemiological scenarios where the infective pressure is not high are scarce. Our study systematically searched for clonally complex infections (mixed infections by more than one strain and simultaneous presence of clonal variants) by applying mycobacterial interspersed repetitive-unit (MIRU)-variable-number tandem-repeat (VNTR) analysis to M. tuberculosis isolates from two population-based samples of respiratory (703 cases) and respiratory-extrapulmonary (R+E) tuberculosis (TB) cases (71 cases) in a context of moderate TB incidence. Clonally complex infections were found in 11 (1.6%) of the respiratory TB cases and in 10 (14.1%) of those with R+E TB. Among the 21 cases with clonally complex TB, 9 were infected by 2 independent strains and the remaining 12 showed the simultaneous presence of 2 to 3 clonal variants. For the 10 R+E TB cases with clonally complex infections, compartmentalization (different compositions of strains/clonal variants in independent infected sites) was found in 9 of them. All the strains/clonal variants were also genotyped by IS6110-based restriction fragment length polymorphism analysis, which split two MIRU-defined clonal variants, although in general, it showed a lower discriminatory power to identify the clonal heterogeneity revealed by MIRU-VNTR analysis. The comparative analysis of IS6110 insertion sites between coinfecting clonal variants showed differences in the genes coding for a cutinase, a PPE family protein, and two conserved hypothetical proteins. Diagnostic delay, existence of previous TB, risk for overexposure, and clustered/orphan status of the involved strains were analyzed to propose possible explanations for the cases with clonally complex infections. Our study characterizes in detail all the clonally complex infections by M. tuberculosis found in a systematic survey and contributes to the characterization that these phenomena can be found to an extent higher than expected, even in an unselected population-based sample lacking high infective pressure.

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.

Characterization of microevolution events in Mycobacterium tuberculosis strains involved in recent transmission clusters.

Under certain circumstances, it is possible to identify clonal variants of Mycobacterium tuberculosis infecting a single patient, probably as a result of subtle genetic rearrangements in part of the bacillary population. We systematically searched for these microevolution events in a different context, namely, recent transmission chains. We studied the clustered cases identified using a population-based universal molecular epidemiology strategy over a 5-year period. Clonal variants of the reference strain defining the cluster were found in 9 (12%) of the 74 clusters identified after the genotyping of 612 M. tuberculosis isolates by IS6110 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive units-variable-number tandem repeat typing. Clusters with microevolution events were epidemiologically supported and involved 4 to 9 cases diagnosed over a 1- to 5-year period. The IS6110 insertion sites from 16 representative isolates of reference and microevolved variants were mapped by ligation-mediated PCR in order to characterize the genetic background involved in microevolution. Both intragenic and intergenic IS6110 locations resulted from these microevolution events. Among those cases of IS6110 locations in intergenic regions which could have an effect on the regulation of adjacent genes, we identified the overexpression of cytochrome P450 in one microevolved variant using quantitative real-time reverse transcription-PCR. Our results help to define the frequency with which microevolution can be expected in M. tuberculosis transmission chains. They provide a snapshot of the genetic background of these subtle rearrangements and identify an event in which IS6110-mediated microevolution in an isogenic background has functional consequences.

Characterization of Mycobacterium tuberculosis Beijing isolates from the Mediterranean area

BACKGROUND. The Beijing lineage of Mycobacterium tuberculosis is causing concern due to its global distribution and its involvement in severe outbreaks. Studies focused on this lineage are mainly restricted to geographical settings where its prevalence is high, whereas those in other areas are scarce. In this study, we analyze Beijing isolates in the Mediterranean area, where this lineage is not prevalent and is mainly associated with immigrant cases. RESULTS. Only 1% (N = 26) of the isolates from two population-based studies in Spain corresponded to Beijing strains, most of which were pan-susceptible and from Peruvian and Ecuadorian patients. Restriction fragment length polymorphism typing with the insertion sequence IS6110 identified three small clusters (2-3 cases). Mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-15) offered low discriminatory power, requiring the introduction of five additional loci. A selection of the Beijing isolates identified in the Spanish sample, together with a sample of Beijing strains from Italy, to broaden the analysis context in the Mediterranean area, were assayed in an infection model with THP-1 cells. A wide range of intracellular growth rates was observed with only two isolates showing an increased intracellular replication, in both cases associated with contained production of TNF-alpha. No correlation was observed between virulence and the Beijing phylogenetic group, clustered/orphan status, or resistance. The Beijing strain responsible for extensive spread on Gran Canaria Island was also identified in Madrid, but did not lead to secondary cases and did not show high infectivity in the infection model. CONCLUSIONS. The Beijing lineage in our area is a non-homogeneous family, with only certain highly virulent representatives. The specific characterization of Beijing isolates in different settings could help us to accurately identify the virulent representatives before making general assumptions about this lineage.

The effect of combined polymorphisms in chemokines and chemokine receptors on the clinical course of HIV-1 infection in a Brazilian population

Polymorphisms in genes that encode chemokines or their receptors can modulate susceptibility to human immunodeficiency virus (HIV) infection and disease progression. The objective of this study was to assess the frequency of polymorphisms CCR5-Δ32, CCR2-64I, CCR5-59029A and SDF1-3'A and their role in the course of HIV infection in a Southern Brazilian population. Clinical data were obtained from 249 patients for an average period of 6.4 years and genotypes were determined by standard polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism. Survival analyses were conducted for three outcomes: CD4+ T-cell counts below 200 cells/µL, acquired immune deficiency syndrome (AIDS) or death. The frequency of the polymorphisms CCR5-Δ32, CCR2-64I, CCR5-59029A and SDF1-3'A were 0.024, 0.113, 0.487 and 0.207, respectively. CCR5-Δ32 was associated with a reduction in the risk for CD4+ T-cell depletion and with an increased risk for death after AIDS diagnosis. CCR2-64I was associated with a reduction in the risk for developing AIDS. SDF1-3'A was also associated with decreased risk for AIDS, but its effect was only evident when CCR2-64I was present as well. These results highlight the possibility of using these markers as indicators for the prognosis of disease progression and provide evidence for the importance of analysing the effects of gene polymorphisms in a combined fashion.

Comparison of spoligotyping, mycobacterial interspersed repetitive units typing and IS6110-RFLP in a study of genotypic diversity of Mycobacterium tuberculosis in Delhi, North India

The aim of the present study was to compare polymerase chain reaction (PCR)-based methods - spoligotyping and mycobacterial interspersed repetitive units (MIRU) typing - with the gold-standard IS6110 restriction fragment length polymorphism (RFLP) analysis in 101 isolates of Mycobacterium tuberculosis to determine the genetic diversity of M. tuberculosis clinical isolates from Delhi, North India. Spoligotyping resulted in 49 patterns (14 clusters); the largest cluster was composed of Spoligotype International Types (SITs)26 [Central-Asian (CAS)1-Delhi lineage], followed by SIT11 [East-African-Indian (EAI) 3-Indian lineage]. A large number of isolates (75%) belonged to genotypic lineages, such as CAS, EAI and Manu, with a high specificity for the Indian subcontinent, emphasising the complex diversity of the phylogenetically coherent M. tuberculosis in North India. MIRU typing, using 11 discriminatory loci, was able to distinguish between all but two strains based on individual patterns. IS6110-RFLP analysis (n = 80 strains) resulted in 67 unique isolates and four clusters containing 13 strains. MIRUs discriminated all 13 strains, whereas spoligotyping discriminated 11 strains. Our results validate the use of PCR-based molecular typing of M. tuberculosis using repetitive elements in Indian isolates and demonstrate the usefulness of MIRUs for discriminating low-IS6110-copy isolates, which accounted for more than one-fifth of the strains in the present study.

Genotypes of Cryptococcus neoformans and Cryptococcus gattii as agents of endemic cryptococcosis in Teresina, Piauí (northeastern Brazil)

Throughout Brazil, Cryptococcus neoformans is the cause of cryptococcosis, whereas Cryptococcus gattii is endemic to the northern and northeastern states. In this study, the molecular types of 63 cryptococcal isolates recovered from the cerebrospinal fluid of meningitis patients diagnosed between 2008-2010 in Teresina, Piauí, Brazil, were analysed. Out of the 63 patients, 37 (58.7%) were human immunodeficiency virus (HIV)-positive and 26 (41.3%) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7%) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5%), and 24/63 (38.1%) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8%). The occurrence of C. gattii VGII in six apparently healthy children and in seven adolescents/young adults in this region reaffirms the endemic occurrence of C. gattii VGII-induced primary cryptococcosis and early cryptococcal infection. Lethality occurred in 18/37 (48.6%) of the HIV-positive subjects and in 13/26 (50%) of the HIV-negative patients. Our results provide new information on the molecular epidemiology of C. neoformans and C. gattii in Brazilian endemic areas.

Hybridism between Biomphalaria cousini and Biomphalaria amazonica and its susceptibility to Schistosoma mansoni

Molecular techniques can aid in the classification of Biomphalaria species because morphological differentiation between these species is difficult. Previous studies using phylogeny, morphological and molecular taxonomy showed that some populations studied were Biomphalaria cousini instead of Biomphalaria amazonica. Three different molecular profiles were observed that enabled the separation of B. amazonica from B. cousini. The third profile showed an association between the two and suggested the possibility of hybrids between them. Therefore, the aim of this work was to investigate the hybridism between B. cousini and B. amazonica and to verify if the hybrids are susceptible to Schistosoma mansoni. Crosses using the albinism factor as a genetic marker were performed, with pigmented B. cousini and albino B. amazonica snails identified by polymerase chain reaction-restriction fragment length polymorphism. This procedure was conducted using B. cousini and B. amazonica of the type locality accordingly to Paraense, 1966. In addition, susceptibility studies were performed using snails obtained from the crosses (hybrids) and three S. mansoni strains (LE, SJ, AL). The crosses between B. amazonica and B. cousini confirmed the occurrence of hybrids. Moreover, hybrids can be considered potential hosts of S. mansoni because they are susceptible to LE, SJ and AL strains (4.4%, 5.6% and 2.2%, respectively). These results indicate that there is a risk of introducing schistosomiasis mansoni into new areas.

Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009)

We examined strains of Trypanosoma cruzi isolated from patients with acute Chagas disease that had been acquired by oral transmission in the state of Santa Catarina, Brazil (2005) and two isolates that had been obtained from a marsupial (Didelphis aurita) and a vector (Triatoma tibiamaculata). These strains were characterised through their biological behaviour and isoenzymic profiles and genotyped according to the new Taxonomy Consensus (2009) based on the discrete typing unities, that is, T. cruzi genotypes I-VI. All strains exhibited the biological behaviour of biodeme type II. In six isolates, late peaks of parasitaemia, beyond the 20th day, suggested a double infection with biodemes II + III. Isoenzymes revealed Z2 or mixed Z1 and Z2 profiles. Genotyping was performed using three polymorphic genes (cytochrome oxidase II, spliced leader intergenic region and 24Sα rRNA) and the restriction fragment length polymorphism of the kDNA minicircles. Based on these markers, all but four isolates were characterised as T. cruzi II genotypes. Four mixed populations were identified: SC90, SC93 and SC97 (T. cruzi I + T. cruzi II) and SC95 (T. cruzi I + T. cruzi VI). Comparison of the results obtained by different methods was essential for the correct identification of the mixed populations and major lineages involved indicating that characterisation by different methods can provide new insights into the relationship between phenotypic and genotypic aspects of parasite behaviour.

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.