Prevalence and genetic characterization of Cryptosporidium spp. and Cystoisospora belli in HIV-infected patients

Cryptosporidium spp. and Cystoisospora belli are monoxenic protozoa that have been recognized as the causative agents of chronic diarrhea in immunocompromised individuals, especially HIV-infected subjects. The objective of this study was to evaluate the frequency of these intestinal protozoa in HIV-positive patients in the Triângulo Mineiro region of Brazil and to correlate the presence of these infections with clinical, epidemiological and laboratory data of the patients. Oocysts were detected in stool samples of 10 (16.9%) of the 59 patients studied, while Cryptosporidium spp. were present in 10.1% (6/59) and C. belli in 6.7% (4/59). The frequency of these parasites was higher among patients with diarrheic syndrome and CD4+ T lymphocyte counts < 200 cells/mm 3 , demonstrating the opportunistic characteristic of these infections. A significant association was observed between the lack of adherence to antiretroviral therapy and the presence of Cryptosporidium spp. and/or C. belli. Parasitism with Cryptosporidium spp. was more frequent in February and April, the months following the period of high rainfall. The same was not observed for C. belli. Genetic characterization of two isolates led to the identification of Cryptosporidium parvum, one of the main species associated with the zoonotic transmission of cryptosporidiosis.

Characterization of mannose-binding lectin plasma levels and genetic polymorphisms in HIV-1-infected individuals

INTRODUCTION: The present study investigated the association between mannose-binding lectin (MBL) gene polymorphism and serum levels with infection by HIV-1. METHODS: Blood samples (5mL) were collected from 97 HIV-1-infected individuals resident in Belém, State of Pará, Brazil, who attended the Special Outpatient Unit for Infections and Parasitic Diseases (URE-DIPE). CD4+ T-lymphocyte count and plasma viral load were quantified. A 349bp fragment of exon 1 of the MBL was amplified via PCR, using genomic DNA extracted from controls and HIV-1-infected individuals, following established protocols. MBL plasma levels of the patients were quantified using an enzyme immunoassay kit. RESULTS: Two alleles were observed: MBL*O, with a frequency of 26.3% in HIV-1-infected individuals; and the wild allele MBL*A (73.7%). Similar frequencies were observed in the control group (p > 0.05). Genotype frequencies were distributed according to the Hardy-Weinberg equilibrium in both groups. Mean MBL plasma levels varied by genotype, with statistically significant differences between the AA and AO (p < 0.0001), and AA and OO (p < 0.001) genotypes, but not AO and OO (p = 0.17). Additionally, CD4+ T-lymphocytes and plasma viral load levels did not differ significantly by genotype (p > 0.05). CONCLUSIONS: The results of this study do not support the hypothesis that MBL gene polymorphism or low plasma MBL concentrations might have a direct influence on HIV-1 infection, although a broader study involving a large number of patients is needed.

Noonan syndrome: a clinical and genetic study of 31 patients

Noonan syndrome is a multiple congenital anomaly syndrome, inherited in an autosomal dominant pattern. We studied 31 patients (18 males and 13 females) affected by this disorder regarding their clinical and genetic characteristics. The most frequent clinical findings were short stature (71%); craniofacial dysmorphisms, especially hypertelorism, ptosis, downslanting of the palpebral fissures; short or webbed neck (87%); cardiac anomalies (65%), and fetal pads in fingers and toes (70%). After studying the probands' first-degree relatives, we made the diagnosis of Noonan syndrome in more than one family member in three families. Therefore, the majority of our cases were sporadic.

Risk factors, biochemical markers, and genetic polymorphisms in early coronary artery disease

OBJECTIVE: To assess the risk factors, lipid and apolipoprotein profile, hemostasis variables, and polymorphisms of the apolipoprotein AI-CIII gene in early coronary artery disease (CAD). METHODS: Case-control study with 112 patients in each group controlled by sex and age. After clinical evaluation and nutritional instruction, blood samples were collected for biochemical assays and genetic study. RESULTS: Familial history of early CAD (64 vs 39%), arterial hypertension (69 vs 36%), diabetes mellitus (25 vs 3%), and previous smoking (71 vs 46%) were more prevalent in the case group (p<0.001). Hypertension and diabetes were independent risk factors. Early CAD was characterized by higher serum levels of total cholesterol (235 ± 6 vs 209 ± 4 mg/dL), of LDL-c (154 ± 5 vs 135 ± 4 mg/dL), triglycerides (205 ± 12 vs 143 ± 9 mg/dL), and apolipoprotein B (129 ± 3 vs 105 ± 3 mg/dL), and lower serum levels of HDL-c (40 ± 1 vs 46 ± 1 mg/dL) and apolipoprotein AI (134 ± 2 vs 146 ± 2mg/dL) [p<0.01], in addition to an elevation in fibrinogen and D-dimer (p<0.02). The simultaneous presence of the rare alleles of the APO AI-CIII genes in early CAD are associated with hypertriglyceridemia (p=0.03). CONCLUSION: Of the classical risk factors, hypertension and diabetes mellitus were independently associated with early CAD. In addition to an unfavorable lipid profile, an increase in the thrombotic risk was identified in this population. An additive effect of the APO AI-CIII genes was observed in triglyceride levels.

Male-female interaction during breeding and non-breeding seasons in Akodon azarae (Rodentia, Muridae)

Dyad encounters between male and female adults of Akodon azarae (Fischer, 1829) were analyzed by means of observational techniques in a natural closure during the breeding and non-breeding seasons. The animals were held in observation during 21 days, with daily 15-minute recordings of interindividual separation distance, relative displacements, characteristics of the male-female interaction, copulation, and construction and exclusive or shared use of nests by each pair. The couples, which bred successfully, showed, on average, the longest separation distance between male and female allowed by the closure. During the first two weeks of gestation the females exhibited more displacements than their respective mates did. The male-pregnant female encounters were significantly more aggressive than those recorded between pairs which did not breed successfully. During the non-breeding season a shorter average distance between individuals and a frequent use of nests shared by the pair were recorded. The results obtained are discussed within the framework of the social system of A. azarae.

Morphometric and genetic differentiation among populations of Eupemphix nattereri (Amphibia, Anura, Leiuperidae) from central Brazil

To assess genetic structure and phenotypic diversity of Eupemphix nattereri Steindachner, 1863, morphometric and molecular analyses were carried out for nine populations from the State of Goiás. A total of 11 morphometric traits were evaluated and genetic information was estimated using RAPD markers. Genetic and phenotypic distances were determined as a function of geographical origin. Correlation among genetic, morphometric, micro, and macroenviromental were analyzed by the Mantel test. Genetic data indicated high levels of genetic diversity (Φst= 0.3) among the nine populations. Mantel tests did not reveal a significant positive correlation between genetic and geographical distances, indicating that locally geographical populations were not genetically similar, even in distances smaller than 50 km. Discriminant analysis on 11 morphometric measurements showed a high divergence among the nine populations. However, a marginally significant correlation (P=0.08) between genetic and morphometric distances was found. The observed correlation was not causal in terms of the relationship between phenotype and genotype, but indicated common spatial structures. Thus, our results suggest that isolation-by-distance processes may explain population divergence in Eupemphix nattereri.

Abundance, breeding and food of the Little Blue Heron Egretta caerulea (Aves, Ardeidae) in the Patos Lagoon estuary, a recently colonized area in southern Brazil

We document the expansion of the breeding distribution of the Little Blue Heron Egretta caerulea (Linnaeus, 1758) to 850 km beyond its previous southern limit in South America. In addition we present data on abundance, breeding biology and food of the species in the Patos Lagoon estuary, the area which the species recently colonized. The maximum abundance recorded in the breeding colony and in a nocturnal roosting site was 53 and 49 individuals respectively. Nesting occurred from September to March. Birds nested in a mixed breeding colony together with about 3,000 breeding pairs of seven other species of Pelecaniformes, in a swampy forest near the margin of the estuary. Five nests were between 1.5 and 4.3 m from the ground, on the shrub Daphnopsis racemosa (Thymelaeaceae), on the trees Sebastiana brasiliensis (Euphorbiaceae) and Mimosa bimucronata (Leguminosae), or on the bamboo Bambusa sp. (Poaceae). Four nests produced two fledglings each, while one nest was abandoned. Of 13 grouped samples of food regurgitated by five nestlings, Pink Shrimp Farfantepenaeus paulensis (Perez-Farfante, 1967) constituted 70% in mass, while total length of ingested fishes and shrimps varied mostly between 20 and 50 mm. Estuarine prey items represented 99% of the total food mass. The recent southward expansion of the breeding range of the Little Blue Heron in South America may be a response to climate warming of the Patos Lagoon estuary. Degradation of estuaries in the southwestern Atlantic may also be forcing the birds to breed in areas outside previous geographical range.

Dried blood spots collected on filter paper: an international resource for the diagnosis and genetic characterization of human immunodeficiency virus Type-1

The collection of dried blood spots (DBS) on filter paper provides a powerful approach for the development of large-scale, population-based screening programs. DBS methods are particularly valuable in developing countries and isolated rural regions where resources are limited. Large numbers of field specimens can be economically collected and shipped to centralized reference laboratories for genetic and (or) serological analysis. Alternatively, the dried blood can be stored and used as an archival resource to rapidly establish the frequency and distribution of newly recognized mutations, confirm patient identity or track the origins and emergence of newly identified pathogens. In this report, we describe how PCR-based technologies are beginning to interface with international screening programmes for the diagnosis and genetic characterization of human immunodeficiency virus type 1 (HIV-1). In particular, we review recent progress using DBS specimens to resolve the HIV-1 infection status of neonates, monitor the genetic evolution of HIV-1 during early infancy and establish a sentinel surveillance system for the systematic monitoring of HIV-1 genetic variation in Asia.

Antigenic and Genetic Characterization of Twenty-six Strains of Human Respiratory Syncytial Virus (Subgroup A) Isolated During Three Consecutive Outbreaks in Havana City, Cuba

Twenty-six human respiratory syncytial virus strains (subgroup A) isolated from three outbreaks in Havana City during the period 1994/95, 1995/96 and 1996/97 were analyzed to determine their antigenic and genetic relationships. Analyses were performed by monoclonal antibodies and restriction mapping (N gene) following amplification of the select region of the virus genome by polymerase chain reaction. All isolated strains were classified as subgroup A by monoclonal antibodies and they showed a restriction pattern NP4 that belonged to subgroup A. Thus the results obtained in this work, showed a close relation (100%) between antigenic and genetic characterization of the isolated strains in our laboratory. These methods permit the examination of large numbers of isolates by molecular techniques, simplifying the researchs into the molecular epidemiology of the virus.

Genetic diversity and genetic exchange in Trypanosoma cruzi: dual drug-resistant "progeny" from episomal transformants

Extensive characterisation of Trypanosoma cruzi by isoenzyme phenotypes has separated the species into three principal zymodeme groups, Z1, Z2 and Z3, and into many individual zymodemes. There is marked diversity within Z2. A strong correlation has been demonstrated between the strain clusters determined by isoenzymes and those obtained using random amplified polymorphic DNA (RAPD) profiles. Polymorphisms in ribosomal RNA genes, in mini-exon genes, and microsatellite fingerprinting indicate the presence of at least two principal T. cruzi genetic lineages. Lineage 1 appears to correspond with Z2 and lineage 2 with Z1. Z1 (lineage 2) is associated with Didelphis. Z2 (lineage 1) may be associated with a primate host. Departures from Hardy-Weinberg equilibrium and linkage disequilibrium indicate that propagation of T. cruzi is predominantly clonal. Nevertheless, two studies show putative homozygotes and heterozygotes circulating sympatrically: the allozyme frequencies for phosphoglucomutase, and hybrid RAPD profiles suggest that genetic exchange may be a current phenomenon in some T. cruzi transmission cycles. We were able to isolate dual drug-resistant T. cruzi biological clones following copassage of putative parents carrying single episomal drug-resistant markers. A multiplex PCR confirmed that dual drug-resistant clones carried both episomal plasmids. Preliminary karyotype analysis suggests that recombination may not be confined to the extranuclear genome.

Differentiation and genetic analysis of Rhodnius prolixus and Rhodnius colombiensis by rDNA and RAPD amplification

Domiciliated Rhodnius prolixus and sylvatic R. colombiensis were analyzed in order to confirm their genetic divergence and verify the risk that the latter represents in the domiciliation process, and to provide tools for identifying the sources of possible reinfestation by triatomines in human dwellings allowing control programs to be undertaken. Comparison of random amplified polymorphic DNA amplification patterns and cluster analysis suggests reproductive discontinuity between the two species. The calculated statistical F value of 0.24 and effective migration rate of 0.6 individuals per generation are insufficient to maintain genetic homogeneity between them and confirm the absence of present genetic flow. R. colombiensis presents higher intrapopulation variability. Polymerase chain reaction of ribosomal DNA supports these findings. The low genetic flow between the two species implies that R. colombiensis do not represent an epidemiological risk for the domiciliary transmission of Trypanosoma cruzi in the Tolima Department. The lower variability of the domiciliated R. prolixus could result in greater susceptibility to the use of pesticides in control programs.

Antiretroviral resistance and genetic diversity of human immunodeficiency virus type 1 isolates from the Federal District, Central Brazil

In the context of universal access to antiretroviral therapy, the surveillance of human immunodeficiency virus type 1 (HIV-1) genetic diversity and resistance becomes pivotal. In this work our purpose was to describe the genetic variability; prevalence of drug-resistance mutations; and genotypic resistance profiles in HIV-1 infected individuals under antiretroviral treatment, from the Federal District, Brasília, Central Brazil. The entire viral protease and codons 19 to 234 of the reverse transcriptase gene from 45 HIV-1 isolates were amplified and sequenced for subtyping and genotyping. By phylogenetic analysis, 96% of the samples clustered with subtype B and the remaining 4% with HIV-1 subtype F sequences. One major protease inhibitor resistance-associated mutation, I50V, was detected in 38% of the samples. Minor mutations were also found at the protease gene: L10I/V (7%), K20M (2%), M36I (11%), L63P (20%), A71T (2%), and V77I (7%). Many mutations associated with reduced susceptibility to nucleoside or non-nucleoside reverse transcriptase inhibitors were detected: M41L (11%), E44D (4%), D67N (11%), T69D (2%), K70R (11%), L74V (2%), L100I (4%), K103N (18%), V118I (9%), Y181C (11%), M184V (18%), G190A (4%), T215Y (4%), and K219E (4%). This study has shown that 84% of the studied population from the Federal District, showing evidences of therapy failure, presented viral genomic mutations associated with drug resistance. The main antiretrovirals to which this population showed resistance were the PI amprenavir (38%), the NNRTIs delavirdine, nevirapine (31%), and efavirenz (24%), and the NRTIs lamivudine (18%), abacavir, and zidovudine (13%).

Variability and genetic differentiation among Anopheles (Ano.) intermedius Chagas, 1908 and Anopheles (Ano.) mattogrossensis Lutz & Neiva, 1911 (Diptera: Culicidae) from the Brazilian Amazon

Anopheles (Anopheles) intermedius and Anopheles (Ano.) mattogrossensis are Brazilian anopheline species belonging to the scarcely studied Anopheles subgenus. Few studies have been done on the genetic differentiation of these species. Both species have been found infected by Plasmodium and are sympatric with other anopheline species from the Nyssorhynchus subgenus. Eighteen enzymatic loci were analyzed in larval specimens of An. intermedius and An. mattogrossensis aiming to estimate the variability and genetic differentiation between these species. An. mattogrossensis population showed higher genetic variability (P = 44.4 and Ho = 0.081 ± 0.031) than that of An. intermedius (P = 33.3 and Ho = 0.048 ± 0.021). Most analyzed loci showed genotypic frequencies according to Hardy-Weinberg equilibrium, except for LAP1 and LAP2 in An. intermedius, and EST1 and PGM loci in An. mattogrossensis. The genetic distance between these species (D = 0.683) was consistent with the inter-specific values reported for Anopheles subgenus. We verified that the polymorphism and heterozygosity percentile values found in both species and compared to those in the literature, showed no relation between the level of isozyme variability and geographical distribution. The low variability found in these two species is probably more related to the niche they occupy than to their geographic distribution.