Characterization of rotavirus P genotypes circulating among paediatric inpatients in Northern Brazil

Between November 1992 and August 1993, twenty-eight rotavirus-positive stool samples obtained from paediatric inpatients in Belém, Brazil, aged less than four years, were tested by RT-PCR to determine the P genotype specificities. With the exception of 7 non-diarrhoeic children, all patients were either diarrhoeic at admission or developed diarrhoea while in hospital. Rotavirus strains with the gene 4 alleles corresponding to P1B[4] and P1A[8] types (both of which bearing G2 specificity) predominated, accounting for 78.6% of the strains. While only one P2A[6] type strain - with (mixed) G1 and 4 type specificities - was detected, the gene 4 allele could not be identified in 4 (14.3%) of the strains. Most (81%) of the specimens were obtained from children during their first 18 months of life. Rotavirus strains bearing single P1B[4] type-specificity were identified in both diarrhoeic (either nosocomial, 28.6% or community-acquired diarrhoea, 28.6%) and non-diarrhoeic (42.8%) children. P1A[8] gene 4 allele, on the other hand, was detected only among diarrhoeic children, at rates of 57.1% and 42.9% for nosocomial- and- community acquired diarrhoea, respectively. Mixed P1A[8],1B[4] type infection was identified in only one case of community-acquired diarrhoea.

Chronic hepatitis C virus infections in Brazilian patients: association with genotypes, clinical parameters and response to long term alpha interferon therapy

The present study assessed the clinical significance of hepatitis C virus (HCV) genotypes and their influence on response to long term recombinant-interferon-alpha (r-IFN-a) therapy in Brazilian patients. One hundred and thirty samples from patients previously genotyped for the HCV and with histologically confirmed chronic hepatitis C (CH-C) were evaluated for clinical and epidemiological parameters (sex, age, time of HCV infection and transmission routes). No difference in disease activity, sex, age or mode and time of transmission were seen among patients infected with HCV types 1, 2 or 3. One hundred and thirteen of them were treated with 3 million units of r-IFN-a, 3 times a week for 12 months. Initial response (IR) was significantly better in patients with genotype 2 (100%) and 3 (46%) infections than in patients with genotype 1 (29%) (p < 0.005). Among subtypes, difference in IR was observed between 1b and 2 (p < 0.005), and between 1b and 3a (p < 0.05). Sustained response (SR) was observed in 12% for (sub)type 1a, 13% for 1b, 19% for 3a, and 40% for type 2; significant differences were found between 1b and 2 (p < 0.001), and between 1b and 3a (p < 0.05). Moreover, presence of cirrhosis was significantly associated with non response and response with relapse (p < 0.05). In conclusion, non-1 HCV genotype and lack of histological diagnosis of cirrhosis were the only baseline features associated with sustained response to treatment. These data indicate that HCV genotyping may have prognostic relevance in the responsiveness to r-IFN-a therapy in Brazilian patients with chronic HCV infection, as seen in other reports worldwide.

Atypical disseminated cutaneous histoplasmosis in an immunocompetent child, caused by an "aberrant" variant of Histoplasma capsulatum var. capsulatum

A case of atypical disseminated cutaneous histoplasmosis in a five-year old, otherwise healthy child, native and resident in São Paulo metropolitan area is reported. Cutaneous lesions were clinically atypical. Histologic examination disclosed a granulomatous reaction but no fungal structures could be demonstrated by specific staining nor by immunohistochemical reaction. The fungus was isolated from biopsy material on two different occasions, confirming diagnosis of an unusual fungal infection. The fungus, originally thought to be a Sepedonium sp. due to the large sized, hyaline or brownish colored tuberculated macroconidia and to lack of dimorphism (yeast form at 37 °C) produce H and M antigens, visualized by the immunodiffusion with rabbit anti-Histoplasma capsulatum hyperimmune serum. Patient’s serum sample was non reactive with H. capsulatum antigen by immunodiffusion, counterimmunoelectrophoresis and complement fixation tests, and immunoenzymatic assay failed to detect the specific circulating antigen. This serum was tested negative by double immunodiffusion when antigen obtained from one of the isolated samples was used. Both cultures were sent to Dr. Leo Kaufman, Ph.D. (Mycoses Immunodiagnostic Laboratory, CDC-Atlanta/USA), who identified them as H. capsulatum by the exoantigen and gen-probe tests. Both clinic and mycologic characteristics of the present case were atypical, suggesting the fungus isolated is an “aberrant variant” of H. capsulatum var. capsulatum, as described by SUTTON et al. in 199719. Treatment with itraconazole 100 mg/day led to cure within 90 days

Human papillomavirus genotypes in women with cervical cytological abnormalities from an area with high incidence of cervical cancer

It has been well demonstrated the relationship between the infection with high-risk human papillomavirus (HPVs) genotypes and cervical cancer. In Northeastern Argentina a high incidence of this pathology has been described and therefore a high prevalence of HPV infection is expected. In order to identify HPV genotypes associated with malignant and pre-malignant cervical lesions present in the area, 53 ecto-endo cervical cell specimens obtained from women with cytohistological alterations were studied by a PCR-RFLP technique. Out of 53 patients, 34 (64.2%) were positive for HPV infection, being HPV-16 (32.3%) the most frequently found genotype, followed by HPV-58 (14.7%), -6, -18 and -45 (5.9%), -33, -52, -53, -54, -56, -66, -MM4 and -LVX100 (2.9%). Also 5 cases of infection caused by multiple genotypes were found, which corresponded to 14.7% of the positive cases. Results indicate that besides HPV-16 and -18, the most prevalent high-risk HPV genotypes worldwide, others like -45 and -58 as well as co-infection cases are frequent between women of Northeastern Argentina, and a particular attention should be paid to this circumstance because it could be an epidemiological feature of regional importance and a useful information for a future vaccination program.

"Multiplex PCR" identification of the atypical and monophasic Salmonella enterica subsp. enterica serotype 1,4,[5],12:i:- in São Paulo State, Brazil: frequency and antibiotic resistance patterns

Salmonella spp. are the etiologic agents of salmonellosis, a worldwide spread zoonoses causing foodborne outbreaks and clinical diseases. By serological identification, Salmonella enterica subsp. enterica serotype 1,4,[5],12:i:- accounted for 8.8% of human and 1.6% of nonhuman Salmonella strains isolated in São Paulo State, during 1991-2000. A total of 28.6% of them amplified a fragment corresponding to H:1,2 (flagellar phase two) through PCR analysis and were further assigned as S. Typhimurium. Antimicrobial resistance was detected in 36.3% of the 369 PCR-negative strains tested, including the multiresistance to ampicillin, chloramphenicol, sulfonamides, tetracycline, and streptomycin.

Evolution of genotypes of Group A streptococci from colonization and infection

Dissertação para obtenção do Grau de Doutor em Biologia

Rapid turnover of Plasmodium falciparum var gene transcripts and genotypes during natural non-symptomatic infections

The var genes of Plasmodium falciparum code for the antigenically variant erythrocyte membrane proteins 1 (PfEMP1), a major factor for cytoadherence and immune escape of the parasite. Herein, we analyzed the var gene transcript turnover in two ongoing, non-symptomatic infections at sequential time points during two weeks. The number of different circulating genomes was estimated by microsatellite analyses. In both infections, we observed a rapid turnover of plasmodial genotypes and var transcripts. The rapidly changing repertoire of var transcripts could have been caused either by swift elimination of circulating var-transcribing parasites stemming from different or identical genetic backgrounds, or by accelerated switching of var gene transcription itself.

Distribution of hepatitis C virus genotypes among blood donors from mid-west region of Brazil

In order to investigate the hepatitis C virus (HCV) genotypes in mid-west region of Brazil, 250 anti-HCV positive blood donors were studied. Among them, the anti-HCV serological status was confirmed in 205 (82%). HCV RNA was detected in 165 samples, which were genotyped. HCV types 1, 2 and 3 were found in 67.9%, 3% and 29.1% of the donors, respectively. In Goiás state, subtype 1a (50%) was the most prevalent, followed by subtypes 3a (30.9%) and 1b (16.7%). In Mato Grosso state, subtype 1a was also predominant (41%), followed by subtypes 1b (29.5%) and 3a (25%). In Mato Grosso do Sul state, subtypes 1a and 1b were detected equally (36.8%), followed by 3a (21.1%). Subtype 2b was rare (2.4%, 4.5% and 5.3%, respectively). In Distrito Federal, subtype 3a (39%) was more frequent than 1a (31.7%) and the remaining (29.3%) belonged to subtype 1b.

Impact of Donor and Recipient Cytokine Genotypes on Renal Allograft Outcome

Allelic differences in gene promoter or codifying regions have been described to affect regulation of gene expression, consequently increasing or decreasing cytokine production and signal transduction responses to a given stimulus. This observation has been reported for interleukin (IL)-10 (-1082 A/G; -819/-592 CT/CA), transforming growth factor (TGF)-beta (codon 10 C/T, codon 25 G/C), tumor necrosis factor (TNF)-alpha (-308 G/A), TNF-beta (+252 A/G), interferon (IFN)-gamma (+874 T/A), IL-6 (-174 G/C), and IL-4R alpha (+1902 G/A). To evaluate the influence of these cytokine genotypes on the development of acute or chronic rejection, we correlated the genotypes of both kidney graft recipients and cadaver donors with the clinical outcome. Kidney recipients had 5 years follow-up, at least 2 HLA-DRB compatibilities, and a maximum of 25% anti-HLA pretransplantation sensitization. The clinical outcomes were grouped as follows: stable functioning graft (NR, n = 35); acute rejection episodes (AR, n = 31); and chronic rejection (CR, n = 31). The cytokine genotype polymorphisms were defined using PCR-SSP typing. A statistical analysis showed a significant prevalence of recipient IL-10 -819/-592 genotype among CR individuals; whereas among donors, the TGF-beta codon 10 CT genotype was significantly associated with the AR cohort and the IL-6 -174 CC genotype with CR. Other albeit not significant observations included a strong predisposition of recipient TGF-beta codon 10 CT genotype with CR, and TNF-beta 252 AA with AR. A low frequency of TNF-alpha -308 AA genotype also was observed among recipients and donors who showed poor allograft outcomes.

Distribution of hepatitis B virus genotypes and viral load levels in Brazilian chronically infected patients in São Paulo city

The objective of the present study was to evaluate the serum viral load in chronically infected Hepatitis B virus (HBV) patients and to investigate the distribution of HBV genotypes in São Paulo city. Quantitative HBV-DNA assays and HBV genotyping have gained importance for predicting HBV disease progression, have been employed for assessing infectivity, for treatment monitoring and for detecting the emergence of drug resistance. Twenty-nine Brazilian patients with suspected chronic hepatitis B were studied, using real time PCR for viral load determination and direct DNA sequencing for the genotyping. The serology revealed chronic HBV infection in 22 samples. The HBV-DNA was positive in 68% samples (15/22). The phylogenetic analysis disclosed that eleven patients were infected with HBV genotype A, two with genotype F and two with genotype D. Thus, the genotype A was the most prevalent in our study.

Five Cases of Atypical Rickettsial Infections

Background: Rickettsia conorii is the most frequent species of RickettsiaI causing disease in Portugal. In general the disease manifests itself by fever, exanthema, headaches and the presence of an eschar. However atypical forms can be present and physicians should be aware. Aims: Analyse the atypical presentation of rickettsiosis. Material and Methods: Children admitted at the CHLC Hospital from 2000 to 2010 with atypical presentation of rickettsiosis. Clinical diagnosis was confirmed by serology and molecular techniques (PCR). Results: Five cases of children with a median age of 2 years, 1 of which female, were admitted between June and August. The diagnoses were: myositis (1), synovitis (1), cholecystitis (1), orchiepididymitis (1) and meningitis (1). Myositis developped with functional disability, CPK 9600 U/L, lower limbs’ edema, hypoalbuminemia (1,6 g/dL) and arterial hypertension. Synovitis developped with functional disability, synovial fluid increase and CRP 16,2 mg/dL. The child with cholecystitis had abdominal pain, intraabdominal fluid increase, leukopenia (1900/μL), thrombocytopenia (75000/μL) and CRP 15,3 mg/dL. Orchiepididymitis developped with testicle’s inflammatory signs, leukopenia (2900/μL), thrombocytopenia (90000/μL) and CRP 14,45 mg/dL. The patient with meningitis, who had pleocytosis (320 cells/μL), hyperproteinorrachia (284 mg/dL), hypoglicorrachia (36 mg/dL), presented only with fever and headaches. The tache noire and the classical triad were present in 3/5 cases. The clinical course was favourable in all cases. Antibodies against Rickettsia of spotted fever group were detected in 3/5 cases. In one patient Rickettsia conorii Malish strain was identified by PCR and sequencing. Conclusions: Rickettsial infection may present itself unusually. In a country of high prevalence, especially during summer months and in the presence of an inoculation eschar, it is of the uttermost importance to study the atypical presentations for a possible rickettsial infection.

Hepatitis C virus genotypes in hemodialysis patients in the Federal District, Brazil

Hepatitis C virus (HCV) genotypes and subtypes were determined in hemodialysis patients in the Federal District, Brazil, by sequencing of the 5' noncoding (NC) and nonstructural 5B (NS5B) regions. From 761 patients, 66 anti-HCV-positive samples were tested for HCV RNA. All 51 HCV RNA-positive samples by PCR of the 5' NC region were genotyped as genotypes 1 (90.2%) and 3 (9.8%). Subtype 1a (82.3%) was the most prevalent, followed by subtypes 3a (9.8%), 1b (5.9%) and 1a/1b (2.0%). Forty-two samples could be amplified and genotyped in the NS5B region: 38 (90.5%) as genotype 1, subtypes 1a, and 8 (9.5%) as genotype 3, subtype 3a. For the 42 samples sequenced in both regions, the genotypes and subtypes determined were concordant in 100% and 95.2% of cases, respectively. Two samples presented discrepant results, with the 5' NC region not distinguishing correctly the subtypes 1a and 1b. These findings indicate that the HCV genotype 1, subtype 1a, is the most prevalent among hemodialysis patients in the Federal District, Brazil.

A real-time quantitative assay for hepatitis B DNA virus (HBV) developed to detect all HBV genotypes

Hepatitis B virus (HBV) is a major cause of chronic liver disease worldwide. Besides genotype, quantitative analysis of HBV infection is extensively used for monitoring disease progression and treatment. Affordable viral load monitoring is desirable in resource-limited settings and it has been already shown to be useful in developing countries for other viruses such as Hepatitis C virus (HCV) and HIV. In this paper, we describe the validation of a real-time PCR assay for HBV DNA quantification with TaqMan chemistry and MGB probes. Primers and probes were designed using an alignment of sequences from all HBV genotypes in order to equally amplify all of them. The assay is internally controlled and was standardized with an international HBV panel. Its efficacy was evaluated comparing the results with two other methods: Versant HBV DNA Assay 3.0 (bDNA, Siemens, NY, USA) and another real-time PCR from a reference laboratory. Intra-assay and inter-assay reproducibilities were determined and the mean of CV values obtained were 0.12 and 0.09, respectively. The assay was validated with a broad dynamic range and is efficient for amplifying all HBV genotypes, providing a good option to quantify HBV DNA as a routine procedure, with a cheap and reliable protocol.

Evaluation of glycoprotein B genotypes and load of CMV infecting blood leukocytes on prognosis of AIDS patients

BACKGROUND: Cytomegalovirus (CMV) remains an important pathogen to immunocompromised patients even in the era of HAART. The present study aimed at evaluating the influence of CMV viral load and its gB genotypes on AIDS patients' outcome. METHODS: Blood samples of 101 AIDS patients were collected and tested for HIV load, CD4 - cell count and opportunistic pathogens, including CMV. Semi-nested PCRs were run to detect CMV genome and in the positive samples, gB genotyping and CMV load were established using enzymatic restriction and real time PCR, respectively. All patients were clinically followed for four years. RESULTS: In thirty patients (31%) CMV was detected and all fatal cases (n = 5) occurred in this group of patients (p = 0.007), but only two patients had CMV disease (1.9%). However, viral load was not statistically associated with any analyzed parameter. The most frequently observed CMV genotype was gB2 (45.16%) followed by gB3 (35.48%). gB2 genotype was more frequently found in patients with CD4-cell counts under 200 cells/mm³ (p = 0.0017), and almost all fatal cases (80%) had gB2 genotype. CONCLUSIONS: Our study suggests that CMV and its polymorphisms in biologically relevant genes, such as the gB encoding ORF, may still influence the prognosis and outcome of AIDS patients. The gB2 genotype was associated to patient's bad outcome.