No evidence of vertical transmission of HTLV-I in bottle-fed children

The most frequent pathway of vertical transmission of HTLV-I is breast-feeding, however bottle fed children may also become infected in a frequency varying from 4 to 14%. In these children the most probable routes of infection are transplacental or contamination in the birth canal. Forty-one bottle-fed children of HTLV-I seropositive mothers in ages varying from three to 39 months (average age of 11 months) were submitted to nested polymerase chain reaction analysis (pol and tax genes). 81.5% of the children were born by an elective cesarean section. No case of infection was detected. The absence of HTLV-I infection in these cases indicates that transmission by transplacental route may be very infrequent.

New prevalence estimate of TT virus (TTV) infection in low- and high-risk population from São Paulo, Brazil

The prevalence of TT virus (TTV) infection was investigated by Polymerase Chain Reaction (PCR) in low- (blood donors and healthy children/adolescents) and high-risk (hemophiliacs) groups from São Paulo, Brazil. Primers based on the untranslated region (UTR) of the viral genome proved to be much more ubiquitous, leading to much higher frequencies for both groups ( > or = 81%) than the earlier N22-PCR directed to the open reading frame 1 (blood donors, 5.5%, and hemophiliacs, 42.3%). The UTR-PCR also revealed an interesting profile for healthy children/adolescents: very high prevalence at the early years and significant decrease in male teenagers. The N22-PCR, in turn, demonstrated higher frequency in hemophiliacs treated with fresh blood products (58%), than in those treated with virus-inactivated clotting factors (9.4%) and blood donors (5.5%).

Assessment of PCR in the detection of Leishmania spp in experimentally infected individual phlebotomine sandflies (Diptera: Psychodidae: Phlebotominae)

DNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei).

Conventional and molecular typing of Salmonella typhi strains from Brazil

Phenotypic and genotypic characteristics of Salmonella Typhi were studied in 30 strains, isolated in different years, from some areas in Brazil. Conventional typing methods were performed by biochemical tests, Vi phage-typing scheme, and antimicrobial susceptibility test. Molecular typing methods were performed by analysis of plasmid DNA and by random amplified polymorphic DNA (RAPD-PCR). For the latter, an optimization step was performed to ensure the reproducibility of the process in genetic characterization of S. Typhi. The predominance of 76.7% of biotype I (xylose +, arabinose -) was noticed in all studied areas. Three phage types were recognized, with prominence for the phage types A (73.3%) and I+IV (23.3%). All the strains were susceptible to the drugs used. However, 36.7% of the strains contained plasmids, with predominance of the 105 Kb plasmid. RAPD was capable of grouping the strains in 8 genotypic patterns using primer 784, in 6, using primer 787 and in 7, using primer 797. Conventional phenotypic typing methods, as well as the DNA plasmid analysis, presented nonsignificant discriminatory power; however, RAPD-PCR analysis showed discriminatory power, reproducibility, easy interpretation and performance, being considered as a promising alternative typing method for S. Typhi.

Molecular characterization of Dengue viruses type 1 and 2 isolated from a concurrent human infection

In 2001, an autochthonous case of dual viremia, resulting from naturally acquired dengue virus DEN-1 and DEN-2 infections was detected during the dengue outbreak that occurred in Barretos, a city with about 105,000 inhabitants in the North region of São Paulo State. Serotype identification was based on virus isolation to C6/36 mosquito cells culture and immunofluorescence assays using type-specific monoclonal antibodies. The double infection was also confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Comparative analysis of the 240-nucleotide sequences of E/NS1 gene junction region between the genome of DEN-1 and DEN-2 isolates of the corresponding reference Nauru and PR 159S1 strains, respectively, showed some nucleotide differences, mainly silent mutations in the third codon position. Results of maximum likelihood phylogenetic analysis of E/NS1 gene sequences indicated that both genotypes of DEN-1 and DEN-2 viruses recovered from double infection in Barretos belonged to genotypes I and III, respectively.

Molecular typing of dengue virus type 2 in Brazil

Strain typing is a critical tool for molecular epidemiological analysis and can provide important information about the spread of dengue viruses. Here, we performed a molecular characterization of DEN-2 viruses isolated in Brazil during 1990-2000 from geographically and temporally distinct areas in order to investigate the genetic distribution of this serotype circulating in the country. Restriction site-specific polymerase chain reaction (RSS)-PCR presented the same pattern for all 52 Brazilian samples, showing the circulation of just one DEN-2 variant. Phylogenetic analysis using progressive pairwise alignments from 240-nucleotide sequences of the E/NS1 junction in 15 isolates showed that they belong to genotype III (Jamaica genotype).

Adenoviruses isolated from civilian and military personnel in the city of Rio de Janeiro, Brazil

Adenovirus are important pathogen primarily associated to respiratory infections of children and military personnel, even though it is also associated to cases of conjunctivitis and keratoconjunctivitis. We analyzed respiratory secretion collected from subjects with and without respiratory infection symptoms, being 181 civilians and 221 military subjects. The samples were inoculated in HEp-2 and/or A549 tissue cultures for viral isolation. Samples presenting cytopathogenic effect (CPE) in any tissue culture were tested by a polymerase chain reaction (PCR) assay to confirm adenovirus isolation. The isolates confirmed as adenovirus were further analyzed by restriction endonuclease assay for determination of viral species. Three isolates were identified as specie A (two from civilian and one from military), one isolate from military was identified as specie C, and one isolate from civilian was identified as specie D. For two isolates the specie could not be identified.

Evidence of active herpesvirus 6 (variant-A) infection in patients with lymphadenopathy in Belém, Pará, Brazil

A total of 323 patients with lymphadenopathy were selected in Belém, Brazil, between January 1996 and December 2001, and screened for the presence of human herpesvirus-6 (HHV-6) IgM- and- IgG antibodies by enzyme-linked immunosorbent assay (ELISA). When seroprevalence is analyzed by gender, similar rates are found for female (60.6%) and male (55.7%) individuals. Seventy-seven (23.8%) patients were HHV-6-IgM-and- IgG-positive (IgM+ subgroup), with positivity rates of 29.7% and 17.7% (p = 0.0007) for female- and male individuals, respectively. Sera from a subgroup (n = 120) of these subjects, with high HHV-6 antibody levels (either IgM+ or IgG+ reactivities), were subsequently processed for the presence of HHV-6 DNA by polymerase chain reaction (PCR)/nested PCR. Active infections (IgM+ and/or IgG+ high levels specific antibodies plus detection of viral DNA) were diagnosed in 20/77 (20.0%) and 8/43 (18.6%); subgroup of the 120 individuals suspected of having HHV-6 suggestive recent infection. All (n = 28) cases of active infection were found to be associated with HHV-6 variant-A (HHV-6A), as detectable by PCR/nested PCR, using variant-specific primer that amplify regions of 195 base pairs (bp) (HHV-6A) and 423 bp (HHV-6B). Rates of HHV-6 DNA detection between female and male patients were similar (p > 0.05) in the IgM+ and IgG+ groups: 20.4% versus 35.7% and 25.0% versus 13.0%, respectively. HHV-6 DNA was detected across < 5 through 41-50-year age-groups for patients whose serum samples were IgM+, with rates ranging from 7.7% (female subjects aged < 5 years) to 80.0% (male, 11-20 years). Among patients whose serological status was IgG+, HHV-6 DNA was detected in < 5, 6-10, 21-30 and > 50 age-groups at rates that ranged from 15.4% (male, < 5 years of age) to 100.0% (female aged 11-20 years). Swelling cervical lymph nodes were the most common sign, accounting for 9 (32.0%) cases in each gender group. Among patients (n = 28) with active infection by HHV-6A variant, duration of symptoms lasted 1-5 days in 35.7% of subjects, whereas in 64.3% of them the disease lasted 6-20 days. Our data suggest that it is worth seeking for HHV-6 infection whenever a patient (infant or adult) presents with lymphadenopathy as a prominent symptom in the course of an acute febrile illness.

Determination of human cytomegalovirus genetic diversity in different patient populations in Costa Rica

Seroprevalence of HCMV in Costa Rica is greater than 95% in adults; primary infections occur early in life and is the most frequent congenital infection in newborns. The objectives of this study were to determine the genetic variability and genotypes of HCMV gB gene in Costa Rica. Samples were collected from alcoholics, pregnant women, blood donors, AIDS patients, hematology-oncology (HO) children and HCMV isolates from neonates with cytomegalic inclusion disease. A semi-nested PCR system was used to obtain a product of 293-296 bp of the gB gene to be analyzed by Single Stranded Conformational Polymorphism (SSCP) and sequencing to determine the genetic polymorphic pattern and genotypes, respectively. AIDS patients showed the highest polymorphic diversity with 14 different patterns while fifty-six percent of HO children samples showed the same polymorphic pattern, suggesting in this group a possible nosocomial infection. In neonates three genotypes (gB1, gB2 and gB3), were determined while AIDS patients and blood donors only showed one (gB2). Of all samples analyzed only genotypes gB1, 2 and 3 were determined, genotype gB2 was the most frequent (73%) and mixed infections were not detected. The results of the study indicate that SSCP could be an important tool to detect HCMV intra-hospital infections and suggests a need to include additional study populations to better determine the genotype diversity and prevalence.

Human herpesvirus-7 as a cause of exanthematous ilnesses in Belém, Pará, Brazil

We screened sera from 370 patients suffering from exanthematous illnesses in Belém, North Brazil, for the presence of human herpesvirus-7 (HHV-7) IgM and IgG antibodies. Samples were obtained from January 1996 to December 2002 and were processed by a HHV-7-specific indirect immunofluorescence assay (IFA). HHV-7-specific IgM and/or IgG antibodies were found in 190 (51.4%) of these patients, with similar prevalence rates (IgM+ and IgG+ subgroups taken together) for female and male subjects: 52.5% and 50.3%, respectively. Serological status as defined by IgG was identified in 135 (36.5%) patients. In 55 (14.9%) of the patients HHV-7 IgM antibodies were detected. HHV-7 IgM- and- IgG antibody rates were similar (p > 0.05) when male and female subjects are compared: 14.4% versus 15.3% and 38.1% versus 35.0%, respectively. Statistically significant difference (p = 0.003) was noted when HHV-7-IgM-positive female and male patients aged 5-8 months are compared. Prevalence rates ranging from 4.6% (female, 5-8 months of age) to 93.3% (female, > 10 years of age) and 12.2% (male, 5-8 months) to 80.0% (male, 8-10 years of age) were noted in the IgG- positive subgroups. A subgroup (n = 131) of patients with IgM or IgG HHV-7 antibodies were examined for the presence of DNA using a polymerase chain reaction/nested PCR. Recent/active HHV-7 infection occurred at a rate of 11.0% (6/55) among patients whose samples presented IgM+ specific antibodies. In a subgroup (n = 76) of patients with high HHV-7-IgG antibody levels (titre > 1:160) DNA could not be detected in sera examined by PCR/nested PCR. Of the six recent/active infections, four subjects with less than 1 year and two with 3 and 6 years of age, presented typical exanthem subitum (E.S), as defined by higher fever (> 38.0 ºC) with duration of 24 to 72 hours, followed by a maculopapular skin rash. Our results underscore the need for searching HHV-7 infection in patients with exanthematous diseases, particularly those presenting with typical E.S. HHV-7 appears therefore to emerge as a newly recognized pathogen of exanthem in our region.

PCR - based diagnosis to evaluate the performance of malaria reference centers

Although the Giemsa-stained thick blood smear (GTS) remains the gold standard for the diagnosis of malaria, molecular methods are more sensitive and specific to detect parasites and can be used at reference centers to evaluate the performance of microscopy. The description of the Plasmodium falciparum, P. vivax, P. malariae and P. ovale ssrRNA gene sequences allowed the development of a polymerase chain reaction (PCR) that had been used to differentiate the four species. The objective of this study was to determine Plasmodium species through PCR in 190 positive smears from patients in order to verify the quality of diagnosis at SUCEN's Malaria Laboratory. Considering only the 131 positive results in both techniques, GTS detected 4.6% of mixed and 3.1% of P. malariae infections whereas PCR identified 19.1% and 13.8%, respectively.

Serological, epidemiological and molecular aspects of hepatitis C virus infection in a population from Londrina, PR, Brazil, 2001-2002

Serological, epidemiological and molecular aspects of hepatitis C virus (HCV) infection were evaluated in 183 subjects from Londrina, Paraná, Brazil, and adjacent areas. Serum samples which tested anti-HCV positive by microparticle enzyme immunoassay (MEIA) obtained from eight patients with chronic hepatitis C, 48 blood donors, and 127 patients infected with the human immunodeficiency virus (HIV) were submitted to another enzyme immunoassay (ELISA) and to the polymerase chain reaction (PCR). About 78.7% of samples were also reactive by ELISA, with the greater proportion (70.8%) of discordant results verified among blood donors. A similar finding was observed for HCV-RNA detection by PCR, with 111/165 (67.3%) positive samples, with higher rates among HIV-positive subjects and patients with chronic hepatitis than among blood donors. Sixty-one PCR-positive samples were submitted to HCV genotyping, with 77.1, 21.3 and 1.6% of the samples identified as types 1, 3 and 2, respectively. Finally, analysis of some risk factors associated with HCV infection showed that intravenous drug use was the most common risk factor among HIV/HCV co-infected patients, while blood transfusion was the most important risk factor in the group without HIV infection. The present study contributed to the knowledge regarding risk factors associated with HCV infection and the distribution of HCV genotypes in the population evaluated.

Molecular identification of Candida dubliniensis isolated from oral lesions of HIV-positive and HIV-negative patients in São Paulo, Brazil

Candida dubliniensis is a new, recently described species of yeast. This emerging oral pathogen shares many phenotypic and biochemical characteristics with C. albicans, making it hard to differentiate between them, although they are genotypically distinct. In this study, PCR (Polymerase Chain Reaction) was used to investigate the presence of C. dubliniensis in samples in a culture collection, which had been isolated from HIV-positive and HIV-negative patients with oral erythematous candidiasis. From a total of 37 samples previously identified as C. albicans by the classical method, two samples of C. dubliniensis (5.4%) were found through the use of PCR. This study underscores the presence of C. dubliniensis, whose geographical and epidemiological distribution should be more fully investigated.

Fecal specimens preparation methods for PCR diagnosis of human taeniosis

Sample preparation and DNA extraction protocols for DNA amplification by PCR, which can be applied in human fecal samples for taeniasis diagnosis, are described. DNA extracted from fecal specimens with phenol/chloroform/isoamilic alcohol and DNAzol® reagent had to be first purified to generate fragments of 170 pb and 600 pb by HDP2-PCR. This purification step was not necessary with the use of QIAmp DNA stool mini kit®. Best DNA extraction results were achieved after eggs disruption with glass beads, either with phenol/chloroform/isoamilic alcohol, DNAzol® reagent or QIAmp DNA stool mini kit®.

Virulence factors of uropathogenic Escherichia coli from a University Hospital in Ribeirão Preto, São Paulo, Brazil

The aim of the study was to determine the occurrence of virulence genes expressing fimbriae, production of hemolysin, colicin and aerobactin among a hundred Escherichia coli isolates obtained from in-and outpatients of a tertiary-care teaching hospital, between July and August 2000, showing clinical and laboratory signs of urinary tract infection (UTI). The presence of genes (pap, afa, sfa) for fimbriae expression was assayed using specific primers in a polymerase chain reaction. Among the isolates studied, the prevalence of the virulence factors was 96.0%, 76.0%, 24.0%, for hemolysin, aerobactin and colicin, respectively; the prevalence of genes coding for fimbrial adhesive systems was 32.0%, 19.0% and 11.0% for pap, sfa and afa respectively. The strains isolated from the outpatients displayed a greater number of virulence factors compared to those from hospitalized subjects, emphasizing the difference between these two kinds of patients.