Active replication of hepatitis B virus (HBV) in HIV type 1 and in HIV type 2 infected patients

To evaluate the effect of concurrent infection by HIV on HBV infection or immunity, we have studied a group of 66 HIV1+ symptomatic Caucasian patients and another of 38 African HIV2+ asymptomatic individuals, concerning their HBV status: serological markers of infection and presence of HBV-DNA in serum, the last taken as sign of hepatitis B virus active replication, were monitored. HIV+ groups were compared with seronegative controls, adequately matched for age, sex and ethnological background. HBV DNA was found in 7.6% of HIV1+ Caucasian patients and 3.2% of seronegative controls; in African HIV2+ individuals 2.6% were also HBV DNA+, a percentage close to that found in HIV2 seronegative controls (2.9%). No correlation was found between HIV infection and HBV active replication. Immunodepression that follows HIV infection over time may be compatible with a degree of T cell function capable of avoiding reinfection with or reactivation of HBV, even in symptomatic stages of acquired immunodeficiency syndrome. Our findings are relevant to the choice of preventive strategies in populations at risk for HIV and HBV infection.

Neutralizing antibodies in Brazilian sera against three strains of human immunodeficiency virus type 1 (HIV-1)

The MN strain of HIV-1 is known to be more prevalent in Brazil, the BRU strain is more prevalent in Europe, and the NDK strain in Africa. It has been suggested in the literature to include different strains in the same vaccine against HIV-1. To contribute to the studies for the development of a universal vaccine, the occurrence of antibodies (Ab) against three HIV-1 strains (MN, BRU and NDK) was determined in serum samples from 85 HIV-1-positive patients, adult volunteers seen at the University Hospital of the Faculty of Medicine of Ribeirão Preto-USP. One-hundred tissue culture infective unit (TCIU) of the viruses reacted with serial dilutions of the sera (2x) and with MT4 cells added at a final concentration of 0.3 × 106 cells/ml, and a cytopathic effect was observed on the 7th and 11th days of incubation. Titres of less than 1/50 were considered to be negative. In 129 tests, the sera were negative for one of the three strains: 40 for MN, 29 for BRU and 60 for NDK. There was a predominance of strains MN and BRU, most of them presenting titres from 1/50 to 1/200. Titres for NDK were detected in 25 sera. We conclude that there seems to be a predominance of strains MN and BRU among the individuals from the region tested; however, the detection of sera with positive NKD titres indicates the need for further studies of this strain in other populations and regions of Brazil

Cytotoxicity in L929 fibroblasts and inhibition of herpes simplex virus type 1 Kupka by estuarine cyanobacteria extracts

The cyanobacteria are known to be a rich source of metabolites with a variety of biological activities in different biological systems. In the present work, the bioactivity of aqueous and organic (methanolic and hexane) crude extracts of cyanobacteria isolated from estuarine ecosystems was studied using different bioassays. The assessment of DNA damage on the SOS gene repair region of mutant PQ37 strain of Escherichia coli was performed. Antiviral activity was evaluated against influenza virus, HRV-2, CVB3 and HSV-1 viruses using crystal violet dye uptake on HeLa, MDCK and GMK cell lines. Cytotoxicity evaluation was performed with L929 fibroblasts by MTT assay. Of a total of 18 cyanobacterial isolates studied, only the crude methanolic extract of LEGE 06078 proved to be genotoxic (IF > 1.5) in a dose-dependent manner and other four were putative candidates to induce DNA damage. Furthermore, the crude aqueous extract of LEGE 07085 showed anti- herpes type 1 activity (IC50 = 174.10 μg dry extract mL−1) while not presenting any cytotoxic activity against GMK cell lines. Of the 54 cyanobacterial extracts tested, only the crude methanolic and hexane ones showed impair on metabolic activity of L929 fibroblasts after long exposure (48–72 h). The inhibition of HSV-1 and the strong cytotoxicity against L929 cells observed emphasizes the importance of evaluating the impact of those estuarine cyanobacteria on aquatic ecosystem and on human health. The data also point out their potential application in HSV-1 treatment and pharmacological interest.

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 analysis of the dengue virus type 1 and 2 in Brazil based on sequences of the genomic envelope-nonstructural protein 1 junction region

The genomic sequences of the Envelope-Non-Structural protein 1 junction region (E/NS1) of 84 DEN-1 and 22 DEN-2 isolates from Brazil were determined. Most of these strains were isolated in the period from 1995 to 2001 in endemic and regions of recent dengue transmission in São Paulo State. Sequence data for DEN-1 and DEN-2 utilized in phylogenetic and split decomposition analyses also include sequences deposited in GenBank from different regions of Brazil and of the world. Phylogenetic analyses were done using both maximum likelihood and Bayesian approaches. Results for both DEN-1 and DEN-2 data are ambiguous, and support for most tree bipartitions are generally poor, suggesting that E/NS1 region does not contain enough information for recovering phylogenetic relationships among DEN-1 and DEN-2 sequences used in this study. The network graph generated in the split decomposition analysis of DEN-1 does not show evidence of grouping sequences according to country, region and clades. While the network for DEN-2 also shows ambiguities among DEN-2 sequences, it suggests that Brazilian sequences may belong to distinct subtypes of genotype III.

Analysis of HIV- type 1 protease and reverse transcriptase in Brazilian children failing highly active antiretroviral therapy (HAART)

The aim of this study was to evaluate the genotypic resistance profiles of HIV-1 in children failing highly active antiretroviral therapy (HAART). Forty-one children (median age = 67 months) receiving HAART were submitted to genotypic testing when virological failure was detected. cDNA was extracted from PBMCs and amplified by nested PCR for the reverse transcriptase and protease regions of the pol gene. Drug resistance genotypes were determined from DNA sequencing. According to the genotypic analysis, 12/36 (33.3%) and 6/36 (16.6%) children showed resistance and possible resistance, respectively, to ZDV; 5/36 (14%) and 4/36 (11.1%), respectively, showed resistance and possible resistance to ddI; 4/36 (11.1%) showed resistance to 3TC and D4T; and 3/36 (8.3%) showed resistance to Abacavir. A high percentage (54%) of children exhibited mutations conferring resistance to NNRTI class drugs. Respective rates of resistance and possible resistance to PIs were: RTV (12.2%, 7.3%); APV (2.4%, 12.1%); SQV(0%, 12.1%); IDV (14.6%, 4.9%), NFV (22%, 4.9%), LPV/RTV (2.4%, 12.1%). Overall, 37/41 (90%) children exhibited virus with mutations related to drug resistance, while 9% exhibited resistance to all three antiretroviral drug classes.

T CD4+ cells count among patients co-infected with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1): high prevalence of tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM)

INTRODUCTION: HIV positive patients co-infected with HTLV-1 may have an increase in their T CD4+ cell counts, thus rendering this parameter useless as an AIDS-defining event. OBJECTIVE: To study the effects induced by the co-infection of HIV-1 and HTLV-1 upon CD4+ cells. MATERIAL AND METHODS: Since 1997, our group has been following a cohort of HTLV-1-infected patients, in order to study the interaction of HTLV-1 with HIV and/or with hepatitis C virus (HCV), as well as HTLV-1-only infected asymptomatic carriers and those with tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM). One hundred and fifty HTLV-1-infected subjects have been referred to our clinic at the Institute of Infectious Diseases "Emílio Ribas", São Paulo. Twenty-seven of them were also infected with HIV-1 and HTLV-1-infection using two ELISAs and confirmed and typed by Western Blot (WB) or polymerase chain reaction (PCR). All subjects were evaluated by two neurologists, blinded to the patient's HTLV status, and the TSP/HAM diagnostic was based on the World Health Organization (WHO) classification. AIDS-defining events were in accordance with the Centers for Disease Control (CDC) classification of 1988. The first T CD4+ cells count available before starting anti-retroviral therapy are shown compared to the HIV-1-infected subjects at the moment of AIDS defining event. RESULTS: A total of 27 HIV-1/HTLV-1 co-infected subjects were identified in this cohort; 15 already had AIDS and 12 remained free of AIDS. The median of T CD4+ cell counts was 189 (98-688) cells/mm³ and 89 (53-196) cells/mm³ for co-infected subjects who had an AIDS-defining event, and HIV-only infected individuals, respectively (p = 0.036). Eight of 27 co-infected subjects (30%) were diagnosed as having a TSP/HAM simile diagnosis, and three of them had opportunistic infections but high T CD4+ cell counts at the time of their AIDS- defining event. DISCUSSION: Our results indicate that higher T CD4+ cells count among HIV-1/HTLV-1-coinfected subjects was found in 12% of the patients who presented an AIDS-defining event. These subjects also showed a TSP/HAM simile picture when it was the first manifestation of disease; this incidence is 20 times higher than that for HTLV-1-only infected subjects in endemic areas.

Evaluation of the human immunodeficiency virus type 1 and 2 antibodies detection in dried whole blood spots (dbs) samples

Human Immunodeficiency Vírus Type 1 and 2 antibodies detection was performed in 457 dried whole blood spots samples (S&S 903). Q-Preven HIV 1+2 was the screening test used. The results were compared with the gold standard serum tests by ELISA (Cobas Core e Axsym HIV1/2 gO) and imunofluorescence was the definitive confirmatory test. The samples were obtained from the Hospital Nossa Senhora da Conceição in Porto Alegre, RS - Brazil, through whole blood transfer to filter paper card and sent to Caxias do Sul, RS - Brazil where the tests were performed. The dried whole blood spot stability was evaluated with two different panels. The first one was composed of five negative and five positive samples stored at room temperature, 4 ºC, -20 ºC and -70 ºC, while the second was composed of two negative and three positive samples stored at 37 ºC (humidity <50%). Each sample was screened every week for six weeks. These measurement results didn't show variation during the study period. The detected sensibility was 100%, specificity was 99.6%, the positive predictive value was 99.5% and negative predictive values were 100%. The results demonstrated high performance characteristics, opening a new perspective of dried whole blood spot utilization in HIV screening diagnosis.

Influence of human t-cell lymphotropic virus type 1 (HTLV-1) Infection on laboratory parameters of patients with chronic hepatitis C virus

Hepatitis C virus (HCV) and human T-cell lymphotropic virus type 1 (HTLV-1) share routes of transmission and some individuals have dual infection. Although some studies point to a worse prognosis of hepatitis C virus in patients co-infected with HTLV-1, the interaction between these two infections is poorly understood. This study evaluated the influence of HTLV-1 infection on laboratory parameters in chronic HCV patients. Twelve HTLV-1/HCV-coinfected patients were compared to 23 patients infected only with HCV, in regard to demographic data, risk factors for viral acquisition, HCV genotype, presence of cirrhosis, T CD4+ and CD8+ cell counts and liver function tests. There was no difference in regard to age, gender, alcohol consumption, smoking habits, HCV genotype or presence of cirrhosis between the groups. Intravenous drug use was the most common risk factor among individuals co-infected with HTLV-1. These patients showed higher TCD8+ counts (p = 0.0159) and significantly lower median values of AST and ALT (p = 0.0437 and 0.0159, respectively). In conclusion, we have shown that HCV/HTLV-1 co-infected patients differs in laboratorial parameters involving both liver and immunological patterns. The meaning of these interactions in the natural history of these infections is a matter that deserves further studies.

TESTING A SUBTYPE-SPECIFIC GP41 AMPLIFICATION METHOD FOR GENOTYPING INDIVIDUALS INFECTED BY HUMAN IMMUNODEFICIENCY VIRUS TYPE-1 IN THE BRAZILIAN POPULATION OF ITAJAÍ, SOUTH BRAZIL

The method used by YAGYU et al. for the subtype-specific polymerase chain reaction (PCR) amplification of the gp41 transmembrane region of the human immunodeficiency virus type-1 (HIV-1) env gene, was tested. HIV-1 proviral DNA from 100 infected individuals in Itajaí, South Brazil was used to analyze this method. Seventy individuals were determined according to this method as having PCR products at the expected size for subtypes B, C, D and F. Of these individuals, 26 (37.1%) were observed as having the expected amplification for subtype C, and 42 (60%) were observed as having the expected products for subtypes B and D. Of the subtype B and D amplicons, 16 (22.9%) were classified as subtype D, and 26 (37.1%) were classified as subtype B. Two individuals (2.9%) had amplicons that were observed after subtype F-specific amplification was performed. Sequencing and comparing the patient sequences to reference sequences confirmed the classification of sequences of subtypes C and B. However, sequences that were falsely determined as being D and F in the PCR assay were determined as being subtypes C and B, respectively, by sequence analysis. For those individuals from whom no amplified products were obtained, a low viral load that was indicated in their patient history may explain the difficulty in subtyping by PCR methods. This issue was demonstrated by the results of ANOVA when testing the effect of viral load on the success of PCR amplification. The alignment of the obtained sequences with HIV-1 reference sequences demonstrated that there is high intra-subtype diversity. This indicates that the subtype-specific primer binding sites were not conserved or representative of the subtypes that are observed in the Brazilian populations, and that they did not allow the correct classification of HIV-1 subtypes. Therefore, the proposed method by YAGYU et al. is not applicable for the classification of Brazilian HIV-1 subtypes.

ORIGIN AND PREVALENCE OF HUMAN T-LYMPHOTROPIC VIRUS TYPE 1 (HTLV-1) AND TYPE 2 (HTLV-2) AMONG INDIGENOUS POPULATIONS IN THE AMERICAS

Human T-lymphotropic virus type 1 (HTLV-1) is found in indigenous peoples of the Pacific Islands and the Americas, whereas type 2 (HTLV-2) is widely distributed among the indigenous peoples of the Americas, where it appears to be more prevalent than HTLV-1, and in some tribes of Central Africa. HTLV-2 is considered ancestral in the Americas and is transmitted to the general population and injection drug users from the indigenous population. In the Americas, HTLV-1 has more than one origin, being brought by immigrants in the Paleolithic period through the Bering Strait, through slave trade during the colonial period, and through Japanese immigration from the early 20th century, whereas HTLV-2 was only brought by immigrants through the Bering Strait. The endemicity of HTLV-2 among the indigenous people of Brazil makes the Brazilian Amazon the largest endemic area in the world for its occurrence. A review of HTLV-1 in all Brazilian tribes supports the African origin of HTLV-1 in Brazil. The risk of hyperendemicity in these epidemiologically closed populations and transmission to other populations reinforces the importance of public health interventions for HTLV control, including the recognition of the infection among reportable diseases and events.

Mutational Analysis of Portuguese Families with Multiple Endocrine Neoplasia Type 1 Reveals Large Germline Deletions

OBJECTIVE: To determine the spectrum of MEN1 mutations in Portuguese kindreds, and identify mutation-carriers. PATIENTS, DESIGN AND RESULTS: Six unrelated MEN1 families were studied for MEN1 gene mutations by single-strand conformational polymorphism (SSCP) and DNA sequence analysis of the coding region and exon-intron boundaries of the MEN1 gene. These methods identified 4 different heterozygous mutations in four families: two mutations are novel (mt 1539 delG and mt 655 ims 11 bp) and two have been previously observed (mt 735 del 46p and mt 1656 del C) all resulting in a premature stop codon. In the remaining two families, in whom no mutations or abnormal MEN1 transcripts were detected, segregation studies of the 5' intragenic marker D11S4946 and codon 418 polymorphism in exon 9 revealed two large germline deletions of the MEN1 gene. Southern blot and tumour loss of heterozygosity analysis confirmed and refined the limits of these deletions, which spanned the MEN1 gene at least from: exon 7 to the 3' untranslated region, in one family, and the 5' polymorphic site D11S4946 to exon 9 (obliterating the initiation codon), in the other family. Twenty-six mutant-gene carriers were identified, 6 of which were asymptomatic. CONCLUSIONS: These results emphasize the importance of the detection of MEN1 germline deletions in patients who do not have mutations of the coding region. Important clues indicating the presence of such deletions may be obtained by segregation studies using the intragenic polymorphisms D11S4946 and at codon 418. The detection of these mutations will help in the genetic counselling of clinical management of the MEN1 families in Portugal.

Pyoderma Gangrenosum Associated with Sclerosing Cholangitis, Type 1 Diabetes Mellitus and Ulcerative Colitis

We describe the case of a 22-year-old black female with type 1 diabetes mellitus diagnosed when she was 12 years old. She first presented (March 1994) with pustules and ulcerations on the upper and lower limbs, trunk and scalp at the age 17. The diagnosis of pyoderma gangrenosum was made. Since presentation, changes in liver function were detected and subsequent study led to the diagnosis of sclerosing cholangitis. The diagnosis of ulcerative colitis was made after colonoscopy. Partial response was obtained with minocycline and clofazimine, but treatment with 5-aminosalicylic acid achieved no improvement of the ulcerations. Liver transplantation, followed by immunosuppressive therapy led to complete regression of the cutaneous lesions.