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Aims: To improve RT-qPCR with an internal control and a synthetic standard curve to detect HEV in HIV co-infected patients. Methods and Results: A single-stranded RNA (ssRNA) and a double-stranded DNA (dsDNA) synthetic curve were designed, compared to the international reference panel for HEV genotypes, and tested to quantify and detect a reference panel for HEV genotypes. The detection limit of the RNA synthetic curve (50 copies per ml) was better than the DNA synthetic curve (100 copies per ml) and the WHO standard curve (250 copies per ml). Then, 280 serum samples from HIV-positive patients were tested for HEV RNA, which was detected in 3 6% of serum samples. The viral load ranged from 2 9 102 copies per ml to 4 78 9 108 copies per ml. HEV IgM/IgG antibodies were not detected in the RNA-positive patients. Sequencing analysis of HEV showed that the virus belongs to genotype 3 (HEV GT3). Conclusions: Real-time PCR was a useful tool to estimate co-infection with HEV/HIV, even in patients with low viral loads and undetectable anti-HEV IgG and IgM antibodies. Significance and Impact of the Study: Hepatitis E virus genotype 3 (HEV GT3) has been associated with silent chronic hepatitis and cirrhosis in HIVpositive subjects worldwide, but there is a lack of data on this co-infection in Brazil.

Shifting of hepatitis A virus (HAV) epidemiology from a high towards an intermediate endemicity pattern and use of antiretroviral therapy increased the risk of HIV/HAV coinfection in developing countries. The aim of this study was to investigate the presence of HAV markers in a cohort of HIV-infected patients from 1988 to 2004. The presence of serum anti-HAV antibodies and HAV-RNA by real-time polymerase chain reaction was investigated in 581 patients. Total anti-HAV antibodies was found in 464/581 (79.8%) patients, however, achanging epidemiologic pattern of hepatitis A among HIV-infected patients from 1988 to 2004 was observed. Among patients susceptible to HAV ( n = 117), 5 (4.2%) were co-infected with HAV, all of them had IgM anti-HAV antibodies and were serum HAV-RNA-positive. The high prevalence of anti-HAV antibodies in HIV-infected patients suggests that screening tests for anti-HAV antibodies should be performed before implementation of hepatitis A vaccination, especially in those patients from endemic countries.

This was a study on the prevalence of HIV-syphilis coinfection among 830 HIV/AIDS patients who were being followed up as outpatients at a hospital in Rio de Janeiro between January and May 2005. The participants underwent laboratory tests at the venereal disease research laboratory (VDRL) consisting of CD4+/CD8+ cell counts and viral load tests. They answered questions about their sociodemographic characteristics and past medical history of syphilis. The prevalence of syphilis was 2.7% (22 patients) and the ratio between men and women with coinfection was approximately 4:1. Homosexual men were the category most affected. We did not find any association between coinfection and age, schooling and the laboratory parameters tested. Out of the total number of patients with syphilis, 73% (16) reported previous treatment; of these, 14 (88%) were reinfected, while two (12%) underwent inappropriate treatment. The presence of HIV-syphilis coinfection among patients undergoing routine follow-up signals the need to advise them to adopt safe sex practices, during their outpatient visits.

We have investigated the phenotypic and genotypic susceptibility of 14 HIV-1 strains isolated from individuals failing HAART therapy to protease inhibitors (PI). Proviral and plasma viral pol gene fragment were amplified, sequenced and subtyped. Nine samples clustered with protease subtype B reference strains and the remaining samples were classified as non-B subtype corresponding to subtype F (n=4) and subtype A (n=1). Although all patients were treated with similar PI drug regimen, the non-B subtype isolates did not present the L90M and I84V mutations and used mainly G48V and V82A/F to achieve drug resistance. A strong cross-resistance phenotype among all four PI was associated with the mutation L90M in the subtype-B isolates, and with G48V and V82A/F in the non-B counterparts. This observation revealed that the non-B viruses tested had specific genotypic characteristics contrasting with the subtype-B isolates.

Development of drug resistance is the inevitable consequence of incomplete suppression of virus plasma levels in HIV-1-infected patients treated with highly active antiretroviral therapy. Resistance mutations previously characterized have been found in B subtype viruses of developed countries. Moreover, mutation profiles for non-B and more divergent B subtype viruses found in developing countries shall be analyzed together with their ex vivo phenotyping in order to establish an exact correlation between the genotyping data and the clinical management counseling for those uncommon virus subtypes. In the present study, we evaluated the mutation profile for individuals infected with B subtype and non-B subtype viruses. Viral DNA fragments corresponding to the RT gene were amplified, sequenced, and subtyped. Phenotyping analysis for reverse transcriptase nucleoside (NRTI) and nonnucleoside inhibitor susceptibility was performed using the recombinant virus assay technology. Brazilian non-B subtypes (subtype F, n = 4, and subtype A, n = 1) isolates showed essentially the same B subtype mutation profile, presenting an NRTI drug resistance with similar MIC50% and MIC90% values for all drugs analyzed regardless of their subtypes. A strong cross-resistance phenotype among AZT, 3TC, and abacavir could be seen in all isolates analyzed. A novel result was that some RT sequences not only revealed the presence of G333D/E mutations but also correlated to the presence of mutation T386I that could abrogate the M184V-surpassing effect of L210W or L210W plus G333D/E. These findings suggest that Brazilian non-B subtype HIV-1 strains use an identical RT drug resistance mutation pattern when compared to B isolates and will contribute to the validation of the genotypic and phenotypic tests in these predominant worldwide-spread viral variants.

OBJECTIVE TLRs (Toll-like receptors) and RLRs (RIG-I-like receptors) mediate innate immune responses by detecting microorganism invasion. RIG-I activation results in the production of interferon (IFN) type 1 and IFN responsive genes (ISGs). As the ubiquitin ligases RNF125 and TRIM25 are involved in regulating RIG-I function, our aim was to assess whether the levels of these three genes vary between healthy and HIV-infected individuals and whether these levels are related to disease progression. DESIGN Gene expression analyses for RIG-I, RNF125, and TRIM25 were performed for HIV-infected adults and the children's peripheral blood mononuclear cells (PBMCs). METHODS Reverse transcription-quantitative PCRs (RT-qPCRs) were performed in order to quantify the expression levels of RIG-I, RNF125 and TRIM25 from PBMCs purified from control or HIV-infected individuals. RESULTS Controls express higher levels of the three genes when compared to HIV-infected patients. These expressions are clearly distinct between healthy and progressors, and are reproduced in adults and children. In controls, RNF125 is the highest expressed gene, whereas in progressors, RIG-I is either the highest expressed gene or is expressed similarly to RNF125 and TRIM25. CONCLUSION A pattern of expression of RIG-I, RNF125, and TRIM25 genes in HIV patients is evident. The high expression of RNF125 in healthy individuals reflects the importance of keeping RIG-I function off, inhibiting unnecessary IFN production. Consistent with this assumption, RNF125 levels are lower in HIV patients and importantly, the RNF125/RIG-I ratio is lower in patients who progress to AIDS. Our results might help to predict disease progression and unveil the role of poorly characterized host genes during HIV infection.

Background The diagnostic investigation of sensitization to dust mites is performed through measurement of specific IgE and cutaneous immediate hypersensitivity tests. The general aim of this study was to comparatively evaluate the diagnostic accuracy of immediate hypersensitivity skin tests and specific IgE for Dermatophagoides pteronyssinus (Dp) and Blomia tropicalis in patients with allergic rhinitis.

Background Atopic asthma occurs in a significant percentage of patients in different age groups and the main sensitizing allergens are dust mites. The aim of this study was to assess the levels of total serum IgE, specific IgE to Dermatophagoides pteronyssinus (Dp) and Blomia tropicalis (Bt) in patients with asthma as well as the influence of gender, age and severity of disease in the total serum IgE levels and in the sensitization to mites.

This was a study on the prevalence of HIV-syphilis coinfection among 830 HIV/AIDS patients who were being followed up as outpatients at a hospital in Rio de Janeiro between January and May 2005. The participants underwent laboratory tests at the venereal disease research laboratory (VDRL) consisting of CD4+/CD8+ cell counts and viral load tests. They answered questions about their sociodemographic characteristics and past medical history of syphilis. The prevalence of syphilis was 2.7% (22 patients) and the ratio between men and women with coinfection was approximately 4:1. Homosexual men were the category most affected. We did not find any association between coinfection and age, schooling and the laboratory parameters tested. Out of the total number of patients with syphilis, 73% (16) reported previous treatment; of these, 14 (88%) were reinfected, while two (12%) underwent inappropriate treatment. The presence of HIV-syphilis coinfection among patients undergoing routine follow-up signals the need to advise them to adopt safe sex practices, during their outpatient visits.

Background The diagnosis of allergic rhinitis (AR) is based on clinical manifestations and supported by a positive result for skin prick test (SPT) or serum specific immunoglobulin E (sIgE) antibodies to aeroallergens. Our objective was to investigate the frequency of patients with clinical manifestations of AR without evidence of specific IgE sensitization. Methods We evaluated patients with clinical manifestations suggestive of AR, other causes of rhinitis excluded, aged >5 years and who had total serum IgE and SPT or sIgE to aeroallergens measured. Skin tests were performed with extracts of Dermatophagoides pteronyssinus, Dermatophagoides farinae, Blomia tropicalis and Aspergillus fumigatus (FDA Allergenic) and total serum IgE and sIgE, for the same allergens, by ImmunoCAP (Phadia). Patients were subdivided into groups according to the results profile, and comparatively analyzed for association with asthma, severity of rhinitis and age. Results We evaluated 116 patients (64% female) aged between 5 and 79 years, including 34 children (29%) and 63 (54%) with bronchial asthma. The observed profiles and frequencies were: high IgE levels and positivity in the SPT or sIgE –55%; normal IgE levels and SPT or sIgE positivity –9%; high IgE levels and SPT and sIgE negativity –3 %; normal IgE levels and negativity in the SPT and sIgE –23%. Among patients with normal levels of total serum IgE and no evidence of specific IgE sensitization, 14% had asthma, while in the remainder the prevalence of asthma was 34% (P = 0.0009). There was no statistical significance in the influence of the rhinitis severity and age in the absence of markers of atopy and allergen sensitization. Conclusions We observed a significant number of patients with clinical manifestations of AR, without evidence of systemic atopy and specific IgE sensitization, indicating the importance of careful research of local allergic rhinitis, as well as other causes of chronic rhinitis. Local allergic rhinitis appears to be less frequent in patients with rhinitis and asthma. The observation of 13% of patients with elevated levels of total IgE without specific sensitization implies the possibility of sensitization to aeroallergens which were not investigated, such as occupational allergens.

Background CVID comprises a variety of clinical phenotypes that may influence the prognosis of the disease. Our goal was to investigate the clinical phenotypes and prognosis of a series of patients with CVID. Methods We evaluated 11 patients with CVID, according to the PAGID criteria in long-term clinical follow-up (> 10 years). Most patients were on regular use of intravenous immunoglobulin (IVIg), provided free of charge by the government. Clinical evaluation was performed monthly and exams every 6 months to 1 year, including immunological evaluation, hematologic, biochemical, autoimmune, stool, urine analysis, chest CT, abdominal ultrasound and specific investigations of infectious diseases and malignancies, when needed. Results The average follow-up was 21.9 years (12–34). Among the 11 patients, the mean current age was 39.8 years (16 to 62), 73% were female and 82% white. The age at symptoms onset ranged from 4 to 31 years (mean = 18) and diagnosis occurred between ages 11 and 47 (mean = 28). Most patients (55%) had the phenotype of infectious complications only, 27% had infections and immune thrombocytopenic purpura and 18% had infections and solid neoplasias. The most common infections were recurrent sinusitis (100%), pneumonia (82%), giardiasis (36%) and tuberculosis (18%). None of the patients developed lymphoproliferative and / or inflammatory complications. With regard to immunological changes, we observed that 4 patients (36%) experienced an increase in CD8 T lymphocytes and inversion of CD4/CD8 ratio. Adherence to the use of IVIg was good in 50% of patients, fair in 38% and unsatisfactory in 12%. All patients have good quality of life, performing their routine activities of study, work and leisure. Conclusions In the population studied, the most frequent phenotypes were infectious complications or infectious complications + autoimmunity. Tuberculosis can be an important infectious complication in patients with CVID in endemic areas. The delay in the diagnosis of CVID, around 10 years, indicates the need to improve the diagnosis of PID in our country. With proper clinical management and good adherence to the use of IVIg, patients with CVID in developing countries may have survival and quality of life similar to those described in developed countries.

A clinical AIDS case definition is needed for surveillance in countries where the CDC case definition is not practical. To derive such a definition, we compared 110 HIV-seropositive and 135 randomly selected HIV-seronegative adult medical-ward inpatients in Brazil. Multivariate analysis of clinical signs and symptoms and simple diagnoses resulted in a discriminant function with sensitivity of 89% and specificity of 96% in predicting for AIDS. These data were the empirical basis for a clinical definition of AIDS in adults drafted in a Caracas, Venezuela, workshop sponsored by the Pan American Health Organization. The revised "Caracas" definition presented here requires a positive HIV serology, the absence of cancer or other cause of immunosuppression, plusor = 10 cumulative points, as follows: Kaposi's sarcoma (10 points); extrapulmonary/noncavitary pulmonary tuberculosis (10); oral candidiasis or hairy leukoplakia (5); cavitary pulmonary/unspecified tuberculosis (5); herpes zoster60 years of age (5); CNS dysfunction (5); diarrheaor = 1 month (2); feveror = 1 month (2); cachexia or10% weight loss (2); astheniaor = 1 month (2); persistent dermatitis (2); anemia, lymphopenia, or thrombocytopenia (2); persistent cough or any pneumonia except TB (2); and lymphadenopathyor = 1 cm ator = 2 noninguinal sites foror = 1 month (2). This definition has a sensitivity of 95% and a specificity of 100% (91% without HIV serology) when applied to the Brazilian patients in this study. The Caracas definition has been adopted by Brazil, Honduras, and Surinam, and is in validation elsewhere. The use of a reasonably sensitive and specific case definition commensurate with available diagnostic resources should facilitate AIDS surveillance in developing countries.

Background Although extensive HIV drug resistance information is available for the first 400 amino acids of its reverse transcriptase, the impact of antiretroviral treatment in C-terminal domains of Pol (thumb, connection and RNase H) is poorly understood. Methods and Findings We wanted to characterize conserved regions in RT C-terminal domains among HIV-1 group M subtypes and CRF. Additionally, we wished to identify NRTI-related mutations in HIV-1 RT C-terminal domains. We sequenced 118 RNase H domains from clinical viral isolates in Brazil, and analyzed 510 thumb and connection domain and 450 RNase H domain sequences collected from public HIV sequence databases, together with their treatment status and histories. Drug-naive and NRTI-treated datasets were compared for intra- and inter-group conservation, and differences were determined using Fisher's exact tests. One third of RT C-terminal residues were found to be conserved among group M variants. Three mutations were found exclusively in NRTI-treated isolates. Nine mutations in the connection and 6 mutations in the RNase H were associated with NRTI treatment in subtype B. Some of them lay in or close to amino acid residues which contact nucleic acid or near the RNase H active site. Several of the residues pointed out herein have been recently associated to NRTI exposure or increase drug resistance to NRTI. Conclusions This is the first comprehensive genotypic analysis of a large sequence dataset that describes NRTI-related mutations in HIV-1 RT C-terminal domains in vivo. The findings into the conservation of RT C-terminal domains may pave the way to more rational drug design initiatives targeting those regions.

We describe a sensitive radioassay for detecting erythrocyte-associated immunoglobulin employing the immunoglobulin-specific reagent Staphylococcus aureus protein A. The assay was used to determine the extent to which erythrocytes from patients with different connective tissue disease bind radiolabelled protein A. Increased amounts of protein A were bound by erythrocytes from patients with systemic lupus erythematosus or with progressive systemic sclerosis when compared to a control group, whereas there was no significant binding to erythrocytes from patients with rheumatoid arthritis or with polymyositis-dermatomyositis. The assay, because of its high sensitivity and of the availability of the reagent in pure form, should be useful for the investigation of cell-bound antibodies and for the differential diagnosis of connective tissue diseases.

Results 33 patients were analyzed: 55% female, 70% Caucasian, 27% Black, and 3% Asian, with ages between 12 and 64 years (mean = 36.7, SD = 11) and length of follow-up between 1 and 17 years (mean = 8.84, SD = 11.29). The profile and frequency of PIDs was as follows: Predominantly antibody deficiencies (55%), Combined immunodeficiencies less profound than generally severe combined immunodeficiency (18%) Combined immunodeficiencies associated with features or syndromic (15%), Complement deficiencies (9%), and Autoinflammatory disorders (3%). The average time between onset of symptoms and diagnosis of PID ranged between 1 and 28 years (mean = 10.83, SD = 7.07). Compliance with treatment was good in 76% of patients, partial in 12% and 12% of treatment dropout. Survival between the onset of clinical manifestations and the end of the study ranged between 1 and 47 years (mean = 21.52, SD =13.79). Throughout the study period, one patient, with Wiskott-Aldrich syndrome and partial adherence to treatment, died due to septicemia and there were two deaths from cardiovascular disease.

Background Atopic dermatitis is a multifactorial disease associated with elevated production of IgE and sensitization to multiple food and inhalant allergens.The aim of this study was to assess the levels of total serum IgE (IgE) and specific IgE (sp IgE ) for Dermatophagoides pteronyssinus (Dp), Blomia tropicalis (Bt), egg white, casein, alpha-lactalbumin and beta-lactoglobulin in patients with atopic dermatitis (AD); and the influence of gender, age and severity of disease in these parameters.

Background The evaluation of the degree of disease control and quality of life of patients with asthma is a challenge for general practitioners and medical specialists. The aim of this study was to assess the degree of disease control and quality of life in patients with moderate and severe asthma in clinical follow-up of Allergy and Immunology clinic as well as comparatively evaluate two instruments for assessing asthma control, versus quality of life evaluation.