Performances of HTLV serological tests in diagnosing HTLV infection in high-risk population of São Paulo, Brazil

Testing problems in diagnosing human T-lymphotropic virus (HTLV) infection, mostly HTLV-II, have been documented in HIV/AIDS patients. Since December 1998, the Immunology Department of Instituto Adolfo Lutz (IAL) offers HTLV-I/II serology to Public Health Units that attend HTLV high-risk individuals. Two thousand, three hundred and twelve serum samples: 1,393 from AIDS Reference Centers (Group I), and 919 from HTLV out-patient clinics (Group II) were sent to IAL for HTLV-I/II antibodies detection. The majority of them were screened by two enzyme immunoassays (EIAs), and confirmed by Western Blot (WB 2.4, Genelabs). Seven different EIA kits were employed during the period, and according to WB results, the best performance was obtained by EIAs that contain HTLV-I and HTLV-II viral lysates and rgp21 as antigens. Neither 1st and 2nd, nor 3rd generation EIA kits were 100% sensitive in detecting truly HTLV-I/II reactive samples. HTLV-I and HTLV-II prevalence rates of 3.3% and 2.5% were detected in Group I, and of 9.6% and 3.6% in Group II, respectively. High percentages of HTLV-seroindeterminate WB sera were detected in both Groups. The algorithm testing to be employed in HTLV high-risk population from São Paulo, Brazil, needs the use of two EIA kits of different formats and compounds as screening, and because of high seroindeterminate WB, may be another confirmatory assay.

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.

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.

Discrepancies and consequences of indirect hemagglutination, indirect immunofluorescence and Elisa tests for the diagnosis of Chagas disease

Using the indirect hemagglutination (IH), indirect immunofluorescence (IIF) and enzyme linked immunosorbent assay (ELISA) tests for the diagnosis of Chagas disease, 4000 serum samples were examined. This study was conducted with different purposes: clinical interest, research support and parasitological monitoring of those patients with Chagas disease who were treated with heart transplantations. The tests occurred without patient selection and in accordance with the medical requests. The results showed discrepancies and brought about several questions, considering the different results that all three methods showed when considered together. What was found brought about concerns and we suggest the adoption of different measures, aiming to avoid these mismatches in the context of this disease.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

EVOLUTION OF PATIENTS WITH AIDS AFTER cART: CLINICAL AND LABORATORY EVOLUTION OF PATIENTS WITH AIDS AFTER 48 WEEKS OF ANTIRETROVIRAL TREATMENT

SUMMARY Combination Antiretroviral Therapy (cART) aims to inhibit viral replication, delay immunodeficiency progression and improve survival in AIDS patients. The objective of this study was to compare two different schemes of cART, based on plasma viral load (VL) and CD4+ T lymphocyte count, during 48 weeks of treatment. For this purpose, 472 medical charts of a Specialized Outpatient Service were reviewed from 1998 to 2005. Out of these, 58 AIDS patients who had received a triple drug scheme as the initial treatment were included in the study and two groups were formed: Group 1 (G1): 47 individuals treated with two nucleoside reverse-transcriptase inhibitors (NRTI) and one non-nucleoside reverse-transcriptase inhibitor; Group 2 (G2): 11 patients treated with two NRTI and one protease inhibitor. In G1 and G2, 53.2% and 81.8% respectively were patients with an AIDS-defining disease. The T CD4+ lymphocyte count increased progressively up until the 24th week of treatment in all patients, while VL became undetectable in 68.1% of G1 and in 63.6% of G2. The study concluded that the evolutions of laboratory tests were similar in the two treatment groups and that both presented a favorable clinical evolution.

The avirulence of the cultivated PF strain of Trypanosoma cruzi: V - the evaluation of parasitologic tests after vaccination of different animal species

After vaccination with the live PF strain of Trypanosoma cruzi, 194 blood cultures were performed in 143 mice, 9 dogs, 5 Cebus monkeys and 7 human subjects. Some of these blood cultures were simultaneously done with xenodiagnosis, subinoculation in baby mice and/or culture of viscerae. The trypanosomes isolated from the few positive cases (6,1%) were incapable of infecting baby mice were considered as cases of immunotolerance. All the other tests were negative.

T cell-dependent immunodepression in vivo in Schistosoma mansoni infected patients

T-cell function was evaluated in 29 patients with either hepatointestinal or hepatosplenic schistosomiasis by intradermal tests to recall antigens. Immunodepression was detected in 26% of the subjects with hepatointestinal schistosomiasis and in 50% of those with the hepatosplenic form. Cellular immunodepression was related to worm load and spleen size. This non specific T-cell immunodepression may represent a serious constraint to the elimination of intracellular pathogens both in hepatosplenic or hepatointestinal schistosomiasis.

Longitudinal study of the indirect immunofluorescence and complement fixation tests for diagnosis of chagas' disease in immunosuppressed patients submitted to renal transplantation

Clinical and serological follow-up of 7 patients submitted to renal transplantation and presenting positive serological reactions to Chagas 'disease before immunossupression did not show significant changes in indirect immunofluorescence and complement fixation titres for Chagas ' disease, or signs and symptoms indicating exacerbation of the disease during follow- up. In addition, 18 of 66 recipients of renal transplants considered to be non-chagasic before immunosuppression showed at least one positive result to the indirect immunofluorescence test for Chagas ' disease during the study period. The results suggest that the immunosuppression State induced in chagasic patients submitted to renal transplant did notpromoted exacerbation of the chronic infection in these patients and not interfere with the serological response of chronic chagasics, thus permitting the use of these serologic reactions for diagnostic purposes in these cases. However, the positive results ofthe indirect immunofluorescence test in non- chagasic patients indicate the needforjudicious interpretation ofthe indirect immunofluorescence test for the diagnosis of Chagas' disease in renal transplanted patients.

Diagnostic tests for amoebic liver abscess: comparison of enzyme - linked immunosorbent assay (Elisa) and counterimmunoelectrophoresis (CIE)

The liver abscess is the most frequent extraintestinal complication of intestinal amoebiasis: its diagnosis is suggested by the clinical picture but it must be confirmed by paraclinic tests. Themost stringent diagnosis requires identification of E. histolytica. But this is possible only in a few cases. Serological tests greatly improve the diagnosis of this severe complication of amoebiasis. We compared the Enzyme Linfed Immunosorbent Assay and the Counterimmunoeletrophoresis techniques. Both techniques were used to detect amoebic antibodies in 50 control patients, 30 patients with liver abscess and 30 patients with intestinal amoebiasis. All the sera from control patients gave negative results iin both techniques. When analysing the sera from patients with intestinal amoebiasis, 10% of them were positive by ELISA but non by CIE. The sera of patients with liver abscess, we found that 90% were positive by the ELISA method and 66.6% by the CIE technique. In patients with amoebic liver abscess, the results showed that the ELISA was more sensitive than the CIE, as it presented a higher sensitivity (100%) than that of the CIE technique (66%).

Assessment of chemiluminescence and PCR effectiveness in relation to conventional serological tests for the diagnosis of Chagas' disease

While testing 414 sera for the diagnosis of Chagas' disease, the conventional reactions of indirect hemagglutination, indirect immunofluorescence and the immunosorbent assay showed a sensitivity of 95.7%, 100% and 98.2% and a specificity of 98%, 98% and 96.4%, respectively, and an excellent association using Fisher's exact test. Chemiluminescence presented 100% sensitivity and 89.6% specificity, while PCR showed 100% specificity and 1.2% sensitivity. It is believed that the three conventional serological reactions are still adequate for diagnosing Chagas' disease.

International meeting: new diagnostic tests are urgently needed to treat patients with Chagas disease

Trypanosoma cruzi infection is often not detected early on or actively diagnosed, partly because most infected individuals are either asymptomatic or oligosymptomatic. Moreover, in most places, neither blood banks nor healthcare units offer diagnostic confirmation or treatment access. By the time patients present clinical manifestations of advanced chronic Chagas disease, specific treatment with current drugs usually has limited effectiveness. Better-quality serological assays are urgently needed, especially rapid diagnostic tests for diagnosis patients in both acute and chronic phases, as well as for confirming that a parasitological cure has been achieved. Some new antigen combinations look promising and it is important to assess which ones are potentially the best, together with their requirements in terms of investigation and development. In August 2007, a group of specialized researchers and healthcare professionals met to discuss the state of Chagas infection diagnosis and to build a consensus for a plan of action to develop efficient, affordable, accessible and easy-to-use diagnostic tests for Chagas disease. This technical report presents the conclusions from that meeting.

Evaluation of laboratory markers of progression of HIV disease to death

INTRODUCTION: One of the important current problems in HIV/AIDS infection is the establishment of epidemiological and laboratorial prognostic parameters during patient follow-up. This study aimed at analyzing the evolution of laboratory tests: CD4 lymphocyte count, viral load, hemoglobin (Hb), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and the epidemiological variables sex and age as prognostic factors for survival in progression to death among AIDS patients. METHODS: A retrospective study was conducted using analysis of medical records, and prospective 24-month follow-up of patients with HIV/ AIDS attended at the President Vargas Hospital Outpatient Clinic, a reference center in HIV/ AIDS attendance in the State of Maranhão, Brazil. The study analyzed patients aged 10 to 60 years old, who manifested AIDS and who were not using antiretroviral therapy or had used it for less than 5 years. The Chi-square test was used for statistical analysis. RESULTS: The sample included 100 patients - 57 were current outpatients, and 43 had died. The variables viral load (p=0.726), ALT (p=0.314), sex (p=0.687), and age (p=0.742) were analyzed, and no evidence of association between them and worst prognosis was observed. CONCLUSIONS: A significant relation was verified between low Hb levels (p=0.000) and CD4 (p=0.000) and shorter survival.

Contributions of culture and antimicrobial susceptibility tests to the retreatment of patients with pulmonary tuberculosis

Introduction This study evaluated the efficacy of retreatment of pulmonary tuberculosis (TB) with regard to treatment outcomes and antimicrobial susceptibility testing (ST) profiles. Methods This retrospective cohort study analyzed 144 patients treated at a referral hospital in Brazil. All of them had undergone prior treatment, were smear-positive for TB and received a standardized retreatment regimen. Fisher's 2-tailed exact test and the χ2 test were used; RRs and 95% CIs were calculated using univariate and multivariate binary logistic regression. Results The patients were cured in 84 (58.3%) cases. Failure was associated with relapsed treatment and abandonment (n=34). Culture tests were obtained for 103 (71.5%) cases; 70 (48.6%) had positive results. ST results were available for 67 (46.5%) cases; the prevalence of acquired resistance was 53.7%. There were no significant differences between those who achieved or not therapeutic success (p=0.988), despite being sensitive or resistant to 1 or more drugs. Rifampicin resistance was independently associated with therapeutic failure (OR: 4.4, 95% CI:1.12-17.37, p=0.034). For those cases in which cultures were unavailable, a 2nd model without this information was built. In this, return after abandonment was significantly associated with retreatment failure (OR: 3.59, 95% CI:1.17-11.06, p=0.026). Conclusions In this cohort, the general resistance profile appeared to have no influence on treatment outcome, except in cases of rifampicin resistance. The form of reentry was another independent predictor of failure. The use of bacterial culture identification and ST in TB management must be re-evaluated. The recommendations for different susceptibility profiles must also be improved.

Serological cross-reactivity of Trypanosoma cruzi, Ehrlichia canis, Toxoplasma gondii, Neospora caninum and Babesia canis to Leishmania infantum chagasi tests in dogs

Introduction: The aim of this study was to evaluate the serological cross-reactivity between Leishmania sp. and other canine pathogens. Methods: Positive serum samples for Ehrlichia canis, Babesia canis, Toxoplasma gondii, Neospora caninum and Trypanosoma cruzi were tested using three serological methods enzyme linked immunosorbent assay (ELISA), indirect immunofluorescent antibody test (IFAT) and Kalazar Detect™, for canine visceral leishmaniasis. Results: Of the 57 dog samples tested, 24 (42.1%) tested positive using one of the three serological methods: 10/57 (17.5%) for ELISA, 11/57 (19.3%) for IFAT and 3/57 (5.3%) for Kalazar Detect™. Conclusions: Our results demonstrated that the presence of other infectious agents may lead to cross-reactivity on leishmaniasis serological tests.