ANTI M. leprae IgM ANTIBODY DETERMINATION BY ULTRAMICROIMMUNOENZYMATIC (UMELISA HANSEN) FOR THE DIAGNOSIS AND MONITORING LEPROSY

The relationship between the IgM antibody response, antigenic load as well as the clinical improvement after chemotherapy was studied in order to obtain useful data for the early diagnosis and monitoring leprosy. A level of 82% (94/115) agreement was obtained between IgM UMELISA HANSEN and slitskin smear examination. Discrepant results were observed in 16 patients who showed positive IgM response despite negative by the skin smear examination. In these patients, the IgM response was seen to be associated to the early signal for bacilli recurrence in the skin. In one of these patients the presence of bacilli was demonstrated in the skin, two months after IgM antibodies being detected by UMELISA HANSEN. Also in one of the treated patients positive by both diagnostic techniques, a remarkable decrease in the IgM antibody levels was seen, correlating with a significant clinical improvement. Moreover it was found a direct relationship between the IgM antibody response and bacterial antigenic load, regardless the time elapsed in the disease's evolution.

PCR-BASED DIAGNOSIS OF A CASE OF HERPETIC WHITLOW IN AN AIDS PATIENT

Herpetic infections are common complications in AIDS patients. The clinical features could be uncommon and antiviral chemotherapy is imperative. A rapid diagnosis could prevent incorrect approaches and treatment. The polymerase chain reaction is a rapid, specific and sensible method for DNA amplification and diagnosis of infectious diseases, especially viral diseases. This approach has some advantages compared with conventional diagnostic procedures. Recently we have reported a new PCR protocol to rapid diagnosis of herpetic infections with suppression of the DNA extraction step. In this paper we present a case of herpetic whitlow with rapid diagnosis by HSV-1 specific polymerase chain reaction using the referred protocol.

TOXOCARIASIS: SEROLOGICAL DIAGNOSIS BY INDIRECT ANTIBODY COMPETITION ELISA

Toxocariasis is caused by infection of man by Toxocara canis and Toxocara cati larvae, the common roundworm of dogs and cats. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. Non specific reactions are observed mainly due to cross-reactivity with Ascaris sp antigens. This investigation aimed at developing and evaluating an indirect antibody competition ELISA (IACE) employing a specific rabbit IgG anti-Toxocara canis excretory-secretory antigens as the competition antibody, in order to improve indirect ELISA specificity performed for toxocariasis diagnosis. For that, the rabbit IgG was previously absorbed by Ascaris suum adult antigens. Sensitivity and specificity of IACE were first evaluated in 28 serum samples of mice experimentally infected with T. canis embryonated eggs. Adopting cut-off value established in this population before infection, sensitivity and specificity were 100% after 20 days post-inoculation. For human population IACE was evaluated using sera from 440 patients with clinical signs of toxocariasis and the cut-off value was established with 60 serum samples from apparently healthy individuals. Using as reference test the indirect ELISA performed by Adolfo Lutz Institute, sensitivity was 60.2%, specificity was 98% and concordance was 77.3%. Repeatability of IACE was evaluated by the inter-reactions variation coefficient (2.4%).

ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE

Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD) other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.

Serological diagnosis of toxoplasmosis: usefulness of IgA detection and IgG avidity determination in a patient with a persistent IgM antibody response to Toxoplasma gondii

We report the detection of specific IgA antibodies and the determination of IgG avidity in sequential serum samples from a patient exhibiting significant levels of Toxoplasma-specific IgM antibodies for seven years after the onset of the clinical symptoms of toxoplasmosis. IgM antibodies were detected by an indirect immunofluorescence test and by three commercial enzyme-linked immunosorbent assays (ELISA). Anti-T. gondii IgA was quantified by the a-capture ELISA technique using a commercial kit. As defined by the manufacturer of the IgA ELISA test used, most patients with acute toxoplasmosis have antibody levels > 40 arbitrary units per ml (AU/mL). At this cut-off level, the patient still had a positive ELISA result (45 AU/mL) in a serum sample taken one year after the beginning of clinical manifestations. The IgG avidity-ELISA test was performed with the Falcon assay screening test (F.A.S.T.®) - ELISA system. Avidity indices compatible with a recent Toxoplasma infection were found only in serum samples taken during the first 5 months after the onset of the clinical symptoms of toxoplasmosis. These results show that the interpretation of positive IgM results as indicative of recently acquired toxoplasmosis requires additional laboratory confirmation either by other tests or by the demonstration of a significant rise in the antibody titers in sequential serum samples.

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.

Clinical and laboratory findings of disseminated Mycobacterium avium complex infection (DMAC) in a pair matched case-control study

A pair matched case/control study was conducted from January 1991 to 30 June 1992 in order to define clinical and laboratory findings associated with DMAC infection in AIDS patients. Since DMAC infection is usually associated with advanced immunodeficiency, and therefore also with other opportunistic illnesses, in addition to the number of CD4+ lymphocytes, cases and controls were matched using the following criteria: date of AIDS diagnosis and antiretroviral therapy, number and severity of associated opportunistic infections and, whenever possible, type of Pneumocystis carinii prophylaxis, age and gender, in this order of relevance. Cases (defined as patients presenting at least one positive culture for MAC at a normally sterile site) and controls presented CD4+ lymphocyte counts below 50 cel/mm3. A significantly higher prevalence of general, digestive and respiratory signs, increased LDH levels, low hemoglobin levels and CD4+ cell counts were recorded for cases when compared to controls. Increases in gGT and alkaline phosphatase levels seen in cases were also recorded for controls. In conclusion, the strategy we used for selecting controls allowed us to detect laboratory findings associated to DMAC infection not found in other advanced immunossupressed AIDS patients without DMAC.

Hantavirus pulmonary syndrome in Brazil: clinical aspects of three new cases

Hantavirus pulmonary syndrome (HPS) has been recognized recently in Brazil, where 28 cases have been reported as of September 1999. We report here the clinical and laboratory findings of three cases whose diagnoses were confirmed serologically. All the patients were adults who presented a febrile illness with respiratory symptoms that progressed to respiratory failure that required artificial ventilation in two of them. Laboratory findings were most of the time consistent with those reported in the United States in patients infected with the Sin Nombre virus, and included elevated hematocrit and thrombocytopenia; presence of atypical lymphocytes was observed in one patient. The chest radiological findings observed in all the patients were bilateral, diffuse, reticulonodular infiltrates. Two patients died. Histopathological examination of the lungs of these patients revealed interstitial and alveolar edema, alveolar hemorrhage, and mild interstitial pneumonia characterized by infiltrate of immunoblasts and mononuclear cells. In the epidemiologic investigation of one of the cases, serologic (ELISA) tests were positive in 3 (25%) out of 12 individuals who shared the same environmental exposure. HPS should be included in the differential diagnosis of interstitial pneumonia progressing to acute respiratory failure.

The use of cyclosporine modifies the clinical and histopathological presentation of tuberculosis after renal transplantation

Tuberculosis is one of the most frequent opportunistic infections after renal transplantation and occurred in 30 of 1264 patients transplanted between 1976 and 1996 at Hospital São Paulo - UNIFESP and Hospital Dom Silvério, Brazil. The incidence of 2.4% is five times higher than the Brazilian general population. The disease occurred between 50 days to 18 years after the transplant, and had an earlier and worse development in patients receiving azathioprine, prednisone and cyclosporine, with 35% presenting as a disseminated disease, while all patients receiving azathioprine and prednisone had exclusively pulmonary disease. Ninety percent of those patients had fever as the major initial clinical manifestation. Diagnosis was made by biopsy of the lesion (50%), positivity to M. tuberculosis in the sputum (30%) and spinal cerebral fluid analysis (7%). Duration of treatment ranged from 6 to 13 months and hepatotoxicity occurred in 3 patients. The patients who died had a significant greater number of rejection episodes and received higher doses of corticosteroid. In conclusion, the administration of cyclosporine changed the clinical and histopathological pattern of tuberculosis occurring after renal transplantation.

hsp65 PCR-restriction enzyme analysis (PRA) for identification of mycobacteria in the clinical laboratory

More than 70 species of mycobacteria have been defined, and some can cause disease in humans, especially in immunocompromised patients. Species identification in most clinical laboratories is based on phenotypic characteristics and biochemical tests and final results are obtained only after two to four weeks. Quick identification methods, by reducing time for diagnosis, could expedite institution of specific treatment, increasing chances of success. PCR restriction-enzyme analysis (PRA) of the hsp65 gene was used as a rapid method for identification of 103 clinical isolates. Band patterns were interpreted by comparison with published tables and patterns available at an Internet site (http://www.hospvd.ch:8005). Concordant results of PRA and biochemical identification were obtained in 76 out of 83 isolates (91.5%). Results from 20 isolates could not be compared due to inconclusive PRA or biochemical identification. The results of this work showed that PRA could improve identification of mycobacteria in a routine setting because it is accurate, fast, and cheaper than conventional phenotypic identification.

Diagnosis of cytomegalovirus infections by qualitative and quantitative PCR in HIV infected patients

A high incidence of cytomegalovirus (CMV) infections is observed in Brazil. These viruses are causatives of significant morbidity and mortality among patients with advanced human immunodeficiency virus (HIV) infection. This work, shows the application of a PCR on determination of CMV load in the buffy coat and plasma. We analyzed the samples of 247 HIV infected patients in order to diagnose CMV infection and disease. We developed a semi-quantitative PCR that amplifies part of the glycoprotein B (gB) gene of CMV. The semi-quantitative PCR was carried out only in positive clinical samples in a qualitative PCR confirmed by a nested-PCR. CD4 lymphocyte count, HIV viral load and CMV disease symptom were correlated with CMV load. CMV genome was detected in the buffy coat of 82 of 237 (34.6%) patients, in 10 of these the CMV load was determined varying between 928 and 332 880 viral copies/mug DNA. None of these 237 patients developed any suggestive manifestation of CMV disease. For the other 10 HIV infected patients selected based on the suspicion of CMV disease, CMV genome was detected in only one case. This patient presented a high CMV load, 8 000 000 copies/mug DNA, and developed a disseminated form of CMV disease including hepatitis and retinitis. Our results were greatly influenced by the impact of the highly active antiretroviral therapy that reduced incidence of CMV viremia and occurrence of CMV disease in the HIV infected patients.

Clinical and epidemiological features of definitive and presumed loxoscelism in São Paulo, Brazil

A retrospective study analysed 359 proven or presume cases of loxoscelism seen at the Hospital Vital Brazil, Instituto Butantan, São Paulo, Brazil, between 1985 and 1996. The spider was identified in 14%. The bites occurred predominantly in the urban areas (73%) between September and February. Patients > 14 years were commonest inflicted (92%) and 41% were bitten while getting dressed. Only 11% sought medical care within the first 12 hours post bite. Cutaneous loxoscelism was the commonest form presenting (96%); commonest manifestations were: pain (76%), erythema (72%), edema with enduration (66%), ecchymosis (39%). Skin necrosis occurred in 53% of patients, most frequently seen on trunk, tigh and upper arm, and when patients seek medical care more than 72 hours after bite. Local infection was detected in 12 patients (3%). Hemolysis was confirmed in 4 cases (1.1%). Generalised cutaneous rash, fever and headache were also observed in 48% of the total of patients. None of them had acute renal failure or died. Treatment usually involved antivenom administration (66%), being associated with corticosteroids (47%) or dapsone (30%). Presumptive diagnosis of loxoscelism may be established based on clinical and epidemiological findings. Further investigations are required to prove the value of antivenom and other treatment schedules.

Diagnosis of pulmonary pneumocystosis by microscopy on wet mount preparations

We have compared the searching of the presence of "honeycomb" structures by direct microscopy on wet mount preparations with the direct immunofluorescence (DIF) for the diagnosis of Pneumocystis carinii pneumonia (PCP) in 115 bronchoalveolar (BAL) fluids. The samples belonged to 115 AIDS patients; 87 with presumptive diagnosis of PCP and 28 with presumptive diagnosis other than PCP. The obtained results were coincident in 114 out of 115 studied samples (27 were positive and 87 negative) with both techniques. A higher percentage of positive results (32.18%) among patients with presumptive diagnosis of PCP with respect to those with presumptive diagnosis other than PCP (3.57%) was observed. One BAL fluid was positive only with DIF, showed scarce and isolated P. carinii elements and absence of typical "honeycomb" structures. The searching for "honeycomb" structures by direct microscopy on wet mount preparations could be considered as a cheap and rapid alternative for diagnosis of PCP when other techniques are not available or as screening test for DIF. This method showed a sensitivity close to DIF when it was applied to BAL fluids of AIDS patients with poor clinical condition and it was performed by an experienced microscopist.

Pancreatic involvement in fatal human leptospirosis: clinical and histopathological features

Hyperamylasemia has been reported in more than 65% of patients with severe leptospirosis, and the true diagnosis of acute pancreatitis is complicated by the fact that renal failure can increase serum amylase levels. Based on these data we retrospectively analyzed the clinical and histopathological features of pancreas involvement in 13 cases of fatal human leptospirosis. The most common signs and symptoms presented at admission were fever, chills, vomiting, myalgia, dehydratation, abdominal pain and diarrhea. Trombocytopenia was evident in 11 patients. Mild increased of AST and ALT levels was seen in 9 patients. Hyperamylasemia was recorded in every patient in whom it was measured, with values above 180 IU/L (3 cases). All patients presented acute renal failure and five have been submitted to dialytic treatment. The main cause of death was acute respiratory failure due to pulmonary hemorrhage. Pancreas fragments were collected for histological study and fat necrosis was the criterion used to classify acute pancreatitis. Histological pancreatic findings were edema, mild inflammatory infiltrate of lymphocytes, hemorrhage, congestion, fat necrosis and calcification. All the patients infected with severe form of leptospirosis who develop abdominal pain should raise the suspect of pancreatic involvement.

Cerebral Sinovenous Thrombosis in Children: Clinical Presentation and Extension, Localization and Recanalization of Thrombosis

Many important questions regarding pathophysiology and treatment of cerebral sinovenous thrombosis need clarification and may depend on further knowledge on the etiology, site, extension and recanalization of the thrombosis. We studied these variables in a cohort of children and adolescents from seven Portuguese Centers. We conclude from our results that the deep venous system and the superior longitudinal sinus are less frequently affected with thrombosis but have a greater potential for serious neurologic disease and for major sequelae. Non-recanalization, at least in the long term, is not an adverse prognostic factor. Extensive propagation of the thrombus from the initial site of origin seems to be common. The early identification of risk factors and their treatment coupled with an aggressive attitude towards diagnosis and treatment for thrombosis involving the deep venous system would be warranted.