The relevance of laboratory diagnosis of human cryptosporidiosis and other coccidia

Human infection by Cryptosporidium spp and other coccidia are due to opportunist non-host specific microorganisms. In HIV seropositive patients, the gastrointestinal symptoms accompanying such infections may be serious and prolonged and may include nausea, low-grade fever, abdominal cramps, anorexia and watery diarrhoea. We studied 188 stool samples from 111 patients (84 men and 27 women) with diarrhoea. A modified Ziehl-Nielsen technique for the detection of Cryptosporidium spp and Isospora belli was employed. The mean age of the patients was 31 years. Cryptosporidium spp was seen in 18% (n=20) of the patients, 90% (n=18) of whom were HIV seropositive. Isospora belli was recorded only from HIV seropositive patients (5.4% of all the patients studied and 6.5% of those who were HIV seropositive). These data confirm the good results obtained with this technique for the identification of Cryptosporidium spp and other coccidia and also reaffirm the clinical importance of correctly diagnosing the cause of diarrhoea, particularly in HIV seropositive patients.

Human parvovirus B19 infection: clinical and epidemiological study of 24 cases

From March 1994 to November 1995 24 cases of human parvovirus B19 infection were seen at the Infectious Diseases Department of the Hospital Universitário Antônio Pedro, Niterói - RJ. Serum samples for IgM detection (capture enzyme immunoassay) were positive from the 1st to the 27th day after the onset of the exathema. The classical features of erythema infectiosum (slapped cheecked syndrome) were observed in 8 (33.3%) cases all of them children. Eight patients (6 adults and 2 children) presented a symmetrical polyartropathy, seen more frequently in women. These results show that B19 infection diagnosis is difficult when the disease does not present the classical features and because of the frequent involvement of the joints this infection should be considered in the differential diagnosis of early rheumatoid arthritis.

Dot-ELISA for the detection of anti-Cysticercus cellulosae antibodies in cerebrospinal fluid using a new solid phase (resin-treated polyester fabric) and Cysticercus longicollis antigens

A dot-ELISA was developed for the detection of antibodies in CSF in the immunologic diagnosis of human neurocysticercosis, using antigen extracts of the membrane and scolex of Cysticercus cellulosae (M+S-Cc) and, alternately, membrane (M) and vesicular fluid (VF) of Cysticercus longicollis (Cl) covalently bound to a new solid phase consisting of polyester fabric treated with N-methylol-acrylamide resin (dot-RT). The test was performed at room temperature, with reduced incubation times and with no need for special care in the manipulation of the support. The sensitivity rates obtained were 95.1% for antigen Cc and 97.6% for antigen Cl. Specificity was 90.6% when Cc was used, and 96.9% and 100% when M-Cl and VF-Cl were used, respectively. No significant differences in titer were observed between tests carried out with homologous and heterologous antigens. The low cost and easy execution of the dot-RT test using antigen extracts of Cysticercus longicollis indicate the test for use in the immunodiagnosis of human neurocysticercosis.

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

IgM and IgA Antibody Responses in 12 Cases of Human Acquired Toxoplasmosis

The persistence, in some subjects, of specific IgM antibodies to Toxoplasma gondii for several months after the acute phase of infection has complicated the interpretation of serological test results for toxoplasmosis. Several reports have emphasized the value of the detection of Toxoplasma-specific IgA antibodies for the diagnosis of acute toxoplasmosis. In this article, we report the follow-up profiles of Toxoplasma-specific IgM and IgA antibodies in serum samples obtained from 12 patients at various intervals after the onset of the clinical manifestations of infection. IgM antibodies were detected by the indirect immunofluorescence (IIF) test, antibody capture enzyme-linked immunosorbent assay (cELISA) and enzyme-mediated chemilluminescent technique (CmL). IgA antibodies were quantified by the direct ELISA (dELISA) and cELISA procedures. As defined by the manufacturer of the cELISA test for IgA used, most patients with acute toxoplasmosis have antibody levels > 40 arbritary units per ml (AU/ml). At values > 40 AU/ml, the cELISA for IgA detected significant antibody levels for a shorter time than the other techniques used for IgM and IgA detection. However, IgA levels £ 40 AU/ml do not exclude the possibility of acute toxoplasmosis since such levels can be reached very soon after infection with T. gondii. The results obtained in the present study show that the serological diagnosis of acute toxoplasmosis may not be such an easy task. Our data suggest that use of the IgA-cELISA concomitantly with IgM antibody screening could permit, in some circumstances, a more efficient diagnosis of acute acquired toxoplasmosis

DETECTION OF HTLV-I ANTIBODIES AND DNA IN BLOOD SAMPLE OF A PATIENT WITH MYELOPATHY IN NIGERIA

We describe a case of human T-lymphotropic virus type I associated myelopathy in a 50-year old woman in Nigeria. The patient presented with progressive loss of tone to the two lower limbs and later inability to walk. The HTLV-I antibody presence in the plasma collected from the patient was repeatedly detected by enzyme immunoassays (Abbott HTLV-I EIA and Coulter SELECT-HTLV I/II) and confirmed by Western blot technique. In addition, HTLV-I DNA was amplified from the genomic DNA isolated from the peripheral blood mononuclear cells of the patient by the polymerase chain reaction technique. This finding is significant being the first report of association of HTLV-I with myelopathy in Nigeria.

FETO-PLACENTARY PATHOLOGY IN HUMAN PARVOVIRUS B19 INFECTION

In view of the scarce references concerning the histological data in congenital parvovirus human B19 infection, we intend to provide a description of the pathological features observed in six autopsies.The virus was detected by DNA hybridization (ISH-DBH),PCR and electronmicroscopy (EM) in paraffin-embedded feto-placentary tissues.These cases constitute a subset from 86 Non Immunologic Hydrops Fetalis (NIHF) cases, in which a systemic complex of inflammatory/degenerative lesions of unknown etiology was visualized by optical microscopy. In one case a syphilitic process was detected, typefying a double infection. All fetuses showed a similar pathology - hydrops, hepato-splenomegaly, lung hypoplasia and erythroblastemia, the specific histological feature being the presence of intranuclear inclusions in the erythroid progenitors, in the erythropoietic visceral tissue and in blood marrow. Complex cardiopathy allied to abnormal lung lobulation and polisplenia were observed once; in 2 cases endocardial fibroelastosis was diagnosed. The pulmonary lesions were represented by dysmaturity allied to interstitial mononuclear infiltration. The hepatic consisted of cholestasis, portal fibrosis, canalicular proliferation, hemossiderosis, focal necroses and giant cell transformation. The central nervous system lesions were predominantly anoxic although the autolysis impaired a correct diagnosis.

Efficiency of indirect immunofluorescence assay as a confirmatory test for the diagnosis of human retrovirus infection (HIV-1 and HTLV-I/II) in different at risk populations

We compared the indirect immunofluorescence assay (IFA) with Western blot (Wb) as a confirmatory method to detect antibodies anti retrovirus (HIV-1 and HTLV-I/II). Positive and negative HIV-1 and HTLV-I/II serum samples from different risk populations were studied. Sensitivity, specificity, positive, negative predictive and kappa index values were assayed, to assess the IFA efficiency versus Wb. The following cell lines were used as a source of viral antigens: H9 ( HTLV-III b); MT-2 and MT-4 (persistently infected with HTLV-I) and MO-T (persistently infected with HTLV-II). Sensitivity and specificity rates for HIV-1 were 96.80% and 98.60% respectively, while predictive positive and negative values were 99.50% and 92.00% respectively. No differences were found in HIV IFA performance between the various populations studied. As for IFA HTLV system, the sensitivity and specificity values were 97.91% and 100% respectively with positive and negative predictive values of 100% and 97.92%. Moreover, the sensitivity of the IFA for HTLV-I/II proved to be higher when the samples were tested simultaneously against both antigens (HTLV-I-MT-2 and HTLV-II-MO-T). The overall IFA efficiency for HIV-1 and HTLV-I/II-MT-2 antibody detection probed to be very satisfactory with an excellent correlation with Wb (Kappa indexes 0.93 and 0.98 respectively). These results confirmed that the IFA is a sensitive and specific alternative method for the confirmatory diagnosis of HIV-1 and HTLV-I/II infection in populations at different levels of risk to acquire the infection and suggest that IFA could be included in the serologic diagnostic algorithm.

Detection of Toxoplasma gondii soluble antigen, SAG-1(p30), antibody and immune complex in the cerebrospinal fluid of HIV positive or negative individuals

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.

Outbreaks of human-herpes virus 6 (HHV-6) infection in day-care centers in Belém, Pará, Brazil

A total of 730 children aged less than 7 years, attending 8 day-care centers (DCCs) in Belém, Brazil were followed-up from January to December 1997 to investigate the occurrence of human-herpes virus 6 (HHV-6) infection in these institutional settings. Between October and December 1997 there have been outbreaks of a febrile- and -exanthematous disease, affecting at least 15-20% of children in each of the DCCs. Both serum- and- plasma samples were obtained from 401 (55%) of the 730 participating children for the detection of HHV-6 antibodies by enzyme-linked immunosorbent assay (ELISA), and viral DNA amplification through the nested-PCR. Recent HHV-6 infection was diagnosed in 63.8% (256/401) of them, as defined by the presence of both IgM and IgG-specific antibodies (IgM+/IgG+); of these, 114 (44.5%) were symptomatic and 142 (55.5%) had no symptoms (p = 0.03). A subgroup of 123 (30.7%) children were found to be IgM-/IgG+, whereas the remaining 22 (5.5%) children had neither IgM nor IgG HHV-6- antibodies (IgM-/IgG-). Of the 118 children reacting strongly IgM-positive ( > or = 30 PANBIO units), 26 (22.0%) were found to harbour the HHV-6 DNA, as demonstrated by nested-PCR. Taken the ELISA-IgM- and- nested PCR-positive results together, HHV-6 infection was shown to have occurred in 5 of the 8 DCCs under follow-up. Serological evidence of recent infections by Epstein-Barr virus (EBV) and parvovirus B19 were identified in 2.0% (8/401) and 1.5% (6/401) of the children, respectively. Our data provide strong evidence that HHV-6 is a common cause of outbreaks of febrile/exanthematous diseases among children attending DCCs in the Belém area.

Association between human parvovirus B19 and arthropathy in Belém, Pará, north Brazil

A total of 220 patients with arthropathy were selected in Belém, Pará between January 1994 and December 2000, and screened for the presence of human parvovirus B19 IgM and IgG antibodies by enzyme-linked immunosorbent assay (ELISA). A subgroup (n = 132) of patients with high levels of antibodies (either IgM+/IgG+ or IgM-/IgG+) were examined for the presence of DNA by polymerase chain reaction/nested PCR. Recent/active infection (detection of IgM and/or IgG-specific antibodies and presence of viral DNA) was identified in 47.7% of the 132 individuals with arthropathy. In our study, women were significantly more affected (59.7%) than men (35.4%) (P = 0.0006). The age group of 11-20 years (84.6%), among female patients, and 21-30 years (42.1%), among male, were those with the highest incidence rates. The analysis of the temporal distribution of B19-associated arthropaties showed a cyclic pattern, with peak incidence rates occuring at 3-5 year intervals. Significant diference (P = 0.01) was observed when comparing both the highest (39.0%) and the lowest (11.0%) seropositivity rates for the years of 1995 and 2000, respectively. The interfalangial joints of hands and feet were mostly affected, with 50.0% and 48.0% of cases among both women and men, respectively. In a smaller proportion, other joints such as those of knee, ankle, pulse and shoulder were affected. As for the duration, symptoms lasted 1 to 5 days in 54.0% of the individuals, whereas in 46.0% of them the disease lasted 6-10 days, if considered the subgroup (n = 63) of patients with recent/active infection by parvovirus B19. In our study, joint clinical manifestations occurred symmetrically. Our results indicate that B19 may be an important agent of arthropathies in our region, and this underscores the need for specific laboratory diagnosis when treating patients suffering from acute arthropathy, mainly pregnant women.

Study of chronic hemolytic anaemia patients in Rio de Janeiro: prevalence of anti-human parvovirus B19 IgG antibodies and the developement aplastic crises

The prevalence of anti-human parvovirus B19 IgG antibodies was determined in sera from 165 chronic hemolytic anemia patients, receiving medical care at Instituto Estadual de Hematologia (IEHE), Rio de Janeiro, during the year of 1994. This sample represents around 10% of the chronic hemolytic anemia patients attending at IEHE. Most of these patients (140) have sickle cell disease. Anti-B19 IgG antibodies were detected in 32.1% of patients. No statistically significant difference (p > 0.05) was seen between IgG antibody prevalence in male (27.8%) and female (35.5%) patients. Anti-B19 IgG antibodies were more frequent in older (37.6%) than younger (28.2%) than 20 years old patients, although this difference had no statistical significance (p > 0.05). Anti-B19 IgG antibody prevalence showed that 67.9% of patients enrolled in the study were susceptible to B19 acute infection. With the aim to detect acute B19 infection, patients follow up continued until February 1996. During this period four patients presented transient aplastic crisis due to human parvovirus B19 as confirmed by the detection of specific IgM antibodies. All four patients were younger than 20 years old, and 3 were younger than 10 years old. Three of them were sickle cell disease patients. Three of the four acute B19 infection occurred during 1994 springtime.

Human toxocariasis: a seroepidemiological survey in the municipality of Campinas (SP), Brazil

The occurrence of human Toxocara infection was evaluated in three neighborhoods of the periphery of the Campinas municipality (Jardim Santa Mônica, Jardim São Marcos and Jardim Campineiro) in 1999. Forty residences and 138 residents were randomly selected by drawing lots and were submitted to a seroepidemiological survey, which included blood collection for the immunoenzymatic detection (ELISA) of anti-Toxocara antibodies and a blood count, and the application of a semi-structured questionnaire for the evaluation of epidemiological data. Significant levels of anti-Toxocara antibodies were detected in 23.9% of the 1999 samples. No significant difference in the frequency of infection according to age was observed. Environmental contamination with Toxocara eggs was observed in 12.3 and 14.0% of 57 soil samples collected in the same region in December 1998 and July 1999, respectively. Univariate analysis and multiple logistic regression of the data obtained from the questionnaires and of the results of the serological tests, suggest a significant influence of socioeconomic variables on the frequency of human infection with Toxocara under the conditions prevalent in the study area.

Comparison between immunomagnetic separation, coupled with immunofluorescence, and the techniques of Faust et al. and of Lutz for the diagnosis of Giardia lamblia cysts in human feces

In the present study, the performance of Immunomagnetic Separation technique, coupled with Immunofluorescence (IMS-IFA), was compared with the FAUST et al. and Lutz parasitological techniques for the detection of Giardia lamblia cysts in human feces. One hundred and twenty-seven samples were evaluated by the three techniques at the same time showing a rate of cyst detection of 27.5% by IMS-IFA and 15.7% by both Faust et al. and Lutz techniques. Data analysis showed a higher sensitivity of IMS-IFA for the detection of G. lamblia cysts in comparison with the techniques of FAUST et al. and Lutz. The use of this methodology as a routine procedure enables the processing of many samples simultaneously, in order to increase recovery rate of G. lamblia cysts and reduce the time of sample storage.

The influence of the human genome on chronic viral hepatitis outcome

The mechanisms that determine viral clearance or viral persistence in chronic viral hepatitis have yet to be identified. Recent advances in molecular genetics have permitted the detection of variations in immune response, often associated with polymorphism in the human genome. Differences in host susceptibility to infectious disease and disease severity cannot be attributed solely to the virulence of microbial agents. Several recent advances concerning the influence of human genes in chronic viral hepatitis B and C are discussed in this article: a) the associations between human leukocyte antigen polymorphism and viral hepatic disease susceptibility or resistance; b) protective alleles influencing hepatitis B virus (HBV) and hepatitis C virus (HCV) evolution; c) prejudicial alleles influencing HBV and HCV; d) candidate genes associated with HBV and HCV evolution; d) other genetic factors that may contribute to chronic hepatitis C evolution (genes influencing hepatic stellate cells, TGF-beta1 and TNF-alpha production, hepatic iron deposits and angiotensin II production, among others). Recent discoveries regarding genetic associations with chronic viral hepatitis may provide clues to understanding the development of end-stage complications such as cirrhosis or hepatocellular carcinoma. In the near future, analysis of the human genome will allow the elucidation of both the natural course of viral hepatitis and its response to therapy.