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.

Analysis of the genetic polymorphism of Paracoccidioides brasiliensis and Paracoccidioides cerebriformis "Moore" by random amplified polymorphic DNA (RAPD) and 28S ribosomal DNA sequencing: Paracoccidioides cerebriformis revisited

Our purpose was to compare the genetic polymorphism of six samples of P. brasiliensis (113, 339, BAT, T1F1, T3B6, T5LN1), with four samples of P. cerebriformis (735, 741, 750, 361) from the Mycological Laboratory of the Instituto de Medicina Tropical de São Paulo, using Random Amplified Polymorphic DNA Analysis (RAPD). RAPD profiles clearly segregated P. brasiliensis and P. cerebriformis isolates. However, the variation on band patterns among P. cerebriformis isolates was high. Sequencing of the 28S rDNA gene showed nucleotide conservancy among P. cerebriformis isolates, providing basis for taxonomical grouping, and disclosing high divergence to P. brasiliensis supporting that they are in fact two distinct species. Moreover, DNA sequence suggests that P. cerebriformis belongs in fact to the Aspergillus genus.

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

PRELIMINARY REPORT ON THE PUTATIVE ASSOCIATION OF IL10 -3575 T/A GENETIC POLYMORPHISM WITH MALARIA SYMPTOMS

Only a small percentage of individuals living in endemic areas develop severe malaria suggesting that host genetic factors may play a key role. This study has determined the frequency of single nucleotide polymorphisms (SNPs) in some pro and anti-inflammatory cytokine gene sequences: IL6 (-174; rs1800795), IL12p40 (+1188; rs3212227), IL4 (+33; rs2070874), IL10 (-3575; rs1800890) and TGFb1 (+869; rs1800470), by means of PCR-RFLP. Blood samples were collected from 104 symptomatic and 37 asymptomatic subjects. Laboratory diagnosis was assessed by the thick blood smear test and nested-PCR. No association was found between IL6 (-174), IL12p40 (+1188), IL4 (+33), IL10 (- 3575), TGFb1 (+869) SNPs and malaria symptoms. However, regarding the IL10 -3575 T/A SNP, there were significantly more AA and AT subjects, carrying the polymorphic allele A, in the symptomatic group (c2 = 4.54, p = 0.01, OR = 0.40 [95% CI - 0.17- 0.94]). When the analysis was performed by allele, the frequency of the polymorphic allele A was also significantly higher in the symptomatic group (c2 = 4.50, p = 0.01, OR = 0.45 [95% CI - 0.21-0.95]). In conclusion, this study has suggested the possibility that the IL10 - 3575 T/A SNP might be associated with the presence and maintenance of malaria symptoms in individuals living in endemic areas. Taking into account that this polymorphism is related to decreased IL10 production, a possible role of this SNP in the pathophysiology of malaria is also suggested, but replication studies with a higher number of patients and evaluation of IL10 levels are needed for confirmation.

Two competitive enzyme immunoassays for the detection of IgG class antibodies to hepatitis a antigen

Two competitive enzyme immunoassays (EIA) techniques were developed: in the first (COMP-1), test sera were added together with HAV antigen on anti-HAV IgG-coated wells followed by an anti-HA VHRP conjugate; in the second (COMP-2), test sera and anti-HA VHRP conjugate competed for HAV epitopes previously adsorbed to anti-HA V IgG-coated wells. Both procedures used tetramethylbenzidine (TMB) as a substrate. Both competitive tests were shown to be reproducible and suitable for routine diagnosis and research purposes.

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%).

Evaluation of Leishmania antigens preparation and storage for use in enzyme immunoassays

The influence of time and temperature on the storage of an alkaline antigen of L.major-like and L.(V.) braziliensis promastigotes added or not of a proteases inhibitor (PMSF) was evaluted by means of an IgG-ELISA. Antibodies in assays using L. major-like antigen stored at -20oC for 6 monsths had a statistically lower geometric mean titer (GMT) and different 95% confidence interval limits (CL) than antigens stored otherwise, as assessed by the "t" statistic. The PMSF L. major-like antigen after storage for 6 months at a temperature of 4oC had the same GMT and 95% CL displayed at time zero as well as when storage for 4 and 6 months at -20oC. Significant diferences were not found when L.(V.) braziliensis antigens were stored at times and temperatures mentioned; the PMSF antigen stored for 2 months at -70oC resulted in a lower serum GMT and 95% CL than any other, as assessed by the "t" statistic. Antigen performance did not show any statistical difference associated to the addition of PMSF within the same species; the largest difference between antigens was that between PMSF-L. (V.) braziliensis and L. major-like without PMSF.

Dot enzyme-linked immunosorbent assay (dot-ELISA) for schistosomiasis diagnosis using dacron as solid-phase

Dacron and nitrocellulose were evaluated as matrices for the dot enzyme linked immunosorbent assay (dot-ELISA) for schistosomiasis and compared to indirect immunofluorescence (IMF). Titration of sera from 18 schistosomiasis patients against soluble worm antigen preparation (SWAP) was carried out and sera from healthy individuals from non-endemic areas were used as controls. The IMF was less sensitive than the dot-ELISAs, although the difference was not statistically significant (p > 0.05). The dot-ELISA based on nitrocellulose was as sensitive as that using dacron. Stability did not differ between nitrocellulose and dacron. Specificity was lower when dacron was used than when nitrocellulose was used, although the difference was not statistically significant (p > 0.05). In conclusion, this work showed that nitrocellulose and dacron performed similarly in dot-ELISA, suggesting that they may be used alternatively in population surveillance in endemic areas.

Identification of Leishmania chagasi from skin in Leishmania/HIV co-infection: a case report

A case of HIV/Leishmania co-infection presenting both visceral and cutaneous manifestations is reported. Leishmania infection was confirmed by conventional methods (parasitological approach and serology) and by PCR. Leishmania chagasi isolated from the skin lesion was characterized by enzyme electrophoresis and by restriction fragment length polymorphism of the internal transcribed spacer of the ribosomal gene.

Evaluation of an enzyme immunoassay for clinical diagnosis of neurocysticercosis in symptomatic patients

INTRODUCTION: Neurocysticercosis is an infection of the human central nervous system caused by the metacestode larvae of Taenia solium. Neurocysticercosis is the most common parasitic disease in developing countries. Epilepsy is the most common clinical manifestation. Difficulties in confirming the diagnosis motivated the evaluation of the enzyme-linked immunosorbent assay on cerebral spinal fluid (CSF). METHODS: Twenty-two patients with NCC and 44 control patients were studied. CSF was analyzed using a commercial ELISA kit developed for NCC. Sensitivity and specificity were measured and a multivariate logistic regression was performed. RESULTS: Sensitivity and specificity of ELISA were 31.8% and 100%, respectively, with accuracy of 77.3%. Only the size of the lesions proved to be important for performance of the test. CONCLUSIONS: The results showed that ELISA contributes to the diagnosis of neurocysticercosis if the result is negative or if the patient has a lesion of 2 cm or more.

Characterization of mannose-binding lectin plasma levels and genetic polymorphisms in HIV-1-infected individuals

INTRODUCTION: The present study investigated the association between mannose-binding lectin (MBL) gene polymorphism and serum levels with infection by HIV-1. METHODS: Blood samples (5mL) were collected from 97 HIV-1-infected individuals resident in Belém, State of Pará, Brazil, who attended the Special Outpatient Unit for Infections and Parasitic Diseases (URE-DIPE). CD4+ T-lymphocyte count and plasma viral load were quantified. A 349bp fragment of exon 1 of the MBL was amplified via PCR, using genomic DNA extracted from controls and HIV-1-infected individuals, following established protocols. MBL plasma levels of the patients were quantified using an enzyme immunoassay kit. RESULTS: Two alleles were observed: MBL*O, with a frequency of 26.3% in HIV-1-infected individuals; and the wild allele MBL*A (73.7%). Similar frequencies were observed in the control group (p > 0.05). Genotype frequencies were distributed according to the Hardy-Weinberg equilibrium in both groups. Mean MBL plasma levels varied by genotype, with statistically significant differences between the AA and AO (p < 0.0001), and AA and OO (p < 0.001) genotypes, but not AO and OO (p = 0.17). Additionally, CD4+ T-lymphocytes and plasma viral load levels did not differ significantly by genotype (p > 0.05). CONCLUSIONS: The results of this study do not support the hypothesis that MBL gene polymorphism or low plasma MBL concentrations might have a direct influence on HIV-1 infection, although a broader study involving a large number of patients is needed.

Evaluation of Leishmania (Leishmania) chagasi strains isolated from dogs originating from two visceral leishmaniasis-endemic areas in Brazil using multilocus enzyme electrophoresis

INTRODUCTION: Domestic dogs are the most important reservoir in the peridomestic transmission cycle of Leishmania (Leishmania) chagasi. The genetic variability of subpopulations of this parasite circulating in dogs has not been thoroughly analyzed in Brazil, even though this knowledge has important implications in the clinical-epidemiological context. METHODS: The objective of this study was to evaluate and compare the phenotypic variability of 153 L. chagasi strains isolated from dogs originating from the municipalities of Rio de Janeiro (n = 57) and Belo Horizonte (n = 96), where the disease is endemic. Strains isolated only from intact skin were selected and analyzed by multilocus enzyme electrophoresis using nine enzyme systems (6PG, GPI, NH1 and NH2, G6P, PGM, MDH, ME, and IDHNADP). RESULTS: The electrophoretic profile was identical for all isolates analyzed and was the same as that of the L. chagasi reference strain (MHOM/BR/74/PP75). Phenetic analysis showed a similarity index of one for all strains, with the isolates sharing 100% of the characteristics analyzed. CONCLUSIONS: The results demonstrate that the L. chagasi populations circulating in dogs from Rio de Janeiro and Belo Horizonte belong to a single zymodeme.

Genetic polymorphism for IFNγ +874T/A in patients with acute toxoplasmosis

INTRODUCTION: A single nucleotide polymorphism (SNP) in the gene encoding gamma interferon influences its production and is associated with severity of infectious diseases. This study aimed to evaluate the association of IFNγ+874T/A SNP with duration of disease, morbidity, and development of retinochoroiditis in acute toxoplasmosis. METHODS: A case-control study was conducted among 30 patients and 90 controls. RESULTS: Although statistical associations were not confirmed, A-allele was more common among retinochoroiditis cases and prolonged illness, while T-allele was more frequent in severe disease. CONCLUSIONS: Despite few cases, the results could indicate a relation between IFNγ+874T/A single nucleotide polymorphism and clinical manifestations of toxoplasmosis.

Laboratory diagnosis of amebiasis in a sample of students from southeastern Brazil and a comparison of microscopy with enzyme-linked immunosorbent assay for screening of infections with Entamoeba sp.

Introduction: Epidemiological studies on amebiasis have been reassessed since Entamoeba histolytica and E. dispar were first recognized as distinct species. Because the morphological similarity of these species renders microscopic diagnosis unreliable, additional tools are required to discriminate between Entamoeba species. The objectives of our study were to compare microscopy with ELISA kit (IVD®) results, to diagnose E. histolytica infection, and to determine the prevalence of amebiasis in a sample of students from southeastern Brazil. Methods: In this study, diagnosis was based on microscopy due to its capacity for revealing potential cysts/trophozoites and on two commercial kits for antigen detection in stool samples. Results: For 1,403 samples collected from students aged 6 to 14 years who were living in Divinópolis, Minas Gerais, Brazil, microscopy underestimated the number of individuals infected with E. histolytica/E. dispar (5.7% prevalence) compared with the ELISA kit (IVD®)-based diagnoses (15.7% for E. histolytica/E. dispar). A comparison of the ELISA (IVD®) and light microscopy results returned a 20% sensitivity, 97% specificity, low positive predictive value, and high negative predictive value for microscopy. An ELISA kit (TechLab®) that was specific for E. histolytica detected a 3.1% (43/1403) prevalence for E. histolytica infection. Conclusions: The ELISA kit (IVD®) can be used as an alternative screening tool. The high prevalence of E. histolytica infection detected in this study warrants the implementation of actions directed toward health promotion and preventive measures.

Impact of the IL-18 gene polymorphism in response to antiviral therapy in chronic HCV genotype 4 patients

^a Introduction Interleukin (IL)-18 is a well-known major proinflammatory cytokine with broad biological effects. The major immunomodulatory functions of IL-18 include enhancing T cell and natural killer cell cytotoxicity. Serum levels of this cytokine were shown to increase in chronic hepatitis C patients compared to non-infected healthy people. An association between IL-18 gene promoter polymorphisms and pegylated interferon (PEG-IFN) and ribavirin treatment outcomes has been reported for individuals with chronic hepatitis C virus genotype 1 (HCV-1). In this study, HCV genotype 4 (HCV-4) patients were assessed for IL-18 gene polymorphisms and treatment outcomes or severity of liver disease because data concerning the impact of IL-18 gene polymorphisms on patients with HCV-4 infections are limited. Methods This study included 123 chronic HCV-4 Egyptian patients and 123 apparently healthy volunteer blood donors who served as a control group. HCV genotyping was performed using the line probe assay. IL-18 genotyping was performed using the TaqMan Real-Time PCR method in all 246 patient and control samples. Results In our study, all patients had HCV-4. IL-18 gene single nucleotide polymorphism (SNP) (-607C/A) genotype distributions and allele frequencies did not differ between HCV patients and normal healthy subjects or between patient groups when compared according to the therapeutic response. Moreover, the presence of an IL-18 SNP was not associated with histological disease severity. We conclude that the presence of the IL-18 SNP rs1946518 does not affect the outcome of chronic HCV-4 treatment in Egyptian patients. Conclusions The IL-18 SNP rs1946518 does not affect response to treatment in chronic HCV-4 patients.