Kirsten A. Visoná

Role of saliva, urine and feces in the transmission of type B hepatitis.

Abstract To identify vehicles of transmission of Type B hepatitis virus, feces, urine and saliva of chronic carriers of the hepatitis antigens and patients with acute Type B hepatitis were examined...

Genotype F prevails in HBV infected patients of hispanic origin in Central America and may carry the precore stop mutant.

The distribution of HBV genotypes and the presence of the precore stop mutation were investigated in HBV strains from Central America. 333 HBsAg positive sera from chronic HBsAg carriers and acute hepatitis B cases from five different countries (Costa Rica, Nicaragua, Honduras, El Salvador and Guatemala) were tested for HBV DNA by nested PCR. Genotyping by limited sequencing within the S gene was performed on 90 strains, 66 from sera with a high level of HBV DNA, and another 24 from sera positive for HBV DNA only after nested PCR. 23 of the samples were anti‐HBe positive. Genotype F was found in 71 (79%), A in 13 (14%), D in 5 (6%) and C in one of the 90 sera. 18 patients with genotype F infection had anti‐HBe and HBV DNA in serum. Since the three published precore sequences of genotype F strains have a C1858, which is known to prevent the precore stop mutation from G to A at position 1896, the precore and part of the core genes were sequenced from 19 anti‐HBe positive sera with HBV DNA, 17 with genotype F and 2 with genotype A. The A1896 mutation was found in 11 of the 17 genotype F strains. All these had a T1858, which was also present in 5 of the 6 genotype F strains with G1896. The precore region was therefore sequenced from genotype F strains from 5 HBeAg positive sera from the five different Central American countries. These also had a T1858, which thus is the wild type substitution in genotype F in Central America. A number of mutations were recorded between residues 57 and 68 in the core protein corresponding to a unique clustering region of the genotype F strains. The predominance of genotype F in Central American populations of Hispanic origin was not anticipated since this genotype is regarded as indigenous to the Amerindian populations of the New World. J. Med. Virol. 51:305–312, 1997.

Molecular Epidemiology of Hepatitis B Virus in Central America Reflected in the Genetic Variability of the Small S Gene

The S genes of 31 Central American hepatitis B virus (HBV) strains belonging to genotypes A, C, D, and F (4, 1, 4, and 22 strains, respectively) were compared with 104 published S genes. According to the deduced S gene product, 21 genotype F strains encoded adw4, while 1 encoded ayw4. Three clusters were revealed within genotype F, which correlated with substitutions at residue 45. In a cluster of 18 Central American and 1 Alaskan strain, all had Thr45. One cluster included 2 Central American strains and 6 strains from South America and Europe, which had Leu45. Two Nicaraguan strains differed by five substitutions, including a Pro45 in the S gene product from other F strains. In conclusion, the dominating HBV genotype was F, which might be the reason for a low prevalence of HBV in the area, despite high prevalence of hepatitis A. These infections otherwise vary in parallel and are considered to reflect socioeconomic conditions.

Evaluation of a pooling method for routine anti‐HCV screening of blood donors to lower the cost burden on blood banks in countries under development

A pooling system was developed for use in anti‐HCV screening of voluntary blood donors at the local Central American Red Cross blood banks, in Nicaragua, El Salvador and Honduras. The commercially available second generation anti‐HCV screening kit from Abbott Laboratories (North Chicago, IL) was used with a modification in the initial serum dilution procedure. Pools of five sera were selected for routine screening, based on comparative studies of individual samples and of pools with different sample sizes.

High prevalence of GB virus C strains genetically related to strains with Asian origin in Nicaraguan hemophiliacs

The presence of hepatitis GB virus C (GBV‐C), also known as hepatitis G virus (HGV), and hepatitis C virus (HCV) were investigated in sera from 45 hemophiliacs from nine locations in Nicaragua using a nested polymerase chain reaction (PCR). Primers used to detect GBV‐C and HCV derived from the helicase region and 5′UTR, respectively. Seventeen (38%) patients were positive for GBV‐C RNA in serum by PCR. Twelve (27%) patients were positive for HCV RNA by PCR. Six (13%) of these were coinfected with GBV‐C. Anti‐HCV was detected in all the 12 HCV RNA positive hemophiliacs and in another 14 (31%) individuals, in whom GBV‐C RNA was found in 2. Ten patients (22%) lacked markers for both GBV‐C and HCV. The mean age of the patients positive for GBV‐C but negative for HCV by PCR was significantly lower than for those negative for GBV‐C but positive for HCV by PCR (P < 0.05; Students t‐test), indicating that the risk for this group of hemophiliacs to acquire GBV‐C infection is higher as compared to the risk of acquiring HCV infection. Eleven GBV‐C strains were sequenced in the 5′UTR. Sequence comparison to previously published GBV‐C strains revealed that all 11 strains were more similar to Asian strains than to strains of European and African origin. Sequences in the NS5‐B region were available for 8 HCV strains, all of which were found to belong to genotype 1a. The similarity of the Nicaraguan GBV‐C strains to strains from Asia indicates that the GBV‐C strains in the region presumably have an Amerindian origin. It is also considered that the HTLV II strains in the New World aboriginal populations are ancient and brought there by the ancestral Amerindian populations from Asia. Further, the genotype F of hepatitis B virus, known to represent the strains in populations with Amerindian background, predominates in Central American populations with Hispanic background. It remains to be clarified why Amerindian strains of GBV‐C as well as of HBV predominate also in populations with mixed ethnic background in Central America. J. Med. Virol. 52:149–155, 1997.

HBx M130K and V131I (T-A) mutations in HBV genotype F during a follow-up study in chronic carriers

BackgroundAround 400 million people worldwide are chronically infected with Hepatitis B virus (HBV). An estimated 10% of these chronic patients develop progressive liver damage including cirrhosis and Hepatocellular Carcinoma (HCC). The HBx gene encodes a protein of 154 amino acids which is a transactivator and has been associated with HBV pathogenesis. A change in the amino acid sequences at positions 130 and 131 in the HBV-X protein (M130K and V131I) produced by T-A point mutations at the nucleic acids level has been associated with severe liver damage and HCC in patients from China and Africa. Further, such changes have been proposed as a prognostic marker for progressive liver damage and HCC. The purpose of this study was to determine if T-A mutations are present in HBV chronic carriers with genotype F (the major genotype in Costa Rica) and further, if these mutations are associated with HBV disease progression in Costa Rica HBV patients from 1972 to 1985.ResultsSerum samples from 50 HBV positive individuals were amplified and directly sequenced, 48 belonged to genotype F, 1 from genotype D and another was classified as D or E.T-;A mutations were absent in 17 acute patients who recovered, but was present in 12 of 29 chronic carrier samples (42.8%), in one sample the T-A mutations were detected as early as 29 days after clinical onset of disease. In 17 carriers with available liver biopsies, T-;A mutations were found in 8 sera of 13 (61.5%) classified as moderate or severe, and none in 4 biopsies with mild liver damage. However, it was not possible to demonstrate a statistical association between the presence of T-A mutations and moderate/severe liver damage, using a Fischer exact test, 1 tail, p = 0.05.In 4 patients HCC was diagnosed, and 2 of them presented the T-A mutations in their sera.ConclusionT-A mutations were found in HBV genotype F in chronic carriers but not in patients who recovered from acute infection. These mutations could be developing early during infection although the possibility of infection with the mutant virus could not be excluded.More studies are necessary to establish if the T-A mutation can be used as a prognostic marker for severity of liver disease in patients infected with HBV.

Determination of human cytomegalovirus genetic diversity in different patient populations in Costa Rica

Seroprevalence of HCMV in Costa Rica is greater than 95% in adults; primary infections occur early in life and is the most frequent congenital infection in newborns. The objectives of this study were to determine the genetic variability and genotypes of HCMV gB gene in Costa Rica. Samples were collected from alcoholics, pregnant women, blood donors, AIDS patients, hematology-oncology (HO) children and HCMV isolates from neonates with cytomegalic inclusion disease. A semi-nested PCR system was used to obtain a product of 293-296 bp of the gB gene to be analyzed by Single Stranded Conformational Polymorphism (SSCP) and sequencing to determine the genetic polymorphic pattern and genotypes, respectively. AIDS patients showed the highest polymorphic diversity with 14 different patterns while fifty-six percent of HO children samples showed the same polymorphic pattern, suggesting in this group a possible nosocomial infection. In neonates three genotypes (gB1, gB2 and gB3), were determined while AIDS patients and blood donors only showed one (gB2). Of all samples analyzed only genotypes gB1, 2 and 3 were determined, genotype gB2 was the most frequent (73%) and mixed infections were not detected. The results of the study indicate that SSCP could be an important tool to detect HCMV intra-hospital infections and suggests a need to include additional study populations to better determine the genotype diversity and prevalence.

Variability in HIV-1 partial genomic sequences in Costa Rican patients: analysis with different bioinformatics tools

OBJECTIVE To estimate subtype and genomic variability in the HIV pol gene of Costa Rican patients by using different bioinformatics tools and to use this information to establish new policies to better manage these patients. METHODS A total of 113 pol sequences available from Costa Rican patients under highly active antiretroviral therapy were analyzed by using the Genotyping, REGA, Stanford, and MEGA programs. The pol sequences came from 77 virologic failures (VF) and 36 basal samples (BS). Of the 77 VF, 22 also were sequenced in the env region. RESULTS No major differences were found among the variables studied. However, there was a tendency for more variability in VF patients with a high baseline viral load. In the pol gene, 75%-83% of BS and 66%-75% of VF samples were pure B subtype by Genotyping and REGA, respectively. The other samples presented variations related mainly to circulating recombinant form CRF12 by genotyping or to CRF17 or -29 by phylogenetic analysis or a new possible BD recombinant with all programs. In the Stanford program, all variable samples showed a subtype B with high polymorphism. The variability in the env sequences was lower than that in the pol region. CONCLUSION The B subtype is predominant in Costa Rican HIV-positive patients. There is high variability within sequences with potential recombination between B and F or D subtypes. The BD recombinant has not been previously reported. This high variability is likely the result of possible recombinant events, nonadherence to antiretroviral therapy, sexual intercourse without protection, and many sexual partners. Similar studies should be done in other countries in the Region, in particular in those places with extensive immigration, in order to decrease the possibility of virus variability as well as the cost of antiretroviral therapy.

Impact of Hepatitis B and Hepatitis C Virus Infections in a Hematology-Oncology Unit at a Children's Hospital in Nicaragua, 1997 to 1999

ABSTRACT The risk of acquiring both hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in patients with hematological-oncological disorders has been documented. However, the impact and risk factors for such infections from different geographical areas vary, and the use of both immunological and molecular assays to determine HCV infections has been our approach. Children from a hematology-oncology unit (HOU) in Nicaragua were studied for both HBV and HCV serological markers; studies for the latter used both immunological (anti-HCV) and molecular (HCV RNA) assays. The children from the HOU included patients with leukemia, lymphoma, other neoplasias, and anemia and a smaller group with other hematological diseases. As a control group, children from other units at the same hospital were enrolled, as well as health care workers attending both patient populations. Pertinent clinical and personal data for each child at the HOU were obtained for statistical analysis. Of the 625 children from the HOU enrolled in this study 53.3% were infected with HCV and 29.4% had a prior or present HBV infection. In the child patient control group 3.2% had HBV markers and all were negative for HCV. The group of children with leukemia had the highest infection rate for both HBV and HCV. However, the determination of anti-HCV was found to have an overall low sensitivity in children from HOU, and a retest consisting of a molecular assay to determine HCV RNA was performed to better establish the total number of HCV-infected subjects in this group. The highest independent risk factor for infection was hospitalization. The very high prevalence rates for both HBV and HCV infection in this patient group indicate an urgent need to implement better control of known risk factors and to consider the use of both immunological and molecular assays for HCV diagnostic purposes.

Development of low cost peptide‐based anti‐hepatitis C virus screening and confirmatory assays: Comparison with commercially available tests

Screening and confirmatory low cost reagent tests have been developed for detection of anti‐hepatitis C virus (HCV). Assays are based on the use of specific synthetic peptides from several structural and non‐structural viral proteins. The efficacy of the screening anti‐HCV EIA‐Spep assay was compared with both Abbott EIA 2.0 (Abbott Laboratories, North Chicago, IL) and Ortho EIA 2.0 (Ortho Diagnostic Systems, Raritan, NJ) anti‐HCV detection kits and the confirmatory EIA‐Cpep assay was compared with the Abbott Matrix anti‐HCV confirmation test. In the EIA‐Spep, a pool of 3 peptides was added to each well of a microtiter plate. In EIA‐Cpep, each well was separately coated with 1 of 4 peptides and 1 recombinant protein. A total of 867 blood donor samples from Costa Rica tested simultaneously with the 3 screening assays yielded the same specificity and negative predictive values of ≥99.9% and 100%, respectively. A comparative study on voluntary blood donor samples from Honduras, Nicaragua, and El Salvador using the 2 anti‐HCV confirmatory assays revealed different patterns that are 46% positive, 24% indeterminate, and 30% negative with the EIA‐Cpep assay vs. 31% positive, 48% indeterminate, and 21% negative with the Matrix assay. A study of 71 patient samples from Costa Rica showed a higher correlation between initially reactive samples when analyzed by the Abbott and Ortho kits, than when the assay results were compared between the Abbott and EIA‐Spep kits; the latter detected 7 and 15 non‐reactive samples, respectively. These results could reflect the use of a similar antigen source for the 2 commercial assays. The presence of HCV RNA in a group of 29 samples analyzed was related to the simultaneous reactivity in all 3 screening assays. None of the discordant samples had detectable levels of HCV RNA. Economic difficulties for health care services in the developing countries of Central America have prevented implementation of routine anti‐HCV blood donor screening tests. This is likely to be the primary reason for uncontrolled dissemination of HCV, and the lack of identification of potential high risk groups. Alternative low cost reagents developed locally as described in this article could be a useful tool in the control of HCV spread throughout the developing world. J. Med. Virol. 58:221–226, 1999.