Identification of mutations in the S gene of hepatitis B virus in HIV positive Mexican patients with occult hepatitis B virus infection.

INTRODUCTION AND AIM: Occult hepatitis B virus infection (OBI) is characterized by the presence of replication-competent hepatitis B virus (HBV) DNA in the liver and/or serum of patients with undetectable levels of the HBV surface antigen (HBsAg). Due to the shared infection routes HIV positive patients are at higher risk of developing OBI, thus, the aim of this study was to determine the frequency of OBI in Mexican HIV-infected patients and to identify mutations in the HBV S gene that could be associated to the development of OBI. MATERIALS AND METHODS: Plasma samples from 50 HIV-infected patients with undetectable levels of the HBsAg were obtained and analyzed. The Core, PreS and S genes were amplified by nested PCR and sequenced by the Sanger method. To analyze HBV diversity in the OBI-positive patients, ten sequences of 762bp from the HBV S gene were selected, cloned, and subsequently sequenced for mutational analyses. RESULTS: OBI infection was found with a frequency of 36% (18/50). All the HBV sequences corresponded to the H genotype. The most common mutations were: C19Y, Q129H, E164D, and I195M, with a frequency of 44%, 36%, 39% and 48% respectively. CONCLUSIONS: In this study, we report the presence of OBI in a cohort of Mexican HIV-infected patients with an overall prevalence of 36%. Mutational analyses revealed that four non-silent mutations were frequent in different regions of the HBsAg gene, suggesting that they might be associated to the development of OBI in this population, nevertheless, further studies are required to determine their role in the pathogenesis of OBI.

Genotypic testing for HIV-1 drug resistance using dried blood samples.

In third-world countries, dried blood samples (DBS) are a convenient alternative to plasma for monitoring viral load during HIV-1 therapy. In this study, we evaluated the feasibility of using DBS to perform HIV-1 drug resistance genotyping in a ViroSeq assay in which the protease and reverse transcriptase regions of the pol gene are analyzed. Fifty-seven antiretroviral genotypes from plasma samples were tested, and drug resistance genotypes were determined. Only 38.6% paired DBS samples were sequenced. Failure to amplify DNA from DBS samples generally correlated with plasma viral loads below log(10) 5.1. The majority of the mutations identified in plasma pol sequences were also found in their DBS counterpart, with a concordance in genotype interpretation of 96.4%. Several factors were identified that could potentially improve both the sensitivity and the quality of genotype data, such as sample storage conditions and sequence analysis. Therefore, DBS sampling is useful to determine viral load and drug resistance genotypes in HIV patients.

Use of dried blood samples for monitoring hepatitis B virus infection.

BACKGROUND: Hepatitis B virus (HBV) infection is a problem in several regions of the world with limited resources. Blood samples dried on filter paper (DBS) have been successfully used to diagnose and monitor several infectious diseases. In Mexico there is an urgent need for an affordable and easy sampling method for viral load (VL) testing and monitoring of chronic HBV infection. The purpose of this work was to validate the utility of DBS samples for monitoring HBV infection in patients from Mexico City. METHODS: Matched samples of plasma and DBS on filter paper from 47 HBV infected patients from the Instituto Mexicano del Seguro Social (IMSS), were included. To evaluate the DNA stability and purity from DBS stored at different temperature conditions, samples from ten patients were stored at 4 degree, 25 degree, and 37 degree C for 7 days. After DBS elution and DNA extraction, the purity of these samples was determined measuring the O.D. rate 260/280. The DBS utility for molecular studies was assessed with PCR assays to amplify a 322 bp fragment from the "a" determinant region of the HBV "S" gene. The VL from all samples was determined to evaluate the correlation between plasma and DBS matched samples. RESULTS: The quality of the DNA from DBS specimen is not adversely affected by storage at 4 degree, 25 degree and 37 degree C for up 7 days. Statistical ANOVA analyses did not show any significant difference. The same amplification efficiency was observed between DNA templates from samples stored at different temperatures. The Pearson correlation between the VL from DBS and plasma matched samples was 0.93 (p = 0.01). The SD was 1.48 for DBS vs.1.32 for Plasma, and an average of log10 copies/mL of 5.32 vs. 5.53. ANOVA analysis did not show any statistically significant difference between the analyzed groups (p = 0.92). CONCLUSION: The results provide strong evidence that the isolation and quantification of DNA-HBV from DBS is a viable alternative for patient monitoring, and molecular characterization of the virus variants circulating in Mexico.

[Epidemiologic and situational panorama of hepatitis C in Mexico]. / Panorama epidemiológico y situacional de la hepatitis C en México.

OBJECTIVE: To determine the epidemiological situation of Chronic Hepatitis C (CHC) in our country. BACKGROUND DATA: Chronic Hepatitis C affects 170 million people worldwide, and about 0.7% of Mexican population. There is no enough epidemiological information about CHC in our country, and it is very probable that some cases are not even detected. METHODS: An investigation poll was performed. Age, gender, birthday, weight, race, residence and birth place, routes of transmission, ALT levels, histological, serological and molecular diagnosis, evidence of complications and previous treatments were recorded. A data recollection sheet was dispatched to different country provinces; they had 6 months to answer it, in order to recollect all information. RESULTS: 831 patients were analized (58.6% female and 41.4% male) with the following distribution in our country provinces: Aguascalientes 15, Chihuahua 12, Distrito Federal 495, Durango 10, Jalisco 89, Guanajuato 78, Yucatán 8, Querétaro 11, Sonora 40, Tabasco 15, Baja California 5, Veracruz 13, Tamaulipas 2 and 38 patients of Nuevo León. The highest incidence of CHC was found at fifth and sixth decade of life (28.5% y 26.7% respectively. The weight distribution was 36.2% < 65kg, 34.6% 65-75 kg and 29.2% > 75 kg. 86.5% had chronic hepatitis and 13.2% cirrhosis. The risk factors for HCV infection analysis showed that the main route of transmission was via contaminated blood (64.2%); when we excluded the patients that were exposed before 1995, the incidence was lowered to 4.5%. The higher incidence was showed between 1970 and 1990 (68%). The intravenous drug users were predominantly male and on those patients in the provinces near the north border line of our country. The predominant genotype was gen- 1 no matter the province (72.2%), in the intravenous drug users genotype 3 was found in 25%. The viral load was similar in all the provinces. 75% of the patients had have treatment and 22.5% had have two cycles, 50% of cirrhotic patients had have treatment whereas only 28% of the patients with late complications had have it. The most common treatment was pegylated alpha-2a interferon plus ribavirine. CONCLUSIONS: 1. The main route of transmission was blood transfusion. There is a marked decrease in the incidence of post-transfusional hepatitis since the introduction of anti-VHC antibody screening of blood donors (4.5%). 2. The time between the infection and diagnosis was 23 years for chronic hepatitis and 26 years for cirrhosis. 3. Intravenous drugs use was an important route of transmission in the north of our country. 4. The predominant genotype was gen-1. 5. Almost all the patients with chronic hepatitis received treatment, the most common used was pegylated interferon alpha-2a and ribavirin. 6.50% of the patients with CHC have late complications.

Use of Dried Plasma Spots for HIV-1 Viral Load Determination and Drug Resistance Genotyping in Mexican Patients.

Monitoring antiretroviral therapy using measurements of viral load (VL) and the genotyping of resistance mutations is not routinely performed in low- to middle-income countries because of the high costs of the commercial assays that are used. The analysis of dried plasma spot (DPS) samples on filter paper may represent an alternative for resource-limited settings. Therefore, we evaluated the usefulness of analyzing DPS samples to determine VL and identify drug resistance mutations (DRM) in a group of HIV-1 patients. The VL was measured from 22 paired plasma and DPS samples. In these samples, the average VL was 4.7 log10 copies/mL in liquid plasma and 4.1 log10 copies/mL in DPS, with a correlation coefficient of R = 0.83. A 1.1 kb fragment of HIV pol could be amplified in 14/22 (63.6%) of the DPS samples and the same value was amplified in plasma samples. A collection of ten paired DPS and liquid plasma samples was evaluated for the presence of DRM; an excellent correlation was found in the identification of DRM between the paired samples. All HIV-1 pol sequences that were obtained corresponded to HIV subtype B. The analysis of DPS samples offers an attractive alternative for monitoring ARV therapy in resource-limited settings.

Occult hepatitis B virus infection among Mexican human immunodeficiency virus-1-infected patients.

AIM: To determine the frequency of occult hepatitis B infection (OHBI) in a group of human immunodeficiency virus (HIV)-1+/ hepatitis B surface antigen negative (HBsAg)- patients from Mexico. METHODS: We investigated the presence of OHBI in 49 HIV-1+/HBsAg- patients. Hepatitis B virus (HBV) DNA was analyzed using nested PCR to amplify the Core (C) region and by real-time PCR to amplify a region of the S and X genes. The possible associations between the variables and OHBI were investigated using Pearson's χ(2) and/or Fisher's exact test. RESULTS: We found that the frequency of OHBI was 49% among the group of 49 HIV-1+/HBsAg- patients studied. The presence of OHBI was significantly associated with the HIV-1 RNA viral load [odds ratio (OR) = 8.75; P = 0.001; 95%CI: 2.26-33.79] and with HIV-antiretroviral treatment with drugs that interfere with HBV replication (lamivudine, tenofovir or emtricitabine) (OR = 0.25; P = 0.05; 95%CI: 0.08-1.05). CONCLUSION: The OHBI frequency is high among 49 Mexican HIV-1+/HBsAg- patients and it was more frequent in patients with detectable HIV RNA, and less frequent in patients who are undergoing HIV-ARV treatment with drugs active against HBV.