Helena Medina Cruz

Assessment of dried blood spot samples as a simple method for detection of hepatitis B virus markers

Detection of hepatitis B virus (HBV) serological markers in dried blood spot (DBS) samples by enzyme immunoassay (ELISA) has not yet been fully optimized. In this study, the ability to detect three HBV markers (HBsAg, anti‐HBc, and anti‐HBs) was evaluated in DBS samples using a modified commercial ELISA. Matched serum and DBS samples were obtained from individuals with or without a past history of HBV infection. Sera samples were tested according to the manufacturers instructions, but for DBS testing, paper diameters, elution buffer, volume of input sample, and cut‐off values were evaluated to optimize the assay. Stability studies were done on DBS stored at for up to 180 days at different temperatures. The absorbance values that yielded the maximum sensitivity and specificity were determined based on the area under the ROC curve (AUROC) and chosen as the cut‐off value. Using this parameter, sensitivity was 90.5%, 97.6%, and 78% for anti‐HBc, HBsAg, anti‐HBs assays, respectively. Specificity was 92.6%, 96.7%, and 97.3% for anti‐HBc, HBsAg, and anti‐HBs assays, respectively. HBV markers could be detected in DBS samples until 63 days after sample collection at most temperatures, but storage at −20°C yielded more consistent results. These results indicate that modified ELISA can be used to detect HBV markers in DBS samples, particularly if the samples are stored appropriately. J. Med. Virol. 83:1522–1529, 2011.

Update on hepatitis B and C virus diagnosis.

Viral hepatitis B and C virus (HBV and HCV) are responsible for the most of chronic liver disease worldwide and are transmitted by parenteral route, sexual and vertical transmission. One important measure to reduce the burden of these infections is the diagnosis of acute and chronic cases of HBV and HCV. In order to provide an effective diagnosis and monitoring of antiviral treatment, it is important to choose sensitive, rapid, inexpensive, and robust analytical methods. Primary diagnosis of HBV and HCV infection is made by using serological tests for detecting antigens and antibodies against these viruses. In order to confirm primary diagnosis, to quantify viral load, to determine genotypes and resistance mutants for antiviral treatment, qualitative and quantitative molecular tests are used. In this manuscript, we review the current serological and molecular methods for the diagnosis of hepatitis B and C.

Performance of rapid hepatitis C virus antibody assays among high- and low-risk populations

BACKGROUND Rapid tests for the detection of antibodies to hepatitis C virus (anti-HCV) can facilitate access to diagnosis. OBJECTIVES This study aimed to evaluate the performance of rapid tests for anti-HCV detection in the sera, whole blood, and oral fluid samples from individuals with different endemicity profiles and risk behaviors. STUDY DESIGN Three groups donated biological samples that were tested using three anti-HCV rapid tests (WAMA, Bioeasy and OraSure): (I) suspected cases of hepatitis C, (II) individuals who were living in remote areas in Brazil and (III) crack users and beauty professionals. Reproducibility, repeatability and cross-reactivity to other infectious agents (dengue, HIV, malaria, and syphilis) were also evaluated. RESULTS In group I, specificities varied from 93.75% to 100% and sensitivities varied from 76.03% to 93.84% according to the EIA results. When anti-HCV/HCV RNA-reactive sera samples were considered true-positive HCV cases, the sensitivities and specificities varied from 86.3% to 99.09% and 93.75% to 100%, respectively. In group II, the OraSure rapid test presented the best performance. In group III, the Bioeasy assay performed best using saliva and whole blood and the OraSure assay performed best using oral fluid samples. The reproducibility and repeatability of the WAMA and Bioeasy tests were excellent. The level of concordance between the HCV EIAs and the rapid tests using samples that were reactive for other infectious agents varied from 82.35% to 100% for the WAMA assay and 94.11% to 100% for the Bioeasy assay. CONCLUSION All of the rapid tests could be used to identify active HCV infection among individuals with different endemicity profiles and risk behaviors.

Evaluation of Saliva Specimens as an Alternative Sampling Method to Detect Hepatitis B Surface Antigen

In this study, a modified enzyme immunoassay (EIA) was evaluated for the Hepatitis B surface antigen (HBsAg) among whole saliva and oral fluid samples. Specimens were collected from 115 individuals who gave serum and oral fluid using Salivette (Sarstedt, Nümbrecht, Germany) and whole saliva. Saliva specimens were tested following a modified ELISA, and the results were compared with paired serum specimens that were tested according to the suppliers instructions. Transport buffer for the oral fluids, sample volume for assay, incubation period of sample with conjugate as well as cut‐off values were evaluated to optimize the assay. The highest sensitivity and specificity were obtained by increasing the incubation of sample and conjugate to 16 hr and using the area under the receiver operating characteristic curve to calculate cut‐off values. HBsAg was detected in 40 oral fluids and 44 whole saliva samples out of 47 paired positive serum specimens and not detected in 64 oral fluids and 63 whole saliva samples out of 68 matched negative sera samples by the ELISA assay. There was excellent agreement between the results for the serum and saliva specimens kappa value (κ): 0.80 for oral fluid and κ: 0.87 for whole saliva and there was excellent reproducibility. Using an optimized protocol, the sensitivities of whole saliva and oral fluid were 93.6 and 85.1%, respectively, whereas specificities of whole saliva and oral fluid were 92.6 and 94.1%, respectively. Our data showed a significant promise for the use of whole saliva and oral fluid together with the modified commercial EIA for Hepatitis B virus infection surveillance. J. Clin. Lab. Anal. 25:134–141, 2011.

An evaluation of different saliva collection methods for detection of antibodies against hepatitis C virus (anti-HCV)

BACKGROUND Saliva samples can be used as an alternative fluid for against hepatitis C virus (anti-HCV) detection owing to the ease of collection and excellent acceptability. This study was conducted to optimize a commercial enzyme immunoassay (EIA) to detect anti-HCV in saliva samples. METHODS Ninety-six individuals donated paired serum and saliva samples that were obtained, using a commercial device (Salivette) and spitting into a sterile container. Initially, elution buffer for the Salivette samples, sample volume, incubation time and temperature, and two different anti-HCV EIAs were evaluated. Using the optimized assay, three methods for cut-off calculation were also evaluated. RESULTS A 20-fold increase in the sample volume for both collection methods was needed. Moreover, the Radim assay was the most appropriate assay for anti-HCV detection in saliva samples, and the quality parameters were increased when a ROC curve was used to determine the cut-off value. Using this optimized assay, the sensitivities, specificities, accuracies, positive and negative predictive values were above 90% for saliva obtained using both the Salivette and spitting methods. Using this assay, discordant false-negative results were obtained for only two Salivette samples and five spitting samples. The concordance kappa was 93% for the Salivette method and 86.1% for the spitting method, demonstrating excellent performance. CONCLUSIONS Saliva samples obtained for both methods can be employed for anti-HCV detection among HCV-infected or HCV-suspected cases, but several modifications must be performed on commercial EIAs to obtain good results. Moreover, samples obtained with commercial devices are more appropriate for anti-HCV detection in saliva samples.

A Cross Section Study to Determine the Prevalence of Antibodies against HIV Infection among Hepatitis B and C Infected Individuals

(1) Background: There are limited data regarding human immunodeficiency virus (HIV) prevalence among hepatitis B virus (HBV) or hepatitis C virus (HCV) infected individuals. The aim of this cross-sectional study is to determine the prevalence of HBV and HCV infection among HIV individuals; (2) Methods: A total of 409 patients (126 HBV+ and 283 HCV+) referred to the Brazilian Reference Laboratory for Viral Hepatitis from 2010 to 2013 donated serum samples. Anti-HIV, HBsAg, anti-HBc, anti-HBs, anti-HBcIgM, anti-HBe, HBeAg, and anti-HCV antibodies were measured, and anti-HCV positive samples were tested for viral RNA and genotype; (3) Results: The anti-HIV antibody prevalence was 10.31% and 4.59% among HBV+ and HCV+ patients, respectively. The HCV mean (SD) viral load was log 5.14 ± 1.64 IU/mL, and genotype I was most prevalent (163/283). Anti-HBs and anti-HBc were detected in 40% and 26% of HCV+ individuals, respectively. Among the HBV+ population, the presence of anti-HIV antibodies was associated with male gender, marital status (married), tattoo, sexual orientation, sexual practices (oral sex and anal sex), history of sexually transmitted diseases (STDs), history of viral hepatitis treatment, and a sexual partner with hepatitis or HIV. For the HCV+ group, the presence of anti-HIV antibodies was associated with female gender, marital status (married), anal intercourse, previous history of STDs, and number of sexual partners; (4) Conclusion: A high prevalence of anti-HIV antibodies was found among individuals with HBV and HCV, showing the importance of education programmes towards HIV infection among HBV- and HCV-infected individuals.

Prevalence of hepatitis B and C virus infections among military personnel

BACKGROUND Data regarding Hepatitis B and C viruses (HBV and HCV) prevalence among military personnel in Brazil are lacking, but the work-related risk of exposure can be high. The objective of this study was to estimate the seroprevalence of HBV and HCV and the risk factors associated to HBV exposure among Brazilian military personnel. METHODS A cross-sectional study was conducted and included 433 male military adults aged 18-25 years old working in Rio de Janeiro during October 2013. All individuals completed a questionnaire to assess their risk of exposure and provided a blood sample to HBV and HCV testing. RESULTS None of the participants presented HBsAg or anti-HBc IgM, 18 (4.1%) were positive for total anti-HBc, 247 (57.0%) were positive for anti-HBs, and 3 (0.7%) were anti-HCV reactive. The majority of military personnel with past HBV infection (anti-HBc reactive) and HBV immunity (anti-HBs reactive) had a history of prior dental procedures (88.9% and 77.3%), consumption of alcohol at least once a week (50% and 55.9%), and practiced oral sex (61.1% and 58.3%, respectively). In addition, anti-HBc positivity was common among individuals with a history of surgery (44.4%) and practice of anal sex (50%). At univariate analysis, age group was associated to anti-HBc and anti-HBs positivity. CONCLUSIONS Low rates of HBV and HCV infection were observed among Brazilian military personnel in comparison to the general Brazilian population. HBV immunity rates were relatively low indicating the need for vaccination campaigns in this group.

Poor sensitivity of rapid tests for the detection of antibodies to the hepatitis B virus: implications for field studies

Rapid tests (RTs) can be used as an alternative method for the conventional diagnosis of hepatitis B virus (HBV). This study aims to evaluate antibodies to HBsAg (anti-HBs) and antibodies to HBeAg (anti-HBe) RTs under different Brazilian settings. The following three groups were included: GI: viral hepatitis outpatient services; GII: low resource areas; and GIII: crack users and beauticians. Imuno-rápido anti-HBsAg™ and Imuno-rápido anti-HBeAg™ RTs were evaluated and showed specificities greater than 95% in all groups. The sensitivity values to anti-HBs were 50.38%, 51.05% and 46.73% and the sensitivity values to anti-HBe were 76.99%, 10.34% and 11.76% in the GI, GII and GIII groups, respectively. The assays had a low sensitivity and high specificity, which indicated their use for screening in regions endemic for HBV.

Importance of Collection Methods and Stability of Oral Fluid Samples for Hepatitis B Surface Antigen Detection

Oral fluid (OF) sample collection and stability for HBsAg detection are not fully established. This study aims to investigate the applicability of OF collectors and sample stability for Hepatitis B virus surface antigen detection.

A Cross-Sectional Study of Viral Hepatitis Perception among Residents from Southeast and North Regions of Brazil

Few data are available regarding viral hepatitis perception among the general global population. The present study aims to estimate the perception of viral hepatitis in a cohort of individuals living in two geographical regions of Brazil: North (Manaus city (MA)) and Southeast (Rio de Janeiro city (RJ)). A cross-sectional, descriptive study was carried out among 287 subjects recruited in MA (134) and RJ (153). All individuals answered a questionnaire assessing socio-demographic characteristics and viral hepatitis awareness. Participants’ responses were scored and divided using interquartile values. Associations between socio-demographic characteristics and knowledge were also evaluated. Interquartile analysis scored 0–21 correct answers as “Very Weak”; 22–27 as “Weak”; 28–31 as “Intermediate”; and 32–47 as “Desirable”. Mean ± standard deviations (SD) of correct responses were weak in both MA (24.1 ± 7.0) and RJ (26.3 ± 7.3). Bivariate analysis showed an association between viral hepatitis awareness and both education level (p < 0.001) and family income (p < 0.01). Desirable scores were more common in female participants (61%), those aged between 21–30 years (40%), those with a secondary education (51.7%), those who received high income (31.6%), and those from RJ (70.0%). Health education campaigns in these cities are recommended to increase knowledge and reduce the transmission of these viruses.