Correction: Hemostasis in elderly patients with human immunodeficiency virus (HIV) infection-Cross-sectional study.

[This corrects the article DOI: 10.1371/journal.pone.0227763.].

Hemostasis in elderly patients with human immunodeficiency virus (HIV) infection-Cross-sectional study.

INTRODUCTION: Aging and chronic HIV infection are clinical conditions that share the states of inflammation and hypercoagulability. The life expectancy of the world population has increased in the last decades, bringing as complications the occurrence of diseases that undergoing metabolic, bone, cardiological, vascular and neurological alterations. HIV-infected patients experience these changes early and are living longer due to the success of antiretroviral therapy. The objectives of this study was to evaluate some changes in the plasma hemostatic profile of 115 HIV-reactive elderly individuals over 60 years old in the chronic phase of infection, and compare with 88 healthy uninfected elderly individuals. Plasma determinations of D-dimers, Fibrinogen, von Willebrand Factor, Antithrombin, Prothrombin Time, Activated Partial Thromboplastin Time, and platelet count were performed. In the HIV-reactive group, these variables were analyzed according to viral load, protease inhibitor use and CD4+ T lymphocyte values. After adjusted values for age and sex, the results showed higher levels of Antithrombin (103%; 88%, p = 0.0001) and Prothrombin Time activities (92.4%; 88.2%, p = 0.019) in the HIV group compared to the control group. We observed higher values of Fibrinogen in protease inhibitor users in both the male (p = 0.043) and female (p = 0.004) groups, and in the female HIV group with detected viral load (p = 0.015). The male HIV group with a CD4+ count> 400 cells / mm3 presented higher von Willebrand Factor values (p = 0.036). D-Dimers had higher values in the older age groups (p = 0.003; p = 0.042, respectively). CONCLUSION: Our results suggest that the elderly with chronic HIV infection with few comorbidities had a better hemostatic profile than negative control group, reflecting the success of treatment. Protease inhibitor use and age punctually altered this profile.

Longitudinal Study of Hepatitis A Infection by Saliva Sampling: The Kinetics of HAV Markers in Saliva Revealed the Application of Saliva Tests for Hepatitis A Study.

Despite the increasing numbers of studies investigating hepatitis A diagnostic through saliva, the frequency and the pattern of hepatitis A virus (HAV) markers in this fluid still remains unknown. To address this issue, we carried on a longitudinal study to examine the kinetics of HAV markers in saliva, in comparison with serum samples. The present study followed-up ten patients with acute hepatitis A infection during 180 days post diagnosis (dpd). Total anti-HAV was detected in paired serum and saliva samples until the end of the follow-up, showing a peak titer at 90th. However, total anti-HAV level was higher in serum than in saliva samples. This HAV marker showed a probability of 100% to be detected in both serum and saliva during 180 dpd. The IgM anti-HAV could be detected in saliva up to 150 dpd, showing the highest frequency at 30th, when it was detected in all individuals. During the first month of HAV infection, this acute HAV marker showed a detection probability of 100% in paired samples. The detection of IgM anti-HAV in saliva was not dependent on its level in serum, HAV-RNA detection and/or viral load, since no association was found between IgM anti-HAV positivity in saliva and any of these parameter (p>0.05). Most of the patients (80%) were found to contain HAV-RNA in saliva, mainly at early acute phase (30th day). However, it was possible to demonstrate the HAV RNA presence in paired samples for more than 90 days, even after seroconversion. No significant relationship was observed between salivary HAV-RNA positivity and serum viral load, demonstrating that serum viral load is not predictive of HAV-RNA detection in saliva. Similar viral load was seen in paired samples (on average 104 copies/mL). These data demonstrate that the best diagnostic coverage can be achieved by salivary anti-HAV antibodies and HAV-RNA tests during 30-90 dpd. The long detection and high probability of specific-HAV antibodies positivity in saliva samples make the assessment of salivary antibodies a useful tool for diagnosis and epidemiological studies. The high frequency of HAV-RNA in saliva and the probability of detection of about 50%, during the first 30 dpd, demonstrate that saliva is also useful for molecular investigation of hepatitis A cases, mainly during the early course of infection. Therefore, the collection of saliva may provide a simple, cheap and non-invasive means of diagnosis, epidemiological surveys and monitoring of hepatitis A infection purposes.

Genotypic Characterization of Herpes Simplex Virus Type 1 Isolates in Immunocompromised Patients in Rio de Janeiro, Brazil.

Herpes simplex virus type 1 (HSV-1) is a prevalent human pathogen that causes a variety of diseases, including an increased risk of developing more severe disease in HIV-infected individuals. In Brazil, there is no information about the molecular epidemiology of HSV-1 infection, especially in HIV-infected individuals. The aim of this study was to perform the genotypic characterization of HSV-1 among HIV-infected patients. A total of 214 serum samples from HIV-positive patients without HSV infection symptoms were enrolled in one of two reference hospitals for HIV infection managing in Rio de Janeiro. The gG and gI genes were analyzed by restriction fragment length polymorphism (RFLP) and full nucleotide sequencing of the US8 (1601 bp), UL44 (1996 bp), and UL23 (1244 bp) regions was performed. A total of 38.3% (82/214) and 32.7% (70/214) of the serum samples tested positive for gG and gI genes, respectively. RFLP analysis classified the HSV-1 as belonging to genotype A. Phylogenetic analysis of the Brazilian samples for the US8, UL44, and UL23 regions demonstrated that the nucleotide identity between Brazilian samples was higher than 97% for all genes. No acyclovir mutation was detected in the patients. The shedding of HSV in the serum samples from HIV-positive patients who were asymptomatic for HSV infection was detected in this work. This is the first report of molecular characterization of HSV-1 in Brazilian samples since there is no previous data available in the literature concerning the genotypic classification and stable distribution of Brazilian strains of HSV-1 in Rio de Janeiro, Brazil.

Cross-Sectional Study of Hepatitis A Virus Infection in the Pantanal Population before Vaccine Implementation in Brazil: Usage of Non-Invasive Specimen Collection.

Population-based prevalence studies are essential tools for screening of hepatitis A and provide important data on susceptible groups. However, surveillance in isolated communities is difficult because of the limited access to these areas and the need for blood sample collection. This study aimed to determine the anti-HAV prevalence using oral fluid samples to provide an alternative tool for epidemiological studies that might be useful for vaccination-related decisions. The study population was composed of 224 volunteers from South Pantanal, aged 3 to 86 years old. This study was performed using oral fluids, previously standardized for anti-HAV antibody detection, which were collected using a ChemBio device. Eluates were tested using modified commercial EIA to detect anti-HAV antibodies. The overall prevalence was 79.1%, corresponding to 178 reactive EIA tests out of 224 samples. The age stratified data revealed a prevalence of 47.8% between 0-10 years, 84% in 11-20 years and 91.9% in subjects older than 21 years. Results indicate that hepatitis A prevalence was higher in adolescents and adults, corroborating the literature reports. Thus, oral fluid samples could replace serum in HAV epidemiological studies in isolated communities as they are efficient at detecting anti-HAV antibodies.

NS5A inhibitor resistance-associated polymorphisms in Brazilian treatment-naive patients infected with genotype 1 hepatitis C virus.

OBJECTIVES: Several promising NS5A protein inhibitors for hepatitis C virus (HCV) treatment, showing good antiviral activity, are currently being evaluated in clinical trials. However, viral breakthroughs associated with resistant variants have been observed, especially in patients infected with HCV-1a. We aimed to evaluate the occurrence of potential resistance mutations in the NS5A gene of HCV among Brazilian treatment-naive patients. METHODS: Direct sequencing of the HCV NS5A gene was performed in serum samples of 106 treatment-naive patients infected with subtypes 1a (n = 52) and 1b (n = 54). The sequence variability, signature patterns in amino acid sequences and variants associated with NS5A inhibitors were evaluated. RESULTS: The M28T and Y93H mutations were found in the subtype 1a sequences of two (3.85%) patients, and seven (13.46%) other patients presented the secondary mutation(s) H58P, E62D or H58P-E62D. For subtype 1b, the Y93H mutation was found in two (3.70%) patients and the substitutions R30Q, L31M, P58S and I280V were found in eight (14.81%) patients. Two distinct HCV-1a clades were distinguished by a phylogenetic analysis performed along with representative HCV-1a sequences and sequences containing HCV NS5A inhibitor resistance mutations retrieved from the Los Alamos database. All Brazilian sequences formed a large group of related sequences inside clade 1. It is noteworthy that 65.85% of sequences with substitution at sites 28, 30, 31 and 93 were found in clade 1. CONCLUSION: Brazilian HCV-1a sequences presented a peculiar pattern of amino acid composition, mutations and frequencies, which is distinct from other previously characterized sequences from other locations. The association of these findings with the outcome of treatment with NS5A inhibitors awaits further analysis.

Low prevalence of hepatitis B and C virus markers among children and adolescents.

This study aimed to determine the prevalence of HBV and HCV among children and adolescents attending schools and daycare centres in Rio de Janeiro State, located in southern Brazil. Serum samples from 1,217 individuals aged 0 to 18 years were collected from 1999 to 2012 and tested for HBsAg, total anti-HBc, anti-HBs, and anti-HCV by ELISA. Reactive HBsAg and anti-HBc samples were tested for HBV DNA. Reactive anti-HCV samples were tested for HCV RNA and genotyped by RFLP. HBsAg was detected in 1.8% of individuals, and total anti-HBc was detected among 3.6% of individuals. Anti-HBs reactivity was found among 25.3% (322/1,217) of the individuals and increased from 6.28% in the years 1999-2000 to 76.2% in the years 2001-2012 (P < 0.0001). HBV DNA was detected in 18 of 51 individuals who presented with HBsAg or isolated anti-HBc, and nine were considered occult hepatitis B cases. Three individuals were anti-HCV- and HCV RNA-positive: two of them were infected with genotype 1, and the other was infected with genotype 3. Low levels of HBV and HCV markers were observed in children and adolescents. HBV immunity increased during the period of study, indicating that childhood universal HBV vaccination has been effective for controlling HBV infection in Brazil.

Evidence of hepatitis A virus person-to-person transmission in household outbreaks.

The person-to-person transmission of the hepatitis A virus primarily occurs in enclosed spaces, particularly in the presence of inadequate hygiene conditions and a high proportion of susceptible individuals. Thus, intimate family contact stands out as a risk factor for HAV infection dissemination. The present study aimed to evaluate the occurrence of household HAV transmission. Blood samples were collected from patients with hepatitis A (index cases) and their family members (contacts) that were referred to an ambulatory care clinic specializing in viral hepatitis. A total of 97 samples were collected from 30 families with a confirmed hepatitis A case (index case). Serological and molecular techniques for the diagnosis of hepatitis A were conducted on all samples. HAV infection (anti-HAV IgM + and/or HAV RNA +) was detected in 34.3% (23/67) of the contacts; 34.3% (23/67) of the contacts were immune to HAV, and 31.4% (21/67) were susceptible. In the household contacts, HAV immunity was significantly associated with older age; susceptibility to infection and HAV infection were associated with younger age. Household outbreaks were detected in 16/30 families studied. Co-circulation of subgenotypes IA and IB was found in the household outbreaks, and person-to-person transmission was evidenced in six of the household outbreaks, with 100% homology between the index case and contact strains. The results demonstrated the relevance of HAV household transmission, reaffirming the need for hepatitis A vaccine administration in susceptible contacts and effective infection control procedures to prevent the extension of household outbreaks.

Knowledge and prevalence of viral hepatitis among beauticians.

Beauty treatments, such as tattooing, piercing, manicures, pedicures, and barbershop shaving, can pose an important risk of virus transmission. This study was conducted to determine hepatitis A virus (HAV), hepatitis B virus (HBV), and hepatitis C virus (HCV) prevalence in a sample of beauticians from Rio de Janeiro (Southeast Brazil) and to assess the knowledge and attitudes of these professionals regarding viral hepatitis and their practices during their activities. One hundred nineteen beauticians were recruited in September 2010. Serum samples were tested for total anti-HAV, total anti-HBc, HBsAg, anti-HBs, and anti-HCV reactivity. A questionnaire was administered to identify socio-demographic risk factors and to determine knowledge and attitudes regarding viral hepatitis. Prevalence was 73.9% for total anti-HAV, 0% for HBsAg, 5.9% for anti-HBc, 23.6% for anti-HBs, and 0.8% for anti-HCV. Most professionals (81.5%) were well informed (4-7 correct answers) and reported the use of disposable sandpaper and nail sticks and sterilized pliers, but only 40% of them reported adequate processes of disinfection/sterilization. In conclusion, a high prevalence of HAV infection and a low prevalence of HBV and HCV infection were observed among beauticians. In addition, most of these individuals were not immune to HBV, indicating the need for vaccination campaigns targeting these professionals. Most of these professionals were well informed regarding viral hepatitis, although there was a gap in knowledge regarding disinfection and sterilization procedures. Public health prevention strategies should be adopted to improve education about disinfection/sterilization procedures for manicures and pedicures.

Could oral fluid be used to evaluate anti-hepatitis A virus status in individuals living in difficult-to-access areas?

A strategy adopted by different countries to reduce the number of new cases of hepatitis A is the vaccination. However, the mosaic of the epidemiological profile in developing countries has hampered the establishment of a unified nationwide vaccination program. To determinate national vaccination policies, the results of epidemiological studies need to be carefully considered. For this monitoring, the use of oral fluid is very important due to the painless and non invasive collection characteristics. There are few studies investigating which oral fluid collection device is optimal to detect low antibody levels and its use in selecting individuals for vaccination. So, the present study aimed to evaluate different oral fluid collection devices to detect humoral immune response against hepatitis A virus and its application in epidemiological studies. Therefore, 90 matched serum and oral fluid samples were collected from volunteers with different immune status, under ideal conditions of collection (optimization panel); and 224 matched samples in difficult-to-access areas (epidemiological study). Serum was collected by venipuncture and the oral fluid was obtained using three commercial devices: Salivette(®), OraSure(®) and ChemBio(®). Serum and oral fluid were submitted to a commercial immunoblot to detect total anti-HAV antibodies. The optimization panel demonstrated that ChemBio(®) device had the best performance (100% agreement), followed by OraSure(®) (95.4%) and Salivette(®) (90.8%). The optimal collection device (ChemBio(®)), tested in a difficult-to-access area and evaluated under precarious conditions of collection, showed similar prevalence of total anti-HAV between serum and oral fluid, 80.8% and 79%, respectively. A follow-up was performed to evaluate the stability of oral fluid and it was observed that 210 days after the collection it was possible to detect anti-HAV antibodies. Oral fluid can be used to detect low levels of specific-antibody, being important to select age groups to be vaccinated. Therewith, the choice of proper collection device is essential to evaluate HAV antibodies in the epidemiological scenario.

Mutations in hepatitis C virus NS3 protease domain associated with resistance to specific protease inhibitors in antiviral therapy naïve patients.

The prevalence of naturally occurring mutations in hepatitis C virus associated with resistance to protease inhibitors in chronically infected patients has not been reported in Brazil. The NS3 serine protease domain was sequenced in 114 therapy-naïve patients infected with subtype 1a (n = 48), 1b (n = 53), or 3a (n = 13). A V36L mutation was observed in 5.6% patients infected with subtype 1b and in all isolates of the 3a subtype, and a T54S mutation was detected in 4.1% of isolates of subtype 1a. In conclusion, the presence of variants carrying mutations associated with resistance to protease inhibitors in therapy-naïve patients may be important for future therapeutic strategies.

Detection of hepatitis C virus in platelets: evaluating its relationship to antiviral therapy outcome.

BACKGROUND/AIMS: Detection of HCV has been documented in extrahepatic sites such as platelets. However, its influence on antiviral therapy outcome is unknown. In this study, we investigated the relationship between the detection of HCV in platelets from a cohort of 48 chronically HCV-infected patients and response to antiviral therapy. METHODOLOGY: This study comprised of 19 males and 29 females, mean age 54.9 +/- 8.72 years, followed-up in Rio de Janeiro, Brazil, between August 2004 and October 2006. HCV-RNA was detected in serum and platelets (pre-treatment, end-of-treatment and 24 weeks after completion of therapy) by reverse transcription-nested polymerase chain reaction. Patients with genotype 1 or 4 were treated with peginterferon-alfa/ribavirin for 48 weeks, and patients with genotype 3 received interferon-alfa/ribavirin for 24 weeks. RESULTS: Baseline detection of HCV in platelets was found not to be related to therapy outcome. However, significant associations between detection rates of HCV in platelets and serum at the end-of-treatment (p = 0.0203), and 24 weeks after completion of therapy (p = 0.0016) were observed. Interestingly, HCV was detected in platelets from two patients with normal ALT who lost detectable serum HCV at the end-of-treatment and, after 24 weeks of followup, relapsed virologically in serum. CONCLUSIONS: Our data suggest that patients with HCV persistence in platelets by the end-of-treatment appear to be at an increased risk of recurrent HCV infection.

Normal plasma antithrombin activity in patients with relapsing-remitting and secondary progressive multiple sclerosis.

OBJECTIVE: Multiple sclerosis (MS) is an inflammatory disease characterized by multifocal areas of central nervous system (CNS) demyelination. Activation of coagulation factors and fibrin deposition are observed around CNS blood vessels in experimental autoimmune encephalomyelitis, an animal model of MS. Antithrombin (AT) is a potent anticoagulant with remarkable anti-inflammatory properties, and its inhibitory effects on coagulation and inflammation may play a role in the pathogenesis and clinical course of MS. We studied the association between plasma AT activity and clinical forms of MS. PATIENTS AND METHODS: A total of 69 patients, 37 with relapsing-remitting and 32 with secondary progressive MS, were included in the study. A control group (CG) of 34 normal subjects was also studied. Plasma AT activity (Stachrom ATIII) was quantified using a chromogenic activity assay with normal reference values ranging from 70% to 120% of AT activity. RESULTS: We found no difference between plasma AT levels in patients and those in CG. We also found no association of AT levels with activity of disease, duration of disease progression, level of neurological disability, and treatment. CONCLUSION: We found no association between plasma AT activity and RRMS or SPMS. It remains to be studied whether exists or not an association between abnormal plasma AT activity and other MS forms.

Occult hepatitis B virus infection in HIV-infected patients: Evaluation of biochemical, virological and molecular parameters.

AIM: To determine the prevalence of occult hepatitis B virus (HBV) infection in a group of human immunodeficiency virus (HIV)-infected Brazilian patients and to investigate its association with biochemical, virological and molecular features. METHODS: Sera from 43 patients positive for HBV core antibody and negative for HBV surface antigen (HBsAg) were tested for HBV DNA positivity by semi-nested PCR. HBV loads were assessed by real-time PCR. S gene was cloned and sequenced for HBV isolates from 3 patients. HBsAg expression of these cases was performed in HuH7 cells. RESULTS: HBV DNA was found in 6/43 (14%) samples, all except one associated with low viral loads. Occult HBV infection was further correlated with anti-hepatitis C virus (anti-HCV) antibodies positivity, but not with alanine aminotransferase (ALT) elevated levels. S gene sequences derived from three patients were determined. Two of them displayed mutations that may explain HBsAg negativity. In the first one, a stop codon mutation was found at position 216 in the C-terminal end of HBsAg. In the second patient, E164D and I195M substitutions in HBsAg, associated with lamivudine-resistance mutations in the polymerase were identified. As expected, all clones showing those mutations displayed undetectable or very low levels of HBsAg. CONCLUSION: Occult HBV infection was frequent in HIV-infected patients, was not associated with ALT elevation but significantly correlated with HCV seropositivity. The low viremia and the detection of HBsAg mutants confirm that multifactorial mechanisms are involved in occult HBV infection. HBV molecular monitoring should be employed for an adequate management of HBV/HIV co-infected patients.

Detection of hepatitis C virus in platelets: evaluating its relationship to viral and host factors.

BACKGROUND/AIMS: Interaction of platelets with HCV is presumed to be one of the pathogenic mechanisms implicated in HCV-associated thrombocytopenia. Nevertheless, analysis of factors influencing the detection of HCV in platelets is not well understood. In this study, we investigated the relationship between the detection of HCV in platelets from a cohort of 39 chronically HCV-infected patients and several viral and host factors. METHODOLOGY: This study comprised of 14 males and 25 females with a median age of 53 years, followed-up in Rio de Janeiro, Brazil, between August 2003 and December 2004. HCV-RNA was detected in serum and platelet samples by reverse transcription-nested polymerase chain reaction. Genotypes were determined by using direct nucleotide sequencing of the PCR products and plasma viral loads by using HCV-Amplicor Monitor 2.0. RESULTS: When compared on the basis of the results of the detection of HCV-RNA in platelets, patients did not differ significantly in relation to viral load and genotype, platelet count, aminotransferases and degree of hepatic fibrosis. CONCLUSIONS: Our data suggest that HCV can be detected in platelets of chronically HCV-infected patients independent of these cofactors, including circulating HCV load. Studies on HCV dynamics are needed to provide new insights into HCV binding to platelets.

Hepatitis C virus-associated thrombocytopenia: a controlled prospective, virological study.

Chronic hepatitis C virus (HCV) infection has also been associated with the development of several extrahepatic alterations, including thrombocytopenia, and a variety of pathogenic mechanisms are reported to be implicated in this hematological abnormality. Different studies have succeeded in detecting HCV in platelets with discrepant results. Moreover, most of the studies on HCV-associated thrombocytopenia have failed to provide data concerning the infecting genotype, a factor with prognostic implication in chronically HCV-infected patients. To determine whether thrombocytopenia is an extrahepatic alteration dependent on particular HCV genotypes, and to assess the relationship between thrombocytopenia and detection of HCV-RNA (positive strand) in platelets from patients with chronic HCV infection, 106 anti-HCV+/HCV-RNA+ patients (57 thrombocytopenic and 49 non-thrombocytopenic) were prospectively studied. The infecting genotype was analyzed from sera by using direct nucleotide sequencing of the polymerase chain reaction (PCR) products from core region. Genotypes 1a, 1b, and 3a were more prevalent in our patients, and no association between these genotypes and thrombocytopenia was observed ( p=0.891). HCV-RNA was detected in platelets by reverse transcriptase (RT)-nested PCR in the 5' non-coding region with a higher frequency (60%) in thrombocytopenic patients than in non-thrombocytopenic subjects (35%, p=0.017), suggesting that HCV is directly involved in the process that, at least in part, leads to thrombocytopenia.