Kátia Luz Torres

Hepatitis C virus in blood donors, Brazil.

To the Editor: The Fundacao de Hematologia e Hemoterapia do Amazonas is a public health service in Manaus, Brazil, that is responsible for serologic screening of all blood donations in the region. In the state of Amazon, 9.0% of donated blood is discarded on the basis of serologic findings; discarding because of hepatitis C virus (HCV) antibodies declined from 1.25% in 1995 to 0.32% in 2007. The aim of this study was to characterize the serologic and molecular profile of HCV-antibody–positive blood donors from the Fundacao de Hematologia e Hemoterapia do Amazonas. For the study, 154 donors were selected from a routine database of voluntary blood donors who had donated from September 2005 through April 2007 (82,851 donations). Fresh plasma samples were sent to the laboratory in Manaus through the usual transportation systems for regular donations; i.e., samples from 27 cities are transported by air for ≈2 hours, and samples from 21 localities are transported by boat or road, all under refrigerated conditions. An in-house standardized nested-PCR was used to detect HCV RNA (1). Genotype assignment was based on type-specific motifs on the sequenced amplicons delimited by primers HC11/HC18 from the 5′ untranslated region (2). Viral load was determined by commercial assay (HCV Monitor, Roche Molecular Systems, Inc., Branchburg, NJ, USA). An association was observed between HCV RNA and donor age; the same trend was seen in the first-time blood donor group. Associations between HCV-RNA detection and gender (p = 0.875) and place of donation (p = 0.989) were not significant. Using 18–25 years of age as the reference group, we found that odds ratios (ORs) for having HCV viremia were higher for those 45–55 years of age (OR 8.19, p<0.001) and 35–45 years of age (OR 3.49, p = 0.003). We observed increasing rates of RNA detection according to the signal-to-cutoff (S/CO) ratio. However, some donors had a weak S/CO ratio (between 1 and 2) with positive nested-PCR tests (Figure). Although adopting an S/CO ratio as a criterion for referring for further testing by recombinant immunoblot assay (RIBA) has been advocated by some groups (3), our data show that this criterion may be misleading and would deny a confirmatory diagnosis by giving false-negative results for many persons. Figure Distribution of hepatitis C virus (HCV) enzyme immunoassay signal-to-cutoff (S/CO) values by recombinant immunoblot assay (RIBA) interpretations among HCV-RNA–positive [PCR (+)] and HCV-RNA–negative [PCR (−)] donated blood samples. ... A total of 113 samples were analyzed by RIBA; among 48 RIBA-reactive samples, 9 (18.8%) were negative for HCV RNA in plasma. However, because PCR results may sometimes be negative for persons who are actually infected, a single negative PCR result should not be relied on as evidence that virus has cleared from plasma. Such patients must be observed for years before they may be declared cured (4). Among 97 RIBA-positive or -indeterminate samples, viral load was detectable in only 33 samples: 27 (81.8%) RIBA-positive samples and 6 (18.2%) RIBA-indeterminate samples. Only HCV genotypes 1 (87.1%) and 3 (12.9%) were found. Geographic distribution shows genotypes 1 and 3 in Manaus and only genotype 1 in other Amazon cities. This genotype geographic distribution is similar to that found for many Brazilian cities and Eastern countries and may reflect the route of HCV introduction into the Amazon; the virus was probably brought to the Amazon region by European immigrants and blood-derived medicines imported to Brazil. This hypothesis is corroborated by the finding of genotype 3 exclusively in Manaus, suggesting that this city is the point of arrival of HCV and that new strains were disseminated from Manaus to inner localities. Historical reconstruction of HCV in Amazon could be attempted by using these isolates as well as others from hepatitis patients in the region, including genotype 2 (5). We found a higher-than-expected rate of 50% for indeterminate immunoblot results among samples that were HCV-RNA positive by nested PCR. The presence of HCV RNA in plasma samples from 70%–75% of blood donors with indeterminate immunoblot results has also been reported by other groups in Brazil (6,7); however, in contrast, other investigators have reported RNA prevalence in such samples to be ≈2.5% (1,8). Indeterminate RIBA test results can indicate seroconversion or seroreversion or, occasionally, a chronic infection when RNA HCV is detected in plasma (9,10). To provide better understanding of the meaning of these indeterminate results, ongoing follow-up studies are examining the immune status of these persons. Our data offer insights for counseling of donors who have repeatedly HCV-reactive results. We suggest that Amazon region blood banks screen by enzyme immunoassay and use molecular testing as the first supplemental test and that immunoblot be applied to the remaining HCV-RNA nonreactive samples to distinguish between true and false anti-HCV carriers. This new algorithm would save considerable resources currently spent on immunoblot-indeterminate persons in addition to HCV-RNA reactive persons who do not require further testing for confirmation. Moreover, according to current policy, those with false-positive results may be reinstated as donors if they have negative retesting results after 6 months.

Immunological/virological peripheral blood biomarkers and distinct patterns of sleeping quality in chronic Hepatitis C patients

The rational of this study we intended to investigate whether the peripheral blood immunological/virological biomarkers were associated with distinct patterns of sleeping quality in patients with chronic hepatitis C‐(HCV). Distinct well‐established indexes/scores were used to categorize the sleeping quality of HCV patients, including the Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale and Fatigue Severity Scores. Our findings demonstrated that HCV patients classified as ‘good sleeper’ displayed an enhanced frequency of circulating CD8+ T cells, lower frequency of activated (CD69+) neutrophils and eosinophils but enhanced frequency of activated lymphocytes besides lower seric levels of IL‐4/IL‐8/IL‐10 but higher levels of IL‐12, besides lower HCV virus load and lower anti‐HCV IgG levels. In contrast, HCV patients classified as ‘poor sleeper’ displayed enhanced levels of activated neutrophils and eosinophils but lower frequency of activated lymphocytes, higher seric levels of IL‐6/TNF‐α/IL‐10 but lower levels of IL‐12 besides higher HCV virus load and increased anti‐HCV IgG levels. Positive correlation was further confirmed by the relationship between the leucocyte activation status, the cytokine levels, the HCV viral load and the anti‐HCV IgG reactivity with the PSQI indexes. Analysis of cytokine signature curves demonstrated that lower frequency of IL‐10 was observed in HCV patients classified as ‘good sleepers’, whereas enhanced frequency of IL‐6 was found HCV patients classified as ‘poor sleepers’. In conclusion, our data suggest that immunological biomarkers (leucocytes activation status and seric cytokines levels) are likely to be associated with sleeping quality patterns in HCV patients, suggesting their putative use for clinical monitoring purposes.

Occult hepatitis B virus infection among blood donors from the Brazilian Amazon: implications for transfusion policy

Brazil requires the performance of both a test for hepatitis B surface antigen (HBsAg) and a test for antibodies to the core of hepatitis B for blood donor screening. Blood centres in regions of high HBV endemicity struggle to maintain adequate stocks in face of the high discard rates due to anti‐HBc reactivity. We evaluated the potential infectivity of donations positive for anti‐HBc in search of a rational approach for the handling of these collections.

Hepatitis C virus screening and clinical monitoring of biomarkers in patients undergoing hemodialysis.

In this study, 395 volunteers were enrolled to investigate the seroprevalence of hepatitis C virus, the immunological and the alanine aminotransferase (ALT) biomarkers amongst hemodialysis patients, living in Manaus, Brazil. An overall seroprevalence of 13.9% was found in the hemodialysis patients. Analysis of seroconversion patterns demonstrated that most patients with HCV seroconverted up to 10 years following the first hemodialysis session. Anti‐NS5 antibody was detectable in 60.4% of patients with HCV. A lower percentage of circulating CD3+ and CD4+ T‐cells was found in patients seronegative for HCV, whereas a higher frequency of CD8+ T‐cells was the hallmark of patients with HCV. An overall low activation state of monocytes and eosinophils were observed in hemodialysis patients. In contrast, a higher frequency of activated neutrophils was observed in patients with HCV, selectively in the NS5+ subgroup. All hemodialysis patients had a higher percentage of activated lymphocytes, with the higher activation state in patients with NS5− reactivity. Higher ALT levels were observed in patients with HCV, especially in the NS5+ subgroup. Interestingly, the ALT levels were correlated negatively with the lymphocyte activation state, selectively in the NS5− subgroup, suggesting a protective role of these activated lymphocytes in patients with HCV. These findings reinforce the importance of the transmission of HCV among hemodialysis patients, suggesting that apart from the HCV screening, the serological and ALT biomarkers may represent important predictors of morbidity and/or mortality among patients undergoing hemodialysis. J. Med. Virol. 81:1220–1231, 2009.

Anti-inflammatory/regulatory cytokine microenvironment mediated by IL-4 and IL-10 coordinates the immune response in hemophilia A patients infected chronically with Hepatitis C virus†

In the past decades patients with hemophilia were infected commonly by hepatitis C virus (HCV) and a significant number of patients are infected chronically. Focusing on the role of the immune system for controlling and or maintaining HCV infection, the leukocyte and cytokine profiles of peripheral blood from hemophilia A patients and other patients with and without HCV infection were studied. The results demonstrated that hemophilia A is characterized by a general state of circulating leukocytes activation along with an overall increase in the frequency of IL‐6 and IL‐10 with decrease of IL‐8 and IL‐12. HCV infection of patients with hemophilia A does not influence further the activation state of circulating leukocytes but is accompanied by lower levels of alanine transaminase (ALT) and a prominent anti‐inflammatory/regulatory serum cytokine pattern, mediated by IL‐4 and IL‐10. Additionally, the results demonstrated that hemophilia A patients infected with HCV displaying No/Low antibody response to C33c and C22 have significant lower viral load and higher serum levels of IL‐12 and IL‐4. This finding suggests that the differential RIBA reactivity to C33c/C22 HCV core proteins may have a putative value as a prognostic biomarker for the infection in hemophilia A patients. J. Med. Virol. 85: 1009–1018, 2013.

Transfusion-transmitted malaria in endemic zone: epidemiological profile of blood donors at the Fundação HEMOAM and use of rapid diagnostic tests for malaria screening in Manaus

Objective With 99% of the cases in Brazil, malaria is endemic in the Amazon region. Transfusion-transmitted malaria, an important risk in endemic areas, has been reported. The aim of this study was to describe the epidemiological profile of blood donor candidates at the Fundação de Hematologia e Hemoterapia do Amazonas and evaluate the efficacy of rapid diagnostic tests used for malaria screening of blood donors within endemic regions. Methods Between May 2008 and May 2009, 407 blood donor candidates were selected and grouped based on the Malaria Annual Parasite Index of the geographic area in which they originated: Group 1 (eligible donors – n = 265) originated from areas of low to medium risk of exposure to malaria and Group 2 (ineligible donors – n = 142) originated from high-risk areas. All samples were concurrently screened using two immunochromatic antigen-based rapid tests and by the thick smear test. Results All samples were negative by all three methods. The demographic profile indicated that the majority of participants were male, ages ranged from 18 to 39 years and less than half the candidates had only elementary schooling. Two issues need to be addressed: one is the ineligibility of donors and its impact on blood donor centers as, in this study, 22.7% of the donors were considered ineligible. The other is the limited sensitivity of the parasitological tests used, allowing a risk of false-negative results. Conclusion New methods are needed to ensure transfusion safety without rejecting potential donors, which would ensure safe transfusion without harming the blood supply.

Self-sampling coupled to the detection of HPV 16 and 18 E6 protein: A promising option for detection of cervical malignancies in remote areas

Objective To evaluate both the performance and acceptability of a method coupling self-sampling with detection of cervical malignancy via elevated HPV 16 and 18 E6 oncoproteins (OncoE6™ Cervical Test) in remote areas in Brazil. Methods Women living in rural villages in proximity to Coari city, Amazonas, Brazil were invited to participate in a cervical cancer screening study. 412 subjects were enrolled; there were no refusals. In addition to E6 protein detection, DNA was extracted from the brushes and evaluated for HPV genotypes by PCR (PGMY09/11), followed by typing by the Papillocheck™ if positive. Subjects who were found to be positive for OncoE6 or HPV-DNA were referred for colposcopy. Results For 110 subjects (27%) this was the first cervical cancer exam. Overall the HPV-DNA prevalence was 19.1% (n = 79); 1.4% (n = 6) were positive by the OncoE6 Test. Fifty-six women attended the invitation for colposcopy where nine had an abnormal cervix and were subsequently biopsied. Histopathological analysis revealed 2 CIN3, 2 carcinomas and 5 CIN1. OncoE6 called two out of the three HPV 16 or 18 associated CIN3+ lesions. Conclusions The findings suggest that self-administered sample collection in combination with OncoE6 Test is feasible in this population. This could enable expanded screening coverage while ensuring a high specificity which is imperative given the remote geographic location, since women bearing abnormal test results would necessitate travel and logistical burden to access colposcopy and treatment.

Sustained virological response in patients with coinfection by hepatitis C virus genotypes 1 and 2, after just nine weeks of antiviral therapy: case report

A report of a 67 year-old male patient with positive serology for HCV. PCR revealed the presence of HCV RNA, viral load of 2,000 copies/mL and genotypes 1 and 2. The patient was treated with peginterferon alfa-2a at 180 mcg/week and ribavirin at 1,000 mg/day. In week four of treatment, HCV viral load was undetectable. In week nine, the patient developed hematemesis, worsening of asthenia, anorexia and impaired general condition, so the treatment was discontinued. The PCR was negative six months and one year after the cessation of treatment. The patient remains asymptomatic.

Immune response characterization in HIV/HCV co-infected patients of medicine tropical foundation

Immune response characterization in HIV/HCV coinfected patients of medicine tropical foundation Adriana Malheiro, Liziara Silva Fraporti, Flamir Victoria, Katia Luz Torres, Joao Paulo Diniz Pimentel, Andrea Tarrago, Laura Patricia Viana Maia, Felicien Vasquez, Jose Eduardo Levi, Marilu Victoria From 16 International Symposium on HIV and Emerging Infectious Diseases Marseille, France. 24-26 March 2010

Evaluation of immune response profiles of individuals with chronic Hepatitis C treated with interferon alpha and ribavirin, in the foundation of tropical medicine of Amazonas

Evaluation of immune response profiles of individuals with chronic Hepatitis C treated with interferon alpha and ribavirin, in the foundation of tropical medicine of Amazonas Ana Ruth Araujo, Liziara Silva Fraporti, Katia Luz Torres, Joao Paulo Diniz Pimentel, Tatiane Amabile, Andrea Tarrago, Laura Patricia Viana Maia, Nadja Garcia, Walter Luiz Neves, Adriana Malheiro From 16 International Symposium on HIV and Emerging Infectious Diseases Marseille, France. 24-26 March 2010