Diagnosis of hepatitis C virus in Brazilian blood donors using a reverse transcriptase nested polymerase chain reaction: comparison with enzyme immunoassay and recombinant protein immunoblot assay

Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA > or = 3, ALT<1), group B (EIA > or = 3, ALT>1), group C (1<=EIA<3, ALT<1), group D (1<=EIA<3, ALT>1) and group E (EIA<=0.7). HCV sequences were detected by RT-nested PCR, using primers for the most conserved region of viral genome. RIBA-2 was applied to the same samples. In group A (n=6), all samples were positive by RT-nested PCR and RIBA-2. Among 124 samples in group B, 120 (96.8%) were RIBA-2 positive and 4 (3.2%) were RIBA-2 indeterminate but were seropositive for antigen c22.3. In group B, 109 (87.9%) of the RIBA-2 positive samples were also RT-nested PCR positive, as well as were all RIBA-2 indeterminate samples. In group C, all samples (n=9) were RT-nested PCR negative: 4 (44.4%) were also RIBA-2 negative, 4 (44.4%) were RIBA-2 positive and 1 (11.1%) was RIBA-2 indeterminate. HCV-RNA was detected by RT-nested PCR in 3 (37.5%) out of 8 samples in group D. Only one of them was also RIBA-2 positive, all the others were RIBA-2 indeterminate. All of the group E samples (controls) were RT- nested PCR and RIBA-2 negative. Our study suggests a strong relation between anti-HCV EIA-2 ratio > or = 3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%), especially when the c22.3 band was detected.

Immunofluorescence assay reactivity patterns of serum samples presenting indeterminate Western blot results for antibodies to HIV-1 and HTLV-I/II in Cordoba, Argentina

Serum samples (n: 110) from blood donors and high risk individuals from Cordoba, Argentina with indeterminate HIV-1 and HTLV-I/II Wb profiles were studied for specific antibodies to HTLV-I/II and HIV-1 by indirect immunofluorescence assay (IFA) and for the presence or absence of HIV-1 and HTLV-I/II specific bands by Wb. This study was carried out in order to characterize their putative reactions with HIV-1 and HTLV-I/II proteins and to resolve the retrovirus infection status of these individuals. Results indicated that blood donors sera displaying indeterminate HIV-1 or HTLV-I/II Wb patterns were not immunoreactive to HTLV-I/II and HIV-1 on IFA. However, a high rate of indeterminate HIV-1 and HTLV-I/II Wb samples from high risk individuals had positive HTLV-I/II and HIV-1 IFA results respectively. Our study supports the growing evidence that HTLV-HIV indeterminate seroreactivity in low risk population is due to a cross reaction against nonviral antigens, and in high risk populations the indeterminate samples show serological cross-recognition between HIV-1 proteins and HTLV-I/II proteins on Wb. These results point out the necessity to investigate the HTLV-I/II reactivity in indeterminate HIV-1 samples and viceversa in order to confirm the diagnosis. Finally, this study shows the potential usefulness of IFA in elucidating the status of HIV-1 and HTLV-I/II infection of individuals with indeterminate Wb profiles, thus enabling resolution of retrovirus infection status.

Evaluation of surrogate markers for human immunodeficiency virus infection among blood donors at the blood bank of "Hospital Universitário Regional Norte do Paraná", Londrina, PR, Brazil

This study evaluated the usefulness of the anti-HBc, hepatitis C virus antibodies (anti-HCV), human T cell lymphotropic virus I and II antibodies (anti-HTLV I/II), serologic tests for syphilis, and surface antigen of hepatitis B virus (HBsAg) as surrogate markers for the risk for HIV infection in 80,284 serum samples from blood donors from the Blood Bank of "Hospital Universitário Regional Norte do Paraná", Londrina, Paraná State, Brazil, analyzed from July 1994 to April 2001. Among 39 blood donors with positive serology for HIV, 12 (30.8%) were anti-HBc positive, 10 (25.6%) for anti-HCV, 1 (2.6%) for anti-HTLV I/I, 1 (2.6%) was positive for syphilis, and 1 (2.6%) for HBsAg. Among the donors with negative serology for HIV, these markers were detected in 8,407 (10.5%), 441 (0.5%), 189 (0.2%), 464 (0.6%), and 473 (0.6%) samples, respectively. The difference was statistically significant (p < 0.001) for anti-HBc and anti-HCV. Although the predictive positive value for these surrogate markers were low for HIV infection, the results confirmed the anti-HBc and anti-HCV as useful surrogate markers for HIV infection thus reinforcing the maintenance of them in the screening for blood donors contributing to the prevention of the small number of cases in which HIV is still transmitted by transfusion.

Detection of EBV-DNA in serum samples of an immunosuppressed child during a three years follow-up: association of clinical and PCR data with active infection

Twenty-four whole blood and serum samples were drawn from an eight year-old heart transplant child during a 36 months follow-up. EBV serology was positive for VCA-IgM and IgG, and negative for EBNA-IgG at the age of five years old when the child presented with signs and symptoms suggestive of acute infectious mononucleosis. After 14 months, serological parameters were: positive VCA-IgG, EBNA-IgG and negative VCA-IgM. This serological pattern has been maintained since then even during episodes suggestive of EBV reactivation. PCR amplified a specific DNA fragment from the EBV gp220 (detection limit of 100 viral copies). All twenty-four whole blood samples yielded positive results by PCR, while 12 out of 24 serum samples were positive. We aimed at analyzing whether detection of EBV-DNA in serum samples by PCR was associated with overt disease as stated by the need of antiviral treatment and hospitalization. Statistical analysis showed agreement between the two parameters evidenced by the Kappa test (value 0.750; p < 0.001). We concluded that detection of EBV-DNA in serum samples of immunosuppressed patients might be used as a laboratory marker of active EBV disease when a Real-Time PCR or another quantitative method is not available.

The epidemiologic profile and prevalence of cardiopathy in Trypanosoma cruzi infected blood donor candidates, Londrina, Paraná, Brazil

To describe the epidemiologic profile and prevalence of cardiopathy in 163 Trypanosoma cruzi serum positive blood donor candidates, a descriptive study was carried out between August, 1996 and November, 1997 at the Londrina State University Chagas Disease Outpatient Clinic. The profile found was: young, average age 42.95 ± 8.62 years; male (65%); Caucasian (84%); low level of schooling; low family income; agricultural worker (26%); born in the state of Paraná (67%); from rural areas (85%); migrated to the city (85%); and the vector as the main mechanism of transmission. During the clinical characterization a chronic cardiac form was found in 38% of the patients and classified as cardiac suggestive form in 21% and little suggestive of Chagas disease in 17%. No significant difference was found among age group distribution, sex and the presence of cardiac symptoms in patients with or without cardiopathy. This study emphasizes the importance of expanding medical services to areas with a greater prevalence of infected individuals, in a hierarchical manner and aiming at decentralization.

Seroprevalence for hepatitis E virus (HEV) infection among volunteer blood donors of the Regional Blood Bank of Londrina, State of Paraná , Brazil

A cross-sectional study was carried out among 996 volunteer blood donors enrolled from May 1999 to December 1999 to determine the seroprevalence of hepatitis E virus (HEV) infection among volunteer blood donors of the Regional Blood Bank of Londrina, State of Paraná, Brazil, and to evaluate whether the rate of seroprevalence of IgG anti-HEV antibodies is associated with sociodemographic variables and with seropositivity for hepatitis A virus (HAV) infection. All participants answered the questionnaire regarding the sociodemographic characterisitcs. Serum samples were tested for IgG antibodies to HEV (anti-HEV) by an enzyme linked immunoassay (ELISA). All serum samples positive for anti-HEV IgG and 237 serum samples negative for anti-HEV were also assayed for IgG anti-HAV antibodies by ELISA. Anti-HEV IgG was confirmed in 23/996 samples, resulting in a seroprevalence of 2.3% for HEV infection, similar to previous results obtained in developed countries. No significant association was found between the presence of anti-HEV IgG antibodies and the sociodemographic variables including gender, age, educational level, rural or urban areas, source of water, and sewer system (p > 0.05). Also, no association with seropositivity for anti-HAV IgG antibodies was observed (p > 0.05). Although this study revealed a low seroprevalence of HEV infection in the population evaluated, the results showed that this virus is circulating among the population from Londrina, South Brazil, and point out the need of further studies to define the clinical and epidemiological importance of HEV infection and to identify additional risk factors involved in the epidemiology and pathogenesis of this infection in this population.

Evaluation of the human immunodeficiency virus type 1 and 2 antibodies detection in dried whole blood spots (dbs) samples

Human Immunodeficiency Vírus Type 1 and 2 antibodies detection was performed in 457 dried whole blood spots samples (S&S 903). Q-Preven HIV 1+2 was the screening test used. The results were compared with the gold standard serum tests by ELISA (Cobas Core e Axsym HIV1/2 gO) and imunofluorescence was the definitive confirmatory test. The samples were obtained from the Hospital Nossa Senhora da Conceição in Porto Alegre, RS - Brazil, through whole blood transfer to filter paper card and sent to Caxias do Sul, RS - Brazil where the tests were performed. The dried whole blood spot stability was evaluated with two different panels. The first one was composed of five negative and five positive samples stored at room temperature, 4 ºC, -20 ºC and -70 ºC, while the second was composed of two negative and three positive samples stored at 37 ºC (humidity <50%). Each sample was screened every week for six weeks. These measurement results didn't show variation during the study period. The detected sensibility was 100%, specificity was 99.6%, the positive predictive value was 99.5% and negative predictive values were 100%. The results demonstrated high performance characteristics, opening a new perspective of dried whole blood spot utilization in HIV screening diagnosis.

Confirming the presence of HTLV-1 infection and the absence of HTLV-2 in blood donors from Arequipa, Peru

Epidemiological studies conducted in Peru disclosed HTLV-1 to be prevalent in different ethnic groups, and found HTLV-2 in some Amazonian Indians and in men who have sex with men. No data concerning HTLV-1/2 infection in blood donors from Arequipa, a highlands region in southern Peru, is available. We searched for the presence of HTLV-1 and HTLV-2 antibodies in 2,732 serum samples obtained from blood donors from this geographic area. HTLV-1/2-specific antibodies were detected using an enzyme-linked immunosorbent assay (ELISA) and were confirmed by Western blot (WB). Reactive sera had their blood bags discarded from donation, and the demographic characteristics of the donors were analyzed. Thirty-five sera (1.2%) were HTLV seroreactive by ELISA, and 25 were confirmed HTLV-1-positive by WB. One serum disclosed HTLV-positivity, and the remaining nine serum samples showed indeterminate results by WB; three of which had an HTLV-1 indeterminate Gag profile. The median age of HTLV-positive individuals was 34.6 years; 27 were male and eight were female. All individuals were from southern Peru: 27 from Arequipa, five from Puno, and three from Cuzco. HTLV co-positivity with hepatitis B (five sera) and syphilis (one serum) were detected. Previous transfusion and tattooing were observed in two and one individuals, respectively. No serum was positive for HTLV/HIV co-infection. This study confirmed, for the first time, HTLV-1 infection and the absence of HTLV-2 infection in blood donors from Arequipa, Peru and suggests vertical transmission as the major route of HTLV-1 transmission and acquisition in this geographic region.

Differences in exoenzyme production and adherence ability of Candida spp. isolates from catheter, blood and oral cavity

Phospholipase and proteinase production and the ability of adhesion to buccal epithelial cells (BEC) of 112 Candida isolates originated from oral cavity of HIV infected patients and from blood and catheter of intensive care unit patients were investigated. The proteinase production was detected by inoculation into bovine serum albumin (BSA) agar and the phospholipase activity was performed using egg yolk emulsion. A yeast suspension of each test strain was incubated with buccal epithelial cells and the number of adherence yeast to epithelial cells was counted. A percentage of 88.1% and 55.9% of Candida albicans and 69.8% and 37.7% of non-albicans Candida isolates produced proteinase and phospholipase, respectively. Non-albicans Candida isolated from catheter were more proteolytic than C. albicans isolates. Blood isolates were more proteolytic than catheter and oral cavity isolates while oral cavity isolates produced more phospholipase than those from blood and catheter. C. albicans isolates from oral cavity and from catheter were more adherent to BEC than non-albicans Candida isolates, but the adhesion was not different among the three sources analyzed. The results indicated differences in the production of phospholipase and proteinase and in the ability of adhesion to BEC among Candida spp. isolates from different sources. This study suggests that the pathogenicity of Candida can be correlated with the infected site.

Canine visceral leishmaniasis: study of methods for the detection of IgG in serum and eluate samples

The Brazilian Ministry of Health recommends the culling and euthanasia of dogs with a positive serological test for canine visceral leishmaniasis (CVL). In the Municipality of Rio de Janeiro, the technique used for the diagnosis of CVL is the indirect fluorescent antibody test (IFAT), using blood samples eluted on filter paper (eluate). A dog survey was conducted over a period of one year in the region of Carapiá, in order to evaluate the diagnosis of CVL in this region. All animals underwent clinical examination, and blood samples (serum and eluate) were collected for analysis by enzyme immunoassay (ELISA) and IFAT. A skin biopsy was obtained for parasitological examination (culture). A total of 305 animals were studied and Leishmania chagasi was isolated from nine animals. Sensitivity and specificity were 100% and 96.6% for ELISA, respectively, 100% and 65.5% for IFAT (cut-off at a 1:40 dilution), 100% and 83.4% for IFAT (cut-off at a 1:80 dilution), and 22.2% and 97.0% for eluate IFAT. In conclusion, ELISA was the best tool for the diagnosis of CVL among the serological techniques tested. The present results suggest the need for a better evaluation of filter paper IFAT as the only diagnostic method for CVL in the Municipality of Rio de Janeiro.

Seroepidemiological study of human cysticercosis with blood samples collected on filter paper, in Lages, State of Santa Catarina, Brazil, 2004-2005

INTRODUCTION: Human serofrequency of antibodies against Taenia solium antigens was determined and risk factors for cysticercosis transmission were identified. METHODS: Individuals (n=878) from periurban and rural locations of Lages, SC, were interviewed to gather demographic, sanitary and health information. Interviews and blood sample collections by finger prick on Whatman filter paper were performed from August 2004 to May 2005. Observation determined that 850 samples were suitable for analysis and were tested by ELISA using vesicular fluid of Taenia crassiceps heterologous antigen. To ensure the reliability of the results, 77 samples of the dried blood were matched with sera. The reactive samples were submitted to a serum confirmatory immunoblot (IB) test using purified Taenia crassiceps glycoproteins. RESULTS: The ELISA results for the dried blood and serum samples were statistically consistent. ELISA was positive in 186 (21.9%) out of 850 individuals. A group of 213 individuals were asked to collect vein blood for IB (186 with positive result in ELISA and 27 with inappropriate whole blood samples) and 130 attended the request. The IB was positive in 29 (3.4%) out of 850 individuals. A significant correlation (p = 0.0364) was determined among individuals who tested positive in the IB assay who practiced both pig rearing and kitchen gardening. CONCLUSIONS: ELISA with dried blood eluted from filter paper was suitable for cysticercosis population surveys. In Lages, human infection was associated with pig rearing and kitchen gardening. The prevalence index was compatible with other Latin American endemic areas.

Molecular detection of Treponema pallidum sp. pallidum in blood samples of VDRL-seroreactive women with lethal pregnancy outcomes: a retrospective observational study in northern Brazil

INTRODUCTION: Although control measures of maternal and congenital syphilis are available in Brazil, difficulties exist within the healthcare network in providing a laboratory diagnosis of the infection during the prenatal period. The objective of this study was to confirm the presence of Treponema pallidum by PCR in women with positive VDRL serology and lethal pregnancy outcomes, i.e., abortion, stillbirth and neonatal death. METHODS: A retrospective study was conducted on VDRLseroreactive women with lethal pregnancy outcomes admitted to the Fundação Santa Casa de Misericórdia do Pará (FSCM-PA) between January and July 2004. Serum samples and DNA from whole blood were obtained at the time of screening by the VDRL test. These samples were analyzed by IgG ELISA, IgM FTA-Abs and simple PCR (polA). RESULTS: During the study period, 0.7% (36/4,912) of women with lethal pregnancy outcomes presented a positive VDRL test. The polAgene was amplified in 72.7% (24/33) of these women, with 55.6% (20/36) and 94.4% (34/36) presenting IgM and IgG antibodies against T. pallidum, respectively. Comparison of these results showed a significant difference, with agreement between the PCR and IgM FTA-Abs results, suggesting that maternal syphilis was an active infection. No basic cause of death of the conceptus was reported in 97.2% (35/36) of cases. Among women who were submitted to the VDRL test during the prenatal period, only four of the nine seroreactive patients underwent treatment. CONCLUSIONS: The high frequency of syphilis in the group studied indicates the fragility of the service of infection diagnosis, treatment and monitoring, compromising epidemiological control.

Prevalence of Chagas disease in blood donors at the Uberaba Regional Blood Center, Brazil, from 1995 to 2009

INTRODUCTION: A retrospective study was conducted to assess the occurrence of blood donations that were ineligible due to Chagas disease infection from 1995 to 2009 at the Uberaba Regional Blood Center (HRU), Brazil, verify the tendency of this ineligibility, and describe the epidemiologic profile of the donors. METHODS: Retrospective studies of serological ineligibility due to Chagas disease, statistical analysis by means of the chi-square test and odds ratio, study of the tendencies using a dispersion graph and the linear correlation coefficient (r) were performed. RESULTS: In the period under study, a 0.2% serum prevalence of ineligibility due to Chagas disease was found, with a significant drop in ineligible donations from 2001 to 2009. Among the serum positive-donors, there was a significant predominance among those aged 30 years or above and non-single individuals. CONCLUSIONS: The results show a rate of occurrence that is lower than that described in literature, as well as a progressive drop during the 15 years under assessment. Such results are a consequence of systematic combat of the vector since the 70s and the progressive and consistent increase of returning donors, resulting in a drop of the contamination risk factor by means of blood transfusion and in the improvement of the quality of hemotherapy practices in the HRU.

Sodium serum levels in hypoalbuminemic adults at general medical wards

Hypoalbuminemia may cause interstitial edema and hemodilution, which we hypothesized may influence serum sodium levels. Our purpose was to compare serum sodium levels of hospitalized adults with or without hypoalbuminemia. All sodium and albumin serum levels of 142 adults hospitalized at general medical wards over a six-month period were searched at a University Hospital mainframe computer. Relevant laboratory data and clinical details were also registered. Hypoalbuminemia was defined by serum albumin concentration < 3.3 g/dl Fisher, Mann-Whitney, and Student's t tests were applied to compare groups with or without hypoalbuminemia. Ninety-nine patients, classified as hypoalbuminemic, had lower blood hemoglobin (10.68 ± 2.62 vs. 13.54 ± 2.41), and sodium (135.1 ± 6.44 vs. 139.9 ± 4.76mEq/l) and albumin (2.74 ± 0.35 vs. 3.58 ± 0.28g/dl) serum levels than non-hypoalbuminemic (n=43). Pearson's coefficient showed a significant direct correlation between albumin and sodium serum levels (r=0.40) and between serum albumin and blood hemoglobin concentration (r=0.46). Our results suggest that hypoalbuminemic adults have lower serum sodium levels than those without hypoalbuminemia, a phenomenon that may be at least partially attributed to body water retention associated with acute phase response syndrome.

Effects of a novel fermented soy product on the serum lipids of hypercholesterolemic rabbits

OBJECTIVE: To assess the effect of a new feed soy product fermented by Enterococcus faecium and Lactobacillus jugurti on the serum lipid levels of rabbits with induced hypercholesterolemia. METHODS: Thirty-two rabbits were divided into 4 groups as follows: 1) control (C); 2) hypercholesterolemic (H); 3) hypercholesterolemic + fermented product (HPF); and 4) control + fermented product (CPF). The H and HPF groups were fed with a diet with 0.15% (p/p) cholesterol in the first 15 days. C and CPF groups received regular food preparation. The HPF and CPF groups received 10 mL daily of the fermented 30 days. Blood samples were drawn at the beginning of the study and at the 15th and 30th days. Concentrations of total cholesterol, HDL-cholesterol, and triglycerides were analyzed. RESULTS: After 15 days, the HPF group showed a total cholesterol concentration lower (18.4%) than that of the H group (p=0.05), but this difference disappeared after 30 days. No change was observed in total cholesterol levels of C and CPF groups. After 15 days, the HDL-cholesterol was higher (17.8%) in the HPF group, but the triglyceride levels remained unchanged in all groups during the same period of time. CONCLUSION: The soy fermented product caused an 18.4% reduction in total cholesterol and a 17.8% increase in the HDL-fraction. It may, therefore, be a possible coadjutor in the treatment of hypercholesterolemia.