Exploration of an Efficient Simultaneous Molecular Detection Method of HIV, HCV, and Syphilis from a Single Dried Blood Spot / 生物医学与环境科学（英文）

Objective@#The aim of the present study was to evaluate the performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one dried blood spot (DBS) as an alternative sample to plasma.@*Method@#A total of 571 paired DBS/plasma samples were collected from men who have sex with men (MSM) and injection drug users (IDUs), and serological and molecular assays were performed. Using plasma results as the reference standard, the performance of DBS tests for HIV-1 RNA, HIV-1 DNA, and HCV RNA was evaluated. Pearson's correlation coefficients and Bland-Altman analysis were performed to assess the correlation and concordance between DBS and plasma.@*Results@#Among paired plasma/DBS samples with detectable HIV-1 RNA and HCV RNA, five samples (5/32) were not detectable in DBS, while measurable HIV-1 RNA levels were present in plasma (1.44 to 3.99 log @*Conclusion@#The performance of the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA using one DBS was acceptable. DBS, as an alternative sample to plasma, may be a viable option for the simultaneous detection of HIV-1 RNA, HIV-1 DNA, and HCV RNA in resource-limited settings or for individuals living in areas that are difficult to access.

Prevalence of HCV infection in a prison population of the greater Florianópolis area

Abstract INTRODUCTION The present study aimed to estimate the prevalence of Hepatitis C virus (HCV) infection in a prison population. METHODS: A total of 147 individuals were interviewed and subjected to venipuncture for collection of blood sample. The study population consisted of male individuals who attended the health unit of the state penitentiary of Florianópolis. RESULTS: The prevalence of HCV infection was 5.4%. Regarding behavioral variables, 95 (64.6%, p<0.0507) subjects reported consuming alcohol and 7 (4.8%, p<0.0476) reported having already used injectable drugs. CONCLUSIONS: The prevalence of HCV infection in the studied population was higher than that in the general populations.

One-step real-time PCR assay for detection and quantification of RNA HCV to monitor patients under treatment in Brazil

ABSTRACT The Brazilian Public Health Service provides freely αPEG-IFN to treat patients infected with HCV. The primary goal of HCV therapy is the long-term elimination of HCV from the blood to reduce the risk of HCV associated complications and death. Patient viremia affects the treatment duration and response, thus influencing clinical decisions. We developed a high-throughput method to perform the quantification of RNA hepatitis C virus (HCV) virus load in plasma samples to monitor patients under treatment. The method is based on a duplex detection, in a one-step real-time RT-PCR assay and it has been validated according to the rules established by the official Brazilian regulatory agency (ANVISA). This new method was compared to a commercial kit (Cobas/Taqman HCV Test v2.0 - Roche), showing virus load results with significant correlation between them (p= 0,012) using commercial and clinical panels. In addition, 611 samples from patients treated with peguilated alfa-interferon (αPEG-IFN) from different regions of Brazil were analyzed. Our one-step real-time RT-PCR assay demonstrated good performance in viral load measurement and in treatment course monitoring, with acceptable sensitivity and specificity values.

Hepatitis C viral load in HCV-monoinfected and HCV/HIV-1-, HCV/HTLV-1/-2-, and HCV/HIV/HTLV-1/-2-co-infected patients from São Paulo, Brazil

ABSTRACT Co-infections of hepatitis C virus (HCV) and either human immunodeficiency virus type 1 (HIV-1), human T-cell lymphotropic virus type 1 (HTLV-1) or type 2 (HTLV-2) have been described as having an impact on HCV viremia and subsequent disease progression. HCV load in serum samples from 622 patients (343 males, 279 females; median age 50.8 years) from São Paulo/southeast Brazil was analyzed using the Abbott Real Time HCV assay (Abbott Molecular Inc., IL, USA). Samples were obtained from HCV-monoinfected (n = 548), HCV/HIV-1- (n = 41), HCV/HTLV-1- (n = 16), HCV/HTLV-2- (n = 8), HCV/HIV/HTLV-1- (n = 4), and HCV/HIV/HTLV-2-co-infected (n = 5) patients, and results were compared among the groups and according to sex. The median HCV load in HCV-monoinfected patients was 5.23 log10 IU/mL and 0.31 log10 higher in men than in women. Increases in viral load of 0.51 log10, 0.54 log10, and 1.43 log10 IU/mL were detected in HCV/HIV-1-, HCV/HTLV-1- and HCV/HIV/HTLV-1-co-infected individuals, respectively, compared with HCV-monoinfected counterparts. In contrast, compared to HCV/HIV co-infected patients, HCV/HTLV-2-co-infected patients had an HCV load of 5.0 log10 IU/mL, whereas HCV/HIV/HTLV-2-co-infected patients had a median load 0.37 log10 IU/mL lower. Significant differences in HCV loads were detected, with males and HCV/HIV-1- and HCV/HIV/HTLV-1-co-infected patients presenting the highest values. Conversely, females and HCV/HTLV-2-co-infected patients exhibited lower HCV loads. Overall, HCV viremia is increased in HIV and/or HTLV-1-co-infection and decreased in HTLV-2 co-infection.

Asociación del antecedente transfusional antes de 1994 con hepatitis vírica tipo C en mujeres / Linking hepatitis C virus infection to pre-1994 blood transfusions in female patients

INTRODUCCIÓN La mayor parte de las transfusiones se llevan a cabo en mujeres. La introducción en los bancos de sangre de las técnicas serológicas disminuyó la incidencia de infección por virus de hepatitis C después de una transfusión. En México, las pacientes que se transfundieron antes de 1994 están en riesgo de presentar una infección por virus de hepatitis C. El objetivo de este estudio fue medir la asociación entre el antecedente transfusional antes de 1994 e infección por virus de hepatitis C en mujeres atendidas en la zona metropolitana de Guadalajara, México. MÉTODOS Estudio observacional, analítico, de casos y controles, en el que se incluyeron mujeres sanas y mujeres con infección por hepatitis vírica tipo C, en las cuales se determinó el antecedente transfusional antes y después de 1994. El grupo de casos lo conforman 150 mujeres con diagnóstico serológico y confirmatorio de hepatitis C, en tanto el grupo control son 150 mujeres sanas con serología negativa. RESULTADOS Se encontró un odds ratio de 9,07 (intervalo de confianza 95% 5,37 ­ 15,3; p<0,001), una proporción de casos expuestos de 0,72, de controles expuestos de 0,22, una fracción atribuible poblacional de 0,64 (intervalo de confianza 0,53 ­ 0,73) y una fracción atribuible en expuestos de 0,88 (intervalo de confianza 0,81 ­ 0,93). CONCLUSIONES En las mujeres, el haber tenido una transfusión antes de 1994 en la zona metropolitana de Guadalajara, representa un riesgo 9,07 veces mayor de infección por virus de la hepatitis C que no tener antecedente transfusional en esa fecha.

INTRODUCTION Most blood transfusions occur in female patients. The introduction of serologic screening practices by blood banks reduced the transfusion-related rate of infection with hepatitis C virus (HCV). In Mexico patients with pre-1994 transfusion history are at high risk of being detected with HCV infection. We aimed at establishing an interrelationship between two variables: pre-1994 transfusion history and rate of infection in women treated in the Guadalajara Metropolitan Area hospitals, in Mexico. METHODS Analytical observational case-control study which included both non-infected women and patients diagnosed with hepatitis C virus infection, in whom the pre-1994 transfusion history was determined. The cases were 150 women with confirmed hepatitis C virus serologic diagnosis. The controls were 150 women whose hepatitis C virus-detection serologic tests had yielded negative results. RESULTS An odds ratio of 9.07 (95% CI: 5.37 ­ 15.3; p< 0.001) was found where the rate of infection for the case group was 0.72 while the control group had a ratio of 0.22; population attributable risk (PAR) was 0.64 (95% CI: 0.53 ­ 0.73), while etiologic fraction was 0.88 (95% CI: 0.81 ­ 0.93). CONCLUSIONS Among women, having been exposed to pre-1994 blood transfusion means a risk 9.07 times higher than not being exposed to blood transfusion in the same time frame.

Seroprevalence of HCV markers among HIV infected patients from Curitiba and metropolitan region / Soroprevalência de marcadores do vírus da hepatite C (HCV) em pacientes infectados com HIV de Curitiba e Região Metropolitana

SUMMARY Objective: to determine the prevalence and epidemiological factors associated with hepatitis (HCV) coinfection in human immunodeficiency virus (HIV) patients from Curitiba and the metropolitan region. Methods: a study with 303 HIV+ patients, mean age 41.2 years (18-73); 50.5% men, followed at the Hospital de Clínicas, Universidade Federal do Paraná, between April 2008 and March 2009. Clinical and epidemiological data were obtained through questionnaires and retrospective analysis of medical records. Anti-HCV antibodies were detected by chemiluminescence immunoassay. Results: a total of 12.9% of HIV+ patients were positive for anti-HCV antibodies, 64.1% were men and 35.9% women, with mean age of 44.5 years (24-66). The frequency of HCV among men was 16.7% and among women 9.1% (p=0.06). HCV prevalence was associated to HIV infection when compared to the general population (p<10-6, OR=100.4; 95CI=13.7-734.9). The parenteral route of transmission was the most frequent among coinfected patients (46.1%), and the sexual transmission among HIV+/HCV- (71.8%) (p=0.02, OR=0.2; 95CI=0.1-0.7). The frequency of intravenous drug users was higher among the coinfected patients (61.5%) compared to the non coinfected (12.6%) (p<10-6, OR=11.1; 95CI=4.5-27.7). Conclusion: the prevalence of coinfection with HCV in HIV+ patients is 12.9%, 88 times higher than in the general population in Curitiba. The most frequent route of transmission in the coinfected patients is parenteral, but the sexual route is also representative (34.6%).

RESUMO Soroprevalência de marcadores do vírus da hepatite C (HCV) em pacientes infectados com HIV de Curitiba e Região Metropolitana Objetivo: verificar a prevalência e caracterizar fatores epidemiológicos associados à coinfecção por HCV em pacientes HIV+ de Curitiba e Região Metropolitana. Métodos: estudo envolvendo 303 pacientes HIV+, com idade média de 41,2 anos (18-73); 50,5% homens; acompanhados no Hospital de Clínicas da Universidade Federal do Paraná, entre abril de 2008 e março de 2009. Os dados clínico-epidemiológicos foram obtidos por meio de questionários e análise retrospectiva dos prontuários. Os anticorpos anti-HCV foram detectados por ensaio imunoenzimático quimioluminescente. Resultados: dos pacientes HIV+, 12,9% apresentaram sorologia positiva para o HCV, sendo 64,1% homens e 35,9% mulheres, com idade média de 44,5 anos (24-66). A frequência nos homens foi de 16,7%, e nas mulheres, 9,1% (p=0,06). A prevalência do HCV foi significativamente associada à infecção por HIV quando comparada à população geral (p<10-6, OR=100,4; IC95%=13,7-734,9). A via de transmissão parenteral foi a mais frequente entre os coinfectados (46,1%), e a sexual, a mais frequente entre os não coinfectados (71,8%) (p=0,02, OR=0,2; IC95%=0,1-0,7). A frequência de usuários de drogas injetáveis foi maior entre os coinfectados (61,5%) do que entre os não coinfectados (12,6%) (p<10-6, OR=11,1; IC95%=4,5-27,7). Conclusões: a prevalência da infecção por HCV nos pacientes HIV+ é de 12,9%, 88 vezes maior que a infecção na população geral de Curitiba. A via de transmissão mais frequente entre os coinfectados foi a parenteral, porém, a via sexual também é representativa para a transmissão do HCV (34,6%).

A complete molecular biology assay for hepatitis C virus detection, quantification and genotyping

Introduction Molecular biology procedures to detect, genotype and quantify hepatitis C virus (HCV) RNA in clinical samples have been extensively described. Routine commercial methods for each specific purpose (detection, quantification and genotyping) are also available, all of which are typically based on polymerase chain reaction (PCR) targeting the HCV 5′ untranslated region (5′UTR). This study was performed to develop and validate a complete serial laboratory assay that combines real-time nested reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) techniques for the complete molecular analysis of HCV (detection, genotyping and viral load) in clinical samples. Methods Published HCV sequences were compared to select specific primers, probe and restriction enzyme sites. An original real-time nested RT-PCR-RFLP assay was then developed and validated to detect, genotype and quantify HCV in plasma samples. Results The real-time nested RT-PCR data were linear and reproducible for HCV analysis in clinical samples. High correlations (> 0.97) were observed between samples with different viral loads and the corresponding read cycle (Ct - Cycle threshold), and this part of the assay had a wide dynamic range of analysis. Additionally, HCV genotypes 1, 2 and 3 were successfully distinguished using the RFLP method. Conclusions A complete serial molecular assay was developed and validated for HCV detection, quantification and genotyping. .

Optimization of one-step real-time PCR for the X-tail target of HCV as a diagnostic test

Infection with hepatitis C virus (HCV) is a global public health issue. The bloodborne nature of HCV transmission poses a substantial risk to healthcare workers, due to occupational exposure to needlestick injuries and blood and other body fluids containing the virus. Undiagnosed HCV infection, including in healthcare workers, represents a growing problem worldwide as the infected population ages, and HCV-related mortality and morbidity is expected to rise substantially over the coming decades. Consequently, diagnostic tests for HCV play an important role in this scenario. The aim of this study was to standardize a one-step RT-PCR assay for detection of HCV. The test demonstrated reproducibility, sensibility (100%), and the limit of detection was set at 100IU/mL. Our study indicates that this assay can be used as a diagnostic tool to follow up healthcare workers after occupational exposure

simple and rapid method for the detection of HIV-1/HCV in co-infected patients

Due to some limitations of serological methods in diagnosis of patients infected with HIV-1 [human immunodeficiency virus] and HCV [hepatitis C virus], it is profoundly important to use molecular methods for the detecting of these infectious agents. However, the most significant problems are the exorbitant cost of these methods and the need of a thermocycler which is an expensive instrument. The current research recruits a multiplex nucleic acid sequence base amplification [NASBA] in order to simultaneously detect HIV-1 and HCV genomes in patients' plasma samples. Sensitivity and specificity of this method have been evaluated using clinical samples. A multiplex NASBA assay for simultaneous detection of HCV and HIV-1 by the use of specific primers were designed and validated. A well-conserved region in the HIV-1 pol gene and 5'-NCR of HCV genome were used. A total of 40 samples of HIV-1 [20 samples] and HCV [20 samples] were used in the NASBA assay. The specificity and sensitivity of the assay were evaluated. Our results have demonstrated that the primers used in the assay had no interrelation with each other and other possible interfering agents in the assay. The analytical sensitivity of the assay for both HIV-1 and HCV was determined to be 1000 copies/mL and the clinical sensitivity and specificity were 93.3% and 100%, respectively. By exploiting this multiplex NASBA assay, it is possible to detect HIV-1 and HCV infection/co-infection in patients' plasma with a suitable sensitivity and specificity. Furthermore, due to its simplicity and multiplexing feature, it could be used in limited access laboratories in a cost-effective manner.

Occult hepatitis B virus infection among hepatitis C virus seropositive and seronegative blood donors in Alexandria, Egypt

Background and aims: Occult hepatitis B virus infection [OBI, serum hepatitis B surface antigen negative but hepatitis B virus DNA positive] is an emerging problem in the safety of blood transfusion. The introduction of hepatitis B surface antigen in the screening panel for blood bank donors has substantially reduced, but not entirely eliminated, the risk of hepatitis B virus infection. It has been suggested that infection with hepatitis C virus may inhibit hepatitis B virus replication. Therefore, this study aimed at estimating the prevalence and risk factors for OBI among blood donors and determining its relationship with hepatitis C virus infection

Subjects and methods: Presence of hepatitis B virus DNA and hepatitis C virus RNA was investigated for among 508 hepatitis B surface antigen-negative blood donors in Alexandria, Egypt. Half of the donors were identified as hepatitis C virus antibody reactive

Results: OBI was detected in 21 donors [4.1%] from the studied population: eight were among hepatitis C virus antibody positive donors [3.2%], among whom seven [33.3%] had hepatitis C virus RNA in their serum, and 13 [5.1%] were among hepatitis C virus antibody negative donors, with no statistically significant difference. The only significant risk factor for OBI among the studied blood donors was visiting local barbers

Conclusion and recommendations: OBI is a considerable risk in blood banks, making screening for hepatitis B virus infection only on the basis of surface antigenemia insufficient

Hepatitis C virus quantification in serum and saliva of HCV-infected patients

The hepatitis C virus (HCV) can be detected in blood and other bodily fluids, such as saliva, semen and gastric juices. The aim of this study was to compare the HCV viral loads in the serum and saliva of infected patients. Twenty-nine patients with detectable HCV RNA in their serum and saliva were included in this study. The HCV viral loads were determined through quantitative real-time polymerase chain reactions. The median viral RNA levels were 5.78 log10 copies in the serum and 3.32 log10 copies in the saliva. We observed that the salivary HCV viral load was significantly lower than the viral load in the serum. Further studies are required to understand the role of saliva in the diagnosis, management and potential transmission of HCV.

Individual donor nucleic acid testing for blood safety against HIV-1 and hepatitis B and C viruses in a tertiary care hospital.

Background. In July 2010, we started universal individual donor nucleic acid testing (ID-NAT) at our blood bank. This test simultaneously detects human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV) and hepatitis C virus (HCV) in samples of donor blood. We continued to do the enzymelinked immunosorbent assay (ELISA) test for these agents, as per the guidelines of the Drug Controller General of India. We assessed the impact of ID-NAT in preventing transfusionassociated transmission of viruses. Methods. We used fourth generation ELISA to screen blood samples of all voluntary and replacement blood donors. ID-NAT was done by transcription-mediated amplification (TMA). Results. Of the 18 356 donors, ID-NAT could not be performed on 2 samples which were inadequate. Of the 18 354 donors tested by both ID-NAT and fourth generation ELISA, 7 were found to be NAT-positive but ELISA-negative (NAT yield) for HBV and HCV. The prevalence of NAT yield cases among routine donors was 1 in 2622 donations tested (0.038%). Since we supply blood as components (packed red cells, fresh frozen plasma and platelet concentrate), these 7 units of blood would have yielded 21 components and hence 21 patients could have been infected with HBV and HCV viruses. Conclusion. In the vast majority of blood units tested, the results of ELISA and ID-NAT for HIV-1, HBV and HCV were concordant. ID-NAT did detect the presence of viruses missed by ELISA in some blood units. It widespread use in blood banks would ensure safer blood transfusion.

Hepatitis C virus infection in hemodialysis patients in Maracaibo, Venezuela / Infecção pelo vírus da Hepatite C em pacientes em hemodiálise em Maracaibo, Venezuela

Over a two year period, the incidence of hepatitis C virus (HCV) infection was evaluated in 29 hemodialysis patients, aged between 15 and 75 years (mean ± SD: 45 ± 39.5 years), from the University Hospital Hemodyalisis Unit, Maracaibo, Zulia State, Venezuela. Anti-HCV antibodies were determined using a fourth generation ELISA (Innotest HCV Ab IV) kit and positive blood samples were tested using a recombinant assay kit (Inno-LIA HCV Ab III), both kits from Innogenetics N.V., Belgium. The findings indicate a lack of HCV seroconversion in the hemodialysis patients over the study period, confirmed by the recombinant assay. Risk factors for HCV infection were 0.3270 (95 percent confidence interval: 0.01323-8.080) in patients undergoing hemodialysis. The findings suggest a lack of significant sources for HCV infection due to the preventive measures to avoid its transmission in the hemodialysis unit.

Durante período de 2 anos, estudamos a incidência da infecção pelo vírus da hepatite C (VHC) em 29 pacientes em tratamento de diálise, com idades entre 15 e 75 anos (c ± DS; 45 ± 39,5 anos), procedentes da unidade de hemodiálise do Hospital Universitário de Maracaibo, Estado Zulia, Venezuela. Para a detecção dos anticorpos contra o VHC (anti-VHC) utilizamos a técnica de imunoensaio enzimático (ELISA, Innotest HCV Ab IV) e em amostras reativas por ELISA, utilizamos o método de immunoblot recombinante de terceira geração (Inno-LIA HCV Ab III), ambos da casa comercial Innogenetics N.V., Bélgica. Os resultados demonstram ausência de soroconversão ao VHC nos pacientes hemodializados durante o período estudado, o que foi confirmado pelo método de imunoblot recombinante. Os fatores de risco ao VHC foram 0,327 (95 por cento CI: 0,01323 - 8,080) nos pacientes submetidos ao tratamento de diálise. Nossos resultados sugerem ausência de fontes de infecção neste centro de hemodiálise e que as medidas universais de controle de infecção são cumpridas.

[Multiplex RT-PCR assay for detection of Co-infection HIV-1 and HCV viruses in plasma samples]

HIV-1 and HCV infections especially in co-infected forms are among the most important infections transferred during blood transfusion. The screening of the blood products is valuable for preventing the transmission of infections. The aim of this study was to evaluate multiplex RT-PCR assay for detection of Co-infection HIV-1 and HCV Viruses in plasma samples. This laboratory study was done to evaluate the use of multiplex RT-PCR assay for simultaneous detection of HIV-1 and HCV genomes in plasma samples. The amplified genomes were detectable in 3% agarose gel base on difference in the numbers of nucleotides. The sensitivity and specificity of this assay was determined on healthy and infected subjects whome simultanously exhibit HIV-1 and HCV co-infection using plasma samples. The specificity results showed that the primers used in this assay have no interaction with each other and other possible interfering agents. The clinical sensitivity and specificity of the assay has been considered as 90% and 100%, respectively. Multiplex RT-PCR can be used for screening of blood donors due to high sensivity and specificity

Frequency of hepatitis C virus infection and estimation of serum alanine aminotransferase in HCV positive patients

Hepatitis C virus [HCV] has emerged as a leading cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. Hepatitis C is rapidly emerging as a major health problem in developing countries including Pakistan. Serum Alanine Aminotransferase [ALT] is the most frequently utilized screening test in routine evaluation of liver damage. This study was conducted to determine the seroprevalence of hepatitis C virus infection and its association with serum alanine aminotransferase. Determination of the seroprevalence of hepatitis C virus infection and its association with serum alanine aminotransferase in patients at social security hospital, Islamabad. A cross sectional study was conducted at clinical laboratory of social security hospital Islamabad, from May 2009 to October 2009. All samples referred to social security hospital for hepatitis profile were included in the study. All patients were screened serologically for hepatitis C virus antibodies and serum alanine aminotransferase was determined in selected HCV positive patients only. Out of 1006 blood samples 146 were positive for anti-HCV antibodies with an overall positivity of 14.5%. Out of these 55% cases were males and 45% were females. Seroprevalence of HCV was found to be 8% in the age group of <20 years, and almost equal i.e. 46% and 49% in 20-40 years and >40 years age groups respectively. Serum ALT was determined in total 83 patients only: they showed levels less than 30U/L in 25% patients, 30-45U/L in 15% patients, 46-60U/L in 19% patients, 61-100U/L in 29% patients and more than 100U/L in 12% patients. The present study revealed that prevalence of HCV was 14.5% and ALT levels were found to be more than 45U/L in 75% of the anti HCV positive cases, showing the significance of this biochemical marker as diagnostic tool in such patients

Magnetic bead technology for viral RNA extraction from serum in blood bank screening

Nucleic acid amplification testing (NAT) was recently recommended by Brazilian legislation and has been implemented at some blood banks in the city of São Paulo, Brazil, in an attempt to reduce blood-born transmission of human immunodeficiency virus (HIV) and hepatitis C virus. OBJECTIVE: Manual magnetic particle-based extraction methods for HIV and HCV viral nucleic acids were evaluated in combination with detection by reverse transcriptase - polymerase chain reaction (RT-PCR) one-step. METHODS: Blood donor samples were collected from January 2010 to September 2010, and minipools of them were submitted to testing. ELISA was used for the analysis of anti-HCV/HIV antibodies. Detection and amplification of viral RNA was performed using real-time PCR. RESULTS: Out of 20.808 samples screened, 53 samples (29 for HCV and 24 for HIV) were confirmed as positive by serological and NAT methods. CONCLUSION: The manual magnetic bead-based extraction in combination with real-time PCR detection can be used to routinely screen blood donation for viremic donors to further increase the safety of blood products.