Use Of Nucleic Acid Testing Research Articles

Modeling the effects of vaccination, nucleic acid testing, and face mask wearing interventions against COVID-19 in large sports events.

The transmission of SARS-CoV-2 leads to devastating COVID-19 infections around the world, which has affected both human health and the development of industries dependent on social gatherings. Sports events are one of the subgroups facing great challenges. The uncertainty of COVID-19 transmission in large-scale sports events is a great barrier to decision-making with regard to reopening auditoriums. Policymakers and health experts are trying to figure out better policies to balance audience experiences and COVID-19 infection control. In this study, we employed the generalized SEIR model in conjunction with the Wells-Riley model to estimate the effects of vaccination, nucleic acid testing, and face mask wearing on audience infection control during the 2021 Chinese Football Association Super League from 20 April to 5 August. The generalized SEIR modeling showed that if the general population were vaccinated by inactive vaccines at an efficiency of 0.78, the total number of infectious people during this time period would decrease from 43,455 to 6,417. We assumed that the general population had the same odds ratio of entering the sports stadiums and becoming the audience. Their infection probabilities in the stadium were further estimated by the Wells-Riley model. The results showed that if all of the 30,000 seats in the stadium were filled by the audience, 371 audience members would have become infected during the 116 football games in the 2021 season. The independent use of vaccination and nucleic acid testing would have decreased this number to 79 and 118, respectively. The combined use of nucleic acid testing and vaccination or face mask wearing would have decreased this number to 14 and 34, respectively. The combined use of all three strategies could have further decreased this number to 0. According to the modeling results, policymakers can consider the combined use of vaccination, nucleic acid testing, and face mask wearing to protect audiences from infection when holding sports events, which could create a balance between audience experiences and COVID-19 infection control.

An outbreak of leptospirosis associated with cattle workers during the wet season, in the Northern Territory of Australia, 2021.

An outbreak of leptospirosis occurred in the Top End of the Northern Territory, Australia, during the wet season in early 2021. There were 14 outbreak cases; most were male (12/14; 86%) and non-Indigenous (13/14; 93%) with a median age of 22 years (range 19-52 years). We conducted a descriptive case series to investigate the outbreak. We determined that the outbreak was most likely due to higher than usual rainfall in a workplace with exposure to cattle, heightened by wearing clothing and footwear which offered little protection, with limited use of personal protective equipment (PPE). Increased and ongoing education for cattle industry workers, and promotion of the use of appropriate clothing and PPE, may minimise the risk of future outbreaks. Australia's national surveillance case definition for leptospirosis should be reviewed to incorporate the use of nucleic acid testing in the detection of leptospirosis.

Rapid, instrument-free colorimetric quantification of DNA using Nile Blue.

The use of nucleic acid tests (NAT) for sensitive and rapid detection of pathogens relevant to human health has increased due to the ubiquity of nucleic acid amplification techniques such as polymerase chain reaction. The use of such tools for detection of amplified nucleic acid (NA) in field and clinical settings is limited by the need for complex instrumentation and trained users. To address these limitations we developed a rapid, robust, and instrument-free colorimetric detection method for nucleic acids using a visible region dye, Nile Blue (NB). NB is a cationic benzophenoxazine dye with well-known binding interactions with NA and has been used in instrumental methods for DNA quantification. When combined with dsDNA, the color of NB shifts from blue to purple. Images of this color shift are collected and are subjected to image analysis. Observed changes in the red and green colorimetric intensities are linked to the ratio of dsDNA to NB. By titrating solutions of dsDNA against a series of NB concentrations, we found it possible to quantitate dsDNA at concentrations ranging from 10-100 μg mL-1 using a k-means cluster analysis method. This range is comparable to that of NA concentrations quantified using gold-standard UV-Visible spectroscopy and to the concentrations of NA in biological samples after amplification. Unknown concentrations of dsDNA from yeast extracts were correctly identified within ±5 μg mL-1 of true concentration. Preliminary experiments demonstrate use of the developed NB method on paper-based analytical devices. As an instrument-free detection method, NB allows for rapid and robust quantification of dsDNA in field settings.

Potential Unintended Consequences of National Infectious Disease Screening Strategies in Deceased Donor Kidney Transplantation: A Cost-Effectiveness Analysis.

In order to counter the lack of sufficient kidney donors, there has been interest in expanding the utilization of organs from increased infectious-risk donors. Negative nucleic acid testing of increased infectious-risk organs has been shown to increase their use as compared to only enzyme-linked immunosorbent assay negativity. However, it is not known how the expanded use of nucleic acid testing on a national scale might affect total donor utilization. The objective of this paper was to determine if a national screening policy requiring the use of nucleic acid testing in both increased infectious-risk and non-increased infectious-risk renal transplant donors would increase the donor organ pool. This study used decision-tree analysis to determine the cost-effectiveness of four US national screening policies based on an increasingly expansive use of nucleic acid testing for increased infectious-risk and non-increased infectious-risk kidneys. Parameters were taken from the literature. All costs were reported in 2020 US dollars using a Medicare payer perspective and a life-time horizon. The use of nucleic acid screening solely for increased infectious-risk organs was the dominant strategy. Our results were robust to deterministic and probabilistic sensitivity analyses. One of the main driving factors of cost-effectiveness was the false-positive rate of nucleic acid testing. Before implementing nucleic acid screening outside of increased infectious-risk organs, its false-positivity rate should be directly studied to ensure that its use does not detrimentally affect transplantation numbers, quality-adjusted life-years, and costs.

Hepatitis C–Positive Donors in Cardiac Transplantation: Problems and Opportunities

With the growing need for donor hearts and longer transplant waiting lists, there is a growing interest in expanding the donor pool by reconsidering previously excluded donor candidates. There has been an increase in solid organ availability due to drug overdose deaths in the setting of the recent opioid epidemic. However, these donors often have transmissible infections such as hepatitis C. In this review, we discuss the challenges associated with heart transplantation from hepatitis C-infected donors as well as the recent advancements that are making the use of these organs possible. With the introduction and widespread use of nucleic acid testing (NAT), the ability to distinguish viremic donors and those that have cleared the virus has become a reality. In addition, with the emergence of direct antiviral agents, there is an increase in data showing the short-term outcomes and success of hepatitis C treatment for recipients of viremic donor hearts. As techniques to distinguish donor hepatitis C infection status and successful treatments emerge, the percentage of accepted hepatitis C donor hearts is increasing. A number of studies showing success with hepatitis C organ transplants present a promising new avenue for organ procurement essential to meet the increasing demand for donor hearts.

Prevalence, incidence and residual risk of transfusion-transmitted hepatitis B virus infection in Italy from 2009 to 2018.

In Italy, the use of nucleic acid testing for hepatitis B virus (HBV) in donor screening has allowed the detection of infections in the window phase, as well as the presence of occult infections which could potentially be transmitted. The aim of this study was to analyse the trends of epidemiological data focused on HBV infection in blood donors and to estimate the residual risk of transmitting HBV from both the window phase and occult infection over a 10-year period in Italy. Data were obtained from the Italian Haemovigilance System which includes the results of screening tests for transfusion transmissible infections. During the period of this survey (2009-2018), the molecular methods used for HBV screening were transcription-mediated amplification and polymerase chain reaction tests. Prevalence and incidence were calculated. The residual risk was estimated by applying the incidence-window period model for acute cases and a more recently reported model for estimating the risk due to occult infections. A total of 17,424,535 blood donors and 30,842,794 donations were tested for HBV. Altogether, 6,250 donors tested positive for HBV markers: 4,782 (175.6×105) were first time donors and 1,468 (10.0×105) were repeat donors. The prevalence of HBV markers in first time donors was 275.9×105 in 2009, declining to 143.6×105 in 2018. The incidence of new infections was 3.37×105 in 2009 and 2.17×105 in 2018. The overall residual risk for HBV amounted to 1 in 2,566,854 donations calculated as the sum of risks of both acute infections in the window period (1 in 5,835,306 donations) and occult infections (1 in 4,582,270 blood units). In Italy, the residual risk of transfusing a blood unit infected with HBV, both from window phase and occult infections, is currently very low, amounting to levels that can be considered tolerable.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics.

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test.OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses.METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection.FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

Modeling complete removal of risk assessment questions in the USA predicts the risk of HIV exposure in blood recipients could increase despite the use of nucleic acid testing.

The safety of the blood supply in a number of countries is achieved by interventions that include behaviour-based time-limited or indefinite deferrals and screening of donated units for transfusion-transmitted infections. The relatively high sensitivity of nucleic acid testing (NAT) used in blood donor screening has raised the question of whether such time-based deferrals can be eliminated in favour of individual risk assessment. Data on the annual number of incident human immunodeficiency virus (HIV) infections associated with various behaviours and on the performance characteristics of NAT applied to donor screening were used to model the number of potentially infected units that might escape detection in the worst-case scenario in which individual risk assessment was implemented, but was not effective as a screening tool, and donors did not otherwise self-select for lower risk. In the absence of effective individual risk-based screening or donor self-selection, the model predicts that in the United States, an additional 39 (95% CI 35-43) HIV-infected units would escape detection by nucleic acid testing, potentially capable of exposing approximately 68 (95% CI 61-75) individuals to the risk of HIV infection through the administration of prepared blood components. Despite some inherent uncertainty, the worst-case scenario of completely ineffective individual risk assessment, absence of donor self-selection and increased reliance on NAT for blood screening is estimated to be associated with an approximately fourfold increase in the risk of HIV exposure through transfusion in the United States.

Development and evaluation of a loop-mediated isothermal amplification assay for detection of Ehrlichia canis DNA in naturally infected dogs using the p30 gene.

Canine monocytic ehrlichiosis (CME) is a common tick-borne disease caused by the rickettsial bacterium Ehrlichia canis (Rickettsiales: Anaplasmataceae). In view of the different stages and variable clinical signs of CME, which can overlap with those of other infections, a conclusive diagnosis can more readily be obtained by combining clinical and hematological evaluations with molecular diagnostic methods. In this study, a loop-mediated isothermal amplification (LAMP) assay targeting the p30 gene of E. canis was developed. The assay was developed using DNA extracted from E. canis-infected cultures of the macrophage cell line DH82 and samples from dogs testing positive for E. canis DNA by PCR. The LAMP assay was compared to a p30-based PCR assay, using DNA extracted from EDTA-anticoagulated blood samples of 137 dogs from an endemic region in Brazil. The LAMP assay was sensitive enough to detect a single copy of the target gene, and identified 74 (54.0%) E. canis DNA-positive samples, while the p30 PCR assay detected 50 positive samples (36.5%) among the field samples. Agreement between the two assays was observed in 42 positive and 55 negative samples. However, 32 positive samples that were not detected by the PCR assay were identified by the LAMP assay, while eight samples identified as E. canis-positive by PCR showed negative results in LAMP. The developed E. canis LAMP assay showed the potential to maximize the use of nucleic acid tests in a veterinary clinical laboratory, and to improve the diagnosis of CME.

Multiplex Screening for Blood-Borne Viral, Bacterial, and Protozoan Parasites using an OpenArray Platform

The use of nucleic acid tests for detection of pathogens has improved the safety of blood products. However, ongoing pathogen emergence demonstrates a need for development of devices testing for multiple pathogens simultaneously. One approach combines two proven technologies: Taqman chemistry for target identification and quantification and the OpenArray nanofluidic real-time PCR platform for spatial multiplexing of assays. A panel of Taqman assays was developed to detect nine blood-borne pathogens (BBPs): four viral, two bacterial, and three protozoan parasites. The custom BBP OpenArray plate with 18 assays was tested for specificity and analytical sensitivity for nucleic acid from each purified pathogen and with pathogen-spiked human blood and plasma samples. For most targets, the limits of detection (10 to 10,000 copies/mL) were comparable with existing real-time platforms. The testing of the BBP OpenArray with pathogen-spiked coded human plasma or blood samples and negative control specimens demonstrated no false-positive results among the samples tested and correctly identified pathogens with the lowest concentration detected ranging from 10 cells/mL (Trypanosoma cruzi) to 10,000 cells/mL (Escherichia coli). These results represent a proof of concept that indicated the BBP OpenArray platform in combination with Taqman chemistry may provide a multiplex real-time PCR pathogen detection method that points the way for a next-generation platform for infectious disease testing in blood.

Deceased Organ Donor Screening for HIV, Hepatitis B, and Hepatitis C Viruses: A Survey of Organ Procurement Organization Practices

Although Organ Procurement and Transplantation Network (OPTN) policy requires that all potential deceased organ donors are screened for human immunodeficiency (HIV), hepatitis B (HBV) and hepatitis C (HCV) viruses by serology, no current policy requires the use of nucleic acid testing (NAT) for organ donor screening. An electronic survey was sent to 58 organ procurement organizations (OPO) in the United States to assess current screening practices of potential deceased organ donors. Fifty-seven responses were collected for data analysis; not all respondents answered all questions. All OPOs performed required HIV, HBV and HCV serology screening and 48 (84%) performed confirmatory testing for seropositive donors. Ninety-eight percent, 75% and 97% of OPOs performed prospective HIV, HBV and HCV NAT, respectively. Fifty-two percent and 47% used a transcription-mediated amplification assay for HIV and HCV NAT, respectively. Of the 56 respondents that performed HIV NAT and 55 respondents that performed HCV NAT, 39 tested all donors. Seventeen (32%) OPOs performed confirmatory testing for all HIV-positive NAT results, and 15 (27%) OPOs performed confirmatory testing for all HCV-positive NAT results. Since 2008, the number of OPOs performing NAT has increased and more OPOs are testing all donors.

Pandemic (H1N1) 2009 and Seasonal Influenza A (H3N2) in Children’s Hospital, Australia

To the Editor: We read with interest the report by Carcione et al. of clinical features of pandemic influenza A (H1N1) 2009 and comparison of these with 2009 seasonal influenza infection in a population-based study from Western Australia (1). Here we share our experience of hospitalizations for influenza in a tertiary care children’s hospital in Sydney, New South Wales, Australia, during the 3 peak influenza seasons of the last decade. During the 2009 Southern Hemisphere single influenza wave (June–September), we prospectively studied every child <15 years of age who was hospitalized with laboratory-confirmed influenza (74% had proven pandemic [H1N1] 2009) in Children’s Hospital at Westmead (CHW), Sydney, as part of a collaboration between the National Centre for Immunisation Research and Surveillance and the Australian Paediatric Surveillance Unit. The study was approved by the Human Research Ethics Committee at CHW and supported by the state (New South Wales) health department. Data from hospitalizations for seasonal influenza at CHW in 2003 and 2007 (previous peaks in the last decade) were analyzed by using our previous studies and medical records (2–4). To compare pneumonia rates, we used the same case definitions in 2007 and 2009 (radiologic changes consistent with pneumonia). Proportions were compared by using the χ2 statistic. In 2009, the numbers of children with laboratory-confirmed influenza admitted to the hospital and to the pediatric intensive care unit (PICU) at CHW (226 and 22, respectively) were nearly double those admitted in 2007 (122 and 12) but similar to the number in 2003 (257 and 22). The proportion of case-patients admitted to the PICU, the length of hospital stay, and the length of PICU stay were similar in 2003, 2007, and 2009. In 2009, among the 226 influenza-associated hospitalizations, 167 (74%) were for pandemic (H1N1) 2009 infection; in 2007, 119 of 122 influenza-associated hospitalizations were for seasonal influenza A (H3N2) infection (Table). During the 2009 pandemic wave, of all children admitted with laboratory-confirmed influenza, the proportion hospitalized with pandemic (H1N1) 2009 who were 5 years of age were significantly higher (61 [37%] and 15 [13%]; p = 0.0001). However, the proportion of those >5 years of age admitted to PICU in 2009 was less than in 2007 (10 [16%] of 61 vs. 3 [20%] of 15; p = 0.71). Similar percentages of children with preexisting conditions were admitted in 2009 and 2007 (47% and 49%, respectively). However, pneumonia was a more frequent complication in 2009 than in 2007 (42 [25%] of 167 vs. 15 [13%] of 119; p = 0.01). In 2009, the proportion of children with pandemic (H1N1) 2009 who needed mechanical ventilation (7 [4%] of 167) was similar to the proportion in 2007 who had seasonal influenza (H3N2) (6 [5%] of 119; p = 0.77). Furthermore, no child at CHW in 2007 or in 2009 received extracorporeal membrane oxygenation. Table Comparison of influenza-related hospitalizations, Children’s Hospital at Westmead, Sydney, Australia, 2003, 2007, and 2009 influenza seasons* Vomiting occurred much more frequently in 2009 than in 2007 (59 [35%] of 167 vs. 16 [13%] of 119; p = 0.0001). In 2009, of 62 children who did not exhibit vomiting when first examined and who were subsequently treated with antiviral drugs, only 1 had vomiting develop in the hospital. This condition resolved within hours, and the 5-day course of antiviral treatment was completed. Chart review showed that in no child did antiviral treatment exacerbate vomiting, and no children required antiemetic treatment or intravenous rehydration. These data suggest that oseltamivir was uncommonly associated with vomiting in hospitalized children. In macaque monkeys, pandemic (H1N1) 2009 virus is more pathogenic than seasonal influenza A (H1N1), particularly affecting the lungs (5). The significantly higher proportion (and number) of pneumonia patients at CHW in 2009 than in 2007 (Table) seems to provide additional evidence of the pathogenecity of pandemic (H1N1) 2009 virus. However, the number of children with laboratory-confirmed influenza admitted to the hospital was similar in 2009 and 2003. Given that the sensitivity of influenza laboratory tests has improved over time (e.g., greater use of nucleic acid tests), these data suggest that the incidence of hospitalization during the 2009 pandemic was not greater than the incidence during the 2003 influenza season. Although pneumonia appeared more likely to be diagnosed in hospitalized children in 2009 than in 2007, we observed no increase in the risk for serious outcomes (PICU admission or ventilation rate, length of admission, or death) in children hospitalized with pandemic (H1N1) 2009 infection than in children hospitalized with seasonal influenza (H3N2) during the 2 peak years studied. The 2009 pandemic had an effect on children’s services that was comparable to the busiest interpandemic influenza season of the previous decade. The large number of admissions and complications, including in children with no existing medical condition, supports the need for universal influenza vaccination.

Nucleic Acid Testing to Detect HBV Infection in Blood Donors

The detection of hepatitis B virus (HBV) in blood donors is achieved by screening for hepatitis B surface antigen (HBsAg) and for antibodies against hepatitis B core antigen (anti-HBc). However, donors who are positive for HBV DNA are currently not identified during the window period before seroconversion. The current use of nucleic acid testing for detection of the human immunodeficiency virus (HIV) and hepatitis C virus (HCV) RNA and HBV DNA in a single triplex assay may provide additional safety. We performed nucleic acid testing on 3.7 million blood donations and further evaluated those that were HBV DNA-positive but negative for HBsAg and anti-HBc. We determined the serologic, biochemical, and molecular features of samples that were found to contain only HBV DNA and performed similar analyses of follow-up samples and samples from sexual partners of infected donors. Seronegative HIV and HCV-positive donors were also studied. We identified 9 donors who were positive for HBV DNA (1 in 410,540 donations), including 6 samples from donors who had received the HBV vaccine, in whom subclinical infection had developed and resolved. Of the HBV DNA-positive donors, 4 probably acquired HBV infection from a chronically infected sexual partner. Clinically significant liver injury developed in 2 unvaccinated donors. In 5 of the 6 vaccinated donors, a non-A genotype was identified as the dominant strain, whereas subgenotype A2 (represented in the HBV vaccine) was the dominant strain in unvaccinated donors. Of 75 reactive nucleic acid test results identified in seronegative blood donations, 26 (9 HBV, 15 HCV, and 2 HIV) were confirmed as positive. Triplex nucleic acid testing detected potentially infectious HBV, along with HIV and HCV, during the window period before seroconversion. HBV vaccination appeared to be protective, with a breakthrough subclinical infection occurring with non-A2 HBV subgenotypes and causing clinically inconsequential outcomes. (Funded by the American Red Cross and others.).

Provider Response to a Rare but Highly Publicized Transmission of HIV Through Solid Organ Transplantation

On November 13, 2007, the first reported case in 20 years of HIV (human immunodeficiency virus) transmission from a Centers for Disease Control and Prevention high-risk donor (HRD) made national headlines. We sought to characterize change in the practice of transplant surgeons resulting from this rare event. We performed a survey between January 17, 2008, and April 15, 2008, assessing attitudes and practices of transplant surgeons regarding HRDs. Descriptions of changes in practice after the event were categorized, and associations between responses and regional-, center-, and physician-level factors were studied. Transplant centers in the United States. Four hundred twenty-two transplant surgeons in current practice. Changing practice following the 2007 HIV transmission event. Among surgeons who responded to the survey, 31.6% changed their practice following the event. Also, 41.7% decreased use of HRDs, 34.5% increased emphasis on informed consent, 16.7% increased use of nucleic acid testing, and 6.0% implemented a formal policy. Ranking fear of being sued or hospital pressure as important disincentives to HRD use was associated with more than 2-fold higher odds of changing practice. Ranking medical risks of HIV as an important disincentive was associated with 8.29-fold higher odds of decreasing HRD use. The most common responses to this rare event were avoidance (decreased HRD use) and assurance (increased emphasis on informed consent) behaviors rather than patient safety measures (increased use of nucleic acid testing and implementation of formal policies), suggesting that fear of legal or regulatory consequences was the biggest driver of physician decision making and that the current litigious environment is failing to protect patient interests.

Advances in HIV laboratory testing

Laboratory tests are readily available for the diagnosis of HIV infection. These are based on the detection of HIV-specific antibodies and HIV p24 antigen in combination screening assays, followed by confirmation by Western blot. Managing established HIV infection, including the use of antiretroviral drugs, has been facilitated by the use of nucleic acid tests that measure HIV RNA load in plasma or detect mutations associated with drug resistance. Quality assurance programs ensure high-level performance of HIV assays.

Donor-Derived Infections in Transplant Patients

Numerous cases of donor-derived infections (DDI) after solid-organ transplantation have been reported in recent times. While some are expected (often due to cytomegalovirus or Epstein-Barr virus), others have been clinically unexpected. Pre-transplant screening is quite helpful in abrogating the risks of DDI but could be optimized further to increase patient safety, reduce risk, augment the number of organs transplanted, and save more lives. Testing thus far has largely been serologic; advances in testing technology have allowed the use of nucleic acid testing, as well as other new testing modalities. The optimal use of such testing in the solid organ transplant setting remains to be determined. Increased awareness of DDI has augmented and will continue to augment our ability to effectively diagnose and care for solid organ transplant recipients.

Laboratory testing for hepatitis C

Serological diagnosis of patients infected with the hepatitisC virus (HCV) can be performed using two categories of tests:indirect tests, which detect antibodies against HCV; and directtests, which detect, quantify, or characterize components ofthe viral particle, such as HCV RNA testing and testing fordetection of the HCV core antigen.Anti-HCV antibodies are usually detected using third- andfourth-generation immunoenzymatic assays – enzymeimmunoassay (EIA)/enzyme-linked immunosorbent assay(ELISA) 3 and EIA/ELISA 4, respectively – which containHCV core antigens and HCV nonstructural genes. Thespecificity of the EIA tests available on the market that detectanti-HCV was determined to be higher than 99%, whereastheir sensitivity, which was more difficult to determine due tothe lack of gold standard tests with high sensitivity, was 95-99% [1]. However, false-positive results for anti-HCV canoccasionally occur, especially in populations with prevalencerates below 10% [2-4].There are many reasons why laboratories do not routinelyuse a supplementary test based on immunoblot analysis, suchas the recombinant immunoblot assay, to complement thediagnosis of HCV infection. In addition to the high cost ofsuch a test, the lack of laboratory standards that can evaluateits performance and interpretation, in conjunction with itsactual accuracy, is among the principal reasons. Furthermore,this type of test does not distinguish past from presentinfection, and its use is only indicated for confirmation of EIAresults.In contrast, the use of nucleic acid testing (NAT) makes itpossible to differentiate between viremic and nonviremicindividuals by detection of HCV RNA, allowing the clinician adifferentiated approach to anti-HCV-positive individuals.However, there can be situations in which HCV RNA is notdetected (negative HCV RNA) and the individual has activeinfection with HCV. This can occur in individuals in whomanti-HCV antibody titers are high and RNA titers are low [5].Therefore, HCV RNA might not be detectable in certainindividuals in the acute phase of the disease. However, thesefindings are transient, and chronic infection can develop [6].In addition, HCV RNA intermittent positivity has beenobserved in individuals chronically infected with HCV [6-8].Negativity of HCV RNA results can indicate resolved infection.In 15 to 25% of those anti-HCV positive individuals whoacquired the infection after 45 years of age, the infectionresolves spontaneously. This percentage increases to 40-45%in those who acquired the HCV infection in childhood oryoung adulthood [9].Different tests based on polymerase chain reaction (PCR)have been developed to directly detect the viral particle. Onecharacteristic of real-time PCR is amplification coupled withdetection, which allows the evaluation of the number of viralgenomes at the onset of and throughout the reaction.Qualitative detection of HCV RNA by reverse transcriptase(RT)-PCR is generally accepted as the most sensitive andstandardized test to date [10,11]. Nevertheless, there is variabilityamong the results from different laboratories, as evidenced bythe use of international panels of proficiency. The accuracyand reliability of the results are directly related to the laboratoryprocedures adopted in the performance of the tests [12]. Thelack of preliminary care in sample collection, in conjunctionwith the time involved in preparing and separating the samples,can result in incorrect results. It is extremely important that alllaboratory procedures comply with Good Laboratory Practiceand strictly follow the protocols standardized by themanufacturers of the diagnostic kits and reagents.The gold standard consists of the careful use of NAT,standardized for detection of HCV RNA, together with EIAs(specificity in conjunction with sensitivity).An alternative to aid diagnosis is the use of the ratiobetween optical density and cut-off value (OD/COV) or thesample/cut-off ratio as an indicator of the true positivity ofthe test. Studies carried out in Brazil show that, in EIAs,reagents with OD/COV greater than 3 are repeatedly associatedwith 100% true-positive results (positive predictive value) andpresent approximately 92% positivity for HCV RNA by RT-PCR [13]. In terms of the population studied, the positivepredictive value is increased when accompanied by riskfactors, high levels of alanine aminotransferase (ALT), or liverdisease.In immunocompetent patients, EIAs present excellentreproducibility; however, in hemodialyzed orimmunocompromised patients, EIA sensitivity is significantlyreduced [14].In low-risk populations, such as blood donors, or inrandom population screening, i.e., in populations that do notpresent risk factors for the acquisition of HCV infection,negative EIA results are sufficient to rule out the presence ofHCV. However, false-positive results can occur in thesepopulations. In such cases, a qualitative study of HCV RNAshould be performed to confirm the diagnosis.In high-risk populations, when there is clinical suspicion ofHCV infection, positive EIA results confirm the exposure toHCV. A qualitative study of HCV RNA should be performed todistinguish individuals with chronic infection from those whohave eliminated the HCV spontaneously.In patients with chronic hepatitis of unknown cause andnegative anti-HCV EIA results, especially inimmunocompromised patients [14], a qualitative study of HCVRNA should be performed. The presence of HCV RNA confirmsthe diagnosis, although a negative result does not rule outHCV infection. In such cases, it is recommended that a newHCV RNA study be performed six months after the first study.Detection of the HCV core antigen by EIA can be an alternativefor early diagnosis of HCV infection.

A chemiluminescent, magnetic particle-based immunoassay for the detection of hepatitis C virus core antigen in human serum or plasma

Hepatitis C virus (HCV) exposure in blood donors is determined serologically by the detection of anti-HCV antibodies in serum or plasma. However, a "window" period of 30-70 days after exposure exists where specific antibodies to HCV antigens are not detected. The use of nucleic acid testing for the detection of HCV RNA or antigen testing for the detection of HCV core protein have resulted in dramatic reductions in the pre-seroconversion window period. In this study, an automated HCV core antigen detection test was developed. This magnetic microparticle-based assay utilizes anti-HCV core monoclonal antibody to capture antigen present in human serum or plasma. Captured antigen is then detected using an anti-HCV core monoclonal antibody conjugated with a chemiluminescent compound. The specificity of this assay was established at 99% upon testing a population of normal volunteer blood donors. Sensitivity was determined by testing 16 commercially available HCV seroconversion panels representing genotypes 1a, 1b, 2b, and 3a. In each panel tested, HCV core antigen was detected prior to anti-HCV antibody, resulting in a reduction of the window period by greater than 23 days on average, and greater than 34 days on panels initially NAT negative. In addition, HCV core antigen was detected in >97% of HCV RNA positive/antibody negative specimens, exhibiting sensitivity nearly equivalent to nucleic acid testing in the pre-seroconversion window period for the panels examined.