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.