Routine diagnosis of viral infections often begins with an initial serology screen and may necessitate nucleic acid testing (NAT) to confirm infection status or to determine the best course of patient management. This common serology-to-molecular cascade in clinical virology has triggered interest by many clinical laboratories in using a single specimen for testing of both serological and molecular markers. Three examples are the diagnosis of HCV, HBV, and HIV, which in many cases all run on the same serology and molecular platforms.
According to the Centers for Disease Control and Prevention (CDC) HCV screening guidelines, a reactive serology screen for HCV antibody may indicate a current HCV infection, a past infection that has resolved, or a false-positive screen.1 Therefore, it is recommended that a reactive screen be followed by an Food and Drug Administration (FDA)–approved NAT for HCV RNA to determine if the infection is current (RNA detected) or a resolved infection/false-positive antibody result (RNA not detected). Unlike HCV, diagnosis of current HBV infection is established by the presence of hepatitis B surface antigen without any need to confirm HBV DNA status.2 However, once HBV diagnosis is established, HBV DNA quantification is a critical component in the early evaluation of patients, informing decisions for treatment initiation. In the case of HIV, a reactive serology screen by HIV-1/2 antigen/antibody immunoassay may indicate an acute infection, an established infection, or a false-positive screen.3 The CDC recommends that a reactive HIV screen be followed by an HIV-1/HIV-2 antibody differentiation test to confirm infection, distinguish the HIV type, and assess if the infection is in the immunogenic (i.e., established) phase. Specimens that are reactive on the initial HIV screen and nonreactive or indeterminate by the HIV-1/HIV-2 antibody test should be tested with a FDA–approved HIV-1 NAT to establish acute infection (RNA detected) or a potential false-positive HIV screen result (RNA not detected). Although diagnosis of HCV, HBV, or HIV involves serological screening followed by confirmation/treatment decision by use of NATs, there are limitations in the testing workflow that restrict the laboratory's ability to test in this sequence using a single specimen.
Serology devices effectively manage high sample throughput and deliver cost-effective immunoassay results in a timely fashion. However, these instruments are not typically designed for molecular applications, which require strict attention to contamination prevention measures because of their sensitivity. In the event of carryover contamination from a virus-positive patient sample into a negative patient sample, nonamplification methods like immunoassay are much less likely to detect trace amounts of antibodies or antigens in the contaminated specimen because the concentration will be far below the test's limit of detection. In contrast, amplification methods like NAT are designed to exponentially enrich for the target at very low concentration levels that can be introduced by carryover contamination. The clinical consequence of these biochemical differences is that most laboratories require either a second blood draw from patients or a preserology aliquot from the primary specimen for supplemental molecular testing because of the potential risk for cross-contamination on serology devices. Such constraints might jeopardize patient follow-up rates and place additional workflow burden on the laboratory so the ability to streamline the process and allow for single specimen vial use for both testing procedures is of high importance.
Currently available serology systems use either a fixed needle for sample transfer into the reaction vessel, which undergoes a buffer washing step between each new sample transfer process, or a disposable sample transfer tip, which is discarded after the sample is transferred to the reaction vessel and replaced with a new sterile tip before the next sample is accessed. Recent studies have suggested that different serology devices may have varying levels of susceptibility to contamination, depending on these on-board pipetting dynamics.4,5 Specifically, a fixed needle sample transfer system was found to be susceptible to HCV cross-contamination at a range of 4% to 23%, depending on the study, whereas serology systems that use disposable tips for sample transfer resulted in no observable contamination event.4,5
Our study investigated the potential risk of HCV, HBV, and HIV nucleic acid cross-contamination using a serology instrument that uses disposable tips rather than a fixed needle for sample transfer before NAT testing.
MATERIALS AND METHODS
Three positive sets of plasma specimens were generated by diluting armored HCV RNA (Roche Molecular Systems, Pleasanton, CA), HBV linearized plasmid (Roche Molecular Systems), or cultured supernatant of HIV 8E5/LAV cell line (SeraCare Life Sciences, Gaithersburg, MD) at either 6 Log10 IU/mL, 8 Log10 IU/mL, or 6 Log10 copies/mL, respectively. For each analyte, negative (n = 60/run) and positive (n = 60) plasma specimens were loaded onto the cobas e 602 module of the cobas 8000 System in an alternating fashion and tested up to three times with the Elecsys Anti-HCV II, Elecsys HBsAg, or Elecsys HIV combi PT assays (Roche Diagnostics GmbH, Mannheim, DE). Volume available in each set of positive plasma specimens permitted either 2 (HCV) or 3 (HBV and HIV) consecutive runs. Fresh negative plasma specimens (Human K3 EDTA Plasma; SeraCare Life Sciences) were loaded with each serology run. Processed negative plasma samples were subsequently tested with the cobas HCV test (n = 120), the cobas HBV test (n = 180), or the cobas HIV test (n = 180) on the cobas 6800 System (Roche Molecular Systems, Branchburg, NJ). False positivity rate was calculated per run and based on any detectable or quantifiable NAT result (95% confidence interval [CI]).
After checkerboard serology processing with high-titer HIV positive specimens (Table 1), 180 of 180 replicates of the negative plasma samples were tested with the cobas HIV test, and were negative (“target not detected”) for the presence of HIV-1 RNA. Based on these results, the cross-contamination rate was 0.00%. The one-sided 95% exact CIs were 0.00% for the lower bound and 0.02% for the upper bound (0%:0.02%). After checkerboard serology processing with high-titer HCV specimens (Table 1), 120 of 120 replicates of the negative plasma samples were negative (“target not detected”) for the presence of HCV RNA, resulting in a cross-contamination rate of 0.00%. One-sided 95% exact CIs were 0.00% for the lower bound and 0.03% for the upper bound (0%:0.03%). After checkerboard serology processing with high-titer HBV specimens (Table 1), 180 of 180 replicates of the negative plasma samples were negative (“target not detected”) for the presence of HBV DNA, resulting in a cross-contamination rate of 0.00%. One-sided 95% exact CIs were 0.00% for the lower bound and 0.02% for the upper bound (0%:0.02%).
Our study found no evidence of cross-contamination in 480 negative specimens on the cobas e 602 serology module after processing alongside of high-titer HCV, HBV, and HIV-spiked specimens. This finding is consistent with a previous study investigating contamination risk on either a serology device using a disposable transfer pipette tip or a device with a fixed needle. Contamination was only observed with the fixed needle device.5 Taken together, these data suggest that pipetting dynamics are a central risk factor for on-board contamination and, furthermore, that serology devices using disposable transfer tips may be most suitable for processing samples intended for subsequent molecular testing.
The concept of reflex NAT testing after a reactive serology result, although already in the CDC guidelines for HCV diagnosis, is under investigation by the CDC for HIV diagnosis as well and is likely to prompt laboratory interest in single-sample reflex strategies.6 In addition, recent CDC recommendations to identify more persons living with chronic HCV (aka birth cohort screening) as well as initiatives like Ending the HIV Epidemic: A Plan for America place great emphasis on quickly identifying HCV- or HIV-infected persons and linking them to care, which—among many other factors—relies on quicker and more accurate diagnosis.7,8 Faster diagnosis of HIV, HCV, or HBV may cut down the need for patient return visits to collect additional specimens and hence may reduce patient loss to follow-up and shorten the window to treatment initiation.8,9 From the laboratory perspective, using a single specimen for reflex testing may reduce the burden of storing an unprocessed preserology aliquot for potential supplemental testing by NAT, especially given the high rate of nonreactive serology screen results. Taken together, these public health and clinical laboratory drivers further emphasize the need for studies focused on streamlining the diagnostic cascade.
Our study challenged the cobas e 602 module by alternating only high-titer specimens with negative specimens, as opposed to alternating with a range of low and high viral titers. Although this represents a more rigorous approach, it does not reflect a typical clinical laboratory presentation, which may include lower viral titers in a more randomized pattern. As such, this study involved greater risk for cross-contamination than an average laboratory testing scenario.
This investigation was not performed in a clinical laboratory and was therefore not subject to an environment that runs high specimen volumes or that has instrument operators with varying levels of molecular training. This protocol required manual interventions, such as loading open specimen tube onto the cobas e 602 module and transferring specimens to the cobas 6800 system, and these interventions may introduce opportunities for unintentional carryover in a clinical laboratory setting without suitable training. Specifically, proper molecular technique would ensure that gloves were worn and frequently changed, that tubes were uncapped before serology testing in a manner that minimized sample transfer or aerosolization, and that tubes were either resealed with sterile caps or otherwise transferred to the molecular platform with careful attention to sample transfer and aerosol prevention after serology testing was complete.10 However, given the notable challenge facing laboratory medicine of finding and retaining personnel with adequate training in molecular diagnostics, automation of serology preanalytics and postanalytic transfer to the molecular test system may help reduce these inherent risks.11 Systems that automate sorting, uncapping, and loading of samples before serology, as well as the transfer of specimens after serology to the molecular platform may avoid human error and contribute to a reduction in contamination.
The results herein demonstrate that specimens analyzed by the cobas e 602 serology module may be suitable for direct, primary specimen reflex testing by a sensitive NAT for confirmation or treatment decision. Automated processes that minimize the need for manual intervention during specimen transfer, either before or after cobas e 602 assessment, may further reduce the chance of a contamination event. Further studies are warranted.
1. Centers for Disease Control and Prevention. Testing for HCV infection: An update of guidance for clinicians and laboratorians. MMWR Morb Mortal Wkly Rep 2013; 62:362–365. Available at: https://www.cdc.gov/mmwr/pdf/wk/mm62e0507a2.pdf
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4. Rondahl E, Gruber M, Joelsson S, et al. The risk of HCV RNA contamination in serology screening instruments with a fixed needle for sample transfer. J Clin Virol 2014; 60:172–173.
5. Tejada-Strop A, McNamara L, Mixson-Hayden T, et al. Feasibility of using same serum/plasma sample tubes for HCV antibody and reflex HCV RNA testing. Presented at: 70th AACC Scientific Annual Meeting & Clinical Lab Expo 2018; Chicago, IL. Available from: https://books.google.com/books?id=vKxjDwAAQBAJ&lpg=PT371&ots=hj1-P8bEcP&dq=tejada%20strop%20a-75&pg=PT372#v=onepage&q=tejada%20strop%20a-275&f=false
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6. Pitasi MA, Patel SN, Wesolowski LG, et al. Performance of an alternative laboratory-based HIV diagnostic testing algorithm using HIV-1 RNA viral load. Sex Transm Dis 2020; 47(Suppl 1):S18–S25.
7. Smith BD, Morgan RL, Beckett GA, et al. Recommendations for the identification of chronic hepatitis C virus infection among persons born during 1945–1965. MMWR Recomm Rep 2012; 61(RR04):1–18. Available at: https://www.cdc.gov/mmwr/preview/mmwrhtml/rr6104a1.htm
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8. Ending the HIV epidemic: A plan for America (CDC website). Available at: https://www.cdc.gov/endhiv/index.html
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9. Yehia BR, Schranz AJ, Umscheid CA, et al. The treatment cascade for chronic hepatitis C virus infection in the United States: A systematic review and meta-analysis. PLoS One 2014; 9:e101554.
10. Establishing Molecular Testing in Clinical Laboratory Environments: CLSI Document MM19-A (ISBN 1-56238-773-1). Wayne, PA: Clinical and Laboratory Standards Institute, 2011.
11. Ledeboer NA, Dallas SD. The automated clinical microbiology laboratory: Fact or fantasy? J Clin Microbiol 2014; 52:3140–3146.