The evaluation of low cut‐off index values of Elecsys® HIV combi PT assay in predicting false‐positive results

Abstract Objective To analyze the results of different cut‐off index (COI) values of Elecsys® HIV combi PT assay and to assess the role of COI in reducing the frequency of false‐positive results. Methods We conducted a retrospective study of samples analyzed by Elecsys® HIV combi PT assay, a 4th‐generation ECLIA, between 2016 and 2017. A total amount of 379 122 samples were collected for HIV (Human Immunodeficiency Virus) screening. Results A total of 379 122 samples were analyzed. 2528 (0.67%) were positive by Elecsys® HIV combi PT. Of these, 468 were false‐positive results, and most of them (94.87%) were in samples with 1 < COI < 15. The false‐positive rate was 0.12%. Patients with false‐positive samples were more distributed in elder (P < .001) and female (P < .001) than true‐positive specimens. The median COI in true‐positive specimens was (385.20), which is significantly higher than false‐positive specimens (2.08). The consistency between Elecsys® HIV combi PT assay and 3rd‐generation and positive predictive value (PPV) increased with higher COI values. Cancer, infection, and neurological diseases were considered the potential confounding factors of HIV false‐positive results (19.44%, 11.11%, and 6.62%, respectively). Conclusion Samples with low COI values, especially those contain confounding factors, need to be further scrutinized to determine whether the confounding factors may cause false‐positive problem. In addition, the hypothesis that low COI values may predict false‐positive results is valid.

90% of infected people should know their status, 90% of diagnosed cases should be on antiretroviral medication, and 90% of those being treated should have fully suppressed viral load by 2020. 3 Novel 4th-generation screening and confirmatory assays are now commercially available and have been incorporated into new diagnostic algorithms. 4,5 The 4th-generation immunoassay can identify HIV-1 p24 antigen, anti-HIV IgM, and IgG antibodies simultaneously with sufficient sensitivity and specificity, thus narrowing the "window period" into approximately two weeks, and enabling the detection of acute and early HIV infection. 6,7 Due to the extremely high sensitivity of these techniques, the positive predictive value (PPV) in settings with a low prevalence of HIV may not be optimal, thus potentially leading to false-positive results, arousing unnecessary concerns and interfering the progress of clinical diagnosis. 8,9 This study was carried out to analyze results for different COI values and the use of COI to distinguish between false-positive and true-positive results in a low-prevalence setting through the presented hypothesis, we suggest that low COI values could predict false-positive HIV results.

| Settings
We conducted a retrospective study in a large general teaching hospital with 4300 beds and a catchment population of approximately 16.33 million inhabitants in Sichuan, China. We included serum samples delivered to our laboratory, from inpatients and outpatients, for HIV testing between 2016 and 2017.

| The screening tests
Samples were first analyzed using Elecsys ® HIV combi PT assay, performed on the MODULAR ANALYTICS E170 or Cobas e 601 platform (Roche Diagnostics), following the manufacturer's instructions. Samples were considered as reactive (COI ≥ 1.0), borderline (0.9 ≤ COI < 1.0), or non-reactive (COI < 0.9). All initially reactive or borderline samples were redetermined in duplicate with 3rd-generation assay-colloidal gold method-Anti-HIV (Livzon Diagnostics Inc) or 3rd-enzyme-linked immunosorbent assay (ELISA) which was run on the TECAN (freedom evolyzer, Switzerland) according to the national guide of China. Samples were considered repeatedly reactive (control line and test line both appear/ COI ≥ 0.9 in either of the determinations) and non-reactive (only control line appear or COI < 0.9) by 3rd-generation assay. These assays were all conducted following the manufactures' instructions.

| The supplemental tests
Repeatedly, reactive samples must be confirmed according to recommended confirmatory algorithms. Western Blot HIV Blot 2.2 (MP Diagnostic) detects IgG antibodies specific to viral antigens. On the basis of manufacturer's criteria, the results of individual specimens were reported as positive (the presence of at least two bands, including two env bands, or one env band plus p24 band), indeterminate (reactivity to any of the bands but not compatible with the criteria for a positive interpretation), or negative (the absence of any of the specific bands). As a negative result only means that no IgG antibody detected, patients with indeterminate or negative results required further tests or follow-up.
Samples were considered as confirmed positive if they were reactive by immunoblot, HIV-RNA, or follow-up. HIV p24 Ag was used to help determine the infection status.

| Blocking test
With sufficient sample size, a total amount of 193 samples with initially and repeatedly reactive, uncertain, or negative in antibody confirmation tests stepped into blocking test. HIV Ag/Ab was rerun, before and after blocking the samples with 40 μL Heterophilic Blocking Reagent (HBR, 20 μg/mL) added to each sample and reacted for 1 hour under room temperature. SPSS 23.0 software was used for statistical analysis, and OriginPro 2016 was used for plotting. Quantitative data were expressed as median and interquartile range (IQR). Comparisons between continuous variables were made using the t test or variance analysis, depending on the normality of the distribution. If not, the non-parametric test was adopted for analysis. The data were counted by chisquare test or Fisher's exact probability test. A P-value less than or equal to .05 was considered significant.  When COI value climbed, the consistency and PPV increased simultaneously, while the false-positive specimens decreased (

| The clinical characteristics of false-positive samples and blocking test
The three leading clinic diagnoses among false-positive patients were cancer, infection, and neurological diseases, with the proportions of 19.44%, 11.11%, and 6.62%, respectively ( Table 2)

| D ISCUSS I ON
The 4th-generation assay is capable for detecting both HIV-1 p24 antigen and antibody simultaneously, which can narrow the "window period" into about two weeks, making it widely used as screening assay. 11  The Elecsys ® HIV combi PT assay, a 4th-generation assay, is currently recognized and recommended as HIV screening test internationally. It has a special pre-treatment of samples to release antigen and can evaluate the diagnostic performance. 12,13 According to our study, the specificity and sensitivity of the Elecsys ® HIV combi PT were 99.88% and 100%. The false-positive rate and positive predictive value were 0.12% and 71.38%. The results were similar to previous studies that the Elecsys ® HIV combi PT performed well in specificity (>99.50%) and sensitivity (nearly 100%) ( Table 3).
Considering all these merits of the 4th-generation assay, it is more appropriate for routine clinical screening and HIV early infection screening in high-risk groups. 17 In this study, false-positive specimens showed some similarities. The COI value was significantly lower in the false-positive group than that in the true-positive group. Nearly, all false-positive samples were distributed at a low level of COI (1-15), and a higher COI value accompanied by fewer false-positive samples.
Results also showed that most patients with false-positive results were elders and females, which was consistent with previous studies. 17,18 Recent studies revealed that many factors, such as cancers, rheumatoid factors, autoimmune diseases, and pregnancy, might lead to poor specificity in HIV screening by the 4th-generation assay and cause an indeterminate result for the WB test. [19][20][21][22][23][24] More elderly patients in the false-positive group are more likely to suffer from underlying diseases. These diseases and some potential confounding substances in patients may blamed for some false-positive results. Although follow-up visit and use of HBR for blocking can eliminate interference to certain extent, proceeding HIV-RNA test as soon as possible, the history of epidemiology and confounding factors should also be taken into consideration.

| E THIC AL CONS IDER ATIONS
The study was conducted in full compliance with the principles of the Helsinki Declaration and local regulations. The study protocol was approved by ethics committee of the West China Hospital of Sichuan University. Exemption for obtaining informed consents from subjects was granted as a retrospective analysis of routinely collected programmatic data, and there was no direct contact with patients. Abbreviations: FP, false-positive; NP, not provided; PPV, positive predictive value.