False-Positive Result from a Bayer Versant Human Immunodeficiency Virus Type 1 Branched-DNA Viral Load Assay, with a Possible Role for Light Leakage after Inadequate Maintenance of the Analyzer

Multiple reports have noted the occurrence of false-positive results when human immunodeficiency virus (HIV) viral load assays were used qualitatively to diagnose HIV infection (1-3, 5, 6). These false positives have been reported for both the PCR-based RNA amplification and the branched-DNA (bDNA)-based signal amplification techniques. Suggested causes for these erroneous results include cross-contamination, nonspecific hybridization, and incorrect specimen processing. Here, we report the occurrence of a false-positive HIV viral load result from the Bayer Versant HIV-1 RNA 3.0 bDNA assay. Testing and maintenance of the Bayer System 340 analyzer used in the performance of this assay led us to conclude that leakage of the chemiluminescent signal, or “cross-talk,” between sample wells in an inadequately maintained analyzer likely led to the false-positive result (4).

The patient was a 28-year-old male who was referred for HIV testing after a needle stick injury from a needle contaminated by blood from a newly diagnosed HIV-positive patient. He was given postexposure prophylaxis consisting of zidovudine, lamivudine, and nelfinavir for 4 weeks. Baseline and 6-week HIV-1/2 antibody enzyme immunoassay (EIA) testing produced negative results. An HIV viral load assay was ordered at the same time as the 6-week EIA, for which the patient's blood was collected in a Greiner K₂ EDTA tube. This tube was centrifuged and then frozen at −70°C within 65 min of collection. The following day, the Versant HIV-1 RNA 3.0 bDNA assay was performed according to the manufacturer's instructions, using the Bayer System 340 analyzer, and produced a result of 955 copies/ml. No error code was generated by the analyzer during this test.

Immediately after this test was performed, a Bayer technician examined the analyzer by prior arrangement (see below). It was discovered that the read head of the instrument was not adequately compressing the tops of the sample wells, potentially resulting in light leakage between wells. Maintenance was performed to correct this problem. A repeat viral load bDNA assay on the same serum 2 days later produced a result of <75 copies/ml. Additional EIAs performed 3 days and 45 days after the initial viral load assay were repeatedly negative (7 and 12 weeks following the needle stick exposure). An additional viral load assay using new serum collected 2 days after the first viral load also produced a result of <75 copies/ml. In the 4.5 months since the needle stick exposure, the patient continues to be asymptomatic, with negative HIV testing, and is presumed to be HIV negative.

For several weeks prior to this test, the analyzer generated intermittent error reports due to abnormally high luminescence intensities for certain standard specimens. The analyzer did not indicate problems with the read head during our regularly scheduled operator-performed maintenance. We notified the manufacturer, and a maintenance visit was arranged, as described above. Meanwhile, we sought to determine whether light leakage could be occurring by placing an empty strip of sample wells just below the strip of wells containing the standard specimens. Table 1 shows the luminescence results for the standard-specimen and empty wells in the first and second rows under the before-maintenance heading, respectively. The third row contains the luminescence results for routine-patient samples in the strip immediately below the empty strip. There was a strong correlation between the luminescence intensities of the empty wells and those of the adjacent wells immediately above, below, and diagonal to each empty well (P < 0.01). It is notable that the sample well immediately below our patient's initial (presumed false-positive) well had a very high luminescence intensity, corresponding to a viral load of >500,000 copies/ml (data not shown).

TABLE 1.

Luminescence intensities

Sample group	Relative luminescence of well:												rsa
	1	2	3	4	5	6	7	8	9	10	11	12
Before maintenance													0.944b
Standard specimensd	7,870	8,602	651.6	60.63	7.709	1.826	2.075	1.318	0.800	0.388	8.445	2,200
Empty wells	6.107	6.554	1.728	0.253	0.073	0.072	0.211	0.081	0.053	0.054	0.334	1.614
Routine-patient samplese	317.0	86.99	1.495	3.871	5.831	6.629	184.2	1.280	11.68	0.948	13.67	40.58
After maintenance													0.572c
Standard specimensd	8,384	7,980	518.3	57.79	6.318	3.326	3.213	1.233	1.312	0.558	7.227	2,341
Empty wells	0.023	0.014	0.022	0.002	0.006	0.000	0.002	0.006	0.002	0.006	0.015	0.000
Routine-patient samplese	1,617	1,027	5.258	91.37	28.75	1.135	0.770	22.33	0.888	180.3	1.619	1.971

^aSpearman rank order correlation coefficient, for correlation between the empty-well and total luminescence intensities for the standard- and routine-patient-sample wells immediately above, below, and diagonal to the empty well.

^bP < 0.01.

^cP > 0.05.

^dThe standard-specimen rows under the before-maintenance and after-maintenance headings represent different sets of standard specimens.

^eThe routine-patient-sample rows under the before-maintenance and after-maintenance headings represent different sets of patient specimens.

After preventive maintenance was performed and the read head compression was corrected, we again tested the assay for possible light leakage as described above, using empty wells in the strip below the standard-specimen wells. The results are shown for standard-specimen, empty, and routine-patient wells in the first, second, and third rows under the after-maintenance heading in Table 1, respectively. There was only a marginal correlation between the results for the empty and adjacent wells (P > 0.05).

In summary, we present a case of a false-positive HIV result obtained using the Versant HIV-1 bDNA viral load assay, apparently caused by inadequate maintenance resulting in light leakage between sample wells during luminescence reading by a Bayer System 340 analyzer. While we present one way that a false-positive result may be generated by a viral load assay, it is important to note that other false positives previously reported with multiple assay types show that there can be diverse causes. Clinicians using HIV viral load assays for HIV diagnosis must be cognizant of the potential for false-positive results and prepare their patients for this possibility. In addition, the phenomenon we report here could conceivably cause known HIV-positive patients' viral loads to be erroneously reported above certain critical values, indicating treatment failure where none may be occurring and leading to unwarranted changes in clinical management. This case underscores the need for the proper maintenance of the apparatus used in a chemiluminescent assay to ensure that light leakage is not occurring between samples. Even isolated deviations of the luminescence intensities of standard specimens should prompt a thorough evaluation of the assay, and preventive maintenance of the analyzer, before further clinical use of the assay. Future design modifications of these assays may need to incorporate improved detection of light leakage.