|Viral load||False viral load||Inconsistency with antibody tests||RNA|
|Performance characteristics of the bDNA 3.0 assay for quantitation of HIV-1 RNA in plasma|| ||Erice A et al.
| ||“Negative results (<50 copies/mL), were obtained in 30/32 (94%) [bDNA 3.0] assays [meaning that false-positive results were obtained in 2/32 or 6% of cases]”|
| ||7th Conf. Retroviruses and Opp Infections. 2000 Jan 30-Feb 2.||2000|
|HIV type 1 RNA level and CD4 count as prognostic markers and surrogate end points.|| ||HIV Surrogate Marker Collaborative Group.
| ||A meta-analysis of 96 different studies that looked at the specificity and sensitivity of the polymerase chain reaction (PCR) in diagnosing HIV infection : they found that the specificity of PCR varied widely in these studies from a low of 40% to a high of 100%, which means that false positive rates varyed from 60% to 0%. They would have had even higher false positive rates if they had included "indeterminate" PCR results as being positive. In the studies of highest quality, according to the authors, the false positive rate ranged from 5% to 0%. The authors also found that studies using more recent PCR technology were no more accurate than older studies, and that publication bias may have prevented studies with worse results from being published. The PCR test result is called positive or negative based on a threshhold value, and is not a "yes or no" result : "Because both sensitivity and specificity are determined by the choice of the threshhold for an abnormal test result, there is an inherent tradeoff between them. The threshhold can be chosen so that PCR is 100% sensitive or so that it is 100% specific, but not normally both (unless the test is perfect...). Thus, a study that only evaluates the sensitivity of PCR or only evaluates the specificity of PCR provides insufficient information for evaluation of the test's performance."|
| ||AIDS Research and Human Retroviruses; 2000, 16(12); 1123-1133.||2000|
|Quantitation of HIV-1 RNA viral load using nucleic acid sequence based amplification methodology and comparison with other surrogate markers for disease progression.|| ||Sitnik R, Pinho JR
| ||"In this study, HIV-1 viral blood quantitation determined by Nucleic Acid Sequence Based Amplification (NASBA) was compared with other surrogate disease progression markers (antigen p24, CD4/CD8 cell counts and beta-2 microglobulin) in 540 patients followed up at Sao Paulo, SP, Brazil. HIV-1 RNA detection was statistically associated with the presence of antigen p24, but the viral RNA was also detected in 68% of the antigen p24 negative samples, confirming that NASBA is much more sensitive than the determination of antigen p24."|
| ||Mem Inst Oswaldo Cruz 1998 May-Jun;93(3):411-5||1998|
|Genital tract Human Immunodeficiency Virus Type 1 (HIV-1) shedding and inflammation and HIV-1 env diversity in perinatal HIV-1 transmission.|| ||Panther LA et al.
| ||“In the CVL [cervico-vaginal lavage] samples, 9 (41%) of 22 yielded culturable HIV-1, 16(67%) of 24 were PCR positive for proviral HIV-1 DNA, 7 (30%) of 23 were positive for cell-free HIV-1 RNA, and 11 (45%) of 24 were positive for cell-associated HIV-1 RNA.”|
| ||JID. 2000 Feb;181:555-63.||2000|
|Utility of an HIV-1 RNA assay in the diagnosis of acute retroviral syndrome.|| ||More D et al.
| ||“Because of the RNA assay's 1.9% to 3.0% false-positive rate, results must be carefully interpreted and compared to HIV-1 viral load levels seen during proven HIV-1 seroconversion. We report the case of a sexually active woman with symptoms suggestive of ARS who had a false-positive HIV-1 RNA assay result.”|
| ||S Med J. 2000 Oct;93(10):1004-6.||2000|
|Pitfalls of HIV RNA testing in the San Francisco post-exposure prevention (PEP) project.|| ||Roland ME et al.
| ||“18 subjects had 1 or 2 positive results with v.2.0 and an undetectable confirmatory test for a false positive rate of 4.4%. The rate is similar at baseline (9/183 subjects = 4.9%), wk. 4 (7/162= 4.3%) and wk. 26 (2/44 = 4.5%). Of the 18 pos. specimens, 9 tested pos. once and 9 twice. With version 3.0, 11 of 67 samples tested were pos. (16.4%). 6 were pos. once and 5 twice. The range of false pos. rates was 9.1% at wk. 4 (total of 22 specimens) to 26.7% at wk. 26 (total of 15 specimens). A week 4 sample with two values of 8,000 copies/ml on v.2.0 was neg. by DNA PCR, p24 antigen and Western Blot. Follow-up testing of this subject at wk. 26 was negative for HIV antibody and RNA. Discussion: The emotional impact of a false positive screening RNA test in a recently exposed person is significant. With the high false positive rate, we do not advocate the routine use of HIV RNA tests to screen asymptomatic people. The high rate of repeat false positive tests in a given sample (50%) suggests a possible biologic mechanism.”|
| ||Conf Retroviruses Opportunistic Infect. 1999 Jan 31-Feb 4;6(101):Abstract no. 179.||1999|
|Effect of HIV-1 genetic diversity on HIV-1 RNA quantification in plasma: comparative evaluation of three commercial assays.|| ||Coste J et al.
| ||“We observed that clade A strains [of HIV] were not detected by RT-PCR [Reverse Transcriptase-Polymerase Chain Reaction] and that clade G-strains were not detected by NASBA [Nucleic Acid Sequence-Based Amplification]. However, the copy number detected by RT-PCR in one clade E (CM235) and in one clade F (163.3070) was much lower than the copy number detected by bDNA [branched DNA] and NASBA...for clades B and D (UG270), the HIV-1 RNA levels measured by bDNA were lower than those obtained by RT-PCR and NASBA”|
| ||JAIDS. 1997;15:174.||1997|
|Manufacturer's notice|| ||Roche
| ||“The AMPLICOR HIV-1 MONITOR Test is an in vitro nucleic acid amplification test for the quantitation of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in human plasma...[It] is not intended to be used as a screening test for HIV or as a diagnostic test to confirm the presence of HIV infection...”|
| ||Amplicor HIV-1 Monitor Test||1996|
|Diagnosis of primary HIV-1 infection.|| ||Daar ES et al.
| ||“Primary infection was defined as a confirmed positive virologic test result with either a negtaive HIV antibody assay result or an indeterminate Western blot. Because there is no virologic gold standard, we assumed that levels of plasma HIV RNA had a sensitivity of 100% for diagnosing primary infection [bonus marks for detecting the flaw in this logic].False-positive HIV RNA measurements were defined as those that were negative on repeated testing and those obtained in patients who did not undergo seroconversion [note the contradiction with the previous sentence]...Eight of 303 uninfected patients (2.6%) had false-positive results on HIV RNA testing”|
| ||Ann Int Med. 2001 Jan 2;134(1).||2001|
|DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells.|| ||Ou CY et al.
| ||“The number of peripheral blood lymphocytes expressing viral RNA, as detected by in situ hybridization in an infected person is less than 1 in 10,000 cells...Defective provirus would be detected by the PCR technique provided the region targeted for amplification was preserved [viral load/PCR tests cannot distinguish between defective and infectious HIV!]”|
| ||Science. 1988 Jan 15;239(4837):295-7.||1989|
|False positive viral loads : what are we measuring?|| ||Irwin Matt
| ||"Most people, including many clinicans, believe that the numbers generated by a viral load test represent the number of active viruses present in each milliter of a person's blood, but this is not what viral loads actually represent. The viral load test is used to measure the quantity of RNA fragments that are believed to be specific to HIV which are present in each milliliter of a person's blood... What actually happens is that probes are used to identify short RNA sequences thought to come from HIV. Then whatever is found by the probes is amplified exponentially by a string of replication steps. Only after all of these amplifications are completed can the RNA fragments be detected and counted. Then a complex mathematical estimation is used to try to acertain how many RNA fragments were present in the original sample of blood, which finally generates the number which represents a person's "viral load". Each one of these steps introduces the potential for inaccurate results, from the assumption that only RNA from HIV will be identified and amplified, to the assumption that the mathematical formula will accurately reveal how many of such RNA copies were originally present. The human genome has about 3 billion base pairs, while that of HIV has only about 10,000. Because of this difference, human cells produce a great deal more RNA than HIV does. RNA from human cells could be released in large quantities during times of rapid cell death, which is what occurs during the infectious and inflammatory processes commonly present in people diagnosed HIV-positive... This argument is strengthened by the fact that typical RNA assays look for only about 3% of HIV's genetic material, or about 300 base pairs. Another fact that increases the risk of false positive viral loads is that these tests use RNA sequences that are based on the antibody proteins detected by the ELISA and Western Blot antibody tests. This means that if a person has a false positive or indeterminate result on either of the antibody tests they are also very likely to have a false positive result on the viral load test... Because anti-HIV medications reduce RNA synthesis in a wide variety of cells, the reductions in viral load that accompany the use of these medications may indicate a non-specific reduction in total RNA burden, as opposed to a specific reduction in HIV RNA. This argument is supported by the finding ... that most people with high viral loads do not have culturable/infectious virus, and that even in people who do have culturable virus, between 99.99% and 99.9999% of the viruses are non-culturable and non-infectious..."|