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APTIMA HIV-1 RNA QUALITATIVE ASSAY (APTIMA - HIV-1 RNA QUALITATIVE ASSAY, 2010) Part 2
Dec 5, 2010

To the good Dr. Bob,

I have submitted a donation on-line, and will submit a heftier holiday donation as well, but in the interim I was hoping you might field another inquiry from me regarding a post of mine a few weeks ago pertaining to the Aptima HIV 1 RNA Qualitative Assay. I have a bit more specific questions regarding the general replication of the HIV virus with reference to RNA copies per milliliter during the first several weeks/months after infection.

I've read the package insert for the Gen-Probe Aptima HIV 1 RNA Qualitative Assay with regards to their own study of clinical sensitivity. The study involved 1,041 specimens from four commercial vendors (867 for HIV-1 only and 174 for HIV-1 & HCV). Very important to note of course, during the study, specimens known to contain less than 100 copies/ml of viral RNA were excluded from the analysis and therefore the sensitivity cited was for samples with viral RNA concentration equal to or greater than 100 copies/ml. On all 1,041 specimens, the assay returned reactive results; or 100% for Gen-Probe's particular study.

Analytical sensitivity was also performed by serial dilution of negative human plasma spiked with HIV-1. This study yielded 100% reactive results at 300 copies/ml (715/715), 100% reactive results at 100 copies/ml (718/718), 98.5% reactive results at 30 copies/ml (702/713), 82.6% reactive at 10 copies/ml (592/717), 42.5% at 3 copies/ml (305/717) and 19.4% at 1 copy/ml (139/718).

Understanding that viremia and HIV viral replication is likely very different from one person to the next, however also assuming there have been studies performed on the "average" viral replication after acute infection which you would likely be very familiar with, and considering I received a non-reactive qualitative RNA test at 32 days post potential exposure, I have the following questions:

1) What is the likelihood, if infected, a person would have less than 100 copies/ml of HIV RNA, or even 30 copies/ml, after one month post exposure? I read in one study posted on The Body the following: "On entry to the study, the median duration of HIV infection for those with acute HIV was estimated to be 28 days. At the initial screening visit, blood viral load was significantly higher in men with acute HIV (1,000,000 copies/ml) than men with chronic HIV (100,000 copies/ml)".

I also read on the Johns Hopkins HIV guide the following: "the range of RNA copies/ml can be very wide during acute infection. If the blood is obtained during the symptomatic phase and not everyone is symptomatic, the viral load can be between 100,000 and ten million. It can even be as high as 90 million copies/ml. If the blood is obtained from the individual after seroconversion, the viral load may be as low as 1,000 copies to as high as 500,000 copies."

2) If a person becomes infected, quickly seroconverts and develops antibodies, is it possible that without interference (i.e. no antiretrovirals or PEP) the virus could have spiked already and then been suppressed to under 100 copies/ml of HIV RNA by the body's natural immune system within a month's time - or any amount of time for that matter? In other words, if an RNA qualitative assay becomes reactive within a month, would you expect it to permanently remain reactive without the use of antiretrovirals? I believe I read somewhere, and it wasn't a doctor that posted the information, that if you're going to take a PCR, a DNA PCR is better post 28 days than the RNA PCR because of the potential of the body's natural immune system to suppress the virus to undetectable levels. However with what I've read about the virus, that doesn't seem very plausible. So now I'm confused and wondering if I took the right test or waited too long for the RNA PCR to be accurate at 32 days. What is your opinion?

3) If one is/was presenting with potential ARS syptoms, would you expect that the viral load should then be high enough to produce a reactive result on a qualitative RNA PCR at that time?

4) I read on the CDC the following "Another type of test is an RNA test, which detects the HIV virus directly. The time between HIV infection and RNA detection is 9-11 days." I also read on AVERT the following: "A PCR test can detect the genetic material of HIV rather than the antibodies to the virus, and so can identify HIV in the blood within two or three weeks of infection." So aside from price (which I've already paid), and the chance of a false positive (which didn't occur), what is the downfall of the test for me at this point in time? False negative due to low viral amounts? Technical error? Is my non-reactive result at 32 days pretty reliable? Dr. Bob, I've read your words over and over in the post you provided me stating if a four-week Aptima PCR is undetectable, it would be extremely extremely unlikely a three-month HIV antibody test would be reactive in your experience. Can I relax? The infectious disease doctor performing the test advised that with my one-time exposure risk, combined with the non-reactive qualitative PCR results post 32 days, I could be nearly 99.9% certain I wasn't infected, but for my own peace of mind, take the antibody test > three months.

5) Would any kind of drugs, besides antiretrovirals, or other co-infections, halt the replication of HIV RNA?

Sheesh!!! I've turned into a perpetual worrier...and questioner. Anxiety and stress are through the roof and taking their physical tolls. What do you recommend I do from here regarding testing? In your experience, how accurate do you think a negative qualitative PCR is one month post exposure? Any insights you can offer are, as always, greatly appreciated.

Warm regards and a happy and healthy holiday season to you and yours. Signed, Aptimistaken

Response from Dr. Frascino

Hi Aptimistaken,

You're going to qualify for an advanced degree in nucleic acid polymerase chain reaction laboratory analysis before long. Dude, it's time to turn off your inner scientific geek and come back to planet earth. I can't believe you actually read the Gen-Probe package insert for APTIMA HIV-1 RNA Qualitative Assay! That's got to be more painful than Palin's latest ghost-written babble.

I'll try to briefly respond to your many questions:

1. It would be very unlikely to have less than 30 or 100 copies/ml of HIV RNA one month after infection. I agree with what you read on The Body and on the Johns Hopkins HIV information site.

2. This would be extremely unlikely within a month's time. It can occur in the rare cases called "elite controllers." As for the "right test," the gold standard for HIV-diagnostic testing remains an HIV-antibody test taken outside the window period. The APTIMA HIV-1 RNA Qualitative Assay is the only nucleic acid test approved by the FDA for diagnostic testing.

3. Yes.

4. You don't really have a downside. False negatives are not a problem with PCR testing. Technical error is always possible, but extremely rare. I would assume your 32 day result is indeed "pretty reliable." I absolutely agree with the assessment and advice you received from the infectious disease physician.

5. Antiretroviral drugs can decrease HIV replication, thereby decreasing HIV RNA viral load levels. Co-infections or other drugs would not decrease HIV replication. (Please note HIV replication is quite different from anti-HIV-antibody production.)

If indeed your anxiety and stress are "through the roof" (and I agree they are!), you should seek help for this very real medical problem. Anti-anxiety medications and/or psychotherapy (counseling) can be extremely beneficial.

As for additional testing, I'd recommend you get a three-month HIV-antibody test primarily for peace of mind. I'm confident the result will be negative. As for the accuracy of qualitative PCR testing at one month, none of us have a great deal of personal experience in evaluating the sensitivity or specificity of this test at a specific point in time. As you can probably imagine, it would be difficult to evaluate as you would need to exactly when someone became infected and evaluate this testing technology against others, etc. I would trust the FDA recommendation and guidelines.

Thanks for your ongoing support of The Robert James Frascino AIDS Foundation (www.concertedeffort.org). It's very much appreciated during this holiday season. In return I'm sending you my best good-luck/good-health karma that you are now and will forever be HIV free!

Good luck and Happy Thankschristkwanzaakkah.

Dr. Bob

APTIMA HIV-1 RNA QUALITATIVE ASSAY (APTIMA - HIV-1 RNA QUALITATIVE ASSAY, 2010) Nov 18, 2010

Dear Dr. Bob,

I thank you kindly and sincerely for your efforts, support and the invaluable knowledge you provide to the questioners on this forum. I have been reading this website extensively (over a month now) since my potential exposure date, and have been wavering as to whether or not I should post a question; at least certainly not before being as proactive as I can on my end with the tests currently available to me given the elapsed time. I wont even delve into what I believe to have been the onset and continuation of many ARS symptoms as I recognize actual test results always eclipse symptoms, many of which Im optimistically contributing to stress and anxiety.

My exposure risk, being a male, is unprotected insertive vaginal sex with a female stripper. The circumstances are tired, regrettable and the same; bachelor party, Im not much of a drinker, went back for what I thought would be a lap dance and left the establishment with dreaded horror. I have no way of knowing this womans HIV status or of getting in contact with her. Every day is a living nightmare now especially considering the questionable CDC per act risk of exposure guidelines in light of the Kimberly Powers North Carolina study.

So, seven days post exposure I went to my local Internist and got baseline tested for all STDs including HIV (ELISA). All test results came back negative although I know only Chlamydia and Gonorrhea would be reliable after such a short period of time. I was told to come back to test for HIV (and the remaining STDs) at six weeks, three months, six months and one year. Well, the anxiety has gotten the better of me

I just saw an Infectious Disease doctor (HIV Specialist) in the urban city in which I live. At 32 days post exposure, I took the Aptima HIV-1 RNA Qualitative Assay and just received the results, which were negative. I believe this is the only FDA approved test for diagnostic screening. Although Ive read your comments stating The problem with HIV PCRs is the rate of false-positive results and cost as well as The test, however, is not meant to be used as a stand-alone test for the diagnosis of HIV-1 infection. A positive nucleic acid test should be viewed as a unconfirmed test result, indicating probable infection, and should be followed up later with traditional EIA antibody testing to confirm infection with the Human Immunodeficiency Virus.

Please correct me as this is purely supposition from what Im reading, but is the concern of using these tests for diagnostic purposes the high false positive rate, which would need to be confirmed with further testing; vice receiving a negative qualitative result after 28 daysor 32 days in my case, which some seem to deem conclusive? Or rather, it seems that negative test results for the Aptima HIV-1 RNA Qualitative Assay are potentially more reliable than positive results for diagnostic testing purposes, and Ive read on a few different websites that a negative test at 28 days is conclusive.

Can you kindly provide comment regarding this last paragraph? If nothing else, is this negative result an encouraging sign for me and/or are you able to assign an estimated percentage of the accuracy? The GEN-PROBE website cites a sensitivity confidence interval of 99.6%-100%.

Again, I thank you kindly for any response you can provide. If you would be so kind as to provide a link for donations in your response I will send one promptly.

All the best and continued good health and happiness. Signed, Aptimistaken???

Response from Dr. Frascino

Hi Aptimistaken,

Thanks for your kind comments! You are correct: the Aptima HIV-1 RNA Qualitative Assay is the only RNA PCR test approved by the FDA for HIV-diagnostic screening. You are also correct: the biggest problem with PCR assays is false-positive results and that is why a positive Aptima HIV-1 RNA test is considered a preliminary (unconfirmed) test result and requires a reactive (positive) "traditional" EIA (ELISA) HIV-antibody test before someone is given the diagnosis of HIV positive. False-negative results are not considered a significant problem with PCR assays. As for the exact window period for an Aptima test to be considered conclusive, yes, some Web sites and testing centers claim 28 days. I have not seen convincing scientific evidence that this is completely reliable. Confirming a definitive window period cutoff is extremely difficult, because there are a number of potentially confounding variables. Certainly we've learned from studying the natural history of HIV disease that those who have contracted the virus should have a positive HIV nucleic acid test by day 28. However, there are always unusual situations and rare complicating factors (such as technical or clerical errors) to consider. Nucleic acid testing is also much more difficult and costly to perform. All things considered, the HIV-antibody test taken outside the window period remains the gold standard for HIV diagnostic testing, at least for now.

PCR/nucleic acid testing can be very helpful in diagnosing acute HIV disease (ARS) within the window period. This is where I find the most appropriate use for this type of testing. I'll reprint below some information from the archives about the APTIMA test.

Donation information for The Robert James Frascino AIDS Foundation can be found on the foundation's Web site at www.concertedeffort.org. Thank you for your support. In return I'm sending my good-luck karma that you are now and will always be HIV free!

Be well. WOO-HOO on your negative APTIMA!

Dr. Bob

Another Aptima HIV PCR question Apr 19, 2010

Dr. Vegas here again. Thanks for the info re the Aptima PCR test. Could you answer another couple questions? When is the optimal time (to avoid false negatives) after exposure to do the test? I've been doing PCR's 4 weeks after exposure for both my occupational patients and those with sexual contacts. If the PCR is negative at whatever time is optimal what is the chance of the 3 month antibody test becoming positive? We do 3 month antibody tests on occupational exposures anyways but if the PCR is negative on the others is the 3 month antibody test still necessary?

Response from Dr. Frascino

Hi,

Unfortunately there is no temporal relationship regarding the likelihood of obtaining a false-positive HIV plasma RNA PCR. The problem relates to the test sensitivity and assay methods rather than to anomalies in the plasma.

There have been no controlled studies to scientifically answer your question concerning an undetectable PCR RNA at a specific time followed by a reactive anti-HIV antibody test at a later date. The anti-HIV antibody test at three months remains the gold standard. The Aptima PCR test is FDA approved for diagnosis, and other quantitative HIV PCR RNA tests and qualitative HIV PCR DNA tests have been used to identify infected patients during the acute retroviral syndrome. If a four-week Aptima PCR is undetectable, it would be extremely extremely unlikely a three-month HIV antibody test would be reactive in my experience.

Dr. Bob

Role of HIV PCR (APTIMA HIV-1 RNA QUALITATIVE ASSAY FDA APPROVED FOR DIAGNOSTIC USE) Apr 14, 2010

I am a family physician and first I wanted to commend your frank and frequently humorous answers to your readers questions. The appropriate use of humor when taking care of patients humanizes us as physicians. Continue your good work.

I am the medical director of a Las Vegas occupational/urgent care medicine clinic. We frequently see and manage blood borne pathogen exposures. (as well as the occasional patient who did something in Vegas that they want to stay in Vegas) Most are exceedingly low risk for HIV (needle sticks from diabetes syringes left in trash, etc) Some are slightly higher risk (HIV patients as source). Hepatitis B is a bigger concern but thanks to vaccine it doesn't present a huge problem.

In cases where the risk is slightly higher or the patient is exceptionally anxious I have been doing a 4 week HIV PCR. Our local Quest lab says this test is greater than 95% sensitive at 4 weeks. There are of course different PCR's. Judging from some of your answers it appears you don't recommend them. I agree that the 3 month HIV antibody test is definitive and seals the deal. Is there any role for the PCR's in my setting?

Response from Dr. Frascino

Hi,

Medical director of an urgent care facility in Vegas??? Now that must really be an entertaining position. I love Vegas and visit frequently. I'm always amazed at the often bizarre-appearing folks I see lumbering in and out of casinos on The Strip. I often have the urge to walk up to some of them and just say, "I demand an explanation." But, I digress . . . .

The problem with HIV PCRs is the rate of false-positive results and cost. Only one HIV-1 RNA PCR test is FDA approved for diagnostic screening (so far). It's the Gen-Probe APTIMA assay. (See below.)

Other HIV PCR tests are sometimes used to help diagnose someone who presents with a history of significant HIV exposure and is developing acute retroviral syndrome symptoms while still within the window period (defined as the first three months after exposure).

Hope that helps. Now can you help me with my craps game? I'm still a bit uncertain about all those onetime bets in the center of the table.

Be well. Thanks for appreciating my admittedly twisted sense of humor. Then again, you do live in Vegas, so . . . .

Dr. Bob

Approval of HIV-1 RNA qualitative assay for diagnostic use

The Food and Drug Administration (FDA) announced the approval, on October 5, 2006, of the APTIMA(r) HIV-1 RNA Qualitative Assay, manufactured by Gen-Probe Incorporated of San Diego, California, for use in clinical laboratories and public health facilities to detect primary (early) HIV-1 infection. The APTIMA® HIV-I RNA Qualitative Assay is an in vitro nucleic acid test (NAT) for the detection of human immunodeficiency virus (HIV-1) in human plasma intended for use as an aid in the diagnosis of HIV-I infection, including acute or primary infection, before the appearance of antibodies to HIV-1. Traditional detection and diagnosis of HIV-I infection is based on testing for anti-viral antibodies by enzyme immunoassay (EIA) with confirmation by supplemental antibody tests such as Western blot or immunofluorescence assays (IFA). Although sensitivity of HIV-1 antibody detection has increased in the last few years, a window period between infection and detectable serological markers still exists. Following a recent exposure to HIV-I, it may take several months for the antibody response to reach detectable levels, during which time, testing for antibodies to HIV-I, including the use of rapid antibody tests, will not indicate true infection status. The newly approved test may provide earlier diagnosis of infection because it detects nucleic acid of the human immunodeficiency virus (HIV-1) in human plasma, rather than the antibody response to the virus. Presence of HIV-I RNA in the plasma of patients without antibodies to HIV-I is indicative of acute or primary HIV-1 infection. The test, however, is not meant to be used as a stand-alone test for the diagnosis of HIV-1 infection. A positive nucleic acid test should be viewed as a unconfirmed test result, indicating probable infection, and should be followed up later with traditional EIA antibody testing to confirm infection with the Human Immunodeficiency Virus. In addition, the APTIMA HIV-1 RNA Qualitative Assay may be used as an additional test to confirm HIV-I infection in an individual whose specimen is repeatedly reactive for HIV-1 antibodies. This is important because the Western blot can, in some instances, be difficult to interpret and may not always provide a conclusive result. The APTIMA test can be used instead of the traditional Western blot test or IFA for confirmation of HIV-1 infection when the screening test result for HIV-1 antibodies is positive. The sensitivity of the APTIMA(r) HIV-1 RNA Qualitative Assay is comparable to that of FDA approved viral load assays that measure the amount of HIV-1 virus circulating in the blood of patients with established HIV-1 infection to monitor the treatment and progression of AIDS. Unlike the viral load tests, however, the APTIMA test has been approved for the diagnosis of primary HIV-1 infection, as well as for confirming HIV-1 infection when tests for antibodies to HIV-1 are positive. The product labeling for this test will be available soon on the list of FDA Licensed/Approved HIV, HTLV and Hepatitis Tests on the FDA web site. Richard Klein Office of Special Health Issues Food and Drug Administration



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