window

“Hey Doc, all these tests are killing me! I’ve done like 8 HIV tests in the last 2 months, but I’m so confused. So when will the tests be conclusive? Some sites are saying there’s a 6 months window period, while some are telling me that 28 days with the HIV Combo or Duo test is conclusive. So what’s your take?”

If you have read my previous short article, you would know by now when is the right time for the right test.

As promised, the following paragraphs would contain the technical, microbiological details on the HIV window period. And hopefully, you’ll understand the science behind the discussion on HIV tests and the various window periods.

Much of the information here is collated from the latest guidelines and publications on HIV testing and research.

 

Window Period for the various HIV tests

Days after exposure to HIV:

Eclipse Period – Day 0-9

This is the initial time period after HIV infection when no laboratory markers are consistently detectable. There are no HIV tests available that can detect HIV infection during this window period. You are strongly advised to consult your Family Physician concerning HIV Post Exposure Prophylaxis (HIV PEP) especially in the first 72 hours after a high-risk exposure (for example, unprotected sexual intercourse with a suspected HIV source).

HIV RNA detectable at Day 10

Approximately 10 days after infection, HIV­1 RNA becomes detectable by NAAT (Nucleic Acid Amplification Test) in blood plasma. Subsequently, its quantity increases to very high levels within the human body.[1] [2] [3] [4] [5]

Your doctor might order a HIV RNA PCR test during this time frame. This test can detect HIV RNA from day 10. Low levels of the HIV virus (a commonly cited cutoff is <5000 copies/mL) may be indicative of a false-positive result and should not be considered diagnostic of primary HIV infection.

This test, however, is not recommended as a standalone test. A standard 3rd generation HIV antibody test should be repeated in 3 to 6 weeks.

HIV p24 Antigen detectable at Day 14-20

Next, HIV-1 p24 antigen is expressed as the HIV virus multiples, and quantities of p24 rise to levels that can be detected by 4th generation tests within 4 to 10 days after the initial detection of HIV-1 RNA.[6] [7]

The HIV p24 antigen peaks at about 20-30 days after exposure.[8] However, the p24 antigen titer continuously decreases after reaching its peak, and subsequently becomes undetectable.

The detection of p24 antigen is transient or temporary because, as the body begins to produce antibodies against the HIV virus, the antibodies bind to the p24 antigen and form immune complexes. This process of antigen-antibody immune complex formation interferes with p24 assay detection.

Therefore, a negative result for p24 antigen assay after antibody seroconversion does not exclude the possibility of HIV infection.

More discussion concerning this test will be found below.

Immunoglobulin M (IgM) antibodies detectable at Day 20-23

The human body eventually produces antibodies against the invading HIV virus. These IgM antibodies can be detected in the body about 3 to 5 days after the p24 antigen is first detectable.[9] [10] This is about 10 to 13 days after the appearance of HIV RNA.

These IgM antibodies can be detected by 3rd and 4th generation HIV tests ordered by your doctor.

Immunoglobulin G (IgG) antibodies detectable at Day 28 to 48

Finally, IgG antibodies are produced by the human body, and persist throughout the course of HIV infection. These IgG antibodies are normally detectable by 1st and 2nd generation tests by the 28th to 48th day after exposure to HIV.[11] [12]

With the current third-generation tests which detect both IgM and IgG antibodies, HIV antibodies can be detected in most individuals within 3 to 4 weeks of viral transmission.[13]

If you are planning to do a 3rd generation HIV test, you can be tested at 4 to 6 weeks following a possible exposure to HIV, with testing repeated at 3 months if negative. It is estimated that >95% of individuals have detectable HIV antibodies by 4 to 6 weeks after infection, with >99% having detectable antibodies by 3 months post-exposure.

Most clinics in Singapore now use 3rd and 4th generation HIV tests, so the window period for HIV detection is much shorter than before.

 

Limitations of the 4th Generation HIV P24 Antigen/Antibody Test

In 4th generation HIV tests, there exist a second window period, also (unsurprisingly) known as the “second window.” This second diagnostic window exists in which viral p24 antigen and antibody levels can fall below the diagnostic threshold for current 4th generation tests, following an initial positive result.

These rare cases of a “second window” occur during early HIV seroconversion. This is when the HIV p24 antigen/antibody combination test result transiently reverts to negative.[14] [15] [16]

Concerning this “second window,” The Centers for Disease Control and Prevention (CDC) report:

“One case of a “second window” of 8 days’ duration was observed in a study of 28 patients in Africa and Thailand with acute, non-B subtype HIV infections identified by frequent RNA testing.[17] Subsequent testing was conducted with immunoassays and viral load assays at frequent intervals. In this case, an FDA-approved 4th generation assay became reactive for antigen at day 9 after RNA detection, was subsequently nonreactive between days 17-25, and became reactive again at day 29 when antibody levels, detected by a 3rd generation assay, began to rise. Presumably, this phenomenon was due to the short interval when antibodies begin to appear during which antigen bound to antibody inhibits detection of either antigen or antibody by the assays. Experience with the FDA-approved 4th generation immunoassays is too limited to predict whether or how often transient seroreversion might occur in patients with subtype B infections.” – Laboratory Testing for the Diagnosis of HIV Infection: Updated Recommendations, page 23

This is one of the reasons why a negative rapid 4th generation HIV test result should be interpreted cautiously, and repeat testing done if necessary.

There also exists the possibility of a variant strain of HIV that does not react to the specific antigens used in the 4th generation assay. In addition, individuals with severe immunosuppression, as well as those taking highly active antiretroviral therapy (HAART), can have negative screening results.

Currently, both the UK and CDC’s guidelines do not consider the rapid HIV p24 antigen/antibody test (also known as the rapid HIV Combo or rapid HIV Duo test) conclusive at 28 days post-exposure. An early negative result at 28 days (1 month) needs to be confirmed with a second test 90 days (3 months) after the exposure.

Screening and supplemental test results should always be interpreted in conjunction with the person’s clinical presentation, medical history, and other laboratory results.

 

Footnotes

[1] Keele  BF,  Giorgi  EE,  Salazar-­Gonzalez  JF,  et  al.  Identification  and  characterization  of  transmitted  and  early  founder  virus   envelopes  in  primary  HIV-­1  infection.  Proc  Natl  Acad  Sci  U  S  A.  2008;;105(21):7552-­7.  Available  at:   http://www.ncbi.nlm.nih.gov/pubmed/18490657.

[2] Lee  HY,  Giorgi  EE,  Keele  BF,  et  al.  Modeling  sequence  evolution  in  acute  HIV-­1  infection.  J  Theor  Biol.  2009;;261(2):341-­ 60.  Available  at:  http://www.ncbi.nlm.nih.gov/pubmed/19660475.

[3] Lindback  S,  Karlsson  AC,  Mittler  J,  et  al.  Viral  dynamics  in  primary  HIV-­1  infection.  Karolinska  Institutet  Primary  HIV   Infection  Study  Group.  AIDS.  2000;;14(15):2283-­91.  Available  at:  http://www.ncbi.nlm.nih.gov/pubmed/11089616.

[4] Lindback  S,  Thorstensson  R,  Karlsson  AC,  et  al.  Diagnosis  of  primary  HIV-­1  infection  and  duration  of  follow-­up  after  HIV   exposure.  Karolinska  Institute  Primary  HIV  Infection  Study  Group.  AIDS.  2000;;14(15):2333-­9.  Available  at:   http://www.ncbi.nlm.nih.gov/pubmed/11089621.

[5] Vermeulen  M,  Coleman  C,  Mitchel  J,  et  al.  Comparison  of  human  immunodeficiency  virus  assays  in  window  phase  and  elite   controller  samples:  viral  load  distribution  and  implications  for  transmission  risk.  Transfusion.  2013;;53(10  Pt  2):3284-­98.   Available  at:  http://www.ncbi.nlm.nih.gov/pubmed/23445273.

[6] Masciotra  S,  McDougal  JS,  Feldman  J,  Sprinkle  P,  Wesolowski  L,  Owen  SM.  Evaluation  of  an  alternative  HIV  diagnostic   algorithm  using  specimens  from  seroconversion  panels  and  persons  with  established  HIV  infections.  J  Clin  Virol.   2011;;52(Suppl  1):S17-­22.  Available  at:  http://www.ncbi.nlm.nih.gov/pubmed/21981983.

[7] Fiebig  EW,  Wright  DJ,  Rawal  BD,  et  al.  Dynamics  of  HIV  viremia  and  antibody  seroconversion  in  plasma  donors:   implications  for  diagnosis  and  staging  of  primary  HIV  infection.  AIDS.  2003;;17(13):1871-­9.  Available  at:   http://www.ncbi.nlm.nih.gov/pubmed/12960819.

[8] Busch MP, Satten GA. Time course of viremia and antibody seroconversion following human immunodeficiency virus exposure. Am J Med. 1997;102:117–124. discussion 125-6.

[9] Owen  SM,  Yang  C,  Spira  T,  et  al.  Alternative  algorithms  for  human  immunodeficiency  virus  infection  diagnosis  using  tests   that  are  licensed  in  the  United  States.  J  Clin  Microbiol.  2008;;46(5):1588-­95.  Available  at:   http://www.ncbi.nlm.nih.gov/pubmed/18322061.

[10] Tomaras  GD,  Yates  NL,  Liu  P,  et  al.  Initial  B-­cell  responses  to  transmitted  human  immunodeficiency  virus  type  1:  virion-­ binding  immunoglobulin  M  (IgM)  and  IgG  antibodies  followed  by  plasma  anti-­gp41  antibodies  with  ineffective  control  of  initial   viremia.  J  Virol.  2008;;82(24):12449-­63.  Available  at:  http://www.ncbi.nlm.nih.gov/pubmed/18842730.

[11] Louie  B,  Wong  E,  Klausner  JD,  et  al.  Assessment  of  rapid  tests  for  detection  of  human  immunodeficiency  virus-­specific   antibodies  in  recently  infected  individuals.  J  Clin  Microbiol.  2008;;46(4):1494-­7.  Available  at:   http://www.ncbi.nlm.nih.gov/pubmed/18234875.

[12] Delaney  KP,  Branson  BM,  Uniyal  A,  et  al.  Evaluation  of  the  performance  characteristics  of  6  rapid  HIV  antibody  tests.  Clin   Infect  Dis.  2011;;52(2):257-­63.  Available  at:  http://www.ncbi.nlm.nih.gov/pubmed/21288853.

[13] Constantine NT, van der Groen G, Belsey EM, Tamashiro H. Sensitivity of HIV-antibody assays determined by seroconversion panels. AIDS. Dec 1994;8(12):1715-20.

[14] Niederhauser C, Strohle A, Stolz M, Muller F, Tinguely C. The risk of a second diagnostic window with 4th generation HIV assays: Two cases. J Clin Virol. 2009;45(4):367-9. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19546027.

[15] Speers D, Phillips P, Dyer J. Combination assay detecting both human immunodeficiency virus (HIV) p24 antigen and anti- HIV antibodies opens a second diagnostic window. J Clin Microbiol. 2005;43(10):5397-9. Available at: http://www.ncbi.nlm.nih.gov/pubmed/16208030.

[16] Meier T, Knoll E, Henkes M, Enders G, Braun R. Evidence for a diagnostic window in fourth generation assays for HIV. J Clin Virol. 2001;23(1-2):113-6. Available at: http://www.ncbi.nlm.nih.gov/pubmed/11595590.

[17] Eller LA, Manak M, Malia JA, et al. Reduction of HIV window period by 4th gen HIV combination tests. Poster O-142, Conference on Retroviruses and Opportunistic Infections 2013; http://hivresearch.org/media/pnc/7/media.737.pdf. Accessed April 16, 2014.