HIV AIDS laboratory findings: Difference between revisions

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__NOTOC__
__NOTOC__
[[Image:TEM HIV.jpg|thumb|200px|Thin-section transmission electron micrograph (TEM) depicting the ultrastructural details of two ”human immunodeficiency virus” (HIV) virions]]
{{AIDS}}
{{AIDS}}
{{CMG}} '''Associate Editors-in-Chief:''' [[User:Ujjwal Rastogi|Ujjwal Rastogi, MBBS]] [mailto:urastogi@perfuse.org]
{{CMG}}; '''Associate Editors-in-Chief:''' Ujjwal Rastogi, MBBS,  {{Ammu}}; {{JH}}
==Overview==
==Overview==
A number of laboratory tests are important for initial evaluation of HIV-infected paients. Two surrogate markers ([[AIDS laboratory tests#CD4 T-Cell Count|CD4 T-cell count (CD4 count)]], [[AIDS laboratory tests#Plasma HIV RNA Testing|plasma HIV RNA]]) are routinely used to asses immune function and level of viral [[viremia]].
Important laboratory tests for the initial evaluation of patients with suspected HIV infection include screening tests with high sensitivity such as ELISA, dot blot, and latex agglutination assays, and confirmatory tests with high specificity such as western blot assays, P24 antigen assays, and nucleic acid testing. Two surrogate markers, the CD4 T-cell count (CD4 count) and the plasma HIV RNA viral load, are routinely used to asses immune function and levels of [[viremia]]. Resistance testing is becoming of greater importance in the management of HIV/AIDS patients given the increased resistance to certain antiretroviral agents.


==Laboratory Findings==
==Laboratory Findings==
A number of laboratory tests are important for initial evaluation of HIV-infected patients as follows:
<center>'''Sequence of Appearance of Laboratory Markers for HIV-1 Infection'''</center>
#Upon entry into care.
[[File:Sequence of appearance of laboratory markers for HIV-1 infection.jpg|thumb|center|800px|Note. Units for vertical axis are not noted because their magnitude differs for RNA, p24 antigen, and antibody. Modified from MP Busch, GA Satten (1997)<ref name="pmid9845513">{{cite journal| author=Busch MP, Satten GA| title=Time course of viremia and antibody seroconversion following human immunodeficiency virus exposure. | journal=Am J Med | year= 1997 | volume= 102 | issue= 5B | pages= 117-24; discussion 125-6 | pmid=9845513 | doi= | pmc= | url=http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=9845513  }} </ref> with updated data from Fiebig (2003),<ref name="pmid12960819">{{cite journal| author=Fiebig EW, Wright DJ, Rawal BD, Garrett PE, Schumacher RT, Peddada L et al.| title=Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection. | journal=AIDS | year= 2003 | volume= 17 | issue= 13 | pages= 1871-9 | pmid=12960819 | doi=10.1097/01.aids.0000076308.76477.b8 | pmc= | url=http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=12960819  }} </ref> Owen (2008),<ref name="pmid18322061">{{cite journal| author=Owen SM, Yang C, Spira T, Ou CY, Pau CP, Parekh BS et al.| title=Alternative algorithms for human immunodeficiency virus infection diagnosis using tests that are licensed in the United States. | journal=J Clin Microbiol | year= 2008 | volume= 46 | issue= 5 | pages= 1588-95 | pmid=18322061 | doi=10.1128/JCM.02196-07 | pmc=PMC2395119 | url=http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=18322061  }} </ref> and Masciotra (2011, 2013). <ref name="pmid21981983">{{cite journal| author=Masciotra S, McDougal JS, Feldman J, Sprinkle P, Wesolowski L, Owen SM| title=Evaluation of an alternative HIV diagnostic algorithm using specimens from seroconversion panels and persons with established HIV infections. | journal=J Clin Virol | year= 2011 | volume= 52 Suppl 1 | issue=  | pages= S17-22 | pmid=21981983 | doi=10.1016/j.jcv.2011.09.011 | pmc= | url=http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=21981983  }} </ref><ref name="pmid23911678">{{cite journal| author=Masciotra S, Luo W, Youngpairoj AS, Kennedy MS, Wells S, Ambrose K et al.| title=Performance of the Alere Determine™ HIV-1/2 Ag/Ab Combo Rapid Test with specimens from HIV-1 seroconverters from the US and HIV-2 infected individuals from Ivory Coast. | journal=J Clin Virol | year= 2013 | volume= 58 Suppl 1 | issue=  | pages= e54-8 | pmid=23911678 | doi=10.1016/j.jcv.2013.07.002 | pmc= | url=http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=23911678  }} </ref><br> - Source: https://www.cdc.gov/]]]
#During follow-up if antiretroviral therapy (ART) has not been initiated.
#Prior to and after initiation or modification of therapy to assess virologic and immunologic efficacy of ART.
#To monitor for laboratory abnormalities that may be associated with antiretroviral (ARV) drugs.


*Two surrogate markers are used routinely to assess the immune function and level of HIV viremia:
===Types of HIV Tests===
#[[AIDS laboratory tests#CD4 T-Cell Count|CD4 T-cell count (CD4 count)]]
*'''Diagnostic Testing'''
#[[AIDS laboratory tests#Plasma HIV RNA Testing|Plasma HIV RNA]] (viral load).
# Screening tests
# Supplemental tests
# Confirmatory tests
 
*'''Follow-up Testing'''
# [[CD4]] count
# HIV [[viral load#HIV viral load test|viral load]]
 
*'''Resistance Testing'''
# Phenotypic tests
# Genotypic tests
# Tropism assays
 
===Testing Schedule===
Laboratory testing is important at several stages during an HIV infection:
#At diagnosis
#During follow-up if antiretroviral therapy (ART) has not been initiated
#Prior to and after initiation or modification of therapy to assess virologic and immunologic efficacy of ART
#To monitor for laboratory abnormalities that may be associated with ART
<br>
[[Image:Aids3.jpg|500 px|thumb|center|By WHO infographics]]
[[Image:Aids6.jpg|500 px|thumb|center|By WHO infographics]]
 
==Diagnostic Testing==
====Screening Tests====
Most HIV tests used to screen for HIV infection detect the presence of antibodies against HIV. Detectable antibodies usually develop within 2–8 weeks after infection, but may take longer. There are three different screening tests:
# [[ELISA]] test (based on antigen-antibody and enzyme substrate reactions).
# Rapid Tests ([[Dot blot]] and [[Latex fixation test|Latex Agglutination Tests]]).
# Simple Tests (Particle [[agglutination]] tests).


*[[AIDS laboratory tests#Drug-Resistance Testing|Resistance testing]] should be used to guide selection of an ARV regimen in both ART-naive and ART-experienced patients:
Both simple and rapid tests are readily available and cheaper as compared to ELISA although they may not be as sensitive.
# A viral [[AIDS laboratory tests#Coreceptor Tropism Assays|tropism]] assay should be performed prior to initiation of a CCR5 antagonist.
# [[AIDS laboratory tests#HLA-B*5701 Screening|HLA-B*5701 testing]] should be performed prior to initiation of abacavir (ABC).


Many laboratories  use ''fourth generation'' screening tests which detect anti-HIV  antibody (IgG and IgM) and the HIV p24 antigen. The detection of HIV antibody or antigen in a patient previously known to be negative  is evidence of HIV infection. Individuals whose first specimen indicates evidence of HIV infection will have a repeat test on a second blood sample to confirm the results.
====Supplemental Tests====
These are used to validate results obtained by the screening tests and are of two types:
# [[Western blot]] tests
# [[Immunofluorescence]] tests


==Testing during Window Period==
====Confirmatory Tests====
The window period (the time between initial infection and the development of detectable antibodies against the infection) can vary since it can take 3&ndash;6&nbsp;months to [[seroconversion|seroconvert]] and to test positive. Detection of the virus using polymerase chain reaction ([[PCR]]) during the window period is possible, and evidence suggests that an infection may often be detected earlier than when using a fourth generation EIA screening test. Positive results obtained by PCR are confirmed by antibody tests.<ref name="pmid16706742">{{cite journal
These test aim at the following:
# Demonstration of Viral Antigen (P24).
# Isolation of HIV.
# Detection of viral nucleic acid.
 
The confirmatory tests can diagnose HIV infection even during the [[window period]] (initial two to three weeks of infection), in which both the screening and the supplemental tests fail to diagnose the infection. However these are done in the reference centers thus time consuming and costly.
 
===Important Considerations===
*The window period is the time between the initial infection and the development of detectable antibodies against the virus. This period varies between 1 to 6 months prior to which antibody testing is generally negative and a possible HIV infection can be missed. Detection of the virus using polymerase chain reaction ([[PCR]]) during the window period is preferable, and evidence suggests that an infection may often be detected earlier than when using a fourth generation enzyme immunoassay screening test. Positive results obtained by PCR are later confirmed by antibody tests.<ref name="pmid16706742">{{cite journal
|author=Weber B
|author=Weber B
|title=Screening of HIV infection: role of molecular and immunological assays
|title=Screening of HIV infection: role of molecular and immunological assays
Line 35: Line 68:
|doi=10.1586/14737159.6.3.399
|doi=10.1586/14737159.6.3.399
|url=http://dx.doi.org/10.1586/14737159.6.3.399
|url=http://dx.doi.org/10.1586/14737159.6.3.399
}}</ref> HIV tests are usually performed on venous blood.
}}</ref>  


Routinely used HIV tests for infection in [[neonates]], born to HIV-positive mothers, have no value because of the presence of maternal antibody to HIV in the child's blood. HIV infection can only be diagnosed by PCR, testing for HIV pro-viral DNA in the children's [[lymphocyte]]s.<ref name="pmid11791341">{{cite journal
*Routinely used HIV tests cannot be used in [[neonates]] born to HIV-positive mothers because of the presence of maternal antibodies to HIV in the child's blood. Prior to 18 months, HIV infection can only be diagnosed by PCR, testing for HIV pro-viral DNA.<ref name="pmid11791341">{{cite journal
|author=Tóth FD, Bácsi A, Beck Z, Szabó J
|author=Tóth FD, Bácsi A, Beck Z, Szabó J
|title=Vertical transmission of human immunodeficiency virus
|title=Vertical transmission of human immunodeficiency virus
Line 46: Line 79:
|year=2001
|year=2001
|pmid=11791341
|pmid=11791341
}}</ref>  
}}</ref>
===Importance of Testing Donor Blood Sample===
Many people are unaware that they are infected with HIV.<ref name=Kumaranayake>
 
{{cite journal
| author=Kumaranayake L, Watts C | title=
Resource allocation and priority setting of HIV/AIDS interventions: addressing the generalized epidemic in sub-Saharan Africa |journal=J. Int. Dev. | year=2001 | pages=451&ndash;466 | volume=13 | issue=4 | id={{DOI|10.1002/jid.798}}
 
}}</ref> Less than 1% of the sexually active urban population in Africa has been tested, and this proportion is even lower in rural populations. Furthermore, only 0.5% of pregnant women attending urban health facilities are counseled, tested or receive their test results. Again, this proportion is even lower in rural health facilities.<ref name=Kumaranayake>
 
{{cite journal
| author=Kumaranayake L, Watts C | title=
Resource allocation and priority setting of HIV/AIDS interventions: addressing the generalized epidemic in sub-Saharan Africa |journal=J. Int. Dev. | year=2001 | pages=451&ndash;466 | volume=13 | issue=4 | id={{DOI|10.1002/jid.798}}
 
}}</ref> Therefore, donor blood and blood products used in medicine and medical research are screened for HIV.
 
==Historical Perspective of HIV testing==
 
: 1981: First AIDS case reported
: 1984: Human immunodeficiency Virus (HIV) identified
: 1985: First test for HIV licensed (ELISA)
: 1987: First Western Blot blood test kit
: 1992: First rapid test
: 1994: First oral fluid test
: 1996: First home and urine tests
: 2002: First rapid test using finger prick
: 2003: Rapid finger prick test granted CLIA waiver
: 2004: First rapid oral fluid test (also granted CLIA waiver)
: 2006: CDC recommends routine HIV screening in U.S. health-care settings
: 2007: CDC launches Expanded HIV Testing Initiative in U.S.
: 2007: WHO/UNAIDS global guidelines recommend routine HIV screening in health-care settings
: 2010: First HIV diagnostc test approved that detects both antigen and antibodies
 
==Types==
Laboratory tests for detecting HIV infection are of three types:
# Screening tests
# Supplemental tests
# Confirmatory tests


After confirmation of the diagnosis, severity of disease and rate of progression are estimated by measurement of:
==Follow-up Testing==
# [[CD4]] count.
===CD4 T-Cell Count===
# HIV [[viral load#HIV viral load test|viral load]].
The [[CD4]] count serves as the major laboratory indicator of immune function in patients who have HIV infection. It is one of the key factors in deciding whether to initiate ART and prophylaxis for [[opportunistic infection]]s, and it is the strongest predictor of subsequent disease progression and survival according to clinical trials and cohort studies.<ref name="pmid9182471">Mellors JW, Muñoz A, Giorgi JV, Margolick JB, Tassoni CJ, Gupta P et al. (1997) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=9182471 Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection.] ''Ann Intern Med'' 126 (12):946-54. PMID: [http://pubmed.gov/9182471 9182471]</ref><ref name="pmid12126821">Egger M, May M, Chêne G, Phillips AN, Ledergerber B, Dabis F et al. (2002) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=12126821 Prognosis of HIV-1-infected patients starting highly active antiretroviral therapy: a collaborative analysis of prospective studies.] ''Lancet'' 360 (9327):119-29. PMID: [http://pubmed.gov/12126821 12126821]</ref>


==AIDS Screening Tests==
A significant change (2 standard deviations) between two tests is approximately a 30% change in the absolute count or an increase or decrease in CD4 percentage by 3 percentage points. The CD4 count is one of the most important factors in the decision to initiate ART and/or prophylaxis for opportunistic infections. All patients should have a baseline CD4 count at entry into care.  An adequate CD4 response for most patients on therapy is defined as an increase in CD4 count in the range of 50–150 cells/mm3 per year, generally with an accelerated response in the first 3 months. Subsequent increases in patients with good virologic control show an average increase of approximately 50–100 cells/mm3 per year for the subsequent years until a steady state level is reached.<ref name="pmid14557216">Kaufmann GR, Perrin L, Pantaleo G, Opravil M, Furrer H, Telenti A et al. (2003) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=14557216 CD4 T-lymphocyte recovery in individuals with advanced HIV-1 infection receiving potent antiretroviral therapy for 4 years: the Swiss HIV Cohort Study.] ''Arch Intern Med'' 163 (18):2187-95. [http://dx.doi.org/10.1001/archinte.163.18.2187 DOI:10.1001/archinte.163.18.2187] PMID: [http://pubmed.gov/14557216 14557216]</ref> Patients who initiate therapy with a low CD4 count or at an older age may have a blunted increase in their count despite virologic suppression.  The CD4 cell count response to ART varies widely, but a poor CD4 response is rarely an indication for modifying a virologically suppressive ARV regimen. In patients with consistently suppressed [[viral load]]s who have already experienced ART-related immune reconstitution, the CD4 cell count provides limited information, and frequent testing may cause unnecessary anxiety in patients with clinically inconsequential fluctuations. Thus, for the patient on a suppressive regimen whose CD4 cell count has increased well above the threshold for opportunistic infection risk, the CD4 count can be measured less frequently than the viral load. In such patients, CD4 count may be monitored every 6 to 12 months, unless there are changes in the patient’s clinical status, such as new HIV-associated clinical symptoms or initiation of treatment with [[interferon]], [[corticosteroid]]s, or anti-neoplastic agents.


HIV testing is  mandatory in the U.S. in certain cases:
=====Frequency of CD4 Count Monitoring=====
 
* Blood and organ donors
* Military applicants and active duty personnel
* Federal and state prison inmates under certain circumstances
* Newborns in some states
* As of January 2010, HIV testing is no longer mandatory for those wishing to emigrate to the United States or for refugees
 
Immunodeficient state and side-effects of HIV medication can itself cause various complications which should be monitored during the course of treatment. Monitoring for the development of these complications includes several laboratory and serological tests.
 
Most HIV tests used to screen for HIV infection detect the presence of antibodies against Human Immunodeficiency Virus. Detectable antibodies usually develop within 2–8 weeks after infection, but may take longer; the period after initial infection with HIV before detectable antibodies develop is the “window period”. These are of three types.
# [[ELISA]] test (based on antigen-antibody and enzyme substrate reactions).
# Rapid Tests ([[Dot blot]] and [[Latex fixation test|Latex Agglutination Tests]]).
# Simple Tests (Particle [[agglutination]] tests).
 
Both Simple and Rapid Tests are ready available and cheaper as compared to ELISA.
 
===Home Test===
 
Home Access HIV-1 Test System, the only home HIV test currently approved by the FDA, may be purchased from many drug stores and online. '''Procedure:''' Individual performs the test by pricking finger with a lancet, placing drops of blood on treated card, and mailing to lab for testing. Identification number on card is used when phoning for results; counseling and referral available by phone. '''Results:''' in as little as three days.<ref name="pmid9040298">{{cite journal |author=Frank AP, Wandell MG, Headings MD, Conant MA, Woody GE, Michel C |title=Anonymous HIV testing using home collection and telemedicine counseling. A multicenter evaluation |journal=Arch. Intern. Med. |volume=157 |issue=3 |pages=309–14 |year=1997 |month=February |pmid=9040298 |doi= |url=http://archinte.ama-assn.org/cgi/pmidlookup?view=long&pmid=9040298 |accessdate=2012-02-13}}</ref>
 
===Urine Test===
 
'''Procedure:''' Urine sample collected by health care provider and is tested at lab. Calypte is the only FDA-approved HIV urine test. '''Results:'''  a few days to two weeks.<ref name="pmid11365516">{{cite journal |author= |title=FDA approves urine-based Western blot test for HIV. Food and Drug Administration |journal=AIDS Policy Law |volume=13 |issue=12 |pages=10 |year=1998 |month=June |pmid=11365516 |doi= |url= |accessdate=2012-02-13}}</ref>
 
==AIDS Supplemental Tests==
These are used to validate results obtained by the screening tests and are of two type:
# [[Western blot]] test
# [[Immunofluorescent]] tests.
 
==AIDS confirmatory tests==
These test aim at the following:
# Demonstration of Viral Antigen (P24).
# Isolation of HIV.
# Detection of viral nucleic acid.
 
The confirmatory tests can diagnose HIV infection even during the [[window period]] (initial two to three weeks of infection), in which both the screening and the supplemental tests fail to diagnose the infection. However these are done in the reference centers thus time consuming and costly.
 
==HIV testing in Pregnancy==
*All pregnant women in the United States should be tested for HIV infection as early during pregnancy as possible. A second test during the third trimester, preferably at <36 weeks’ gestation, should be considered for all pregnant women.
 
* A second test is recommended for women in the following conditions:
** Known to be at [[AIDS risk factors#Risk factors and Transmission|high risk]] for acquiring HIV. Women at high risk are:
***Women who use illicit drugs.
*** Women who have STDs during pregnancy.
*** Women who have have multiple sex partners during pregnancy.
*** Women who live in areas with high HIV prevalence.
*** Women who have HIV-infected partners
** Women receiving health care in jurisdictions with elevated incidence of HIV or AIDS among women.
** Women living in facilities in which prenatal screening identifies at least one HIV-infected pregnant women per 1,000 women screened.
*Rapid HIV screening should be performed on any woman in labor who has an undocumented HIV status unless she declines.If a rapid HIV test result is positive in these women, antiretroviral prophylaxis should be administered without waiting for the results of the confirmatory test.
 
===Importance of testing===
*Testing pregnant women is particularly important not only to maintain the health of the patient, but because interventions (i.e., antiretroviral and obstetrical) can reduce the risk for perinatal transmission of HIV.
 
* Evidence indicates that, in the absence of antiretroviral and other interventions, 15%–25% of infants born to HIV-infected mothers will become infected with HIV; such evidence also indicates that an additional 12%–14% of infants born to infected mothers who breastfeed into the second year of life will become infected.<ref name="pmid16088819">{{cite journal |author=Bulterys M, Weidle PJ, Abrams EJ, Fowler MG |title=Combination antiretroviral therapy in african nursing mothers and drug exposure in their infants: new pharmacokinetic and virologic findings |journal=J. Infect. Dis. |volume=192 |issue=5 |pages=709–12 |year=2005 |month=September |pmid=16088819 |doi=10.1086/432490 |url=http://www.jid.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=16088819 |accessdate=2012-02-22}}</ref>
 
===Screening protocol===
*The patient should first be informed that she will be tested for HIV as part of the panel of prenatal tests, unless she declines, or opts-out, of screening.<ref name="pmid15998754">{{cite journal |author=Chou R, Smits AK, Huffman LH, Fu R, Korthuis PT |title=Prenatal screening for HIV: A review of the evidence for the U.S. Preventive Services Task Force |journal=Ann. Intern. Med. |volume=143 |issue=1 |pages=38–54 |year=2005 |month=July |pmid=15998754 |doi= |url=http://www.annals.org/cgi/pmidlookup?view=long&pmid=15998754 |accessdate=2012-02-22}}</ref>
 
*For women who decline, providers should continue to strongly encourage testing and address concerns that pose obstacles to testing.
 
*Women who decline testing because they have had a previous negative HIV test should be informed about the importance of retesting during each pregnancy.
 
===Laboratory test===
*An RNA test should be used in conjunction with an HIV antibody test for women who have signs or symptoms consistent with acute HIV infection.
 
==HIV infection among Infants and Children==
School-age children exposed to HIV before birth are at increased risk for language problems and could benefit from early diagnosis and classroom intervention, according to a new study.
 
*Diagnosis of HIV infection in a pregnant woman indicates the need to consider whether the woman’s other children might be infected.
 
*Infants and young children with HIV infection differ from adults and adolescents with respect to the diagnosis, clinical presentation, and management of HIV disease.
 
*Antibody tests for HIV are expected to be positive in the sera of both infected and uninfected infants born to seropositive mothers.
===Laboratory test===
A definitive determination of HIV infection for an infant aged <18 months is usually based on HIV nucleic acid testing. <ref name="pmid18055670">{{cite journal |author=Read JS |title=Diagnosis of HIV-1 infection in children younger than 18 months in the United States |journal=Pediatrics |volume=120 |issue=6 |pages=e1547–62 |year=2007 |month=December |pmid=18055670 |doi=10.1542/peds.2007-2951 |url=http://pediatrics.aappublications.org/cgi/pmidlookup?view=long&pmid=18055670 |accessdate=2012-02-22}}</ref>
==Laboratory Monitoring Schedule for Patients Prior to and After Initiation of Antiretroviral Therapy ==
{| class=wikitable border=1
!
!Entry into care
!Follow-up before ART
!ART initiation or modification <sup>a</sup>
!2–8 weeks post-ART initiation or modification
!Every 3–6 months
!Every 6 months
!Every 12 months
!Treatment failure
!Clinically indicated
|-
|CD4 count
|style="text-align:center"|√
|every 3–6 months
|style="text-align:center"|√
|style="text-align:center"|--
|style="text-align:center"|√
|colspan=2|In clinically stable patients with suppressed viral load, CD4 count can be monitored every 6–12 months
|style="text-align:center"|√
|style="text-align:center"|√
|-
|Viral load
|style="text-align:center"|√
|every 3–6 months
|style="text-align:center"|√
|style="text-align:center"|√ <sup>b</sup>
|style="text-align:center"|√ <sup>c</sup>
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|√
|style="text-align:center"|√
|-
|Resistance testing
|style="text-align:center"|√
|style="text-align:center"|--
|style="text-align:center"|√ <sup>d</sup>
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|√
|style="text-align:center"|√
|-
|HLA-B*5701 testing
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|√ (if considering ABC)
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|-
|Tropism testing
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|√ (if considering a CCR5 antagonist)
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|if considering a CCR5 antagonist or for failure of regimen
|style="text-align:center"|√
|-
|Hepatitis B serology <sup>e</sup>
|style="text-align:center"|√
|style="text-align:center"|--
|style="text-align:center"|may repeat if HBsAg (-) and HBsAb (-) at baseline
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|√
|-
|Basic chemistry <sup>f</sup>
|style="text-align:center"|√
|style="text-align:center"|every 6–12 months
|style="text-align:center"|√
|style="text-align:center"|√
|style="text-align:center"|√
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|√
|-
|ALT, AST, bilirubin
|style="text-align:center"|√
|style="text-align:center"|every 6–12 months
|style="text-align:center"|√
|style="text-align:center"|√
|style="text-align:center"|√
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|√
|-
|CBC with differential
|style="text-align:center"|√
|style="text-align:center"|every 6–12 months
|style="text-align:center"|√
|style="text-align:center"|√ (if on ZDV)
|style="text-align:center"|√
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|√
|-
|Fasting lipid profile
|style="text-align:center"|√
|style="text-align:center"|if normal annually
|style="text-align:center"|√
|style="text-align:center"|√ (consider 4–8 weeks after starting new ART)
|style="text-align:center"|--
|style="text-align:center"|√ (if abnormal at last measurement)
|style="text-align:center"|√ (if normal at last measurement)
|style="text-align:center"|--
|style="text-align:center"|√
|-
|Fasting glucose
|style="text-align:center"|√
|style="text-align:center"|if normal annually
|style="text-align:center"|√
|style="text-align:center"|--
|style="text-align:center"|√ (if abnormal at last measurement)
|style="text-align:center"|√ (if normal at last measurement)
|style="text-align:center"|--
|style="text-align:center"|--
|style="text-align:center"|√
|-
|Urinalysis <sup>g</sup>
|style="text-align:center"|√
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|Pregnancy test
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*'''a''' : ARV modification may be done for treatment failure, adverse effects, or simplification.
*'''b''' : If HIV RNA is detectable at 2–8 weeks, repeat every 4–8 weeks until suppression to <200 copies/mL, then every 3–6 months.
* '''c''' : For adherent patients with suppressed viral load and stable clinical and immunologic status for >2–3 years, some experts may extend the interval for HIV RNA monitoring to every 6 months.
*'''d''' : For ART-naive patients, if resistance testing was performed at entry into care, repeat testing is optional; for patients with viral suppression who are switching therapy for toxicity or convenience, resistance testing will not be possible and therefore is not necessary.
*'''e''': If HBsAg is positive at baseline or prior to initiation of ART, TDF + (FTC or 3TC) should be used as part of ARV regimen to treat both HBV and HIV infections. If HBsAg and HBsAb are negative at baseline, hepatitis B vaccine series should be administered.
 
*'''f''': Serum Na, K, HCO<sub>3</sub>, Cl, BUN, creatinine, glucose (preferably fasting); some experts suggest monitoring phosphorus while on TDF; determination of renal function should include estimation of creatinine clearance using Cockcroft-Gault equation or estimation of glomerular filtration rate based on MDRD equation.
*'''g''': For patients with renal disease, consult “Guidelines for the Management of Chronic Kidney Disease in HIV-Infected Patients: Recommendations of the HIV Medicine Association of the Infectious Diseases Society of America”.<ref name="pmid15889353">{{cite journal |author=Gupta SK, Eustace JA, Winston JA, Boydstun II, Ahuja TS, Rodriguez RA, Tashima KT, Roland M, Franceschini N, Palella FJ, Lennox JL, Klotman PE, Nachman SA, Hall SD, Szczech LA |title=Guidelines for the management of chronic kidney disease in HIV-infected patients: recommendations of the HIV Medicine Association of the Infectious Diseases Society of America |journal=Clin. Infect. Dis. |volume=40 |issue=11 |pages=1559–85 |year=2005 |month=June |pmid=15889353 |doi=10.1086/430257 |url=http://www.cid.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=15889353 |accessdate=2012-05-07}}</ref>
*'''h''': More frequent monitoring may be indicated for patients with increased risk of renal insufficiency, such as patients with diabetes, hypertension, etc.
==CD4 T-Cell Count==
 
The CD4 count serves as the major laboratory indicator of immune function in patients who have HIV infection. It is one of the key factors in deciding whether to initiate ART and prophylaxis for opportunistic infections, and it is the strongest predictor of subsequent disease progression and survival according to clinical trials and cohort studies.<ref name="pmid9182471">Mellors JW, Muñoz A, Giorgi JV, Margolick JB, Tassoni CJ, Gupta P et al. (1997) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=9182471 Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection.] ''Ann Intern Med'' 126 (12):946-54. PMID: [http://pubmed.gov/9182471 9182471]</ref><ref name="pmid12126821">Egger M, May M, Chêne G, Phillips AN, Ledergerber B, Dabis F et al. (2002) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=12126821 Prognosis of HIV-1-infected patients starting highly active antiretroviral therapy: a collaborative analysis of prospective studies.] ''Lancet'' 360 (9327):119-29. PMID: [http://pubmed.gov/12126821 12126821]</ref>
 
A significant change (2 standard deviations) between two tests is approximately a 30% change in the absolute count or an increase or decrease in CD4 percentage by 3 percentage points.
*'''Use of CD4 Count for Initial Assessment.''' The CD4 count is one of the most important factors in the decision to initiate ART and/or prophylaxis for opportunistic infections. All patients should have a baseline CD4 count at entry into care ('''AI''').
*'''Use of CD4 Count for Monitoring Therapeutic Response.''' An adequate CD4 response for most patients on therapy is defined as an increase in CD4 count in the range of 50–150 cells/mm3 per year, generally with an accelerated response in the first 3 months. Subsequent increases in patients with good virologic control show an average increase of approximately 50–100 cells/mm3 per year for the subsequent years until a steady state level is reached.<ref name="pmid14557216">Kaufmann GR, Perrin L, Pantaleo G, Opravil M, Furrer H, Telenti A et al. (2003) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=14557216 CD4 T-lymphocyte recovery in individuals with advanced HIV-1 infection receiving potent antiretroviral therapy for 4 years: the Swiss HIV Cohort Study.] ''Arch Intern Med'' 163 (18):2187-95. [http://dx.doi.org/10.1001/archinte.163.18.2187 DOI:10.1001/archinte.163.18.2187] PMID: [http://pubmed.gov/14557216 14557216]</ref> Patients who initiate therapy with a low CD4 count or at an older age may have a blunted increase in their count despite virologic suppression.
 
'''Frequency of CD4 Count Monitoring.'''
In general, CD4 counts should be monitored every 3–4 months to:
In general, CD4 counts should be monitored every 3–4 months to:
# Determine when to start ART in untreated patients.
# Determine when to start ART in untreated patients.
# Assess immunologic response to ART.
# Assess immunologic response to ART.
# Assess the need for initiation or discontinuation of prophylaxis for opportunistic infections ('''AI''').
# Assess the need for initiation or discontinuation of prophylaxis for opportunistic infections .
 
The CD4 cell count response to ART varies widely, but a poor CD4 response is rarely an indication for modifying a virologically suppressive ARV regimen. In patients with consistently suppressed viral loads who have already experienced ART-related immune reconstitution, the CD4 cell count provides limited information, and frequent testing may cause unnecessary anxiety in patients with clinically inconsequential fluctuations. Thus, for the patient on a suppressive regimen whose CD4 cell count has increased well above the threshold for opportunistic infection risk, the CD4 count can be measured less frequently than the viral load. In such patients, CD4 count may be monitored every 6 to 12 months, unless there are changes in the patient’s clinical status, such as new HIV-associated clinical symptoms or initiation of treatment with interferon, corticosteroids, or anti-neoplastic agents ('''CIII''').
 
'''Factors that affect absolute CD4 count.''' The absolute CD4 count is a calculated value based on the total white blood cell (WBC) count and the percentages of total and CD4+ T lymphocytes. This absolute number may fluctuate among individuals or may be influenced by factors that may affect the total WBC and lymphocyte percentages, such as use of bone marrow–suppressive medications or the presence of acute infections. Splenectomy <ref name="pmid7795071">Zurlo JJ, Wood L, Gaglione MM, Polis MA (1995) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=7795071 Effect of splenectomy on T lymphocyte subsets in patients infected with the human immunodeficiency virus.] ''Clin Infect Dis'' 20 (4):768-71. PMID: [http://pubmed.gov/7795071 7795071]</ref><ref name="pmid10195261">Bernard NF, Chernoff DN, Tsoukas CM (1998) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=10195261 Effect of splenectomy on T-cell subsets and plasma HIV viral titers in HIV-infected patients.] ''J Hum Virol'' 1 (5):338-45. PMID: [http://pubmed.gov/10195261 10195261]</ref> or coinfection with human T-lymphotropic virus type I (HTLV-1) <ref name="pmid17823752">Casseb J, Posada-Vergara MP, Montanheiro P, Fukumori LM, Olah I, Smid J et al. (2007) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=17823752 T CD4+ cells count among patients co-infected with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1): high prevalence of tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM).] ''Rev Inst Med Trop Sao Paulo'' 49 (4):231-3. PMID: [http://pubmed.gov/17823752 17823752]</ref> may cause misleadingly elevated absolute CD4 counts. Alpha-interferon, on the other hand, may reduce the absolute CD4 number without changing the CD4 percentage.<ref name="pmid1672701">Berglund O, Engman K, Ehrnst A, Andersson J, Lidman K, Akerlund B et al. (1991) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=1672701 Combined treatment of symptomatic human immunodeficiency virus type 1 infection with native interferon-alpha and zidovudine.] ''J Infect Dis'' 163 (4):710-5. PMID: [http://pubmed.gov/1672701 1672701]</ref> In all these cases, CD4 percentage remains stable and may be a more appropriate parameter to assess the patient’s immune function.
=== Use of CD4 in deciding treatment initiating in treatment-naive patients===
Antiretroviral therapy (ART) is recommended for all HIV-infected individuals. The strength of this recommendation varies on the basis of pretreatment CD4 cell count:
* CD4 count <350 cells/mm3 ('''AI''')
* CD4 count 350 to 500 cells/mm3 ('''AII''')
* CD4 count >500 cells/mm3 ('''BIII''')
 
==Plasma HIV RNA Testing==
 
Plasma HIV RNA (viral load) should be measured in all patients at baseline and on a regular basis thereafter, especially in patients who are on treatment, because viral load is the most important indicator of response to antiretroviral therapy (ART) ('''AI'''). Analysis of 18 trials that included more than 5,000 participants with viral load monitoring showed a significant association between a decrease in plasma viremia and improved clinical outcome.<ref name="pmid10357378">Murray JS, Elashoff MR, Iacono-Connors LC, Cvetkovich TA, Struble KA (1999) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=10357378 The use of plasma HIV RNA as a study endpoint in efficacy trials of antiretroviral drugs.] ''AIDS'' 13 (7):797-804. PMID: [http://pubmed.gov/10357378 10357378]</ref> Thus, viral load testing serves as a surrogate marker <ref name="pmid9182469">Hughes MD, Johnson VA, Hirsch MS, Bremer JW, Elbeik T, Erice A et al. (1997) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=9182469 Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response. ACTG 241 Protocol Virology Substudy Team.] ''Ann Intern Med'' 126 (12):929-38. PMID: [http://pubmed.gov/9182469 9182469]</ref> for treatment response and can be useful in predicting clinical progression.<ref name="pmid9419168">Marschner IC, Collier AC, Coombs RW, D'Aquila RT, DeGruttola V, Fischl MA et al. (1998) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=9419168 Use of changes in plasma levels of human immunodeficiency virus type 1 RNA to assess the clinical benefit of antiretroviral therapy.] ''J Infect Dis'' 177 (1):40-7. PMID: [http://pubmed.gov/9419168 9419168]</ref><ref name="pmid10853978">Thiébaut R, Morlat P, Jacqmin-Gadda H, Neau D, Mercié P, Dabis F et al. (2000) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=10853978 Clinical progression of HIV-1 infection according to the viral response during the first year of antiretroviral treatment. Groupe d'Epidémiologie du SIDA en Aquitaine (GECSA).] ''AIDS'' 14 (8):971-8. PMID: [http://pubmed.gov/10853978 10853978]</ref> The minimal change in viral load considered to be statistically significant (2 standard deviations) is a threefold, or a 0.5 log 10 copies/mL change.
 
Optimal viral suppression is generally defined as a viral load persistently below the level of detection (<20–75 copies/mL, depending on the assay used). However, isolated “blips” (viral loads transiently detectable at low levels, typically <400 copies/mL) are not uncommon in successfully treated patients and are not thought to represent viral replication or to predict virologic failure.<ref name="pmid11448280">{{cite journal |author=Havlir DV, Bassett R, Levitan D, Gilbert P, Tebas P, Collier AC, Hirsch MS, Ignacio C, Condra J, Günthard HF, Richman DD, Wong JK |title=Prevalence and predictive value of intermittent viremia with combination hiv therapy |journal=JAMA |volume=286 |issue=2 |pages=171–9 |year=2001 |month=July |pmid=11448280 |doi= |url=http://jama.ama-assn.org/cgi/pmidlookup?view=long&pmid=11448280 |accessdate=2012-05-08}}</ref>
 
In addition, low-level positive viral load results (typically <200 copies/mL) appear to be more common with some viral load assays than others, and there is no definitive evidence that patients with viral loads quantified as <200 copies/mL using these assays are at increased risk for virologic failure.<ref name="pmid19113986">{{cite journal |author=Gatanaga H, Tsukada K, Honda H, Tanuma J, Yazaki H, Watanabe T, Honda M, Teruya K, Kikuchi Y, Oka S |title=Detection of HIV type 1 load by the Roche Cobas TaqMan assay in patients with viral loads previously undetectable by the Roche Cobas Amplicor Monitor |journal=Clin. Infect. Dis. |volume=48 |issue=2 |pages=260–2 |year=2009 |month=January |pmid=19113986 |doi=10.1086/595707 |url=http://www.cid.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=19113986 |accessdate=2012-05-08}}</ref><ref name="pmid17715371">{{cite journal |author=Damond F, Roquebert B, Bénard A, Collin G, Miceli M, Yéni P, Brun-Vezinet F, Descamps D |title=Human immunodeficiency virus type 1 (HIV-1) plasma load discrepancies between the Roche COBAS AMPLICOR HIV-1 MONITOR Version 1.5 and the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 assays |journal=J. Clin. Microbiol. |volume=45 |issue=10 |pages=3436–8 |year=2007 |month=October |pmid=17715371 |pmc=2045351 |doi=10.1128/JCM.00973-07 |url=http://jcm.asm.org/cgi/pmidlookup?view=long&pmid=17715371 |accessdate=2012-05-08}}</ref><ref name="pmid20611035">{{cite journal |author=Willig JH, Nevin CR, Raper JL, Saag MS, Mugavero MJ, Willig AL, Burkhardt JH, Schumacher JE, Johnson VA |title=Cost ramifications of increased reporting of detectable plasma HIV-1 RNA levels by the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 version 1.0 viral load test |journal=J. Acquir. Immune Defic. Syndr. |volume=54 |issue=4 |pages=442–4 |year=2010 |month=August |pmid=20611035 |pmc=2901889 |doi=10.1097/QAI.0b013e3181d01d1d |url=http://meta.wkhealth.com/pt/pt-core/template-journal/lwwgateway/media/landingpage.htm?issn=1525-4135&volume=54&issue=4&spage=442 |accessdate=2012-05-08}}</ref> For the purposes of clinical trials the AIDS Clinical Trials Group (ACTG) currently defines virologic failure as a confirmed viral load >200 copies/mL, which eliminates most cases of apparent viremia caused by [[AIDS medical therapy#Viral blips|blips]] or assay variability. This definition may also be useful in clinical practice.


==Drug-Resistance Testing==
=====Factors that affect absolute CD4 count=====
Genotypic and phenotypic resistance assays are used to assess viral strains and inform selection of treatment strategies. Standard assays provide information on resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs). Testing for integrase and fusion inhibitor resistance can also be ordered separately from several commercial laboratories. No genotypic assays for assessing resistance to CCR5 antagonists are currently commercially available for clinical use in the United States.
The absolute CD4 count is a calculated value based on the total [[white blood cell]] (WBC) count and the percentages of total and CD4+ T lymphocytes. This absolute number may fluctuate among individuals or may be influenced by factors that may affect the total WBC and [[lymphocyte]] percentages, such as use of [[bone marrow]]–suppressive medications or the presence of [[acute]] infections. [[Splenectomy]] <ref name="pmid7795071">Zurlo JJ, Wood L, Gaglione MM, Polis MA (1995) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=7795071 Effect of splenectomy on T lymphocyte subsets in patients infected with the human immunodeficiency virus.] ''Clin Infect Dis'' 20 (4):768-71. PMID: [http://pubmed.gov/7795071 7795071]</ref><ref name="pmid10195261">Bernard NF, Chernoff DN, Tsoukas CM (1998) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=10195261 Effect of splenectomy on T-cell subsets and plasma HIV viral titers in HIV-infected patients.] ''J Hum Virol'' 1 (5):338-45. PMID: [http://pubmed.gov/10195261 10195261]</ref> or coinfection with human T-lymphotropic virus type I ([[HTLV-1]]) <ref name="pmid17823752">Casseb J, Posada-Vergara MP, Montanheiro P, Fukumori LM, Olah I, Smid J et al. (2007) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=17823752 T CD4+ cells count among patients co-infected with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1): high prevalence of tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM).] ''Rev Inst Med Trop Sao Paulo'' 49 (4):231-3. PMID: [http://pubmed.gov/17823752 17823752]</ref> may cause misleadingly elevated absolute CD4 counts. Alpha-interferon, on the other hand, may reduce the absolute CD4 number without changing the CD4 percentage.<ref name="pmid1672701">Berglund O, Engman K, Ehrnst A, Andersson J, Lidman K, Akerlund B et al. (1991) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=1672701 Combined treatment of symptomatic human immunodeficiency virus type 1 infection with native interferon-alpha and zidovudine.] ''J Infect Dis'' 163 (4):710-5. PMID: [http://pubmed.gov/1672701 1672701]</ref> In all these cases, CD4 percentage remains stable and may be a more appropriate parameter to assess the patient’s immune function.


===Genotypic Assays===
===Plasma HIV RNA Testing (HIV Viral Load)===
Genotypic assays detect drug-resistance mutations present in relevant viral genes. Most genotypic assays involve sequencing of the RT and PR genes to detect mutations that are known to confer drug resistance. Genotypic assays that assess mutations in the integrase and gp41 (envelope) genes are also commercially available. Genotypic assays can be performed rapidly with results available within 1–2 weeks of sample collection. Interpretation of test results requires knowledge of the mutations that different ARV drugs select for and of the potential for cross resistance to other drugs conferred by certain mutations. The International AIDS Society-USA (IAS-USA) maintains a list of updated significant resistance-associated mutations in the RT, PR, integrase, and envelope genes.<ref name="pmid10815085">{{cite journal |author=Hirsch MS, Brun-Vézinet F, D'Aquila RT, Hammer SM, Johnson VA, Kuritzkes DR, Loveday C, Mellors JW, Clotet B, Conway B, Demeter LM, Vella S, Jacobsen DM, Richman DD |title=Antiretroviral drug resistance testing in adult HIV-1 infection: recommendations of an International AIDS Society-USA Panel |journal=JAMA |volume=283 |issue=18 |pages=2417–26 |year=2000 |month=May |pmid=10815085 |doi= |url=http://jama.ama-assn.org/cgi/pmidlookup?view=long&pmid=10815085 |accessdate=2012-05-08}}</ref>


Various tools are now available to assist the provider in interpreting genotypic test results. <ref name="pmid17053360">{{cite journal |author=Flandre P, Costagliola D |title=On the comparison of artificial network and interpretation systems based on genotype resistance mutations in HIV-1-infected patients |journal=AIDS |volume=20 |issue=16 |pages=2118–20 |year=2006 |month=October |pmid=17053360 |doi=10.1097/01.aids.0000247585.46567.2d |url= |accessdate=2012-05-08}}</ref><ref name="pmid16782210">{{cite journal |author=Vercauteren J, Vandamme AM |title=Algorithms for the interpretation of HIV-1 genotypic drug resistance information |journal=Antiviral Res. |volume=71 |issue=2-3 |pages=335–42 |year=2006 |month=September |pmid=16782210 |doi=10.1016/j.antiviral.2006.05.003 |url=http://linkinghub.elsevier.com/retrieve/pii/S0166-3542(06)00130-6 |accessdate=2012-05-08}}</ref><ref name="pmid16619162">{{cite journal |author=Gianotti N, Mondino V, Rossi MC, Chiesa E, Mezzaroma I, Ladisa N, Guaraldi G, Torti C, Tarquini P, Castelli P, Di Carlo A, Boeri E, Keulen W, Kenna PM, Lazzarin A |title=Comparison of a rule-based algorithm with a phenotype-based algorithm for the interpretation of HIV genotypes in guiding salvage regimens in HIV-infected patients by a randomized clinical trial: the mutations and salvage study |journal=Clin. Infect. Dis. |volume=42 |issue=10 |pages=1470–80 |year=2006 |month=May |pmid=16619162 |doi=10.1086/503568 |url=http://www.cid.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=16619162 |accessdate=2012-05-08}}</ref><ref name="pmid15909273">{{cite journal |author=Torti C, Quiros-Roldan E, Regazzi M, De Luca A, Mazzotta F, Antinori A, Ladisa N, Micheli V, Orani A, Patroni A, Villani P, Lo Caputo S, Moretti F, Di Giambenedetto S, Castelnuovo F, Maggi P, Tinelli C, Carosi G |title=A randomized controlled trial to evaluate antiretroviral salvage therapy guided by rules-based or phenotype-driven HIV-1 genotypic drug-resistance interpretation with or without concentration-controlled intervention: the Resistance and Dosage Adapted Regimens (RADAR) study |journal=Clin. Infect. Dis. |volume=40 |issue=12 |pages=1828–36 |year=2005 |month=June |pmid=15909273 |doi=10.1086/429917 |url=http://www.cid.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=15909273 |accessdate=2012-05-08}}</ref> Clinical trials have demonstrated the benefit of consultation with specialists in HIV drug resistance in improving virologic outcomes. Clinicians are thus encouraged to consult a specialist to facilitate interpretation of genotypic test results and the design of an optimal new regimen.
*[[Plasma]] HIV RNA (viral load) should be measured in all patients at baseline and on a regular basis thereafter, especially in patients who are on treatment, because viral load is the most important indicator of response to antiretroviral therapy (ART) ('''AI''').
* Analysis of 18 trials that included more than 5,000 participants with viral load monitoring showed a significant association between a decrease in plasma [[viremia]] and improved clinical outcome.<ref name="pmid10357378">Murray JS, Elashoff MR, Iacono-Connors LC, Cvetkovich TA, Struble KA (1999) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=10357378 The use of plasma HIV RNA as a study endpoint in efficacy trials of antiretroviral drugs.] ''AIDS'' 13 (7):797-804. PMID: [http://pubmed.gov/10357378 10357378]</ref>
*The viral load testing serves as a surrogate marker <ref name="pmid9182469">Hughes MD, Johnson VA, Hirsch MS, Bremer JW, Elbeik T, Erice A et al. (1997) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=9182469 Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response. ACTG 241 Protocol Virology Substudy Team.] ''Ann Intern Med'' 126 (12):929-38. PMID: [http://pubmed.gov/9182469 9182469]</ref> for treatment response and can be useful in predicting clinical progression.<ref name="pmid9419168">Marschner IC, Collier AC, Coombs RW, D'Aquila RT, DeGruttola V, Fischl MA et al. (1998) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=9419168 Use of changes in plasma levels of human immunodeficiency virus type 1 RNA to assess the clinical benefit of antiretroviral therapy.] ''J Infect Dis'' 177 (1):40-7. PMID: [http://pubmed.gov/9419168 9419168]</ref><ref name="pmid10853978">Thiébaut R, Morlat P, Jacqmin-Gadda H, Neau D, Mercié P, Dabis F et al. (2000) [http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&retmode=ref&cmd=prlinks&id=10853978 Clinical progression of HIV-1 infection according to the viral response during the first year of antiretroviral treatment. Groupe d'Epidémiologie du SIDA en Aquitaine (GECSA).] ''AIDS'' 14 (8):971-8. PMID: [http://pubmed.gov/10853978 10853978]</ref>
*The minimal change in viral load considered to be statistically significant (2 standard deviations) is a threefold, or a 0.5 log 10 copies/mL change.
*Optimal viral suppression is generally defined as a viral load persistently below the level of detection (<20–75 copies/mL, depending on the [[assay]] used).
*However, isolated “blips” (viral loads transiently detectable at low levels, typically <400 copies/mL) are not uncommon in successfully treated patients and are not thought to represent viral replication or to predict virologic failure.<ref name="pmid11448280">{{cite journal |author=Havlir DV, Bassett R, Levitan D, Gilbert P, Tebas P, Collier AC, Hirsch MS, Ignacio C, Condra J, Günthard HF, Richman DD, Wong JK |title=Prevalence and predictive value of intermittent viremia with combination hiv therapy |journal=JAMA |volume=286 |issue=2 |pages=171–9 |year=2001 |month=July |pmid=11448280 |doi= |url=http://jama.ama-assn.org/cgi/pmidlookup?view=long&pmid=11448280 |accessdate=2012-05-08}}</ref>
*In addition, low-level positive viral load results (typically <200 copies/mL) appear to be more common with some viral load assays than others, and there is no definitive evidence that patients with viral loads quantified as <200 copies/mL using these assays are at increased risk for virologic failure.<ref name="pmid19113986">{{cite journal |author=Gatanaga H, Tsukada K, Honda H, Tanuma J, Yazaki H, Watanabe T, Honda M, Teruya K, Kikuchi Y, Oka S |title=Detection of HIV type 1 load by the Roche Cobas TaqMan assay in patients with viral loads previously undetectable by the Roche Cobas Amplicor Monitor |journal=Clin. Infect. Dis. |volume=48 |issue=2 |pages=260–2 |year=2009 |month=January |pmid=19113986 |doi=10.1086/595707 |url=http://www.cid.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=19113986 |accessdate=2012-05-08}}</ref><ref name="pmid17715371">{{cite journal |author=Damond F, Roquebert B, Bénard A, Collin G, Miceli M, Yéni P, Brun-Vezinet F, Descamps D |title=Human immunodeficiency virus type 1 (HIV-1) plasma load discrepancies between the Roche COBAS AMPLICOR HIV-1 MONITOR Version 1.5 and the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 assays |journal=J. Clin. Microbiol. |volume=45 |issue=10 |pages=3436–8 |year=2007 |month=October |pmid=17715371 |pmc=2045351 |doi=10.1128/JCM.00973-07 |url=http://jcm.asm.org/cgi/pmidlookup?view=long&pmid=17715371 |accessdate=2012-05-08}}</ref><ref name="pmid20611035">{{cite journal |author=Willig JH, Nevin CR, Raper JL, Saag MS, Mugavero MJ, Willig AL, Burkhardt JH, Schumacher JE, Johnson VA |title=Cost ramifications of increased reporting of detectable plasma HIV-1 RNA levels by the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 version 1.0 viral load test |journal=J. Acquir. Immune Defic. Syndr. |volume=54 |issue=4 |pages=442–4 |year=2010 |month=August |pmid=20611035 |pmc=2901889 |doi=10.1097/QAI.0b013e3181d01d1d |url=http://meta.wkhealth.com/pt/pt-core/template-journal/lwwgateway/media/landingpage.htm?issn=1525-4135&volume=54&issue=4&spage=442 |accessdate=2012-05-08}}</ref>  
*For the purposes of clinical trials the AIDS Clinical Trials Group (ACTG) currently defines virologic failure as a confirmed viral load >200 copies/mL, which eliminates most cases of apparent viremia caused by [[AIDS medical therapy#Viral blips|blips]] or assay variability. This definition may also be useful in clinical practice.


===Phenotypic Assays===
==Resistance Testing==
Phenotypic assays measure the ability of a virus to grow in different concentrations of ARV drugs. RT and PR gene sequences and, more recently, integrase and envelope sequences derived from patient plasma HIV RNA are inserted into the backbone of a laboratory clone of HIV or used to generate pseudotyped viruses that express the patient- derived HIV genes of interest. Replication of these viruses at different drug concentrations is monitored by expression of a reporter gene and is compared with replication of a reference HIV strain. The drug concentration that inhibits viral replication by 50% (i.e., the median inhibitory concentration [IC]<sub>50</sub>) is calculated, and the ratio of the IC<sub>50</sub> of test and reference viruses is reported as the fold increase in IC<sub>50</sub> (i.e., fold resistance).
===Phenotypic Testing===
Phenotypic assays measure the ability of a virus to grow in different concentrations of ARV drugs. RT and PR gene sequences and, more recently, integrase and envelope sequences derived from patient plasma HIV RNA are inserted into the backbone of a laboratory clone of HIV or used to generate pseudotyped viruses that express the patient-derived HIV genes of interest. Replication of these viruses at different drug concentrations is monitored by expression of a reporter gene and is compared with replication of a reference HIV strain. The drug concentration that inhibits viral replication by 50% (i.e., the median inhibitory concentration [IC50]) is calculated, and the ratio of the IC50 of test and reference viruses is reported as the fold increase in IC50 (i.e., fold resistance).


Automated phenotypic assays are commercially available with results reported in 2–3 weeks. However, phenotypic assays cost more to perform than genotypic assays. In addition, interpretation of phenotypic assay results is complicated by incomplete information regarding the specific resistance level (i.e., fold increase in IC<sub>50</sub>) that is associated with drug failure, although clinically significant fold increase cutoffs are now available for some drugs. Again, consultation with a specialist can be helpful for interpreting test results.
===Genotypic Testing===
Genotypic assays detect drug-resistance mutations present in relevant viral genes. Most genotypic assays involve sequencing of the RT and PR genes to detect mutations that are known to confer drug resistance. Genotypic assays that assess mutations in the integrase and gp41 (envelope) genes are also commercially available. Genotypic assays can be performed rapidly and results are available within 1 to 2 weeks of sample collection. Interpretation of test results requires knowledge of the mutations selected by different antiretroviral (ARV) drugs and of the potential for cross resistance to other drugs conferred by certain mutations.


Further limitations of both genotypic and phenotypic assays include lack of uniform quality assurance for all available assays, relatively high cost, and insensitivity for minor viral species. Despite being present, drug resistant viruses constituting less than 10%–20% of the circulating virus population will probably not be detected by available assays. This limitation is important because after drugs exerting selective pressure on drug-resistant populations are discontinued, a wild-type virus often reemerges as the predominant population in the plasma. As a consequence, the proportion of virus with resistance mutations decreases to below the 10%–20% threshold.12-14 For some drugs, this reversion to predominantly wild-type virus can occur in the first 4–6 weeks after drugs are stopped. Prospective clinical studies have shown that, despite this plasma reversion, reinstitution of the same ARV agents (or those sharing similar resistance pathways) is usually associated with early drug failure, and the virus present at failure is derived from previously archived resistant virus. Therefore, resistance testing is of greatest value when performed before or within 4 weeks
===Coreceptor Tropism Assays===
after drugs are discontinued ('''AII'''). Because detectable resistant virus may persist in the plasma of some patients for longer periods of time, resistance testing beyond 4 to 6 weeks after discontinuation may still reveal mutations. However, the absence of detectable resistance in such patients must be interpreted with caution in designing subsequent ARV regimens.
* HIV enters cells by a complex process that involves sequential attachment to the [[CD4]] [[receptor]] followed by binding to either the [[CCR5]] or [[CXCR4]] molecules and fusion of the viral and cellular membranes.<ref name="pmid15000703">{{cite journal| author=Moore JP, Kitchen SG, Pugach P, Zack JA| title=The CCR5 and CXCR4 coreceptors--central to understanding the transmission and pathogenesis of human immunodeficiency virus type 1 infection. | journal=AIDS Res Hum Retroviruses | year= 2004 | volume= 20 | issue= 1 | pages= 111-26 | pmid=15000703 | doi=10.1089/088922204322749567 | pmc= | url= }} </ref>
*CCR5 inhibitors (i.e., [[maraviroc]] [MVC]), prevent HIV entry into target cells by binding to the CCR5 receptor.<ref name="pmid16205738">{{cite journal |author=Fätkenheuer G, Pozniak AL, Johnson MA, Plettenberg A, Staszewski S, Hoepelman AI, Saag MS, Goebel FD, Rockstroh JK, Dezube BJ, Jenkins TM, Medhurst C, Sullivan JF, Ridgway C, Abel S, James IT, Youle M, van der Ryst E |title=Efficacy of short-term monotherapy with maraviroc, a new CCR5 antagonist, in patients infected with HIV-1 |journal=Nat. Med. |volume=11 |issue=11 |pages=1170–2 |year=2005 |month=November |pmid=16205738 |doi=10.1038/nm1319 |url=http://dx.doi.org/10.1038/nm1319 |accessdate=2012-05-10}}</ref>
*Phenotypic and, to a lesser degree, genotypic assays have been developed that can determine the coreceptor tropism (i.e., CCR5, CXCR4, or both) of the patient’s dominant virus population.
*The vast majority of patients harbor a CCR5-utilizing virus (R5 virus) during acute/recent infection, which suggests that the R5 variant is preferentially transmitted compared with the CXCR4 (X4) variant.
*Viruses in many untreated patients eventually exhibit a shift in coreceptor tropism from CCR5 to either CXCR4 or both CCR5 and CXCR4 (i.e., dual- or mixed-tropic; D/M-tropic).  
*This shift is temporally associated with a more rapid decline in CD4 T-cell counts, although whether this shift is a cause or a consequence of progressive immunodeficiency remains undetermined.<ref name="pmid9034141">{{cite journal |author=Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR |title=Change in coreceptor use correlates with disease progression in HIV-1--infected individuals |journal=J. Exp. Med. |volume=185 |issue=4 |pages=621–8 |year=1997 |month=February |pmid=9034141 |pmc=2196142 |doi= |url=http://www.jem.org/cgi/pmidlookup?view=long&pmid=9034141 |accessdate=2012-05-10}}</ref><ref name="pmid8096374">{{cite journal |author=Koot M, Keet IP, Vos AH, de Goede RE, Roos MT, Coutinho RA, Miedema F, Schellekens PT, Tersmette M |title=Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS |journal=Ann. Intern. Med. |volume=118 |issue=9 |pages=681–8 |year=1993 |month=May |pmid=8096374 |doi= |url= |accessdate=2012-05-10}}</ref>


===National Institute of Health (NIH) Recommendations for Drug-Resistance Testing===
'''Phenotypic Tropism Assays'''
[http://www.aidsinfo.nih.gov/contentfiles/lvguidelines/AA_Recommendations.pdf]
*There are at least two high-throughput phenotypic assays that can quantify the coreceptor characteristics of [[plasma]]-derived virus.  
*HIV drug-resistance testing is recommended for persons with HIV infection when they enter into care regardless of whether antiretroviral therapy (ART) will be initiated immediately or deferred ('''AIII'''). If therapy is deferred, repeat testing at the time of ART initiation should be considered ('''CIII''').
*Both involve the generation of laboratory viruses that express patient-derived envelope proteins (i.e., [[gp120]] and [[gp41]]).  
*Genotypic testing is recommended as the preferred resistance testing to guide therapy in antiretroviral (ARV)-naive patients ('''AIII''').
*These pseudoviruses are either replication competent (Phenoscript assay, VIRalliance, Paris, France) or replication defective (Trofile assay, Monogram Biosciences, Inc.).  
* Standard genotypic drug-resistance testing in ARV-naive persons involves testing for mutations in the reverse transcriptase (RT) and protease (PR) genes. If transmitted integrase strand transfer inhibitor (INSTI) resistance is a concern, providers may wish to supplement standard genotypic resistance testing with genotypic testing for  resistance to this class of drug ('''CIII''').
*These pseudoviruses then are used to infect target cell lines that express either CCR5 or CXCR4. In the Trofile assay, the coreceptor tropism of the patient-derived virus is confirmed by testing the susceptibility of the virus to specific CCR5 or CXCR4 inhibitors in vitro.  
* HIV drug-resistance testing should be performed to assist in the selection of active drugs when changing ARV regimens in persons with virologic failure and HIV RNA levels >1,000 copies/mL ('''AI'''). In persons with HIV RNA levels >500 but <1,000 copies/mL, testing may be unsuccessful but should still be considered ('''BII''').
*The Trofile assay takes about 2 weeks to perform and requires a plasma HIV RNA level ≥1,000 copies/mL.
* Drug-resistance testing should also be performed when managing suboptimal viral load reduction ('''AII''').
* In persons failing INSTI-based regimens, genotypic testing for INSTI resistance should be considered to determine whether to include a drug from this class in subsequent regimens ('''BIII''').
* Drug-resistance testing in the setting of virologic failure should be performed while the person is taking prescribed ARV drugs or, if not possible, within 4 weeks after discontinuing therapy ('''AII''').
* Genotypic testing is recommended as the preferred resistance testing to guide therapy in patients with suboptimal virologic responses or virologic failure while on first or second regimens ('''AIII''').
* Addition of phenotypic to genotypic testing is generally preferred for persons with known or suspected complex drugresistance mutation patterns, particularly to protease inhibitors (PIs) ('''BIII''').
* Genotypic resistance testing is recommended for all pregnant women prior to initiation of therapy ('''AIII''') and for those entering pregnancy with detectable HIV RNA levels while on therapy ('''AI''').


==HLA-B*5701 Screening==
'''Genotypic Tropism Assays'''
The Hypersensitivity Reaction to Abacavir (ABC HSR) is a multiorgan clinical syndrome typically seen within the initial 6 weeks of ABC treatment. This reaction has been reported in 5%–8% of patients participating in clinical trials when using clinical criteria for the diagnosis, and it is the major reason for early discontinuation of ABC. Discontinuing ABC usually promptly reverses HSR, whereas subsequent rechallenge can cause a rapid, severe, and even life threatening recurrence.<ref name="pmid11726000">{{cite journal| author=Hetherington S, McGuirk S, Powell G, Cutrell A, Naderer O, Spreen B et al.| title=Hypersensitivity reactions during therapy with the nucleoside reverse transcriptase inhibitor abacavir. | journal=Clin Ther | year= 2001 | volume= 23 | issue= 10 | pages= 1603-14 | pmid=11726000 | doi= | pmc= | url=http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=11726000  }} </ref>
*Genotypic determination of HIV-1 coreceptor usage is based on sequencing the V3-coding region of HIV-1 env, the principal determinant of coreceptor usage.
*A variety of algorithms and bioinformatics programs can be used to predict coreceptor usage from the V3 sequence. When compared to the phenotypic assay, genotypic methods show high [[specificity]] (~90%) but only modest [[sensitivity]] (~50%–70%) for the presence of a CXCR4-utilizing virus.
*Given these performance characteristics, these assays may not be sufficiently robust to completely rule out the presence of an X4 or D/M variant.<ref name="pmid20048714">{{cite journal |author=Lin NH, Kuritzkes DR |title=Tropism testing in the clinical management of HIV-1 infection |journal=Curr Opin HIV AIDS |volume=4 |issue=6 |pages=481–7 |year=2009 |month=November |pmid=20048714 |pmc=2874683 |doi=10.1097/COH.0b013e328331b929 |url=http://meta.wkhealth.com/pt/pt-core/template-journal/lwwgateway/media/landingpage.htm?issn=1746-630X&volume=4&issue=6&spage=481 |accessdate=2012-05-10}}</ref>


Studies that evaluated demographic risk factors for ABC HSR have shown racial background as a risk factor, with white patients generally having a higher risk (5%–8%) than black patients (2%–3%). Several groups reported a highly significant association between ABC HSR and the presence of the major histocompatibility complex (MHC) class I allele HLA-B*5701. <ref name="pmid11888582">{{cite journal| author=Mallal S, Nolan D, Witt C, Masel G, Martin AM, Moore C et al.| title=Association between presence of HLA-B*5701, HLA-DR7, and HLA-DQ3 and hypersensitivity to HIV-1 reverse-transcriptase inhibitor abacavir. | journal=Lancet | year= 2002 | volume= 359 | issue= 9308 | pages= 727-32 | pmid=11888582 | doi= | pmc= | url=http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=11888582  }} </ref><ref name="pmid11943262">{{cite journal| author=Hetherington S, Hughes AR, Mosteller M, Shortino D, Baker KL, Spreen W et al.| title=Genetic variations in HLA-B region and hypersensitivity reactions to abacavir. | journal=Lancet | year= 2002 | volume= 359 | issue= 9312 | pages= 1121-2 | pmid=11943262 | doi=10.1016/S0140-6736(02)08158-8 | pmc= | url=http://www.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed&tool=sumsearch.org/cite&retmode=ref&cmd=prlinks&id=11943262  }} </ref> Because the clinical criteria used for ABC HSR are overly sensitive and may lead to false-positive ABC HSR diagnoses, an ABC skin patch test (SPT) was developed as a research tool to immunologically confirm ABC HSR.<ref name="pmid12409746">{{cite journal |author=Phillips EJ, Sullivan JR, Knowles SR, Shear NH |title=Utility of patch testing in patients with hypersensitivity syndromes associated with abacavir |journal=AIDS |volume=16 |issue=16 |pages=2223–5 |year=2002 |month=November |pmid=12409746 |doi= |url=http://meta.wkhealth.com/pt/pt-core/template-journal/lwwgateway/media/landingpage.htm?issn=0269-9370&volume=16&issue=16&spage=2223 |accessdate=2012-05-10}}</ref> A positive ABC SPT is an ABC-specific delayed HSR that results in redness and swelling at the skin site of application. All ABC SPT–positive patients studied were also positive for the HLA-B*5701 allele. The ABC SPT could be falsely negative for some patients with ABC HSR and, at this point, is not recommended for use as a clinical tool.  The PREDICT-1 study randomized patients before starting ABC either to be prospectively screened for HLA-B*5701 (with HLA-B*5701–positive patients not offered ABC) or to standard of care at the time of the study (i.e., no HLA screening, with all patients receiving ABC) <ref name="pmid18256392">{{cite journal |author=Mallal S, Phillips E, Carosi G, Molina JM, Workman C, Tomazic J, Jägel-Guedes E, Rugina S, Kozyrev O, Cid JF, Hay P, Nolan D, Hughes S, Hughes A, Ryan S, Fitch N, Thorborn D, Benbow A |title=HLA-B*5701 screening for hypersensitivity to abacavir |journal=N. Engl. J. Med. |volume=358 |issue=6 |pages=568–79 |year=2008 |month=February |pmid=18256392 |doi=10.1056/NEJMoa0706135 |url=http://dx.doi.org/10.1056/NEJMoa0706135 |accessdate=2012-05-10}}</ref> The overall HLA-B*5701 prevalence in this predominately white population was 5.6%. In this cohort, screening for HLA-B*5701 eliminated immunologic ABC HSR (defined as ABC SPT positive) compared with standard of care (0% vs. 2.7%), yielding a 100% negative predictive value with respect to SPT and significantly decreasing the rate of clinically suspected ABC HSR (3.4% vs. 7.8%). The SHAPE study corroborated the low rate of immunologically validated ABC HSR in black patients and confirmed the utility of HLA-B*5701 screening for the risk of ABC HSR (100%
'''Recommendations for Coreceptor Tropism Assays'''
sensitivity in black and white populations).<ref name="pmid18444831">{{cite journal |author=Saag M, Balu R, Phillips E, Brachman P, Martorell C, Burman W, Stancil B, Mosteller M, Brothers C, Wannamaker P, Hughes A, Sutherland-Phillips D, Mallal S, Shaefer M |title=High sensitivity of human leukocyte antigen-b*5701 as a marker for immunologically confirmed abacavir hypersensitivity in white and black patients |journal=Clin. Infect. Dis. |volume=46 |issue=7 |pages=1111–8 |year=2008 |month=April |pmid=18444831 |doi=10.1086/529382 |url=http://www.cid.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=18444831 |accessdate=2012-05-10}}</ref>
*Coreceptor tropism assay should be performed whenever the use of a CCR5 inhibitor is being considered.
*Coreceptor tropism testing might also be considered for patients who exhibit virologic failure on a CCR5 inhibitor.


On the basis of the results of these studies, the Panel recommends screening for HLA-B*5701 before starting patients on an ABC-containing regimen ('''AI'''). HLA-B*5701–positive patients should not be prescribed ABC ('''AI'''), and the positive status should be recorded as an ABC allergy in the patient’s medical record ('''AII'''). HLA-B*5701 testing is needed only once in a patient’s lifetime; thus, efforts to carefully record and maintain the test result and to educate the patient about its implications are important. The specificity of the HLAB*5701 test in predicting ABC HSR is lower than the sensitivity (i.e., 33%–50% of HLA-B*5701–positive patients would likely not develop confirmed ABC HSR if exposed to ABC). HLA-B*5701 should not be used as a substitute for clinical judgment or pharmacovigilance, because a negative HLA-B*5701 result does not absolutely rule out the possibility of some form of ABC HSR. When HLA-B*5701 screening is not readily available, it remains reasonable to initiate ABC with appropriate clinical counseling and monitoring for any signs of ABC HSR ('''CIII''').
==Gallery==
===NIH Recommendations for HLA-B*5701 Screening===
The Panel recommends screening for HLA-B*5701 before starting patients on an abacavir (ABC)-containing regimen to
reduce the risk of hypersensitivity reaction (HSR) ('''AI''').
* HLA-B*5701-positive patients should not be prescribed ABC ('''AI''').
* The positive status should be recorded as an ABC allergy in the patient’s medical record ('''AII''').
* When HLA-B*5701 screening is not readily available, it remains reasonable to initiate ABC with appropriate clinical counseling and monitoring for any signs of HSR ('''CIII''').


==Coreceptor Tropism Assays==
<gallery>
HIV enters cells by a complex process that involves sequential attachment to the CD4 receptor followed by binding to either the CCR5 or CXCR4 molecules and fusion of the viral and cellular membranes.<ref name="pmid15000703">{{cite journal| author=Moore JP, Kitchen SG, Pugach P, Zack JA| title=The CCR5 and CXCR4 coreceptors--central to understanding the transmission and pathogenesis of human immunodeficiency virus type 1 infection. | journal=AIDS Res Hum Retroviruses | year= 2004 | volume= 20 | issue= 1 | pages= 111-26 | pmid=15000703 | doi=10.1089/088922204322749567 | pmc= | url= }} </ref> CCR5 inhibitors (i.e., maraviroc [MVC]), prevent HIV entry into target cells by binding to the CCR5 receptor.<ref name="pmid16205738">{{cite journal |author=Fätkenheuer G, Pozniak AL, Johnson MA, Plettenberg A, Staszewski S, Hoepelman AI, Saag MS, Goebel FD, Rockstroh JK, Dezube BJ, Jenkins TM, Medhurst C, Sullivan JF, Ridgway C, Abel S, James IT, Youle M, van der Ryst E |title=Efficacy of short-term monotherapy with maraviroc, a new CCR5 antagonist, in patients infected with HIV-1 |journal=Nat. Med. |volume=11 |issue=11 |pages=1170–2 |year=2005 |month=November |pmid=16205738 |doi=10.1038/nm1319 |url=http://dx.doi.org/10.1038/nm1319 |accessdate=2012-05-10}}</ref> Phenotypic and, to a lesser degree, genotypic assays have been developed that can determine the coreceptor tropism (i.e., CCR5, CXCR4, or both) of the patient’s dominant virus population. One assay (Trofile, Monogram Biosciences, Inc., South San Francisco, CA) was used to screen patients who were participating in studies that formed the basis of approval for MVC, the only CCR5 inhibitor currently available. Other assays are under development and are currently used primarily for research purposes or in clinical situations in which the Trofile assay is not readily available.
===Background===
The vast majority of patients harbor a CCR5-utilizing virus (R5 virus) during acute/recent infection, which suggests that the R5 variant is preferentially transmitted compared with the CXCR4 (X4) variant. Viruses in many untreated patients eventually exhibit a shift in coreceptor tropism from CCR5 to either CXCR4 or both CCR5 and CXCR4 (i.e., dual- or mixed-tropic; D/M-tropic). This shift is temporally associated with a more rapid decline in CD4 T-cell counts, 3-4 although whether this shift is a cause or a consequence of progressive immunodeficiency remains undetermined.<ref name="pmid9034141">{{cite journal |author=Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR |title=Change in coreceptor use correlates with disease progression in HIV-1--infected individuals |journal=J. Exp. Med. |volume=185 |issue=4 |pages=621–8 |year=1997 |month=February |pmid=9034141 |pmc=2196142 |doi= |url=http://www.jem.org/cgi/pmidlookup?view=long&pmid=9034141 |accessdate=2012-05-10}}</ref><ref name="pmid8096374">{{cite journal |author=Koot M, Keet IP, Vos AH, de Goede RE, Roos MT, Coutinho RA, Miedema F, Schellekens PT, Tersmette M |title=Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS |journal=Ann. Intern. Med. |volume=118 |issue=9 |pages=681–8 |year=1993 |month=May |pmid=8096374 |doi= |url= |accessdate=2012-05-10}}</ref> Antiretroviral (ARV)-treated patients who have extensive drug resistance are more likely to harbor detectable X4- or D/M-tropic variants than untreated patients who have comparable CD4 T-cell counts.<ref name="pmid16960780">{{cite journal |author=Hunt PW, Harrigan PR, Huang W, Bates M, Williamson DW, McCune JM, Price RW, Spudich SS, Lampiris H, Hoh R, Leigler T, Martin JN, Deeks SG |title=Prevalence of CXCR4 tropism among antiretroviral-treated HIV-1-infected patients with detectable viremia |journal=J. Infect. Dis. |volume=194 |issue=7 |pages=926–30 |year=2006 |month=October |pmid=16960780 |doi=10.1086/507312 |url=http://www.jid.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=16960780 |accessdate=2012-05-10}}</ref> The prevalence of X4- or D/M-tropic variants increases to more than 50% in treated patients who have CD4 counts <100 cells/mm.


===Phenotypic Assays===
Image: HIV_lab01.jpeg| Digitally-colorized scanning electron micrograph (SEM) depicts a single, red-colored H9-T cell that had been infected by numerous, spheroid-shaped, mustard-colored human immunodeficiency virus (HIV) particles. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
There are now at least two high-throughput phenotypic assays that can quantify the coreceptor characteristics of plasma-derived virus. Both involve the generation of laboratory viruses that express patient-derived envelope proteins (i.e., gp120 and gp41). These pseudoviruses are either replication competent (Phenoscript assay, VIRalliance, Paris, France) or replication defective (Trofile assay, Monogram Biosciences, Inc.). These pseudoviruses then are used to infect target cell lines that express either CCR5 or CXCR4. In the Trofile assay, the coreceptor tropism of the patient-derived virus is confirmed by testing the susceptibility of the virus to specific CCR5 or CXCR4 inhibitors in vitro. The Trofile assay takes about 2 weeks to perform and requires a plasma HIV RNA level ≥1,000 copies/mL.


The performance characteristics of these assays have evolved. Most, if not all, patients enrolled in premarketing clinical trials of MVC and other CCR5 inhibitors were screened with an earlier, less sensitive version of the Trofile assay.<ref name="pmid17116663">{{cite journal |author=Whitcomb JM, Huang W, Fransen S, Limoli K, Toma J, Wrin T, Chappey C, Kiss LD, Paxinos EE, Petropoulos CJ |title=Development and characterization of a novel single-cycle recombinant-virus assay to determine human immunodeficiency virus type 1 coreceptor tropism |journal=Antimicrob. Agents Chemother. |volume=51 |issue=2 |pages=566–75 |year=2007 |month=February |pmid=17116663 |pmc=1797738 |doi=10.1128/AAC.00853-06 |url= |accessdate=2012-05-10}}</ref> This earlier assay failed to routinely detect low levels of CXCR4-utilizing variants. As a consequence, some patients enrolled in these clinical trials harbored low, undetectable levels of CXCR4-utilizing viruses at baseline and exhibited rapid virologic failure after initiation of a CCR5 inhibitor.<ref name="pmid16641282">{{cite journal |author=Westby M, Lewis M, Whitcomb J, Youle M, Pozniak AL, James IT, Jenkins TM, Perros M, van der Ryst E |title=Emergence of CXCR4-using human immunodeficiency virus type 1 (HIV-1) variants in a minority of HIV-1-infected patients following treatment with the CCR5 antagonist maraviroc is from a pretreatment CXCR4-using virus reservoir |journal=J. Virol. |volume=80 |issue=10 |pages=4909–20 |year=2006 |month=May |pmid=16641282 |pmc=1472081 |doi=10.1128/JVI.80.10.4909-4920.2006 |url=http://jvi.asm.org/cgi/pmidlookup?view=long&pmid=16641282 |accessdate=2012-05-10}}</ref> This assay has since been revised and is now able to detect lower levels of CXCR4-utlizing viruses. In vitro, the assay can detect CXCR4-utilizing clones with 100% sensitivity when those clones make up 0.3% of the population. Although this more sensitive assay has had limited use in prospective clinical trials, it is now the only one that is commercially available. For unclear reasons, a minority of samples cannot be successfully phenotyped with either generation of the Trofile assay. In patients with plasma HIV-1 RNA below the limit of detection, coreceptor usage can be determined from proviral DNA obtained from peripheral blood mononuclear cells; however, the clinical utility of this assay remains to be determined.
Image: HIV_lab02.jpeg| Digitally-colorized transmission electron micrograph (TEM) depicts a single human immunodeficiency virus (HIV). <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>


===Genotypic Assays===
Image: HIV_lab03.jpeg| Thin section transmission electron micrograph (TEM) depicts numerous virions revealed in a preparation of HIV (human immunodeficiency virus). <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
Genotypic determination of HIV-1 coreceptor usage is based on sequencing the V3-coding region of HIV-1 env, the principal determinant of coreceptor usage. A variety of algorithms and bioinformatics programs can be used to predict coreceptor usage from the V3 sequence. When compared to the phenotypic assay, genotypic methods show high specificity (~90%) but only modest sensitivity (~50%–70%) for the presence of a CXCR4-utilizing virus. Given these performance characteristics, these assays may not be sufficiently robust to completely rule out the presence of an X4 or D/M variant.<ref name="pmid20048714">{{cite journal |author=Lin NH, Kuritzkes DR |title=Tropism testing in the clinical management of HIV-1 infection |journal=Curr Opin HIV AIDS |volume=4 |issue=6 |pages=481–7 |year=2009 |month=November |pmid=20048714 |pmc=2874683 |doi=10.1097/COH.0b013e328331b929 |url=http://meta.wkhealth.com/pt/pt-core/template-journal/lwwgateway/media/landingpage.htm?issn=1746-630X&volume=4&issue=6&spage=481 |accessdate=2012-05-10}}</ref>


Recent studies in which V3 genotyping was performed on samples from patients screening for clinical trials
Image: HIV_lab04.jpeg| Highly magnified transmission electron micrographic (TEM) image reveals the presence of mature forms of the human immunodeficiency virus (HIV) in a tissue sample. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
of MVC suggest that genotyping performed as well as phenotyping in predicting the response to MVC.<ref name="pmid20736814">{{cite journal |author=McGovern RA, Thielen A, Mo T, Dong W, Woods CK, Chapman D, Lewis M, James I, Heera J, Valdez H, Harrigan PR |title=Population-based V3 genotypic tropism assay: a retrospective analysis using screening samples from the A4001029 and MOTIVATE studies |journal=AIDS |volume=24 |issue=16 |pages=2517–25 |year=2010 |month=October |pmid=20736814 |doi=10.1097/QAD.0b013e32833e6cfb |url= |accessdate=2012-05-10}}</ref> On the basis of these data, accessibility, and cost, European guidelines currently favor genotypic testing for determining coreceptor usage. An important caveat to these results is that the majority of patients who received MVC were first shown to have R5 virus by a phenotypic assay (Trofile). Consequently, the opportunity to assess treatment response to MVC in patients whose virus was considered R5 by genotype but D/M or X4 by phenotype was limited to a relatively small number of patients. It is also important to note that the genotyping approaches used in these studies are not routinely available from clinical laboratories in the United States at this time.


Given the uncertainty regarding the genotypic assays and fewer logistical barriers to obtaining a phenotype
Image: HIV_lab05.jpeg| Electron micrograph reveals the presence of the human immunodeficiency virus (HIV-1) which had been co-cultivated with human lymphocytes. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
in the United States than elsewhere, the Panel recommends that a phenotype be used as the preferred
coreceptor tropism screening test in the United States.


Other potential clinical uses for the tropism assay are for prognostic purposes or for assessment of tropism
Image: HIV_lab07.jpeg| Histopathologic changes seen in human skin biopsy specimen due to Kaposi’s sarcoma. <SMALL><SMALL>''[http://phil.cdc.gov/phil/home.asp From Public Health Image Library (PHIL).] ''<ref name=PHIL> {{Cite web | title = Public Health Image Library (PHIL) | url = http://phil.cdc.gov/phil/home.asp}}</ref></SMALL></SMALL>
prior to starting antiretroviral therapy (ART), in case a CCR5 inhibitor is required later (e.g., in a regimen
change for toxicity). Currently, sufficient data do not exist to support these uses.


===NIH Recommendations for Coreceptor Tropism Assays===
</gallery>
*Coreceptor tropism assay should be performed whenever the use of a CCR5 inhibitor is being considered ('''AI''').
*Coreceptor tropism testing might also be considered for patients who exhibit virologic failure on a CCR5 inhibitor ('''CIII''').


==References==
==References==
{{reflist|2}}
{{reflist|2}}


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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1]; Associate Editors-in-Chief: Ujjwal Rastogi, MBBS, Ammu Susheela, M.D. [2]; Jesus Rosario Hernandez, M.D. [3]

Overview

Important laboratory tests for the initial evaluation of patients with suspected HIV infection include screening tests with high sensitivity such as ELISA, dot blot, and latex agglutination assays, and confirmatory tests with high specificity such as western blot assays, P24 antigen assays, and nucleic acid testing. Two surrogate markers, the CD4 T-cell count (CD4 count) and the plasma HIV RNA viral load, are routinely used to asses immune function and levels of viremia. Resistance testing is becoming of greater importance in the management of HIV/AIDS patients given the increased resistance to certain antiretroviral agents.

Laboratory Findings

Sequence of Appearance of Laboratory Markers for HIV-1 Infection
Note. Units for vertical axis are not noted because their magnitude differs for RNA, p24 antigen, and antibody. Modified from MP Busch, GA Satten (1997)[1] with updated data from Fiebig (2003),[2] Owen (2008),[3] and Masciotra (2011, 2013). [4][5]
- Source: https://www.cdc.gov/

]

Types of HIV Tests

  • Diagnostic Testing
  1. Screening tests
  2. Supplemental tests
  3. Confirmatory tests
  • Follow-up Testing
  1. CD4 count
  2. HIV viral load
  • Resistance Testing
  1. Phenotypic tests
  2. Genotypic tests
  3. Tropism assays

Testing Schedule

Laboratory testing is important at several stages during an HIV infection:

  1. At diagnosis
  2. During follow-up if antiretroviral therapy (ART) has not been initiated
  3. Prior to and after initiation or modification of therapy to assess virologic and immunologic efficacy of ART
  4. To monitor for laboratory abnormalities that may be associated with ART


By WHO infographics
By WHO infographics

Diagnostic Testing

Screening Tests

Most HIV tests used to screen for HIV infection detect the presence of antibodies against HIV. Detectable antibodies usually develop within 2–8 weeks after infection, but may take longer. There are three different screening tests:

  1. ELISA test (based on antigen-antibody and enzyme substrate reactions).
  2. Rapid Tests (Dot blot and Latex Agglutination Tests).
  3. Simple Tests (Particle agglutination tests).

Both simple and rapid tests are readily available and cheaper as compared to ELISA although they may not be as sensitive.

Supplemental Tests

These are used to validate results obtained by the screening tests and are of two types:

  1. Western blot tests
  2. Immunofluorescence tests

Confirmatory Tests

These test aim at the following:

  1. Demonstration of Viral Antigen (P24).
  2. Isolation of HIV.
  3. Detection of viral nucleic acid.

The confirmatory tests can diagnose HIV infection even during the window period (initial two to three weeks of infection), in which both the screening and the supplemental tests fail to diagnose the infection. However these are done in the reference centers thus time consuming and costly.

Important Considerations

  • The window period is the time between the initial infection and the development of detectable antibodies against the virus. This period varies between 1 to 6 months prior to which antibody testing is generally negative and a possible HIV infection can be missed. Detection of the virus using polymerase chain reaction (PCR) during the window period is preferable, and evidence suggests that an infection may often be detected earlier than when using a fourth generation enzyme immunoassay screening test. Positive results obtained by PCR are later confirmed by antibody tests.[6]
  • Routinely used HIV tests cannot be used in neonates born to HIV-positive mothers because of the presence of maternal antibodies to HIV in the child's blood. Prior to 18 months, HIV infection can only be diagnosed by PCR, testing for HIV pro-viral DNA.[7]

Follow-up Testing

CD4 T-Cell Count

The CD4 count serves as the major laboratory indicator of immune function in patients who have HIV infection. It is one of the key factors in deciding whether to initiate ART and prophylaxis for opportunistic infections, and it is the strongest predictor of subsequent disease progression and survival according to clinical trials and cohort studies.[8][9]

A significant change (2 standard deviations) between two tests is approximately a 30% change in the absolute count or an increase or decrease in CD4 percentage by 3 percentage points. The CD4 count is one of the most important factors in the decision to initiate ART and/or prophylaxis for opportunistic infections. All patients should have a baseline CD4 count at entry into care. An adequate CD4 response for most patients on therapy is defined as an increase in CD4 count in the range of 50–150 cells/mm3 per year, generally with an accelerated response in the first 3 months. Subsequent increases in patients with good virologic control show an average increase of approximately 50–100 cells/mm3 per year for the subsequent years until a steady state level is reached.[10] Patients who initiate therapy with a low CD4 count or at an older age may have a blunted increase in their count despite virologic suppression. The CD4 cell count response to ART varies widely, but a poor CD4 response is rarely an indication for modifying a virologically suppressive ARV regimen. In patients with consistently suppressed viral loads who have already experienced ART-related immune reconstitution, the CD4 cell count provides limited information, and frequent testing may cause unnecessary anxiety in patients with clinically inconsequential fluctuations. Thus, for the patient on a suppressive regimen whose CD4 cell count has increased well above the threshold for opportunistic infection risk, the CD4 count can be measured less frequently than the viral load. In such patients, CD4 count may be monitored every 6 to 12 months, unless there are changes in the patient’s clinical status, such as new HIV-associated clinical symptoms or initiation of treatment with interferon, corticosteroids, or anti-neoplastic agents.

Frequency of CD4 Count Monitoring

In general, CD4 counts should be monitored every 3–4 months to:

  1. Determine when to start ART in untreated patients.
  2. Assess immunologic response to ART.
  3. Assess the need for initiation or discontinuation of prophylaxis for opportunistic infections .
Factors that affect absolute CD4 count

The absolute CD4 count is a calculated value based on the total white blood cell (WBC) count and the percentages of total and CD4+ T lymphocytes. This absolute number may fluctuate among individuals or may be influenced by factors that may affect the total WBC and lymphocyte percentages, such as use of bone marrow–suppressive medications or the presence of acute infections. Splenectomy [11][12] or coinfection with human T-lymphotropic virus type I (HTLV-1) [13] may cause misleadingly elevated absolute CD4 counts. Alpha-interferon, on the other hand, may reduce the absolute CD4 number without changing the CD4 percentage.[14] In all these cases, CD4 percentage remains stable and may be a more appropriate parameter to assess the patient’s immune function.

Plasma HIV RNA Testing (HIV Viral Load)

  • Plasma HIV RNA (viral load) should be measured in all patients at baseline and on a regular basis thereafter, especially in patients who are on treatment, because viral load is the most important indicator of response to antiretroviral therapy (ART) (AI).
  • Analysis of 18 trials that included more than 5,000 participants with viral load monitoring showed a significant association between a decrease in plasma viremia and improved clinical outcome.[15]
  • The viral load testing serves as a surrogate marker [16] for treatment response and can be useful in predicting clinical progression.[17][18]
  • The minimal change in viral load considered to be statistically significant (2 standard deviations) is a threefold, or a 0.5 log 10 copies/mL change.
  • Optimal viral suppression is generally defined as a viral load persistently below the level of detection (<20–75 copies/mL, depending on the assay used).
  • However, isolated “blips” (viral loads transiently detectable at low levels, typically <400 copies/mL) are not uncommon in successfully treated patients and are not thought to represent viral replication or to predict virologic failure.[19]
  • In addition, low-level positive viral load results (typically <200 copies/mL) appear to be more common with some viral load assays than others, and there is no definitive evidence that patients with viral loads quantified as <200 copies/mL using these assays are at increased risk for virologic failure.[20][21][22]
  • For the purposes of clinical trials the AIDS Clinical Trials Group (ACTG) currently defines virologic failure as a confirmed viral load >200 copies/mL, which eliminates most cases of apparent viremia caused by blips or assay variability. This definition may also be useful in clinical practice.

Resistance Testing

Phenotypic Testing

Phenotypic assays measure the ability of a virus to grow in different concentrations of ARV drugs. RT and PR gene sequences and, more recently, integrase and envelope sequences derived from patient plasma HIV RNA are inserted into the backbone of a laboratory clone of HIV or used to generate pseudotyped viruses that express the patient-derived HIV genes of interest. Replication of these viruses at different drug concentrations is monitored by expression of a reporter gene and is compared with replication of a reference HIV strain. The drug concentration that inhibits viral replication by 50% (i.e., the median inhibitory concentration [IC50]) is calculated, and the ratio of the IC50 of test and reference viruses is reported as the fold increase in IC50 (i.e., fold resistance).

Genotypic Testing

Genotypic assays detect drug-resistance mutations present in relevant viral genes. Most genotypic assays involve sequencing of the RT and PR genes to detect mutations that are known to confer drug resistance. Genotypic assays that assess mutations in the integrase and gp41 (envelope) genes are also commercially available. Genotypic assays can be performed rapidly and results are available within 1 to 2 weeks of sample collection. Interpretation of test results requires knowledge of the mutations selected by different antiretroviral (ARV) drugs and of the potential for cross resistance to other drugs conferred by certain mutations.

Coreceptor Tropism Assays

  • HIV enters cells by a complex process that involves sequential attachment to the CD4 receptor followed by binding to either the CCR5 or CXCR4 molecules and fusion of the viral and cellular membranes.[23]
  • CCR5 inhibitors (i.e., maraviroc [MVC]), prevent HIV entry into target cells by binding to the CCR5 receptor.[24]
  • Phenotypic and, to a lesser degree, genotypic assays have been developed that can determine the coreceptor tropism (i.e., CCR5, CXCR4, or both) of the patient’s dominant virus population.
  • The vast majority of patients harbor a CCR5-utilizing virus (R5 virus) during acute/recent infection, which suggests that the R5 variant is preferentially transmitted compared with the CXCR4 (X4) variant.
  • Viruses in many untreated patients eventually exhibit a shift in coreceptor tropism from CCR5 to either CXCR4 or both CCR5 and CXCR4 (i.e., dual- or mixed-tropic; D/M-tropic).
  • This shift is temporally associated with a more rapid decline in CD4 T-cell counts, although whether this shift is a cause or a consequence of progressive immunodeficiency remains undetermined.[25][26]

Phenotypic Tropism Assays

  • There are at least two high-throughput phenotypic assays that can quantify the coreceptor characteristics of plasma-derived virus.
  • Both involve the generation of laboratory viruses that express patient-derived envelope proteins (i.e., gp120 and gp41).
  • These pseudoviruses are either replication competent (Phenoscript assay, VIRalliance, Paris, France) or replication defective (Trofile assay, Monogram Biosciences, Inc.).
  • These pseudoviruses then are used to infect target cell lines that express either CCR5 or CXCR4. In the Trofile assay, the coreceptor tropism of the patient-derived virus is confirmed by testing the susceptibility of the virus to specific CCR5 or CXCR4 inhibitors in vitro.
  • The Trofile assay takes about 2 weeks to perform and requires a plasma HIV RNA level ≥1,000 copies/mL.

Genotypic Tropism Assays

  • Genotypic determination of HIV-1 coreceptor usage is based on sequencing the V3-coding region of HIV-1 env, the principal determinant of coreceptor usage.
  • A variety of algorithms and bioinformatics programs can be used to predict coreceptor usage from the V3 sequence. When compared to the phenotypic assay, genotypic methods show high specificity (~90%) but only modest sensitivity (~50%–70%) for the presence of a CXCR4-utilizing virus.
  • Given these performance characteristics, these assays may not be sufficiently robust to completely rule out the presence of an X4 or D/M variant.[27]

Recommendations for Coreceptor Tropism Assays

  • Coreceptor tropism assay should be performed whenever the use of a CCR5 inhibitor is being considered.
  • Coreceptor tropism testing might also be considered for patients who exhibit virologic failure on a CCR5 inhibitor.

Gallery

References

  1. ↑ Busch MP, Satten GA (1997). "Time course of viremia and antibody seroconversion following human immunodeficiency virus exposure". Am J Med. 102 (5B): 117–24, discussion 125-6. PMID 9845513.
  2. ↑ Fiebig EW, Wright DJ, Rawal BD, Garrett PE, Schumacher RT, Peddada L; et al. (2003). "Dynamics of HIV viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary HIV infection". AIDS. 17 (13): 1871–9. doi:10.1097/01.aids.0000076308.76477.b8. PMID 12960819.
  3. ↑ Owen SM, Yang C, Spira T, Ou CY, Pau CP, Parekh BS; et al. (2008). "Alternative algorithms for human immunodeficiency virus infection diagnosis using tests that are licensed in the United States". J Clin Microbiol. 46 (5): 1588–95. doi:10.1128/JCM.02196-07. PMC 2395119. PMID 18322061.
  4. ↑ Masciotra S, McDougal JS, Feldman J, Sprinkle P, Wesolowski L, Owen SM (2011). "Evaluation of an alternative HIV diagnostic algorithm using specimens from seroconversion panels and persons with established HIV infections". J Clin Virol. 52 Suppl 1: S17–22. doi:10.1016/j.jcv.2011.09.011. PMID 21981983.
  5. ↑ Masciotra S, Luo W, Youngpairoj AS, Kennedy MS, Wells S, Ambrose K; et al. (2013). "Performance of the Alere Determine™ HIV-1/2 Ag/Ab Combo Rapid Test with specimens from HIV-1 seroconverters from the US and HIV-2 infected individuals from Ivory Coast". J Clin Virol. 58 Suppl 1: e54–8. doi:10.1016/j.jcv.2013.07.002. PMID 23911678.
  6. ↑ Weber B (2006). "Screening of HIV infection: role of molecular and immunological assays". Expert Rev. Mol. Diagn. 6 (3): 399–411. doi:10.1586/14737159.6.3.399. PMID 16706742.
  7. ↑ Tóth FD, Bácsi A, Beck Z, Szabó J (2001). "Vertical transmission of human immunodeficiency virus". Acta Microbiol Immunol Hung. 48 (3–4): 413–27. PMID 11791341.
  8. ↑ Mellors JW, Muñoz A, Giorgi JV, Margolick JB, Tassoni CJ, Gupta P et al. (1997) Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection. Ann Intern Med 126 (12):946-54. PMID: 9182471
  9. ↑ Egger M, May M, Chêne G, Phillips AN, Ledergerber B, Dabis F et al. (2002) Prognosis of HIV-1-infected patients starting highly active antiretroviral therapy: a collaborative analysis of prospective studies. Lancet 360 (9327):119-29. PMID: 12126821
  10. ↑ Kaufmann GR, Perrin L, Pantaleo G, Opravil M, Furrer H, Telenti A et al. (2003) CD4 T-lymphocyte recovery in individuals with advanced HIV-1 infection receiving potent antiretroviral therapy for 4 years: the Swiss HIV Cohort Study. Arch Intern Med 163 (18):2187-95. DOI:10.1001/archinte.163.18.2187 PMID: 14557216
  11. ↑ Zurlo JJ, Wood L, Gaglione MM, Polis MA (1995) Effect of splenectomy on T lymphocyte subsets in patients infected with the human immunodeficiency virus. Clin Infect Dis 20 (4):768-71. PMID: 7795071
  12. ↑ Bernard NF, Chernoff DN, Tsoukas CM (1998) Effect of splenectomy on T-cell subsets and plasma HIV viral titers in HIV-infected patients. J Hum Virol 1 (5):338-45. PMID: 10195261
  13. ↑ Casseb J, Posada-Vergara MP, Montanheiro P, Fukumori LM, Olah I, Smid J et al. (2007) T CD4+ cells count among patients co-infected with human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type 1 (HTLV-1): high prevalence of tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). Rev Inst Med Trop Sao Paulo 49 (4):231-3. PMID: 17823752
  14. ↑ Berglund O, Engman K, Ehrnst A, Andersson J, Lidman K, Akerlund B et al. (1991) Combined treatment of symptomatic human immunodeficiency virus type 1 infection with native interferon-alpha and zidovudine. J Infect Dis 163 (4):710-5. PMID: 1672701
  15. ↑ Murray JS, Elashoff MR, Iacono-Connors LC, Cvetkovich TA, Struble KA (1999) The use of plasma HIV RNA as a study endpoint in efficacy trials of antiretroviral drugs. AIDS 13 (7):797-804. PMID: 10357378
  16. ↑ Hughes MD, Johnson VA, Hirsch MS, Bremer JW, Elbeik T, Erice A et al. (1997) Monitoring plasma HIV-1 RNA levels in addition to CD4+ lymphocyte count improves assessment of antiretroviral therapeutic response. ACTG 241 Protocol Virology Substudy Team. Ann Intern Med 126 (12):929-38. PMID: 9182469
  17. ↑ Marschner IC, Collier AC, Coombs RW, D'Aquila RT, DeGruttola V, Fischl MA et al. (1998) Use of changes in plasma levels of human immunodeficiency virus type 1 RNA to assess the clinical benefit of antiretroviral therapy. J Infect Dis 177 (1):40-7. PMID: 9419168
  18. ↑ Thiébaut R, Morlat P, Jacqmin-Gadda H, Neau D, Mercié P, Dabis F et al. (2000) Clinical progression of HIV-1 infection according to the viral response during the first year of antiretroviral treatment. Groupe d'Epidémiologie du SIDA en Aquitaine (GECSA). AIDS 14 (8):971-8. PMID: 10853978
  19. ↑ Havlir DV, Bassett R, Levitan D, Gilbert P, Tebas P, Collier AC, Hirsch MS, Ignacio C, Condra J, Günthard HF, Richman DD, Wong JK (2001). "Prevalence and predictive value of intermittent viremia with combination hiv therapy". JAMA. 286 (2): 171–9. PMID 11448280. Retrieved 2012-05-08. Unknown parameter |month= ignored (help)
  20. ↑ Gatanaga H, Tsukada K, Honda H, Tanuma J, Yazaki H, Watanabe T, Honda M, Teruya K, Kikuchi Y, Oka S (2009). "Detection of HIV type 1 load by the Roche Cobas TaqMan assay in patients with viral loads previously undetectable by the Roche Cobas Amplicor Monitor". Clin. Infect. Dis. 48 (2): 260–2. doi:10.1086/595707. PMID 19113986. Retrieved 2012-05-08. Unknown parameter |month= ignored (help)
  21. ↑ Damond F, Roquebert B, Bénard A, Collin G, Miceli M, Yéni P, Brun-Vezinet F, Descamps D (2007). "Human immunodeficiency virus type 1 (HIV-1) plasma load discrepancies between the Roche COBAS AMPLICOR HIV-1 MONITOR Version 1.5 and the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 assays". J. Clin. Microbiol. 45 (10): 3436–8. doi:10.1128/JCM.00973-07. PMC 2045351. PMID 17715371. Retrieved 2012-05-08. Unknown parameter |month= ignored (help)
  22. ↑ Willig JH, Nevin CR, Raper JL, Saag MS, Mugavero MJ, Willig AL, Burkhardt JH, Schumacher JE, Johnson VA (2010). "Cost ramifications of increased reporting of detectable plasma HIV-1 RNA levels by the Roche COBAS AmpliPrep/COBAS TaqMan HIV-1 version 1.0 viral load test". J. Acquir. Immune Defic. Syndr. 54 (4): 442–4. doi:10.1097/QAI.0b013e3181d01d1d. PMC 2901889. PMID 20611035. Retrieved 2012-05-08. Unknown parameter |month= ignored (help)
  23. ↑ Moore JP, Kitchen SG, Pugach P, Zack JA (2004). "The CCR5 and CXCR4 coreceptors--central to understanding the transmission and pathogenesis of human immunodeficiency virus type 1 infection". AIDS Res Hum Retroviruses. 20 (1): 111–26. doi:10.1089/088922204322749567. PMID 15000703.
  24. ↑ Fätkenheuer G, Pozniak AL, Johnson MA, Plettenberg A, Staszewski S, Hoepelman AI, Saag MS, Goebel FD, Rockstroh JK, Dezube BJ, Jenkins TM, Medhurst C, Sullivan JF, Ridgway C, Abel S, James IT, Youle M, van der Ryst E (2005). "Efficacy of short-term monotherapy with maraviroc, a new CCR5 antagonist, in patients infected with HIV-1". Nat. Med. 11 (11): 1170–2. doi:10.1038/nm1319. PMID 16205738. Retrieved 2012-05-10. Unknown parameter |month= ignored (help)
  25. ↑ Connor RI, Sheridan KE, Ceradini D, Choe S, Landau NR (1997). "Change in coreceptor use correlates with disease progression in HIV-1--infected individuals". J. Exp. Med. 185 (4): 621–8. PMC 2196142. PMID 9034141. Retrieved 2012-05-10. Unknown parameter |month= ignored (help)
  26. ↑ Koot M, Keet IP, Vos AH, de Goede RE, Roos MT, Coutinho RA, Miedema F, Schellekens PT, Tersmette M (1993). "Prognostic value of HIV-1 syncytium-inducing phenotype for rate of CD4+ cell depletion and progression to AIDS". Ann. Intern. Med. 118 (9): 681–8. PMID 8096374. Unknown parameter |month= ignored (help); |access-date= requires |url= (help)
  27. ↑ Lin NH, Kuritzkes DR (2009). "Tropism testing in the clinical management of HIV-1 infection". Curr Opin HIV AIDS. 4 (6): 481–7. doi:10.1097/COH.0b013e328331b929. PMC 2874683. PMID 20048714. Retrieved 2012-05-10. Unknown parameter |month= ignored (help)
  28. ↑ 28.0 28.1 28.2 28.3 28.4 28.5 "Public Health Image Library (PHIL)".
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