Acquired Immunodeficiency Syndrome - Analytical Chemistry (ACS

Acquired Immunodeficiency Syndrome. Max R. Proffitt. Anal. Chem. , 1993, 65 (12), pp 396–400. DOI: 10.1021/ac00060a605. Publication Date: June 1993...
0 downloads 0 Views 2MB Size
CLINICAL CHEMISTRY

Acquired Immunodeficiency - Syndrome -

Max R. Proffitt Departments of Clinical Pathology and Immunolog , The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, &hi0 44295-5140 Acquired immunodeficiencysyndrome (AIDS) is acomplex disease whose root cause is immunological degeneration mediated by a retrovirus, the human immunodeficiencyvirus (HIV). The diagnosis AIDS is based on clinical data supplemented with laboratory documentation of HIV infection (FZ). Inthe followingdiscussion1will firstreviewaspects of the epidemiology and clinical manifestations of HIV infection and AIDS. Becauseofthe vastness of the literature, I have made no attempt toencompass the many opportunistic diseases that in the presence of documented HIV infection define AIDS. This has been done recently in great detail elsewhere (F2-F6). The accurate detection of HIV infection is the key component in defining AIDS and AIDS-associated conditions. I will, thus, focus the remainder of my review on current testing options and algorithms for diagnosing HIV infection and discuss some of the dilemmas that may he encountered in attempting to properly perform and interpret the tests. The laboratory diagnosis of HIV infection relies primarily on serological methods. All of the tests thus far licensed by the FDA are for the detection of antibodies to HIV with the exception of a qualitative test for an HIV core protein, p24. The latter is also a serological test, intended to detect the antigen in serum or plasma. Most other tests, especially those for the detection of the virus, its genes, or viral gene products, are doneprimarilyinresearchlaboratories or highlyspecialized reference laboratories. There are several recent sources for this information (F7-FZO). For the purposes of this review, only the serological diagnosis of HIV infection with FDA-licensed tests will he discussed. This review will bring the reader up to date (as of October 1992) on the aforementioned topics. EPIDEMIOLOGY AND CLINICAL MANIFESTATIONS O F AIDS Within the last decade we have observed the evolution of a new disease, acquired immunodeficiency syndrome. We have seen AIDS progress from a disease affecting a few homosexual males in San Francisco and New York (FZZFZ4) to an alarming global andemic affecting men, women, and children of all ages an of all major ethnic groups (FZ5FZ9). Today,thediseaseAIDS,andHIV, thevirusthatcauses AIDS, affect not only youn homosexual males, but hemophiliacs, past recipients of lood transfusions, intravenous drug users (IVDUs), sexual partners of an HIV-infected person, and infants born to mothers from either of the latter two groups. More alarmingly, HIV infection is beginning to appear in parts of the rural United States in non-drug-using, pregnant women who have no other sexually transmitted diseases (STD) and who have no known high-risk sex partners (FZO). As in many other parts of the world (e.&, Africa), HIV now bas clearly shown its potential for spread in the United States by heterosexual routes not linked with other risk factors. Evidence indicates that well over 1 million cases of AIDS have already occurred worldwide. Moreover, the Harvard Global AIDS Policy Coalition estimates that an additional 12.9 million men, women, and children are now infected with HIV (FZ9). The Coalition projects that between 38 million and 110 million adults and more than 10 million children will he infected with the virus by the year 2000. A vaccine to prevent spread of HIV may he years away (FZ9),and in the absence of more effective therapies, few doubt that the majority of those infected will develop a variety of HIVassociated conditions and eventually AIDS. When first seen in young homosexual males, AIDS presented as Pneumbqstrs rorinii pneumonia (PCPJ or PCP and Kaposi's 3arcoma (KSJ. Prior to the AIDS era, these opportunistic diseases occurred mainly in immunosuppressed patients such as organ transplant recipients, those undergoing chemotherapy, or those with genetically determined immune deficiencies. RarelywerePCPor KSseeninotherwise healthy young individuals (FZZ-FZ4). However, these were not otherwise healthy men, for they also were found to have a

B %

386R

ANALYTICAL CHEMISTRY, VOL. 65. NO. 12. JUNE 15. 1993

Max R. PrMM has been a member of the Drofessional staff of The Cleveland Clinic

since 1976. He presently is responsible for the activlties of the laboratories of Clinical Virology and Retrovirology In the Department of Cllnical Pathology. He holds a secondary appointment in the Department of Immunology of the Research InstBute of The Cleveland Clinic. He received his Ph.D. from the University of Tennessee and his postgraduate training in the Infectious Disease Unit at the Massachusetts General Hospital and HarvardMedicalSchool. HeisaformerFelbw @ ' of the Cancer Research Fund, Damon Runyon-Walter Wincheli Foundation, and a former Fellow and Scholar of The Leukemia Society of America. He is a member of several professional societies including the American Association of Immunologists. American Society for Microbiology. American Association for the Advancement of Science, New York Academy 01 Sciences. International Association for Comparative Research in Leukemia and Related Diseases. and Pan American Group for RapM Vlral Diagnosis. Hi3 majwresearch interests reiatetocllnicaldiagnosisof viral Infections, to retroviruses, and to viral interactions with the immune system.

1 3

profound depression, without known cause, of a vital subset of immunoregulatorylymphocytesknownasT4 (CD4+)helper cells without known cause (FZZ). Depression of CD4+T cells is now a hallmark of advanced HIV infection and AIDS (FZ, FZZ). As the medical community discerned that this was a new disease syndrome-immune depression accompanied by a depressed CD4+ T-cell numbers in the presence of an opportunistic infection or a cancer such as Kaposi's sarcoma and in the absence of other apparent causes of immunodeficiency-it was designated "acquired immunodeficiency syndrome". The formal definition of AIDS has since been revised by the Centers for Disease Control (CDC) to include a much broader range of opportunistic infections and malignancies in the presence of laboratory-documented HIV infection (FZ). In January 1993, the Centers for Disease Control further expanded the definition of AIDS to include all HIV-infected persons with CD4+ T-lymphocyte counts helow 200Imm3 and added pulmonary tuberculosis, recurrent pneumonia, and invasive cervical cancer to the current list of AIDS-defining conditions (FZZ). With awareness that certain groups of individuals were a t highriskofdeveloping AIDScamerealization thattherewere also those who had persistent generalized lymphadeno athy (PGL), but were otherwise healthy, and those with &irelated complex (ARC) who had constitutional symptoms and infections that were not AIDS-defining. In most instances, those conditions eventually progressed to definable AIDS (F23, F24). Because the terms PGL. ARC. and AIDS have not alwavs been uniformly used in the sake way, and because HfV infection results in a much broader range of conditions than was originally appreciated, classification schemes that stratify HIV infection by the severityof associated medical conditions haveheencreated (F25,F26). Thisisactuallyamorepractical way of viewing AIDS since the course of HIV disease can range from an acute flulike syndrome characterized by viremia, to an often prolonged asymptomatic phase, to an array of clinical conditions that may he mild or may he severe and life-threatening ( F 2 7 4 3 2 ) . Within a short time of its appearance in the United States, AIDS was recognized as a major problem in Africa and in Haiti as well (F33,F34). Evidence that AIDS was caused by an infectious agent, probably one transmitted by blood and by sexual contact, accumulated rapidly. ISOLATION A N D CHARACTERIZATION OF HIV In 1983and 1984scientists in France and the United States isolated and characterized cytopathic retroviruses from

CLINICAL CHEMISTRY

atients with AIDS and lymphadenopathy s drome (F35$37). Various names given to the isolatesb t c i r discoverers human T-cell included lymphadenopath virus (LAV) (I$$), lymphotropic virus type If1 (HTLV-111) (F36),and AIDSassociated retrovirus (ARV) 0737). Subsequently, a subcommittee of the International committee on Taxonomy of Viruses recommended that the various isolates be uniformly designated human immunodeficiency virus (8'38). Once isolated, HIV was grown in vitro in sufficient quantities for further characterization and for use as antigen in the preparation of serologic tests to detect HIV infection (F39). Because recipients of blood products had been identified early in the epidemic as being at risk of HIV infection, screening tests for the detection of antibodies to HIV based on enzyme immunoassay (EIA) techno10 were ra idly developed, and the first of these was licensefiy the UIFDA for use in blood banking in 1985 (F40).Since the implementation of this testin the threat to the blood supply has been reduced dramaticaby (F41-F43). In 1986, a distinctly different subtype of HIV, HIV-2 was isolated from West African AIDS patients (F44).Altho h much less prevalent than HIV-1, HIV-2 ap ears to?e transmitted by the same routes and is aduafy becoming globally disseminated. It is also provigng new challenges for the blood supply and for diagnosis (F45).

LABORATORY DIAGNOSIS OF HIV INFECTIONS The number and variety of highly sensitive and specific tests to assist the accurate diagnosis of HIV infection have increased substantially since the first tests were licensed. To date, the test of only one manufacturer (Abbott) has been licensed by the FDA for the qualitative detection of p24 antigen, a core protein of HIV. Other tests for the detection of the virus, its roducts, or its enes are currently undergoing or pending F A review for icensure. It is important to emphasize that among the testa that are licensed, all are serolo ical assa 8. With the exception of the test for HIV p24, tfie other {censed tests detect antibodies to HIV and only indirectly indicate that infection has occurred. The antibody tests assist diagnosis, but in the absence of other indicators may not always be definitive. Because the results of these tests are only one component of a di osis of HIV infection, it is incorrect to refer to them as A ' %S tests". All repeatably reactive antibody screenin tests must be confirmed with su plementary tests, ande!t diagnosis of actual disease shouldte accompanied by additional clinical followu and assessment of immune status (e.g., T-cell studies) (J22,F46) Screening by EIA for HIV Antibodies. An enzymebased immunoassay (EIA or ELISA) desi ed to detect antibodies to the virus usually is the first test g n e to 'screen" an individual for HIV infection (F47). Although originally designed for use in screenin blood products donated for transfusion, the HIV ELIS! has become an important diagnostic tool for initially determining the HIV status of patients as well. The recommended laboratory al orithm of testing for antibodies to HIV calls for an initiaftest to be done on the specimen (serum or plasma). If that test is "nonreactiven, nothing further is done and the result is reported. If the specimen is initially "reactive", the same specimen should be retested in duplicate in a subsequent test run. If one or both duplicate tests are reactive, the specimen is considered "repeatably reactive" and a supplementary test is required to confirm that result. A Western blot test or an indirect immunofluorescence assay (IFA) are the most widely used supplementary tests, and both will be discussed shortly. Most commercially available HIV ELISAs are extremely reliable tests (F47). Because they were designed for use in blood banking, they are highly sensitive (>99%1. Although false ositive and false negative results are relatively rare, they l o occur (Fa). The predictive value of an ELISA. screening test depends on the prevalence of HIV infection m the opulation being tested. In a population at low risk of Hdinfection where the prevalence of HIV is low (0.1 % or less), a reactive test is much more likely to signify a fahe positive than a true positive; in that population the predictive value of a reactive

8

test is low whereas that of a nonreactive test is high. In contrast, in the high-risk populations, among whom the prevalence of HIV infection is relatively high, a reactive test is much more likely to signify a true positive than a false positive; in these individuals,the predictivevalue of a reactive test is high. Because false negative results are more likely to occur in this grou ,the predictive value of a negative test is F49lower than would {e true for a low-risk population (F40, F51,Fa). Predictive values notwithstanding, false positive HIV ELISA results have been reported with serum from some atients with conditions includinghematologicmalignancies, NA viral infections, autoimmune diseases, multiple myeloma, primary biliary cirrhosis,and alcoholic hepatitis, among others (F52).How often falsely positive results mi ht occur in populations of patients with various other me&cal conditions unrelated to HIV is unclear but should be anticipated as wider testin for HIV infection in clinic and hospital patients inevitally occurs ( F a ) . The true HIV status of such individuals usually can be resolved with su plementary tests such as the Western blot, indirect immuno&orescence assa or ELISAs containing recombinant or synthetic pepti& antigens. Done in tandem with reactive ELISA screening tests, these tests contribute to specificity. False positive HIV ELISA results occur because of human or technical errors associated with doing the tests or because of antibodies cross-reactivewithviral or nonviral components in the tests. With ELISAs that utilize, as antigen, a lysate of virus grown in human cells, there may be cross-reactions with contaminatingcellular proteins that remain in the 1 sate, even after purification steps (F47). Notable causes offalse positive reactions have been anti-HLA-DR antibodies that sometimes are produced in multiparous women and in multiply transfused patients (F534'56).Likewise,antibodies may be present that are cross-reactivewith HIV determinants that coincidentallyalso ha pen to be present on an unrelated antigen to which the iniividual has been exposed. For example, antibodies to proteins of other viruses have been reported to cross-react with HIV determinants (3'57,F54). FalselyreactiveHIV ELISAs alsohave been reported recently in persons who received vaccines for influenza (F58)and hepatitis B viruses (F59).In many instances, false reactions are relatively weak, transient, and cannot be confirmed to be HIV-specific with supplementary testing. Moreover, such specimens often are ne ative when subjected to a different manufacturer's HIV E%SA. Cross-reactivitywith cellular proteins has been essentially eliminated with the introduction of a new eneration of improved ELISAs employing synthetic HI$ pe tides or recombinant HIV peptides (F60).However,a n t i b d e s crossreactive with a cloning vector (e.g., Escherichia coli) occasionallymay be responsible for false reactivity in recombinant antigen-based tests (F61,F62). False positive HIV ELISAa also can occur as a conse uence of passively transferred antibodies. This occurs in $abies who acquire antibodies transplacentally from HIV-infected mothers (F63)or in individuals given therapeutic or prophylactic human y- lobuliis. Indeed, some lots of 7-lobulin prepared from 00 of human serum have been s!own to contain antibdies to HIV (F64-F66). No evidence of infectious HIV has been found in these pools. Presumably the process' stepsthe pools undergo are sufficient to destroy % that may be present. As HIV vaccine trials infectious H get under way, it also will be important for physicians to be aware of individuals participatin in these trials, for they can be expected to develop HIV antitodies, but they will not be infected. False ne ative HIV-1 ELISAs sometimes occur in popu.lations SUC! as male homosexuals or IVDUs who are at high risk of HIV infection and among whom the revalence of HIV infection is high (F67). Usually this can ge attributed to collectionof a specimen too soon after initial HIV infection. Several weeks may elapse before someone who has been infected mounts an immune response and produces HIV antibodies in quantities sufficient for detection (seroconversion). It takes longer for some individuals to complete seroconversion than it does for others; however, the vast majority do so within a period of less than 6 months (F68). Seroconversion actually commences as soon after infection as B-lymphocytesbegin to produce antibodies to the various

b

t

ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993

SO7R

CLINICAL CHEMISTRY

HIV-2 ELISAs does not necessaril mean that the individual component proteins of the virus. Initially, small amounts of being tested is infected with HI82. To clearly document relativelylow affinity antibodies are produced. After primary HIV-2-specificantibodies, the test bein used should contain infection, antibodies to viral core and envelope proteins (e. no epitopes with which HIV-1 antibodiies are reactive at a p24 and gp41 or gp120/160, res ectively) usually can %; detectable level; the test should discriminate between HIV-1 detected earlier than can antibor8es to other viral roteins and HIV-2 antibodies. An algorithm for combined H1V-U (F69,F70). If sufficient antibody has not been proiuced at HIV-2 EIA testing and confirmation has been recently the time a specimen is collected, a false negative result wll recommended (F84). be obtained. When this occurs in someone who is at risk of SupplementaryTests for HIV Antibodies. Regardless HIV infection, especially if there is current or recent evidence of symptoms consistent with an acute viral illness (e.g., sore of the type of test(s) done to screen for antibodies to HIV, F71)), throat, fevedsweats, headache, lethargy, myalgia (F70, a specimen that is repeatably reactive in a screening test a fresh blood specimen should be obtained and tested after should have that reactivity verified with a supplementary a few weeks to ensure that infection does not go undetected. test. Although an HIV-1 Western blot is the supplementary test that has been used most often to confiim a positive With the introduction of ELISA and other tests for screening test for HIV-1 antibodies (F47,F85),an ELISA detecting HIV antibodies, there has been concern that there employingrecombinant HIV peptides as antigen (Cambrid e might be individuals infected with HIV variants lacking Biotech) and an indirect immunofluorescence assay (Walicritical epitopes present on the viral peptides used for a heim Pharmazeutika) have been licensed by the FDA for use articular s thetic- or recombinant peptide-based assay. as supplementary tests (F60,F86). 8hould this so, antibodies stimulated in the patient might fail to react with key viral epitopes present in the assay, The Western blot detects antibodies to individual HIV-1 resulting in a falsely negative test result (F72). proteins and glycoproteinsthat have been electro horetically separated accordingto their molecular weight an&ransferred In addition to testing serum and plasma, approaches that onto strips of nitrocellulose paper. The pa er stri serves as utilize ELISA to test for HIV antibodies in dried blood spots an antigen-impregnated matrix which can f e incufated with collected on special filter paper have been developed (F73, F74). The test kit of at least one manufacturer (Genetic a patient’s serum or plasma. When antibodies to HIV-1 are present in a specimen, the bind to the separated proteins Systems) has received FDA licensure for this application. and with the assistance of &IA technology are visualized as This approach has been articularly useful in situations where colored “bands” on the paper (F47). The constellation of it is difficult to collect bkod in sufficient quantity for routine testing. Newborn babies born to mothers at risk of HIV bands that appears is used to interpret the test result. If no bands are detected, the specimen is considered negative. If infection are good examples (F75, F76).There are also reports bands are present, the specimen may be declared “positive” that HIV ELISA can be used to screen urine and saliva for or “indeterminate” (F87,F47). It should be stressed that antibodies (F77-F79);however, no manufacturer’s test has there has not been uniform agreement concerning the yet been licensed for this application. interpretation of this band pattern, and different consensus Rapid Screening Tests. Various a proaches to the rapid groups have roposed and publicized different criteria for detection of antibodies to HIV-1 have gee, re orted. These interpreting k1V-1 Western blots. Indeed, not even the includethe agglutinationof HIV antigen-coate latex particles interpretation guidelines in the brochures of each FDAas well as various EIA-based and immunoblot procedures licensed manufacturer of HIV-1 Western blots are the same. (F8O-FS3).Two of these, a latex agglutination assay emHowever, a majority of laboratories now have adopted the ploying recombinant HIV antigens (Cambridge Biotech) and recommendations of ASTPHLD/CDC for the interpretation an immunoblot assay em loying a combination of both HIV of HIV Western blots (F87).Following those recommendalysate- and recombinant-Kased peptides (Murex Corp.) have tions, a negative Western blot would have no bands; a positive received FDA licensure. When done precisely as directed by would have at least two of the key bands, p24, gp41, gp120/ the manufacturer, these rapid tests perform well and dem160, whereas an indeterminate would have a single band or onstrate sensitivities and specificities approaching those of combination of bands that does not fit the interpretation of ELISA. They are not convenient for screening large numbers positive. of specimens, where ELISA is a better choice. However, they When assessing the results of HIV tests, physicians and are particularly useful for diagnostic testing in third world others who may be counseling patients should note the countries where economics, equipment, electricity, refrigerotential fallibility of the Western blot (F88).They should ation, and ersonnel constraints may reclude the perfore s ecifically aware how the laboratory they use interprets mance of EeISA. As the prevalence of h I V increases, there its &estern blots and the quality control measures taken. It undoubtedly will be increased demand for rapid testa that is possible for someone whose spectrum of HIV antibodies is can be easily performed in urgent care settings and physicians’ incomplete, or whose titer is weak, to receive a positive offices. This will of course require that appropriate pre- and Western blot result from one laboratory and an indeterminate posttest HIV counseling is available; that qualified, knowlresult from another. The dilemma is compounded by the edgeable, and well-trained personnel are available to do the fact that even in the same laboratory, an occasionalspecimen tests; that test proficiency is documented; and that appromay yield slightly different patterns when the Western blot priate quality control measures are taken. is repeated or when serial samples from the same individual HIV-2 and CombinationHIV-1/HIV-2 Tests. A second are tested (F89,F90). Fortunately, Western blot results on human immunodeficiency virus, HIV-2, has been described most specimensfrom individualswho have repeatably reactive (F44).Closely related to a simian immunodeficiency virus ELISAs and who have identifiable risk of infection are usually (SIV),HIV-2 also causes AIDS. HIV-1 and HIV-2 are related unequivocal, and diagnosing their HIV infection is not but distinctly different retroviruses; however, HIV-2 may be difficult. Greater than 95% of those infected develop less virulent than HIV-1 and disease may take longer to occur. sufficient antibodies to qualify them as “reactivenwithin six Their overall nucleotide sequence homolo is approximately months, and most do so within just a few weeks of infection 505% ;they are more homologousin theirpo andgag sequences (F68,F91,F92). (60%)butleas homologous(40%)intheirenusequences(F45). Dependin on the manufacturer, and ossibly the geographic With re ard to both the ELISA and the Western blot, it source of t%e specimen bein tested, ELISASfor HIV-1 may is those wfo are infected, but have not completed serocondetect 8-90% of those infected with HIV-2 (F45,F84). version who present the greatest diagnostic challenge (F47, Although HIV-2 infection is found primarily in certain F68,F93).If a blood specimen is collected so close to the time countries of West Africa, more than 32 cases have been of infection that sufficient antibodies have not developed, documented in North America. In most instances, HIV-2 the Western blot result may be indeterminate at best and infection detected outside of Africa has occurred in individuals falsely negative at worst. In this case, the screeningtest could who have traveled to or lived in West Africa or have had be repeatably reactive, yet the specimen, lacking the approsexual contact with someone from that part of Africa. priate spectrum of antibodies to significant viral proteins, could be indeterminate by Western blot. Thus, with seroBecause HIV-2 and HIV-1 both pose threats to the blood conversion in progress but incomplete, the Western blot may ly ELISAs for HIV-2, and more recently combination be indeterminate. In most instances of true infection, EZ&Ls that reliably permit the simultaneous detection of however, a Western blot done on a second specimen collected antibodies to both viruses, have been developed and licensed a few weeks later will be positive. An occasional patient may (F84).It should be noted that reactivity in currently licensed

a

E

Y

S98R

ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993

CLINICAL CHEMISTRY

present so late in the course of infection that with the overall decline in immunocompetence, antibodies to certain HIV proteins have waned as well, Depending on the interpretation scheme used by a particular laboratory, if key antibodies required for the interpretation “positive” are among those that have waned, an indeterminate Western blot result may be obtained even when infection has been previously documented unequivocally. In cases where an HIV-1 Western blot exhibits the unusual indeterminate pattern of gag (p55, p24, or p17) plus pol (p66, p51 or p31) bands in the absence of enu (gp160, gp120 or gp41) bands, the Centers for Disease Control has recommended that the specimen be tested for HIV-2 (F84).This should be done because there is sufficient cross-reactivity between roteins encoded b the gaglpol genes of HIV-1 and HIV-2 (i45)to cause an AIV-1 ELISA to be repeatably reactive, but the Western blot to have only one or two gag/ pol bands. Problems also may be encountered when an HIV Western blot is done on someone at no identifiable risk of infection. For example, recent studies of blood donors in whom no risk of HIV infection could be ascertained, who were nonreactive for HIV antibodies by ELISA, and for whom all other testa for HIV were negative revealed that 20-40% might have an F90). Physiciansand others indeterminate Western blot (F89, involved in testing and counselin those who seek testing or are rovided testing to rule out ! IIV infection as part of a meical examination or for insurance, should be aware of this potential problem. For this reason, it is prudent to reserve the Western blot as a confirmatory test for specimens that already have tested repeatably reactive in a screening test. As reviously pointed out, althou h uncertainty associated witi an indeterminate Western bfot often can be resolved with follow-up testing, the anxiety an indeterminate result creates in a test subject is understandably intense. Moreover, the additional testing and counseling required to resolve the uncertainty can be both emotionall and financiallyexpensive. Given our current knowledge,the enters for Disease Control has recommended that “a person whose Western blot test results continue to be consistently indeterminate for at least six months-in the absence of any known risk factors, clinical symptoms, or other findin s may be considered to be negative for antibodies to H!Tl” (F94). The diagnostic dilemma has been made more complex by reporta describin individuals who were infected with HIV but did not compfete seroconversion for up to 3 years (F95 F96). This led to fears there might be unknown numbers 0; individuals who are infected, but seronegative. However, there have been relatively few other studies supporting this. In fact, conclusionsdrawn from one of the above studies (F96) were subsequently called into uestion by the authors themselves when they were unatle to substantiate their original findings in follow-upstudies (F97).I n addition, data from another recent study involving low-risk blood donors with persistently indeterminate Western blob provided no evidence of HIV infection in the absence of antibodies to HIV (F98).The reasons for the discrepancies in some of these studies are unclear. Whether a seronegativeHIV carrier state exista in some individuals remains a question without a definitive answer. If it does occur, it must be relatively infrequent. A thorough medical history, including a sexual history, should reveal behaviors or other risk factors (e being a sexual partner of a hi h-risk individual) that woJd raise the index of suspicion of%IV infection even in the face of indeterminate results. However, as heterosexual spread of the virus increases it will become more difficult for physicians and counselors to determine risk. Indirect immunofluorescence assay for HIV antibodies is a distinctly different assay compared with the Western blot. IFA utilizes uninfected and HIV-infectedhuman cells affixed to glass slides (F86).The cells, rather than partially purified viral proteins on nitrocellulose paper, serve as the substrate on which testing for antibodies to HIV is done. Fluoresceinated anti-human immunoglobulin is used as a secondary reagent to detect human antibodies that bind to infected cells. A particular pattern of ap le een fluorescence on infected but not on uninfected ce& ingcates the presence of HIV specific antibodies. In contrast to the Western blot, IFA provides no information concerning the spectrum of antibodies to the indi-

8

vidual viral proteins. Nevertheless, IFA can provide clues to the nature of falsely reactive specimens contamingantibodies reactive with cellular rather than viral componenta. Because the specimen is placed on both HIV-infectedand noninfected cells, reactivitywith both can indicate that reactivity detected in a screenin ELISA mi ht not be caused by HIV-specific antibodies. boreover, t e pattern of fluorescence can be informative as well. For example, nuclear fluorescence or particular patterns of c plasmic fluorescence on both infected and noninfecte(Yt“ cells may be attributable to autoantibodies reactive with DNA or cytoplasmic organelles respectively. Fortunately, most truly positive specimens are not difficult to interpret. Nevertheless,some s ecimens may require additional testin since reactivity w i d noninfected cells does not rule out Hdinfection; some infected individuals also may produce autoantibodies to cellular constituents as well. Tests for HIV-1p24 Antigen. HIV-1 p24 antigen is a gag- ene-encoded core protein of the virus. A number of simifar testa desi ed for the detection of this product in tissue culture fluigas well as in serum and plasma have been described (F99).Most have been des’ ed as antigen capture testa in an ELISA format. The only%% antigen assay thus far licensed (Abbott HIVAG-1) utilizes human anti-HIV antibodies coated on plastic beads as the solid phase for “capturing”p24 from serum. After incubationwith specimen, the bead is immersed in a second antibody to HIV (rabbit), washed, then immersed in an enzyme-labeled goat antibody to rabbit IgG. Finally, the bead is incubated with substrate that produces a color change upon reacting with the enzyme bound to the goat antibody. The intensity of the color chan e is proportional to the amount of antigen present over t i e linear portion of a standard curve. However, this test is not licensed for the quantitative measurement of p24 in serum. Neutralization of the reaction with an HIVAG-1 blocking antibody is recommended to confirm a repeatably reactive specimen. The neutralizationstep is important, because there have been reporta of falsely positive p24 antigen assays in certain clinical conditions (F100). As previously discussed, some HIV-infected sub’ecta may be antigenemic prior to their production of antibodies to the virus (seroconversion), whereas others may be antigenemic late in infection when some of their antibodies may wane. These are the times when testa for antigenemia may be the most valuable for diagnosis or prognosis (F47,F99). A modification of the HIV p24 antigen assay has been reported that appears to make the test much more sensitive than were earlier formats, including the Abbott HIVAG-1 ELISA. This modification includes an acidification step to dissociate immune complexes (F101,F102). Acidification appears to disrupt preexisting immune complexes, freeing antigen for ‘capture” by the antibodies in the test system. This modification may further assist the detection and diagnosis of HIV-1 infection in infanta where definitive diagnosis may be significantly delayed and difficult (F63, F103). Acid-mediated immune complex dissociation (ICD) is likely to become an important consideration as various manufacturers refine HIV antigen assays and approach the FDA for licensure. In summary, development of more sensitive,more specific, and more rapid testa for the detection and diagnosis of HIV infectionsundoubtedlywill continueat a rapid pace. At times, diagnosis of infection based primarily on the serologic testa thus far licensed can be inconclusive and frustrating. Most diagnostic laboratories are poorly equipped and lack staff adequate1 trained in retrovirology to provide the sophisticated a n i ex ensive esoteric testing that is sometimes required. As &IV infection expands to an ever larger number of women and children, the demand for licensure of a broader ran e of testa that can reliably detect viral genes, viral gene prducta, and the virus itself, yet are easy to erform, can only increase. Likewise, as the prevalence of HIF-2 infection rises, licensed supplementary testa that can accurately distinguish between antibodies to HIV-1 and HIV-2 will be needed as will testa to detect this virus and ita products. Antiviraldru s to combat HIV infection are also being rapidly developed. Cfonse uently, testa for the accurate quantitation of these viruses b o 8 before and after the initiation of antiviral therapies will be needed as will accurate methods to monitor

f

ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993

SBBR

CLINICAL CHEMISTRY

the emergence of viral strains that are resistant to specific antiviral drugs. LITERATURE CITED (FI) Centers for Disease Control. Mwb. Mortal. Weekly Rep. 1987, 38(1S), 3s-15s. (F2) DeVlta, V. T.; Hellman, S.;Rosenberg, S. A. AIDS: E-, D&gnosk, r m m t and prevent&n, 3rd ed.;J. B. uppincott: philadeiphla, 1992. (F3) Qlandon, J. D.; Tlmpone, J. Q.; Schnittman, S. M. Cln. Infect. Ob. 1992, 15, 134-157. (F4) Felnberg, J.; Hoth. D. F., Jr. Hosp. PTact. 1992, 27, 181-184, 187-189, 173-174. (F5) CWry, A.; Twner, A. J.; LMS, S. J. C h . Pethd. 1991, 44, 182-193. (F8) Strand, C. L. Arch. Pathd. Lab. M.1990, 114. 277-283. (F7) Constantine, N. T.; Calehan,J. D.;Watts, D. M. RefrovtelTesLlrg: EssenW fOfC&dtY ControlandLaboraroryD&gnosls; CRC Press: B o a Raton, FL, 1992. (F8) Bylund, D. J.; Zlegner. U. H. M.; Hooper, D. 0. Clln. Lab. M.1992. 12. 305-333. (F9) F'hak, J. P.; Wollnsky, S. Clh. Infect. Dls. 1992, 15, 13-16. (FIO) Davey, R. T., Jr.; Lane, C. Rev. Infect Dls. 1990, 12, 912-930. (F11) Centers for Disease Control. Mwb. Mortal. Weekly Rep. 1981.30,250252. (F12) Centers for Disease Control. M.MOlz81. WeeklyRep. 1981,30,305308. (F13) Gottlbb, M. S.; Schroff, H. M.; Schanker, H. M.; et el. N. €ngL J. M. 1981, 305, 1425-1430. (F14) Jaffe, H.; Bregman, D.; Sellk, R. J. Infect. Dls. 1983, 148, 339-345. (F15) Centers for Dlsease Control. Mwb. Mortal. WeeklyRep. 1989, 38,229238. (FIB) Plot, P.; Plummer, F. A.; Mhalu, F. S.; et el. Sclence 1988,239,573-579. (F17) Chin, J. Lancet 1990, 336, 221-224. (F18) Palace, J. Science 1991, 252, 372-373. (Fig) Ehrhardt, A. A. Am. J. pub. Health 1992, 82, 1459-1481. (F20) Ellerbrock, T. V.; Lleb, S.;Harrlngton,P. E.; et al. N. €ng/.J. M.1992, 327, 1704-1709. (F21) Rosenberg, 2. F.; Faucl, A. S. AIDS: €t/o&gy, Dkgnosk, Treatmentand PreWtkn, 3rd ed.;J. B. Llpplncott: Philadelphia, I 9 9 2 Chapter 4. (F22) Centers for Dlsease Control. M.Mort. Weekly Rep. 1993,41.981982. (F23) Abrams, D. I.Clln. Immund. Allergy 1988, 6, 581-599. (F24) Centers for Dlseaw Control. Mwb. Mortal. Weakly Rep. 1987, 38(S-8). 1-48. (F25) Centers for Disease Control. Mwb. Mortcll. Weekly Rep. 1988,35,334339. (F28) Redtleld, R. R; Wright, C. D.; Tramont, E. C. N. €ng/. J. M.1988, 3 14, 13I- 132. (F27) Cooper, D. A.; Qold, J.; MacLean, P.; et el. lancet 1985, 1, 537-540. (F28) Tlndall, B.; Barker, S.; Donovan, B. Arch. Intern. M.1988, 148,945949. (F29)Clark, S. J.; Saag, M. S.; Decker, W. D.; et al. N. Engl. J. M.1991, 324. 954-980. (FlO)daar, E. S.; Moudgll, T.; Meyer, R. D.; et ai. N. €ng/. J. M.1991, 324, 961-984. (F31) Levy, J. A. Leboretwy D&& of InfecnrwS Dlseeses, Vd.11. V M , RMettslelandCh/amyd&lDlseamSpringer-Verlag: New York, 1988;pp 877-891. (F32) MOSS, A. R. AIDS 1989, 3,55-81. (F33) Lang, W. R.; Jaffe, J. H. N. €ngl. J. M. 1987, 316, 1409-1410. (F34) auinn, T. c.; Mann, J. M.; Curran, J. w. sabnce 1908, 234,955-963. (F35) Bane-Slnoussl, F.; Chermenn, J. C.; Rey, F.; et al. Sclence 1983, 220. 888-87 1. (F38) Gelb, R. C.; Salahuddin, S. 2.; Popovlc, M.; et el. Sclence 1984, 224. 500-503. (F37) Levy, J. A.; Hoffman, A. D.; Kramer, S.; et el. Sclence 1984, 225, 840842. (F38) Coffln, J.; Haase, A.; Levy, J. A.; et el. Sclence 1988. 232, 697. (F39) Web, S. H.; (kedert, J. J.; Sarngadharan, M. Q.; et el. J. Am. M. A W . 1985, 1253. 221-225. (F40) Petricclnl, J. C. Ann. Intern. M.1985, 103, 728-729. (F41) Dodd, R. Y. N. EngL J. M. 1992, 327, 419-421. (F42) Ndson, K. E.; Donahue, J. G.; Munor. A.; et el. Ann. Intern. M.1992, 717, 554-559. (FM) k Y t l l M l f l . S. J.: Brewer,. T. F.:. Flneberp. H. V.;- et el. Ann. Intern. M. . 1892; 117, 612-813. (F44) Clavel, F.; Ouetard, D.; Brun-Vezlnet, F.; et al. Sclence 1988, 233,343346. (F45) Obrlen, T. R.; Wge,J. R.; Holmberg, S. D. J. Am. M.Assoc. 1992, 267, 2775-2779. (F48) Fahey, J. L.; Taylor, J. M. G.; Detels, R.; et al. N. Engl. J. h4s-d. 1990, 322, 188-172. (F47) Sloend, E. M.; PM. E.; Chlarello, R. J.; et al. J. Am. M.Assoc. 1991, 266, 2881-2888. (F48) Schwartz. J. S.; Dana, P. E.; Klnoslan. B. P. J. Am. M.Assoc. 1988. 259. 2574-2579. (F49) Ward, J. W.; Holmberg, S. D.; Allen, J. R.; et ai. N. fngl. J. M.1988, 316. 473-478. (F50) k h , M.P.; Taylor, P. E.; Lenes, B. A.; et al. N. €ng/. J. h4s-d. 1890, 323, 1308-1312. (F51)Councllon Sclentlfic Affalrs. J. Am. M.Assoc. 1985,254,1342-1345. (F52) Stedtelberg, J. M.; Cockerlll, F. R., 111 Map Clln. proc. 1988, 63.373380. (F53) Mortlmsr, P. P.; Parry, J. V.; Mortlmer, J. Y. Lancet 1985, 2, 873-877.

--

400R

ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993

rmwm

(F54) DO&, N. L.; bmberson, H. v.; o'Brlen, T. A. 1888, 28, 4 12-41 8 . (F55) Yu, S. K.; Fong, C. K. Y.; Lam, M. L. N. €t@. J. W .1989, 320, 1495-1498. (F56) Kihnl, Pa;Seldl. S.; H o l z b e r ~ ,Q. Lancet 1985, I, 1222-1223. (F57) Gonzalez-Scarano, F.; Waxham, M. N.; Ross, A. M. AIDS Ras. Mm. RefrOvkupecr 1987, 3, 245-252. (F58) MacKenzle, W. R.; Davls, J. P.; Peterson, D. E.; et ai. J. Am. M.Assoc. 1992, 268, 1015-1017. (F59) Lee, D. A.; Eby, W. C.; Mollnaro, Q. A. Lancet 1992, 330,1080. (F80) Johnson, J. E. J. C h . Mcfobkf, 1992, 30, 218-218. (F81) Trlbe, D. E.; Reed, D. L.; Llndell, P.; et ai. J. ckh. WrdW. 1988, 26, 841-847. (F82) Bkmberg, J.; Vlnclc, E.; Honsson, C.; et ai. AIDS Res. tfum. Retmvhrsss 1990, 6, 1383-1372. (F83) Sison, A. V.; Campos, J. M. CIn. Mcfobkf. Rev. 1992, 5, 236-247. (F84) Wood, C. C.; Wllllams, A. E.; McNamara, J. Q.; et ai. Ann. Intern. W. 1988, 105, 536-538. (F65)LeU;oldman, M.; McBride,J. H.; Howanltz, P. J.; et el. Am. J. CUn. Peihd. 1987, 87, 835-839. (F66) Schlech, W. F.; Lee, S. H. S.; W,J.; et el. J. Am. M.Assoc. 1989, 261, 411-413. (F87) Centers for Dlseem Control. Mwb. Mortal. W&& Rep. 1990,38(RRl8), 1-31. (F68) Jackson, J. B. rranstuekn 1992, 32, 497499. (F89) Cooper. D. A.; Imrle, A. A.; Penny, R. J. Infect. DIP. 1987, 155, 11131118. (F70) Gurley, R. J.; Qroopman, J. E. RetmMhrs -and Hbman Dbaw Marcel Dekker: New York, 1990, pp 359-377. (F71) Tlndell, B.; Barker, S.; Donovan, B et el. Arch. Intern. W ,1988, 146, 945-949. (F72) Moore, J. P.; Wallace, L. H.; Follett, E. A. C.; et ai. AIDS 1988, 155-183. (F73) Vamler, 0. E.; Ulb, F. B.; Relna, S.; et al. AIDS Res. tfum.Retmnhpss 1988, 4, 132-138. (F74) Lllb, F.; Varnler, 0. E.; Mantle, E.; et el. BUOY. WorldHsellh Org, 1992, 70, 323-328. (F75) Hoff, R.; Berardl, V. P.; Welblen, B. J.; et el. N. fng/. J. M.1988, 318, 525-530. (F78) Beebe, J. L.; B r l g ~L. ~ ,C. J. ckh. MkfoWoL 1990, 28, 806810. (F77) Connell, J. A,; Paw, J. V.; Mortlmer, P. P.; et el. Lancet 1990, 335, 1386- 1389. (F78) Desal, S.; Bates, H.; Mlchalskl, F. J. Lencst 1991, 337, 183-184. (F79) MaJw. C. J.; Read, S. E.; hates, R. A.; et ai. J. Infect. DIP. 1991, 183, 899-702. (F80) Starkey, C.; YenCieberman, B.; Proffltt, M. R. J. C h . Mkw6W. 1990, 28, 819-822. (F81)Mitchell, S. W.; Mboup, S.; Mingle, J.; et al. Lancet 1991, 337, 13281330. (F82) Leonard, G.; V d k , M.; Sangare, A.; et ai. €u. J. Mcfobkf, Infect. Dk. 1991, 10. 838-840. (F83) Aubert, V.; Erb, P.; Pwin. L. H.; et el. Lab, W .1991, 22, 889-872. (F84) Centers for Disease Control. Mwb. Mortal. W&ly Rw.1992, 41(RR12). 1-9. (F85) Valdisenl, R. 0.; Taylor. R. N.; Heam, T. L.; et al. Arch. Peihd. lab. W . 1990, 114, 263-287. (F86) Sulllvan, M. T.; Mucke, H.; Kadey, S. D.; et ai. J. CIn. WrdW. 1992, 30, 2509-2510. (F87) Centers for Disease Control. Mwb. Mortal. W&&Rq. 1991,40,692895. (F88) Mortimer, P. P. Lancet 1991, 337, 288-287. (F89)Qenesca,J.;Shlh,J. W.K.;Jett,B. W.;etal.lancet1989,1,1023-1025. (F90) Midthun, K.; Qarrlson, L.; Clements, M. L.; et ai. J. In-. DIP. 1990, 162, 1379-1382. (F9l) Horsburgh, C. R.; Ou, C.-Y.; Jason, J.; et ai. Lancet 1989, 2, 637-840. (F92) Esteben, J.; Shih, J.; Tal, C.; et al. Lencet 1985, 2, 1083-1088. (F93) Agbede, 0. 0. Clln. Mcfobkf. Newsleft. 1892, 14, 121-128. (F94)CentersforMseaseControl. MofbH. kkrtel. W&hRep. 1989, 3B(Suppl. 5 7 ) , 1-7. (F95) Wollnsky, S. M.; R l n a k , C. R.; Kwok. S.; et ai. Ann. Intern. M.1888, 111, 981-972. (F98) Imagawa, D. T.; Lee, M. H.; Wollnsky, S. M.; et ai. N. €I@. J. W .1989, 320, 1458-1482. (F97) Imagawa, D.; Detels, R. N. €f@. J. Msd. 1991, 325, 1250-1251. (F98) Ebb, 8. E.; Busch. M. P.; KhayamBashl, H.; et al. Transfu&bn 1992, 32,

503-508. (F99) Harry, D. J.; Jennlngs, M. B.; Yee. J. A.; et el. C h . Mcfobkf, Rev. 1989, 2, 241-249. (F100) Agballka, F.; Ferchal. F.; Qarnler, J.P.; et al. AIDS 1992, 6, 959-962. (FIOI) Nishenlan, P.; Huskins, K. R.; Stehn, S.; et al. J. Infect. Dk. 1990, 762, 21-28. (F102) Ascher, D. P.; Roberts, C.; Fowler, A. J. Acq&edImmme&#c, 1992, 5, 1080-1083. (F103) Kllne, M. W.; Shearer, W. T. Infect. DIP. Clh. North Am. 1992. 6, 1-17.

w.