Special Section: Polymerase Chain Reaction (PCR) - A New Tool in Clinical Medicine

Detection of Human Retroviruses by PCR

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Introduction

protein (p24) to become detectable in culture supernatants. Direct detection of p24 antigens in patient sera has been used but the assay lacks sensitivity, presumably both due to the complexing of the antigens by antibodies and the paucity of protein in peripheral blood (16). The application of the polymerase chain reaction (PCR) was first reported in the detection of HIV-1 (17) and the HTLVs (18) in 1987 and 1988. The procedure provides exquisite sensitivity, is rapid, and requireslittle specimen. In addition, the amplified products are non-infectious and therefore the hazards which accompany virus cultivation are circumvented. This article will focus on the major applications of PCR to the dectection of human retrovirus and recent advances in the field of PCR which will help advance the technology from research to the clinical setting.

The family of human retroviruses consists of the Human Tcell Lymphotropic Virus types -1(1) and -11(2) (HTLV-I and II) and the Human ImmunodeficiencyVirus types -1 (3, 4) and -2 (5).HTLV-I and -11 are members of the Oncovirinae subfamily which immortilizes and transforms normal T lymphocytes. HIV-1 and -2 are members of the Lentivirinae subfamily. In contrast to the HTLVs, HIV-1 and -2 have a cytolytic effect on the infected lymphocytes. The four retroviruses share a similar genomic organization although the HlVs containseveral additional regulatoryelements that are not present in the HTLVs (4, 5). HTLV-I and -11 share approximately 60 % overall nucleotide sequency similarity, HIV-1 and -2 approximately 40-50 %. Nucleotide sequence alignments indicate that these two viral subfamilies are only distantly related. HTLV-Iis the etiologicagent of bothadult-T-cellleukemia (ATL) (6) and a neurological disorder known as HTLV-I Application of PCR to Diagnosis of Human associated myelopathy (7) HAMhropicalspastic parapareRetroviral Infection sis (TSP) (8). The virus is endemic in southwesternJapan, the Caribbean, and parts of Africa (6-9). Defection of Virus in Neonates HTLV-II has been isolated from two individuals with atypical hairy cell leukemia (2, 10). However, this virus has Approximately 15-30 % of babies born to HIV-1 infected yet to be definitively associated with this or any other mothers are themselves infected. The time scale for disdisease. Several studies have revealed a high incidence of ease progression is often short and, therefore, early diagnoHTLV-II infection among intravenous drug users in the sis of HIV infection is essential for early therapeutic interUnited States (11,12) and in Italy (13). HTLV-IIinfectionhas vention. Because maternal antibodies can persist up to 18 also been reported among Guaymi Indians in Panama (14) months, conventionalserological assays cannot distinguish and in individualsin Gabon (15) who had no history of blood between maternal and neonatal infection. Virus cultivation transfusion or intravenous drug use. The significance of and PCR have been used to evaluate specimens collected these findings remains to be determined. from infants. Although the reported sensitivity of PCR for HIV-1 and -2 are recognized as the etiology agents of this populationof patients varies (19-21 ),the consensusis acquired immunodeficiency syndrome (AIDS). Infectionwith that PCR shows great promise in the early identification of HIV-1 has been reported worldwide, while HIV-2 is essen- infected infants. tially confined to western Africa. Serologic assays for HTLV-1/41 and HIV-1 are widely Resolution of lndeterminafe Serology used in large-scale screening of blood and blood products for antibodies to the virus. However, the specificity of these A small percentage of low risk blood donors have tested serological assays can be hampered by crossreactivity of positivefor the presenceof antibodiesto HIV-1by the ELlSA antibodies to host haptens and to haptens of relatedviruses assay but positivity could not be confirmed by the Western (i.e. HTLV-I and -11, and HIV-1 and -2). In addition, the blot assay. Positivity by Western blot requires the presence protracted time between acute infection and antibody pro- of antibodiesto two of the following proteins: p24, gp41, and duction can impede early detection. Moreover, serological either gp120 or gp160 (22). Negativity is defined as the assays are, by definition, indirect, as they measure the absence of reactivity to any bands. Individualswho possess host’s response to an invading pathogen, rather than the antibodieswhich react nonspecificallyto one or more of the pathogen itself. HIV core proteins (p17, p24, or p55) or to other non-HIV Conventional methods for direct detection of these proteins do not fulfill the criteria for either HIV positivity or retroviralagents require either cocultivationof the virus with negativity and their seerological status is classified as a susceptible cell and/or detection of viral antigens. Refine- indeterminate. Given the serious implications of HIV infecments in culture techniques have greatly improved virus tion, a method to resolve the HIV status of these individuals isolation rates. Nonetheless, virus cultivation procedures is necessary. are laborious, time consuming, costly, and require the PCR has been used to resolve the infection status of handling of large amounts of infectious agents. Further- individuals with indeterminate Western blots. Jackson et al more, it can take up to 4 weeks for the viral nucleocapsid (23) and Celum et al (24) reported the absence of HIV-1

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proviral sequences in the peripheral blood mononuclear cells (PBMCs) in low risk individuals with indeterminate Western blots. On the other hand, the presence of antibodies to p24 in high risk individuals may be indicative of early infection (24). Likewise, HTLV-1 Western blot indeterminate samples have also been evaluated by PCR. As with HIV-1, low risk individuals with an HTLV-1 indeterminateWestern blot did not harbor HTLV-I or -11 sequences (25).

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Early Detection The window between the time of HIV infection and seroconversion is typically several weeks to 6 months although longertimes have been reported (26). PCR can be used to identify individuals who are infected but who have not seroconverted (26,27). The procedure has been used to screen sexual partners of infected hemophiliacs (28) and seronegative homosexual men (29) who are at higher risk for acquiring the infection. Because of its ability to identify early and low-level infections, PCR has been used to screen for the presence of HIV-1 proviral DNA in health care workers who sustained needlestickinjuries (30) and is currently being evaluated by tissue banks for use in screening donor organs and tissues.

Virus Typing Specific serologic diagnosis of HIV-1 and HIV-2 is complicated by cross-reactivityof antibodies against heterologous antigens (31). Although both HIV-1 and -2 are associated with AIDS, there are some indications that HIV-2 may be less pathogenic than HIV-1 (32). Differentiation between the two virus types will be important in following the epidemiology and pathogenicity of these two agents. Amplification with type-specific primers has been used to confirm infection with the respectivevirus types and PCR was used to confirm the first HIV-1/-2 co-infection (33). Similarly, serologic assays that use whole virus antigen cannot distinguish between HTLV-Iand -11. Individualswith antibodiesto HTLV-Iare at risk for developingATL and TSP of HAM, while the risk factors associatedwith antibodiesto HTLV-II have yet to be established. Identification of the specificvirustype will help direct the clinical management of these individuals. Although HTLV-I and -11 are clearly distinct, the two viruses share approximately 60 % nucleotide sequence similarity. By identifyingvariable regions that are flanked by common regions, a single primer pair has been usedto amplify both targets while differentiationwas achieved with type specific probes (18). Recently, synthetic peptide-basedtests which recognize antibodies to the transmembrane antigens of HIV-1 and -2 have also shown promise in differentiating the two viruses (34). Likewise, synthetic peptides from the env gene products of HTLV-I and -11 have been used to discriminate between HTLV-I and -11 in patients (35).

Characterizationof Genetic Variants HIV-1 and -2 are genetically highly variable, particularly in the surface envelope protein sequences (36-37). Considerable interest has been generated in the last few years to understand the role of variant isolates in disease progression. The ability to amplify, clone, and sequence proviral DNA directly from patient specimen has greatly facilitated

these analyses. In addition to eliminatingthe laborious task of virus cultivation, the sequences obtained directly from patient sample more accurately reflect the virus population as cultivation can lead to strain selection (38). These types of analysis may help define the genetic basis for biological differences among sequential isolates.

Molecular Epidemiology The rapidly evolving nature of HIV-1, particularly in the variable regions of gp120, has provideda tool with which to evaluate the relatedness of viral strains isolated from different individuals as well as to follow the evolution of HIV-1 sequences diversity among close contacts. The transmission of HIV from a dentist to 5 of his patients during invasive dental procedures was recently reported based on sequence similarity between the viral DNA sequences amplified from the dentist and his patients (39). McClutchan et al recently used mispaired primers to identify divergent HIV-1 isolates (40). In their study, each isolate was amplified with 40 HIV-1 specific primers using an “anchored” PCR approachin which one primer was kept constant while the other was varied. The pattern of PCR failures as a result of mismatches with the varaible primer, provideda PCR “fingerprint” of the isolates. These types of analysis should help determine the geographic distribution of HIV-1 variants, albeit with much lower resolution than sequencing.

ldentificafion of Drug-ResistantMutants Prolongedtreatment with zidovudine (AZT) can lead to the emergence of drug-resistant strains of HIV-1 (41). The reduced resistance is accompanied by the appearance of specific mutations in the gene encoding the reverse transcriptase (42). The most resistant strains have four amino acid substitutions in the reverse transcriptase at residues 67, 70, 215, and 219; partially resistant isolates have subsets of these mutations. By designing primers with mismatches to either the mutant or wild-type isolates, PCR has been used to rapidly assess for the presence or absence of these mutations. Mutations which result in resistance to DDI (43) and non-nucleosideanalogues (44) have been identified in a similar manner.

RNA and DNA Quantification Rapid and accurate methods to quantitate HIV expression is essential for following disease progression, monitoring antiviral therapy, and understandingthe pathogenesisand transmission of the virus. Some investigators have used PCR to quantitate levels of integrated and unintegrated proviral DNA in PBMCs and various tissues (45,46). Other investigators have targeted viral genomic and/or messenger RNA in plasma serum or in PBMCs for quantitation (47-49). At present, it is unclear which of these targets is most appropriate for assessing progression and antiviral efficacy. Since HIV, like all retroviruses, replicates through a DNA intermediate, quantitation of both RNA and proviral DNA may be required to monitor the effect of therapy on various aspects of the virus’ life cycle. In developing a quantitative PCR assay, every aspect of the procedure, from sample preparation to product detection must be carefully controlled. Critical to quantitation by PCR is the inclusionof internalstandards in the samples, so

Detection of Human Retroviruses by PCR that variability in reaction conditions, machine cycling, sample preparation, and sample integrity can be controlled.

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Advances in PCR Inthe last two years, significant advancements in PCR have been made. First, primer-template mismatch studies provided guidelines for designing primers for amplification of heterogenous targets. Model systems with HIV established that some 3’-terminal mismatches were detrimentalto PCR while others had no discernible effect (50). To maximize efficient amplification of variant isolates, primers which terminate in a T should be considered since mismatches of T with either A, C, or T are efficiently extended. The substitution of inosine at positions of sequence ambiguity has also been reported to improve detection of HIV-1 sequences (51). Second, “hot-start” PCR has been developed to increase specificity of amplifications (52). In this procedure, a critical reaction component is withheld until the reaction temperature approaches 80°C. Consequently, non-specificannealing and extensionswhich typically occur during PCR set up are minimized. The increased specificity conferredby “hotstart” PCR has enabled Nuovo et al to successfully perform in situ PCR with dramatic improvements in sensitivity (53). Third, new enzymes have been isolated and used in PCR. The DNA polymerase from Thermus thermophilus (Tfh) has both reverse transcription and DNA polymerization activities (54). Because reverse transcription is performed at elevated temperature, Tth polymerase should facilitate transcription through secondary structures and GC-rich regions. A 61 KDa modified form of recombinant Taq polymerase, Stoffel fragment, has twice the thermostability of Taq polymerase and may be particularly useful for amplification of templates that are GC-rich or contain complex secondary structure (S. Stoffel, personal communication). The DNA polymerase from Thermococcus litoralis has been demonstrated to facilitate amplification of meth~’5dC-substit~ted DNA (55). Fourth, two carryover prevention protocols have been developed to address false positive PCRs. The first procedure uses isopsoralen to cross-link the amplified DNA rendering it unsuitable as template for subsequent amplification (56).The second procedure uses uracil-N-glycosylase. In this procedure dUTP is substituted fordTTP in PCR. Any dU-containing PCR products which are inadvertently carried over to new reactions are cleaved with UNG prior to amplification. (57, 58). Finally, the development of sensitive non-isotopicdetection systems have eliminatedthe needfor radioactive probes (59). One system that is particularly amenable to large scale analysis uses a colorimetric (HRPRMB) microtiter detection format (60). To facilitate analysis of samples, amplifications are performed in a faster and more precise thermocycler that accommodates up to 96 samples in a microtiter plate array (TC9600, Perkin-Elmer). With this setup, amplification and detection can be accomplished in aboutfour hours. Equally important, the UNGMUTPcarryover preventionprotocol has been successfullyincorporatedinto this assay. The development of carryover prevention protocols, the engineering of faster and more precise thermocyclers and the development of a sensitive colorimetric microtiter plate detection assay, perhaps represent the most significant

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recent technologicaladvancements in PCR.Together, these developments are expected to facilitate the transfer of assays from research to the clinical laboratories. I thank John Sninsky and Beverly Dale for reviewing the manuscript and Helen Chin for preparing the manuscript.

Shirley Kwok, B.S. Roche Molecular Systems, Inc. 1400 Fifty-Third Street Emeryville, CA 94608 USA

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Detection of human retroviruses by PCR.

Special Section: Polymerase Chain Reaction (PCR) - A New Tool in Clinical Medicine Detection of Human Retroviruses by PCR Ann Med Downloaded from in...
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