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The detection of HIV-1 proviral DNA by PCR in clotted blood specimens
Journal of Virological Methods ELSEVIER
Journal of Virological
Methods 52 (1995) 87-94
The detection of HIV-l proviral DNA by PCR in clotted blood specimens Yu.S. Boriskin aY *, J.C. Booth a, S. Fernando a, D. Carrington a, M.R. Evans b, P. Hay ‘, A.R.M. Coates a aDepartment of Medical Microbiology, St George’s Hospital Medical School, Cranmer Terrace, London SW1 7 ORE, UK b Communicable Diseases, St George’s Hospital Medical School, London, UK ’ Genitourinary Medicine, St George’s Hospital Medical School, London, UK Accepted
22 September
1994
Abstract Six hundred and ninety-two specimens, each consisting of a suspension of the residual free cells in samples of clotted blood (but not the clots themselves) from 680 patients at high risk for exposure to human irnmunodeficiency virus (HIV), were tested by a nested polymerase chain reaction (PCR) procedure which had been optimized to give a sensitivity of detection of one copy of HIV-l proviral plasmid DNA, and the results were compared to those of testing for antibody to HIV on the same specimens. Fifty-one of the specimens were positive for antibody to HIV and 49 of these were also positive by the PCR; the two samples which gave discordant results were found to be PCR-positive when the test was repeated on DNA extracted from the clots themselves. Two specimens were found to be negative for antibody to HIV but were positive by the PCR (53 positive specimens in all). Direct sequencing of the PCR DNA products confirmed their specificity in all cases and demonstrated that no two patients gave the same predicted amino acid sequence for the V3 loop region. The sequences revealed both European/North American and African motifs at the crown of the V3 loop thus indicating a diversity of HIV strains in the SW Thames Region of South London. The results show that the confirmatory PCR for HIV-l can be carried out efficiently on the same clotted blood specimens as used for routine HIV serology on patients undergoing diagnostic evaluation. Keywords:
HIV-l
PCR; Clotted blood specimens;
V3 loop sequence variation
* Corresponding author. Fax: +44 81 6821320. 0166-0934/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-0934(94)00146-4
88
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of Virological Methods 52 (1995) 87-94
1. Introduction The PCR is a sensitive tool for detecting HIV-l proviral DNA in peripheral blood mononuclear cells (PBMC) (reviewed by Lewis and Gibbs, 1993) and in a few cases has given a positive result in the weeks before seroconversion and before the detection of specific antibodies (Horsburgh et al., 1989; Farzadegan et al., 1993); hence, its value in longer-term seronegative high-risk HIV patients is controversial (Sheppard et al., 1991, 1993; Read et al., 1992; Luque et al., 1993). The HIV PCR is usually carried out on PBMC DNA recovered from anticoagulated blood, however, specimens collected for routine HIV serology are normally in the form of clotted blood. To avoid the need for both kinds of specimen, the possibility was investigated of performing the HIV PCR on the residual uncoagulated cells in clotted blood specimens after serum has been withdrawn for HIV antibody testing. This procedure was evaluated in an investigation into the value of the PCR for detecting proviral DNA in the circulation, in advance of seroconversion, as has been reported by others (Horsburgh et al., 1989; Farzadegan et al., 1993). An important requirement was for the PCR procedure to be carefully optimized in terms of its sensitivity for proviral DNA.
2. Materials
and methods
2.1. Blood specimens The clotted blood specimens were obtained from the diagnostic virology laboratory of St George’s Hospital. All had originated from the Genitourinary Medicine Clinic, mostly from individuals who had had casual hetero- or homosexual intercourse, or who had been sexually active in a country with a high prevalence of HIV infection. After testing the serum by a commercial enzyme immunoassay for antibody to HIV (Abbott HIV 1 + 2 third-generation EIA; Abbott Diagnostics, Maidenhead, Berkshire, UK), the rest of the sample was processed for the isolation of DNA, and by the PCR, usually within 9 days of storage at 4°C. All the clotted blood samples were tested by the PCR without knowing the patients’ serological status to HIV. Anticoagulated (ethylene diamine tetraacetic acid: EDTA) whole blood samples were obtained from known HIV-seropositive patients who were attending the outpatients’ clinic of St George’s Hospital. 2.2. Isolation of PBMC DNA from blood 1 ml of uncoagulated blood or, in the case of clotted blood, 1 ml of the suspension of cells remaining in the original specimen collection tubes, was mixed with 1 ml of lysis buffer (0.32 M sucrose, 10 mM Tris, pH 8.0, 5 mM MgCl, and 1% Triton X-100) and, after 5 min incubation at room temperature, centrifuged at 10000 X g for 5 min followed by two 1 min washes of the pellet with 1 ml of lysis buffer. The pellet of nuclei that remained was then digested with 100 ~1 of proteinase K extraction buffer (50
Yu.S. Boriskin et al. /Journal
of Virological Methods 52 (1995) 87-94
89
mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl,, 0.01% gelatin, 0.45% NP-40, 0.45% Tween 20, 60 pug/ml proteinase K) at 60°C for 1 h and the proteinase K was inactivated by boiling for 5 min. The DNA content was measured in the Hoefer TKO 100 fluorimeter and ranged from below the threshhold of detection of the instrument up to 700 ng/pl (median 145 ng/& 1 pg of DNA or less was taken for each PCR reaction. Some of the clotted specimens contained less than 1 ml of cell suspension, therefore smaller volumes, e.g. 150 ~1, were processed by mixing with equal volumes of lysis buffer. In a few cases, all that was available for DNA extraction were traces of blood cells, which were washed off the bottom of the specimen collection tube with the lysis buffer. As a precaution, the clots for all the specimens were stored at - 20°C so that in the case of discrepancy arising between the results of the PCR and the serological test, the PCR was repeated on DNA which was extracted from the clot using silica particles as described by Zeillinger et al. (1993). 2.3. PCR procedure
and DNA product analysis and validation
A two-step nested PCR was used in which both steps entailed 30 cycles of denaturation at 94°C for 30 s (5 min 30 s in the first cycle) followed by annealing at 59°C for 30 s and extension at 72°C for 90 s (8 min 30 s on the last cycle). The primers were kindly supplied by the MRC AIDS Directed Programme and were as follows: the em primers ADP 825/834 (outer) and ADP 826/828 (internal) amplified a 361 bp fragment between nucleotides 6971 and 7332 of the HIV-l em V3 region; the gag primers ADP 816/817 (outer) and ADP 812/813 (internal) amplified a 115 bp fragment between nucleotides 1551 and 1666 of the HIV-l gag gene. The standard PCR mixture (50 ~1) was composed of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl,, 0.01% gelatin (i.e. Boehringer-Mannheim 10 X PCR reaction buffer), 0.2 mM each dNTP, 0.5 PM each primer and 1 U Taq polymerase. Using the PCR Optimizer Kit (Invitrogen BV, NV Leek, The Netherlands) the PCR conditions were modified for each set of primers as follows: em primers: 60 mM Tris-HCl, pH 8.5, 15 mM (NH,j2S0,, 2 mM MgCl,, 2.5 mM each dNTP, 0.5 PM each primer; gag primers: as for em except that the pH was 9.0 and the MgCl, concentration was 1.5 mM. All PCR runs included a positive control containing 10 copies of HIV-l plasmid DNA (Bootman and Kitchin, 1992) and a negative control in the form of human DNA extracted from the PBMC of a HIV-l antibody-negative and PCR-negative volunteer. The quality of the DNA that was extracted from each of the clotted blood specimens was evaluated in a separate PCR which was always put up at the same time as the virus-specific PCR but using primers specific for a 360 base-pair region of the human histidyl tRNA synthetase gene as described previously (Taylor-Wiedeman et al., 1991; Fernando et al., 1994); all samples proved positive by this test. On completion of the PCR the entire 50 ~1 reaction volume was loaded onto a 6% polyacrylamide gel (Boriskin et al., 1993). The visible band of PCR product DNA was cut out of the gel and the DNA was eluted and sequenced directly using the Sequenase 2.0 kit (Amersham International plc, UK) as described by Boriskin et al. (1994).
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3. Results 3.1. Optimization
of the PCR for sensitivity
with the em and gag primers
The optimization of the PCR procedure was carried out on DNA extracted from EDTA-anticoagulated blood specimens from 14 known HIV-l antibody-positive patients. The sensitivity of the nested PCR was evaluated using the MRC ADP PCR Reference Reagents Set (No. ADP956, Bootman and Kitchin, 1992) which provided a range of HIV DNA concentrations containing known numbers of HIV-l DNA copies. When tested with the standard PCR mixture (see Materials and methods) the enu and the gag primers detected 5 and 10 copies of HIV-l plasmid DNA, respectively. When applied to testing the patient’s DNA, the gag primers failed to detect 2 out of the 14 DNA specimens all of which were positive in the PCR with the enu primers. Simmonds et al. (1990) also noted discordant results with gag and enu primers at the level of detection of single HIV DNA molecules, and Gibson et al. (1991) reported a higher sensitivity of the PCR with env than with gag primers when both were tested under the same reaction conditions. A sensitivity of detection of one copy of HIV proviral DNA for the nested PCR has been reported by Simmonds et al. (1990) and by Zazzi et al. (1993). This level was attained in our PCR after adjusting the conditions as described in Materials and methods. The optimized PCR reactions for the eno primers yielded a single, 361 bp, DNA product whereas those for the gag primers gave a major band of 115 bp as well as several spurious bands of lower intensity (not shown). For practical reasons the gag primers were abandoned and the enu primers alone were used for the rest of the study. When the same 14 blood specimens were re-extracted for DNA and retested by PCR using the enu primers after having been stored for 3 weeks at 4’C, again all were positive. In all instances the specificity of the PCR product DNA was confirmed by sequencing. 3.2. PCR on DNA recouered from clotted blood specimens Six hundred and ninety-two specimens of clotted blood from 680 patients attending the Genitourinary Medicine Clinic were tested for antibody to HIV and for HIV-l DNA by the PCR procedure described above, that is by the examining of DNA extracted from the residue of uncoagulated cells in the specimen tube. Fifty-one of the 692 specimens, from 39 of the patients, proved positive for antibody to HIV and 49 of the same 51 specimens were positive also by the PCR. One of the patients had been found to be negative by both serology and the PCR when first tested, but was positive when retested 2 months later. In the case of the two specimens that gave discordant results, repeating the PCR on DNA that was extracted from the clots themselves (each specimen being extracted on its own in order to avoid the possibility of contamination) gave clear positive results. Two specimens, both of which were from different patients, were shown to be negative for antibody to HIV but were positive when tested by the PCR; sequencing of
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Table 1 Predicted amino acid sequences for the V3 loop region (amino acids 300-336 inclusive of flanking glycosylation sites) for the PCR product DNA obtained from the blood specimens of HIV-l-positive patients in the SW Thames Region NCTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHCN --_-_--------~~_~~~~~~-~~N~~----L-_--~~ -____------R-TL---_“I,----------K--__ -_---S __-I-G---_-__------Q”----_-__-_ _------_----_P_-------A-_A---N_--_-__
------_--_-_-R--------A-_R----_-_-----_--------_-_--G-m----_-_A-_D-_--___--_-----_--_-G_-_----------_--R*-__--_--------_--____-_--D--M----_--“-_------_D-I-y_-_-------------_A-_D---NP-K_Y---Np-K-y____-L_-----G----_-_--_A----_------_--__ _-~~~~~~~~~G-_----_--“-------_----_-___ _-_--S-----R-T-_--_“‘“-A-KT-T--_-_--_-___-__--_----G-_-_--_--------T-_-_--R-_-_-_--------G-_-_------A-_D----__-_________ H-I__-__---G-TM---_“---------N-_-__-__-----------G-------~~~A-*D--_---_-__-_----S------T--------R--Q--Q----__ -----S-------P-------VIATDC--_~~~~~_~_--------------N-~~~-~~~~~~D-_~~-_----_-_____-_------_----_------K-_---_--_-___-_----------S-------________________ ___-------R--_----_T--A-_------_-__--_--I-------R-----------A-_D----_---_--_-_--
_-_______-_G-__--__----------N-_----_-_-_----------R----_--_---D-_------_---_
_-_-_-___-_G-R--___-----------_______ _-_--S-_---G-_-_--_-L-_--------_----_--_-
_________----_----__--A--Q----------_
y____s_____________-_-_-_--“-_--__-_T---------_--Q---------------_----_-~--~~~~~~~QGT~~~~~~~w-_~~N-V-~-----_~
NCTRPNNNTRKSIHIGPGQAFYATGDIIGDIRQAHCN “---------_-_R-----------R”F_-T_--_-“------_----_R-----T---------N--L---_ “_----__-_---R---------_--_R-_T_---_-
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Table 1 (continued) --------___-_R_____T--_______________ ~_~__________~_----~---A-_--__-_--_-_______S____Q_~R_____SLFT-*N---___K-F_T “__--------_m----_T_________________ ----------RG”-----_____IA*-T----________ ~_~________-“__----------A-_------_-___ -____Y_____-“___________________E-_____ Consensus sequences for the North American/European HIV-1 isolates were derived from LaRosa et al. (1990, top line) and from Myers et al. (1993, line 39). Lines 2-12 and 40-42 represent anticoagulated blood samples, the rest of the sequences were obtained from clotted blood specimens. Asterisks indicate deleted amino acids.
the PCR product DNA in both cases confirmed the specificity for HIV; repeat specimens were requested but were not forthcoming. In all, 51 blood specimens were positive by both serology and PCR and another two specimens were PCR-positive but antibody-negative. 3.3, Sequencing
information
of the PCR product DNA
Direct sequencing of the PCR DNA product from all 53 positive specimens of clotted blood confirmed their specificity for HIV-l; 41 of these sequences, all from different patients, were different from one another on the basis of their predicted amino acid sequences (Table 1). This finding provided reassurance that the positive results were true for each individual patient and had not arisen as a consequence of cross-contamination. Moreover, when three of the positive patients were each tested repeatedly at weekly intervals on three separate occasions, and when six others were tested on two occasions, the sequences for each patient were completely reproducible at the nucleotide level. The positive control DNA in the PCR was a plasmid which contained the V3 sequence for the reference strain BHlO; the sequence for this was shown to be exactly as reported by Myers et al. (1993) following amplification on several occasions. Among strains of HIV-l the four amino acids at the crown of the V3 loop tend to be more conserved than the amino acids on either side, particularly among geographically related strains, but the differences that do occur in this central tetrapeptide are of value for classifying isolates. In all our PCR-positive samples, including both the clotted bloods and the 14 anticoagulated bloods which were used in optimizing the PCR, a range of conserved tetrapeptide sequences was detected, of which 80% were representative of European and North American strains of HIV @Rosa et al., 1990); the other tetrapeptide sequences were those more often associated with strains from Central Africa (Albert et al., 1992). The GPGR sequence, which is found in most North American and European isolates, was detected in 37 of the patients; a variant of this sequence, namely, GPGQ, which is more characteristic of African isolates (Myers et al., 1993), was found in 16 patients (Table 1). The sequences obtained from 15 of these patients suggested a mixed population of viral variants on the basis of two bands being detected at the same level in the sequencing gel, however, this only involved the central tetrapeptide in five cases giving the possible alternative combinations of GPGr/q, Gp/lGR, Gp/aGQ, GPGr/s and g/aPGR.
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4. Discussion The results obtained on using the PCR procedure, to test for proviral HIV-l DNA in clotted blood specimens, showed good agreement with the results of serological testing in 692 specimens. Thus, 51 specimens were positive by both methods and two more were positive by PCR alone. This prevalence of discordant results is comparable to that in other studies (Sheppard et al., 1991, 1993; Read et al., 1992; Luque et al., 1993). Our finding that the PCR can be performed successfully on clotted blood simplifies the use of the PCR for diagnostic purposes by doing away with the need to collect a specimen of anticoagulated blood at the same time. In two out of the 51 seropositive samples tested, the PCR gave negative results probably because of an insufficient number of HIV-l bearing cells being present in the specimens. However, in both of these cases HIV-l proviral DNA was detected in the clot itself. The use of the enu primers, with their ability to amplify the hypervariable V3 loop region, provided a reliable control for the absence of cross-contamination of DNA from one specimen to another, in that different V3 sequences were found in all the patients tested (Table 1) whereas those obtained in follow-up samples from the same patient were perfectly identical. The various predicted amino acid sequences detected by the PCR are summarized in Table 1 and show that there is a great diversity of HIV strains in the SW Thames Region of South London, undoubtedly a reflection of its cosmopolitan population. In addition to the characteristic V3 loop sequence for North American and European strains, which retains the amino acid sequence GPGR at the crown of the loop, we have identified a diversity of other motifs that are more characteristic of African strains. This agrees with the fact that many of the HIV-positive patients attending St George’s Hospital are from East Africa. Some of the sequences identified were unusual, although not globally unique &Rosa et al., 1990; Myers et al., 1993). The study shows that the PCR is unlikely to replace serological testing as a first-line diagnostic option. However, the PCR can serve as a confirmatory method for HIV antibody-reactive sera, able to replace the Western blot as a more rapid procedure which can be performed either on a small quantity of the residual cellular material in a clotted blood specimen or on the clot itself. The use of this usually discarded material leaves serum available for other tests, e.g., p24 or HIV-specific IgM that may be required as prognostic markers. The methodology has yet to be evaluated on a large series of long-standing HIV-positive patients with low CD4 counts and may be less suitable for diagnosing infection in these patients.
Acknowledgements We thank Andy Shipp for collecting the clotted blood specimens and for performing the antibody testing. The HIV-l-specific primers and the PCR reference reagents were supplied by the Medical Research Council AIDS Directed Programme. The study was supported by the South West Thames Regional Health Authority HIV/AIDS Ringfenced Allocation 1992/1993.
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References Albert, J., Franzen, L., Jansson, M., Scarlatti, G., Kataaha, P.K., Katabira, E., Mubiro, F., Rydaker, M., Rossi, P., Petterson, U. and Wigzell, H. (1992) Ugandan HIV-1 V3 loop sequences closely related to the U.S./European consensus. Virology 190, 674-681. Bootman, J.S. and Kitchin, P.A. (1992) An international collaborative study to assess a set of reference reagents for HIV-1 PCR. J. Virol. Methods 37, 23-42. Boriskin, Yu.S., Booth, J.C. and Yamada, A. (1993) Rapid detection of mumps virus by the polymerase chain reaction. J. Virol. Methods 42, 23-32. Boriskin, Yu.S., Booth, J.C. and Yamada, A. (1994) Sequence variation within carboxyl terminus of the nucleoprotein gene of mumps virus strains. Clin. Diagn. Virol. 2, 79-85. Farzadegan, H., Vlahov, D., Solomon, L., Munoz, A., Astembrovski, J., Taylor, E., Burnley, A. and Nelson, K.E. (1993) Detection of human immunodeficiency virus type 1 infection by polymerase chain reaction in a cohort of seronegative intravenous drug users. J. Infect. Dis. 168, 327-331. Fernando, S., Booth, J., Boriskin, Y., Butcher, P., Carrington, D., Steel, H., Tryhom, Y., Corbishley, C., Keeling, P., Murday, A. and McKenna, W. (1994) Association of cytomegalovirus infection with post-transplantation cardiac rejection as studied using the polymerase chain reaction. J. Med. Virol. 42, 396-404. Gibson, K.M., McLaren, K.A. and Clewley, J.P. (1991) A simple and rapid method for detecting human immunodeficiency virus by PCR. J. Virol. Methods 32, 277-286. Horsburgh, C.R., Jason, J., Longini, L.M., Mayer, K.H., Schochetman, G., Rutherford, G.W., Seage, G.R., Ou, C.Y., Holmberg, S.D., Evatt, B.L. and Jaffe, H.W. (1989) Duration of human immunodeficiency virus infection before detection of antibody. Lancet ii, 637-639. LaRosa, G.J., Davide, J.P., Weinhold, K., Waterbury, J.A., Profy, A.T., Lewis, J.A., Langlois, A.J., Dreesman, G.R., Boswell, N., Shadduck, P., Holley, H., Karplus, M., Bolognesi, D.P., Matthews, T.J., Emini, E.A. and Putney, S.D. (1990) Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science 249, 932-935. Lewis, D.E. and Gibbs, R.A. (1993) Detection and significance of HIV sequences in HIV infection. Progr. Med. Virol. 40, 19-47. Luque, F., Leal, M., Pineda, J.A., Torres, Y., Aguado, I., Olivera, M., Hernandez-Quero, J., Sanchez-Quijano, A., Rey, C. and Lissen, E. (1993) Failure to detect silent HIV infection by polymerase chain reaction in subjects at risk for heterosexually transmitted HIV type 1 infection. Eur. J. Clin. Microbial. Infect. Dis. 12, 663-667. Myers, G., Berzofsky, B., Korber, R., Smith, F. and Pavlakis, G.N. (1993) Human Retroviruses and AIDS. Theoretical Biology and Biophysics, Group T-10, Los Alamos, NM. Read, S., Cassol, S., Coates, R., Major, C., McLaughlin, B., Salas, T., Francis, A., Fanning, M., MacFadden, D., Shepherd, F., Calzavara, L., Johnson, K. and O’Shaughnessy, M. (1992) Detection of incident HIV infection by PCR compared to serology. J. Acquir. Immune Defic. Syndromes 5, 1075-1079. Sheppard, H.W., Dondero, D., Anton, J. and Winkelstein, W. (1991) An evaluation of the polymerase chain reaction in HIV-1 seronegative men. J. Acquir. Immune Defic. Syndromes 4, 819-823. Sheppard, H.W., Busch, M.P., Louie, P.H., Madej, R. and Rodgers, G.C. (1993) HIV-l PCR and isolation in seroconverting and seronegative homosexual men: absence of long-term immunosilent infection. J. Acquir. Immune Defic. Syndromes 6, 1339-1346. Simmonds, P., Balfe, P., Peutherer, J.F., Ludlam, CA., Bishop, J.O. and Leigh Brown, A.J. (1990) Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J. Virol. 64, 864-872. Taylor-Wiedeman, J., Sissons, J.G.P., Borysiewicz, L.K. and Sinclair, J.H. (1991) Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells. J. Gen. Virol. 72, 2059-2064. Zazzi, M., Romano, L., Brasini, A. and Valensin, P.E. (1993) Simultaneous amplification of multiple HIV-1 DNA sequences from clinical specimens by using nested-primer polymerase chain reaction. AIDS Res. Hum. Retrovirus. 9, 315-320. Zeillinger, R., Schneeberger, C. and Speiser, P. (1993) A simple method for isolation of DNA from blood clots suited for use in PCR. BioTechniques 14, 202-203.
Journal of Virological
Methods 52 (1995) 87-94
The detection of HIV-l proviral DNA by PCR in clotted blood specimens Yu.S. Boriskin aY *, J.C. Booth a, S. Fernando a, D. Carrington a, M.R. Evans b, P. Hay ‘, A.R.M. Coates a aDepartment of Medical Microbiology, St George’s Hospital Medical School, Cranmer Terrace, London SW1 7 ORE, UK b Communicable Diseases, St George’s Hospital Medical School, London, UK ’ Genitourinary Medicine, St George’s Hospital Medical School, London, UK Accepted
22 September
1994
Abstract Six hundred and ninety-two specimens, each consisting of a suspension of the residual free cells in samples of clotted blood (but not the clots themselves) from 680 patients at high risk for exposure to human irnmunodeficiency virus (HIV), were tested by a nested polymerase chain reaction (PCR) procedure which had been optimized to give a sensitivity of detection of one copy of HIV-l proviral plasmid DNA, and the results were compared to those of testing for antibody to HIV on the same specimens. Fifty-one of the specimens were positive for antibody to HIV and 49 of these were also positive by the PCR; the two samples which gave discordant results were found to be PCR-positive when the test was repeated on DNA extracted from the clots themselves. Two specimens were found to be negative for antibody to HIV but were positive by the PCR (53 positive specimens in all). Direct sequencing of the PCR DNA products confirmed their specificity in all cases and demonstrated that no two patients gave the same predicted amino acid sequence for the V3 loop region. The sequences revealed both European/North American and African motifs at the crown of the V3 loop thus indicating a diversity of HIV strains in the SW Thames Region of South London. The results show that the confirmatory PCR for HIV-l can be carried out efficiently on the same clotted blood specimens as used for routine HIV serology on patients undergoing diagnostic evaluation. Keywords:
HIV-l
PCR; Clotted blood specimens;
V3 loop sequence variation
* Corresponding author. Fax: +44 81 6821320. 0166-0934/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0166-0934(94)00146-4
88
YuS. Boriskin et al. /Journal
of Virological Methods 52 (1995) 87-94
1. Introduction The PCR is a sensitive tool for detecting HIV-l proviral DNA in peripheral blood mononuclear cells (PBMC) (reviewed by Lewis and Gibbs, 1993) and in a few cases has given a positive result in the weeks before seroconversion and before the detection of specific antibodies (Horsburgh et al., 1989; Farzadegan et al., 1993); hence, its value in longer-term seronegative high-risk HIV patients is controversial (Sheppard et al., 1991, 1993; Read et al., 1992; Luque et al., 1993). The HIV PCR is usually carried out on PBMC DNA recovered from anticoagulated blood, however, specimens collected for routine HIV serology are normally in the form of clotted blood. To avoid the need for both kinds of specimen, the possibility was investigated of performing the HIV PCR on the residual uncoagulated cells in clotted blood specimens after serum has been withdrawn for HIV antibody testing. This procedure was evaluated in an investigation into the value of the PCR for detecting proviral DNA in the circulation, in advance of seroconversion, as has been reported by others (Horsburgh et al., 1989; Farzadegan et al., 1993). An important requirement was for the PCR procedure to be carefully optimized in terms of its sensitivity for proviral DNA.
2. Materials
and methods
2.1. Blood specimens The clotted blood specimens were obtained from the diagnostic virology laboratory of St George’s Hospital. All had originated from the Genitourinary Medicine Clinic, mostly from individuals who had had casual hetero- or homosexual intercourse, or who had been sexually active in a country with a high prevalence of HIV infection. After testing the serum by a commercial enzyme immunoassay for antibody to HIV (Abbott HIV 1 + 2 third-generation EIA; Abbott Diagnostics, Maidenhead, Berkshire, UK), the rest of the sample was processed for the isolation of DNA, and by the PCR, usually within 9 days of storage at 4°C. All the clotted blood samples were tested by the PCR without knowing the patients’ serological status to HIV. Anticoagulated (ethylene diamine tetraacetic acid: EDTA) whole blood samples were obtained from known HIV-seropositive patients who were attending the outpatients’ clinic of St George’s Hospital. 2.2. Isolation of PBMC DNA from blood 1 ml of uncoagulated blood or, in the case of clotted blood, 1 ml of the suspension of cells remaining in the original specimen collection tubes, was mixed with 1 ml of lysis buffer (0.32 M sucrose, 10 mM Tris, pH 8.0, 5 mM MgCl, and 1% Triton X-100) and, after 5 min incubation at room temperature, centrifuged at 10000 X g for 5 min followed by two 1 min washes of the pellet with 1 ml of lysis buffer. The pellet of nuclei that remained was then digested with 100 ~1 of proteinase K extraction buffer (50
Yu.S. Boriskin et al. /Journal
of Virological Methods 52 (1995) 87-94
89
mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl,, 0.01% gelatin, 0.45% NP-40, 0.45% Tween 20, 60 pug/ml proteinase K) at 60°C for 1 h and the proteinase K was inactivated by boiling for 5 min. The DNA content was measured in the Hoefer TKO 100 fluorimeter and ranged from below the threshhold of detection of the instrument up to 700 ng/pl (median 145 ng/& 1 pg of DNA or less was taken for each PCR reaction. Some of the clotted specimens contained less than 1 ml of cell suspension, therefore smaller volumes, e.g. 150 ~1, were processed by mixing with equal volumes of lysis buffer. In a few cases, all that was available for DNA extraction were traces of blood cells, which were washed off the bottom of the specimen collection tube with the lysis buffer. As a precaution, the clots for all the specimens were stored at - 20°C so that in the case of discrepancy arising between the results of the PCR and the serological test, the PCR was repeated on DNA which was extracted from the clot using silica particles as described by Zeillinger et al. (1993). 2.3. PCR procedure
and DNA product analysis and validation
A two-step nested PCR was used in which both steps entailed 30 cycles of denaturation at 94°C for 30 s (5 min 30 s in the first cycle) followed by annealing at 59°C for 30 s and extension at 72°C for 90 s (8 min 30 s on the last cycle). The primers were kindly supplied by the MRC AIDS Directed Programme and were as follows: the em primers ADP 825/834 (outer) and ADP 826/828 (internal) amplified a 361 bp fragment between nucleotides 6971 and 7332 of the HIV-l em V3 region; the gag primers ADP 816/817 (outer) and ADP 812/813 (internal) amplified a 115 bp fragment between nucleotides 1551 and 1666 of the HIV-l gag gene. The standard PCR mixture (50 ~1) was composed of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl,, 0.01% gelatin (i.e. Boehringer-Mannheim 10 X PCR reaction buffer), 0.2 mM each dNTP, 0.5 PM each primer and 1 U Taq polymerase. Using the PCR Optimizer Kit (Invitrogen BV, NV Leek, The Netherlands) the PCR conditions were modified for each set of primers as follows: em primers: 60 mM Tris-HCl, pH 8.5, 15 mM (NH,j2S0,, 2 mM MgCl,, 2.5 mM each dNTP, 0.5 PM each primer; gag primers: as for em except that the pH was 9.0 and the MgCl, concentration was 1.5 mM. All PCR runs included a positive control containing 10 copies of HIV-l plasmid DNA (Bootman and Kitchin, 1992) and a negative control in the form of human DNA extracted from the PBMC of a HIV-l antibody-negative and PCR-negative volunteer. The quality of the DNA that was extracted from each of the clotted blood specimens was evaluated in a separate PCR which was always put up at the same time as the virus-specific PCR but using primers specific for a 360 base-pair region of the human histidyl tRNA synthetase gene as described previously (Taylor-Wiedeman et al., 1991; Fernando et al., 1994); all samples proved positive by this test. On completion of the PCR the entire 50 ~1 reaction volume was loaded onto a 6% polyacrylamide gel (Boriskin et al., 1993). The visible band of PCR product DNA was cut out of the gel and the DNA was eluted and sequenced directly using the Sequenase 2.0 kit (Amersham International plc, UK) as described by Boriskin et al. (1994).
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3. Results 3.1. Optimization
of the PCR for sensitivity
with the em and gag primers
The optimization of the PCR procedure was carried out on DNA extracted from EDTA-anticoagulated blood specimens from 14 known HIV-l antibody-positive patients. The sensitivity of the nested PCR was evaluated using the MRC ADP PCR Reference Reagents Set (No. ADP956, Bootman and Kitchin, 1992) which provided a range of HIV DNA concentrations containing known numbers of HIV-l DNA copies. When tested with the standard PCR mixture (see Materials and methods) the enu and the gag primers detected 5 and 10 copies of HIV-l plasmid DNA, respectively. When applied to testing the patient’s DNA, the gag primers failed to detect 2 out of the 14 DNA specimens all of which were positive in the PCR with the enu primers. Simmonds et al. (1990) also noted discordant results with gag and enu primers at the level of detection of single HIV DNA molecules, and Gibson et al. (1991) reported a higher sensitivity of the PCR with env than with gag primers when both were tested under the same reaction conditions. A sensitivity of detection of one copy of HIV proviral DNA for the nested PCR has been reported by Simmonds et al. (1990) and by Zazzi et al. (1993). This level was attained in our PCR after adjusting the conditions as described in Materials and methods. The optimized PCR reactions for the eno primers yielded a single, 361 bp, DNA product whereas those for the gag primers gave a major band of 115 bp as well as several spurious bands of lower intensity (not shown). For practical reasons the gag primers were abandoned and the enu primers alone were used for the rest of the study. When the same 14 blood specimens were re-extracted for DNA and retested by PCR using the enu primers after having been stored for 3 weeks at 4’C, again all were positive. In all instances the specificity of the PCR product DNA was confirmed by sequencing. 3.2. PCR on DNA recouered from clotted blood specimens Six hundred and ninety-two specimens of clotted blood from 680 patients attending the Genitourinary Medicine Clinic were tested for antibody to HIV and for HIV-l DNA by the PCR procedure described above, that is by the examining of DNA extracted from the residue of uncoagulated cells in the specimen tube. Fifty-one of the 692 specimens, from 39 of the patients, proved positive for antibody to HIV and 49 of the same 51 specimens were positive also by the PCR. One of the patients had been found to be negative by both serology and the PCR when first tested, but was positive when retested 2 months later. In the case of the two specimens that gave discordant results, repeating the PCR on DNA that was extracted from the clots themselves (each specimen being extracted on its own in order to avoid the possibility of contamination) gave clear positive results. Two specimens, both of which were from different patients, were shown to be negative for antibody to HIV but were positive when tested by the PCR; sequencing of
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Table 1 Predicted amino acid sequences for the V3 loop region (amino acids 300-336 inclusive of flanking glycosylation sites) for the PCR product DNA obtained from the blood specimens of HIV-l-positive patients in the SW Thames Region NCTRPNNNTRKSIHIGPGRAFYTTGEIIGDIRQAHCN --_-_--------~~_~~~~~~-~~N~~----L-_--~~ -____------R-TL---_“I,----------K--__ -_---S __-I-G---_-__------Q”----_-__-_ _------_----_P_-------A-_A---N_--_-__
------_--_-_-R--------A-_R----_-_-----_--------_-_--G-m----_-_A-_D-_--___--_-----_--_-G_-_----------_--R*-__--_--------_--____-_--D--M----_--“-_------_D-I-y_-_-------------_A-_D---NP-K_Y---Np-K-y____-L_-----G----_-_--_A----_------_--__ _-~~~~~~~~~G-_----_--“-------_----_-___ _-_--S-----R-T-_--_“‘“-A-KT-T--_-_--_-___-__--_----G-_-_--_--------T-_-_--R-_-_-_--------G-_-_------A-_D----__-_________ H-I__-__---G-TM---_“---------N-_-__-__-----------G-------~~~A-*D--_---_-__-_----S------T--------R--Q--Q----__ -----S-------P-------VIATDC--_~~~~~_~_--------------N-~~~-~~~~~~D-_~~-_----_-_____-_------_----_------K-_---_--_-___-_----------S-------________________ ___-------R--_----_T--A-_------_-__--_--I-------R-----------A-_D----_---_--_-_--
_-_______-_G-__--__----------N-_----_-_-_----------R----_--_---D-_------_---_
_-_-_-___-_G-R--___-----------_______ _-_--S-_---G-_-_--_-L-_--------_----_--_-
_________----_----__--A--Q----------_
y____s_____________-_-_-_--“-_--__-_T---------_--Q---------------_----_-~--~~~~~~~QGT~~~~~~~w-_~~N-V-~-----_~
NCTRPNNNTRKSIHIGPGQAFYATGDIIGDIRQAHCN “---------_-_R-----------R”F_-T_--_-“------_----_R-----T---------N--L---_ “_----__-_---R---------_--_R-_T_---_-
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Table 1 (continued) --------___-_R_____T--_______________ ~_~__________~_----~---A-_--__-_--_-_______S____Q_~R_____SLFT-*N---___K-F_T “__--------_m----_T_________________ ----------RG”-----_____IA*-T----________ ~_~________-“__----------A-_------_-___ -____Y_____-“___________________E-_____ Consensus sequences for the North American/European HIV-1 isolates were derived from LaRosa et al. (1990, top line) and from Myers et al. (1993, line 39). Lines 2-12 and 40-42 represent anticoagulated blood samples, the rest of the sequences were obtained from clotted blood specimens. Asterisks indicate deleted amino acids.
the PCR product DNA in both cases confirmed the specificity for HIV; repeat specimens were requested but were not forthcoming. In all, 51 blood specimens were positive by both serology and PCR and another two specimens were PCR-positive but antibody-negative. 3.3, Sequencing
information
of the PCR product DNA
Direct sequencing of the PCR DNA product from all 53 positive specimens of clotted blood confirmed their specificity for HIV-l; 41 of these sequences, all from different patients, were different from one another on the basis of their predicted amino acid sequences (Table 1). This finding provided reassurance that the positive results were true for each individual patient and had not arisen as a consequence of cross-contamination. Moreover, when three of the positive patients were each tested repeatedly at weekly intervals on three separate occasions, and when six others were tested on two occasions, the sequences for each patient were completely reproducible at the nucleotide level. The positive control DNA in the PCR was a plasmid which contained the V3 sequence for the reference strain BHlO; the sequence for this was shown to be exactly as reported by Myers et al. (1993) following amplification on several occasions. Among strains of HIV-l the four amino acids at the crown of the V3 loop tend to be more conserved than the amino acids on either side, particularly among geographically related strains, but the differences that do occur in this central tetrapeptide are of value for classifying isolates. In all our PCR-positive samples, including both the clotted bloods and the 14 anticoagulated bloods which were used in optimizing the PCR, a range of conserved tetrapeptide sequences was detected, of which 80% were representative of European and North American strains of HIV @Rosa et al., 1990); the other tetrapeptide sequences were those more often associated with strains from Central Africa (Albert et al., 1992). The GPGR sequence, which is found in most North American and European isolates, was detected in 37 of the patients; a variant of this sequence, namely, GPGQ, which is more characteristic of African isolates (Myers et al., 1993), was found in 16 patients (Table 1). The sequences obtained from 15 of these patients suggested a mixed population of viral variants on the basis of two bands being detected at the same level in the sequencing gel, however, this only involved the central tetrapeptide in five cases giving the possible alternative combinations of GPGr/q, Gp/lGR, Gp/aGQ, GPGr/s and g/aPGR.
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4. Discussion The results obtained on using the PCR procedure, to test for proviral HIV-l DNA in clotted blood specimens, showed good agreement with the results of serological testing in 692 specimens. Thus, 51 specimens were positive by both methods and two more were positive by PCR alone. This prevalence of discordant results is comparable to that in other studies (Sheppard et al., 1991, 1993; Read et al., 1992; Luque et al., 1993). Our finding that the PCR can be performed successfully on clotted blood simplifies the use of the PCR for diagnostic purposes by doing away with the need to collect a specimen of anticoagulated blood at the same time. In two out of the 51 seropositive samples tested, the PCR gave negative results probably because of an insufficient number of HIV-l bearing cells being present in the specimens. However, in both of these cases HIV-l proviral DNA was detected in the clot itself. The use of the enu primers, with their ability to amplify the hypervariable V3 loop region, provided a reliable control for the absence of cross-contamination of DNA from one specimen to another, in that different V3 sequences were found in all the patients tested (Table 1) whereas those obtained in follow-up samples from the same patient were perfectly identical. The various predicted amino acid sequences detected by the PCR are summarized in Table 1 and show that there is a great diversity of HIV strains in the SW Thames Region of South London, undoubtedly a reflection of its cosmopolitan population. In addition to the characteristic V3 loop sequence for North American and European strains, which retains the amino acid sequence GPGR at the crown of the loop, we have identified a diversity of other motifs that are more characteristic of African strains. This agrees with the fact that many of the HIV-positive patients attending St George’s Hospital are from East Africa. Some of the sequences identified were unusual, although not globally unique &Rosa et al., 1990; Myers et al., 1993). The study shows that the PCR is unlikely to replace serological testing as a first-line diagnostic option. However, the PCR can serve as a confirmatory method for HIV antibody-reactive sera, able to replace the Western blot as a more rapid procedure which can be performed either on a small quantity of the residual cellular material in a clotted blood specimen or on the clot itself. The use of this usually discarded material leaves serum available for other tests, e.g., p24 or HIV-specific IgM that may be required as prognostic markers. The methodology has yet to be evaluated on a large series of long-standing HIV-positive patients with low CD4 counts and may be less suitable for diagnosing infection in these patients.
Acknowledgements We thank Andy Shipp for collecting the clotted blood specimens and for performing the antibody testing. The HIV-l-specific primers and the PCR reference reagents were supplied by the Medical Research Council AIDS Directed Programme. The study was supported by the South West Thames Regional Health Authority HIV/AIDS Ringfenced Allocation 1992/1993.
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