standardized nested polymerase chain reaction-based assay for

JOURNAL OF CLINICAL MICROBIOLOGY, May 1993, p. 1066-10740095-1137/93/051066-09$02.00/0Copyright X 1993, American Society for Microbiology

Standardized Nested Polymerase Chain Reaction-Based Assay forDetection of Human Immunodeficiency Virus Type 1

DNA in Whole Blood LysatesS. SAUVAIGO,'* V. BARLET,2 N. GUETFrARI,3 P. INNOCENTI,2 F. PARMENTIER,4 C. BASTARD,4

J. M. SEIGNEURIN,2 J. C. CHERMANN,3 R. TEOULE,S AND J. MARCHAND'

CIS Bio International, BP 32, 91192 Gifsur Yvette,' Virologie Medicale Moleculaire, Faculte deMedecine-Centre Hospitalier Universitaire BP 217X, 38043 Grenoble Cedex,2 Institut National

de la Sant,e et de la Recherche Medicale U-322, Campus Universitaire de Luminy, BP 33,13273 Marseille Cedex 9,3 CRTS et Gene'tique Humaine, BP 58, 76232 Bois-Guillaume

Cedex, and CEN, DRFMC/SESAMIRBIO, 85X, 38041 Grenoble Cede-,S France

Received 5 October 1992/Accepted 21 January 1993

The routine detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in clinical samplesrequires a standardized, simple, and sensitive test. To identify the HIV-1 proviral DNA in blood, we used a

solid-phase assay based on the affinity capture and the gamma counting of the amplified product after a nestedpolymerase chain reaction (AMPLICIS test). In order to simplify the general process, whole-blood lysatesrather than peripheral blood mononuclear cell lysates were used for the amplifications. The solid-phase captureand counting of the final amplified products allowed us to define precise interpretive criteria to determine thepositivity level of the test. Three new primer sets located in the gag and pol structural genes and in the tatregulatory gene of HIV-1 were studied. The results obtained in 54 seropositive and 120 seronegative individualsdemonstrated the ability of the AMPLICIS test to be used for HIV-1 provirus detection: 53 of 54 of theseropositive specimens were found to be positive with at least two primer sets. We also assessed the usefulnessof this test for the estimation of the HIV-1 DNA load by the end point dilution method with serial dilutions ofblood lysates from 26 HIV-1-seropositive patients.

The polymerase chain reaction (PCR) has already beenwidely used for the detection of human immunodeficiencyvirus type 1 (HIV-1) sequences in blood from HIV-1-sero-positive or individuals at risk for HIV-1 infection (15, 27).The overall sensitivity and specificity of the test vary greatlyfrom one group of investigators to another (10, 34) becausethey are dependent on each group's experience and on thereagents and the thermocycler used.

Different factors also affect the overall amplification effi-ciency. The sample preparation and the primer pairs used tomatch the target sequence are two important points.The choice of adapted primers is crucial in the case of

HIV-1 because it is a highly variable virus. The detectionefficiency of a test for HIV-1 will thus greatly depend on theability of the chosen primers to detect all the HIV-1 strains(23). Very few primer pairs have been extensively studied.The primer set SK 38/39 located in the gag gene of HIV-1 iswell characterized and shows good sensitivity (17, 27, 39).Other primers located in thepol or env genes seem to be lessefficient (34, 37).Sample preparation is also an important step, but few

attempts have been made to simplify the specimen treat-ments. The simplest protocol for the detection of HIV-1proviral sequences in blood described in large studies re-quires at least the separation of the peripheral blood mono-nuclear cells (PBMCs) with a Ficoll gradient and subsequentlysis of the cells (8, 22). The presence of residual heparin inthe lysate may, however, lead to a decrease in the amplifi-cation efficiency and thus increases the risk of false-negative

* Corresponding author.

results (4, 38). Moreover, the use of crude cell lysatessometimes leads to an elevated background level (9).For PCR analysis, the nested amplification concept ap-

pears to be the most sensitive approach to the amplificationof HIV-1 sequences (1, 13). Our group has described a novelimprovement of this technique which allows the detection ofthe final amplified products on polystyrene tubes by measur-ing 125I gamma radioactivity (6, 31).The aim of the present study was to define a standardized

detection protocol with precise interpretive criteria for theresults that were obtained by using a solid-phase assay basedon the affinity capture and gamma counting of the amplifiedproduct after a nested PCR (AMPLICIS procedure) fordetection of HIV-1. New primer sets located in the con-served part of the gag, pol, and tat genes of HIV-1 were

selected. We assessed the sensitivity and the specificity ofthis assay using whole-blood lysates from 54 HIV-1-seropos-itive and 120 HIV-1-seronegative individuals. We also eval-uated the usefulness of this test for the estimation of theHIV-1 proviral load using serial dilutions of the whole-bloodlysates.

MATERIALS AND METHODS

Clinical samples. A total of 54 samples from 54 HIV-1-seropositive individuals (confirmed as positive for antibodiesto HIV-1 by enzyme-linked immunosorbent assay [ELISA]and the Western blot [immunoblot] procedure) were col-lected in EDTA tubes (some of them were collected inheparinized tubes). The lysis was performed on fresh orfrozen samples. The cohort for the present study consistedof 35 patients at Centers for Disease Control (CDC) stage II,3 patients at CDC stage III, and 16 patients at CDC stage IV

1066

Vol. 31, No. 5

PCR-BASED ASSAY FOR HIV-1 DETECTION 1067

TABLE 1. Patient characteristics'

Specimen CDC stage Illness

1101ll112113114115b116117118b119120121122b123b124b125b126b127b128129b130b131b132b133b134b135136b137b138139b140b141b142143b144145b146147148149150b151152b153154155b156158159160161162163b164

IVC1

IVC1

IVC1

IVC1

IIIVEIIIIIVAC,IIIVC'

IVC1DIVDII

IIIIII

IIIIIII

IIIIIIIII

II

IIIVC2IIIIIIIIIIIIIIIVE1VC2IIIVDIIIIIIIIIIIIIIIIIII1VC2II

1VC2II

IVAII

II

DCPCPPCPTOX

Thr

PCP, WS

PCP

PCP, KSKS

OC

ThrOC

KS

HZ

OC

WS

450

No. of CD4cells/,u

9017011020

27027080

2701103102903020120250270200260180360140180703068020

22029022020022011011013020038043013045039051044055026019054049077042014050

440520

AVT

AZTAZTAZTAZTAZT

AZTAZTAZT

AZT

AZT

AZTAZT

AZT

AZTAZT

AZTAZTAZTAZTAZTAZT(±)AZTAZTAZT

AZTAZTAZTAZT

AZT

AZT

AZTAZT

AZTAZTAZTAZT

Abbreviations: AVT, antiviral treatment; +, occasional treatment; -, no

treatment; DC, disseminated candidiasis; PCP, pneumocystis caninii pneumo-

nia; Tox, toxoplasmosis; Thr, thrombocytopenia; WS, wasting syndrome;KS, Kaposi's sarcoma;OC, oral candidiasis; HZ, herpes zoster virus; AZT,zidovudine.

Specimens used for the quantification experiments.

(see Table 1 for characteristics of the patients: CD4 cellcounts, antiviral treatment, and clinical status).Blood samples from 120 HIV-1-seronegative (determined

by ELISA) blood donors were taken at random among

healthy individuals at low risk for HIV-1 infection.

Sample preparation. For the preparation of the bloodlysates, we used a modification of a previously describedprotocol (18). Five hundred microliters of whole bloodcollected either in heparinized or in EDTA tubes werecentrifuged (500 x g, 5 min), resuspended in 0.5 ml of TEbuffer (10 mM Tris HCI [pH 8], 1 mM EDTA), and washedthree times with the same buffer. The cells were suspendedin 500 ,ul of the lysis buffer (50 mM KCI, 10 mM Tris HCI [pH8.3], 2.5 mM MgCI2, 0.5% Tween 20, 200 ,ug of proteinase K[Boehringer-Mannheim] per ml) and digested for 1 h at 56°C.The enzyme was inactivated by heating for 10 min at 95°C.The lysates could be stored at -20°C until the amplificationswere performed. The amplifications were performed on theequivalent of 25 ,u1 of blood prepared from 500 ,u1 of wholeblood.The lysis method could be applied to fresh samples as well

as to frozen ones. In the case of frozen blood, the hemoglo-bin appeared to stick to the pellet and the number of washeshad to be increased in order to completely eliminate thehemoglobin, which inhibits the Taq polymerase activity (1).The leukocyte debris remaining after the proteinase K diges-tion was eliminated by centrifugation.

Primers. Primers from three different regions of the HIV-1gene were selected. The gag and pol (except the reversetranscriptase region) structural genes and the tat regulatorygene were chosen because of the low mutation rate observedbetween the different isolates in those regions, as determinedby the alignment program BISANCE (Paris, France) on thesequences of isolates available in GenBank (HIVBRUCG,HIVELICG, HIVHXB2CG, HIVMAL, HIVPV22, HIVSF2CG, HIVHNIVNDDK). The selected primers could haveone or two mismatches compared with the sequences ofsome of the isolates. The mismatches were preferentiallylocalized in the middle or near the 5' ends of the primers.The melting temperature as well as the GC content were thesame in the primers of a pair. The 3' ends of the primer pairwere not complementary to each other, to avoid the forma-tion of primer dimers.The oligonucleotides were synthesized on an Applied

Biosystems A 380 synthesizer and were purified by reversed-phase high-pressure liquid chromatography (HPLC; HPLCMonitor; Merck). Biotinylated (CBIO) and tyraminylated(DTRA) primers were synthesized as described previously(29, 31). Iodination of the tyraminylated primers was per-formed by the chloramine T oxidation method (31). 125I-labeled primers were mixed with nonlabeled primers toobtain the desired specific activity and were stored inlyophilized form. They could be kept for 5 weeks at -20°C.CBIO and DTRA primers have a free 5' end and can be usedas probes after labeling with [_y-32P]ATP (Amersham) byusing polynucleotide kinase (Boehringer-Mannheim).

Amplifications. (i) AMPLICIS process. For the AMPLICISprocess, we used the two-step amplification method de-scribed previously (31) (Fig. 1). The MgCl2, primers, andTaq polymerase concentrations were optimized. The firstPCR was run in a volume of 100,ul of 10 mM Tris HCI (pH8.5)-50 mM KCl-1.5mM MgCl2 containing 35 pmol of eachof the outer primers (A1/A2), 2 U of Taq polymerase(Perkin-Elmer Cetus), and 200,uM (each) deoxynucleosidetriphosphates (Boehringer-Mannheim). The samples wereoverlaid with mineral oil (Sigma), and 25 ,ul of the bloodlysate was added last. Then, the samples were first dena-tured for 10 min at 94°C in a thermal cycler (Perkin-Elmer).This was followed by 30 cycles consisting of 1.30 min at94°C, 1.30 min at 55°C, and 2 min at 72°C. A final 8-minelongation was performed at 72°C. Five microliters of the

VOL. 31, 1993

1068 SAUVAIGO ET AL.

FIRST ROUND AMPLIFICATION PRODUCTS

A2 primer

Al primer

DILUTION

+SECOND ROUND AMPLIFICATION PRODUCTS

1251 labelled primer___-~ (DTRA)

biotinylated primer(CB10)

0p

//-d

/'

-d

SOLID PHASE CAPTURE

zr ~ ~ omll-

GAMMA COUNTING

FIG. 1. Scheme of the AMPLICIS assay. Al and A2, first-roundamplification outer primers; CBIO and DTRA, second-round ampli-fication inner modified primers (CBIO, capture primer; DTRA,detection primer).

(ii) Comparison experiments: amplification followed byprobe hybridization. The one-step amplification consisted,first, of 10 min at 94°C and then 40 cycles of 1 min ofdenaturation at 94°C, 1 min of annealing at 55°C, and 2 minof elongation at 72°C. A final step of 8 min at 72°C was thenperformed. The amplifications were performed in a volumeof 100 ,ul containing 25 ,ul of blood lysate in the same bufferdescribed above: 200 ,uM (each) deoxynucleoside triphos-phates, 50 pmol of each outer primer (A1/A2), and 1.25 U ofTaq polymerase. Twenty microliters of the amplified DNAwas analyzed by electrophoresis in 2% (pol DNA products)or 3% (gag and tat DNA products) Nusieve (two-thirds)-agarose (one-third) (Sigma) gel in TAE buffer (0.04 MTris-acetate [pH 8], 0.001 M EDTA). The products werevisualized under UV light after ethidium bromide staining.Samples were transfered to a nylon membrane (Hybond N+;Amersham) in 0.4 N NaOH as described by the manufac-turer. Filters were prehybridized for 2 h at 40°C in 2x SSC(20x SSC is 3 M NaCl plus 0.3 M sodium citrate [pH7.0])-5x Denhardt-0.2% sodium dodecyl sulfate (SDS) and,were then probed with [_y-32P]dATP-labeled oligonucleotide(106 cpm/ml) in the same buffer for 2 h at 40°C. Excess probewas removed by three washes at room temperature with 5 xSSC for 5 min, 2x SSC-1% SDS for 5 min, and finally, 0.2xSSC-0.1% SDS for 5 min. The membranes were then ex-posed to X-ray film (Hyperfilm; Amersham) for 5 h and wereexposed to intensifying screens at -80°C overnight.

Prevention of contamination and amplification controls. Weused the general specific procedures recommended to avoidcontaminations (20). Positive (8E5 cells lysates) and negative(noninfected DNA) controls were systematically incorpo-rated in the assays.

Cell lines. The 8E5 cells were purchased from the AIDSResearch and Reference Reagent Program (those cells con-tain one copy of HIV-1 integrated DNA per cell). CEM cellswere infected as described earlier (28) with the HIV-1-BRUprototype strain (3), the NDK (11) and PAS (28) isolates, andHIV-2 strain HIV-2-ROD. Blood-derived macrophages wereinfected with the macrophage-tropic strain PAR as describedpreviously (32). Specificity control amplifications were per-formed on 1 ,ug of cell-extracted DNA after 5 days postin-fection for the T-lymphocyte cell lines (5) and after 10 dayspostinfection for the macrophages (32) by using the AMPLI-CIS process. The primer specificities were also checked on 1,ug of DNA extracted from HUT 102 cells (containing sevencopies of human T-cell leukemia virus type I [HTLV-I] DNAper cell).

RESULTS

amplified products was pipetted under the oil, and 995 ,l ofwater was used to dilute the products (dilution, 1/200). Twomicroliters of this solution was submitted to another roundof amplification with the inner modified primers. The secondPCR was carried out in a volume of 25 ,ul containing 2.5 pmolof the biotinylated primer (CBIO), 2.5 pmol (100,000 cpm/pmol) of the 251I-labeled primer (DTRA), 0.75 U of Taqpolymerase, and the other reagents described above for usein the first step. After 30 cycles of amplification (94°C for 1min, 55°C for 1 min, 72°C for 1 min), 20 RI was incubated in500 RI of fixation buffer (50 mM Tris HCl [pH 9.5], 500 mMNaCl) in avidin-coated tubes (31) for 1 h at 37°C. The tubeswere then washed twice with the same buffer containing0.5% Tween 20. Bound radioactivity was measured by directgamma counting of the tubes.

Optimization of the amplification conditions: sensitivity ofthe assay and influence of the blood lysate on the amplificationrate. MgCl2, primer, and enzyme concentrations were testedsystematically. In order to simplify the whole procedure, theamplification conditions chosen were identical for the threesets of primers. A compromise which gave good results withany of the three sets of primers was found.The sensitivity of the assay was determined by using serial

dilutions of 8E5 cells in the presence or absence of a bloodlysate from an HIV-1-seronegative donor.We found that by performing 30 cycles in the first ampli-

fication step and 30 cycles in the second round, we obtainedgood results and that one HIV-1 DNA copy could bedetected even in the presence of the lysate. The kineticcurves obtained in the presence of the lysate were also less

J. CLIN. MICROBIOL.


TABLE 2. HIV-1 primers sequences

Localization Fragment lengthGene Primer 5'-3'sequence..(position no.)a (bp [amplification round])

gag Al GAT TTA AAC ACC ATG CTA AAC ACA GTG G 882-909 322 (I)A2 TTT GGT CCC TGT CTT ATG TCC AAA ATG C 1177-1204DTRA TTT GTT CCT GAA GGG TAC TAG TAG TTC C 1047-1074 125 (II)CBIO TCA ATG AGG AAG CTG CAG AAT GGG ATA G 949-976

pol Al TGC CCA CAC TAA TGA TGT AAA ACA ATT A 3208-3235 558 (I)A2 ATT TCC TCC AAT TCC TTT GTG TGC TGG T 3739-3766DTRA ATT CAC TTT TAT CTG GTT GTG CTT GAA T 3647-3674 231 (II)CBIO AGG AGC AGA AAC TTT CTA TGT AGA TGG G 3436-3463

tat Al TGA GGA GGT CTT CGT CGC TGT CTC CGC T 5563-5590 232 (I)A2 TTG GGT GTC GAC ATA GCA GAA TAG GCG T 5360-5387DTRA TTC CTG CCA TAG GAG ATG CCT AAG GCT T 5533-5560 200 (II)CBIO TTG GGT GTC GAC ATA GCA GAA TAG GCG T 5360-5387

a The localization of the primers on the HIV BRU proviral sequence (GenBank) and the length of the synthesized fragments are indicated.

regular, but no decrease in the final signal level was observed(data not shown).The background noise measured in the presence of the

blood lysate from the HIV-1-seronegative donor was verylow and similar to that obtained with amplifications per-formed on water.Primer specificities and sensitivities. All of the primers

selected for use in the present study were 28 nucleotides inlength (Table 2). The stabilities of the duplexes decreasedaccording to the number of the mismatches, and the effect ofthese mismatches on the melting temperature of a hybrid ismore pronounced for hybrids shorter than 20 bp (see refer-ence 24 for a review). Primers of 28 bases in length formmore stable duplexes than do primers of 20 bases in length incases of mispairing, and thus are more prone to amplify thetarget sequence, especially when this target sequence ishighly variable.The specificity of each primer set was assessed by per-

forming the amplification assay on HIV-2-infected cells andHTLV-I-containing cells. No signal could be observed,whereas strong signals were obtained with CEM cells ormacrophages infected with different HIV-1 isolates (BRU,NDK, PAS, PAR), indicating that the primers can be usedregardless of the variability of the strain (data not shown).The efficiencies of the primer sets during the HIV-1

proviral amplification were checked on two blood lysatesfrom two different infected patients (Fig. 2). Kinetic curveswere plotted during the course of the second round ofamplification. The tat primer set gave the best first amplifi-cation yield, because the measured signals appeared system-atically earlier with that set than with the others. At the endof the second step, the levels reached with the gag or tatprimer set were equivalent. The pol primer set had a loweramplification efficiency, although high values were alsoobtained after the 30 cycles of amplification.Sample collection. The effects of two usual anticoagulants

on the final sensitivity of the whole assay were compared for12 lysates.No significant differences could be observed between the

specimens collected either in EDTA or in heparinized tubes,provided that the lysis was performed on 500 ,ul of blood.However, the screening of the HIV-1-seropositive patientswas performed on blood collected in EDTA tubes.

Screening of seronegative donors and definition of a cutoffvalue for the HIV-1 AMPLICIS procedure. Blood lysates

from 120 HIV-1-seronegative donors taken at random wereexamined by the AMPLICIS assay, which simultaneouslytested for the signals of the primer sets in the three regions.The objective of the study was to check the specificity of theprimer sets and the technique as well as to determine thepositivity level of the test.The background noise values usually varied between 100

and 400 cpm; they could reach 1,000 cpm after long-termstorage of the 125I-labeled primer (5 weeks). Positive signalsvaried between 8,000 and 20,000 cpm, depending on the ageof the DTRA primer. A cutoff value (Co) was estimated asthe average of the background values (x) plus 5 times the

cpm 20000

lysate 225

10000

05 15 25 35

cycles

cpm 20000

lysate 226

10000

5 1 5 25 35cycles

FIG. 2. Second-round amplification kinetic curves obtained withthe three sets of primers for two blood lysates from two HIV-1-seropositive individuals. El, tat gene; *, pol gene; 0, gag gene.

VOL. 31, 1993


standard deviation. The cutoff value was validated whenCoix was greater than 3.Some samples gave a positive signal for one of the three

regions and were retested; none of the positive samples gavea positive signal for more than one gene. Among the speci-mens from 120 donors that were screened, one remainedpositive for the pol region after retesting. This specimenmight have been contaminated by positive controls during itspreparation. A new blood specimen from this donor wastested and gave negative results by the AMPLICIS assay.Comparison between one-step amplification assay and then

hybridization and the AMPLICIS process. The comparisonbetween one-step amplification and then hybridization andthe AMPLICIS process was performed on 15 blood lysates(4 from patients at CDC stage IV, 1 from a patient at CDCstage III, 10 from patients at CDC stage II) after independentamplifications of each lysate. The lysates tested by theAMPLICIS procedure were classified as positive or negativefor each primer set by using the cutoff values. After theone-step PCR and Southern blot hybridization, 15 of 15infected samples were positive with the tat primer set and 10of 15 and 11 of 15, respectively, were positive with the gagand pol primer sets. By the AMPLICIS assay, 15 of 15samples were found to be positive with the gag primer set,14 of 15 were found to be positive with the tat primer set, and13 of 15 were found to be positive with the pol primer set.

In conclusion, 14 of 15 samples were positive in at leasttwo regions after the AMPLICIS assay, whereas 11 of 15samples were positive in at least two regions after theone-step procedure.The results of the comparison were in agreement with the

kinetic curves obtained previously. The tat signal appearedearlier than the gag and pol signals in the course of thesecond amplification, showing that the amplification levelreached at the end of the first round was already high. Afterthe 30 cycles of the second round of amplification, the gagsignal was equivalent to the tat one, whereas the pol signalremained smaller because of a less efficient amplificationrate.AMPLICIS assay on seropositive patients. A total of 54

blood lysates from HIV-1-seropositive patients were sub-jected to the AMPLICIS assay. A specimen was classifiedpositive when at least two viral regions were detected at thefirst examination. Samples positive in only one region wereretested.A total of 52 samples (96%) were found to be positive with

at least two primer pairs after the initial screening (48 of 54with the three sets). The two remaining samples (samples164 and 158) were positive in one region only; results forthese two samples were considered doubtful and the sampleswere then retested.Sample 164, which was positive for the tat region at the

first examination, was positive for the two other regions atthe second screening. It was then considered definitelypositive. This sample was still positive when it was retested;a mishandling of the sample may account for the lack ofHIV-1 detection at the first examination.Sample 158 was positive only for the gag region after the

first screening by the AMPLICIS assay. A total of 12 testsperformed on this sample resulted in three positive signals,once for each region.

Serial dilutions. The use of serial dilutions of PBMCs mayallow estimation of the proviral load in infected samples (7,35). The frequency at which negative results occur for eachdilution is correlated with the number of HIV-1 DNAmolecules in each sample (Poisson distribution). In order to

simplify the experiments, we used serial dilutions of thewhole-blood lysates to evaluate a relative viral gene load inthe HIV-1-seropositive specimen studied. The dilutions of 26of the lysates were amplified with the gag primers (seeexamples in Fig. 3), and the last dilution that gave a positivesignal was considered to contain one copy of the HIV-1 gagsequence.

Estimation of the HIV-1 gag DNA load. CD4 cells havebeen shown to constitute the main reservoir for HIV-1 DNAin peripheral blood (16, 33), and infected CD4 cells arebelieved to contain one copy of integrated proviral DNA percell (33). We can thus attempt to estimate the theoretical rateof infected CD4 cells as a function of the number of CD4 cellcounts for each specimen (Fig. 4). The results show that thepercentage of CD4 cells containing HIV-1 gag sequencesincreases with a decrease in the number of CD4 cells (meanvalue for specimens with a CD4 cell count of <400, 5.06HIV-1 gag sequences per 1,000 CD4 cells; mean value forspecimens with a CD4 cell count of >400, 1.66 HIV-1 gagsequences per 1,000 CD4 cells). The variations observedbetween samples within the same range of CD4 cell countswere high, however, especially when the CD4 cell count wasless than 200. Similar results have been reported by othergroups (12, 26), and we confirmed the fact that the patientswith low CD4 cell counts who harbored a low amount ofHIV-1 proviral DNA were those who were asymptomatic(12). The mean number of CD4-infected cells was 1 in 209 forspecimens from patients at CDC stage IV (lower ratio, 1/360;higher ratio, 1/20) and 1 in 1,118 for specimens from patientsat CDC stages II and III (lower ratio, 1/3,500; higher ratio,1/30). The amount of provirus was clearly higher in patientswith AIDS than in those who were asymptomatic, whatevertheir CD4 cell counts might be.

DISCUSSION

We have previously shown that the use of nested primersin PCR increases both the sensitivity and the specificity ofthe amplification, in particular, when the sample is dilutedfor the second round of PCR (31). This increased sensitivityallows amplifications to be performed on crude blood lysateswithout an increase in the background noise level. Thesimplification of the specimen treatment is important be-cause it saves time and prevents the cross-contaminationbetween samples that results from exhaustive handling.Large-scale preparation of blood samples can be carried outeasily by this protocol.We established that a minimal initial sampling volume of

500 ,ul is necessary for lysate preparation. This was the casewhen the samples were from patients with low CD4 cellcounts because it was necessary to get enough T4 cells tofind at least one that harbored the integrated HIV-1 provirus.It was also important when the samples were from asymp-tomatic patients with high CD4 cell counts and with a lowviral DNA load. The theoretical number of T4 cells con-tained in 25 RI of whole blood (approximately 20,000) issmaller than the corresponding number (approximately70,000) in the generally used PBMC preparations (1, 37).However, in the present study the initial sampling volumewas sufficient to allow the unambiguous detection of HIV-1DNA in all 35 patients at CDC stage II and all but 1 of thesymptomatic patients (specimen 158). More specimens fromasymptomatic patients with high CD4 cell counts (>600)were not available, and we could not confirm these resultswith specimens from a larger population.

In our study of 12 samples, heparin had no effect on the

J. CLIN. MICROBIOL.


M 126 11200

1 2 3 4 5 6 7

cpm

cpm

1 2 3 4 5 6 7

M 1321170

10000

1 2 3 4 5 6 7

cpm

1 2 3 4 5 6 7 1 2 3 4 5 6 7

FIG. 3. HIV-1 gag DNA amplification in serial dilutions of whole-blood lysates from HIV-1-seropositive individuals. The AMPLICISassay was performed on fivefold serial dilutions of the lysates prepared in water (lysate, column 1; dilutions, columns 2 to 5). First- andsecond-amplification-negative controls were incorporated into each test (columns 6 and 7). The results obtained for 6 of the 26 patients testedare reported with the specimen code and patient characteristics (CDC stage and CD4 cell count per microliter of blood). The result obtainedfor each dilution was generally clearly positive or clearly negative. Calculation criteria based on the lysates from HIV-1-seronegative patientswere used, however, when the positivity status of a dilution was in doubt.

final results obtained by the AMPLICIS assay. In fact, theinhibitory effect of this anticoagulant on amplification is notcomplete (14), and the second round of amplification, whichis a high-yield reaction (31), may compensate for the nega-tive effect of heparin. The heparin may also be eliminated

during the cell pellet washing step and thus could not inhibitthe Taq polymerase or bind to the target DNA.The use of solid-phase gamma counting of the final ampli-

fication products allowed for the easy and fast screening ofall samples. The results are given as numbers and the

cpm

10000

0

cpm 2

cpm

VOL. 31, 1993


c

.2

(4

n

0

.0

.E

0

100 -

10 -.

1.

,1

0

a

a

.

Q

0aEQ a

a

a

aa

< 200 200-400 > 400

CD4 cell counts

FIG. 4. Estimated rate of infected CD4 cells (per 1,000) relatedto the CD4 cell count per microliter of blood for 26 HIV-1-seropositive patients. The number of infected CD4 cells was evalu-ated by using the theoretical number of CD4 cells present in the lastblood lysate dilution that gave a positive signal after the AMPLICISassay. The last positive dilution was considered to contain one copyof the proviral HIV-1 gag sequence. 0, patients at CDC stage IV;[l, patients at CDC stage II or III; 0 and *, patients undergoingzidovudine therapy.

samples were classified as clearly positive or clearly nega-tive. Visualization of the second-step amplification productson agarose gels is not adapted for use in large-scale studiesand, as reported earlier (13), is not sensitive enough for thedetection of all positive samples. In addition, this methoddoes not differentiate the specific DNA duplexes from thenonspecific fragments of the same size.

Different primer sets were also studied since two distinctprimer pairs are usually recommended for use, first, to limitthe risk of contamination, which increases with the fre-quency of a given primer set use (34), and, second, to resolveconflicting results that may arise (2). To facilitate the perfor-mance of this assay, the amplification conditions were iden-tical for the three sets of primers.The lower amplification rate observed with thepol primer

set may be related to the length of the synthesized frag-ments. The presence of a T at the 3' end of the tat primer setsequences may help to preserve the amplification rate evenin the case of 3' mispairing (21). Secondary structures withinthe target sequences may also explain the differences in thebehaviors of different primers. However, the final resultswere equivalent with the three sets of primers (at the firstexamination, 51 of 54 of the HIV-1-seropositive sampleswere detected with the pol and gag sets and 52 of 54 weredetected with the tat set).Among the 54 HIV-1-seropositive specimens studied, only

2 needed a confirmatory test after the first screening. Sample164 was easily detected at the second screening, whereasconflicting results were obtained with specimen 158. Thissample came from a patient at CDC stage IV (CD4 cell countper microliter, 770) and should have contained high levels ofintegrated provirus. However, the zidovudine treatmentmay have decreased the HIV-1 DNA load. Because thisspecimen was positive once for each region, it was probablynot infected with a special HIV-1 variant that could not berecognized by the primers. Amplification of this sample with,B-globin primers (30) was positive. However, because it wasnot quantitative, it does not reflect the integrity of the pellet

and the hypothesis that part of the material was lost duringthe sample preparation cannot be rejected. A new samplecould not be obtained but would be required to make aconclusion regarding this patient's status.The comparison, performed on blood lysates, between the

one-step amplification and then hybridization and the AM-PLICIS process shows that the nested technique is moresensitive, especially with the gag and pol primer sets. Thisallows the amplifications to be performed with these primerson crude material without any loss of sensitivity.The usefulness of the AMPLICIS assay for HIV-1 DNA

quantification by the end point dilution method was alsotested. Only one AMPLICIS assay for gag was performedon each lysate dilution; an approximate profile of the gagDNA load in each specimen could thus be obtained. A moreextensive analysis of each dilution would allow a moreprecise calculation of the HIV-1 proviral DNA load in theblood lysates (35). However, the results obtained seem to bein agreement with previously reported data on the HIV-1DNA loads in asymptomatic and symptomatic HIV-1-in-fected patients (22, 25, 26). The variations in the results forthe samples from asymptomatic patients with CD4 cellcounts of <200 may be explained partly by the fact thatcertain patients were undergoing treatment with zidovudine.Moreover, the time at which therapy was started wasdifferent among these patients, and the response to zidovu-dine may also vary from one person to another (36).The AMPLICIS assay applied to the end point dilution

method may be a way to follow the variation of the HIV-1DNA load in blood samples. The method is extremelysensitive, and even in the presence of whole-blood lysate,one HIV-1 DNA copy can easily be detected. The signalsobserved were, in all cases, clearly positive or clearlynegative because they depend only on the presence orabsence of the target DNA. This method does not requirestandard curves or the very precise conditions necessarywhen the amplification must be in the exponential phase(19).

In conclusion, the assay developed for HIV-1 detection isspecific, highly sensitive, and easy to perform. It requires noextensive purification of the samples and only a small initialblood volume. This can be important for the application ofthe technique to screening of newborn infants. It is adaptableto large-scale studies and automation. Interpretation of theresults is easy and depends only on objective criteria.

ACKNOWLEDGMENTS

This work was supported in part by grant 90MO802 from GBM/MRT and in part by the Institut National de la Sante et de laRecherche Medicale.HUT 102 cells were a gift from C. Desgranges (Institut National

de la Sante et de la Recherche Medicale U271, Lyon, France).

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standardized nested polymerase chain reaction-based assay for

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