Journal of Yirolog~~a~ methods, 44 (I 993) 45-56 @ 1993 Elsevier Science Publishers B.V. All rights reserved / 0166-0934~93/~06.00

VIRMET 01530

-~~

Journal of Virological Methods

Quantification of human cytomegalovirus DNA in peripheral blood polymorphonuclear leukocytes

of immuno~ompromised patients by the polymerase chain reaction

Donato Zipeto”2b, Faust0 Baldantib, Davide Zella”, Milena Furioneb, Ada Cavicchini”, Gabriele Milanesi” and Giuseppe Gernab

“lstituto di Cenetica Biochimica ed Evoluzionistica, Consiglio Nazionale delle Ricerche, Paviu (Italy] and bLaboratorio di Virologia. Istituto di Malattie Infettive, Universitri di Pavia, Istituto di Ricovero

e Cura a Carattere Scientifico Policlinico S. Matteo, Pavia (Italy) and “Sorin Biomedica, Saluggia, Vercelli (Italy)

(Accepted 23 March 1993)

Summary

Human cytomegalovirus (HCMV) DNA amplification by the polymerase chain reaction (PCR) was utilized previously for successful monitoring of HCMV infections in immunocompromised patients. However, analysis of an extended series of clinical samples revealed the relatively frequent presence of PCR inhibitors. Hence, the need for availability of an internal control of the reaction allowing identification of false negative results. Similarly, an internal standard appeared necessary for quanti~cation of viral DNA in clinical samples. For this purpose, we constructed a recombinant DNA molecule which could be amplified by the same set of primers used for HCMV DNA amplification. Coamplification of the recombinant DNA molecule and clinical samples proved to be a simple and reliable method for verifying sample competence for amplification. In addition, coamplification of serial known amounts of the same molecule, used as internal standard, and test sample, allowed quantification of viral DNA in polymorphonuclear leukocyte samples. Quantitative monitoring of HCMV infection and antiviral treatment may provide critical indications as to whether and when to initiate or discontinue antiviral treatment in immunocompromised patients with systemic HCMV infections.

correspondence to: G. Gerna, Virus Laboratory, Institute of Infectious Diseases, University of Pavia, IRCCS Policlinico S. Matteo, 27100 Pavia, Italy. FAX: f 39-382-423320.

46

Human cytomegalovirus; Viral DNA quantification; Peripheral blood polymorpho- nuclear leukocytes; Immunocompromised patients; Polymerase chain reaction;

Internal PCR control; Coamplification; Internal standard

Introduction

Disseminated human cytomegalovirus (HCMV) infections are a major complication in organ transplant recipients and AIDS patients (Gerna et al., 1990a; 1991). Monitoring of these infections is achieved routinely by quantitative determination of HCMV antigenemia (Gerna et al., 1992) and viremia (Gerna et al., 1990b). HCMV DNA determination by the polymerase chain reaction (PCR) has proven to be a very sensitive method for viral detection in peripheral blood polymorphonuclear leukocytes, yet presence of viral DNA did not always correlate with viremia or clinical symptoms (Gerna et al., 1991), thus complicating the interpretation of its clinical significance. The need for viral DNA quantification, on the other hand, has arisen in view of monitoring, at a molecular level, HCMV infections and efficacy of antiviral treatment.

Methods developed in the last few years for DNA quantification by means of PCR may be classified into four major groups: (i) end-point sample dilution prior to PCR reaction; (ii) coamplification of target and cellular DNA sequences, such as P-globin, fi-actin, HLA DQ (Kellog et al., 1990; Katz et al., 1990; Ballagi-Pordiny et al., 1991); (“‘) m comparison of sample PCR product with PCR product obtained by serial dilutions of the same target sequence (external standard) (Dickover et al., 1990); (iv) coamplification of target DNA and the same modified target sequence as internal standard, which are amplified by the same primers and differentiated by size (Gilliland et al., 1990; Ozawa et al., 1990; Menzo et al., 1992; Diviacco et al., 1992), presence or absence of a restriction site (Becker-Andrk et al., 1989; Fox et al., 1992) or different electrophoretic mobility in a temperature gradient gel electrophoresis (TGGE) system (Hence et al., 1989). Each proposed method contains some pitfalls, which will be discussed later.

We used a different approach. A recombinant DNA molecule of 365 base pairs (derived from plasmid GEM 4Z), was chosen for its similarity in nucleotide (A + T/G + C) composition to the HCMV IEl gene region (exon 4) currently amplified in our laboratory for HCMV DNA detection (Gerna et al., 1991; Zipeto et al., 1992). This molecule was constructed by adding at its ends the same sequences as HCMV primers. Thus, it could be amplified by the same set of primers used for HCMV DNA detection and was used as both internal control of amplification in each clinical sample (to detect potential PCR inhibitors) and internal standard for HCMV DNA quantification.

47

Materials and Methods

Construction of plasmid pAC

In order to construct a recombinant DNA molecule competent for amplification by the same pair of primers relevant to exon 4 of the IEl gene and currently in use in our laboratory for HCMV DNA detection in clinical samples (Zipeto et al., 1992) a pair of complemen~ry 40mer oligonucleotides consisting in 5’ of 20 bases corresponding to HCMV primer sequences and in 3’ of 20 bases complementary to a region of pGEM4Z (Promega, Madison, WI, USA), was synthesized. The primer used were: forward (S-3’) AGACC TTCAT GCAGA TCTCC GGTTA TTGTC TCATG AGCGG; reverse (5’-3’) GGTGC TCACG CACAT TGATC TGCTC TGATG CCGCA TAGTT. In both primer sequences, nt l-20 were relevant to the HCMV IEl-gene (Zipeto et al., 1992), whereas nt 2140 were relevant to plasmid pGEM4Z. By performing PCR on pGEM4Z with these primers, a recombinant DNA molecule including HCMV primers at its ends was obtained. The pGEM4Z amplified region, which was 365 bp long and differed in size from HCMV IEl region (which was 267 bp long), was chosen on the basis of: (i) its similarity in nucleotide composition with the amplified HCMV IEl region; (ii) the presence in the internal part of the molecule of a sequence similar in A + T/G + C composition to the probe used for hybridization of HCMV ampli~cation products. These properties should result in a similar efficiency of amplification and hybridization for the HCMV IEI region and the recombinant molecule.

In detail, pGEM4Z was first linearized by using endonuclease Barn HI; then, 0.1 pg of the linearized plasmid were added to the PCR mixture and subjected to 40 cycles of amplification. The amplification product was then purified by centrifugation on Centricon 30 (Amicon, Beverly, MA, USA) and spectro- photometrically quantified. Finally, it was ligated to vector PCR 1000 according to manufacturer’s instructions, and then cloned in competent INVaF cells (TA cloning kit, Invitrogen, San Diego, CA). The resulting 3287 bp plasmid, referred to as pAC, was extracted from positive clones using the Qiagen puri~cation system (Promega, USA). Following linearization, pAC was ready for use in coamplification PCR assays on clinical samples to be tested for presence of HCMV DNA.

Coamplfication by PCR

In order to verify that pAC was amplified with the same efficiency as the HCMV IEI region, the HCMV amplification product (267 bp) was cloned in vector PCR 1000. Following linearization, the plasmid, referred to as pCM, was coamplified with linearized pAC in a series of log,, dilutions containing an identical number of copies (1 O6 through 10) of the two plasmids. Clinical samples were ampli~ed (Gerna et al., 1991; Zipeto et al., 1992) in the presence of 100 copies of pAC, to identify negative results due to the presence of PCR inhibitors.

48

Samples competent for amplification were then coamplified in the presence of 5 x lo’, 5 x lo* and 5 x lo3 copies of PAC. PCR was performed for 40 cycles (1 min at 94°C 1 min at WC, 40 s at 72°C for the first 5 cycles and, then, 40 s at 94°C 1 min at 55°C and 3 min plus 5 s/cycle at 72°C for the remaining 35 cycles) using a DNA Thermal Cycler 480 (Perkin Elmer Cetus, Norwalk, CA) and optimized reaction parameters, as well as the Hot-StartTM technique. Under these conditions, synthesis of specific amplification products is obtained, even when starting from a very low input copy number and in presence of cellular DNA. Amplification products were detected by gel electrophoresis followed by ethidium bromide staining and direct band visualization on a UV transillumi- nator. Specificity of amplification products was determined by hybridization using the DNA enzyme immunoassay (DEIA) technique (Mantero et al., 1991).

DNA purzyication

In order to eliminate false negatives due to PCR inhibitors present in some clinical specimens, aliquots of 150 000 polymorphonuclear leukocytes (PMNL) from immunocompromised patients were submitted to DNA extraction using a rapid method based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate, together with nucleic acid-binding properties of silica particles (Boom et al., 1990).

DNA quantlyication

Aliquots of PMNLs from immunocompromised patients, each containing 50000 PMNL, were amplified in the presence of 50, 500 and 5000 copies of PAC. Two equal aliquots of each amplification product were separately immobilized onto nylon filters in a slot-blot apparatus. Filter hybridization was then performed using two separate digoxigenin 3’-end labeled oligonucleotide probes (DIG Oligonucleotide 3’-End Labeling Kit, Boehringer Mannheim Biochemica, Mannheim, Germany) specific for pAC and HCMV sequences respectively. Hybridization products were detected by using chemiluminescence (Dig Luminescent Detection Kit, Boehringer Mannheim Biochemica). The hybridization bands were then digitalized and analyzed by using an appropriate software (Image 1.47 for Apple@ MacintoshTM, NIH, Bethesda, MD, USA) to determine the intensity of bands, which was directly related to the amount of amplification products obtained for pAC and HCMV, respectively. The ratio pAC/HCMV obtained for each of the 3 pAC concentrations (50, 500 and 5000 copies) was plotted against the pAC copy number (loglo) to obtain a standard curve. This was constructed utilizing the computer program CA-Cricket Graph III for Apple’8 MacintoshTM (Computer Associates International Inc. Islandia, NY, USA), the interpolation curve fit method using the natural cubic spline interpolation algorithm. The intersection point of the curve with the line parallel to abscissa corresponding to a pAC/HCMV ratio of 1.00 gave the DNA copy number of the sample.

49

Results

CoampliJi:cation control

The efficiency of amplification of pAC relative to pCM was verified by coamplification of the two plasmids in a series of loglo dilutions containing the same copy number of each (from 1 million copies to 1 copy). Results showed that the two plasmids were amplified practically with the same efficiency, since each dilution gave the same amount of final product for the two molecules (Fig. 1A). In the presence of a low input copy number (< 20), a slight predominance of either amplification product was observed in multiple replicate experiments (n = 8), which was likely to be due to a stochastic dilution error. On the other hand, competition between the two molecules was investigated by coamplifying increasing concentrations of pAC in the presence of decreasing amounts of pCM. Results showed that when initial concentration of one molecule was greater than the other one by 4 logs 10, no amplification product of the less represented molecule was observed (Fig. 1B). In addition, the competition effect was markedly reduced by using slightly prolonged extension times for each cycle.

Use of pAC as internal amplification control

Several hundreds of PMNL samples from immunocompromised patients (AIDS patients and heart transplant recipients) were amplified in the presence of 100 copies of PAC. According to different groups of samples examined, the

PAC

PCM

Copy Number (Log,,)

6543210 MW

0123456

Fig. 1. pAC and pCM coamplification experiments. On the left (A): serial dilutions (lo6 to 1 copy) show the same efficiency of amplification of the 2 plasmids, which are detected in comparable amounts down to 10 input copies. M, Molecular weight markers. On the right (B), increasing log,0 concentrations of pAC (1 to IO6 copies) are coamplified with decreasing log,, concentrations of pCM; the 2 plasmids are amplified with the same efficiency when the copy number of each plasmid is identical (103), whereas the competition effect becomes more evident concomitantly with the incresing difference in input copy number of the 2 plasmids.

50

prevalence of PCR inhibitors appeared to be in the range of 5-20%. Samples containing PCR inhibitors were then extracted using the silica procedure. Results obtained in 5 of these samples are exemplified in Fig. 2, which shows that clinical specimens are partially (2 20% reduction in pAC ampliIication signal as compared to the mean value of 3 pAC controls) or totally non- competent for amplification in A - as demonstrated by the reduction (lanes 1 and 5) or lack (lanes 2-4) of amplification of the internal control (pAC) - and the same specimens after silica extraction in B.

An example of HCMV DNA quantification in a clinical sample according to the procedure described in ‘Materials and Methods’ is reported in Fig. 3. In Fig. 3A, PAGE results of the three coamplification reactions performed using 50, 500 and 5000 copies of pAC, are reported. In Fig. 3B, chemiluminescence hybridization results of the three coamplification products are shown

A

Fig. 2. pAC (100 copies) used as internal control of amplification of 5 clinical samples showing presence of PCR inhibitors. A, lanes l-5 PMNL samples which are partially or totally non-competent for amplification. B. The same specimens after silica extraction. After hybridization, all 5 samples were

HCMV-positive (data not shown).

51

HCMV -

C 186 2 1s

s

is 14 1*3

iii. 1’2 111

jg 1,o ___~c*~~D__~~~~_L~“~~~~~~~**~~~~~~~~ _....________*w.

Y

2

0,a 099

O,?

e 096 I 0s

1s 1,8 2.0 2,2 2,4 2,6 2,8 3,0 3.2 3,4 3,6 3,8

Log,, copy number

Fig. 3. Quantification of HCMV DNA in a clinical sample (50000 PMNL) by coamplification with PAC. hybridization and densitometric analysis. A: polyacrylamide gel electrophoresis of the 3 coamplitication products relevant to a single clinjcal sample using three serial logto dilutions of PAC. B: slot-blot hybridization of the amplification products shown in A. C: following densitometric analysis, pAC/HCMV peak area ratios were plotted against logta copy number, obtaining a standard curve, By interpolation from the standard curve of the pAC/HCMV ratio of 1 .OO, the HCMV copy number present in the sample was

derived (I460 copy number).

separately for pAC and HCMV. The derived curve with the interpolated value for loglo copy number of the sample is reported in Fig. 3C. Using this procedure, HCNV DNA was quantified in sequential blood samples from two heart transplant recipients (Table 1). DNA findings were compared with quantitative levels of viremia and antigenemia by linear regression analysis and correlation was found to be significant 0,<0.05). In patient 1, DNA was first detected in blood 30 days after transplantation, whereas viremia and antigenemia were detected 51 days post-transplant, when viral DNA approximated the peak. In the following days, initiation of antiviral treatment with foscarnet (due to appearance of fever, gastralgia, leukopenia and thrombocytopenia) caused a progressive drop in viremia, antigenemia and number of viral genome equivalents along with resolution of clinical symptoms. In patient 2, a low number of viral DNA copies was detected one week before antigenemia and viremia became positive. Then, all viral parameters progressively increased reaching a peak as follows: viral DNA 76, viremia 81

52

TABLE i

Virologic follow-up of 2 heart transplant recipients.

Patient Days after no. transplant

HCMV quantitation

Antigenemia” Viremiab DNAemia’

Clinical Antiviral symptoms treatment

1 9 0 19 0 30 0 43 0 51 30 55 250 57 270 61 I5 64 8 68 0 71 3

2 6 9

:: 44 73 76

87; 86

100 123

0 0 3

35 70

350 400 650 600 700

10 0

0 0

t: 9

132

; 0 0 0

e 1 8

54 ND

12 56

196 19 0 0

<20 <20

20 25

2,000 3,500

600 50 50 60 60

<20 20 50

950 3,000 3,700 3,800 3,500 7,200 4,000

250 30

- - - - - - + - + + + t _ + - + - -

- - - - -

- - - - - - - - - - - -

“antigenemia: no. HCMV pp65-positive PMNL/200,000 PMNL examined (Gerna et al., 1992). bviremia: no. positive cultured ~broblasts~200,OOO PMNL inoculated (Gerna et al., 1990a). ‘DNAemia: no. of HCMV genome equivalents/SO,000 PMNL tested by PCR.

and antigenemia 86 days after transplantation, respectively. Since no clinical symptom was observed (likely due to late appearance of infection which could be controlled by the recovering immune system), no antiviral treatment was required and recovery from HCMV infection occurred spontaneously. At day 123, when viremia and antigenemia tested negative, viral DNA was still detected at a very low level.

Experiments carried out in order to verify the range of reproducibility of quantitative DNA results showed that a maximal variation of It 30% was found among different amplification assays performed on the same test samples.

Discussion

Comparison of the HCMV DNA quantification method described in this report with quantification methods reported previously indicates some advantages in terms of simplicity of performance and result significance. End

53

point sample dilution methods prior to PCR are in fact semiquantitative assays which allow only comparison of the relative amounts of target sequences in clinical samples without providing the absolute number of input viral DNA copies. Methods based on coamplilication of target and cellular DNA sequences present in the same clinical sample (Kellog et al., 1990; Katz et al., 1990; Ballagi-Pordany et al., 1991) on the other end, display some major disadvantages due to different amplification efliciency caused by the use of two different DNA target sequences and two different pairs of primers, and also by the presence of largely different relative amounts of cellular and viral DNA. Methods based on comparison of sample PCR product with PCR product obtained by serial dilutions of the same target sequence (external standard) (Dickover et al., 1990) do provide comparable amplification efficiency (same sequences and primers), yet in the absence of an internal control of competence for amplification, do not consider the presence of potential sample inhibitors.

Methods based on coamplilication of target DNA and the same modified target sequence as internal standard (which are amplified by the same primers and differentiated by size, presence or absence of a restriction site, or by electrophoretic mobility) show the advantages of an equivalent efficiency of amplification and allow evaluation of the inhibitory effect of samples (Becker- Andre et al., 1989; Hence et al., 1989; Gilliland et al., 1990; Menzo et al., 1992; Fox et al., 1992).

Among different methods using the same target sequence variably modified as internal standard, the method reported by Fox et al. (1992) and based on the presence of a restriction site, deserves particular consideration, since it is, in our knowledge, one of the first proposed PCR method for HCMV DNA quantification. In our opinion this method does not appear to be entirely satisfactory for the following reasons: (i) partial digests can alter relative amounts of competitor and target sequence; (ii) a partial digest of competitor in a negative sample can give a false positive result; iii) the proposed method requires Southern blot, which is a quite cumbersome procedure potentially causing a partial transfer of amplified material (and namely low molecular weight molecules) onto filter, with consequent alteration of the relative amounts of the two molecules.

The quantification method described in this report differs from the latter group of methods, in that it uses a molecule different from target DNA in size and sequence, although similar in A + T/G+ C content. By progressively increasing the extension time during PCR, the competition effect between the two molecules was greatly reduced. This allow restriction of the internal standard to three serial loglo dilutions, which were sufficient to quantitate DNA with an acceptable degree of precision within a wide range and, at the same time, in a relatively large number of samples. In addition, given the high number of false negative results in clinical samples due to the frequent presence of PCR inhibitors, DNA silica extraction became a preliminary essential part of our routine procedure for DNA quantification. DNA quantification was achieved by densitometric analysis of slot-blot hybridization products. Since a

54

major advantage of our method is the opportunity of carrying out hybridization analysis of the same PCR amplification product by using two different probes capable of recognizing amplified standard and target DNA, respectively, other hybridization methods simpler than slot-blot are now under evaluation. Among these, immunoenzymatic methods such as DEIA using a non-radiolabeled biotinylated probe bound to avidin-coated microtiter plates and a MAb specific for double stranded-DNA seem to be one of the most promising with a microtiter plate ELISA format. Automation of these procedures will be a further goal.

Results of HCMV DNA quantification obtained during follow-up of 2 HTRs are particularly interesting. Firstly, quantitative viral DNA levels appear to correlate with levels of viremia and antigenemia. Secondly, a low DNA copy number is present at the onset of infection prior to appearance of HCMV viremia and antigenemia as well as at the end of infection, when viremia and antigenemia have become negative. These findings are in agreement with those previously reported by our group on a more extended series of patients, in whom viral DNA was only qualitatively determined (Gerna et al., 1991). In addition, it is emphasized that even minimal amounts of viral DNA were never detected in PMNL of HCMV-seropositive immunocompetent subjects, when using our PCR method for HCMV DNA quantification. On the other hand, it has been recently documented that PMNL of immunocompetent individuals do not harbour, unlike adherent mononuclear cells, HCMV DNA (Taylor- Wiedeman et al., 1993).

Potential clinical applications of the method described now may include study of the natural history of HCMV infection in the immunocompromised host as well as molecular evaluation of the efficacy of antiviral treatment (ganciclovir, foscarnet). Further simplification of the hybridization method would provide a handy means for quantitative DNA analysis of large numbers of clinical samples.

Acknowledgements

This work was partially supported by Minister0 della Sanita, ISS, Progetto Nazionale AIDS 1993, grant nos. 8205-09 and 7204-71, and by Consiglio Nazionale delle Ricerche, Target Project BTBS, grant no. 92.01201.PF70. We thank Linda D’Arrigo for revision of English.

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