Detection of cytomegalovirus using ‘boosted’ nested PCR

Molecularand CellularProbes(1995) 9, 251-257

Detection of cytomegalovirus using 'boosted' nested PCR Kristin Lund Borg, 1'2. Svein A. N o r d b ~ 7

Per W i n g e 1 and A r e D a l e n 1'2

UNIGEN Center for Molecular Biology, University of Trondheim, 2Department of Microbiology, Regional Hospital of Trondheim, Norway (Received 19 May 1994, Accepted 3 April 1995) As a part of a study of an outbreak of CMV infections in a neonatal care intensive care unit, a modified nested PCR was developed for detection of CMV DNA in clinical specimens. Standard nested PCR involves a critical step; passage of PCR products from the first reaction round to the second round. We have adapted a 'boosted' nested PCR which implies amplification in one single step, thus reducing the contamination problems. Nasopharyngeal aspirates and urine samples from patients with perinatal CMV infections, breast milk from some of their mothers, amniotic fluids, urine samples and lymphocytes from seropositive healthy adults were examined by PCR and culture. In the total of 614 of clinical specimens, the PCR test yielded positive results in 51 samples from 14 patients, whereas CMV was isolated in 25 samples from 11 cases only. All samples from healthy individuals were negative. CMV DNA was detected in all culture-positive samples, but all samples from healthy adults were negative. 29/68 culture negative specimens were positive by PCR. No cross-reactivity to other herpes viruses or to human DNA was observed. Our findings show a high sensitivity and a high specificity of the 'boosted' nested PCR. We conclude that the described PCR method can be used for the rapid detection of CMV in clinical specimens with a greatly reduced risk of contamination, and it has proved to be a very useful tool in diagnostic work. © 1995 Academic Press Limited KEYWORD$: cytomegalovirus infection, 'boosted' nested PCR, alkaline phosphatase, slot-blot

hybridization, chemiluminescent substrate.

INTRODUCTION virus infections, z'lz However, the method involves the passage of the amplified products from the first reaction to a second stage that is both inconvenient and provides an opportunity for contamination. We have adapted a nested PCR with amplification in a single step, thus reducing the contamination problems. 6'11 Both the outer (0.8 pmol) primers and the inner primers (25 pmol) are added to the initial reaction mixture. Because of the length (26-mer), the outer primers have higher melting temperature Cl'm= 65°C) than the inner primers (17-mer with a Tin= 43°C). The initial PCR conditions using a thermal file with a high annealing temperature (65°C), allow only

Cytomegalovirus (CMV) infections are usually asymptomatic, while serious disease with high mortality is seen in premature infants and other immunocompromised individuals. 1'12'1s Diagnosis of CMV is clinically difficult and the standard procedure for isolation of virus in tissue culture is time consuming and may be unreliable. The polymerase chain reaction (PCR) offers the advantages of speed compared to conventional culture, and the sensitivity is far better than commonly used antigen tests.~-s'8'9''6To improve PCR sensitivity and specificity, nested PCR, performed in two stages, has recently been developed for diagnosis of several

* Author to whom correspondenceshould be addressedat: UNIGENCenterfor Molecular Biology,Universityof Trondheim, N-7005 Trondheim, Norway. • 0890-8508195/040251 +07 $12.00/0

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© 1995 Academic Press Limited

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the outer primers to function. This is followed by a second stage with an annealing temperature of 43°C making the inner primers operative. We have chosen to amplify a 184-bp segment from the fourth exon of the CMV immediate early gene. To confirm the identity of CMV PCR products, slot-blot hybridization using an alkaline phosphatase linked oligonucleotide probe was used. In this report we address the use of 'boosted' nested PCR to detect CMV DNA in clinical specimens (urine, amniotic fluid, nasopharyngeal aspirate, breast-milk and serum from pregnant women, premature infants, newborns and their mothers and also lymphocytes from healthy individuals.

MATERIALS AND METHODS Patients Urine samples, nasopharyngeal aspirates and serum samples were collected from 110 premature infants during an outbreak of CMV infections in the neonatal intensive care unit at the University Hospital of Trondheim. Four of the preterm infants (BW 735-1280g) developed grave CMV infections and were treated with antiviral agents, mainly ganciclovir. All infants had respiratory problems, and three of them had patent ductus arteriosus. Two of the infants died, and typical inclusion bodies were found in lungs and kidneys by autopsy. One of them had necrotizing enterocolitis and thrombocytopenia, but died from respiratory failure. The other infant underwent surgery for patent ductus arteriosus, developed hepatosplenomegaly and died from respiratory failure and encephalitis. Fourteen healthy newborns whose mothers were found CMV IgM positive during a predelivery screening was also tested. Breast milk samples from 14 mothers and amniotic fluid from three cases with growth-retarded foetuses (diagnosed by ultrasound) were also tested. In addition urine, lymphocyte and serum samples from 12 healthy individuals were examined by nested PCR and virus isolation or serology.

Nasopharyngeal aspirates, lymphocytes and breast milk samples Nasopharyngeal samples were centrifuged at 1500 rev min -~ for 10 rain. Breast milk samples were centrifuged at 10 000 rev min -~ for 10 rain to remove fat. The supernatants were discarded. The cell pellets were resuspended in 5 ml PBS and centrifugation and washing were repeated. The pellets (from nasoph, or milk) were lysed in 1001.tl of lysis buffer (50 mM potassium chloride, 10 mu Tris-Hcl, ph 8.3, 2.5 mM magnesium chloride, 0.1 (rag gelatin) ml -~, 0.45% Triton 100 (Sigma) and 1.51.d proteinase K (20 mgm1-1, Sigma).8 The pretreated samples were incubated at 65°C for 1 h and then at 95°C for 10 min to inactivate the proteinase K. Lymphoprep (Nycomed Pharma) was used for isolation of lymphocytes from heparinized peripheral blood. DNA was extracted from the lymphocytes using TRIzol reagent followed by ethanol precipitation.

Preparation of PCR primers We amplified a 184-bp segment of the fourth exon of the CMV immediate early gene. The oligonucleotides were prepared on a Cruachem DNA synthesizer, and the primer sequences were as follows: CMV IE outer primer I (2047-2071): 5'-GCT GCG GCA TAG AAT CAA GGA GCAC-3'. CMV IE outer primer 2 (24162441): 5'-GGT TGG TGG TCT TAG GGA AGG CTG AG-3'. The first primer pair allowed amplification of 393bp sequences. CMV IE inner primer I (2089-2105): 5'-CCA GAC GGA AGA GAA AT-3'; CMV IE inner primer 2 (2256-2272): 5'-ACA AGC CAT CCA CAT CT-3'. The second primer pair allowed amplification of 184-bp sequences. In patients being repeatedly positive in CMV PCR and negative in culture, we did an additional confirmatory PCR, amplifying a 1000-bp segment from the CMV immediate early gene. The oligonucleotide primer sequences were as follows: CMV primer 1 (492-512) : 5'-GCA GAG CTC GTT TAG TGA ACC-3'; CMV primer 2 (1527-1547) : 5'TGG CAC CTT GGA GGA AGG GCC-3'.

Urine samples and amniotic fluids Urine samples from newborns and infants and amniotic fluids were centrifuged at 2500 rev min -* for 10 rain to remove cell debris. Two microlitres of the supernatant was directly amplified by PCR without prior DNA extraction.

'Boosted' nested PCR In this study, several procedures were adapted to reduce the risk of contamination: pre-aliquoting reagents, the use of filter pipette tips and physical

Detection of cytomegalovirus separation of the sample preparation from the area of reaction product analysis. The reaction mixture consisted of: 2.5 mM MgCI2, 10 mM Tris-HCI pH 8"85, 50 mM KCI, 1"0 mM EDTA, 0"25% Triton 100 (Sigma), 200 pM each of dNTP, 0.8 pmol of each of the CMV IE outer primers, 25 pmol of each of the CMV iE inner primers and 2 I~1 target DNA in a final volume of 50pl. The tubes were covered with 100 pl mineral oil (Sigma) and then placed at 95°C for 5 min in a heating block. Furthermore, 0.6 U Taq Polymerase (Boehringer Mannheim) was then added to the mix (hot start). The amplification reaction was performed in a DNA thermal cycler (Perkin Elmer Cetus). The initial cycling conditions were as follows: denaturation, 1 min 94°C; annealing, 1 min 30 s at 65°C (for the outer primers); 2 min at 72°C (25 cycles) followed by a second stage cycling consisting of: 1 min 94°C; 1 min 30 s at 43°C (allowing the inner primers to anneal); 2 min at 72°C, repeated for 25 cycles. Each extension period was increased by 1 s. Controls for each run included purified viral DNA extracted from human lung fibroblast (HL) infected cells with the AD 169 strain of CMV (90 ng), DNA from uninfected HL cells (100 ng) and buffer with sterile, distilled H20 replacing target DNA. In several experiments, we checked the quality of DNA preparation by amplifying beta-globin [primer 1 (427-446): 5'-TCT GTC CAC TCC TGA TGC TG3' and primer 2 (592-611): 5'-CCC ATA GAC TCA CCC TGA AG-3'; 1850-bp segment). Twenty microlitres of all the PCR products were analysed by electrophoresis in a 2% agarose gel containing 0.5 I~g ml -* ethidium bromide at 150 V for 2 h using 23 mM Tris-borate buffer, pH 8.3 and 0.5 mM EDTA.

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(100 p.g) was used for the enzyme conjugation. The Elink Plus oligonucleotide labelling kit (Cambridge Research Biochemical) was used to prepare the enzyme conjugated oligonucleotide probe.

Slot-blot hybridization Twenty microlitres of the amplificated product was applied to a Gene-Screen Plus nylon membrane (Dupont) which was specifically recommended for the slot-blot system. The filter was prehybridized with 30ml buffer containing 5 x SSC, 0.5 SDS, 1% BSA (Sigma), 0.1% Ficoll (Pharmacia), 0.1% Polyvinylpyrrilodone (PVP, Sigma) for 1.5 h at 37°C, and then hybridized using the oligonucleotide-alkaline phosphates conjugate in fresh prehybridization solution to a final concentration of 1.0 nM at 37°C overnight. The filter was washed twice in washing buffer containing 1 x SSC, 0-1% SDS for 5 min at 42°C, and then twice in a washing buffer containing 0.25% SDS. The filter was washed in 1 x SSC for 5 min at room temperature, before visualization using the Lumi-Phos substrate according to the method of the producer (Cambridge Biochemical Research).

Virus isolation Nasopharyngeal aspirate, urine, breast-milk and amniotic fluid were inoculated on to human lung fibroblasts (HL). The cultures were observed for Cytopathic effects for 3 weeks. Positive cultures were verified by an indirect fluorescence test using monoclonal antibodies against CMV immediate early antigen.

Standard nested PCR The reaction mixtures and the temperature cycling conditions were similar to that of 'boosted' nested PCR, except the use of the primers. In the first PCR roundonly the outer primers (8 pmol) were used, and a 5-pl portion of the amplified product was subjected to a second PCR round (25 cycles).

Labelling of oligomer probe The following oligonucleotide probe was labelled with alkaline phosphatase: (2109-2129) : 5"-CTG GCG CCT TTA ATA TGA TGG-3'. A C6 aminomodifier was coupled to the 5" end during the DNA synthesises. •One hundred microlitres of aminomodified probe

Serology Determination of CMV IgM and IgG antibodies in serum was performed using a commercial enzyme immunoassay (Murex).

Results In several cases the specificity of the PCR products were verified by slot-blot hybridization using the enzyme-conjugated CMV probe. The PCR products (184-bp fragments) showing the presence of virus in

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K.L. Borg et aL 1 2 3 4 5 6 7 8 9 I0 11 12 1314

184

Fig. 1. Detection of CMV DNA in clinical specimens using 'boosted' nested PCR to amplify a 184-bp segment from the exon 4 of the CMV immediate early gene. Lanes: 2, DNA fragment marker; 3, negative control (uninfected HL cells); 4, positive control (infected HL cells); 5, negative control (no DNA); 6, amniotic fluid (N); 7, breast milk (L); 8, urine (C); 9, nasopharyngeal aspirate (C); 10, Epstein-Barrvirus (Daudi strain); 11, varizella zoster virus (patient isolate); 12, herpes simplex virus 2 (patient isolate); 13, herpes simplex virus 1 (patient isolate).

1 A

2

3 ,

Dept of Microbiology, Ullev~l Hospital, Oslo) which contains the immediate early gene of CMV, was serially diluted and subjected to the 'boosted' nested PCR assay. By direct gel analysis, 1 fg of CMV DNA could be detected which corresponds to about 70 copies of CMV DNA. The sensitivity of our PCR assay was also determined in a clinical specimen. Plasmid DNA with a known amount of CMV DNA was serially diluted in a negative urine sample. We could detect 1 fg CMV DNA corresponding to about 70 copies of CMV DNA. We compared the sensitivity of 'boosted' nested PCR to standard nested PCR and single pair PCR using the same CMV dilutions. The sensitivity of the standard nested PCR was equal to the 'boosted' nested PCR, while the single pair PCR could only detect 10 pg of CMV DNA.

Specificity of 'boosted' nested PCR The specificity of the assay was examined by amplifying DNA from other herpes viruses. 'Boosted' nested PCR did not cross-react with herpes simplex virus 1 and 2, varicella zoster virus and EpsteinBarr virus. Four hundred and forty seven clinical specimens obtained from 14 healthy newborns and 97 premature infants were negative both by 'boosted' nested PCR and virus isolation.

4 ,,~ ,~,:, \

c Fig. 2. Analysis of PCR amplification products by slotblot hybridization. A1, herpes simplex type 1 (patient isolate); A2, herpes simplex virus type 2 (patient isolate); A3, varizella zoster virus (patient isolate); A4, EpsteinBarr virus (Daudi strain); B1, nasopharyngealaspirate (C); B2, urine (C); B3, breast milk (L); B4, amniotic fluid (N); C1, negative control (no DNA); C2, positive control (CMV infected HL cells); C3, negative control (uninfected HL cells). some CMV positive samples and negative reactivities with other herpes viruses are shown in Fig. 1. Sensitivity of the 'boosted' nested PCR For determination of the sensitivity of 'boosted' nested PCR, plasmid pACAYC 184 (kindly provided by the

Detection of CMV in various types of clinical materials In the total clinical specimens, the PCR test yielded positive results in 51 samples from 14 patients, whereas CMV was isolated in 25 samples from 11 cases only. Twenty nine of 68 culture negative specimens were positive by PCR.

Urine specimens A total of 418 urine samples collected from 110 premature infants and 14 newborns were examined by 'boosted' nested PCR and virus isolation. Twenty two urine specimens from four CMV-positive premature infants proved positive by PCR (Table 1). Eighteen of these urine specimens were positive by virus isolation. Eight samples from two premature infants (CMV-IgM-negativeJIgG-positive) were positive by PCR and negative by culture. Nine samples from five newborns were positive by PCR, while four samples from four of these newborns were positive by culture (Table 1). All the five newborns had CMV IgG antibodies, while three of them

Detection of cytomegalovirus

255

Table 1. This table shows the positive results of CMV testing by 'boosted' nested PCR, virus isolation and serology

(total numbers of tested samples are indicated in parentheses) Patients

Urine samples

Naso. aspirates

Amniotic fluid

PCR

Culture

PCR

PCR

A B C D E F G H

2(2) 3(3) 9(11) 6(8) 2(18) 7(8) 3(3) 2(2)

2(2) I(3) 8(11) 0(8) 0(18) 7(8) 1(3) 1(2)

3(3) 2(4) I(13) 1(3) 0(2) 2(2)

I J

1(1) I(I)

0(1) I(I)

K L M N

2(2)

1(2)

Culture

Culture

Breast milk PCR

Culture

Serology IgM

lgG

2(3) 0(4) 0(13) 0(3) 0(2) 0(2)

+ + + + +

+ + + + + + + +

1(1) I(I)

0(1) 0(I)

-+

+ +

2(2)

1(2)

+ -+

+ + + +

2(2) 1(1) 1(1)

1(1)

0(2) 0(1)

Patients:

A-F: premature infants. G-K: healthy newborns. L-M: mothers of infants E and F. N: pregnant woman.

also had CMV IgM antibodies. The mothers of these newborns had both CMV IgM and IgG antibodies. One (CMV-lgM-negativeJIgG-positive) of these five newborns proved positive by PCR and negative by culture. The two premature infants and one newborn being positive by 'boosted' nested PCR and negative by culture were confirmed positive by an additional PCR on a different genomic segment (not shown). From this patient group at least two clinical samples, which were taken from different locations and/or at various times, were found positive by PCR. Twenty two samples were positive by PCR and virus isolation, while 21 samples were negative by PCR and virus isolation (Table 2). Sixteen samples

38 (64.4%) 21 (35"60/0)

Breast milk and amniotic fluid specimens

Comparison between results from virus isolation and PCR in urine samples from patients A-K in Table 1 Virus isolation Virus isolation 422 (37.3%)

16 (27%)

0

21 (53-6%)

PCR -

22 (37.3%)

37 (62.7%)

Nasopharyngeal aspirates

A total of 176 nasopharyngeal aspirates were collected from the same patients as described above. Four of the six nasopharyngeal aspirates from two premature infants with CMV-positive urine were positive by PCR (-Fable I), while two samples from one patient were .positive by virus isolation. Seventeen samples collected from two premature infants with CM-negative urine were negative by virus isolation, while three of these samples were positive by PCR. Eight nasopharyngeal samples from five newborns whose urine were positive by PCR, were examined. Six samples from four of these children were positive by PCR (Table I), while only one of these samples was positive by virus isolation. Three samples were positive by PCR and virus isolation, while 18 samples were negative by both tests (Table 3). Ten samples were negative by virus isolation, but PCR positive (Table 3).

Table 2.

PCR +

were negative by virus isolation, but PCR-positive (Table 2). All urine samples found positive by culture, were positive by PCR.

Seventeen breast milk samples were collected from 14 mothers. Three breast milk samples from two

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K.L. Borg et al.

mothers were positive by PCR but negative by culture. The amniotic fluid obtained from one CMV IgM and IgG positive woman was positive by both virus isolation and PCR (Table 1).

Detection of CMV in specimens from healthy

individuals Urine samples collected from 12 healthy individuals (CMV-lgM-negative and IgG-positive) were negative by 'boosted' nested PCR and virus isolation. The lymphocyte samples from these persons were negative by PCR (virus isolation not done).

Detection of CMV in premature infants during antiviral treatment

Four of the premature infants (patients a,b,c and f in Table I) with symptomatic CMV infections were treated with ganciclovir. Seven of 34 urine samples from these four premature infants, and two of 19 nasopharyngeal aspirates from two of these children (patients c and f) were positive by PCR during treatment. All samples collected during the treatment period were negative by culture.

DISCUSSION AND CONCLUSION DNA amplification techniques are rapidly gaining importance in diagnostic virology. Nested PCR may represent an improvement as to sensitivity and specificity, and being a more practical assay for routine diagnostic use. We developed the 'boosted' nested PCR technique with the purpose of maintaining the speed of the

Comparison between results from virus isolation and PCR in nasopharyngial samples from patients A-K in Table 1

Table 3.

Virus isolation Virus isolation + PCR

+

3 (9.7%)

10 (32.2%)

13 (41 "9%)

PCR -

0

18

18

(58.1%)

(58.1%)

3

28

(9.7%)

(91.3%)

nested PCR technique and reducing the risk of contamination. The critical step, which involves opening the reaction tubes with amplified products, is eliminated using 'boosted' nested PCR. Contamination problems are considerably reduced by the rather rigorous procedure adapted in this study. Using both the outer and inner primers in a single step amplification is simpler and faster as compared to virus isolation and standard nested PCR. The use of a similar technique to overcome problems with 'primer-dimer' formation has been reported by Ruano et al. u At the start a low concentration of the outer primers was used, while the second (inner) primer pair was used at a conventional concentration (20-30 pmol). This procedure also reduced 'mismatch' priming during the first PCR cycles, because of the amount of the starting primers. The high specificity of the 'boosters' nested PCR was shown by sequential monitoring of 111 healthy children which were PCR and culture negative in repeated samples. The PCR assay did not cross-react with other virus types of the herpes family or cellular DNA. The PCR technique was suitable for detection of CMV DNA in a wide variety of biological specimens without prior isolation and purification of the DNA. Our investigations have shown that in culturenegative patients it is important to detect CMV DNA in at least two different specimens or use an additional PCR assay for confirmation before any conclusions are made. The sensitivity of the 'boosted' nested PCR was comparable to the sensitivity of the standard nested PCR (showed by titration of plasmid CMV DNA) and clearly more sensitive than virus isolation. Two of the premature infants and one healthy newborn were PCR-positive both in nasopharyngeal aspirates and in urine samples and negative by culture. They were CMV-lgM-negative and IgG-positive and showed no clinical signs of CMV infections. It is possible that these three individuals represent lowlevel CMV excretors. Monitoring of CMV excretion during gancyclovir treatment showed an immediate reduction in the level of excreted CMV DNA as estimated from the amplification products. However, CMV DNA was shown in the urine at least in one test in all treated children and the CMV excretions were detected again shortly after withdrawal of medication. Demmler et al. s reported that inhibitors can be present in urine samples, and that these problems can be eliminated if a small amount of urine (1-2 #1) is used for analysis. In our study we used only 2 #1 of the urine sample in order to avoid possible interference by inhibition. Because of the low annealing temperature (43°C) in the first 25 cycles, non-specific amplification products

Detection of cytomegalovirus are observed with some clinical specimens. Slot-blot hybridization is required to identify specific PCR products in such cases. The 'boosted' nested PCR showed a high degree of reproducibility; all positive PCR samples were repeatedly reactive. In this study no CMV DNA could be detected in healthy individuals, which may suggest that our PCR assay is not sensitive enough to demonstrate latent CMV genomes in healthy seropositive persons. This makes the assay ideal for clinical diagnostic use. Our findings suggest that the 'boosted' nested PCR is a rapid, sensitive and specific test for detecting CMV DNA in several kinds of clinical specimens. The technique is more practical for routine diagnostic use than the standard nested PCR and may minimize contamination of amplified DNA products as well.

ACKNOWLEDGEMENT We wish to express our thanks to Inger Johanne Haugen and Heidi Sterten at the Department of Microbiology, the University Hospital of Trondheim for excellent technical assistance.

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