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Multiplex polymerase chain reaction for adenovirus and herpes simplex virus in eye swabs
Journalof viiiiological lbwwds Journal of Virological Methods 56 (1996) 41-48
Multiplex polymerase chain reaction for adenovirus and herpes simplex virus in eye swabs R. Jacksonla, D.J. Morrisa,b, R.J. Cooped+*, A.S. Baileyb, P.E. Klapperb, G.M. Cleator”, A.B. Tulle” aDivision of Virology, Department of Pathological Sciences, University of Manchester, 3rd Floor, Clinical Sciences Building, Manchester Royal Infirmary, Oxford Road, Manchester Ml3 9WL, UK ‘Clinical Virology Luborarory, Manchester Royal Infirmary Manchester, UK “Royal Eye Hospital Manchester UK
Accepted 17 July 1995
Abstract Adenoviruses and herpes simplex virus (HSV) can cause clinically indistinguishable episodes of acute eye disease. Adenovirus infection is associated with nosocomial outbreaks and HSV may result in episodes of recurrent ocular inflammation. In a comparison of multiplex PCR for the two viral DNAs and virus isolation in cell culture, identical results were obtained for 18 of 20 specimens (positive for adenovirus in 5, HSV in 5, and negative in 8). One specimen was falsely negative for each viral DNA. Inclusion of human p-globin primers in the adenovirus-HSV reaction was precluded by a consequential lo-lOO-fold reduction in sensitivity for the two viral targets and by the failure of B-globin DNA amplification at the annealing temperature (45°C) required to ensure detection of adenoviruses of serotypes 7 and 11 with the selected adenovirus primers. A single-target /?-globin PCR gave positive results with 19 of the 20 specimens prepared by treatment with proteinase K iysis buffer, indicating the effectiveness of this simple DNA extraction procedure. Nonetheless, the availability of effective antiviral therapy for HSV made monitoring for extraction failure using human primers crucial to avoid false-negative results for HSV DNA. Adenovirus-HSV PCR has considerable potential for the rapid diagnosis of viral eye disease particularly if /3-globin primers can be included in the reaction. Keywords: Polymerase chain reaction (PCR); Multiplex; Eye infection; Adenovirus;
Herpes simplex virus
1. Introduction Viral eye infections 1 Clinical Microbiology Laboratory, 2nd Floor, Clinical !kiences Building, Manchester Royal Infumary, Oxford Road, Manchester Ml3 9WL, UK.
*Corresponding author. 0166-0934/96/$15.00 0 1996 Elsevier Science B.V. AU rights SSDZ 0166-0934(95)01903-D
reserved
are mostly
caused by aden-
or herpes simplex virus (HSV) (Darougar et al., 1989). These two agents can produce clinitally indistinguishable episodes of acute follicular conjunctivitis. Adenoviruses are also associated oviruses
42
R. Jackson et al. ! Journal of Virological Methods 56 (1996) 41-48
with epidemic keratoconjunctivitis and pharyngoconjunctival fever (Wadell, 1987) and HSV is linked with dendritic ulcers and cornea1 opacification (Whitley, 1990). Rapid accurate diagnosis is advantageous for both agents. Laboratory diagnosis of ocular HSV or adenovirus infection relies on virus isolation in cell culture which takes a median of 3-4 (Whitley, 1990) or 14 days (Killough et al., 1990), respectively, or on more rapid, but separate, antigen detection assays for each agent which lack both the sensitivity and specificity of virus isolation (Killough et al., 1990; Whitley, 1990). With adenoviruses early diagnosis is important for the control of nosocomial outbreaks (Ankers et al., 1993) which can involve hundreds of patients attending an eye hospital out-patient clinic (Richmond et al., 1984). Herpetic eye infections merit prompt acyclovir therapy (Whitley, 1990). We recently evaluated a genus-reactive adenovirus polymerase chain reaction (PCR) (Allard et al., 1990) which achieved a sensitivity of 75-90% in comparison with virus isolation when examining conjunctival swabs (Morris et al., 1995). This PCR had the potential to replace virus culture as the primary screening test for ocular adenovirus infections. Detection of HSV could then be the only reason for retaining the cumbersome and expensive technique of virus isolation in cell culture for all conjunctival swabs. In the present study, the development of a multiplex PCR for simultaneous diagnosis of ocular adenovirus and HSV infection is reported. This approach should avoid any need for separate HSV and adenovirus testing of conjunctival swabs which might yield either virus.
2. Materials and methods 2.1. PCR primers The adenovirus primers (Hl, 5’-GCCGCAGTGGTCTTACATGCACATC-3’ and H2, S-CAGCACGCCGCGGATGTCAAAGT3’) were selected from the hexon region DNA sequence of adenovirus types 2 and 5 (product size 300 bp; Kinloch et al., 1984; Allard et al., 1990; Turner et al., 1993; Morris et al., 1995),
whereas the HSV primers were derived from part of the viral thymidine kinase gene (n7 5’CGCGCGGTACCTTTATGGGCAGCATGA-3’ and n8C 5’-CAGGGTAAATAACGTGTCCCCGATATGG-3’; product size 350 bp; Klapper et al., 1990). Inclusion of human P-globin primers (5’-CAACTTCATCCACGTTCACC-3’ and 5’GAAGAGCCAAGGACAGGTAC-3’; product size 268 bp; Bauer et al., 1991) in the multiplex adenovirus-HSV PCR was attempted to provide an internal control for the DNA extraction method used on conjunctival swabs. 2.2. PCR methods Each 50-~1 reaction mixture comprised 10 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, magnesium chloride and glycerol (concentrations specified in Results), 200 PM each deoxynucleoside triphosphate, 0.2 PM each primer, 1.25 U Taq DNA polymerase (Boehringer Mannheim, Lewes, East Sussex, UK), and 5 ~1 DNA extract or sterile distilled water (contamination control) covered with a drop of mineral oil (Turner et al., 1993; Morris et al., 1995). The standard PCR programme comprised denaturation at 94°C for 7 min followed by either 30 cycles of 2 min at 94°C 1.5 min at 45°C 50°C or 60°C and 2 min at 70°C or 30 cycles of 1 min at 94°C 1 min at 45°C and 1 min at 72°C to give more rapid thermal cycling. Annealing at 45°C was essential to ensure detection of subgenus B (type 7 and 11) adenoviruses (Morris et al., 1995), but the optimal annealing temperatures for the HSV and a-globin PCRs were 50-60°C (Klapper et al., 1990; McElhinney et al., 1995). All DNA amplifications were done in a Programmable Dri-Block PHC-1 thermal cycler (Techne, Cambridge, UK). The PCR products were analysed using standard techniques in 3% NuSieve agarose gels (Turner et al., 1993). Strict physical separation of PCR procedures minimised the risk of DNA carry over (Turner et al., 1993). 2.3. DNA standards Three standard DNA targets were used during the development of the multiplex PCR: DNA
R. Jackson et al.
/Journal of Virological Methods 56 (1996) 41-48
extracted from adenovirus type 2 virions banded on a cesium chloride gradient (G&co-BRL), phenol-chloroform extracted human embryo lung fibroblast DNA (kindly provided by L.M. McElhinney), and phenol-chloroform extracted HSV-1 (syn 17 strain)-infected Vero cells DNA (kindly provided by L. Kinsella). The first two preparations were quantitated as genome copies using Avogadro’s number and optical density measurements. As the HSV-infected cell DNA preparation also included host cell DNA, this preparation was quantitated as tissue culture infectious doses50 (TCID,,) by inoculating serial lo-fold dilutions on to 8 cell culture replicates. 2.4. Clinical specimens Cotton-tipped conjunctival swabs were collected in virus transport medium from patients with possible adenovirus or HSV eye disease. They were examined using virus isolation in cell culture (Killough et al., 1990) and the PCR. The DNA was extracted by addition of lysis buffer (20 mM Tris-HCl pH 8.3, 2 mM ethylenediaminetetraacetic acid, 1% v/v Triton X-100, 0.002% sodium dodecylsulphate, and 500 ,ug/ml proteinase K) to an equal volume of conjunctival swab transport medium, with subsequent incubation at 56°C for 2 h and then at 97°C for 15 min.
43
tinct bands for the latter target). With a similar range of magnesium concentrations in the HSV PCR, bands were only detected at 1.5 and 2.5 mM, with the optimum concentration being 2.5 mM. In the adenovirus PCR with standard cycling times and 2.5 mM magnesium chloride, the band intensity increased with the addition of 5% v/v glycerol and increased still further with 10% v/v glycerol, but the sensitivity was unchanged (Fig. la). In the HSV PCR, the addition of 5% v/v glycerol increased the sensitivity, but further increases in the glycerol concentration decreased the intensity of the band (Fig, lb). Alternative addition of 0.1 mg/ml gelatin completely or pafib inhibited these PCRs.
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3. Results 3.1. Optimisation of single-target PCRs Using the shortened PCR programme and appropriate primers in a series of single-target PCRs, the detection limits for adenovirus, human a-globin and, HSV were 400-4000 copies, 70 copies and 500 TCID,,. Increasing the number of cycles to 40 or 50 had no effect on these sensitivities. With magnesium concentrations of 1.5, 2.5, 4.5, 6.5, 8.5 and 10.5 mM, the maximum band intensity and minimal background non-specific staining was achieved at 1.5 and 2.5 mM for a-globin DNA and 1.5 mM for adenovirus DNA (though all magnesium concentrations gave intense dis-
123456 Fig. 1. Glycerol titrations. Thirty cycles of 94°C for 2 min, 50°C for 1.5 min, 70°C for 2 min; 2.5 mM magnesium chloride. (a) Adenovirus PCR. Reactions in lanes 2-7 contained 4000 copies adenovirus DNA. Lane 1, 1 kb ladder; lanes 2 and 3, 0% v/v glycerol; lanes 4 and 5, 5% v/v glycerol; lanes 6 and 7, 10% v/v glycerol; lane 8, negative control (double distilled water). (b) HSV PCR. Reactions in lanes 2-6 contained 5000 TCID,, HSV. Lane 1, 1 kb ladder; lane 2,0% v/v glycerol; lane 3, 5% v/v glycerol; lane 4, 10% v/v glycerol; lane 5, 15% v/v glycerol; lane 6, 20% v/v glycerol.
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44
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1208
bp
Mrrhods 56 (1996) 41-48
the results being identical whether long (annealing temperature 50°C) or short cycling times were used. Therefore, the results obtained in the various experiments in the present study were not affected by the use of these two different sets of PCR cycling conditions. High levels of adenovirus DNA (40000 copies) prevented detection of less than 5000 TCIDS, of HSV, but with smaller quantities of the first target (4000 copies) the detection limit for the second target fell to 500 TCID,,, (Fig. 3) which was identical to that seen in the single target HSV PCR. In contrast, high levels of HSV (50000 TCID,,) had little effect on the adenovirus sensitivity (4000 copies, Fig. 3, lane 7, compared to Fig. 1). An adenovirus-HSV duplex reaction using only one target DNA at once showed unchanging detection limits for the two targets of 4000 copies and 500 TCID,,, re-
Fig. 2. Adenovirus-/?-globin multiplex PCR. Thirty cycles with I min segments of 94°C. 45°C and 72°C: 2.5 mM magnesium chloride; lanes I and 9, 1 kb ladder. Adenovirus DNA present in lanes 2 and IO, 4000000 copies; lanes 3 and II, 400000 copies; lanes 4 and 12, 40000 copies: lanes 5 and 13. 4000 copies: lanes 6 and 14. 400 copies; lanes 7 and 15. 40 copies. Lanes IO-15 also have 700 copirs human DNA. Lane 8, negative control (double distilled water); lane 16, 700 copies human DNA only.
3.2. Mdtipkx
50
bp
PCRs
In the light of these optimisation experiments, all multiplex PCR reaction mixtures contained 2.5 mM magnesium in addition to the other standard components. In the adenovirus-a-globin PCR using the shortened thermal cycling programme, the detection limit for the viral target was 40000 copies when 700 copies of fi-globin DNA were reliably amplified (Fig. 2). A IO-fold decline in sensitivity for HSV DNA was also seen in a HSV-P-globin PCR. These results led us to add 5% glycerol to all subsequent PCR mixtures, though there was no proof that this would increase the sensitivity. Detailed evaluation of the adenovirus-HSV PCR involved independent titration of the two targets,
SOOb
8
7
8
9
10
11
Fig 3. Adenovirus-HSV multiplex PCR. Thirty cycles of 94°C tor 2 min, 45°C for I.5 min, and 70°C for 2 min; 2.5 mM magnesium chloride; 5% v/v glycerol. Lanes I and 6, I kb ladder; lanes 225, 40000 copies adenovirus DNA and, in order, 50000, 5000, 500 or 0 TCID,, HSV; lanes 779, 4000 copies adenovirus DNA and, in order, 50000, 5000, or 500 TCID,,, HSV; lane 10, 5000 TCID,,, HSV; lane 1 I, negative control (double distilled water).
4s
R. Jackson et al. 1 Journal qf Virological Methods 56 (1996) 41-48
spectively. Thus, duplex PCRs with P-globin primers decreased the detection limits for the viral DNAs lo-lOO-fold, but a dual virus PCR showed a lo-fold or less reduction in the detection limit for adenovirus DNA and a similar reduction for HSV only when adenovirus was in great excess. Simultaneous adenovirus-HSV-P-globin PCR amplified all 3 DNAs at an annealing temperature of 60°C (Fig. 4a), but only the viral DNAs and not the human DNA at an annealing temperature of 45°C (Fig. 4b). Given our previous failure to detect adenovirus types 7 and 11 at annealing temperatures of higher than 55°C or 45°C respectively, a panel of 20 conjunctival swabs was tested using the adenovirus-HSV multiplex PCR (annealing temperature 45°C standard cycling times) and /I-globin single-target PCR (annealing temperature 60°C standard cycling times) in parallel (Table 1). The multiplex PCR detected adenovirus DNA in 5 of 6 specimens culture positive for adenovirus, with one false-negative result, and HSV DNA in 5 of 6 specimens culture positive for HSV, again with one falsely negative DNA amplification. There were no false-positive PCR results for either virus. Several adenovirus subgenera (B, C and D) and serotypes were detected in the PCRs (Table 1). A single target adenovirus PCR using a 45°C annealing temperature gave the same number of positive results for adenovirus as the duplex reaction, though two specimens gave discrepant results in the two PCRs. All but one of the specimens yielded amplifiable human /3-globin DNA.
4. Discussion The detection limits for the single-target adenovirus and human a-globin PCRs (400-4000 and 70 copies) were similar to those previously reported from this laboratory (Morris et al., 1995; McElhinney et al., 1995). Though the sensitivity of the HSV PCR (500 TCIDSO) appeared lower than that previously reported (1 plaque forming unit; Klapper et al., 1990), the precision of quantal assays is such that the 95% confidence limits are wide (in this case N 50-1000 TCID,,). Moreover, the absolute sensitivity for individual targets
(4
* 350 bp * 3oobp s 268 bp
123
12345678 Fig. 4. Adenovirus-HSV-/I-globin multiplex PCR. Thirty cycles of 94°C for 2 min, 60°C (a) or 45°C (b) for 1.5 min, and 70°C for 2 min; 2.5 mM magnesium chloride, 5% v/v glycerol. (a) Lane I, I kb ladder; lane 2, 30000 copies adenovirus DNA, 500 copies human DNA, 30000 TCID,, HSV; lane 3, 700 copies human DNA. (b) Lane I, 1 kb ladder; lane 2, 50000 TCID,, HSV; lane 3, 50000 TCID,, HSV, 700 copies human DNA; lane 4, 700 copies human DNA; lane 5, 40000 copies adenovirus DNA; lane 6, 40000 copies adenovirus DNA, 50000 TCID,, HSV; lane 7, 30000 copies adenovirus DNA, 500 copies human DNA, 30000 TCIDSO HSV; lane 8, negative control (double distilled water).
was not important in interpreting the relative sensitivity of the single and multiple target PC: The attempted development of an adenoviru*HSV-/?-globin PCR produced an assay which detected all 3 targets at an annealing temperature of 60°C. Addition of glycerol, but not gelatin, was possibly beneficial in the single-target viral PCRs,
46
R. Jackson et al.
i Journal of Virnlogical Methods 56 (1996) 41-48
Table 1 ResuIts of adenovirus-HSV multiplex and @-globin and adenovirus single-target PCRs on conjunctival swabs”
R. Jackson et al. / Journal of Virological Methods 56 (1996) 41-48
trigeminal ganglion (Whitley, 1990) and conceivably in the cornea (Cleator et al., 1994). Nonetheless, we found no evidence of persistent adenovirus or latent HSV infection detectable by PCR, but not virus isolation. A large scale prospective evaluation of our single-target adenovirus PCR on 576 conjunctival swabs revealed a specificity of 98% in comparison with virus isolation, and a proportion of the false-positive results probably reflected contamination of the specimens during handling on the open bench in a multi-user diagnostic laboratory (Morris et al., 1995). The shared ability of virus isolation and our viral PCRs to discriminate between active and persistent or latent infection could have reflected the relative insensitivity of the latter techniques. All but one of the lysis buffer-treated conjunctival swabs yielded human /3-globin DNA by PCR, indicating successful extraction of amplifiable DNA. This observation suggested that failure of DNA extraction was an unlikely explanation for failure to detect viral DNA in a specimen positive for adenovirus or HSV in cell culture. It did not, however, imply that inclusion of /I-globin primers in a multiplex PCR for diagnosis of ocular adenovirus or HSV infection was unnecessary. The occasional false negative result for adenovirus DNA in eye swabs would be unimportant if the test was done solely to allow identification of outbreaks of epidemic adenoviral keratoconjunctivitis. However, diagnosis of HSV must be accurate because antiviral therapy with acyclovir is likely to be effective, particularly if given early in the disease process (Whitley, 1990). If /3-globin primers were not included in an adenovirus-HSV PCR, failure to remove any inhibitors of DNA amplification during lysis buffer treatment could lead to unidentifiable false negative results for HSV DNA and untreated progressive ocular HSV disease. It is concluded that simultaneous detection of adenovirus and HSV DNA by PCR has considerable potential as a diagnostic approach for ocular disease. Although the standard thermal cycling programme was used in the current clinical evaluation, the shortened cycling protocol would be equally efficient. Ultimately, an assay giving accurate identification of clinically relevant infection
41
with either agent within 24 h of specimen receipt in the laboratory should be feasible.
Acknowledgements
This work was funded in part by the Peel Medical Research Trust. We are grateful to Mrs. Mary Pike for assistance with the preparation of the manuscript.
References Allard, A., C&ones, R., Juto, P. and Wade& G. (1990) Polymerase chain reaction for detection of adenoviruses in stool specimens. J. Clin. Microbial. 28, 2659-2667. Ankers, H.E., Klapper, P.E., Cleator, G.M., Bailey, AS. and Tulle, A.B. (1993) The role of a rapid diagnostic test (adenovirus immune dot-blot) in the control of an outbreak of adenovirus type 8 keratoconjunctivitis. Eye Suppl. 15-17. Bauer, H.M., Ting, Y., Greer, C.E., Chambers, J.C., Tashiro, C.J., Chimera, J., Reingold, A. and Manos, M.M. (1991) Genital human papillomavirus infection in female university students as determined by a PCR-based method. J. Am. Med. Assoc. 265, 412-411. Cleator, G.M., Klapper, P.E., Dennett, C., Sullivan, A.L., Bonshek, R.E., Marcyniuk, B. and Tulle, A.B (1994) Comeal donor infection by herpes simplex: herpes simplex virus DNA in donor corneas. Cornea 13, 294-304. Darougar, S., Monnickendam, M.A. and Woodland, R.M. (1989) Management and prevention of ocular viral and chlamydia! infections. Crit. Rev. Microbial. 16, 369-418. Horwitz, MS. (1990) Adenoviruses. In: B.N. Fields and D.M. Knipe (Eds), Virology, 2nd edn., Vol. 2., Raven Press, New York, pp. 1723-1740. Killough, R., Klapper, P.E., Bailey, A.S., Sharp, I.R., Tulle, A. and Richmond, S.J. (1990) An immune dot-blot technique for the diagnosis of ocular adenovirus infection. J. Virol. Methods 30, 197-203. Kinloch, R., Mackay, N. and Mautner, V. (1984) Adenovirus hexon. Sequence comparison of subgenus C serotype 2 and 5. J. Biol. Chem. 259, 6431-6436. Klapper, P.E., Cleator, G.M., Dennett, C. and Lewis, A.G. (1990) Diagnosis of herpes encephalitis via a Southern blotting of cerebrospinal fluid DNA amplified by polymerase chain reaction. J. Med. Virol. 32, 261-264. McElhinney, L.M., Cooper, R.J. and Morris, D.J. (1995) Multiplex polymerase chain reaction for human herpesvirus-6, human cytomegalovirus, and human /I-globin DNA. J. Virol. Methods 53, 223-233. Morris, D.J., Bailey, A.S., Cooper, R.J., Turner, PC., Jackson, R., Corbitt, G. and Tulle, A.B. (1995) Polymerase
48
R. Jackson et al. 1 Journal of Virological Metho& 56 (1996) 41-48
chain reaction for rapid detection of ocular adenovirus infection. J. Med. Virol. 46, 1266 132. Richmond, S.J., But-man, R., Crosdale, E., Cropper, L., Longson, D., Enoch, B.E. and Dodd, CL. (1984) A large outbreak of keratoconjunctivitis due to adenovirus type 8. J. Hyg. 93, 285-291. Turner, PC., Bailey, A.S., Cooper, R.J. and Morris, D.J. (1993) The polymerase chain reaction for detecting adenovirus DNA in formalin-fixed, paraffin embedded tissue
obtained post mortem. J. Infect. 27, 43346. Wadell, G. (1987) Adenoviruses. In: A.J. Zuckennan, J.E. Banatvala and J.R. Pattison (Eds), Principles and Practice of Clinical Virology, John Wiley and Sons, Chichester, pp. 251-274. Whitley, R.J. (1990) Herpes simplex viruses. In: B.N. Fields and D.M. Knipe (Eds), Virology, 2nd edn., Vol. 2, Raven Press, New York, pp. 1843-1888.
Multiplex polymerase chain reaction for adenovirus and herpes simplex virus in eye swabs R. Jacksonla, D.J. Morrisa,b, R.J. Cooped+*, A.S. Baileyb, P.E. Klapperb, G.M. Cleator”, A.B. Tulle” aDivision of Virology, Department of Pathological Sciences, University of Manchester, 3rd Floor, Clinical Sciences Building, Manchester Royal Infirmary, Oxford Road, Manchester Ml3 9WL, UK ‘Clinical Virology Luborarory, Manchester Royal Infirmary Manchester, UK “Royal Eye Hospital Manchester UK
Accepted 17 July 1995
Abstract Adenoviruses and herpes simplex virus (HSV) can cause clinically indistinguishable episodes of acute eye disease. Adenovirus infection is associated with nosocomial outbreaks and HSV may result in episodes of recurrent ocular inflammation. In a comparison of multiplex PCR for the two viral DNAs and virus isolation in cell culture, identical results were obtained for 18 of 20 specimens (positive for adenovirus in 5, HSV in 5, and negative in 8). One specimen was falsely negative for each viral DNA. Inclusion of human p-globin primers in the adenovirus-HSV reaction was precluded by a consequential lo-lOO-fold reduction in sensitivity for the two viral targets and by the failure of B-globin DNA amplification at the annealing temperature (45°C) required to ensure detection of adenoviruses of serotypes 7 and 11 with the selected adenovirus primers. A single-target /?-globin PCR gave positive results with 19 of the 20 specimens prepared by treatment with proteinase K iysis buffer, indicating the effectiveness of this simple DNA extraction procedure. Nonetheless, the availability of effective antiviral therapy for HSV made monitoring for extraction failure using human primers crucial to avoid false-negative results for HSV DNA. Adenovirus-HSV PCR has considerable potential for the rapid diagnosis of viral eye disease particularly if /3-globin primers can be included in the reaction. Keywords: Polymerase chain reaction (PCR); Multiplex; Eye infection; Adenovirus;
Herpes simplex virus
1. Introduction Viral eye infections 1 Clinical Microbiology Laboratory, 2nd Floor, Clinical !kiences Building, Manchester Royal Infumary, Oxford Road, Manchester Ml3 9WL, UK.
*Corresponding author. 0166-0934/96/$15.00 0 1996 Elsevier Science B.V. AU rights SSDZ 0166-0934(95)01903-D
reserved
are mostly
caused by aden-
or herpes simplex virus (HSV) (Darougar et al., 1989). These two agents can produce clinitally indistinguishable episodes of acute follicular conjunctivitis. Adenoviruses are also associated oviruses
42
R. Jackson et al. ! Journal of Virological Methods 56 (1996) 41-48
with epidemic keratoconjunctivitis and pharyngoconjunctival fever (Wadell, 1987) and HSV is linked with dendritic ulcers and cornea1 opacification (Whitley, 1990). Rapid accurate diagnosis is advantageous for both agents. Laboratory diagnosis of ocular HSV or adenovirus infection relies on virus isolation in cell culture which takes a median of 3-4 (Whitley, 1990) or 14 days (Killough et al., 1990), respectively, or on more rapid, but separate, antigen detection assays for each agent which lack both the sensitivity and specificity of virus isolation (Killough et al., 1990; Whitley, 1990). With adenoviruses early diagnosis is important for the control of nosocomial outbreaks (Ankers et al., 1993) which can involve hundreds of patients attending an eye hospital out-patient clinic (Richmond et al., 1984). Herpetic eye infections merit prompt acyclovir therapy (Whitley, 1990). We recently evaluated a genus-reactive adenovirus polymerase chain reaction (PCR) (Allard et al., 1990) which achieved a sensitivity of 75-90% in comparison with virus isolation when examining conjunctival swabs (Morris et al., 1995). This PCR had the potential to replace virus culture as the primary screening test for ocular adenovirus infections. Detection of HSV could then be the only reason for retaining the cumbersome and expensive technique of virus isolation in cell culture for all conjunctival swabs. In the present study, the development of a multiplex PCR for simultaneous diagnosis of ocular adenovirus and HSV infection is reported. This approach should avoid any need for separate HSV and adenovirus testing of conjunctival swabs which might yield either virus.
2. Materials and methods 2.1. PCR primers The adenovirus primers (Hl, 5’-GCCGCAGTGGTCTTACATGCACATC-3’ and H2, S-CAGCACGCCGCGGATGTCAAAGT3’) were selected from the hexon region DNA sequence of adenovirus types 2 and 5 (product size 300 bp; Kinloch et al., 1984; Allard et al., 1990; Turner et al., 1993; Morris et al., 1995),
whereas the HSV primers were derived from part of the viral thymidine kinase gene (n7 5’CGCGCGGTACCTTTATGGGCAGCATGA-3’ and n8C 5’-CAGGGTAAATAACGTGTCCCCGATATGG-3’; product size 350 bp; Klapper et al., 1990). Inclusion of human P-globin primers (5’-CAACTTCATCCACGTTCACC-3’ and 5’GAAGAGCCAAGGACAGGTAC-3’; product size 268 bp; Bauer et al., 1991) in the multiplex adenovirus-HSV PCR was attempted to provide an internal control for the DNA extraction method used on conjunctival swabs. 2.2. PCR methods Each 50-~1 reaction mixture comprised 10 mM Tris-HCl (pH 8.3), 50 mM potassium chloride, magnesium chloride and glycerol (concentrations specified in Results), 200 PM each deoxynucleoside triphosphate, 0.2 PM each primer, 1.25 U Taq DNA polymerase (Boehringer Mannheim, Lewes, East Sussex, UK), and 5 ~1 DNA extract or sterile distilled water (contamination control) covered with a drop of mineral oil (Turner et al., 1993; Morris et al., 1995). The standard PCR programme comprised denaturation at 94°C for 7 min followed by either 30 cycles of 2 min at 94°C 1.5 min at 45°C 50°C or 60°C and 2 min at 70°C or 30 cycles of 1 min at 94°C 1 min at 45°C and 1 min at 72°C to give more rapid thermal cycling. Annealing at 45°C was essential to ensure detection of subgenus B (type 7 and 11) adenoviruses (Morris et al., 1995), but the optimal annealing temperatures for the HSV and a-globin PCRs were 50-60°C (Klapper et al., 1990; McElhinney et al., 1995). All DNA amplifications were done in a Programmable Dri-Block PHC-1 thermal cycler (Techne, Cambridge, UK). The PCR products were analysed using standard techniques in 3% NuSieve agarose gels (Turner et al., 1993). Strict physical separation of PCR procedures minimised the risk of DNA carry over (Turner et al., 1993). 2.3. DNA standards Three standard DNA targets were used during the development of the multiplex PCR: DNA
R. Jackson et al.
/Journal of Virological Methods 56 (1996) 41-48
extracted from adenovirus type 2 virions banded on a cesium chloride gradient (G&co-BRL), phenol-chloroform extracted human embryo lung fibroblast DNA (kindly provided by L.M. McElhinney), and phenol-chloroform extracted HSV-1 (syn 17 strain)-infected Vero cells DNA (kindly provided by L. Kinsella). The first two preparations were quantitated as genome copies using Avogadro’s number and optical density measurements. As the HSV-infected cell DNA preparation also included host cell DNA, this preparation was quantitated as tissue culture infectious doses50 (TCID,,) by inoculating serial lo-fold dilutions on to 8 cell culture replicates. 2.4. Clinical specimens Cotton-tipped conjunctival swabs were collected in virus transport medium from patients with possible adenovirus or HSV eye disease. They were examined using virus isolation in cell culture (Killough et al., 1990) and the PCR. The DNA was extracted by addition of lysis buffer (20 mM Tris-HCl pH 8.3, 2 mM ethylenediaminetetraacetic acid, 1% v/v Triton X-100, 0.002% sodium dodecylsulphate, and 500 ,ug/ml proteinase K) to an equal volume of conjunctival swab transport medium, with subsequent incubation at 56°C for 2 h and then at 97°C for 15 min.
43
tinct bands for the latter target). With a similar range of magnesium concentrations in the HSV PCR, bands were only detected at 1.5 and 2.5 mM, with the optimum concentration being 2.5 mM. In the adenovirus PCR with standard cycling times and 2.5 mM magnesium chloride, the band intensity increased with the addition of 5% v/v glycerol and increased still further with 10% v/v glycerol, but the sensitivity was unchanged (Fig. la). In the HSV PCR, the addition of 5% v/v glycerol increased the sensitivity, but further increases in the glycerol concentration decreased the intensity of the band (Fig, lb). Alternative addition of 0.1 mg/ml gelatin completely or pafib inhibited these PCRs.
12345678
3. Results 3.1. Optimisation of single-target PCRs Using the shortened PCR programme and appropriate primers in a series of single-target PCRs, the detection limits for adenovirus, human a-globin and, HSV were 400-4000 copies, 70 copies and 500 TCID,,. Increasing the number of cycles to 40 or 50 had no effect on these sensitivities. With magnesium concentrations of 1.5, 2.5, 4.5, 6.5, 8.5 and 10.5 mM, the maximum band intensity and minimal background non-specific staining was achieved at 1.5 and 2.5 mM for a-globin DNA and 1.5 mM for adenovirus DNA (though all magnesium concentrations gave intense dis-
123456 Fig. 1. Glycerol titrations. Thirty cycles of 94°C for 2 min, 50°C for 1.5 min, 70°C for 2 min; 2.5 mM magnesium chloride. (a) Adenovirus PCR. Reactions in lanes 2-7 contained 4000 copies adenovirus DNA. Lane 1, 1 kb ladder; lanes 2 and 3, 0% v/v glycerol; lanes 4 and 5, 5% v/v glycerol; lanes 6 and 7, 10% v/v glycerol; lane 8, negative control (double distilled water). (b) HSV PCR. Reactions in lanes 2-6 contained 5000 TCID,, HSV. Lane 1, 1 kb ladder; lane 2,0% v/v glycerol; lane 3, 5% v/v glycerol; lane 4, 10% v/v glycerol; lane 5, 15% v/v glycerol; lane 6, 20% v/v glycerol.
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Mrrhods 56 (1996) 41-48
the results being identical whether long (annealing temperature 50°C) or short cycling times were used. Therefore, the results obtained in the various experiments in the present study were not affected by the use of these two different sets of PCR cycling conditions. High levels of adenovirus DNA (40000 copies) prevented detection of less than 5000 TCIDS, of HSV, but with smaller quantities of the first target (4000 copies) the detection limit for the second target fell to 500 TCID,,, (Fig. 3) which was identical to that seen in the single target HSV PCR. In contrast, high levels of HSV (50000 TCID,,) had little effect on the adenovirus sensitivity (4000 copies, Fig. 3, lane 7, compared to Fig. 1). An adenovirus-HSV duplex reaction using only one target DNA at once showed unchanging detection limits for the two targets of 4000 copies and 500 TCID,,, re-
Fig. 2. Adenovirus-/?-globin multiplex PCR. Thirty cycles with I min segments of 94°C. 45°C and 72°C: 2.5 mM magnesium chloride; lanes I and 9, 1 kb ladder. Adenovirus DNA present in lanes 2 and IO, 4000000 copies; lanes 3 and II, 400000 copies; lanes 4 and 12, 40000 copies: lanes 5 and 13. 4000 copies: lanes 6 and 14. 400 copies; lanes 7 and 15. 40 copies. Lanes IO-15 also have 700 copirs human DNA. Lane 8, negative control (double distilled water); lane 16, 700 copies human DNA only.
3.2. Mdtipkx
50
bp
PCRs
In the light of these optimisation experiments, all multiplex PCR reaction mixtures contained 2.5 mM magnesium in addition to the other standard components. In the adenovirus-a-globin PCR using the shortened thermal cycling programme, the detection limit for the viral target was 40000 copies when 700 copies of fi-globin DNA were reliably amplified (Fig. 2). A IO-fold decline in sensitivity for HSV DNA was also seen in a HSV-P-globin PCR. These results led us to add 5% glycerol to all subsequent PCR mixtures, though there was no proof that this would increase the sensitivity. Detailed evaluation of the adenovirus-HSV PCR involved independent titration of the two targets,
SOOb
8
7
8
9
10
11
Fig 3. Adenovirus-HSV multiplex PCR. Thirty cycles of 94°C tor 2 min, 45°C for I.5 min, and 70°C for 2 min; 2.5 mM magnesium chloride; 5% v/v glycerol. Lanes I and 6, I kb ladder; lanes 225, 40000 copies adenovirus DNA and, in order, 50000, 5000, 500 or 0 TCID,, HSV; lanes 779, 4000 copies adenovirus DNA and, in order, 50000, 5000, or 500 TCID,,, HSV; lane 10, 5000 TCID,,, HSV; lane 1 I, negative control (double distilled water).
4s
R. Jackson et al. 1 Journal qf Virological Methods 56 (1996) 41-48
spectively. Thus, duplex PCRs with P-globin primers decreased the detection limits for the viral DNAs lo-lOO-fold, but a dual virus PCR showed a lo-fold or less reduction in the detection limit for adenovirus DNA and a similar reduction for HSV only when adenovirus was in great excess. Simultaneous adenovirus-HSV-P-globin PCR amplified all 3 DNAs at an annealing temperature of 60°C (Fig. 4a), but only the viral DNAs and not the human DNA at an annealing temperature of 45°C (Fig. 4b). Given our previous failure to detect adenovirus types 7 and 11 at annealing temperatures of higher than 55°C or 45°C respectively, a panel of 20 conjunctival swabs was tested using the adenovirus-HSV multiplex PCR (annealing temperature 45°C standard cycling times) and /I-globin single-target PCR (annealing temperature 60°C standard cycling times) in parallel (Table 1). The multiplex PCR detected adenovirus DNA in 5 of 6 specimens culture positive for adenovirus, with one false-negative result, and HSV DNA in 5 of 6 specimens culture positive for HSV, again with one falsely negative DNA amplification. There were no false-positive PCR results for either virus. Several adenovirus subgenera (B, C and D) and serotypes were detected in the PCRs (Table 1). A single target adenovirus PCR using a 45°C annealing temperature gave the same number of positive results for adenovirus as the duplex reaction, though two specimens gave discrepant results in the two PCRs. All but one of the specimens yielded amplifiable human /3-globin DNA.
4. Discussion The detection limits for the single-target adenovirus and human a-globin PCRs (400-4000 and 70 copies) were similar to those previously reported from this laboratory (Morris et al., 1995; McElhinney et al., 1995). Though the sensitivity of the HSV PCR (500 TCIDSO) appeared lower than that previously reported (1 plaque forming unit; Klapper et al., 1990), the precision of quantal assays is such that the 95% confidence limits are wide (in this case N 50-1000 TCID,,). Moreover, the absolute sensitivity for individual targets
(4
* 350 bp * 3oobp s 268 bp
123
12345678 Fig. 4. Adenovirus-HSV-/I-globin multiplex PCR. Thirty cycles of 94°C for 2 min, 60°C (a) or 45°C (b) for 1.5 min, and 70°C for 2 min; 2.5 mM magnesium chloride, 5% v/v glycerol. (a) Lane I, I kb ladder; lane 2, 30000 copies adenovirus DNA, 500 copies human DNA, 30000 TCID,, HSV; lane 3, 700 copies human DNA. (b) Lane I, 1 kb ladder; lane 2, 50000 TCID,, HSV; lane 3, 50000 TCID,, HSV, 700 copies human DNA; lane 4, 700 copies human DNA; lane 5, 40000 copies adenovirus DNA; lane 6, 40000 copies adenovirus DNA, 50000 TCID,, HSV; lane 7, 30000 copies adenovirus DNA, 500 copies human DNA, 30000 TCIDSO HSV; lane 8, negative control (double distilled water).
was not important in interpreting the relative sensitivity of the single and multiple target PC: The attempted development of an adenoviru*HSV-/?-globin PCR produced an assay which detected all 3 targets at an annealing temperature of 60°C. Addition of glycerol, but not gelatin, was possibly beneficial in the single-target viral PCRs,
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i Journal of Virnlogical Methods 56 (1996) 41-48
Table 1 ResuIts of adenovirus-HSV multiplex and @-globin and adenovirus single-target PCRs on conjunctival swabs”
R. Jackson et al. / Journal of Virological Methods 56 (1996) 41-48
trigeminal ganglion (Whitley, 1990) and conceivably in the cornea (Cleator et al., 1994). Nonetheless, we found no evidence of persistent adenovirus or latent HSV infection detectable by PCR, but not virus isolation. A large scale prospective evaluation of our single-target adenovirus PCR on 576 conjunctival swabs revealed a specificity of 98% in comparison with virus isolation, and a proportion of the false-positive results probably reflected contamination of the specimens during handling on the open bench in a multi-user diagnostic laboratory (Morris et al., 1995). The shared ability of virus isolation and our viral PCRs to discriminate between active and persistent or latent infection could have reflected the relative insensitivity of the latter techniques. All but one of the lysis buffer-treated conjunctival swabs yielded human /3-globin DNA by PCR, indicating successful extraction of amplifiable DNA. This observation suggested that failure of DNA extraction was an unlikely explanation for failure to detect viral DNA in a specimen positive for adenovirus or HSV in cell culture. It did not, however, imply that inclusion of /I-globin primers in a multiplex PCR for diagnosis of ocular adenovirus or HSV infection was unnecessary. The occasional false negative result for adenovirus DNA in eye swabs would be unimportant if the test was done solely to allow identification of outbreaks of epidemic adenoviral keratoconjunctivitis. However, diagnosis of HSV must be accurate because antiviral therapy with acyclovir is likely to be effective, particularly if given early in the disease process (Whitley, 1990). If /3-globin primers were not included in an adenovirus-HSV PCR, failure to remove any inhibitors of DNA amplification during lysis buffer treatment could lead to unidentifiable false negative results for HSV DNA and untreated progressive ocular HSV disease. It is concluded that simultaneous detection of adenovirus and HSV DNA by PCR has considerable potential as a diagnostic approach for ocular disease. Although the standard thermal cycling programme was used in the current clinical evaluation, the shortened cycling protocol would be equally efficient. Ultimately, an assay giving accurate identification of clinically relevant infection
41
with either agent within 24 h of specimen receipt in the laboratory should be feasible.
Acknowledgements
This work was funded in part by the Peel Medical Research Trust. We are grateful to Mrs. Mary Pike for assistance with the preparation of the manuscript.
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chain reaction for rapid detection of ocular adenovirus infection. J. Med. Virol. 46, 1266 132. Richmond, S.J., But-man, R., Crosdale, E., Cropper, L., Longson, D., Enoch, B.E. and Dodd, CL. (1984) A large outbreak of keratoconjunctivitis due to adenovirus type 8. J. Hyg. 93, 285-291. Turner, PC., Bailey, A.S., Cooper, R.J. and Morris, D.J. (1993) The polymerase chain reaction for detecting adenovirus DNA in formalin-fixed, paraffin embedded tissue
obtained post mortem. J. Infect. 27, 43346. Wadell, G. (1987) Adenoviruses. In: A.J. Zuckennan, J.E. Banatvala and J.R. Pattison (Eds), Principles and Practice of Clinical Virology, John Wiley and Sons, Chichester, pp. 251-274. Whitley, R.J. (1990) Herpes simplex viruses. In: B.N. Fields and D.M. Knipe (Eds), Virology, 2nd edn., Vol. 2, Raven Press, New York, pp. 1843-1888.