Background & objectives: Since fluorescent antibody test (FAT) has low sensitivity in the rapid detection of cytomegalovirus (CMV) in clinical specimens, a nested polymerase chain reaction (nPCR) to detect the CMV-DNA was evaluated.
Methods: nPCR and FAT were carried out to detect CMV in single specimens from 104 patients and dual specimens from 32 patients with suspected active CMV infection. Of the 136 patients, 3 were HIV positive.
Results: CMV was detected by FAT alone in 3 (1.8%) and FAT and nPCR in 16 (9.5%) specimens and by nPCR alone in 84 (50.0%) specimens from 74 (54.4%) patients. nPCR increased the clinical sensitivity by 50.0 per cent in the specimens and 54.4 per cent in the patients (McNemar test, P
Key words Cytomegalic-inclusion disease - cytomegalovirus - nested polymerase chain reaction
The prevalence of human cytomegalovirus (CMV) infection depends on the age of the population and geographic and socio-economic settings. The vast majority of infections are subclinical, including those acquired in vitro and during the first months of postnatal life, but CMV can cause disease of diverse severity1. In India, sero-epidemiological surveys indicate that primary infection occurs by the fifth year of life in up to 60 per cent of the population and about 90 per cent of the middle aged population shows activation ef the virus which may be apparent or inapparent clinically2 3. With the increasing incidence of acquired immunodeficiency syndrome (AIDS) and use of immunosuppressive therapy in malignancy and organ transplants, the prevalence of CMV infections has increased several folds6-8. The clinical syndromes associated with CMV are a heterophile negative mononucleosis-like syndrome in young adults, cytomegalic inclusion disease, the severe form of congenital infection, pneumonitis in young infants, interstitial pneumonia, retinitis, febrile illness among organ transplant recipients and other immunocompromised hosts and a post transfusion syndrome9. The `gold standard' for diagnosis of CMV infection of humans is the virus isolation in vitro cell cultures. Being a slow growing virus, the conventional test tube culture method of CMV isolation is laborious and time consuming. The rapid method of shell vial technique is availablel10. Since the maintenance of diploid cell lines is essential, which though not impossible, is very difficult in most of our laboratories for routine diagnostic purposes, antigen detection by fluorescent antibody test (FAT) in smears using specific fluorescent labeled antibodies and antibody detection by employing ELISA techniques are used commonly as rapid methods. But, FAT is less sensitive and very subjective, while serology is not a reliable diagnostic tool11. With the advances in molecular biology, polymerase chain reaction (PCR), a rapid and highly sensitive technique is being used extensively for the detection of CMV-DNA 12-14. We employed a nested PCR (nPCR) for the detection of CMV and evaluated it against FAT for the rapid detection of CMV in clinical specimens.
Material & Methods
Patients & specimens: The study was carried out at the Clinical Microbiology- Laboratory, Sankara Nethralaya, Chennai during 1998 to 2001. The 136 patients included in this study were categorized based on the clinical information provided as 59 with congenital disease, 31 with acquired disease, 34 with post organ transplant complications and 12 with intrauterine infections attributable to CMV. Of these, 3 patients with acquired central nervous system (CNS) manifestations were HIV positive patients. A total of 168 specimens [53 urine, 62 peripheral blood, 22 nasopharyngeal aspirate, 11 amniotic fluid, 9 bronchial wash, 3 bronchoalveolar lavage, 6 cerebrospinal fluid (CSF), and I each endotracheal aspirate and placental material] from 136 patients suspected to have active CMV infection were investigated. Single specimens were collected from 104 patients and their distribution is shown in Table I. Dual specimens were collected from 32 patients and their distribution is shown in Table II. All the blood, urine and respiratory specimens were collected during a period of 3-7 days after the acute onset of illness particularly in organ transplant patients, HIV positive patients and patients with congenital CMV infection.
The amniotic fluid specimens were collected during the first trimester of pregnancy in mothers having history of miscarriages to rule out CMV infection of the foetus. Placental tissue of a foetus aborted during the first trimester of pregnancy was tested for CMV infection.
Fluorescent antibody technique (FAT) 15.16: Smears made by crush preparations of mucopurulent samples, cytospin (Shandon, USA) smears of centrifuged deposit of fluid samples and the buffy coat layer smears of blood were fixed in cold acetone and stained using monoclonal antibodies. In brief, mouse anti-CMV monoclonal antibodies (DAKO A/S, Denmark) raised against the immediate early antigen of CMV, rabbit anti-mouse fluorescein isothiocyanate (FITC) conjugate (DAKO A/S, Denmark) and 0.5 per cent Evan's blue (HiMedia, India), as counter stain were used for staining. The smears were observed under the fluorescent microscope (Optiphot, Nikon, Japan) using blue filter at 495 nm and barrier filter at 525 nm.
Nested polymerase chain reaction (nPCR): The nPCR was done as standardized and described by us earlier.17 DNA extraction from different types of specimens was done using standard phenolchloroform extraction as described for each type of specimen17-20. The DNA was finally dissolved in 30(mu)l of distilled water. PCR protocol consisted of use of the set of nested primers, which was customsynthesized by Bangalore Genei, India. The sequences of the nested primers coding for the morphological transforming region II of CMV were as described by Yuen et a112. The nested primers of CMV, CMTRI-5' - CTG TCG GTG ATG GTC TCT TC - 3' and CMTR2 - 5' - CCC GAC ACG CGG AAA AGA AA - 3' for the first round and CMTR3 - 5'-TCT CTG GTC CTG ATC GTC TT - 3' and CMTR4 - 5'-GTG ACC TAC CAA CGT AGG TT3' for the second round generated 234 bp and 168 bp products respectively12,17,21.
The amplification was carried out as described by us earlier 17. In brief, the amplification was done using dNTPs, respective primers and Taq DNA polymerase (Bangalore Genei, India) in Hybaid (UK, model no. HBTR3CM) or Perkin Elmer (USA, model no. 480 & 2400) thermalcyclers. The amplified product was then subjected to gel electrophoresis using 2 per cent agarose gel containing 0.5 (mu)g/ml of ethidium bromide.
The nPCR was done on human DNA extracted from the peripheral blood samples of 35 normal blood donors as controls to determine whether CMV-DNA would be found circulating in healthy persons. The plasma samples of these 35 persons were tested for anti-CMV IgG and IgM antibody status using enzyme-linked immunosorbent assay (ELISA) (BIOKIT, Barcelona, Spain).
Prevention of amplicon contamination22.23: Before the addition of template DNA, 2 (mu)g of 8-methoxy psoralen (Sigma, Germany) per 50 V(mu)l reaction of the cocktail was added. These reaction reagents were exposed to ultraviolet light (302nm) on a transilluminator (Pharmacia, USA) for 5 min to inactivate the activity of 8-methoxy psoralen.
Detection of false negatives and false positives lp nPCR: The specimens that were negative by nPCR were spiked to rule out the presence of nPCR inhibitors. To Spl DNA extracted from the specimen, 25 fg/(mu)l of CMY-DNA from the standard strain (AD-169) was added and then subjected to nPCR. nPCR reagent controls (buffer) and DNA extraction reagent controls (water) were also included with every set of reactions carried out to exclude false positive amplification.
Statistical methods: The McNemar and Chi-square tests were used appropriately to determine the statistical significance of the data 24.
Results
Of the single specimens collected from 104 patients, the presence of CMV was detected by FAT alone in 3 (2.9%), FAT and nPCR in 13 (12.5%) and nPCR alone in 48 (46.2%) specimens (Table 1). Of the 64 dual specimens from 32 patients, the presence of CMV was detected by FAT alone in none, FAT and nPCR in 3 (4.7%) specimens from 3 (9.4%) patients and by nPCR alone in 36 (56.3%) specimens from 23 (71.9%) patients (Table II).
Overall, of the 168 specimens collected from 136 patients, CMV was detected by FAT alone in 3 (1.8%) specimens from 3 (2.2%) patients, FAT and nPCR in 16 (9.5%) specimens from 16 (11.8%) patients and by nPCR alone in 84 (50.0%) specimens from 74 (54.4%) patients. Thus nPCR has a significant increase (McNemar test, P
Of the 62 blood samples tested for CMV infection, nPCR alone detected CMV-DNA in 22 (35.5%) specimens while FAT along with nPCR detected CMV in 7 (11.3%) specimens (Tables I and II). This increases the clinical sensitivity of nPCR on blood specimens by 35.5 per cent. Compared to FAT, nPCR on blood samples showed a significant (McNemar test, P
Of the 26 patients from whom both blood and urine were collected, CMV-DNA was detected by nPCR in both blood and urine in 12 (46.2%) patients, in urine alone in 8 (30.8%) and in the blood alone in 1 (3.80/(). FAT detected the presence of CMV antigens in 3 patients in the blood alone, while nPCR detected the presence of CMV-DNA in both blood and urine. Thus CMV was detected in the urine in 20 (76.9%) of 26 patients and in blood in 13 (50%) patients, this difference was significant (McNemar test, P
Of the 136 patients included in the study, CMV-- DNA was detected in 87 (64.0%). patients, "50 (84.7%) of 59 with congenital CMV disease, 15 (48.4%) of- 31 with acquired CMV disease; 18 (52.9%) of 34 with complications following organ transplantation, and 4 (33.3%) of 12 with intrauterine infections. Thus congenital CMV disease (84.7%) appears to be more prevalent than the acquired CMV disease (48.4%) and this difference was significant (Chi square test, P
nPCR did not detect the presence of CMV-DNA in any of the 35 blood specimens from healthy controls (blood donors). ELISA detected anti-CMV IgG and anti-CMV IgM in 22, anti-CMV IgG alone in 8 and anti-CMV IgM alone in 5 of these 35 blood samples.
Discussion
Monitoring of CMV infection is dependent upon the rapidity and accuracy of the diagnostic technique employed to identify patients who would benefit from presumptive therapy25. Our results showed that nPCR had significantly more sensitivity than FAT for the detection of CMV from clinical specimens. nPCR detected CMV in 84 additional specimens from 74 patients showing a statistically significant increase in the clinical sensitivity. Other workers have also found similar results 12,13,26. Two blood and one amniotic fluid specimens were positive for CMV by FAT alone and we believe these may be interpreted as false positive results as relative subjectivity does exist in the interpretation of FAT results. Within the past decade, PCR has established itself as the most promising tool of molecular biology to be applied both for research and diagnostic purposes26. Although CMV pp65 antigenaemia and branched chain DNA methods are more sensitive than the conventional serological methods, they are less sensitive than PCR25.
A statistically significant (McNemar test, P
It may be relevant to note that a large percentage (77.1 %) of healthy blood donors in the study showed the presence of anti-CMV IgM antibodies. Similar results have been reported by others in Japan (0.1%). Germany (4.9%), and USA (14.4%), though not at such high level as in our study27-29. Therefore the presence of anti-CMV IgM antibodies are known to be present in apparently healthy persons and need not always be suggestive of current active disease. Since reports on this from India are sparse, further studies on a large number of serum samples may help in estimation of the prevalence of anti-CMV IgM antibodies in the population.
On analyzing the results of nPCR on blood and urine collected from 26 patients, CMV-DNA was detected more commonly in the urine than in the blood. Though the urine specimens were expected to present problems in PCR testing on account of urine containing excreted breakdown products, we found PCR on urine to be more successful. It is hence emphasized that urine is the specimen of choice for PCR to detect CMV in patients with systemic illness. Collection of urine being a non-invasive procedure it is an ideal specimen especially in children with congenital infections.
Methods such as dialysis and ultrafiltration have been applied to remove PCR inhibitors in mucopurulent clinical specimens-26. Standard phenol chloroform extraction method was used for the removal of PCR inhibitors from intraocular fluid samples3. In our study the standard phenol chloroform extraction method was employed on all the clinical specimens and PCR inhibitors were excluded in this study by performing nPCR after spiking the specimens with CMV-DNA.
The results of nPCR in this study showed that congenital CMV disease (84.7%) was significantly more than acquired CMV disease (48.4%) and this was suggestive of prenatal CMV infection occurring commonly in our country.
Thus PCR can be employed as a more reliable and sensitive diagnostic tool than the routine virological methods. The presence of CMV-DNA in the peripheral blood is suggestive of active CMV infection. Further, quantitation of the DNA copies by a quantitative PCR could help in the monitoring of active CMV infection and in the timely initiation of preemptive antiviral therapy.
References
1. Tyler KL, Fields BN. Introduction to viruses and viral disease. In : Mandell GL, Douglas RG, Bennett JE, editors. Principles and practice of infectious diseases, 3rd ed. New York : Churchill Livingstone; 1990 p. 1124-34.
2. Madhavan HN, Prakash K, Agarwal SC. Cytomegalovirus infection in Pondicherry south India - a serological survey. Indian J Med Res 1974; 62 : 297-300.
3. Broor S, Kapil A, Kishore J, Seth P. Prevalence of rubella virus and cytomegalovirus infections in suspected cases of congenital infections. Indian J Pediatr 1991; 58 : 75-8.
4. Venkitaraman AR, Seigneurin JM, Lenoir GM, John TI. Infections due to the human herpesviruses in southern India: a seroepidemiological survey. Int d Epidemiol 1986; 15 561-.6.
5. Mathur A, Jindal 1, Chaturvedi UC. A serological study of cytomegalovirus infection at Lucknow. Indian J Med Res 1981; 73 : 678-81.
6. Dodt KK, Jacobsen PH, Hofmann B, Meyer C, Kolmos HJ, Skinhoj P, el al. Development of cytomegalovirus (CMV) disease may be predicted in HIV-infected patients by CMV polymerase chain reaction and the antigenaemia test. AIDS 1997; 11 : F21-8.
7. Nigro G,. Krzysztofiak A, Gattinara GC, Mango T, Mazzocco M, Porcaro MA, et al. Rapid progression of HIV disease in children with cytomegalovirus DNAemia. AIDS 1996; 10 : 1127-33.
8. Chattopadhya D, Aggarwal R, Prakash C, Sen S, Kumari S. Sexually-transmitted disease (STD) markers in multitransfused children in relation to human immunodeficiency virus type-1 (HIV-1 ) infection: impact
of STD markers in blood donors. J Trop Pediatr 1997; 43 178-81.
9. Stagno S, Britt WJ, Pass RF. Cytomegalovirus. In: Schmidt NJ. Emmons RW, editors. Diagnostic procedures for viral, rickettsial and chlamydial infections, 6th ed. Washington DC: American Public Health Association, Inc; 1989 p. 321-- 78.
10. Cleaves CA, Smith TF, Shuster EA, Pearson GR. Comparison of standard tube and shell vial cell culture techniques for the detection of cytomegalovirus in clinical specimens. J Clin Microbiol 1985; 21 : 217-21.
11. St George K, Rowe DT, Rinaldo Jr CR. Cytomegalovirus, Varicella-Zoster virus and Epstein-Barr virus. In : Specter S. Hodinke RL, Young SA, editors. Clinical N;-ology manual, 3rd ed. Washington DC : ASM Press; 2000 p. 410-49.
12. Yuen KY, Lo SK, Chiu EK, Wong SS, Lau Y-L, Liang R, et al. Monitoring of leukocyte cytomegalovirus DNA in bone marrow transplant recipients by nested PCR. J Clin Microbiol 1995; 33 : 2530-4.
13. Mansy F, Brancart F, Liesnard C, Bollen A, Godfroid E. A PCR based DNA hybridisation capture system for the detection of human cytomegalovirus. A comparative study with other identification methods. J Virol Methods 1999; 80 : 113-22.
14. Lo SK, Woo PC, Yuen KY. Hot spot mutations in morphological transforming region 11 (ORF 79) of cytomegalovirus strains causing disease from bone marrow transplant recipients. Arch Virol 1999; 144 : 601-12.
15. Minnich LL, Smith TF, Ray CG. In : Rapid detection of 1-truxes by immunofluorescence, Cunritech 24. Washington DC : American Society for Microbiology, 1988 p. 1-13.
16. Schmidt NJ. Cell culture procedures for diagnostic virology. In : Schmidt NJ, Emmons RW, editors. Diagnostic procedures.for viral, rickcttsial and chlamydial infections, Gth ed. Washington DC : American Public Health Association; .1989 p. 51-100..
17. Priya K. Application of rapid aetiological diagnostic methods on intraocular specimens in viral retino-choroiditis. h)dian J Med Microbiol 2001; 19 : 14-9.
18. Madhavan HN, Priya K, Anand AR, Therese KL. Detection of herpes simplex virus (HSV) genome using polymerase chain reaction (PCR) in clinical samples. Comparison of
PCR with standard laboratory methods for the detection of HSV. J Clin Virol 1999; 14 : 145-51.
19. Kox LFF, Rhienthong D, Miranda AM, Udomsantisuk N, Ellis K, van Leeuwen J, et al. A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples. J Clin Microbiol 1994; 32 : 672-8.
20. Narita M, Matsuzono Y, Shibata M, Togashi T. Nested amplification protocol for the detection of Mycobacterium tuberculosis. Acta Paediatr 1992; 81 : 997-1001.
21. Finny GJ, Manayani DJ, Abraham M, Sridharan G. Standardisation of a nested polymerase chain reaction for cytomegalovirus. Indian .J Med Microbiol 1999; 17 : 26-8.
22. Persing DH, Cimino GD. Amplification product inactivation methods. In : Persing DH, Smith TF, Tenover FC, White TJ, editors. Diagnostic molecular microbiology, principles and applications. Washington DC : American Society for Microbiology; 1993 p. 105-21.
23. Hughes MS, Beck LA, Skuce RA. Identification and elimination of DNA sequences in Taq DNA polymerase. J Clin Microbiol 1994; 32 : 2007-8.
24. Rosner B. Hypothesis testing : categorical data. In : Fundamentals of biostatistics 3rd ed. Boston, MA : PWS-- Kent Publishing Co; 1990 p. 318-97.
25. Bowen EF, Griffiths PD, Davey CC, Emery VC, Johnson MA. Lessons from the natural history of cytomegalovirus. AIDS 1996; 10 (Suppl I) : S37-41.
26. Khan G, Kangro HO, Coates PJ, Heath RB. Inhibitory effects of urine on the polymerase chain reaction for cytomegalovirus DNA. J Clin Pathol 1991; 44 : 360-5.
27. Aochi H, Nagamine K, Hayashi S, Oshida M, Tomiyama Y, Kurata Y. Evaluation of IgM anti human cytomegalovirus antibody. Rinsho By>ori 1990; 38 : 789-93.
28, Albert S, Sibrowski W, Kuhnl P, Knodler B, Seidl S, Bohm BO. CMV antibody determination with two enzyme irnmunoassays in each case. Beitr lusionther 1990; 26 40-2.
29. DePalma L, Criss VR, Sullivan MT, Leitman SF, Williams AE, Luban NL. Detection of cytomegalovirus antibody in platelet concentrates by fluorescence immunoassay and latex agglutination. Tranfusion 1991 : 31 : 245-8.
30. Wiedbrauk DL, Werner JC, Drevon AM. Inhibition of PCR by aqueous and vitreous fluids. .1 Clin Alicrobiol 1995: 33 2643-6.
K. Priya & H.N. Madhavan
Vision: Research Foundation, Sankara Nethralaya, Chennai, India
Received October 21, 2001
Reprint requests: Dr H.N. Madhavan, Director of Research & Professor of Microbiology, L & T Microbiology Research Centre Vision Research Foundation, Sankara Nethralaya, 18 College Road, Chennai 600006, India
Copyright Indian Council of Medical Research Jan 2002
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