Clin Biochem, Vol. 23, pp. 267-277, 1990 Printed in Canada. All rights reserved.

0009-9120/90 $3.00 + .00 Copyright © 1990 The Canadian Society of Clinical Chemists.

Nucleic Acid Hybridization in Viral Diagnosis MARIE L. LANDRY Virology Reference Laboratory, Veterans Administration Medical Center, West Haven, CT 06516, USA, and Departments of Laboratory Medicine and Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA Since 1982, numerous studies have been published utilizing a variety of hybridization techniques to detect viral nucleic acid directly in clinical specimens and in tissue sections. However, hybridization techniques are still not widely used in the clinical laboratory. Other recent advances, such as the development of monoclonal antibodies for virus identification and ELISA kits for virus detection, and the introduction of centrifugation cultures for rapid diagnosis, have postponed the clinical application of hybridization techniques. Furthermore, the use of hybridization for diagnosis has been limited by its insensitivity when compared to cell culture, the need for radioisotopes to increase sensitivity, and the difficulties inherent in transferring a basic research tool to the clinical laboratory. Nevertheless, with recently developed amplification techniques and further advances in nonradioactive labelling of probes, it can be expected that nucleic acid hybridization will be an established technique in diagnostic laboratories in the near future.

KEY WORDS: hybridization techniques; virus infection; viral diseases. Introduction ucleic acid hybridization techniques have been widely used in research laboratories for years; they were not, however, applied to the clinical diagnosis of virus infections until the 1980's (1). Numerous reports on the application of hybridization techniques to viral diagnosis have now appeared in the literature. This paper examines the

N

Correspondence: Marie L. Landry, Virology Reference Laboratory 151B, VA Medical Center, West Haven, CT 06516, USA. Manuscript received September 26, 1989; revised January 31, 1989; accepted March 1, 1990. Abbreviations--AIDS: acquired immune deficiency syndrome; ARC: AIDS related complex; CMV: cytomegalovirus; CNS: central nervous system; EA: early antigen; EBV: Epstein-Barr virus; ELISA: enzyme linked immunosorbent assay; IF: immunofluorescence; IP: immunoperoxidase; HBV: hepatitis B virus; HIV-I: human immunodeficiency virus type 1; HIV-2: human immunodeficiency virus type 2; HPV: human papillomavirus; HSV: herpes simplex virus; HTLV-I: human T cell lymphotropic virus type 1; HTLV-II: human T cell lymphotropic virus type 2; PBMC: peripheral blood mononuclear cells; PCR: polymerase chain reaction; TCIDso: 50% tissue culture infectious dose; VZV: Varicella-zoster virus. CLINICAL BIOCHEMISTRY, VOLUME 23, AUGUST 1990

role of nucleic acid hybridization in the clinical laboratory by reviewing the techniques, their applications, the problems, and recent developments. C o n v e n t i o n a l a n d n e w e r m e t h o d s for viral diagnosis

To place hybridization methods in perspective, it is first necessary to understand conventional methods of viral diagnosis. Conventional methods consist of: virus isolation and identification in cell culture, detection of viral antibody, and recognition of viral inclusions by light microscopy. Of these, virus isolation in cell culture remains the "gold standard" for the viral diagnostic laboratory (2). For primary isolation, a variety of cell cultures derived from h u m a n and animal tissues are inoculated with clinical specimens. After inoculation, the cultures are observed daily, under the light microscope, for cellular changes indicative of virus infection. A presumptive diagnosis of infection with a particular virus can be based on the characteristic cellular changes observed and the type of cell culture exhibiting the changes. The presumptive diagnosis is then confirmed by neutralization of virus induced effects by specific a n t i s e r u m - - a lengthy and tedious assay. Alternatively, virus infected cells can be stained with fluorscein or peroxidase labelled antiserum to the virus suspected. However, in the past, virus specific polyclonal antisera were not readily available or they were of variable quality. In the early 1980's, approximately when hybridization was first applied to viral diagnosis, monoclonal antibodies to commonly isolated viruses became commercially available for the rapid identification of isolates in the clinical laboratory. In addition, the availability of high quality antisera allowed centrifugation cultures, used for many years for chlamydia diagnosis, to be applied to viral diagnosis (3,4). By this technique, specimens are centrifuged onto cell culture monolayers in shell vials; the cultures are incubated for 16-48 h and then fixed and stained with specific antisera. The centrifugation step enhances viral infectivity; cultivation allows biologic amplification of the virus, providing a more sensitive assay than direct detection of virus in clinical 267

LANDRY TABLE 1

Viruses Categorized by Ability to Replicate in Cell Culture

Rapid Herpes simplex Influenza Poliovirus Coxsackie B Echovirus

Variable/ Insensitive

Slow Cytomegalovirus Resp. syncytial Parainfluenza Adenovirus

Varicella-zoster Rhinovirus

specimens. Immunofluorescence (IF) or immunoperoxidase (IP) staining detects virus infected cells early in the replication cycle, days or even weeks before viral cytopathic effects can be visualized under the light microscope. Although isolation in culture is the "gold standard" of viral diagnosis, there are relatively few viruses for which most isolates are obtained within 1-5 days of inoculation; for many viruses, isolation is either slow, insensitive, not available, or not yet possible (Table 1). Thus, despite the advances in viral diagnosis described above, more sensitive and/ or more rapid methods are needed. In the 1970's, development of the enzyme linked immunosorbent assay (ELISA)--which is nonradioactive and readily automated--to detect viral antigens provided an important diagnostic tool for certain viruses, such as rotavirus, that do not replicate well in culture. Thus, to be widely applied, hybridization assays must compete with the conventional and newer methods used in the diagnostic laboratory, in terms of sensitivity, cost, and ease of application.

Require Special Techniques/ Not Cultivable Epstein-Barr Rotavirus Hepatitis A Retroviruses Coxsackie A Arbovirus Enteric adenovirus

Papilloma Hepatitis B Parvovirus Norwalk virus

Hybridization techniques: principles and applications Hybridization techniques have several potential advantages. Whereas infectious virus particles are required for cultivation, viral nucleic acid detection methods can detect both infectious and noninfectious viral particles. If transport to the laboratory is delayed or specimens collected late in disease, hybridization techniques may still yield positive results though cultures are negative. In addition, replication is often inefficient, with 100 fold more noninfectious than infectious particles produced. However, both antigen and nucleic acid hybridization will only detect viruses that are "suspected." The unusual, unexpected, or new viral pathogen will not be detected. Three basic techniques are used; the steps involved are outlined in Table 2. The Southern blot or transfer is named after the man who developed it, E.M. Southern (5). For this technique, DNA must first be extracted and purified, cleaved by restriction

TABLE 2

Hybridization Techniques Southern

Dot or Spot

In Situ

Nucleic acid extraction or intact cells

Fixed tissue, cell culture, or cell smear

Transfer to nitrocellulose

Spot onto nitrocellulose

Fix to slide

Pretreatment

Pretreatment

Pretreatment

Add probe and hybridize

Add probe and hybridize

Add probe and hybridize

Wash

Wash

Wash

Detection

Detection

Detection

Nucleic acid extraction Restriction enzyme digestion Gel electrophoresis

268

CLINICAL BIOCHEMISTRY, VOLUME 23, AUGUST 1990

HYBRIDIZATIONIN VIRAL DIAGNOSIS TABLE 3

Examples of Spot Hybridization for the Diagnosis of Viral Infections Virus*

Sample

Label

Result

Reference

CMV

Urine

32p

-> 103 TCIDso/mL detected

10

CMV

Urine, buffy coat

32p

In urine, 92% sensitivity and 88% specificity; in buffy coat, more sensitive than culture

11

HSV

Clinical specimens Stool

32p

78% sensitivity compared to 100% for culture

12

32p

1 of 8 positive stools detected

13

Stool

32p

More sensitive than immunoelectron microscopy

14

Serum

32p

HBV DNA detected in absence of other markers of infection

15

32p

95% sensitivity compared to Southern blot

16

Parvovirus

Cervical specimens Serum

32p

Sensitivity comparable to RIA

17

HIV

PBMC

32p

Sensitivity comparable to culture

18

Enterovirus Rotavirus Group B Hepatitis B HPV

CMV, cytomegalovirus; HSV, herpes simplex virus; HBV, hepatitis B virus; HPV, human papillomavirus; HIV, human immunodeficiency virus; PBMC, peripheral blood mononuclear cells; TCIDso, 50% tissue culture infectious dose.

enzymes, the fragments separated by gel electrophoresis, and then transferred from the gel to nitrocellulose. Prior to hybridization, the blot is treated to reduce nonspecific binding; then the probe, generally labelled with 32p, is added and hybridization takes place. After hybridization, excess probe is removed by washing and the labelled hybrids are detected by autoradiography. For dot blot or spot hybridization, nucleic acid can be extracted by simplified procedures, or intact cells can be used (6). Samples are directly spotted onto nitrocellulose; this avoids restriction enzyme digestion and gel electrophoresis. Speed, simplicity, and ability to assay large numbers of specimens are obvious advantages for clinical use. However, only a spot is visualized, in contrast to Southern blot where restriction fragments of characteristic molecular weights and patterns can be recognized. Therefore, detecting a nonspecific reaction is more difficult; careful attention to reaction conditions, probe specificity, and controls is essential. For in-situ hybridization, intact cells, in the form of fixed or frozen tissue, cell smears, or cell culture, are probed for viral sequences (7). This method is appropriate for the pathology laboratory because histology can also be evaluated, and the cell type infected can be determined. In-situ hybridization can also be applied to infected cell culture in the diagnostic virology laboratory for rapid detection and identification of virus isolates. Initially, probes for hybridization used viral nucleic acids purified from intact virions grown in

CLINICAL BIOCHEMISTRY, VOLUME 23, AUGUST 1990

culture. Such whole virion probes could be contaminated with cellular nucleic acid, causing nonspecific reactions, or they could contain viral sequences that crosshybridized with cellular DNA sequences (8). Cloned probes have largely circumvented these problems. Probes have traditionally been labelled with radioisotopes: ~2p for spot and Southern blots, and 3H, S, or I for in-situ hybridization. The use of radioisotopes is hazardous to personnel and presents waste disposal problems. F u r t h e r development of nonradioactive labels, such as biotin (9), is necessary before hybridization gains wide acceptance for clinical diagnosis. Hybridization techniques can be applied directly to clinical specimens or tissues, after biologic amplification of virus in cell culture, or after in-vitro amplification of genome sequences. SPOT HYBRIDIZATION

Of the methods described, spot hybridization is most suited to the diagnostic laboratory; numerous reports have appeared in the literature. Examples of these reports (10-18) are included in Table 3. Results have varied depending upon how well the virus studied replicates in culture. For viruses t h a t replicate well, such as herpes simplex virus (HSV) and enterovirus, hybridization results--even with 32p label--have been insensitive compared with culture (12,13). Nevertheless, spot hybridization for enterovirus may be useful. Some enteroviruses, such as

269

LANDRY

group A coxsackieviruses, do not grow well in culture. In addition, there are over 67 enterovirus serotypes and no group antigen. Thus, only type specific antisera are available; enterovirus identification remains a very tedious task. However, conserved sequences have been detected in the 5' noncoding region of the genome, which can serve as broadly reactive probes (19,20). With recent innovations to improve sensitivity (21), spot hybridization may become a useful tool for rapid diagnosis and identification of enteroviruses. When spot hybridization was applied to cytomegalovirus (CMV), a virus that commonly requires 1-4 weeks for conventional isolation, the time to detection was shortened to 2 days from a mean of 8 days (10). However, a minimum of 103 tissue culture infectious doses (TCIDso) per mL was required for a positive result. Shortly after this report, centrifugation cultures for CMV were reported with staining 16 h post inoculation with monoclonal antibody to CMV early antigen (EA) (3). In addition, the sensitivity of centrifugation culture was even better than conventional culture for urine specimens. Thus centrifugation cultures, not hybridization, have been widely accepted in clinical laboratories. In another report on spot hybridization for CMV, a positive hybridization result on buffy coat specimens, often in the absence of positive culture results, appeared to correctly predict patients that would develop serious CMV infections (11). This suggested that hybridization was more sensitive for buffy coat than conventional tissue culture techniques. For rotavirus, culture requires specialized techniques; diagnosis is routinely made by ELISA. Hybridization has been found to be equivalent in sensitivity to ELISA (22). Group B rotavirus is not detected by current ELISA kits and antiserum is in short supply. Recently a hybridization assay has been reported (14) that may prove useful. Although hepatitis B virus (HBV) cannot be cultivated routinely, immunologic assays are available to detect a variety of HBV specific antigens and antibodies. Nevertheless, hybridization assays for HBV DNA may be the best marker for infectivity; they have been found positive when all other assays for HBV have been negative (15). However, falsely positive results by hybridization have also been reported (23). For human papillomavirus (HPV), cultivation in vitro has not yet been accomplished. Over 50 types of HPV have been identified; a number of types have been associated with genital warts. Several "high risk" types, in particular types 16 and 18, have been linked to cervical dysplasia and carcinoma (24). Hybridization is the mainstay of HPV detection; commercial probes are now available to screen cervical swab specimens for the presence of HPV and probes to detect those HPV types considered at high risk for progression to malignancy (Figure 1) (25). The commercially available spot hybridization kits use a 32p labelled probe. 270

6,11

D

16,18

31. 33. 35 -Negative - -

6,11-16, 18 - -

g

Q

31. 33, 35 -Negative - -

6.11-16,

18-

31, 3 3 . 3 5 - Negative

Q

--

Figure 1-Spot hybridization of cervical specimens with human papillomavirus (HPV) DNA typing kit. Twentyfive samples and controls have been applied to each of three membranes, which have been hybridized with a unique 32P-labeled probe combination: HPV types 6 and 11; HPV types 16 and 18; or HPV types 31, 33, and 35. On each membrane, the control specimen corresponding to the probe group applied gives a positive result indicated by a black spot on the autoradiograph. Controls are in the first column. (Courtesy of Life Technologies, Inc., Gaithersburg, MD.) H u m a n parvovirus has been recently recognized as a cause of Fifth's disease, hydrops fetalis, and aplastic crisis in patients with hemoglobinopathies. Until recently, it had not been grown in culture. Viremia becomes undetectable shortly after symptoms appear in normal hosts, thus detection of viral IgM antibody is the primary diagnostic method (26). However, in compromised hosts, viremia may last a week or more and antibody may not develop. Because detection of viral DNA is more sensitive than viral antigen (17,26,27), hybridization may be useful for diagnosis in this circumstance. By spot hybridization of peripheral blood mononuclear cells (PBMC), h u m a n immunodeficiency virus (HIV) RNA has been detected in 80% of seropositive individuals (18). Compared with culture for HIV, hybridization is simpler, less expensive, faster, and avoids the hazards associated with cultivation of the virus. However, weak cross reactions with HTLV-I and HTLV-II were noted; more importantly, 3/33 seronegative persons were falsely positive by hybridization. Spot hybridization has also been used to test for antiviral sensitivity (28,29). Instead of waiting for CLINICAL BIOCHEMISTRY, VOLUME 23, AUGUST 1990

HYBRIDIZATION IN VIRAL DIAGNOSIS TABLE 4

Examples of In-Situ Hybridization for Viral Diagnosis Virus

Sample

Label

Result

Reference

CMV

Lung biopsy

Biotin

Sensitivity similar to culture and immunofluorescence

31

CMV

PBMC

ssS

CMV RNA detected in PBMC 1-2 weeks before conventional tests; detected only 68% of culture positive specimens

32

EBV

Oral epithelial Biotin/3H cells

More sensitive than culture; localized EBV to epithelial cells

33

HPV

Cervical biopsies

BiotinPSS

Biotin label more sensitive with less background staining

34

HIV

Cell culture

3sS

HIV RNA detected within 24 h of infection

35

CMV

Cell culture

Biotin

88% sensitive compared with monoclonal antibodies, and slower result

36

HSV

Cell culture

Biotin

Less sensitive than polyclonal antibody

37

CMV, cytomegalovirus; EBV, Epstein-Barr virus; HPV, human papillomavirus; HIV, human immunodeficiency virus; CMV, cytomegalovirus; HSV, herpes simplex virus.

viral plaques to form, the amount of nucleic acid produced in the presence of antiviral agents is quantitated by hybridization followed by densitometer tracing. With recent reports of resistance to antiviral agents (30), sensitivity testing m a y soon become commonplace in clinical virology laboratories. IN-SITU HYBRIDIZATION

As noted in Table 4, the nonradioactive label, biotin, has been widely used for in-situ hybridization (31-37), and biotin labelled probes are now commercially available for m a n y viruses. Although the biotin label generally provides a less sensitive result, better morphology, more rapid detection steps (several hours versus several days), longer probe shelf life, and less hazardous waste, all compensate for reduced sensitivity. This technique can now be performed by both pathology and viral diagnostic laboratories. In tissue sections, viral diagnosis based on inclusions alone m a y give false results (38). Application of viral probes can give specific identification and detect virus infection in cells without inclusions (39). In addition, if fixation is too long or suboptimal, viral antigens m a y be destroyed. Because viral DNA is more stable, it m a y still be detected. In-situ hybridization techniques are comparable in sensitivity to immunologic assays on fixed tissues for CMV (31,40,41). In a recent report, peripheral CLINICAL BIOCHEMISTRY, VOLUME 23, AUGUST 1990

blood mononuclear cells were assayed for messenger RNA to CMV early antigens by in-situ hybridization (32). In 8/18 specimens, a positive hybridization result was obtained 1-2 weeks before conventional culture yielded positive results. However, negative hybridization results were obtained for 32% of specimens that were found positive by conventional culture. For HPV, in-situ hybridization can also be used, with exfoliated cells or tissue biopsies, to detect virus infection and to detect infection with high risk types (Figures 2a and b). Theoretically, since high risk types seem more likely to progress to malignancy, identified patients can be followed more closely and treated more aggressively. Conversely, those infected with low risk types can expect a benign course with spontaneous regression of condylomas. Whether specific diagnosis and type identification ultimately benefit patient management, remains to be determined. In-situ hybridization for Epstein-Barr virus (EBV) provides results more rapid t h a n culture, which remains a lengthy and specialized research tool. It also provided the first evidence t h a t EBV replicated in oral epithelial cells (33). Furthermore, direct detection of HSV in clinical specimens by in-situ hybridization with a biotin labelled probe has been comparable in sensitivity to monoclonal antibodies (42). When cultures for HIV were monitored for evidence of viral replication, viral RNA was detected 271

LANDRY

(a)

(b)

Figure 2--Detection of human papillomavirus (HPV) type 16 DNA in formalin fixed, paraffin embedded biopsy specimens by in-situ hybridization with a biotinylated HPV type 16-specific DNA probe. A reddish brown color in the nuclei indicates the presence of HPV 16 DNA (Magnification 200 × ). (a) HPV 16 DNA detected in cervical tissue exhibiting severe dysplasia; (b) HPV 16 DNA detected in labial tissue exhibiting Bowenoid disease. (Courtesy of Enzo Diagnostics, Inc., New York, NY.)

first by in-situ hybridization with 35S labelled probes

sitivity to monoclonal antibody in a shell vial

(35). Several days later, viral antigens were expressed intracellularly, as detected by monoclonal antibodies. Cell free virus, as measured by reverse transcriptase assays and ELISA for core antigen, were detectable a week later. Commercially available nonradioactive probes have also been used for rapid identification of viruses in infected cell cultures. For CMV, DNA hybridization was less sensitive and much slower t h a n detection of CMV early antigen by monoclonal antibody (36), because early antigens are produced prior to DNA replication. Detection of HSV in cell culture by hybridization was less sensitive t h a n IP staining with polyclonal antiserum (37), but similar in sen-

assay (43). SOUTHERN BLOTHYBRIDIZATION Much valuable information concerning viral pathogenesis has been obtained by Southern blot. A few examples are included in Table 5 (44-49); a detailed discussion is beyond the scope of this review. The Southern blot is too specialized and tedious for a viral diagnostic laboratory to perform, and it continues to require 32p. However, central reference laboratories or molecular diagnostic laboratories could provide Southern blot testing for viruses similar to its use for genetic and forensic testing.

TABLE 5 Examples of Southern Blot for Diagnosis of Viral Infections Virus

Label

Result

Reference

EBV

32p

EBV DNA detected in primary lymphoma of CNS

44

Hepatitis B

32p

HBV DNA integrated into cell DNA in liver disease

45

Hepatitis B

32p

HBV DNA detected in liver and blood in absence of HBsAg

46

HPV

32p

Presence and type of HPV investigated in different lesions

47

HTLV-I

32p

HTLV-I detected in adult T-cell leukemia and other T-cell malignancies

48

HIV

32p

HIV-1 DNA detected in lymphoid tissues of AIDS and ARC patients

49

EBV, Epstein-Barr virus; HBV, hepatitis B virus; HPV, human papillomavirus; HTLV-I, human T cell lymphotropic virus type 1; HIV, human immunodeficiency virus; CNS, central nervous system. 272

CLINICALBIOCHEMISTRY,VOLUME 23, AUGUST 1990

HYBRIDIZATION IN VIRAL DIAGNOSIS TABLE 6

Use of Nucleic Acid Hybridization in Viral Diagnosis Setting

Current Use

Virus

Hybridization Technique

Viral diagnostic laboratory

Limited

HPV

Spot

Pathology laboratory

Potential

HPV, CMV HSV, VZV EBV, HBV

In situ

Adenovirus

Research laboratory

Extensive

Numerous

Southern, spot, in situ

HPV, human papillomavirus; CMV, cytomegalovirus; HSV, herpes simplex virus; VZV, varicella-zoster virus; EBV, Epstein-Barr virus; HBV, hepatitis B virus.

Problems encountered and recent developments Hybridization methodology is commonly used in research laboratories, but in general, clinical laboratories lack the equipment and expertise. Clinical laboratories have experience with IF and IP staining; now that numerous monoclonal viral antibodies are commercially available, use of these techniques has increased tremendously. Since hybridization is an unfamiliar technique, technically demanding, requires radioisotopes for maximum sensitivity, is expensive, and is at best no more sensitive than immunologic staining, it has not displaced immunologic staining for clinical diagnosis. Also, hybridization has lacked the automation available with antigen detection methods such as ELISA; sample preparation, for maximum sensitivity and specificity, may require ultracentrifugation (50). Despite the theoretically specific nature of hybridization, after the initial flurry of reports on hybridization, researchers began reporting problems with specificity. Contaminating plasmid vector sequences were found to hybridize to plasmids in bacteria present in clinical specimens (23,26,50). Viral RNA genomes have been found to hybridize with mammalian ribosomal RNA from uninfected cells (51). Herpes virus genomes have sequences homologous with cellular DNA and with other herpes viruses (8,50,52). In addition, nonspecific trapping of probe in mucus has been noted (53). A number of innovations have been developed to meet these problems. Care has been taken to eliminate vector sequences from probes. More sensitive probes such as single stranded RNA probes or riboprobes, have been introduced (21,54). Single stranded probes do not have a competing probe rehybridizing reaction. RNA-DNA and RNA-RNA hybrids are more stable than DNA-DNA hybrids, allowing more stringent washes and greater specificity. RNA probes have the disadvantage of being CLINICAL BIOCHEMISTRY, VOLUME 23, AUGUST 1990

more readily degraded by ribonucleases (54). Oligonucleotide probes have also been used, and can be readily synthesized to complement specific viral sequences. These probes are short, 30-50 nucleotides long, and diffuse better into specimens (55). Simplified sample preparation has been reported with mixed results. Some researchers report that tedious phenol/chloroform extraction of nucleic acid in samples is not necessary (56), whereas others find that it is essential for maximum sensitivity and specificity (57). One study for detection of fastidious adenoviruses reported that extraction, restriction enzyme digestion, and Southern blot with a nonradioactive probe were essential for sensitivity and specificity (58). Prior to hybridization, the use of an immunoaffinity step to remove contaminating proteins has been recently reported (59). Biotin labelling has generally been found to be approximately 10 fold less sensitive than radioisotopes. However, when used for experimental HSV keratitis, and in a few clinical specimens, hybridization with biotin appeared more sensitive than virus isolation and antigen detection (60). When time for autoradiography is reduced to a few hours, the sensitivity of radioisotope labels is equivalent to biotin (61). More sensitive results with biotinylated probes have been obtained in in-situ hybridization with a streptavidin-alkaline phosphatase detection system (34,40,41). Additional methods for nonradioactive labelling of probes have appeared in the literature, such as probes covalently linked with modified horseradish peroxidase (54,58,61,62); however, in some studies, the sensitivity has again been low when compared with radioisotopes (54). Nevertheless, more commercial reagents are now available, and protocols continue to be shortened and simplified. In addition, a machine has been developed to automate in-situ hybridization of histologic specimens (63). However, the innovation that has been greeted most enthusiastically is the development of in-vitro amplification techniques which 273

LANDRY greatly enhance the sensitivity of viral genome detection methods.

Amplification m e t h o d s Tremendous excitement in basic research and clinical laboratories has been generated by the development of the polymerase chain reaction (PCR). A number of in-vitro genetic amplification techniques have now been described which 1) amplify target sequences using either PCR (64,65) or the transcript amplification system (TAS) (66), 2) amplify probe sequences using the QB replicase system (67,68), or 3) amplify probe generated signals with sandwich or "Christmas tree" probes (69,70). To date, the most widely applied amplification technique has been PCR. By PCR, repeated cycles of oligonucleotide directed DNA synthesis results in amplification of target sequences over a million fold. The amplified sequence can then be detected by gel electrophoresis and ethidium staining, or by Southern blot or spot hybridization which improves sensitivity. In published reports, PCR has been used to detect a number of viruses whose cultivation is difficult (HIV-1, HIV-2, HTLV-I, HTLV-II, h u m a n B lymphtropic virus) (71-78), lengthy (CMV), (79,80), or unavailable (HPV, HBV, parvovirus) (81-84). For h u m a n immunodeficiency virus (HIV), PCR was more sensitive than culture (73-75); it has also been useful in the differentiation ofretrovirus types (72,78). PCR has been used to greatly enhance the sensitivity of hybridization for CMV. Whereas, conventional spot hybridization requires at least 10 3 TCIDso for a positive result (10), with PCR from 1-10 TCIDso of CMV has been detected (79,80). The sensitivity of this technique has led to the problem of distinguishing how m a n y organisms are associated with clinical significance because detection of small amounts of organisms may be normal. Another problem that is more serious for clinical diagnosis is contamination of specimens with amplified fragments, which m a y contaminate glassware, buffers, and air systems, causing false positive results (85).

Summary and conclusions Nucleic acid hybridization is a powerful technique that has only recently been introduced into the clinical setting. It has had to compete with current techniques, which are also undergoing rapid improvements. Its lack of sensitivity has been a major drawback. Although potential uses for hybridization include diagnosis of enteroviruses, parvovirus, and HBV, only HPV is currently being diagnosed by hybridization in the clinical virology laboratory (Table 6). Pathology laboratories also use hybridization to diagnose HPV; in some larger centers with a significant number of immunosuppressed patients, commercially available probes are used to identify a

274

variety of virus infections in tissue sections (86). Significant progress has been made in a short period. With innovations to simplify sample preparation, to improve nonradioactive labelling in probes, and in-vitro amplification to enhance sensitivity, the application of hybridization in the clinical setting will increase in the near future.

References 1. Landry ML, Fong CKY. Nucleic acid hybridization in the diagnosis of viral infections. Clin Lab Med 1985; 5: 513-29. 2. Landry ML, Hsiung GD. Primary isolation of viruses. In: Specter S, Lanz G, Lee I, eds. Clinical virology manual. Pp. 31-51. New York: Elsevier, 1986. 3. Gleaves CA, Smith TF, Shuster EA, Peason 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. 4. Gleaves CA, Wilson DJ, Wold AD, Smith TF. Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16h postinoculation. J Clin Microbiol 1985; 21: 29-32. 5. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975; 98: 503-17. 6. Brandsma J, Miller G. Nucleic acid and spot hybridization: rapid quantitative screening of lymphoid cell lines for Epstein-Barr viral DNA. Proc Natl Acad Sci USA 1980; 77: 6851-5. 7. Gall JG, Pardue ML. Nucleic acid hybridization in cytological preparations. Methods Enzymol 1971; 21: 470-80. 8. Peden K, Mounts P, Hayward GS. Homology between mammalian cell DNA sequences and human herpes virus genomes detected by a hybridization procedure with a high-complexity probe. Cell 1982; 31: 81-7. 9. Brigati DJ, Myerson D, Leary JJ, Fong CKY, Hsiung GD, Ward D. Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. Virology 1983; 126: 32-50. 10. Chou S, Merigan TC. Rapid detection and quantitation of human cytomegalovirus in urine through DNA hybridization. N Engl J Med 1982; 308: 921-5. 11. Spector SAS, Rua JA, Specter DH, McMillan R. Detection of human cytomegalovirus in clinical specimens by DNA-DNA hybridization. ] Infect Dis 1984; 150: 121-6. 12. Redfield DC, Richman DD, Albanil S, Oxman MN, Wahl GM. Detection of herpes simplex virus in clinical specimens by DNA hybridization. Diag Microbiol Infec Dis 1983; 1: 117-28. 13. Hyypia T, Stalhandske P, Vainionpaa R, et al. Detection of enteroviruses by spot hybridization. J Clin Microbiol 1984; 19: 436-8. 14. Eiden JJ, Firoozmand F, Sato S, Vonderfecht SL, Fang ZY, Yolken RH. Detection of group B rotavirus in fecal specimens by dot hybridization with a cloned cDNA probe. J Clin Microbiol 1989; 27: 422-6. 15. Sun CF, Pao CC, Wu SY, Liaw YF. Screening for

CLINICAL BIOCHEMISTRY,VOLUME23, AUGUST 1990

HYBRIDIZATIONIN VIRAL DIAGNOSIS

16.

17. 18.

19. 20.

21. 22. 23.

24.

25.

26. 27.

28.

29.

30.

31. 32.

hepatitis B virus in healthy blood donors by molecular DNA hybridization analysis. J Clin Microbiol 1988; 26: 1848-52. Challberg SS, Scott CL, Russell CL, Ferenczy A,Richart RM. A simple method for the detection of HPV infection in cervical smears. Proceedings of American Society for Microbiology Annual Meeting; 1987; Abstract S-10. Clewley JP. Detection of human parvovirus using a molecularly cloned probe. J Med Virol 1985; 15: 173-81. Richman DD, McCutchan JA, Spector SA. Detecting human immunodeficiency virus RNA in peripheral blood mononuclear cells by nucleic acid hybridization. J Infect Dis 1987; 156: 823-7. Tracy S. Comparison of genomic homologies in the coxsackievirus B group by use of cDNA:RNA dot-blot hybridization. J Clin Microbiol 1985; 21: 371-4. Rotbart HA, Levin MJ, Villarreal LP, Tracy SM, Semler BL, Wimmer E. Factors affecting the detection of enteroviruses in cerebrospinal fluid with coxsachievirus B3 and poliovirus 1 cDNA probes. J Clin Microbiol 1985; 22: 220-4. Cova L, Kopecka H, Aymard M, Girard M. Use of cRNA probes for the detection of enteroviruses by molecular hybridization. J Med Virol 1988; 24: 11-8. Flores J, Boeggeman E, Purcell RH, et al. A dot hybridization assay for detection of rotavirus. Lancet 1983; 1: 555-8. Diegutis PS, Keiruan E, Burnett L, Nightingale BN, Cossart YE. False-positive results with hepatitis B virus DNA dot-hybridization in hepatitis B surface antigen-negative specimens. J Clin Microbiol 1986; 23: 797-9. Durst M, Gissman L, Ikenberg H, zur Hausen H. A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc Natl Acad Sci USA 1983; 80: 3812-5. Melki R, Khoury B, Catalan F. Nucleic acid spot hybridization with nonradioactive labeled probes in screening for human papillomavirus DNA sequences. J Med Virol 1988; 26: 137-43. Mori J, Field AM, Clewley JP, Cohen BJ. Dot blot hybridization assay of B19 virus DNA in clinical specimens. J Clin Microbiol 1989; 27: 459-64. Anderson MJ, Jones SE, Minson AC. Diagnosis of human parvovirus infection by dot-blot hybridization using cloned viral DNA. J Med Virol 1985; 15: 16372. Gadler H. Nucleic acid hybridization for measurement of effects of antiviral compounds on human cytomegalovirus DNA replication. Antimicrob Agents Chemother 1983; 24: 370-4. Gadler H, Larsson A, Solver E. Nucleic acid hybridization, a method to determine effects of antiviral compounds on herpes simplex virus type 1 DNA synthesis. Antiviral Res 1984; 4: 63-70. Swierkosz EM, Scholl DR, Brown JL, Jolick JD, Gleaves CA. Improved DNA hybridization method for detection of acyclovir-resistant herpes simplex virus. Antimicrob Agents Chemother 1987; 31: 1465-69. Myerson D, Hackman RC, Meyers JD. Diagnosis of cytomegaloviral pneumonia by in situ hybridization. J Infect Dis 1984; 150: 272-7. Stockl E, Popw-Kraupp T, Heinz FX, Muhlbacher F,

CLINICAL BIOCHEMISTRY,VOLUME 23, AUGUST 1990

33.

34.

35.

36.

37.

38.

39. 40. 41.

42. 43.

44. 45.

46.

47.

48. 49.

Balcke P, Kunz C. Potential of in situ hybridization for early diagnosis of productive cytomegalovirus infection. J Clin Microbiol 1988; 26: 2536-40. Sixbey JW, Nedrud JG, Raab-Traub N, Hanes RA, Pagano JS. Epstein-Barr virus replication in oropharyngeal epithelial cells. N Engl J Med 1984; 310: 1225-30. Syrjanen S, Partanen P, Mantyjarvi R, Syrjanen K. Sensitivity of in situ hybridization techniques using biotin- and 35S-labeled human papillomavirus (HPV) DNA probes. J Virol Methods 1988; 19: 225-38. Busch MP, Rajagopalan MS, Gantz DM, Fu S, Steimer KS, Vyas GN. In situ hybridization and immunocytochemistry for improved assessment of human immunodeficiency virus cultures. A m J Clin Pathol 1987; 88: 673-80. Gleaves CA, Hursh DA, Rice DH, Meyers JD. Detection of cytomegalovirus from clinical specimens in centrifugation culture by in situ hybridization and monoclonal antibody staining. J Clin Microbiol 1989; 27: 21-3. Landry ML, Zibello TA, Hsiung GD. Comparison of in situ hybridization and immunologic staining with cytopathologyfor detection and identification of herpes simplex virus infection in cultured cells. J Clin Microbiol 1986; 24: 968-71. Landry ML, Fong CKY, Nedderman K, Solomon L, Hsiung GD. Disseminated adenovirus infection in an immunocompromised host: pitfalls in diagnosis. A m J Med 1987; 83: 555-9. Myerson D, Hackman RC, Nelson JA, et al. Widespread presence of histologically occult cytomegalovirus. Hum Pathol 1984; 15: 430-9. Keh WC, Gerber MA. In situ hybridization for cytomegalovirus DNA in AIDS patients. A m J Pathol 1988; 131: 490-6. Wolber RA, Lloyd RV. Cytomegalovirus detection by nonisotopic in situ hybridization and viral antigen immunostaining using a two-color technique. H u m Pathol 1988; 19: 736--41. Langenberg A, Smith D, Brakel CL, et al. Detection of herpes simplex virus DNA from genital lesions by in situ hybridization. J Clin Microbiol 1988; 26: 933-7. Espy M, Smith TF. Detection of herpes simplex virus in conventional tube cell cultures and in shell vials with a DNA probe kit and monoclonal antibodies. J Clin Microbiol 1988; 26: 22-4. Hochberg FH, Miller G, Schooley RT, et al. Central nervous system lymphoma related to Epstein-Barr virus. N Engl J Med 1983; 309: 745-8. Scotto J, Hadchouel M, Hery C, et al. Hepatitis B virus DNA in children's liver diseases: detection by blot hybridization in liver and serum. Gut 1983; 24: 618-24. Brechot C, Degos F, Lugassy C, et al. Hepatitis B virus DNA in patients with chronic liver disease and negative tests for hepatitis B surface antigen. N Engl J Med 1985; 312: 270-6. Gissman L, Wolnik L, Ikenberg H, et al. Human papillomavirus types 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancers. Proc Natl Acad Sci USA 1983; 80: 560-3. Wong-Staal F, Hahn B, Manzari V, et al. A survey of human leukemias for sequences of a human retrovirus. Nature 1983; 302: 626-8. Shaw GM, Hahn BH, Arya SK, et al. Molecular

275

LANDRY

50.

51.

52.

53.

54.

55. 56.

57.

58.

59.

60.

61.

62. 63.

64.

65. 66. 276

characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome. Science 1984; 226: 1165-71. Augustin S, Popow-Kraupp T, Heinz FX, Kunz C. Problems in detection of cytomegalovirus in urine samples by dot blot hybridization. J Clin Microbiol 1987; 25: 1973-7. Eiden J, Sato S, Yolken R. Specificity of dot hybridization assay in the presence of rRNA for detection of rotaviruses in clinical specimens. J Clin Microbiol 1987; 25: 1809-11. Lurain NS, Thompson KD, Farrand SK. Rapid detection of cytomegalovirus in clinical specimens by using biotinylated DNA probes and analysis of cross-reactivity with herpes simplex virus. J Clin Microbiol 1986; 24: 724-30. Stalhandske P, Hyypia T, Gadler H, Halonen P, Pettersson U. The use of molecular hybridization for demonstration of adenoviruses in human stools. Curr Top Microbiol Immunol 1983; 104: 287-98. Schuster V, Matz B, Wiegand H, Traub B, Kampa D, Neumann-Haefelin D. Detection of human cytomegalovirus in urine by DNA-DNA and RNA-DNA hybridization. J Infect Dis 1986; 154: 309-14. Cubie HA, Norval M. Synthetic oligonucleotide probes for the detection of human papilloma viruses by in situ hybridization. J Virol Methods 1988; 20: 239-49. Hammond G, Hannan C, Yeh T, Fischer K, Mauthe G, Straus SE. DNA hybridization for diagnosis of enteric adenovirus infection from directly spotted human fecal specimens. J Clin Microbiol 1987; 25: 1881-5. Kidd AH, Harley EH, Erasmus MJ. Specific detection and typing of adenovirus types 40 and 41 in stool specimens by dot-blot hybridization. J Clin Microbiol 1985; 22: 934-9. Niel C, Gomes SA, Leite JPG, Pereira HG. Direct detection and differentiation of fastidious and nonfastidious adenoviruses in stools by using a specific nonradioactive probe. J Clin Microbiol 1986; 24: 7859. Jansen RW, Newbold JE, Lemon SM. Combined immunoaffinity cDNA-RNA hybridization assay for detection of hepatitis A virus in clinical specimens. J Clin Microbiol 1985; 22: 984-9. Nago R, Hayashi K, Ochiai H, Kubota Y, Niwayama S. Detection of herpes simplex virus type 1 in herpetic ocular diseases by DNA-DNA hybridization using a biotinylated DNA probe. J Med Virol 1988; 25: 25970. Buffone GJ, Schimbor CM, Demmler GJ, Wilson DR, Darlington GJ. Detection of cytomegalovirus in urine by nonisotopic DNA hybridization. J Infect Dis 1986; 154: 163-6. Renz M, Kurz C. A colorimetric method for DNA hybridization. Nucleic Acids Res 1984; 12: 3435-44. Montone KT, Brigati DJ, Budgeon LR. Anatomic viral detection is automated: the application of a robotic molecular pathology system for the detection of DNA viruses in anatomic pathology substrates using immunocytochemical and nucleic acid hybridization techniques. Yale J Biol Med 1989; 62: 141-58. Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of ~-globin genomic sequences and restriction site anlaysis for the diagnosis of sickle-cell anemia. Science 1985; 230: 1350-4. Ehrlich HA, Gelfand DH, Saiki RK. Specific DNA amplification. Nature 1988; 331: 461-2. Kwoh DY, Davis GR, Whitfield DM, Chappelle HL,

67. 68.

69. 70.

71. 72.

73.

74.

75. 76.

77.

78.

79.

80.

81.

82.

DiMichele I_2, Gingeras TR. Transcription-based amplification system and detection of amplified human immunodeficiency virus type 1 with a bead-based sandwich hybridization format. Proc Natl Acad Sci USA.1989; 86: 1173-7. Haruna I, Spiegelman S. Autocatalytic synthesis of a viral RNA in vitro. Science 1965; 150: 884-6. Lizardi PM, Guerra CE, Lomeli H, Tussie-Luna I, Kramer FR. Exponential amplification of recombinant RNA hybridization probes. Biotechnology 1988; 6: 1197-202. Fahrlander PD, Klausner A. Amplifying DNA probe signals: a "Christmas tree" approach. Biotechnology 1988; 6: 1165-8. Sanchez-Pescador R, Stempien MS, Urdea MS. Rapid chemiluminescent nucleic acid assays for detection of TEM-1 beta-lactamase-mediated penicillin resistance in Neisseria gonorrhea and other bacteria. J Clin Microbiol 1988; 26: 1934-8. Ou CY, Kwok S, Mitchell SW, et al. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science 1988; 239: 2236-9. Rayfield M, DeCock K, Heyward W, et al. Mixed human immunodeficiency virus (HIV) infection in an individual: demonstration of both HIV type 1 and type 2 proviral sequences by using polymerase chain reaction. J Infect Dis 1988; 158: 1170-6. Farzadegan H, Polis MA, Wolinsky SM, et al. Loss of human immunodeficiency virus type 1 (HIV-1) antibodies with evidence of viral infection in asymptomatic homosexual men. Ann Intern Med 1988; 108: 785-90. Imagawa DT, Lee MH, Wolinsky SM, et al. Human immunodeficiency virus type 1 infection in homosexual men who remain seronegative for prolonged periods. N Engl J Med 1989; 320: 1458-62. Laure F, Courgnaud V, Rouzioux C, et al. Detection of HIV-1 DNA in infants and children by means of the polymerase chain reaction. Lancet 1988; 2: 596-9. Duggan DB, Ehrlich GD, Davey FP, et al. HTLVI-induced lymphoma mimicking Hodgkin's disease. Diagnosis by polymerase chain reaction amplification of specific HTLV-I sequences in tumor DNA. Blood 1988; 71: 1027-32. Buchbinder A, Josephs SF, Ablashi D, et al. Polymerase chain reaction amplification and in situ hybridization for the detection of human B-lymphotropic virus. J Virol Methods 1988; 21: 191-7. Lee H, Swanson P, Shorty VS, Zack JA, Rosenblatt JD, Chen ISY. High rate of HTLV-II infection in seropositive IV drug abusers in New Orleans. Science 1989; 244: 471-5. Shibata D, Martin WJ, Appleman MD, Causey DM, Leedom JM, Arnheim N. Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus. J Infect Dis 1988; 158: 1185-92. Demmler GJ, Buffone GJ, Schimbor CM, May RA. Detection of cytomegalovirus in urine from newborns by using polymerase chain reaction DNA amplification. J Infect Dis 1988; 158: 1177-84. Shibata D, Fu YS, Gupta JW, Shah KV, Arnheim N, Martin WJ. Detection of human papillomavirus in normal and dysplastic tissue by the polymerase chain reaction. Lab Invest 1988; 59: 555-9. Salimans MMM, Holsappel S, van de Rijke FM, Jiwa NM, Raap AK, Weiland HT. Rapid detection of human parvovirus B19 DNA by dot-hybridization and CLINICAL BIOCHEMISTRY, VOLUME 23, AUGUST 1990

HYBRIDIZATION IN VIRAL DIAGNOSIS the polymerase chain reaction. J Virol Methods 1989; 23: 19-28. 83. Kaneko S, Miller RH, Feinstone SM, et al. Detection of serum hepatitis B virus DNA in patients with chronic hepatitis using the polymerase chain reaction assay. Proc Natl Acad Sci USA 1989; 86: 312-6. 84. Larzul D, Guigue F, Sninsky JJ, Mack DH, Brechot C, Guesdon JL. Detection of hepatitis B virus sequences

CLINICAL BIOCHEMISTRY, VOLUME 23, AUGUST 1990

in serum by using in vitro enzymatic amplification. J Virol Methods 1988; 20: 227-37. 85. Kwok S, Higuchi R. Avoiding false positives with PCR. Nature 1989; 339: 237-8. 86. Kemnitz J, Haverich A, Gubernatis F, Cohnert TR. Rapid identification of viral infections in liver, heart, and kidney allograft biopsies by in situ hybridization. A m J Surg Pathol 1989; 13: 80-2.

277