Molecular infectious disease testing: the hype, the hope, and the hoopla.

Does obsolescence loom for traditional isolation and identification skills now that the age of molecular. diagnostics is upon us?

For the past 15 years, molecular biologists and diagnostics companies have embraced molecular diagnostic methods for their ability to detect infectious agents in clinical specimens. During this time, the potential for molecular pathology appeared boundless. Thousands of articles and hundreds of reviews have been published describing nucleic-acid methods, their performance, and their impact on clinical and laboratory medicine.

Despite (or, perhaps, because of) this wealth of information, many microbiologists and virologists are confused about molecular diagnostic methods and the role they can or should play in the clinical infectious disease laboratory. Other microbiologists have returned from regional or national meetings feeling like technical dinosaurs because their labs do not offer molecular diagnostic tests.

Should every microbiology laboratory perform molecular diagnostic tests? Will traditional isolation and identification skills become obsolete? Should we continue to train clinical microbiologists? This article will address some of the hype, the hope, and the hoopla associated with molecular diagnostic methods and make a case for integrated infectious disease testing that uses both old and new methods.

Amplification methods

Recent advances in nucleic-acid methods have made molecular diagnostic procedures available to an increasing number of clinical labs. To date, three general amplification strategies have been used to improve the sensitivity of nucleic-acid testing: signal, target, and probe amplification.[1,2]

Signal amplification methods, just as the name implies, increase the signal-generating capability of an assay without altering the number of target molecules. While the sensitivity of these techniques is lower than that of other amplification methods, signal amplification procedures generally are simpler, easier to perform, and provide more quantitative test results. The hybridization protection assay from Gen-Probe (San Diego, Calif.) and the branched-chain DNA (bDNA) method from Chiron (Emeryville, Calif.) employ some of the best-known signal amplification procedures available.

Target amplification methods like the polymerase chain reaction (PCR) can produce from [10.sup.6] to [10.sup.8] copies of the target nucleic acids within a few hours.[3] While PCR (Roche Diagnostic Systems, Branchburg, N.J.) is the leading target amplification technology, most of the other target amplification procedures listed in Figure 1 are in clinical trials and should be commercially available within three years.

Probe amplification methods differ from target amplification in that the presence of the appropriate target sequences allows the system to increase the number of specific (e.g., ligated) probes. Several probe amplification methods are being developed or are in clinical trials (Figure 1). The best-known probe amplification system is the ligase chain reaction from Abbott Laboratories (Abbott Park, Ill.).

Both target and probe amplification methods are designed to generate more nucleic acids, thereby allowing the use of less sensitive (and generally less expensive) signal detection methods. in addition, these procedures typically are more complicated than signal amplification methods. However, the commercial availability of high-quality reagents, controls, and test kits has significantly improved their ease of use. On the negative side, the extreme sensitivity of target and probe amplification methods can be a disadvantage, because even minuscule quantities of contaminating nucleic acids can produce false-positive results.

Each target, probe, and signal amplification system has its own strengths and weaknesses. Some methods work better for RNA than for DNA. Some techniques are isothermal, some require thermocyclers, and others provide easily quantifiable results.[1-3] It is imperative that infectious disease labs understand the advantages and disadvantages of each technique so they can choose methods that are appropriate for them.

Molecular technology benefits

Because most routine bacterial cultures are relatively rapid ([is less than]36 hours) and inexpensive (time and materials [is greater than]$15.00), the principal advantage of molecular infectious disease tests will be in the detection of nonculturable agents such as human papilloma virus, human parvovirus, astroviruses, caliciviruses, hepatitis B virus, and hepatitis C virus (Figure 2). These methods are also valuable for detecting fastidious or slow-growing organisms such as Mycobacterium tuberculosis, Mycoplasma pneumoniae, Legionella pneumophiae, and some pathogenic fungi.

Nucleic-acid methods for these agents can provide diagnostic information in a clinically relevant time frame and significantly reduce the number of long-term cultures. Molecular diagnostic methods are especially useful for detecting highly infectious agents and disease organisms that are dangerous to culture, such as Francisella tularensis, Brucella spp., human immunodeficiency virus (HIV), as well as some pathogenic fungi.

Currently PCR is the method of choice for detecting HIV infections in neonates born to HIV-infected mothers. These methods are also useful when trying to determine the HIV status of patients with unusual antibody reactivities (e.g., when a patient is HIV-antibody positive yet only the p24 band is present on the Western blot).

Additionally, DNA methods help clinical laboratorians who are trying to detect viruses present in very low numbers, such as:

* HIV in antibody-negative patients

* Cytomegalovirus (CMV) in transplanted organs

* Herpes simplex virus (HSV) in the cerebrospinal fluid (CSF) of patients with encephalitis.

Figure 3 lists potential applications for molecular diagnostic procedures in the infectious disease laboratory.

Nucleic-acid methodologies also can play an important role when testing extremely small-volume specimens (e.g., intra-ocular fluid or forensic samples). Our lab routinely performs five PCR tests (HSV, CMV, varicella-zoster virus [VZV], Epstein-Barr virus, and human herpes virus 6) on a single, 100-[Mu]l intra-ocular fluid specimen (a specimen volume barely sufficient for a single culture procedure.)

Furthermore, molecular diagnostic methods can detect infectious agents that usually do not grow in culture because they are not viable or are tied up in immune complexes. Molecular methods can also be used to differentiate antigenically similar organisms and detect specific virus genotypes associated with human cancers such as the human papilloma virus. And finally, molecular epidemiologic techniques have been used to identify point sources for hospital- and community-based outbreaks as well as to predict virus virulence.[4,5]

Major challenges

Infectious disease laboratories that offer, or intend to offer, nucleic-acid tests in this decade face three major challenges -- cost control, establishing the clinical relevance of the test, and laboratory organization.

Cost control is a major issue. Labs simply cannot afford to implement diagnostic tests that do not have a documented clinical and financial advantage.

Commercial PCR methods are capital-intensive, requiring dedicated equipment and lab space. The reagent and material costs for commercial PCR assays are two to five times the cost of bacterial culture. Labor costs associated with molecular methods are significant when one considers a trained microbiologist can rule out the presence of hundreds of different microorganisms for $2 to $10 in labor. But labor costs for ruling out the same number of organisms using antibody- or nucleic-acid-based tests are well over $3,000.

Clinical relevance is important in determining the usefulness of any test. Most nucleic-acid tests detect a single organism, so exclusive use of this technology assumes the physician knows exactly which infectious agent may be causing the disease. This assumption cannot be made in most labs. Our laboratory, for example, isolates HSV from 30% of all VZV virus cultures.

Mixed infections also present problems for nucleic-acid tests because mixed microbial infections cannot be detected unless the lab is specifically instructed to look for the appropriate agents. Because nucleic-acid methods are extremely specific, they cannot detect new infectious agents that come into the community. It could be argued diagnosing the hantavirus outbreak in the four-corners region of the Southwestern United States[6] refutes this observation. But the first lab evidence of hantavirus infection came from serological testing.[7] Once the suspect agent was established, PCR and DNA sequencing methods quickly confirmed the diagnosis and determined the infectious agent was a new hantavirus.

Some molecular diagnostic methodologies may be too sensitive for routine clinical use. For instance, a positive PCR test for cytomegalovirus in a blood specimen from a transplant patient may not be sufficient to initiate antiviral therapy because qualitative PCR methods cannot distinguish past infection from active disease. What is the clinical significance of a positive HSV PCR test in an asymptomatic obstetrics patient at the time of delivery? What does a positive PCR test for Pneumocystis carnii mean in an immunocompetent patient with pneumonia?

Several studies have shown PCR methods can detect MTB in some smear-negative patients. To reduce the spread of tuberculosis, our hospital policy calls for isolating smear-positive patients until they are smear-negative on three consecutive days. Hospitals utilizing MTB-PCR methods must decide if smear-negative, PCR-positive patients require isolation and, if so, when they should be released? These hospitals also must reevaluate their criteria for discontinuing isolation procedures because PCR tests may be positive significantly longer than other methods.

Some of these methods will have a significant impact on clinical practice paradigms. The lab can create a significant amount of goodwill by anticipating these problems, working with hospital decision makers, and offering to call other hospitals who perform the test to determine how these problems are handled within their institutions. By participating in the problem-solving process, the lab becomes part of the solution -- not the problem.

Laboratory organization can be impacted significantly by the implementation of molecular diagnostic procedures. Turf wars associated with molecular diagnostic procedures are a reality in many departments,[8-10] with increasing tensions between technology-oriented labs that advocate centralized testing and discipline-oriented laboratories such as microbiology and virology.

Decentralized molecular biology testing in infectious disease labs has a variety of advantages. These labs have long-standing experience with infectious agents and the physicians who order tests for them. Infectious disease labs are methodologically diverse and, as such, are better equipped to handle indeterminant test results and coordinated quality control programs. These labs have both the infectious agents and the gold-standard culture methods.

We employ an alternative implementation model at William Beaumont Hospital. Our molecular probe laboratory performs a "basal" level of clinical testing[10] and serves as a core facility for use in assay development and vali dation, technician training, and assay troubleshooting. Once new assays are developed and validated, they are transferred to the appropriate discipline-oriented laboratories.[2,10]

Future prospects

By the year 2000, the clinical laboratory can expect to see a variety of sensitive and specific nucleic-acid assays for infectious agents. However, vigorous cost-containment programs also are expected to continue into the next century. Test cost will therefore continue to be a major issue. Molecular diagnostic methods must be cost-effective or they will not be widely used.

Diagnostic companies also must reevaluate their current policy of providing proprietary transport media for each test. Manufacturers must begin a cooperative venture to offer a universal transport medium for culture, enzyme immunoassay, and nucleic-acid testing. Our reference laboratory currently provides slides and four different transport systems for the detection of Chlamydia trachomatis. Many diagnostic and reference laboratories will not be able to afford this extravagance in the future.

Although molecular diagnostic methods will have an impact on infectious disease testing, it is unlikely they will replace routine cultures except in specialized situations. Therefore, the demand for trained clinical microbiologists and virologists is expected to continue. To remain competitive in the next century, however, clinical microbiologists and virologists must acquire molecular diagnostic skills or they may relinquish control of molecular infectious disease testing to laboratories that have little experience in handling infectious agents. All in all, the future of infectious disease testing is bright. Molecular diagnostic methods will provide a host of new tools for detecting these agents in clinical specimens.

The final article of this series will explore molecular diagnoses of genetic disease.

References

[1.] Wiedbrauk DL. Molecular methods for virus detection. Lab Med. 1992; 23: 737-742.

[2.] Wiedbrauk DL. Nucleic-acid detection methods. in: Wiedbrauk DL, Farkas DH, eds. Molecular Methods for Virus Detection. San Diego, Calif: Academic Press Inc.; 1995: 1-24.

[3.] Wiedbrauk DL, Farkas DH, eds. Molecular Methods for Virus Detection. San Diego, Calif: Academic Press Inc.; 1995: 131-372.

[4.] Omata M, Ehata T, Yokosuka O, Hosoda K, Ohto M. Mutations in the precore region of hepatitis B virus DNA in patients with fulminant and severe hepatitis. N Engl J Med. 199 1; 324: 699-704.

[5.] Liang TJ, Hasegawa K, Rimon N, Wands JR, Ben-Porath E. A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. N Engl J Med. 199 1; 324: 1705-1709.

[6.] Centers for Disease Control. Outbreak of acute illness -- Southwestern United States. MMWR. 1993; 42; 421-423.

[7.] Le Guenno B. Identifying hantavirus associated with acute respiratory illness: A PCR victory? Lancet. 1993; 342: 1438-1439.

[8.] Diamandis E. The role of clinical chemistry in molecular diagnostics. Clin Chem News. 1993; 19: 4.

[9.] Farkas DH. Molecular diagnostic testing in 1994 and a glimpse at the future. Clin Chem News. 1994; 20: 4.

[10.] Farkas DH. Launching a DNA lab. MLO. 1995; 27: 42-47.

Objectives for this article:

1. List different types of amplification procedures. 2. Compare and contrast amplification procedures. 3. List advantages and disadvantages of amplification procedures. 4. Describe potential applications for molecular testing in the infectious disease lab. 5. Discuss challenges lab managers are confronted with regarding nucleic-acid testing procedures.

Figure 1

Nucleic acid

methodologies to detect

infectious agents

Standard methods Southern blot Slot/dot blot

Target amplification Ligase-activated transcription Nucleic-acid-based sequence amplification Polymerase chain reaction Strand displacement amplification Transcription-mediated amplification

Probe amplification Cycling probe technology Ligase chain reaction Q-beta replicase 

Signal amplification Branched chain DNA detection Chemiluminescence Hybrid (RNA:DNA) detection assay Hybridization protection assay

Figure 2

Leading uses for

nucleic-acid-based tests

Nonculturable agents Fastidious, slow-growing organisms Highly infectious agents that are dangerous to culture In situ detection of infectious agents Agents present in low numbers Organisms present in small-volume specimens Differentiation of antigenetically similar agents Antiviral drug susceptibility testing Non-viable organisms Molecular epidemiology Culture confirmation

Figure 3

Potential applications for molecular

infectious disease procedures

Bacterial and parasites Cat scratch agents Chlamydia pneumoniae Brucella spp. Ehrlichia spp. Francisella tularensis Neisseria gonorrhoeae Bordetella pertussis Helicobacter pylori Legionella pneumophila Mycobacterium tuberculosis Mycobacterium avium intracellulare Mycoplasma pneumoniae Toxoplasma gondii (CSF, amniotic fluid)

Fungi Coccidioides immitis Histoplasma capsulatum Blastomyces dermatiditis

Viruses Astrovirus Enterovirus (CSF) Herpes simplex virus (CSF) Human immunodeficiency virus Human parvovirus Norwalk viruses Rubella virus (amniotic fluid) Varicella-zoster virus (CSF)