Real-time nucleic acid amplification in clinical microbiology

Vol. 26, No. 2 January 15,2004

Real-Time Nucleic Acid Amplification in Clinical Microbiology ii4atthew J. Bankowski, Ph.D., and Steven M. Anderson, Ph.D., ViroMed Laboratories (LubCorp), Minnetonka, MN

Abstract The use of molecular methods in clinical microbiology has increased exponentially over the past two decades. The main reason

for this influx of molecular-based methods has been the continued development and improvement of PCR and the introduction of other nucleic acid amplification formats. The exquisite sensitivity of such methods has surpassed even the traditional “gold standards” for diagnostic testing. However, it has also brought an additional awareness of sample contamination containment to laboratories. The implementation of these methods suffered initially because of longer turnaround time than routine methods, even though they provided a sensitivity and specificity not achievable by classic microbiological methods. The recent development of real-time nucleic acid amplification methods has elevated clinical molecular nucleic acid amplification testing to a new level. Assay turnaround time can now be as short as 1 to 2 h because of the simultaneous product amplification and detection step. Additionally, the closed system has essentially reduced the risk of contamination to negligible levels. This article provides a brief review of the principles of real-time PCR and also provides a discussion of various clinical applications of real-time nucleic acid amplification technology.

Introduction During the more than 20-year history

of PCR, the technology has become an important tool for the clinical laboratory, with many applications in the areas of infectious diseases, genetics, oncology, and forensics. In the past decade, the development of real-time PCR technologies has provided significant improve- ments for these applications, including the ability to quantify specific target nucleic acids. The basic principles of real-time PCR have been adequately covered in many reviews. In this article, we will review briefly the features and limitations of the technology, discuss qualitative and quantitative detection, and provide examples of the use of real-time PCR in infectious-disease testing (l-3).

Real-time PCR assays offer many

Mailing address: Matthew J. Bankowski, Ph.D., ViroMed Laboratories, 6101 Blue Circle Drive, Minnetonka, MN 55343. Tel.: 9.52-563-4014. Fax: 952-939-4215. E-mail: [email protected].

advantages over traditional PCR methods. They are much quicker to perform, with the enhancements in speed attributed to reduced cycle time, reduced amplicon size, and the elimination of an additional step(s) needed for product detection. This feature provides a benefit to the laboratory because of the decrease in hands-on time needed to perform testing. Further enhancements in this area have been achieved by linking real-time PCR technology to automated nucleic acid extractions, resulting in assays that may take as little as 2 h to perform (4). The fluorescence detection methods used also provide increased sensitivity and a broad dynamic range in quantitative assays. The use of a closed system for amplification and detection also minimizes the potential for amplicon carryover contamination. Limitations of this technology include multiplexing capabilities and the inability to determine amplicon size. The latter is potentially critical for determining assay specificity. This potential problem can be overcome by melting-curve analysis,

which can distinguish specific product from unspecific amplicon, including primer:dimer. Table 1 provides a summary of the advantages and limitations of real-time PCR technology.

A variety of methods allow for the detection of amplification products during PCR, as summarized in Table 2. Both direct and indirect detection formats can be used in either qualitative or quantitative assays, with better sensitivity than traditional methods. Clinical microbiology applications for qualitative real-time assays include detection of bacterial, viral, parasitic, and fungal targets; analysis of genes that confer drug resistance and genetic determi- nants of pathogenesis; and detection of potential bioterrorism agents (5,6).

Real-time PCR assays have revolu-

In This Issue

Real-Time Nucleic Acid Amplification in Clinical Microbiology . . . . . . . . . . . . . . . . . . 9

Clinical Microbiology Newsletter 26:2,2004 0 2004 Elsevier 0196.4399100 (see frontmatter) 9

tionized the technique for quantitation of nucleic acid targets. Real-time assays are characterized by the point in time that amplified product is first detected, rather than by determination of the final quantity of PCR product after a given number of assay cycles. With real-time PCR assays, the higher the copy number of the input target nucleic acid, the earlier a detectable fluorescent signal is produced. Fig. 1 provides an example of an amplification plot in which fluorescence is plotted against cycle number. In such plots, an increase in fluorescence above the baseline is an indication of the presence of amplified product. The parameter C, (threshold cycle) is defined as the fractional cycle number in which the fluorescence signal surpasses the fixed threshold level. The number of cycles needed to reach the fluorescence threshold is inversely correlated to the number of target nucleic acid molecules in the original sample. This can be seen in Fig. 1, where differences in CT are seen for Epstein-Barr virus (EBV) genomic concentrations ranging from 5 to 5,000 copies. A standard curve can be devel- oped by plotting the log of the initial target copy number for the set of standards versus C,. An example for a series of EBV copy number controls is shown in Fig. 2. Interpolation from that standard curve allows the quantitative analysis of specimens. Because C, is determined during the exponential phase of the amplification reaction, it is much more reliable than end-point analysis, which is performed in traditional PCR assays. Real-time assays have a quantitation range that is at least 5 orders of magnitude greater than standard quantitative PCR assays. Quantitative assays are routinely used for hepatitis C virus, EBV, and cyto- megalovirus viral load determinations. This value often represents an impor-

Table 1. Advantages and limitations of real-time PCR assays

Advantages Limitations

Analytical sensitivity Limited multiplexing capabilities

Broad dynamic range Cannot monitor amplicon size

Better precision

No post-PCR steps

Increased throughput

Some systems not compatible with fluorogenic chemistry

Quantitation without co-amplification of internal standards

I

I @.I- 8.P

Figure 1. Cycle number and crossing-point analysis for Epstein-Barr virus real-time PCR using the LightCycler technology and SYBR Green I dye for product detection (left to right: 5,000, 500,50, and 5 copies).

tant laboratory test result used in prognosis, disease stratification, and therapeutic decision making (7-14).

Clinical Applications in Infectious Disease Testing Background

The possibilities for applying real- time nucleic acid amplification (RTM- NAA) testing in the areas of clinical microbiology and infectious diseases are plentiful (Table 3). The crucial question is how appropriate it is to use a real-time nucleic acid test as a supple- mental, or even replacement, tech-

nology in today’s health care climate. The obvious uses are for agents that are difficult to culture, biohazardous, or too low in numbers to be detected by other m&hods. In addition, detection of antimicrobial-resistance, toxin, or other genes contributing to pathogenesis are important uses of real-time testing in clinical microbiology. Real-time formats also offer the previously mentioned benefits and the ability to accu- rately and precisely quantify a nucleic acid target (e.g., “quantitation,” “viral load,” and “bacterial load”). Quantita- tion is useful and often necessary for

NOTE: No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. No suggested test or procedure should be carried out unless, in the reader’s judgment, its risk is justified. Because of rapid advances in the medical sciences, we recommend that the independent verification of diagnoses and drug doses should be made. Discussions, views and recommendations as to medical procedures, choice of drugs and drug dosages are the responsibility of the authors.

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monitoring therapy, prognosis, and gene expression and even for concerns about possible development of antimicrobial resistance. Since the late 19XOs, both the types of methodology and the number of applications of RTM-NAA have continued to increase exponentially (15- 17). The real-time PCR formats (e.g., LightCycler [Roche Diagnostics Corp., Indianapolis, IN] and TaqMan [Applied Biosystems, Foster City, CA]) are the most widely described in the literature, and therefore, selected examples of these will be discussed (2).

Bordetella pertussis and Bordetella parapertussis

Clinical sign$cance Bordetella pertussis and Bordetella

parapertussis are the causative agents of whooping cough and whooping cough-like syndrome, respectively (18). The disease is most severe in infants and may have an atypical presentation in children and adults. In addition, par- tially immunized individuals may be infected, which may be missed in the clinical workup. This contagious and treatable disease may have severe and life-threatening complications, such as pneumonia. Laboratory support for the diagnosis relies on direct detection by the direct fluorescent antibody test (DFA), culture, and PCR. Both DFA and culture sensitivities are low (40 to 50% in some cases), as is the specificity of DFA. The use of PCR has dramati- cally increased both the sensitivity and the specificity of detection. Many studies that support the use of molecular amplification over conventional methods for laboratory diagnosis of Bordetella infection have been published (19-36).

Qualitative RTM-NM Many laboratories, including our

own, use a qualitative approach with the LightCycler technology. Either SYBR Green I dye or specific, labeled probes for the direct detection of B. pertussis and B. parapertussis can be used for clinical specimens. Fig. 3 shows a melting curve analysis for B. pertussis real-time PCR using the LightCycler technology and SYBR Green I dye. The primer- dimer bands in the curve at the left of the graph are often seen when SYBR Green I dye is used. In contrast, the melting curve analysis for B. paruper- tussis, which uses labeled probes for product detection, does not show these

Table 2. Examples of detection chemistry for real-time PCR assays Example Description Indirect detection of amplicon DNA binding dyes Fluoresce when associated with double-stranded DNA (i.e., SYBR green) and exposed to a suitable light source

Direct detection of amplicon Linear oligoprobes Pair of adjacent oligonucleotides labeled with donor and

acceptor fluorophores; fluorescence resonance energy transfer (FRET) occurs when probes are hybridized to target; FRET probes often used in LightCycler assays.

5’ Nuclease probes Probe has both fluorophore and quencher; during PCR, hybridized probe is digested by 5’ exonuclease activity of Taq polymerase, producing fluorescence; TaqMan probes.

Molecular beacons Fluorophore and quencher at the termini of probe, held in close proximity by hairpin structure in the unhybridized probe; hybridization to complementary sequence opens up the hairpin and produces fluorescence.

Light-up probes Linear molecules; peptide nucleic acid probe; when hybridized to a nucleic acid target the fluorophore becomes strongly fluorescent; does not interfere with PCR.

Log Concenfration

Figure 2. Epstein-Barr virus quantitation curve analysis by real-time PCR using the LightCycler technology.

Figure 3. Melting curve analysis for Bordetella pertussis real-time PCR using the LightCycler technology and SYBR Green I dye for product detection.

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non-specific products (Fig. 4). Both approaches are sensitive, but specificity should always be supported by melting- curve analysis when the SYBR Green I dye approach is used.

There are many reports of the use of PCR and real-time PCR for the direct detection of Bordetella spp. in clinical specimens. Both the choice of specimen

and method of nucleic acid extraction are important for optimal sensitivity. Nasopharyngeal swabs appear to offer consistently optimal recovery, even with the use of standard phenol-chloro- form extraction. Both LightCycler and Taqman technologies demonstrate low limits of detection over routine microbiological methods (1 to 50 copies per

reaction or 0.1 to 10 CFU per reaction, depending on the Bordetella sp.). The sensitivity of either culture or DFA is usually considerably less than that of PCR (i.e., culture 40 to 50% vs. PCR 80 to 98%). In addition, the turnaround time using real-time PCR can be as short as 45 min following nucleic acid extraction compared to 1 to 2 days with

Table 3. Lists of selected microbes and gene targets for which real-time nucleic acid amplification is used

Parvovims B 19 Streptococcus pyogenes

Respiratory Salmonella syncytial SPP. TT Treponema spp.

Varicella-zoster 16s ribosome “SA-CF, Staphylococcus aureus strains isolated from cystic fibrosis patients.

Gene target Pathogenesis and toxins Enterohemolysin

Bioterrorism agents Bacillus anthracis

Helicobacter pylori

Human herpesvirus E6/E7

Hantavims

Dengue virus

Intimin Ebola virus

SA-CF” Lassa fever virus

Shigalike toxin

Marburg virus

Rift Valley fever virus

Smallpox virus

Yellow fever virus

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conventional PCR and product detection (20,21,23-25,28,32,35-38).

Herpes Simplex Virus (HSV) Clinical significance

Herpesvirus infections account for significant morbidity in all age groups. Mortality is especially high among the immunocompromised and infants exposed to the virus early in life. Fac- tors contributing to and sustaining infection are the relatively high prevalence of HSV in the general population, its latency, and the ability of the virus to reactivate. The range of HSV disease is extensive and includes dermal, ocular, and genital lesions and systemic exten- sion with central nervous system (CNS) involvement (18).

Figure 4. Melting curve analysis for Bordetella parapertussis real-time PCR using the LightCycler technology and labeled probes for product detection.

Antiviral drugs are available for effective pre-emptive and therapeutic management of HSV infection. Early diagnosis and antiviral administration in serious infection, such as encephalitis, are often critical to reduce mortality in these patients; a 70% mortality rate is possible in untreated patients. The clinical laboratory provides major support for the diagnosis, and accuracy and the shortest possible turnaround time are of utmost importance. Nucleic acid amplification technologies have become the standard of care for patients with CNS disease. Real-time PCR provides highly accurate and rapid state-of-the- art testing. A real-time PCR test result is usually available in 1 to 2 h of laboratory time, compared with the times for conventional virology techniques, which may be 1 h for DFA, 1 to 2 days for the shell vial technique, 1 to 4 days for culture, or 6 h to 2 days for classic PCR, depending on the method. In addition, culture recovery may be as low as 2 to 5% in cases of HSV encephalitis (39-42).

Figure 5. Melting curve analysis for herpes simplex virus showing the usefulness of RTM- NAA as a genotyping method. The insert is an example of a restriction endonuclease analysis used for HSV-1 and HSV-2 typing.

Qualitative vs. quantitative HSV RT-NAA

quently, this technology is potentially useful as a replacement for classical culture. However, if antiviral studies are necessary, the isolate would be needed for further testing. Progress in this area is shown by the use of RTM- NAA rather than the traditional plaque reduction assay for shortening the time needed to determine acyclovir resistance (44).

Qualitative RT-NAA offers all In a 3-year study of patients with the advantages previously described, CNS disease, Alberle and Puchhammer- including direct detection of the target Stock1 (39) showed the usefulness of in clinical samples. In many cases, a virus quantitation. In their study, which “present” or “absent” answer for the used TaqMan technology, the prevalence microbiological agent is all that is of HSV was 26.6%. Quantitation added needed to support most clinical differ- to effective prognosis and predicted ential diagnoses. The work of Espy et specific disease manifestations caused al. (43) showed that RTM-NAA with by HSV. The most important use of LightCycler technology offered a 22% determining the viral load, however, increase in the detection rate of HSV would be to monitor HSV disease in directly in clinical samples from genital, patients undergoing specific antiviral dermal, and ocular sources. Conse- therapy.

HSV genotyping Many published examples of real-

time PCR assays describe detection and genotyping of HSV in clinical specimens (43,45). One question that has been addressed concerns the clinical usefulness of HSV typing. Kimura and others (46) have reported that HSV type 2 (HSV-2) produces more severe disease in the neonatal population, which influences patient management. They tested serum and cerebrospinal fluid (CSF) and concluded that the prognosis is poorer in patients with dissemi- nated infection who have significantly higher viral loads in serum.

Fig. 5 shows an example of the LightCycler assay for detecting and genotyping HSV in clinical specimens, using melting curve analysis to discri- minate HSV-1 from HSV-2. The insert shows an example of a restriction endonuclease analysis that may be used as a

Clinical Microbiology Newsletter 26:2,2004 0 2004 Elsevier 0196.4399/00 (see frontmatter) 13

confirmatory test for any questionable LightCycler patterns encountered.

Epstein-Barr virus Clinical significance

Epstein-Barr virus has been associated with a variety of disease states, including infectious mononucleosis; malignancies, such as Burkitt’s lymphoma and nasopharyngeal carcinoma; and lymphoproliferative disorders in immunocompromised individuals. EBV usually produces a mild and self-limit- ing infection in children. EBV can per- sist for life, held in check by the host immune system, primarily cytotoxic T lymphocytes. In immunocompromised or immunosuppressed individuals, EBV- infected cells can proliferate, causing B-cell lymphoma or lymphoproliferative disorder. Nucleic acid methods, such as real-time PCR, have become important tools for distinguishing active or reactivated EBV infections from latency in this patient population.

Quantitative determination of EBV load: prognosis and therapeutic decisions

While baseline determinations of EBV loads using real-time PCR have been correlated with the disease state, the most important use of this technology is to monitor viral load to predict disease progression or therapeutic efficacy. Lymphoproliferative disorders in immunocompromised individuals, i.e., AIDS patients and transplant recipients on immunosuppressive therapy, are conditions for which EBV viral-load measurements have the greatest clinical significance. The best example of the use of this technology is to monitor transplant recipients for evidence of posttransplant lymphoproliferative disorder (PTLD). The efficacy of EBV viral-load monitoring has been demon- strated in multiple studies of solid- organ recipients and allogeneic-stem- cell recipients. For these individuals, increases in the viral load above baseline often precede clinical evidence of PTLD, and therefore the assay assists clinicians in making therapeutic decisions. The therapeutic options include reducing immunosuppression, use of anti-CD20 antibodies, adoptive T-cell transfer, and aggressive chemotherapy. In studies involving viral-load measurements, several specimen types have been evaluated, including serum, plasma,

CSF, and white blood cells. The cell- associated compartment provides the best correlative data, while elevated serum levels may be seen only in patients with systemic disease with high viral loads. In these situations, real-time PCR is a useful tool for diagnosing sympto- matic EBV infection and monitoring patient viral loads (10,11,47-56).

Summary The recent development of RTM-

NAA methods has added molecular testing to the clinical microbiology test menu in a significant way. The advantages of shorter turnaround time (1 to 2 h) and a closed system to reduce the risk of contamination to negligible levels have dramatic implications for patient care. The additional ability of RTM-NAA to provide quantitation over a wide dynamic range potentially offers both prognostic and therapeutic value in detecting a wide range of infectious agents (e.g., viral load and bacterial load). Also, the costs of real-time PCR testing should be defrayed by a reduction or replacement of conventional test methods. This testing should improve the eventual patient outcome and add to the efficacy of patient care.

Acknowledgements We are grateful to the Clinica

Research and Development Department staff, especially Susan W. Belzer and Bryndon Lembke, and the Clinical Molecular Department staff for their excellent technical performance of the real-time PCR work mentioned here.

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Real-time nucleic acid amplification in clinical microbiology