Clinical Applications of Molecular Biology for Infectious Diseases

Abstract

Molecular biological methods for the detection and characterisation of microorganisms have revolutionised diagnostic microbiology and are now part of routine specimen processing. Polymerase chain reaction (PCR) techniques have led the way into this new era by allowing rapid detection of microorganisms that were previously difficult or impossible to detect by traditional microbiological methods. In addition to detection of fastidious microorganisms, more rapid detection by molecular methods is now possible for pathogens of public health importance. Molecular methods have now progressed beyond identification to detect antimicrobial resistance genes and provide public health information such as strain characterisation by genotyping. Treatment of certain microorganisms has been improved by viral resistance detection and viral load testing for the monitoring of responses to antiviral therapies. With the advent of multiplex PCR, real-time PCR and improvements in efficiency through automation, the costs of molecular methods are decreasing such that the role of molecular methods will further increase. This review will focus on the clinical utility of molecular methods performed in the clinical microbiology laboratory, illustrated with the many examples of how they have changed laboratory diagnosis and therefore the management of infectious diseases.

Introduction

The advent of nucleic acid amplification and detection has resulted in a change from conventional laboratory methods that rely on phenotypic expression of antigens or biochemical products, to molecular methods for the rapid identification of a number of infectious agents. Molecular methods have become increasingly incorporated into the clinical microbiology laboratory, particularly for the detection and characterisation of virus infections and for the diagnosis of diseases due to fastidious bacteria. The advantages of rapid turn-around time and high sensitivity and specificity are appealing but must be matched by rigorous validation and quality control.

Molecular detection has mostly come to the clinical microbiology laboratory in the form of PCR technology, initially involving single round or nested procedures with detection by gel electrophoresis. However, with the introduction of automation for the various stages of DNA or RNA extraction, amplification and product detection together with real-time PCR, molecular laboratories will continue to become more efficient and cost-effective. Microarray technology such as the DNA chip will likely further increase the utility of molecular detection in the clinical microbiology laboratory.

This paper will provide an overview of the clinical applications of molecular methods for infectious diseases, these have been summarised in Tables 1 and 2. Applications include the discipline of virology where it has been applied to resistance testing, genotyping and viral load quantification in addition to routine viral detection. In the area of bacteriology molecular methods have been applied to resistance testing, the detection of infection due to fastidious bacteria, the more rapid detection of serious bacterial infections compared to conventional methods and the detection of bacterial infection after antibiotics have been administered. Advances into the areas of parasitology and mycology have also been made such as more rapid diagnosis of fungal infection in neutropenic patients. Other applications such as the detection of biosecurity agents, applications to epidemiology and infection control together with the potential pitfalls with molecular methods are also discussed.

Table 1.

Examples of molecular methods in use for the diagnosis of infectious diseases^*

Discipline	Examples
Virology	Herpes simplex virus, cytomegalovirus, Epstein-Barr virus, varicella zoster virus, human herpes virus type 6, 7, 8 respiratory viruses (such as influenza virus, respiratory syncytial virus, parain uenza virus, adenovirus, rhinovirus) #SARS-CoV, avian in uenza virus @HIV, hepatitis B, hepatitis C human papilloma virus, enterovirus orf virus, mulloscum contagiosum rotavirus, norovirus, enteric adenoviruses
Bacteriology	C. trachomatis, N. gonorrhoeae, B. pertussis, M. tuberculosis, nontuberculous mycobacteria, T. whipplei, B. henselae, genital mycoplasmata, C. burnettii M. pneumoniae, C. pneumoniae, Legionella spp., N. meningitidis, S. pneumoniae
Parasitology	Plasmodium spp., T. gondii
Mycology	P. jiroveci, Aspergillus spp.

^*the complement of molecular diagnostic tests available varies between laboratories

^#severe acute respiratory syndrome-coronavirus

^@human immunodeficiency virus

Table 2.

Examples of uses for molecular methods other than microorganism identification in the clinical microbiology laboratory^*

Test	Examples
Viral load monitoring	Cytomegalovirus, Epstein-Barr virus hepatitis B, hepatitis C, @HIV
Viral genotyping	@HIV, hepatitis B, hepatitis C, human papillomavirus
Bacterial resistance detection	#MRSA, ^VRE, ♦ESBL containing E. coli, K. pneumoniae M. tuberculosis
Bacterial genotyping	M. tuberculosis, N. meningitidis
Broad-range PCR	Infective endocarditis, bacterial meningitis

^*the complement of molecular tests available varies between laboratories

^#methicillin resistant S. aureus

^{^}vancomycin resistant enterococci

^♦extended spectrum beta-lactamase

^@human immunodeficiency virus

Virology

The diagnosis of viral infections has been hampered for many years due to the cost, laboratory time and skilled personnel required for the cell culture systems used, together with the generally low sensitivity and slow growth of many viruses in artificial media. Serology is often unhelpful in the early stages of infection, specific antisera for the serology tests can be difficult to obtain, and the clinical detection of antibodies is relatively insensitive for a number of viruses. PCR technology has therefore improved the detection of a number of these viruses.

Herpes simplex virus (HSV) encephalitis is a serious infection but diagnosis previously required brain biopsy in certain cases due to the low sensitivity of cerebrospinal fluid (CSF) culture and serology.¹ PCR now allows the detection of HSV DNA from CSF with 95% sensitivity² thus avoiding invasive brain biopsy. Viral meningitis, commonly caused by either enteroviruses or HSV, is more reliably detected by PCR when compared to culture³ and in a shorter time (one versus up to five days). HSV PCR can be multiplexed with other pathogens responsible for meningitis.⁴

The detection of blood borne virus infection is also improved by both PCR and non-PCR molecular methods. Active hepatitis C virus (HCV) infections are diagnosed by the presence of HCV RNA since the detection of antibody to HCV cannot distinguish between past and present infection. In terms of infectiousness only those with detectable HCV RNA have a significant risk of transmitting HCV by transfusion, organ transplantation, needle-stick injury or vertically to the child.⁵ Although infection with the human immunodeficiency virus (HIV) is routinely diagnosed by serology, early HIV infection can be detected by HIV pro-viral DNA detection before HIV antibodies are confirmed by Western Blot serology.⁶ Vertical transmission of HIV infection is also detected in the infant using HIV pro-viral DNA detection.⁷ The Australian Red Cross Blood Service screens pooled samples from all donations for HIV and HCV using the Chiron Procleix HIV-1/HCV transcription mediated amplification assay, thus reducing the potentially infectious window period from 22 and 66 days to 9 and 7 days respectively.⁸

Intrauterine infection of the foetus with cytomegalovirus (CMV),⁹ rubella,¹⁰ and varicella zoster virus¹¹ can be detected by PCR testing of amniocentesis fluid. Genital ulceration due to HSV, usually due to HSV type 2 infection, is now routinely detected by PCR in many clinical microbiology laboratories due to its increased sensitivity over viral culture.

Molecular detection of respiratory viral pathogens from both upper respiratory specimens such as nasopharyngeal aspirates or throat swabs and lower respiratory specimens such as sputum or bronchoalveolar lavage fluid is cost-effective due to the prevention of hospitalisation, decreasing unnecessary testing and procedures, directing specific therapy, and reducing unnecessary antibiotic use.¹² Large multiplex or tandem PCR assays testing for all the common respiratory viruses along with fastidious bacterial causes of pneumonia are now feasible providing a thorough yet cost-effective alternative to conventional detection methods. Uncommon yet significant respiratory viruses such as severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) and influenza A/H5N1 (avian influenza) virus can also be incorporated into these assays thus acting as an in-built early detection system.

During the SARS epidemic due to the SARS-CoV, PCR testing of respiratory specimens for other respiratory viruses was crucial to exclude a number of suspected cases which fulfilled the case definition for SARS. PCR detection was most helpful due to the ability to rapidly screen for many respiratory viruses. Subsequently a specific SARS CoV PCR has been developed for the early detection of SARS-CoV infection with a sensitivity of 50–87% early in the disease.¹³ Serology for SARS-CoV is up to 100% sensitive but of limited diagnostic value early in the disease when the risk of transmission is greatest.¹⁴

The recent avian influenza (H5N1) outbreaks in South East Asia and beyond have also illustrated the need for rapid viral diagnosis. Molecular detection methods were developed following the 1997 Hong Kong outbreak¹⁵ and have the advantage of being rapid and able to be performed in many clinical microbiology laboratories. Specific serology needs live virus for the microneutralisation assay which is currently classed as a Biosafety Level 4 organism in Australia. Likewise direct immunofluorescence detection requires influenza type A/H5-specific monoclonal antibodies.¹⁶

Viruses cause more infectious diarrhoea worldwide than bacteria and other pathogens. The diagnosis of viral diarrhoeal disease has improved with the development of PCR detection. The method of choice for microbiological diagnosis of rotavirus from stool samples is PCR. Norovirus, a calicivirus formerly known as Norwalk virus and responsible for large outbreaks both in the community and health care facilities, can be diagnosed by electron microscopy, enzyme immunoassay and PCR but PCR is the most sensitive and rapid method. PCR is also the most sensitive method for the diagnosis of astroviruses and enteric adenoviruses (serotypes 40 and 41).¹⁷

Treatment Monitoring

Monitoring viral DNA or RNA loads has become the standard of care for several chronic viral infections. Measurement of viral load is performed either by competitive PCR systems, branched chain DNA signal amplification or more recently real-time PCR.

HIV viral load testing is an integral component of the management of HIV infection. It is the major tool used to monitor the success of antiretroviral therapy and to detect the emergence of viral resistance, evidenced by a rise in the viral load despite ongoing therapy. HIV viral loads also predict progression of disease, and give prognostic information.¹⁸ Commercial tests are available and more recently ultrasensitive tests such as the Cobas Amplicor HIV- 1 Monitor Ultrasensitive Test have been released reducing the lower limit of viral detection to 50 copies/mL.¹⁹

Viral load testing is also used for the assessment and monitoring of responses to therapy in chronic HCV and hepatitis B virus (HBV) infection. HCV RNA viral loads are assessed in patients with genotype 1 HCV infections when monitoring for responses to combination interferon-alpha and ribavirin therapy. Patients who remain negative for HCV RNA 6 months after completing combination therapy for HCV infection almost always remain free of the virus in the longer term and have achieved a sustained virological response. If the HCV genotype 1 RNA is undetectable after 12 weeks of therapy there is a 75% chance of a sustained virological response. However, even if the HCV RNA remains detectable, a 33% chance of a sustained virological response remains if a 100-fold decrease in the viral load has occurred after 12 weeks of therapy.²⁰ In HBV carriers with active liver disease HBV DNA loads are measured not only to assess patients regarding the need for either interferon-alpha or lamivudine (a DNA polymerase inhibitor) antiviral therapy but also to monitor their effectiveness. An increase in HBV viral load is also used as a marker of the emergence of lamivudine resistant viral mutants.²¹

Cytomegalovirus infection is a serious infection in bone marrow and solid organ transplant recipients together with HIV-infected patients but detection has been limited by the poor sensitivity of traditional culture methods.²² Viral load testing by quantitative PCR is now the accepted standard for monitoring the emergence of CMV infection during immunosuppression and allows pre-emptive therapy prior to the emergence of clinical disease with high sensitivity when compared to culture.²³

Viral Genotyping and Resistance Testing

HIV genotyping for the detection of drug resistance is the standard of care to guide antiretroviral therapy and complements viral load assessment. Several databases are available such as the Stanford reverse transcriptase and protease database (http://hivdb.stanford.edu) where sequences can be checked for resistance mutations.

Genotyping is also critical to the management of chronic viral hepatitis. There are six HCV genotypes geographically distributed throughout the world. The genotype is the single strongest determinant for success with combination therapy and all patients wishing to undergo therapy firstly undergo HCV genotyping. Those with chronic genotype 2 or 3 HCV infections receive 6 months of therapy with a 76% chance of success compared to a 56% chance of success for those with genotype 1 HCV infection receiving 12 months of therapy.²⁰

Active chronic infections with HBV treated with lamivudine require surveillance for the emergence of lamivudine resistant viral mutants. During lamivudine monotherapy point mutations at the active site of the polymerase gene (YMDD variants) occur with a frequency of 14–32% after one year in phase III studies, and in 42% and 52% of Asian patients after two and three years of therapy respectively.²⁴ The emergence of lamivudine resistance is detected by a rise in HBV viral load and confirmed by sequencing of the active site of the DNA polymerase gene.²⁵

The presence of HBV pre-core mutants may cause active liver disease despite the absence of HBeAg, the common marker for active hepatitis in hepatitis B infection. This may be due to either a premature stop codon point mutation in the precore gene (G1896A) or a mutation in the basal core promoter region down-regulating HBeAg production, both of which can only be reliably detected genotypically.²⁶

Human papilloma virus (HPV) is now accepted as the cause of almost all cervical cancers and HPV genotypes are now classified as either low or high-risk for the causation of these cancers. Screening for pre-neoplastic cytological changes has traditionally been performed by the Papanicaou (Pap) screen, but the detection of high-risk HPV infection is a useful adjunct. Since HPV cannot be routinely cultured in vitro, testing for the 15 high-risk genotypes of HPV requires molecular methods. Detection can be achieved by signal amplification, such as the Digene Hybrid Capture 2 assay which is the only diagnostic in vitro test approved by the Federal Drug Administration (FDA). This assay contains specific RNA probes directed toward the high-risk genotype DNA sequences which are detected by an antibody directed against the DNA-RNA hybrids formed.²⁷ Detection of the high-risk genotypes can also be achieved by target amplification such as multiplex PCR, but commercial assays are not yet available. Detection of these genotypes by molecular analysis can help in the assessment of equivocal Pap smears to define those women at risk of developing cervical cancer.²⁸ Alternatively a normal Pap smear with a negative genetic test for the high-risk genotypes may indicate a longer period of time before re-testing.²⁹ With the advent of a genotype 16 HPV vaccine the role of this testing is likely to assume more importance.

Molecular methods have therefore gone beyond simple detection of viral infections to become an integral component of the management of blood borne virus and other viral infections.

Bacteriology

Fastidious Bacteria

Together with virology, the diagnosis of infections due to fastidious bacteria has benefited greatly from molecular detection. Many of these fastidious bacteria have public health implications such as Mycobacterium tuberculosis, Chlamydia trachomatis, Neisseria gonorrheae and Bordetella pertussis. Non-culture-based molecular testing has the advantage of avoiding the delays of days to weeks for conventional culture to allow early recognition and treatment as a public health imperative. Commercial assays are available for M. tuberculosis and Mycobacterium avium complex, C. trachomatosis, and N. gonorrhoeae. Several nucleic acid detection technologies are in use including PCR, transcription based amplification, ligase chain reaction, strand displacement amplification and the Qβ replicase system.³⁰

The introduction of molecular detection for the fastidious sexually transmitted bacteria has led to a large increase in the proportion of laboratory confirmed cases due to its increased sensitivity allowing more effective contact tracing. In the management of sexual health traditional screening methods require speculum examination in women and urethral swabs in men. These require special equipment and cause embarrassment and discomfort, thus reducing compliance. Molecular detection is useful since noninvasive specimens unsuitable for traditional culture, such as initial stream urine and self-collected vaginal swabs can be used. These are more convenient and acceptable increasing the compliance with testing. Although molecular testing for C. trachomatis and N. gonorrhoeae does not allow monitoring of antibiotic resistance or detect other sexually transmitted diseases, urine testing has shown equivalent sensitivity and specificity to invasive specimens for detection of C. trachomatis in men and women, and for detection of N. gonorrhoeae in men when compared to urethral swabs. In women the sensitivity and specificity of the PCR assay for N. gonorrhoeae was lower for urine compared to cervical samples, however self-collected vaginal swabs may help in this regard. The PCR assay for C. trachomatis has equal sensitivity for vaginal and cervical swabs and a transcription mediated amplification assay has been approved by the U.S. FDA for testing C. trachomatis and N. gonorrhoeae from vaginal specimens.³¹ In remote areas, molecular methods have the advantage of being performed on dry swabs with little degradation of the DNA during transit compared to the difficulties of transporting samples in specialised transport medium to preserve viability. In addition, molecular methods can test for multiple genital pathogens such as C. trachomatis, N. gonorrhoeae, the Donovanosis agent and the genital mycoplasmata from the same swab.

Mycobacteriology has been aided by the introduction of molecular methods. However, it is important to note that molecular detection of M. tuberculosis is one of the few examples where conventional culture remains more sensitive. This is possibly due to the difficulty in releasing the DNA from the bacterial cells during the extraction process. Despite this limitation, molecular detection of M. tuberculosis has a definite role as it allows confirmation of acid-fast bacilli seen on microscopy with up to 98% sensitivity in pulmonary tuberculosis within a day compared to two weeks or more by culture. Specimens that are smear-negative have a much lower chance of molecular confirmation, with reported sensitivities as low as 40%.³² In addition to direct detection from clinical specimens, molecular methods can confirm a positive culture within a day compared to approximately four weeks using phenotypic methods. This has shortened the time for laboratory confirmation of suspected tuberculosis even for smear-negative but culture-positive cases.

Mycobacteriology has also advanced through the use of molecular methods for the speciation of the many nontuberculous mycobacterial species. Phenotypic methods are slow and the limited number of tests available is inadequate to differentiate between the large number of species. Genetic sequencing of the 16S rRNA gene has simplified this process in many laboratories.³³ However, some species such as the rapid grower group cannot be distinguished by 16S rRNA gene sequencing alone, and require a multi-gene approach incorporating the hsp65, rpoB and sod genes.³⁴

Due to its significantly enhanced sensitivity, PCR has replaced direct fluorescent-antibody and culture as the “gold standard” method for detection of B. pertussis early in the disease process.³⁵ In one pertussis school outbreak using nasopharyngeal aspirates, PCR detected 48% of clinical cases compared to 5% confirmed by culture.³⁶ For this pathogen of public health significance, a combination of PCR detection early in disease and serology for suspected cases late in the disease process is used for maximal case ascertainment. Other fastidious respiratory pathogens that can be rapidly diagnosed by molecular means include Legionella spp., Mycoplasma pneumoniae and Chlamydia pneumoniae.

Some bacteria can only be detected by molecular means as culture is either extremely difficult or impossible for the routine microbiology laboratory, or represents a significant occupational risk to the laboratory personnel. Whipple’s disease is a rare but ultimately lethal infection due to Tropheryma whipplei which could previously only be diagnosed by characteristic histopathology and electron microscopy, often from post-mortem material. PCR now allows diagnosis of neuro-Whipple’s disease and endocarditis by the detection of T. whipplei from noninvasive specimens.³⁷ Other examples where molecular diagnosis can help in the diagnosis of difficult or uncultivable bacteria include cat scratch disease due to Bartonella henselae, Q fever due to Coxiella burnetii, and male urethritis due to Mycoplasma genitalium. A more detailed discussion on the molecular methods for the diagnosis of fastidious bacteria can be found in Fenollar and Raoult.³⁸

Rapid Bacterial Diagnosis

Meningococcal disease can have devastating consequences and requires early diagnosis for correct antibiotic therapy as well as early provision of chemoprophylaxis for close contacts. PCR methods can now provide same-day detection from sterile site specimens with a superior speed and sensitivity to culture, and when combined with culture and other laboratory methods, PCR maximises the laboratory confirmation of clinically suspected cases.³⁹ Genoserogrouping for serogroup B and C N. meningitidis strains helps with decisions regarding vaccination and can be combined with N. meningitidis detection. In our laboratory as well as others,⁴⁰ combined N. meningitidis detection and genoserogrouping is routinely performed on clinical specimens from suspected cases.

Rapid detection of the other common bacterial causes of meningitis has also been developed. N. meningitidis with Streptococcus pneumoniae and Haemophilus influenzae type B account for 90% of cases of bacterial meningitis and multiplex PCR methods have been developed for their detection.⁴¹

Antibiotic Resistance

Following on from the success of molecular methods for the detection of several bacterial infections, genotypic detection of antibiotic resistance is appealing due to the avoidance of problems such as variable phenotypic resistance expression. Applying rapid and reliable genotypic detection to bacteria with infection control implications such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) is of great potential benefit. The discrimination of MRSA from other S. aureus is confirmed by the detection of the mecA gene responsible for this resistance. The detection of the mecA gene can be multiplexed with the nuc gene to allow rapid molecular detection of S. aureus and confirmation of MRSA from positive blood culture bottles.⁴² This is important to provide information regarding antibiotic selection as early as possible since mortality rates are higher with MRSA infection compared to methicillin-sensitive S. aureus.⁴³ Likewise, detection of VRE is more sensitive and rapid using DNA-based amplification techniques.⁴⁴ Commercial kits are now becoming available for MRSA and VRE detection using real-time PCR instrumentation which will further improve the speed of detection.

Extended spectrum β-lactamases (ESBL) are found in Escherichia coli and Klebsiella pneumoniae and are readily transmitted on plasmids and transposons. ESBL-containing bacteria can spread rapidly in health care facilities to cause wound infections, urinary tract infections and septicaemia. Their detection requires special laboratory tests since routine antibiotic susceptibility testing may not detect strains carrying the resistance gene. Although most clinical microbiology laboratories currently use phenotypic methods to detect ESBL, molecular detection of these point mutations at the active site of the β-lactamase gene can confirm the ESBL and allow the characterisation of the type of ESBL for monitoring of its spread through health care facilities around the world.

Multi-drug resistant tuberculosis (defined as the presence of both rifampicin and isoniazid resistance) is a serious problem in many parts of the world such as Eastern Europe. Traditional methods for detecting rifampicin and isoniazid resistance require additional incubation from culture, delaying the diagnosis and increasing the risk of transmission of resistant disease in the community. A multiplex PCR for the sequencing of rpoB and hsp65 gene targets can facilitate same day detection of the majority of multi-drug resistant strains³³ but reliable resistance testing will require multi-gene and whole of gene sequencing⁴⁵ better suited to microarray technology.

Mycology and Parasitology

Although not as frequently applied to eukaryotic infections, in a number of clinical circumstances molecular testing can be helpful. Pneumocystis jiroveci (a fungus previously called Pneumocystis carinii) can cause a severe pneumonia in HIV-infected patients and other immunosuppressed patients but detection is limited to microscopy of respiratory tract specimens. Microscopy for the detection of P. jiroveci usually involves methenamine silver staining of tissue specimens or calcofluor white staining of induced sputum or bronchoalveolar lavage specimens. Immunofluorescence is more sensitive than these stains but is more expensive and needs specialised facilities. Sensitivity remains an issue, however, especially in HIV-non-infected patients such that the more sensitive PCR can be very useful.⁴⁶ The specificity of PCR is limited, however, as this organism is a ubiquitous commensal and can be detected by PCR in the absence of pneumonia.⁴⁷ Another mycological example is the use of 18S rRNA gene PCR to detect Aspergillus spp. infection in neutropenic haematology patients. This disease is notoriously difficult to diagnose due to the poor sensitivity of culture early in disease and the difficulty in obtaining histopathological specimens in those with reduced platelet counts. Early treatment is essential for the best outcomes resulting in empiric use of costly and toxic antifungal therapy. When performed frequently Aspergillus PCR can reduce the time required for a specific diagnosis,⁴⁸ however, its exact role to improve the management and therefore the outcome of this devastating disease is still unclear.

Parasitological diagnosis is aided by molecular methods since most parasites are not cultured in routine laboratory settings and therefore diagnosis relies mostly on the relatively less sensitive microscopy or serology. Toxoplasma gondii can be detected by PCR from amniocentesis fluid to confirm foetal infection⁴⁹ and from CSF to diagnose toxoplasma encephalitis. Microscopy remains the mainstay of malaria diagnosis but Plasmodium spp. PCR, because of its superior sensitivity compared to microscopy, can diagnose malaria in those with negative thick and thin blood films due to administration of chemoprophylaxis or partial immunity. Plasmodium species PCR can also detect mixed infections that can be difficult to discern microscopically.⁵⁰

Broad-range PCR

Unlike specific PCR testing where a particular organism is being sought, the use of broad-range PCR for the diagnosis of infectious diseases is more of a fishing expedition. Primers complementary to a conserved region of a gene are used, such as the 16S rRNA bacterial gene or the 18S rRNA gene of fungi. Any amplified product is usually sequenced and compared to more than 9,000 sequences from different organisms in Internet databases.⁵¹ There are several comprehensive databases such as GenBank (www.ncbi.nlm.nih.gov/Genbank), EMBL Data Library (www.ebi.ac.uk/embl), and the DNA Data Bank of Japan (www.ddbj.nig.ac.jp) with daily data exchange between them, and more specialised high quality databases such as RIDOM (www.ridom-rdna.de/) for bacterial rDNA sequences used for mycobacterial speciation. Broad-range PCR using 16S rRNA sequences is appealing as it can, in theory, detect bacteria in any sterile site specimen such as blood or cerebrospinal fluid, in other words a “molecular petri dish.” In fact this method was used to identify B. henselae in bacillary angiomatosis and T. whipplei as the bacterium associated with Whipple’s disease.⁵² A good example of its potential use in diagnostic medicine is for the aetiological diagnosis of infective endocarditis.⁵³ Antibiotic regimens for the therapy of this serious disease rely on the identification of the microbiological aetiology which can be problematic when conventional blood cultures are negative due to the prior administration of antibiotics. Broad-range PCR can be performed on the excised heart valves and vegetations or peripheral blood to reveal a diagnosis that would otherwise be missed. Broad-range PCR has also been applied to the diagnosis of bacterial meningitis.⁵⁴ More recently a spectacular use of broad-range PCR was the identification of the novel virological cause of SARS. Broad-based primers were used to detect unknown viruses in specimens from SARS clinical cases. The sequences showed homology to the coronavirus genus, supported by other laboratory results that resulted in a specific SARS CoV PCR within weeks of the first report of the disease.⁵⁵

A major drawback of broad-range PCR is the risk of amplifying DNA that may be contaminating the specimen or the PCR reagents themselves, especially the Taq DNA polymerases, resulting in false positive results.⁵⁶ Also the accuracy of the data available through public databases is difficult to assess and is dependent on the quality of the sequences deposited, a critical factor when comparing an unknown sequence. It is possible that the matching sequence is either inaccurate or is shared by another organism for which data is not currently available.

Public Health Aspects

Since rapid and reliable aetiological diagnosis underpins the effective management of contagious diseases, molecular diagnostics have an important role. The outbreak of SARS CoV illustrated the importance of ruling out other respiratory viruses such as influenza to facilitate the early identification and quarantine of suspected cases of SARS. This proved effective in controlling the outbreak even though a specific diagnostic test was not available during most of the outbreak. Now several PCR-based diagnostic kits are available and we will be much better equipped for early virological diagnosis should the SARS CoV re-emerge. Diarrhoeal viruses such as noroviruses, which spread rapidly through health care facilities and residential care facilities, can now be rapidly diagnosed to facilitate case isolation. The infectiousness of those with blood borne viruses is also determined predominantly by molecular testing. The ability of health care workers who have been infected with hepatitis B and C to perform exposure-prone procedures such as surgery is determined by PCR testing such that workers with detectable hepatitis B DNA or hepatitis C RNA may be restricted from performing such procedures.

The management of bacterial infections of public health significance is also improved by molecular methods. Early diagnosis of B. pertussis, M. tuberculosis, N. meningitidis is important for the early prevention of transmission, an aim that is best achieved by a combination of conventional and molecular testing.

The advent of molecular epidemiology, which allows the tracking of pathogens based on genotyping of the involved strains from outbreaks, has revolutionised how outbreaks are investigated and managed. The problem with strain differentiation using phenotypic methods in bacterial outbreaks, such as meningococcal disease, is the variable expression of the phenotypic markers. Other methods like multilocus enzyme electrophoresis are very labour-intensive and unable to be performed in many laboratories. Multilocus sequence typing (MLST) avoids these problems since every strain can be typed unambiguously. MLST involves the comparison of nucleotide sequences from internal fragments of a number of housekeeping genes. Sequences obtained are submitted to websites such as http://mlst.zoo.ox.ac.uk/ for N. meningitidis to give an allelic profile which can be compared to existing clones from anywhere in the world. This system avoids the problems of interlaboratory interpretation encountered with other genotyping methods yet can be used to investigate local outbreaks⁵⁷ even in some cases where a viable culture is not obtained. This has been applied to N. meningitidis and S. pneumoniae⁵⁸ which has helped map the spread of virulent clones around the world. Similarly genotyping of M. tuberculosis using different methods such as mycobacterial interspersed repetitive unit genotyping is recommended for the evaluation of community and health care facility tuberculosis outbreaks.

In the future it is likely that point-of-care molecular tests will be available allowing reliable results with rapid turnaround time in the field.

Biosecurity

Biological warfare agents such as Bacillus anthracis, variola major virus (smallpox), Clostridium botulinum and Yersinia pestis (plague) are problematic since they may be invisible, may cause no ill-effect for several days, and are communicable with very small amounts affecting many people. For example, 10g of anthrax spores could kill as many people as a ton of the nerve agent sarin.⁵⁹ The rapidity at which such an incident could escalate mandates rapid, reliable and sensitive detection methods. Real-time PCR methods fulfil these criteria best as conventional methods either lack discriminatory power, are slow, or require highly trained personnel. However, PCR systems require the release of DNA from spores, which can be difficult to achieve without inhibiting the PCR process. Spore disruption and PCR can now be achieved in 15 minutes using newly developed hardware.⁶⁰ Battery-powered portable machines using TaqMan^® real-time PCR are being developed with processing times of only 30 minutes.⁵⁹

In addition microarray technologies have a great deal of potential in this area but are restricted by the sample pretreatment required for such microfluidic devices. These problems are likely to be overcome as new technologies are developed.⁶¹

Limitations of Molecular Methods

Despite significant advantages of molecular diagnostics it cannot yet replace conventional methods for a range of infectious diseases since many common tests performed in the clinical microbiology laboratory are rapid and inexpensive. Advances in conventional technologies have resulted in many rapid antigen tests requiring only minutes for results and the modern automated culture systems allow relatively rapid identification and susceptibility testing. Unlike bacterial culture, which can detect a large number of cultivable bacteria without initially knowing the specific organism responsible, all PCR tests except broad-range PCR can only detect the organism whose DNA is complementary to the primers used. Therefore to cover a similar breadth of possible organisms would require the introduction of inexpensive and simple microarray technologies³⁰ that are not yet available.

False Positive and False Negative Results

Another problem restricting the application of molecular techniques to routine diagnosis is that of false positive and false negative results. To avoid false positive results due to laboratory contamination relatively large laboratory areas are required for physical separation of reagent preparation, specimen preparation and product detection areas together with a high level of staff training and skill. Amplicon laboratory contamination can be reduced by ultraviolet light irradiation of reagents and chemical inactivation of surface contamination with sodium hypochlorite. Amplicons can be destroyed by the use of dUTP to replace dTTP for amplification then uracil N-glycosylase (UNG) treatment of preassembled starting reactions to destroy the dUTP-containing amplicons. Intersample contamination can be reduced by the use of disposable equipment and cotton filter tips, and using disposable personal protective equipment such as caps, gowns and gloves. Even with scrupulous technique problems can be encountered, especially with broad-range PCR due to the presence of foreign DNA in the PCR reagents. It is therefore crucial that appropriate negative controls are included in every PCR run to detect any contamination. The advent of real-time PCR has reduced this risk due to single tube PCR reaction and detection systems.

Poor primer design can also lead to erroneously positive results. Primers may be poorly designed such that incidental amplification of microorganisms other than those sought occurs. Also primers are designed based on the known sequences available through international databases but organisms or sequences yet to be discovered can subsequently reduce the specificity of the PCR.⁶²

False negative results may also be a problem. Some organisms such as mycobacteria are difficult to extract DNA from, reducing the sensitivity of the PCR.⁶³ Substances in some clinical specimens such as sputum and faeces can degrade the DNA and RNA and other specimens may contain substances such as polysaccharides, haem and therapeutic drugs that inhibit the PCR enzymes.¹ It is therefore important to include inhibitor checks for each specimen to ensure a negative PCR reaction is not actually an inhibited reaction. This can be done by incorporation of an internal amplification control to check for both inhibitors and successful DNA extraction. This can be achieved by the addition of non-human pathogen DNA, for example equine herpesvirus as used in our laboratory, to the extraction buffer and its subsequent detection from the extracted sample by PCR using complementary primers.⁶⁴ The amount of spiked DNA is titrated to be as sensitive as possible yet allow regular detection in non-inhibitory specimens. Alternatively the human β-globin gene can be detected by PCR following sample extraction without the need for spiking the reagents with foreign DNA.³⁰ A problem with this method is that the amount of human DNA in each specimen cannot be controlled resulting in an inhibitor check of varying sensitivity. The addition of non-human pathogen RNA, such as bacteriophage RNA, can be used as an inhibitor and extraction control for reverse transcription PCR. If inhibitors are detected, they may be overcome by dilution of the DNA extract or treatment of the DNA extract with products such as GeneReleaser before PCR, or by including PCR facilitators such as bovine serum albumin in the PCR step.⁶⁵ If such manoeuvres fail to overcome the inhibition the sample must be reported as inhibitory and a repeat sample requested.

Lack of Uniformity in Molecular Testing

Molecular diagnosis is also complicated by the vast array of in-house PCR tests used in different laboratories. Commercial tests are available for a number of common and important infectious diseases such as HIV, hepatitis C and B, C. trachomatis, and N. gonorrhoeae but many infectious diseases are unlikely to have a commercial PCR test developed due to their rarity. Differences in primer selection (different genes or different sequences within genes), amplification format such as single round, nested or real-time PCR or one of the other nucleic amplification methods, and product detection methods such as ethidium bromide gel electrophoresis, DNA probes or sequencing make comparisons for sensitivity and specificity difficult.

Differentiation between Infection and Disease

Since the presence of nucleic acid does not necessarily mean the presence of viable organisms a problem with interpretation of PCR results can emerge that does not occur with culture. For some infections such as invasive meningococcal disease the presence of meningococcal DNA from a sterile site has a very high positive predictive value. However, the detection of P. jiroveci in suspected PCP may have only a 50% positive predictive value in immunosuppressed patients since it may colonise as well as cause disease. Likewise herpes viruses such as Epstein-Barr virus (EBV), CMV and HSV are intermittently shed following primary infection without causing disease. Less sensitive detection methods such as culture have a higher specificity but quantitative PCR may be helpful in this regard since higher viral loads are usually more specific for disease. Quantification in viral infections such as HIV, CMV, EBV, HBV and HCV is well established for assessing disease severity or monitoring response to treatment.³⁰ In CMV disease viral load testing can monitor either increases of viral loads to threshold levels or rates of viral load increase to improve the positive predictive value for clinical CMV disease. Another approach is to detect RNA species that are usually degraded within minutes of cell death to indicate pathogen viability and replication.⁶⁶

Future Directions of Molecular Technology

PCR coupled with sequencing has become a powerful tool for the identification of previously unknown pathogens and the epidemiological investigation of new and emerging infectious diseases. Molecular methods have helped reveal that over 30 species of bacteria can form uncultivable forms under unfavourable environmental conditions.⁶⁷ This now means that Koch’s postulates cannot be applied to investigate the validity of certain microorganisms in the causation of disease, such as T. whipplei as the cause of Whipple’s disease. Molecular technology has gone beyond the simple identification of causative organisms for infectious diseases and either now or in the near future will be pivotal to the study of the evolution of pathogens, the maintenance of infective cycles in nature, the investigation of causes and mechanisms of new pathogens, the mechanisms of susceptibility of different host groups and the development of DNA and RNA banking of genes encoding pathogenic factors. This will be achieved with new molecular methods such as microarray, microchips, in situ PCR and automation of molecular procedures.

Microarray and gene chip assays, first published in 1991, have the advantages of miniaturisation and automated construction using industrial robots together with sensitivity and rapid reading of large amounts of detailed genetic information. In fact up to 10⁶ different probes per cm² can be attached to specific sites on the microarray platform⁶⁸ of either nylon membrane or glass slide. Microarrays can identify simultaneously a range of pathogens for particular diseases such as infective diarrhoea, pneumonia or meningitis as well as genetic markers of virulence and antibiotic susceptibility. One of the first applications of this technology was in the field of HIV, in which an array was used to detect protease gene resistance.⁶⁹ Microarrays can be used for complete genome sequencing, an example being the development of an array to sequence the SARS virus following the 2003 outbreak. In addition their role in the detection and characterisation of biosecurity agents is rapidly progressing,⁵⁹ an example being the sequencing of hundreds of different variola major strains by the U.S. Centers for Disease Control and Prevention. However, its use at this stage is mainly research-based and is currently very expensive.¹ Issues of reproducibility must be addressed as the technology is highly sensitive and processing conditions must be standardised and followed rigidly. Also, because multiple data points are generated from each array, computer algorithms are needed to analyse the data.⁶⁹ Commercially, initial efforts focussed on applications of microarrays for use in detecting drug resistance and mycobacterial identification but the biotechnology companies are now assessing the market for molecular diagnosis using microarrays in the infectious diseases laboratories. For example, Affymetrix^® produce GeneChip^® microarrays for pathogen identification, virulence factor identification, pathogen response to drugs, and vaccine development. However, these companies will need to compete with the multiplex real-time PCR kits becoming commercially available such as those produced by Prodesse for the detection of the common respiratory pathogens.

Economic pressures will force the development of more automated and less expensive test procedures similar to those in clinical chemistry laboratories. Nucleic acid extraction and purification and the manual loading of the isolated nucleic acids and master mixes into the PCR reaction vessels remain the most labour-intensive parts of molecular technology. However, new technology has been developed to perform these tasks in the form of automated extraction and purification systems and pipetting robots, respectively. One of the first automated extraction systems developed was the COBAS AmpliPrep™ from Roche.⁷⁰ This system uses specific biotinylated oligonucleotide probes that capture released DNA which is then attached to streptavidin-coated magnetic beads. Roche have since released the MagNA Pure LC System which is well suited to the diagnostic laboratory. This system can process up to 32 samples in 60 minutes and has positive pressure pipetting, built-in UV decontamination and HEPA- filtration to avoid cross-contamination. However, reduced efficiency of extraction compared to manual extraction methods resulting in lower PCR sensitivity may be a problem.⁷¹ A number of other automated extraction systems are now available, such as the QIAGEN BioRobot EZ1 and M48/9604 systems, the Abbott m1000 system, the ABI PRISM™ 6100 Nucleic Acid PrepStation and 6700 Automated Nucleic Acid Workstation, and the Corbett Robotics X-tractor Gene.™ There are therefore a number of systems available offering a range of purchase costs, sample capacities and processing times. Automated fluid handling systems such as the Corbett CAS-1200™ Automated DNA Sample Setup allow automated PCR setup, including reagent preparation, dilution series and sample pipetting.

If the performance characteristics of these systems are found to be acceptable, the molecular diagnostic laboratory will be able to analyse more samples with higher throughput in an economic fashion and require less highly trained personnel. This will allow the clinical microbiology laboratory to answer more questions routinely by molecular methods than just the detection and quantification of microorganisms.

As with all new technologies new questions arise which can limit the clinical utility of the test. For example how long should we expect DNA to persist after recovery or treatment and in what body fluids or tissues will they persist, how can we distinguish between colonisation and active infection, and is the detection of DNA from microorganisms from so-called sterile sites a normal variant?

Although molecular methods have already replaced a number of traditional methods in the virology laboratory, until they can quickly and inexpensively analyse many genetic markers to determine aetiology and susceptibility, conventional culture and susceptibility testing using traditional methods will be required for some time to come.