Clinical developments for testing respiratory tract infections

Peter V Coyle
MD FRC Path
Consultant Virologist/Head of Microbiology
E: [email protected]

Derek Fairley
PhD
Research Scientist
Regional Virus Laboratory
Royal Hospitals Trust
Belfast, UK

Respiratory tract infections (RTIs) are well known and include many common conditions, ranging from the cold to acute pneumonia. They are caused by a large number of both viruses and bacteria, and their routine diagnosis has changed little in 50 years; for the majority of these infections a causative agent is never identified. This diagnostic uncertainty impairs optimum patient management and has led to an overuse of antibiotics, fuelling the development of antibiotic-resistant bacteria.

Clinical problem
Among the top 10 global causes of disability- adjusted life years (DALYs), five of the 10 are infectious diseases, with lower respiratory infections as the number one cause. Community-acquired infections are the largest proportion of these illnesses. Three-quarters of all antibiotic use is for RTIs; the recognition of their importance and the unexplained variation in the associated quality of care has resulted in the development of specialist society guidelines.(1) Most severe respiratory infections are assumed to be bacterial, and almost all patients will receive antibiotics. Two serious categories of respiratory disease in acute adult hospital admissions are community-acquired pneumonia (CAP) and acute exacerbations of chronic obstructive pulmonary disease (AE-COPD). However, definitive microbiological detection rates are only in the order of 50%, and the delay of 48–72 hours for results limits their usefulness for patient management and affecting outcomes. In AE-COPD the role of viruses has been underestimated. Several researcher groups, including our own, have found that 35–60% of exacerbations are associated with acute viral infection. Improving patient treatment and reducing antibiotic resistance should be a focus of future developments in microbial diagnosis.

Childhood RTIs represent the most common cause of hospital admission in the UK. The majority of these are of viral aetiology, including pneumonia. Children therefore represent a large group that may be unnecessarily treated with antibiotics. The annual incidence of CAP in children younger than five years of age is 40 cases per 1,000 in Europe and North America, higher than at any other time of life. In older children, the most common bacterial cause is Streptococcus pneumoniae, with Mycoplasma pneumoniae also more prevalent in this age group. In 20–60% of cases a pathogen is not identified by routine microbiological methods and the decision to use antimicrobial agents is again a clinical one. Signs and symptoms in upper and lower respiratory tract infections are not pathognomonic for identifying a viral or bacterial aetiology. The time delay associated with current microbiology test results means that early antibiotic therapy is inevitably prescribed blindly to patients with acute viral infections. The concern that concomitant bacterial infections may be missed, particularly in children, is a major factor for including an antibiotic regimen in the patient’s treatment – so influencing the prescribing practice of doctors is not straightforward. One study on the use of educational interventions in Kentucky, US – covering 124,092 episodes of care – demonstrated little, if any, impact on promoting appropriate antibiotic prescribing among 216 physicians.(2) The ability to rapidly differentiate clinically relevant bacterial and viral respiratory infections must therefore become a major goal for laboratory medicine.(3) To this end there are promising developments – but also major obstacles – to achieving this aim.

New test strategies
Polymerase chain reaction (PCR) was the first nucleic acid amplification test (NAAT) to accomplish the amplification of DNA in 1985. Subsequently, RNA amplification was made possible by incorporating a preliminary reverse transcriptase step. Many other amplification methods have since been developed, including isothermal and thermocycling-dependent protocols. For multiple targets either multiplex PCR or simultaneous amplification of targets with identical thermocycling parameters is used. Amplified microbial gene targets can be detected by gel-electrophoresis, where products are distinguished by size, or alternatively by using fluorescent reporter dyes. The former can usually resolve nine to 10 different targets – provided that suitable products with distinct sizes can be amplified and resolved – while currently dyes can resolve only four separate emission peaks.

One solution described for this, mass tag PCR, involves primers labelled by a photocleavable link to molecular tags of known molecular weight. Tags can then be released by UV irradiation and the microbe identified from the mass spectroscopy profile. Microarrays incorporating short (10–20mer) or long (50–70mer) oligonucleotides can also be used to resolve a greater number of products, but they have the same difficulties in the simultaneous amplification of multiple targets. In addition to multiplex PCR, microarrays can be used in conjunction with sequence-independent amplification. However, that approach tends to lower sensitivity and requires presubtraction of contaminating host DNA and ribosomal RNA. While the cost of microarrays is currently regarded as prohibitive, when coupled with sequence determination they can provide additional phylogenetic and antimicrobial resistance signatures that currently require complex sequencing or labour-intensive classical bacteriology methods respectively. The cost differential could therefore be less than imagined. Arrays will also be the subject of economy of scale as their numbers increase and their size reduces. Further miniaturisation using micromachining technology is likely to further affect the availability of these technologies.

Diagnostic alternatives
Although molecular tests are currently improving matters, they still have real problems to overcome. The sheer number of viral and bacterial pathogens that can cause RTIs is a problem for molecular assays in particular. The most common respiratory viruses and bacteria that need to be addressed are: influenza virus types A and B; respiratory syncytial virus type A and B; para-influenza virus type 1-4, adenovirus (>50 types); human rhinovirus (>100 types); coronavirus (five types); metapneumovirus; bocavirus; S pneumoniae; S aureus; S pyogenes; M catarrhalis; M pneumoniae; and L pneumophila.

This list is not exhaustive. We have recently improved the diagnosis of viral RTIs in Northern Ireland by introducing a molecular strip for the diagnosis of viral respiratory infections.(4) This methodology has highlighted both the deficiencies of standard methods and the extent of mixed infections (20–30%).

A further problem is determining the clinical significance of detecting a respiratory pathogen with a molecular test. For both molecular and culture-based approaches it is assumed that higher levels of infection are more likely to be clinically significant, although there is no definitive diagnostic level linked with disease. Culture techniques include a dilution step of 1/10(-5) to aid the diagnostic significance of pathogen recovery, while this is not the case with molecular tests. Qualitative NAATs can frequently detect the presence of pathogenic bacteria that can also harmlessly colonise the respiratory tract. It is therefore essential that molecular assays used for diagnosing significant infection should be capable of some degree of quantification. In fact, our experience would suggest that quantification is also necessary for noncolonising microorganisms to differentiate low-copy numbers from the high-copy numbers seen in symptomatic infection. While studies have shown that rapid viral diagnosis has lessened antibiotic usage and hospital stay for patients admitted with acute community-acquired respiratory infection, it has not to date been extended to the coordinated and rapid quantification of the viral and bacterial causes. However, this is an area of interest that will stimulate work leading to both improved patient care and an approach to the problem of suboptimal antibiotic use.

While diagnostic approaches to RTIs have changed little over the past 50 years, there has been an explosion in the development of genomic-based methodologies – the underlying principles of some of these systems have been touched on in this article. These technologies are not unique to microbiology but affect all branches of clinical and laboratory medicine. The selection of appropriate platforms and the skill mix for their routine use in service delivery will require careful planning and selective investment.

Event
Third European Congress of Virology Nürnberg, Germany 1–5 september 2007
W: www.eurovirology.org

Resources
European Society of Clinical Microbiology and Infectious Diseases W: www.escmid.org
European Society for Clinical Virology
W: www.escv.org
European surveillance network for vigilance against viral resistance (VIRGIL)
W: www.virgil-net.org
Federation of European Microbiological
Societies (FEMS)
W: www.fems-microbiology.org

References

1. British Thoracic Society guidelines for the management of community acquired pneumonia in childhood. Thorax 2002;57 Suppl 1:i1-24.
2. Mainous AG 3rd, Hueston WJ, Love MM, et al. An evaluation of statewide strategies to reduce antibiotic overuse. Fam Med 2000;32:22-9.
3. Goossens H, Little P. Community acquired pneumonia in primary care. BMJ 2006;332: 1045-6.
4. Coyle PV, Ong GM, O’Neill HJ, et al. A touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections. BMC Microbiol 2004;4:41.
5. Barker I, Brownlie J, Peckham C, et al. Foresight. Infectious diseases: preparing for the future. A vision of future detection,identification and monitoring systems.London: Office of Science and Innovation; 2006.