key: cord-0034768-awanrm9u
authors: Fox, Julie D.; Tilley, Peter A.
title: Respiratory Pathogens
date: 2007
journal: Molecular Pathology in Clinical Practice
DOI: 10.1007/978-0-387-33227-7_41
sha: 7049dcb36d8b7721648a0abff81ee474f4ff6721
doc_id: 34768
cord_uid: awanrm9u

Respiratory tract infections are among the most common presenting complaints of patients in both hospital and community settings. They are a considerable burden in terms of both patient morbidity and public health interventions. Laboratory diagnosis of respiratory tract infections should provide guidance in therapy and prognosis, as well as useful epidemiological information reflecting trends in the community. Understanding and monitoring such trends facilitates early recognition of new infectious agents in a population. A summary of the common viruses and bacteria causing respiratory tract infections and their clinical relevance is given in Tables 41–1 and 41–2, respectively.

Respiratory tract infections are among the most common presenting complaints of patients in both hospital and community settings. They are a considerable burden in terms of both patient morbidity and public health interventions. Laboratory diagnosis of respiratory tract infections should provide guidance in therapy and prognosis, as well as useful epidemiological information reflecting trends in the community. Understanding and monitoring such trends facilitates early recognition of new infectious agents in a population. A summary of the common viruses and bacteria causing respiratory tract infections and their clinical relevance is given in Tables 41-1 and 41-2,  respectively. Even with a significant clinical effort and analysis of multiple specimens, current laboratory methods fail to diagnose approximately half of lower respiratory tract infections. In fact, laboratory diagnosis of communityacquired pneumonia (CAP) is so poor that current clinical practice guidelines do not recommend testing for all but the most severely affected patients and advise use of empiric therapy. 1 This pragmatic approach fails to address issues of antimicrobial overuse and resistance, public health surveillance, and advancement of medical knowledge.

Many "atypical" bacteria are known to cause severe respiratory symptoms, but lack of good diagnostic procedures has hampered the measurement of the real impact of such infections in the community. Despite vaccination policies, Bordetella pertussis infection remains relatively common in children and adults and is associated with chronic cough in adults. 2 Mycoplasma pneumoniae, Legionella pneumophila, and Chlamydophilia (previously Chlamydia) pneumoniae are all recognized causes of lower respiratory tract infections, but again, their impact has not been studied in detail. In addition, despite the well-recognized association of viral infections with upper and lower respiratory tract infections, the current diagnostic virology procedures do not provide an answer rapidly enough to with parainfluenza virus type 4, human coronaviruses, rhinoviruses, and some enteroviruses would not ordinarily be identified without RNA detection methods. The impact of such infections is only just being realized, and there is probably an underestimation of their clinical importance, particularly for immunocompromised individuals, the elderly, or those with underlying conditions such as asthma. 5, 6 Human metapneumovirus has been confirmed as an important cause of severe lower respiratory tract infection. The virus has been circulating for more than 50 years, and studies using molecular assays have confirmed its wide distribution, 7,8 but many laboratories have not been successful in isolating this virus. The recent identification of the agent causing severe acute respiratory syndrome (SARS), known as SARS-CoV, has illustrated the need for expansion of diagnostic testing to encompass new emerging viruses and the limitations of conventional virological laboratory approaches to respiratory pathogen diagnosis. 9, 10 Even if it is possible to culture viruses efficiently, isolation and confirmation of the cause of a cytopathic effect can take days to weeks, depending on the pathogen. Waiting for a culture-positive result can take many days, during which time the patient may be inappropriately treated and, if hospitalized, infection control measures may not be initiated. Also, the use of primary primate cells in culture (which gives the best yields of influenza and parainfluenza viruses) is unlikely to be sustainable in the long term.

When available, monoclonal antibodies are useful in direct virus-specific antigen detection tests, and these can be used for rapid diagnosis. Many laboratories are able to provide diagnostic testing for influenza, parainfluenza (types 1-3), respiratory syncytial virus (RSV), and respiratory adenoviruses. A respiratory specimen containing cells is necessary for sensitive detection of viruses by immunofluorescence or other antigen-detection methods.

Good-quality diagnostic samples (often lavage or aspirate samples) can usually be obtained from young, immunocompetent, hospitalized individuals, but in other circumstances the ideal sample may not be available. Delays in transportation may reduce specimen quality and compromise assay results. The most-difficult (and least-efficient) specimens for diagnostic testing are swab samples containing minimal cellular material taken from largely asymptomatic individuals in the community. Smears from these samples can be difficult to interpret in a direct antigen test and thus culture of the sample is usually required for pathogen identification. Bacterial antigen detection by a direct fluorescent antibody (DFA) test is similarly compromised by poor sensitivity and has the added concern of artifacts leading to false-positive results, particularly for B. pertussis and L. pneumophila.

Serological assays of an antibody response to infection are available for some respiratory pathogens. Although useful for retrospective evidence of infection in a community, the results are not timely enough for patient management. For some cell-associated or intracellular bacteria and viruses, antibody responses develop slowly, if they develop at all, and convalescent sera taken many weeks after disease onset are required to make a definitive diagnosis (e.g., for C. pneumoniae, Legionella species, RSV). For other infections such as influenza, antibody responses are brisk, but frequent reinfection reduces the IgM response and convalescent sera are required to demonstrate changing titers.

The limitations of conventional testing are well recognized for viral 11, 12 and bacterial 13,14 pathogens, and some of the key differences between nucleic acid and culture or direct antigen testing methods are summarized in Table 41 -3. Utilization of rapid viral diagnostic procedures, such as may be provided by molecular amplification methods, could help to reduce the emergence of antibiotic-resistant bacteria. One study demonstrated that a 52% reduction in antibiotic use was possible using molecular methods for viral diagnosis. 15 Despite the obvious economic burden of CAP, we do not have accurate data on how many of these infections are viral in origin or caused by the atypical bacteria for which routine diagnostic testing is not readily available. The identification of emerging human viral infections (such as H5N1 influenza and SARS-CoV) has heightened awareness of the gaps in respiratory pathogen diagnosis. Nucleic acid detection assays are likely to be utilized more widely to identify novel emerging pathogens that could result in worldwide outbreaks. Molecular amplification assays are being used successfully for the identification of organisms associated with pneumonia. 16 The potential for nosocomial spread of respiratory pathogens is well recognized for patients admitted to the hospital with RSV, parainfluenza, or other infections, serving as a reservoir for transmission to vulnerable patients and leading to possible outbreaks. 17 Previously, available diagnostic methods were not sensitive enough to identify sources of outbreaks, but the advent of molecular amplification methods has allowed even environmental sampling to be helpful in confirming outbreak sources and linking clustered cases of infection. 18 

Samples used for detection of respiratory pathogens include swabs (usually nasopharyngeal or throat), aspirates (nasopharyngeal or tracheal), sputum (usually from individuals presenting with pneumonia), or bronchoalveolar lavage specimens. For infections involving the entire respiratory tract, nasopharyngeal specimens are practical for diagnosis. For other infections, which are more focal, Melting-curve analysis of PCR products using the intercalating dye SYBR Green differentiates between specific and nonspecific products without further manipulation of the PCR products. Specific sequence information required for "Catch-all" approach, which may be advantageous when novel design; triage of testing can be difficult; pathogens need to be identified generic primers may be used to identify novel pathogens Sensitivity

Exquisite sensitivity but can be prone to Generally less sensitive than nucleic acid detection methods cross-contamination problems Specificity

Careful handling required to avoid Careful handling required to avoid contamination, but contamination (common problem); less-common problem than for molecular methods; DFA primer/probe design crucial subject to over interpretation Strain typing

Most definitive method Limited serotyping (e.g., influenza, Legionella) Automation

Automated extraction equipment becoming Difficult to automate available; automated detection commonplace Safety Inactivated before analysis, but antimicrobial Isolates useful for antimicrobial sensitivity testing and sensitivity information requires knowledge of phenotyping, but specialized safety requirements needed for genotypic mutation culture of category 3/4 pathogens Quality assurance

Proficiency and validation of methods not well Culture depends critically on cell line or medium quality; established maintaining quality can be difficult lower respiratory tract specimens are required (e.g., for Legionella). Sample preparation is a critical step for the detection and analysis of organisms. Numerous methods, from simple boiling to sophisticated automated protocols, are available for disruption of the organism and purification of the nucleic acids. Many studies have demonstrated inhibitors in respiratory specimens, making some form of extraction (with or without freezing) necessary to avoid frequent false negative results. Commercial kits for preparation of samples are available and should reduce interlaboratory variation in results. Simultaneous extraction of RNA and DNA facilitates assays for both viruses and bacteria.

Molecular techniques for the detection and analysis of pathogens associated with respiratory infection provide specific diagnoses for individual cases and for outbreaks. Currently, FDA-cleared molecular tests are not available for the detection of respiratory pathogens, with the exception of Mycobacterium tuberculosis (see chapter 43). Molecular tests are performed using either validated laboratorydeveloped procedures or commercial testing reagents. Published diagnostic methods for detection of respiratory pathogen DNA or RNA directly from clinical specimens utilize target amplification procedures such as polymerase chain reaction (PCR) or nucleic acid sequence-based amplification (NASBA).Although direct detection methods based on nucleic acid hybridization would be theoretically possible, the amount of target nucleic acid in specimens may be minimal and such methods would lack sensitivity compared to amplification methods, unless the organism was propagated before analysis. Thus, the molecular amplification procedures reported for direct detection of respiratory pathogens in clinical samples include PCR (e.g., Reference 19 and Figure 41 assays have utilized bacterial ribosomal RNA (rRNA; e.g., Reference 22 ). For cellular samples tested for respiratory pathogens, targeting messenger RNA (mRNA) or genomic antisense RNA may enhance diagnostic sensitivity.

A variety of formats have been utilized for the detection of amplified products. Procedures that separate target amplification from the detection phase (agarose gel analysis or endpoint hybridization) are well established 24, 25 and may allow multiple targets to be analyzed in a single reaction, providing added typing information.

For ease of use and incorporation into diagnostic laboratories, most laboratory-developed assays for detection of respiratory pathogens utilize real-time amplification methods in which the amplification and detection steps are combined. Some methods use intercalating dyes with the analysis of PCR product melting temperatures (e.g., as described previously 26 and illustrated in Figure  41 -1), whereas others use fluorogenic primers or probes (e.g., TaqMan, hybridization format, and molecular beacons 19, [21] [22] [23] 27 ) to ensure the specificity of the reaction. Figure 41 -2 illustrates a real-time RT-PCR assay for SARS-CoV using probe-specific detection of amplified products (one fluorescence measurement per cycle). The range of real-time PCR or RT-PCR and NASBA methodologies used for respiratory targets is diverse given the fact that assays are laboratory developed. 28, 29 The difficulty with diagnosis of respiratory infections is the wide range of pathogens with similar presentations. The nucleic acid technologies utilized currently in the majority of diagnostic laboratories are real-time PCR single-target assays. In some cases, generic primers can be designed to pick up several related pathogens. Such generic primers may be based on conserved protein coding sequences (such as those essential for enzyme function) or noncoding regions for which variation is limited because of the need for maintenance of secondary structure. The use of primer sets to pick up genera or even families of organisms has shown promise in limited studies, including analysis of Legionella and Mycobacteria. Generic assays have the ability to detect many related organisms and may be used to characterize previously undescribed species in respiratory infection.

Limited multiplex procedures have been reported for detection of related organisms using real-time PCR or NASBA procedures, but such assays are difficult to set up and control. For many respiratory pathogens, there is sufficient variation that multiplex approaches have been developed to detect, for example, all possible respiratory adenovirus, 19 influenza, 20 or parainfluenza 21 types. A simple, dual-labeled multiplex NASBA assay is shown in Figure 41 -5 that uses separate primer sets and specific molecular beacon probes for parainfluenza types 2 and 3 (HPIV2 and HPIV3) in the same reaction mix. Each probe is labeled with a different fluorophore, allowing detection and differentiation of both viruses in a single reaction. An ambitious multiplex nested RT-PCR procedure with gel analysis of the amplicons detects influenza A, B, and C viruses, RSV (A and B subtypes) , and adenoviruses in a single assay. 30 The procedure, while complex to set up and validate, was reported to have good specificity and better sensitivity than antigen/culture procedures.

Interpretation and validation of a negative result are important parts of diagnostic tests based on nucleic acid amplification. Some assays incorporate an internal control system to distinguish true-negative from false-negative results. The internal control may amplify with the pathogen-specific primers but result in an amplicon with a different size or internal sequence from the pathogen amplicon. Alternatively, the internal control may be an external sequence spiked into the reaction (heterologous control) and amplified with a primer set different from the pathogen primers. In the example shown in Figure 41 -3, amplification of the RNA heterologous control is consistent across many clinical samples and ensures that there are no gross inhibitors present in the reactions. For cellular samples obtained for detection of respiratory pathogens, the internal control reaction can utilize human DNA, rRNA, or mRNA detection. Such approaches have the added value of assessing for sample collection and integrity, as well as amplification inhibitors.

The relative merits of commercial versus laboratorydeveloped tests depend on the laboratory facilities, the technical expertise available, and the clinical need for expanded diagnosis. Commercial testing reagents provide quality controls and procedure standardization that facilitate clinical studies. 24, 32 Many companies are focusing on providing analyte-specific reagents (ASRs) for respiratory pathogen assays. ASRs will provide the laboratory with quality-controlled primers and probes, while allowing them the flexibility to test for currently known circulating pathogens or according to a local testing algorithm. Microarray-based detection of multiplexed PCR products also has been reported. 31 

Classically, typing of bacteria or viruses has used serological techniques that rely on antibody-antigen interactions. One benefit of approaches based on DNA or RNA detection is the more-detailed, quantitative assessment of the relationship between organisms, providing valuable data relevant to outbreak investigations and community health. Variation at the nucleic acid sequence level is not necessarily reflected in altered protein sequence or function; thus, additional sequence variation information may not correlate with conventional typing methods.

Restriction fragment length polymorphism (RFLP) analysis by pulsed field gel electrophoresis (PFGE), either with or without blot hybridization, has been utilized for analysis of complex DNA genomes from a variety of respiratory pathogens. RFLP analysis also has been applied to PCR or RT-PCR products from respiratory bacteria and viruses. In general, such methods can provide resolution down to the subtype level and have proven useful in outbreak investigation, as illustrated in Figure 41-6 for B. pertussis isolates. The difficulty with gel-based typing assays, such as PFGE, is standardizing results and sharing data between laboratories. Amplified fragment length polymorphism (AFLP) analysis represents an alternative method with better discriminatory power and portability, but this approach has not been used extensively for respiratory isolates to date.

For respiratory viruses, other methods have been used for typing, including heteroduplex mobility assay (HMA), single-strand conformation polymorphism (SSCP), and RFLP analysis of amplified PCR products. In general, HMA is considered technically complex but has the capacity to distinguish viral quasispecies with >3% nucleotide differences. SSCP and RFLP, while technically easier, generally can resolve viruses only to the subtype level, and RFLP has the added constraint of assessing only sequence differences in restriction sites.

The use of sequencing to assess the relationship among viruses is well established, and molecular phylogenetic knowledge is expanding, allowing modeling of viral populations and prediction of new outbreaks. 33 The level of resolution using primary sequence is at one genome, and point mutations can be identified. Sometimes this provides more information than originally sought and creates problems in interpretation; while in other circumstances even small sequence variations can confer important changes in viral transmissibility and disease outcome. Identification of emerging viruses, which may have been recently introduced into the human population (e.g., SARS-CoV and influenza A H5N1 types), is critical to public health. Analysis of such novel viruses has relied heavily on sequencing of isolates or amplicons. 9, 10, 34 Human influenza A viruses are associated with enhanced morbidity and mortality compared with influenza B or influenza C viruses. Differences in pathogenicity for subtypes of influenza A also have been reported; for example, H3N2 is associated with more severe infection than H1N1. Such types and subtypes of influenza A can circulate independently, and their identification is important for assessment of current vaccine efficacy. Reassortment of the two predominant influenza subtypes infecting humans in recent times has been reported, and analysis of main hemagglutinin (HA) types has been undertaken by a range of molecular and nonmolecular methods. 35 (1997-1998 and 2003-2004 34,38 ) emphasize the need for detailed surveillance of influenza viruses and vigilance in identification of emerging viruses of importance to public health. Detailed analysis of avian H5N1 viruses that have infected humans to date have confirmed that all genes are of avian origin and are associated with minimal or very inefficient humanto-human spread. The potential for reassorted viruses that could more easily spread among humans is clear, and molecular methods are now an important part of influenza surveillance.

Recent studies of human metapneumovirus have identified two main lineages, with sequence diversity within each group (Figure 41-8 40 ) , thus displaying a similar pattern to RSV isolates that are classified into two major groups,A and B. 39 Further studies will confirm whether this distinction is associated with differences in virulence.

Sequence analysis for typing of bacteria has been slower to develop than that for viruses but has been utilized for investigation of some atypical bacteria associated with outbreaks of respiratory infection (e.g., Legionella 41 ). Due to the problem of recombination, characterization of a single bacterial gene often does not reflect the organism as a whole. Multilocus sequence typing is a strategy that addresses this and appears useful for analysis of B. pertussis. 42 Microarray hybridization methods have been used to identify and differentiate related pathogens. 31 Such an approach was useful in first identifying the agent of SARS as a coronavirus. 43 Molecular methods provide additional information about the virulence and type of infectious organism, as illustrated by recent experience with SARS-CoV and influenza types.

Molecular tests have advantages over conventional procedures, but the sensitivity of molecular amplification methods can lead to problems with interpretation of results. For many organisms, a gold standard method is not available that accurately reflects the enhanced sensitivity of molecular methods, as has been seen with PCR testing for B. pertussis or C. pneumoniae in clinical samples. Studies confirm that PCR tests are very sensitive, and PCR-positive individuals may be culture negative or asymptomatic, so that results must always be interpreted in the clinical context.

Inhibitors of amplification are common in respiratory specimens, so a negative result must be interpreted in the context of the nucleic acid extraction method and the control results to monitor for nucleic acid degradation and amplification inhibition. When assays for the detection of respiratory pathogens are designed, primers and probes should not cross-react with normal respiratory flora or other respiratory pathogens.

The triage of molecular testing for respiratory infection diagnosis is difficult. Currently, a single respiratory pathogen test detects only one or a few related pathogens. Also, bacterial testing and viral testing are not combined. Thus, many molecular tests must be used to screen for all appropriate pathogens, which increases testing costs. Thus, a laboratory that embarks on using molecular methods for the diagnosis of respiratory infections may require a complex testing algorithm. One approach is use a multiplex amplification procedure to identify multiple pathogens in a single assay, with certain assays now commercially available. 24, 32 Unfortunately, such tests tend to be expensive and, if developed by the laboratory, are very difficult to control and ensure equal sensitivity and specificity for all pathogens. Thus, despite the potential for replacement of many culture and antigen procedures with nucleic acid amplification assays,such a molecular diagnostic revolution has not yet happened. The exception is for new pathogens when nucleic acid amplification and detection methods are clearly far superior to alternatives (e.g., metapneumovirus, SARS-CoV) or for testing of samples that are suboptimal for routine procedures (e.g., in surveillance situations).

Respiratory infections are currently underdiagnosed, despite the fact that accurate pathogen identification is important to ensure appropriate patient management and monitor infectious trends in the community. The major stumbling blocks in the diagnosis and investigation of respiratory infections are the complexity of testing algorithms and the number of potential targets that cause both upper and lower respiratory tract symptoms. Real-time PCR methods have vastly improved the sensitivity for detection and recognition of some difficult-to-culture organisms, and will likely become standard practice in the clinical laboratory in the next few years. There is, however, a limit to how many organisms can be "multiplexed" in a single test.

Microarray hybridization of randomly amplified PCR products from respiratory cultures and clinical samples has shown some success. 31 If the promise of early experiments is maintained when applied to large-scale clinical studies, this could answer some of the technical problems surrounding the use of multiplex systems. Microarray hybridization, while not currently as convenient as realtime PCR detection methods, potentially has the benefit of being able to resolve complex product mixtures and provide clinically valuable information.

The use of molecular methods for typing and outbreak investigation of respiratory pathogens of public health importance is well established and is likely to expand. Future directions will incorporate the use of microarray systems for respiratory pathogen detection and analysis to allow crossing of the barriers between conventional virology and bacteriology (and mycology/parasitology). Once microarray systems have been developed and validated, the costs of this enhanced technology may be reduced and justifiable.

Identification of novel viruses, which have presumably only recently been introduced to humans, has reinforced the need for careful surveillance of emerging respiratory pathogens and institution of appropriate infection control measures. Lessons should be learned from the continuous sensitive surveillance and typing of organisms such as influenza and B. pertussis to direct the use and efficacy of available vaccines.

Molecular techniques developed for detection and analysis of the microbes responsible for respiratory infections will be vital to our understanding of pathogenic mechanisms, appropriate management, and prevention of outbreaks in the future. Gel-based typing procedures (PFGE, HMA, SSCP) slowly will be replaced by sequencebased alternatives (e.g., multilocus sequence typing or MLST), which are more amenable to standardization and sharing of data among laboratories.

Phylogenetic tree illustrating the relationships among fusion gene sequences for 12 human metapneumovirus strains (from 2002). af371337 is the GenBank accession number for the prototype human metapneumovirus strain, and HMPV 35 is a Canadian strain

Canadian guidelines for the initial management of community-acquired pneumonia: an evidence-based update by the Canadian Infectious Diseases Society and the Canadian Thoracic Society. The Canadian Community-Acquired Pneumonia Working Group

Bordetella pertussis in the aetiology of chronic cough in adults. Diagnostic methods and clinic

Clinical and financial benefits of rapid detection of respiratory viruses: An outcomes study

Contribution of influenza and respiratory syncytial virus to community cases of influenza-like illness: an observational study

Respiratory tract viral infections in inner-city asthmatic adults

Polymerase chain reaction is more sensitive than viral culture and antigen testing for the detection of respiratory viruses in adults with hematological cancer and pneumonia

Human metapneumovirus infections in young and elderly adults

Human metapneumovirus as a cause of community-acquired respiratory illness

Identification of severe acute respiratory syndrome in Canada

Identification of a novel coronavirus in patients with severe acute respiratory syndrome

Clinical assessment of a generic DNA amplification assay for the identification of respiratory adenovirus infections

A comparison of nested polymerase chain reaction and immunofluorescence for the diagnosis of respiratory infections in children with bronchiolitis, and the implications for a cohorting strategy

Detection of Bordetella pertussis in a clinical laboratory by culture, polymerase chain reaction, and direct fluorescent antibody staining; accuracy, and cost

Polymerase chain reaction as a sensitive and rapid method for specific detection of Mycoplasma pneumoniae in clinical samples

Cost-effectiveness of rapid diagnosis of viral respiratory tract infections in pediatric patients

Nucleic acid amplification tests for the diagnosis of pneumonia

Molecular epidemiology of two consecutive outbreaks of parainfluenza 3 in a bone marrow transplant unit

Detection of adenoviruses (AdV) in culture-negative environmental samples by PCR during an AdV-associated respiratory disease outbreak

Multiplexed, real-time PCR for quantitative detection of human adenovirus

Rapid detection of influenza A and B viruses in clinical specimens by Light Cycler real time RT-PCR

Development and evaluation of NucliSens basic kit NASBA for diagnosis of parainfluenza virus infection with "end-point" and "real-time" detection

Detection of Mycoplasma pneumoniae by real-time nucleic acid sequence-based amplification

Development and evaluation of nucleic acid sequence based amplification (NASBA) for diagnosis of enterovirus infections using the NucliSens Basic Kit

Rapid simultaneous diagnosis of infections with respiratory syncytial viruses A and B, influenza viruses A and B, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (Hexaplex)

Multiplex PCR for typing and subtyping influenza and respiratory syncytial viruses

Multiplex real-time PCR assay for detection of influenza and human respiratory syncytial viruses

Development and clinical evaluation of an internally controlled, single-tube multiplex real-time PCR assay for detection of Legionella pneumophila and other Legionella species

Real-time PCR in virology

NASBA and other transcription-based amplification methods for research and diagnostic microbiology

Simultaneous detection of influenza A, B, and C viruses, respiratory syncytial virus, and adenoviruses in clinical samples by multiplex reverse transcription nested-PCR assay

Microarray-based detection and genotyping of viral pathogens

Evaluation of the Prodesse Hexaplex multiplex PCR assay for direct detection of seven respiratory viruses in clinical specimens

The application of molecular phylogenetics to the analysis of viral genome diversity and evolution

Molecular aspects of avian influenza (H5N1) viruses isolated from humans

Molecular diagnosis of influenza

Influenza virus types and subtypes detection by single step single tube multiplex reverse transcription-polymerase chain reaction (RT-PCR) and agarose gel electrophoresis

Influenza AH1N2 viruses, United Kingdom, 2001-02 influenza season

Molecular changes associated with the transmission of avian influenza A H5N1 and H9N2 viruses to humans

Circulation patterns of genetically distinct group A and B strains of human respiratory syncytial virus in a community

Human metapneumovirus infections in hospitalized children

Detection of Legionella species in respiratory specimens using PCR with sequencing confirmation

Multilocus sequence typing of Bordetella pertussis based on surface protein genes

Characterization of a novel coronavirus associated with severe acute respiratory syndrome