Microbiological Diagnosis of Respiratory Illness : Recent Advances
Abstract
A wide variety of microorganisms are potential respiratory pathogens, and the spectrum of known pathogens for each respiratory infection syndrome has not changed markers over recent years. Detection of likely etiologic agents of respiratory infections can help direct management and can also play an important role in disease surveillance. For this purpose, we are still reliant on many traditional diagnostic tools that have been used for decades in order to determine the microbial etiology of respiratory infections. However, these tools have been increasingly supplemented by newer methods, particular molecular diagnostic techniques, which have enabled the more rapid detection of many pathogens that were previously difficult to detect. These advances have particularly lead to improvements in the ability to detect respiratory viruses and also other microorganisms that do not normally colonize the respiratory tract. Recognition of the existence of the lung microbiome has challenged the traditional views of pneumonia pathogenesis and may provide the opportunity for new diagnostic tools that are focused on more than just detection of specific known pathogens. Continued liaison between clinicians and laboratory staff is vital in order to facilitate the most cost-effective use of laboratory diagnostics.
Infections of the respiratory tract are among the most common health problems in children worldwide, and are associated with substantial morbidity and mortality. A wide variety of microorganisms are potential respiratory pathogens; knowledge about the likely etiologic agents of respiratory infections can help direct management and can also play an important role in disease surveillance. Beyond the identification of specific pathogens, the clinical microbiology laboratory can also provide valuable information on antimicrobial susceptibility and strain typing. Continued liaison between clinicians and laboratory staff is vital to facilitate the most cost-effective use of laboratory diagnostics.
Presently, we are still reliant on many traditional diagnostic tools that have been used for decades to determine the microbial etiology of respiratory infections. However, these tools have been increasingly supplemented by newer methods, particular molecular diagnostic techniques, which have enabled the more rapid detection of many pathogens that were previously difficult to detect. These advances have particularly led to improvements in the ability to detect respiratory viruses and other microorganisms that do not normally colonize the respiratory tract. Moreover, recent discussions about the existence of a lung microbiome have challenged traditional paradigms about the pathogenesis of respiratory infections. The concept that the healthy lung may not be a sterile organ is reshaping our interpretation of laboratory diagnostics.
This chapter focuses on the use of the clinical microbiology laboratory to determine the microbial causes of respiratory infections in children. Diagnostic aspects of some specific respiratory infections, such as tuberculosis and pertussis, are also covered in other chapters.
Respiratory Pathogens and Syndromes
Tables 22.1–22.17 show the etiologic agents associated with respiratory infections broken down by respiratory syndrome. These lists represent our current understanding and have changed little over recent decades; there have been only a relatively small number of newly discovered pathogens. The latter include human bocavirus, human metapneumovirus, and a variety of coronaviruses (SARS-CoV, CoV-NL63, CoV-HKU1 and MERS-CoV). Pathogen discovery efforts using unbiased next-generation sequencing methods have shown considerable promise but have not yet identified major new respiratory pathogens.
Table 22.1
Etiologic Agents Associated With Pharyngitis
Viral
Bacterial
Fungal
Adenoviruses
Streptococcus pyogenes
Candida species
Coronaviruses
Other β-hemolytic streptococci
Parainfluenza viruses
Corynebacterium diphtheriae
Respiratory syncytial virus
Corynebacterium ulcerans
Human metapneumovirus
Arcanobacterium haemolyticum
Rhinoviruses
Neisseria gonorrheae
Influenza viruses
Mixed anaerobes
Epstein-Barr virus
Treponema pallidum
Enteroviruses
Chlamydophila pneumoniae
Herpes simplex viruses
Mycoplasma pneumoniae
Measles
Streptobacillus moniliformis
Rubella
Cytomegalovirus
HIV
HIV, Human immunodeficiency virus.
Table 22.2
Etiologic Agents Associated With Croup
Viral
Bacterial
Parainfluenza viruses
Mycoplasma pneumoniae
Influenza viruses
Respiratory syncytial virus
Human metapneumovirus
Coronaviruses
Human bocavirus
Adenoviruses
Measles
Rhinoviruses
Enteroviruses
Herpes simplex viruses
Table 22.3
Etiologic Agents Associated With Sinusitis
Viral
Bacterial
Fungal
Rhinoviruses
Haemophilus influenzae
Aspergillus species
Influenza viruses
Streptococcus pneumoniae
Alternaria species
Parainfluenza viruses
Anaerobes
Penicillium species
Adenoviruses
Moraxella catarrhalis
Zygomycetes
Staphylococcus aureus
Streptococcus pyogenes
Mycoplasma pneumoniae
Table 22.4
Etiologic Agents Associated With Acute Bronchitis
Viral
Bacterial
Adenoviruses
Mycoplasma pneumoniae
Influenza viruses
Bordetella pertussis
Parainfluenza viruses
Bordetella parapertussis
Respiratory syncytial virus
Chlamydophila pneumoniae
Rhinoviruses
Haemophilus influenzae
Coronaviruses
Streptococcus pneumoniae
Human metapneumovirus
Moraxella catarrhalis
Herpes simplex viruses
Streptococcus pyogenes
Enteroviruses
Measles
Mumps
Human bocavirus
Table 22.5
Etiologic Agents Associated With Bronchiolitis
Viral
Bacterial
Respiratory syncytial virus
Mycoplasma pneumoniae
Parainfluenza viruses
Adenoviruses
Influenza viruses
Human metapneumovirus
Rhinoviruses
Enteroviruses
Mumps
Herpes simplex viruses
Table 22.6
Etiologic Agents Associated With Pneumonia
Viral
Bacterial
Fungal
Respiratory syncytial virus
Streptococcus pneumoniae
Pneumocystis jiroveci
Parainfluenza viruses
Haemophilus influenzae
Aspergillus species
Influenza viruses
Staphylococcus aureus
Zygomycetes
Coronaviruses
Mycoplasma pneumoniae
Coccidioides immitis
Adenoviruses
Bordetella pertussis
Cryptococcus neoformans
Human metapneumovirus
Legionella species
Histoplasma capsulatum
Rhinoviruses
Enterobacteriaceae
Epstein-Barr virus
Pseudomonas aeruginosa
Enteroviruses
Acinetobacter species
Human bocavirus
Mixed anaerobes
Herpes simplex viruses
Streptococcus agalactiae
Varicella zoster virus
Chlamydophila pneumoniae
Measles
Chlamydia psittaci
Rubella
Chlamydia trachomatis
Cytomegalovirus
Burkholderia pseudomallei
HIV
Streptococcus pyogenes
Neisseria meningitidis
Coxiella burnetii
Mycobacterium species
HIV, Human immunodeficiency virus.
Table 22.10
Etiologic Agents Associated With Lung Abscess
Bacterial
Parasitic
Staphylococcus aureus
Entamoeba histolytica
Anaerobes
Streptococcus pneumoniae
Other gram-negative bacilli
α-Hemolytic streptococci
Table 22.12
Respiratory Specimens and Diagnostic Testing
Specimen Type
Microbiological Investigations
Comment
Sputum/induced sputum
Microscopy; culture; susceptibilities; DFA; PCR
Provided it is a good-quality specimen, it can be a highly informative specimen; can be difficult to obtain in children
Nasopharyngeal aspirate/swab
Microscopy; culture; susceptibilities; DFA; PCR
Most useful in viral infections; requires a skilled operator to obtain specimen; in some ways, it is easier to obtain than a throat swab, because the nares are always accessible
Nasal swab
Microscopy; culture; susceptibilities; DFA; PCR
Limited usefulness as it only recovers organisms present in the nasal cavity and not beyond
Throat swab
Microscopy; culture; susceptibilities; DFA; PCR
Probably the most representative specimen for disease of the upper respiratory tract; many bacterial pathogens are also common colonizers at various stages of childhood; can be difficult to obtain without child and parent cooperation; may represent organisms present in the nose as well as the oropharynx
Endotracheal aspirate
Microscopy; culture; susceptibilities; DFA; PCR
Invasive specimen, but is likely to represent pathogens from the lower respiratory tract; can be contaminated by organisms present in the oropharynx that can make result interpretation difficult
Bronchoalveolar lavage fluid
Microscopy; culture; susceptibilities; DFA; PCR
Invasive specimen but is likely to represent pathogens from the lower respiratory tract; can be contaminated by organisms present in the oropharynx, which can make result interpretation difficult
Transthoracic needle aspiration
Microscopy; culture; susceptibilities; DFA; PCR
Highly invasive specimen; risk of complications; microbiologically of high value provided the correct area has been biopsied
Lung tissue
Microscopy; culture; susceptibilities; DFA; PCR
Highly invasive specimen; risk of complications; microbiologically of high value provided the correct area has been biopsied
Pleural fluid
Microscopy; culture; susceptibilities; DFA; PCR
Invasive specimen but is the specimen of choice in a child with empyema
Blood cultures
Microscopy; culture; susceptibilities;
Very helpful if positive, but the positivity rate in pneumonia is relatively low
Serum/whole blood
Immunoassays; DFA; PCR
Serology per se is of limited value, since a diagnosis is dependent on paired sera that then makes it a retrospective tool; a single high titer can occasionally be obtained in acute disease; PCR on whole blood may be helpful in severe disease to detect viremia, but viremia is generally short lived
Urine
Antigen detection tests; microscopy; culture
Antigen detection tests are of limited value in children; pathogen is rarely cultured from urine
DFA, Direct fluorescent antibody; PCR, polymerase chain reaction.
Table 22.13
Gram Stain Appearance of Bacterial Respiratory Pathogens
Pathogen
Typical Gram Stain Appearance
Likely to Be Significant
Streptococcus pneumoniae
Gram-positive lancet-shaped diplococci
Predominant pathogen in Gram stain with abundant neutrophils
Staphylococcus aureus
Gram-positive cocci in clumps
Haemophilus influenzae
Small pleomorphic gram negative coccobacilli
Streptococcus pyogenes
Gram-positive cocci in chains
Arcanobacterium haemolyticum
Gram-positive diphtheroid-shaped bacilli
Corynebacterium diphtheriae
Pleomorphic diphtheroid gram-positive bacilli; special stain (Loeffler’s methylene blue stain) demonstrates typical club-shaped ends)
Mycoplasma pneumoniae
Absence of organisms as they lack a cell wall and cannot be visualized on Gram stain
Table 22.14
Screening of Respiratory Specimen Quality
Specimen
Acceptable for Culture
Sputum
<10 SEC/average 10× field
Endotracheal aspirate
<10 SEC/average 10× field and bacteria seen in at least 1 of 20 oil immersion fields
Bronchoalveolar lavage fluid
<1% of cells present are SEC
SEC, Squamous epithelial cells.
This table has been modified from Jorgensen JH, Pfaller MA, Carroll KC, et al. Manual of Clinical Microbiology , 11th ed. Washington, DC: American Society of Microbiology; 2015.
Table 22.16
Molecular Assays Commonly in Use for the Diagnosis of Respiratory Diseases
Molecular Assay
Principle
Main Use
Comment
Singleplex PCR
Single DNA or RNA target that is amplified
Can be designed for the detection of any known DNA or RNA sequence
Generally higher sensitivity than multiplex PCR as the targets are not competing
Multiplex PCR
Simultaneous amplification of several DNA or RNA targets
Respiratory pathogens; immunocompromised protocols; detection of various pathogens in blood cultures
Wide coverage of pathogens in a single test informs clinical management in a timely manner
16S rRNA sequencing
Amplification of 16S ribosomal RNA followed by sequencing of the product
Used to detect bacterial species in a clinical specimen that has failed to detect pathogens in culture.
Covers a wide range of pathogens listed in accessible sequence databases
Next-generation sequencing
Sequencing of a whole bacterial or viral genome or simultaneous sequencing of multiple bacterial or viral genes
Resistance testing and outbreak investigations
Can offer multiple gene sequences simultaneously or whole genome sequencing as well as de novo sequencing; currently, high cost prohibits routine use
PCR, Polymerase chain reaction.
Table 22.17
Molecular Terms Commonly Used in Diagnostics
Molecular Term
Explanation
PCR
An in vitro chemical reaction that leads to the synthesis of large quantities of a target nucleic acid sequence.
Reverse transcriptase PCR
RNA targets are converted into cDNA that is then amplified. This is needed for the amplification of RNA viruses (most common respiratory viruses).
RT PCR
The target amplification and the detection step occur simultaneously in the same tube. These assays require special thermal cyclers.
SNPs
Useful markers of genetic differences between strains, e.g., in outbreak investigations.
Target amplification techniques
Copies of a specific target nucleic acid are synthesized, and the products of amplification are detected by specifically designed oligonucleotide primers that bind to the complementary sequence on opposite strands of the double-stranded targets.
Signal amplification techniques
The target itself is not amplified; instead, the concentration of labeled molecules attached to the target nucleic acid is increased and measured
