Laboratory diagnosis of respiratory infections
Peter Gilligan
Feb 29,2008
Overview
In our laboratory, we offer tests for the diagnosis of upper respiratory tract
infections-pharyngitis, sinusitis, ocular and ear infections, and lower tract
infections including pneumonia and bronchitis. Today we will focus on the
diagnosis of common bacterial causes of pharyngitis and lower respiratory tract
infections. Dr Miller will cover mycobacterial, fungal, and viral causes of lower
respiratory tract infections with a special emphasis on the use of molecular
methods to detect these organisms.
Upper respiratory tract infections
Pharyngitis- Group A streptococci is the most important bacterial cause of
pharyngitis. Although pharyngitis is typically a self-limited disease, the diagnosis
of group A streptococci may be attempted. The reasons may be to prevent the
overuse of antimicrobials to treat pharyngitis (GAS negative patients are not
treated) and to prevent ping-ponging of GAS in family with susceptible children.
1. GAS diagnostic strategy- Rapid Antigen Detection Test (RADT)- can be
used in adults; test has high specificity (99%+) but sensitivity ranging from
80-90% (dependent upon test used and quality of specimen collection).
Antigen positive patients would be treated but antigen negative patients
would not.
a. What about back-up culture- pro-can detect other agents of
pharyngitis including Group C and G strep which is a common
cause of bacterial pharyngitis as well as Arcanobacterium.
i. Arcanobacterium-weakly beta hemolytic, gram positive rod
can be confused with group F streptococci-seen in adults
causes a scarlet fever like syndrome with a scarlantiform-like
rash
b. Con- culture adds expense and positive cultures require follow up;
cases of rheumatic fever are vanishing rare in adult-ED rarely does
back up culture
2. Neisseria gonorrhoeae is the other agents of bacterial pharyngitis that we
can attempt to diagnose. Must be done by culture; molecular methods
have not been validated.
Lower respiratory tract infections
Overview- Lower respiratory tract specimens are submitted to the laboratory for
the diagnosis of airway infections including but not limited to community and
hospital acquired pneumonia including ventilator associated pneumonia,
bronchitis, chronic airway infection in cystic fibrosis patients, lung abscess, and
tuberculosis. Specimen types submitted for the detection of etiology of these
infections include: sputum, induced sputum, tracheal aspirates, nonbronchoscopic bronchoalveolar lavage (BAL) from intubated patients, and
bronchoscopically obtained specimens including bronchial washes and BAL, and
pleural fluid. Other specimens discussed in the literature but essentially never
done at UNC Hospitals include transbronchial biopsy for detection of PCP, fungi,
mycobacterium, and viruses; protected bronchial brush for anaerobic bacteria,
fungi, viruses, transthoracic needle aspiration and open lung biopsy. Importantly
only 60% of patients with CAP can produce sputum. Chest tube drainage is
sometimes sent but we don’t know what to do with it.
Pre-analytical stage1. Specimen quality determined by gram staina. Sputum gram stain screening-ratio of WBC to epithelial cells as
seen on low power field must be greater than 1. Screening is not
done in cystic fibrosis patients (sputum producers almost always
have purulent sputa and we are looking for a limited number of
organisms); patients suspected of either M. tuberculosis or
Legionella pneumophilia. Try to tell morphology and what it is
consistent with. For example, Streptococcus pneumoniae has a
distinctive morphology, lancet shaped diplococcus; we should
report: gram positive, lancet shaped diplococci consistent with
Streptococcus pneumoniae. Recent study of pneumococcal
pneumonia showed 16% had inadequate specimens, gram stain
was positive in 63% and culture in 86%.
Other organisms with distinctive gram stains include:
Gram positive cocci in clusters-Staphylococcus aureus
Thin gram negative rods- Pseudomonas aeruginosa
Pointed gram negative rods-Fusobacterium
Barnching, beaded gram positive rods- Nocardia
Mixed gram positive cocci and thin gram negative rods-mixed
anaerobic infections
Pleiomorphic gram negative coccobacilli-Haemophilus
Gram negative diplococci-Moraxella
b. Tracheal aspirate screening-as per sputum with the additional
proviso that the specimen must have organisms seen on oil
immersion. Over 90% of specimens that have “no organisms seen”
on gram stain will not grow a pathogen, so they are only cultured by
request. Likewise, if a specimen shows only yeast, it too is not
cultured because it is likely to represent endotracheal tube
colonization which is common. Candida pneumonia is so rare that it
needs to be diagnosed by other means as tracheal aspirate culture
will have poor positive predictive value.
2. What specimen when trying to detect specific organism.
a. CAP organisms- sputum and tracheal aspirates culture of good
quality specimen will be useful for detection of most bacterial
agents
b. Legionella-induced sputum may help if high index of suspicion
such as positive urinary antigen test otherwise sputum, even of
poor quality is OK-BCYE added to all BAL specimens but must ask
for it on tracheal aspirates and sputum
c. Pneumocystis- induced sputum OK in HIV positive patients;
otherwise use BALs: not a fan of tracheal aspirates, rarely positive
d. Nocardia- should grow from sputum specimen; will grow on most
laboratory isolation method but holding BCYE works best
e. Anaerobes- gram stain that showed mixed gram positive and
negative organisms and grows oropharyngeal flora only is a tip off.
Can look for them on BAL but must be requested as is not routinely
done. Protected bronchial brush probably the optimal specimenrarely done but if anaerobes sought this is the specimen of choice.
Will also culture pleural fluids but rarely positive
f. Anthrax-organism will be detected in blood cultures not respiratory
specimens
3. Culture set-up of different specimens
a. Sputum/tracheal aspirates-HBA, CNA, MacConkey
b. Pleural fluid- as with sputum + anaerobic medium; very low yield in
part because specimens are typically obtained when patients have
been on antimicrobial therapy. May be a good specimen for
bacterial detection by direct sequencing
c. BAL- cultured quantitatively; work up organisms >104 cfu/ml if
pathogens add BCYE (will grow pertussis, Nocarida, Francisella)
and Inhibitory Mould agar to plates used in sputum culture
d. Protected bronchial brush- quantitative on aerobic and anaerobic
plates otherwise like BAL; use 103 cfu/ml as our cut-off
e. Bronchial wash-cultured like a sputum
f. CF specimens-as above with BCSA (for Burkholderia cepacia
complex organism) and Mannitol salts (for Staphylococcus aureus
Analytical stage
1. Key organisms
a. Streptococcus pneumoniae- can be seen in patients with bronchitis
and both hospital and community pneumonia; infrequent in CF
patients. Characteristic gram stain can be of value. Other important
facts- pneumococcal penicillin breakpoints have changed from 1 to 2
ug/ml. blood cultures positive in approximately 30% of patients with
pneumococcal pneumonia- antigen test for pneumococcal urinary
antigen has a sensitivity of 50 to 80%; false positives can occur in
nasopharyngeal carriers
b. Haemophilus influenzae/Moraxella catarrhalis- important agent of
bronchitis especially in those folks with COPD; 90%+ of Moraxella are
beta-lactamase positive- H. influenzae- 20% are ampicillin resistance
with most of the resistance beta-lactamase mediated; non-typable
strains predominant; H. infuenzae uncommon cause of CAP- ranges
of 2 to 12% have been reported
c. Legionella pneumophilia- will grow in 3 to 5 days on BCYE agar;
isolated infrequently- urinary antigen has a sensitivity of 80% at our
institution and very good specificity but numbers or low so data should
be considered carefully; test can remain positive for months
nosocomial infections are rare at our institution; only know of 1
documented nosocomial case since I have been here
d. Staphylococcus aureus- important CF pathogen, CAP secondary to
influenza, nosocomial infection; CA-MRSA found in 3% of CF patients
in a 2 year prospective study-negative gram stain/cultures reported to
be adequate to rule out MRSA
e. Pseudomonas aeruginosa- key organism in chronic airway
infections in CF patients- highly adaptive, can survive some
disinfectants; makes a unique morphotype called “mucoid.” Mucoid
variant grow in a biofilm; organisms growing in biofilm are highly
adaptive to CF lung environment; organisms are refractory to
antimicrobial therapy and immune clearance; organism may be seen
in older women with COPD; also the most common agent of ventilator
associated pneumonia; strains may become resistant to all known
antimicrobials with the exception of colistin- colistin resistance is rare.
f. Burkholderia cepacia complex- important agent in cystic fibrosis
and chronic granulomatous disease patients; environmental bacteria;
highly adaptable and resistant to many antimicrobials and
disinfectants requires special medium for isolation (BCSA is the one
we use here); organism is resistant to wide array of antimicrobials and
some strains develop resistance to all available antimicrobials; limited
antimicrobials for which susceptibility guidelines are available
g. Stenotrophomonas/Achromobacter-glucose non-fermenters of low
virulence; environmental bacteria; highly adaptable and resistant to
many antimicrobials and disinfectants. Can colonize CF patients and
are likely colonizers rather than pathogens in patients on ventilators.
Outside the CF population, found most common in tracheal aspirate
cultures. As with B. cepacia complex, few antimicrobials have
susceptibility guidelines for Stenotrophomonas
h. Acinetobacter- becoming an important agent of nosocomial
pneumonia- glucose non-fermenting gram negative rod; can appear as
a diplococcus on gram stain. Strains being found in BICU and SICU
that are only susceptible to colistin.
i. Mycoplasma/Chlamydophila pneumoniae- important agents of
CAP; no satisfying diagnostic test so is primarily a diagnosis of
exclusion
Post analytical stage
1. Call the floor when specimen of poor quality is collected so recollection can be attempted
2. Try to limit the organisms that we report so that the report will make
sense