JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2011, p.

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0095-1137/11/$12.00 doi:10.1128/JCM.00789-11
Copyright 2011, American Society for Microbiology. All Rights Reserved.

VOL. 49, NO. 9 SUPPL.

The Clinical Microbiology Laboratory in the Diagnosis of

Lower Respiratory Tract Infections
Sheldon Campbell1* and Betty A. Forbes2
Department of Laboratory Medicine, Yale School of Medicine, and Pathology and Laboratory Medicine, VA Connecticut Healthcare,
West Haven, Connecticut 06516,1 and Pathology and Medicine, Virginia Commonwealth University Medical Center,
Richmond, Virginia 232982

1. What is the real value of the Gram stain of expectorated

sputum?
2. What is the value of quantitative culture techniques on
bronchoalveolar lavage (BAL) and mini-BAL specimens, endotracheal (ET) aspirates, and transbronchial biopsy specimens?
3. What is the role of the clinical microbiology lab in the
diagnosis of acute exacerbations of chronic bronchitis
(AECB)?
4. How do we optimize the microbiological evaluation of
cystic fibrosis (CF) patients with exacerbations?
5. What is the optimum laboratory evaluation of patients
with possible pulmonary tuberculosis?
There were discussants from both industry and clinical practice and from a wide variety of clinical practice settings. Surprisingly, there were comparatively few areas of controversy or
serious disagreement.

QUANTITATIVE CULTURES

THE SPUTUM GRAM STAIN

The microbiological assessment of pulmonary samples obtained during bronchoscopic procedures is complicated not
only by the diversity of patients who undergo such procedures
and the variety of specimen types obtained but also by the
presence of colonizing flora, particularly in intubated patients.
Current quantitative culture procedures vary by specimen type.
In general, colony counts of 104/ml suggest contamination,
counts of 104 to 105/ml represent a gray zone result, and counts
of 105/ml of a major isolate suggest a potential pathogen.
Bronchial brushings are placed in 1 ml of saline, and then
quantitative culture is performed by plating10 l of the material. Counts of 103 CFU/ml correlate with disease in suspected pneumonia (20).
Discussion. Although quantitative culture standardizes the
laboratory assessment of these valuable, complex samples, the
discussants emphasized significant problems: (i) the methods
are labor-intensive, (ii) collection methods, including the vol-

The sputum Gram stain, a standard procedure in clinical

microbiology, is used for assessment of specimen quality, for
preliminary, rapid diagnostic information, and for laboratory
quality assurance.
Several systems are used to assess specimen quality using the
sputum Gram stain. A number of quantitative criteria have
been developed to screen for specimen quality, all of which are
based on the premise that an abundance of squamous epithelial cells is indicative of superficial oropharyngeal contamination (18). Samples judged by Gram stain to consist predominantly of upper respiratory tract material are rejected for
routine bacterial culture. The Gram stain in this case has two

* Corresponding author. Mailing address: P&LMS/113, VA Connecticut Healthcare, West Haven, CT 06516. Phone: (203) 932-5711,
ext. 2908. Fax: (203) 937-4746. E-mail: sheldon.campbell@yale.edu.
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functions: cost-effectiveness and avoidance of reporting of misleading information to the clinician, which may result in misdiagnosis, leading to either misguided or unnecessary treatment. Reporting of misleading clinical information is also
avoided by rejecting sputa for culture that is contaminated with
upper respiratory flora because many of the potential pathogens which cause pneumonia can also colonize the upper respiratory tract.
The value of the sputum Gram stain for preliminary diagnosis of respiratory disease is well established. Of significance,
criteria for interpretation and reporting of microorganisms in
Gram-stained smears of lower respiratory tract (LRT) secretions are variable. In addition, recommendations have been
made that sputum culture results be correlated with direct
Gram stain results (9, 11, 19) in order to provide more clinically relevant information in light of the limitations of culture.
Finally, the Gram stain is a useful tool in laboratory quality
assurance. Comparison of Gram stain and culture results can
reveal errors in procedure, specimen collection and/or transport issues, or specimen identification and tracking errors.
Discussion. The discussion centered on making the sputum
Gram stain an even more valuable clinical tool by reporting
more clinically useful information than currently reported in
most laboratories; see Table 1 for examples. Because such
guided reporting carries some risk of misdirecting providers,
guideline development aimed at supporting laboratories in
consistent, evidence-based reporting schemes would be valuable.

Lower respiratory tract infections (LRTIs) produce between

5 and 10% of all deaths reported to the CDC via the 122 Cities
Mortality Reporting System (5). The clinical laboratory plays a
vital role in the diagnosis of these infections but faces numerous challenges due to the complexity of LRTIs, including specimen quality and diversity; contamination of specimens with
oropharyngeal flora; a diverse pathogen population that includes bacteria, viruses, and fungi; and the complex pathophysiology of respiratory tract infections, especially in special populations. Five current questions in the clinical microbiology
(CM) of LRTI were discussed:

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ROLE OF THE CM LABORATORY IN DIAGNOSIS

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TABLE 1. Interpretive reporting of sputum Gram stains

Descriptive report

Interpretive report

2 lancet-shaped Gram-positive cocci in pairs....................................................Consistent with Streptococcus pneumoniae

3 Gram-positive cocci in clusters, 2 Gram-positive cocci in
pairs and chains, 2 Gram-negative rods ........................................................Consistent with Staphylococcus aureus and normal respiratory flora
3 Gram-negative rods...........................................................................................Consistent with Pseudomonas spp.
3 Gram-negative coccobacillary rods, 1 Gram-positive cocci
in pairs and chains ...............................................................................................Consistent with Haemophilus spp.
2 Gram-positive cocci in pairs and chains, 2 Gram-negative
rods, 2 Gram-positive rods ..............................................................................No change from current practice, as this is not a pattern that
suggests a specific pathogen or group of pathogens

WHAT IS THE ROLE OF THE CLINICAL

MICROBIOLOGY LAB IN THE
DIAGNOSIS OF AECB?
The chronic bronchitis syndrome consists of impaired exercise tolerance, dyspnea on exertion, and chronic productive
cough without a specific identified etiology. Patients are usually older with a significant smoking history and may have
other comorbidities. They are at risk for severe pneumonias,
ischemic cardiac disease, and other illnesses, some of whose
symptomatologies overlap significantly with acute exacerbation
of chronic bronchitis (AECB).
AECB is a poorly understood condition. Clinically, it consists of an acute increase in symptoms beyond normal day-today variation, typically in one or more of the main symptoms of
(i) increased frequency and/or severity of cough, (ii) increases
in volume and/or change in character of sputum production,
and (iii) increased dyspnea.
While 70 to 80% of cases of AECB are infection related, it
is unclear whether microbiological laboratory diagnosis contributes significantly to management of the syndrome. The
laboratory is rarely informed that a specimen is from a patient
with AECB. Potential bacterial pathogens, such as Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis, all of which can contribute to exacerbations, are also
isolated from patients between exacerbations. Although these
organisms most likely contribute to the underlying pathology,
their isolation from sputum in the setting of an exacerbation is
of little or no significance. Similarly, organisms such as Staphylococcus aureus, though infrequently isolated, may have little
to do with the exacerbation when found (17). AECB is strongly
associated with acquisition of new strains of some of these

organisms, but strain typing is currently expensive, slow, and

limited in availability. Antibiotics improve outcomes in AECB,
particularly in the sickest patients, but therapy is empirical
rather than pathogen driven. Respiratory viruses, including
rhinovirus, influenza virus, parainfluenza virus, coronavirus,
adenovirus, respiratory syncytial virus, and human metapneumovirus, are frequently associated with AECB; however, therapeutic options are limited, with the exception of modestly
effective therapies for influenza. Thus, the value of viral testing
is also dubious. On the face of it, it appears that the laboratory
has little role in managing chronic obstructive pulmonary disease (COPD), and cost-effective practice would involve limiting microbiological workup. The 2007 Global Initiative for
Chronic Obstructive Lung Disease (GOLD) guidelines and
2001 guidelines from the American College of Physicians state
that sputum cultures should not be performed during most
exacerbations of COPD.
Discussion. Some laboratory tests show value in AECB.
Testing for respiratory syncytial virus has prognostic significance, and for patients admitted to the hospital, results of viral
diagnostics have infection control significance. It might be expected that detection of a viral etiology would allow discontinuation of antibiotics, but critically, the safety of this approach
is yet unproven by clinical trials.
A barrier to limiting laboratory use is that the diagnosis of
AECB is rarely straightforward. Other illnesses, including
pneumonia, enter the differential diagnosis. Of patients with
COPD who died within 24 h of admission with a COPD exacerbation, 28% died of pneumonia (21). So, while microbiological testing is of limited value in managing AECB, it may still
be incumbent on the laboratory to test sputa from these patients as a part of a comprehensive evaluation that includes
other diagnoses, at least in the sickest patients.
It was not felt that sputum culture is overutilized in AECB,
but the microbiologists in the session acknowledged that such
utilization might not come to their attention.
HOW DO WE OPTIMIZE THE MICROBIOLOGICAL
EVALUATION OF CF PATIENTS WITH
EXACERBATIONS?
In common with COPD patients, patients with cystic fibrosis
(CF) have underlying lung disease which typically results in
continual symptoms and important alterations in lung physiology and microbiota (10). Similarly, both environmental and
microbial alterations can trigger increased symptoms in patients with CF.

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ume of saline used in the procedure, are not standardized; and

(iii) the time of specimen transport to the laboratory, especially
from referral sites and outpatient procedure centers, frequently impacts the subsequent organism load in the sample.
Significantly, the studies on which our current quantitative
culture practices are based are approximately 2 decades old
and fail to reflect todays patient populations, particularly the
large number of solid organ and bone marrow transplant recipients undergoing bronchoscopy. The group recommended
that new studies assessing the performance and interpretation
of quantitative cultures of bronchoscopic specimens, with standardization of both the collection and microbiological analysis,
in specific patient populations are necessary to document the
value of these labor-intensive procedures.

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CAMP CLIN MICRO

The complex bacterial flora requires selective media and

complex protocols for isolation of possible pathogens.
It is rarely possible to eradicate colonizing pathogens such
as Pseudomonas and S. aureus, although early eradication
protocols for P. aeruginosa are successful in some patients.
The goal of therapy is often clinical improvement rather
than eradication of infection.
Inhaled therapies that achieve high local antibiotic concentrations are employed both for chronic suppression
and for treatment of exacerbations.
Antibiotic pharmacokinetics are altered in patients
with CF.
Unusual or altered strains of common bacteria (e.g., mucoid P. aeruginosa [8]) and uncommon pathogens (Burkholderia cepacia complex [13], rapidly growing mycobacteria) provide challenges for bacterial identification and
for routine antibiotic susceptibility testing.

Discussion. There was comparatively little discussion on this

topic. Current guidelines, while complex and labor-intensive,
are well crafted, evidence based, and effective for the current
state of our capabilities in clinical microbiology and management of CF, especially if surveillance cultures can be limited to
4/year, as the guidelines recommend. CLSI is currently developing guidelines for antibiotic susceptibility testing of organisms isolated from CF patients, which should clarify some of
the issues, or at least provide standard procedures for further
assessment.
WHAT IS THE OPTIMUM LABORATORY EVALUATION
OF PATIENTS WITH POSSIBLE PULMONARY
TUBERCULOSIS?
The tubercle bacillus was described by Koch in 1882, but the
diagnosis of pulmonary tuberculosis (TB) is still a complex,
slow, uncertain, and expensive process. Optimal laboratory

diagnosis requires multiple specimens taken at intervals for

acid-fast staining, possibly molecular assays for direct detection
of Mycobacterium tuberculosis, culture in rapid broth and on
solid medium, subsequent identification employing either phenotypic or molecular methods, and where appropriate, antimicrobial susceptibility testing.
The canonical three sputa rule for ruling out pulmonary
tuberculosis has come under fire from at least two directions.
Shorter hospital stays, high hospital occupancy rates, and cost
controls drive providers to move patients out of isolation
rooms with airborne precautions as rapidly as possible. Increasingly, accessible molecular diagnostics promise better
sensitivity than acid-fast smear and microscopy in rapid detection of M. tuberculosis, potentially decreasing the number of
specimens required (2), but at a significant cost and with a still
unproven impact on overall diagnostic yield and public health
(15). The latest CDC guidelines (3) recommend that all patients suspected of having pulmonary tuberculosis have at least
one sample tested by a nucleic acid amplification test (NAAT).
The development of gamma interferon release assays
(IGRAs) has provided a new tool for assessing the TB infection status of patients (1, 3, 4). In contrast to the venerable
tuberculin skin test (TST), which requires a carefully performed injection, a return visit, and a highly subjective reading
procedure, clinicians need to perform only a simple venipuncture to collect specimens for IGRA. Conversely, from a laboratory perspective, IGRAs are awkward, requiring specialized
collection materials and/or time-limited and often labor-intensive specimen processing prior to testing. IGRAs provide some
performance advantages compared to the TST, as there is
limited cross-reactivity with nontuberculous mycobacteria and
none with Mycobacterium bovis BCG vaccine strains. Some
IGRAs may be more sensitive than the TST, although the lack
of a gold standard for latent TB infection makes this determination difficult (7). The relative sensitivity of IGRAs and the
TST in immunocompromised populations and in young children is still unclear (12). Also, for individual patients, results of
the TST and the two available IGRAs may disagree, further
complicating the interpretation of these tests. Like the NAATs
for pulmonary tuberculosis, IGRAs provide clinical convenience and some improvement in performance at significant
cost to the laboratory.
Discussion. None of the discussants in the small group and
few in the meeting at large have implemented routine testing
by NAAT for all patients with a suspicion of TB, regardless of
the acid-fast bacillus (AFB) smear result. Only one NAAT is
FDA approved for smear-negative specimens, so access to
appropriate testing is an issue, but the major concerns voiced
were doubts about the effectiveness of the intervention and the
cost to laboratories. Numerous studies have been published on
the ability of nucleic acid amplification (NAA) assays to directly detect M. tuberculosis complex in clinical specimens.
However, drawing definitive conclusions regarding the performance sensitivity, specificity, predictive values, and clinical utility of NAA assays from the myriad of studies is a daunting task.
It is a given that the most significant advantage of NAA assays
is their rapid turnaround time for the direct detection of M.
tuberculosis, which may have important implications for patient
management and tuberculosis control. However, assessing the
performance of these tests on the basis of the current literature

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A relatively predictable series of pathogens colonize the

respiratory tract of CF patients. Nontypeable Haemophilus influenzae and Staphylococcus aureus are typically found early in
life, with S. aureus being one of the most common bacterial
pathogens recovered from the respiratory tract of persons with
CF (14). On the other hand, the prevalence of infection caused
by Pseudomonas aeruginosa varies significantly by age, ranging
from about 25% for children age 5 years and younger to 80%
for adults 25 to 34 years of age (6). Other bacterial opportunists include Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Burkholderia cepacia complex, Ralstonia species,
Cupriavidus species, Pandoraea species, and nontuberculous
mycobacteria (NTM), particularly rapid growers. In addition
to bacteria, fungi such as Aspergillus species, Scedosporium
species, and Exophiala dermatidis have been implicated as
causes of chronic colonization or infection of the airways (14).
A set of evidence-based guidelines for the use of selective
media and procedures is available (16). Proper testing of CF
specimens is materials and labor-intensive, with testing performed for both symptomatic disease and organism-specific
surveillance.
The role of the microbiology laboratory in management of
CF patients is complicated by the chronic nature of the illness
and by the intricacy of the host-microbe interactions:

J. CLIN. MICROBIOL.

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ROLE OF THE CM LABORATORY IN DIAGNOSIS

is problematic. More clinical correlative studies are needed so

that a better understanding of when and how to use NAATs in
conjunction with available clinical information may become
evident, such that these assays are utilized in a cost-effective
manner with optimum patient management. The performance
characteristics of most NAATs performed on smear-negative
specimens have not been defined and during routine use might
be expected to be poorer than in carefully controlled and
limited-duration studies due to accumulating contamination
and operator errors. The costs would likely be borne by laboratories, unless provider ordering can be efficiently driven by
an electronic medical record or similar system; convincing hospitals to bear those costs is a daunting task. The cost to find
one undiagnosed TB patient in most centers will be extremely
high. The public health benefits are predominantly theoretical,
on the basis of estimates of the fraction of smear-negative
patients who would spread tuberculosis. The laboratory is
rarely, if ever, informed of the diagnosis, so it is unclear how
testing would be confined to patients tested for tuberculosis, as
opposed to those in whom the likely diagnosis is NTM infection. This guideline will meet continued resistance from the
laboratory community, unless more compelling data are provided, more streamlined and less expensive tests are FDA
approved, ordering and utilization can be controlled, and reimbursement is provided. None of these conditions currently
applies.
IGRAs clearly have a recognized role in detection of tuberculosis infection in TST-positive, BCG-immunized individuals.
However, more clinical data as to their performance are required before conclusions regarding their usefulness in children and various immunocompromised populations can be
made. From a laboratory perspective, the role of IGRAs will
also require recognition of the increased costs in labor and
reagents to the laboratory, as weighed against the ease of use
for clinics and improved compliance with screening and exposure management due to elimination of follow-up visits.

A number of needs were identified during this session:

Evidence-based guidelines for interpretive reporting of

the sputum Gram stain will allow laboratories to provide
accessible, clinically relevant information to guide the
management of pneumonia patients.
Updated studies of the utility and performance of quantitative cultures of lower respiratory tract specimens in
current patient populations with standardized sampling
and analytical methods are required to document the
value (if any) of these procedures.
Clinical trials are needed to determine whether patients
with AECB and in whom a viral agent is detected still
benefit from empirical antibiotic therapy; the importance
of routine diagnosis of respiratory viral infection in this
population is currently undocumented.
Standardized protocols for susceptibility testing in CF patients, currently in development, will allow standardization
of practice and will support further research on the therapeutic microbiology of CF.

Outcome and cost-effectiveness studies of the routine employment of IGRAs for employee health and risk-based
screening for latent tuberculosis will provide evidence to
establish their appropriate use.
Outcome studies of the public health impact of routine
testing by NAATs on AFB smear-negative patients for
tuberculosis are necessary to document the value, if any,
of this extremely expensive, unfunded intervention.

Session discussants: Matthew Binnicker, Paul Bourbeau, Karen

Carroll, Christian Coogan, David T. Durack, George Goedesky,
Amanda T. Harrington, Nathan A. Ledeboer, Michael Loeffelholz,
Linda M. Mann, Alexander J. McAdam, Michael A. Saubolle, Sharon
Shinn, and B. Jane Smith.
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SUMMARY OF NEEDS

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