JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2011, p. S30–S33 VOL. 49, NO. 9 SUPPL.0095-1137/11/$12.00 doi:10.1128/JCM.00789-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

The Clinical Microbiology Laboratory in the Diagnosis ofLower 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

Lower respiratory tract infections (LRTIs) produce between5 and 10% of all deaths reported to the CDC via the 122 CitiesMortality Reporting System (5). The clinical laboratory plays avital role in the diagnosis of these infections but faces numer-ous challenges due to the complexity of LRTIs, including spec-imen quality and diversity; contamination of specimens withoropharyngeal flora; a diverse pathogen population that in-cludes bacteria, viruses, and fungi; and the complex pathophys-iology of respiratory tract infections, especially in special pop-ulations. Five current questions in the clinical microbiology(CM) of LRTI were discussed:

1. What is the real value of the Gram stain of expectoratedsputum?

2. What is the value of quantitative culture techniques onbronchoalveolar lavage (BAL) and mini-BAL specimens, en-dotracheal (ET) aspirates, and transbronchial biopsy speci-mens?

3. What is the role of the clinical microbiology lab in thediagnosis of acute exacerbations of chronic bronchitis(AECB)?

4. How do we optimize the microbiological evaluation ofcystic fibrosis (CF) patients with exacerbations?

5. What is the optimum laboratory evaluation of patientswith possible pulmonary tuberculosis?

There were discussants from both industry and clinical prac-tice and from a wide variety of clinical practice settings. Sur-prisingly, there were comparatively few areas of controversy orserious disagreement.

THE SPUTUM GRAM STAIN

The sputum Gram stain, a standard procedure in clinicalmicrobiology, is used for assessment of specimen quality, forpreliminary, rapid diagnostic information, and for laboratoryquality assurance.

Several systems are used to assess specimen quality using thesputum Gram stain. A number of quantitative criteria havebeen developed to screen for specimen quality, all of which arebased on the premise that an abundance of squamous epithe-lial cells is indicative of superficial oropharyngeal contamina-tion (18). Samples judged by Gram stain to consist predomi-nantly of upper respiratory tract material are rejected forroutine bacterial culture. The Gram stain in this case has two

functions: cost-effectiveness and avoidance of reporting of mis-leading information to the clinician, which may result in mis-diagnosis, leading to either misguided or unnecessary treat-ment. Reporting of misleading clinical information is alsoavoided by rejecting sputa for culture that is contaminated withupper respiratory flora because many of the potential patho-gens which cause pneumonia can also colonize the upper re-spiratory tract.

The value of the sputum Gram stain for preliminary diag-nosis of respiratory disease is well established. Of significance,criteria for interpretation and reporting of microorganisms inGram-stained smears of lower respiratory tract (LRT) secre-tions are variable. In addition, recommendations have beenmade that sputum culture results be correlated with directGram stain results (9, 11, 19) in order to provide more clini-cally relevant information in light of the limitations of culture.Finally, the Gram stain is a useful tool in laboratory qualityassurance. Comparison of Gram stain and culture results canreveal errors in procedure, specimen collection and/or trans-port issues, or specimen identification and tracking errors.

Discussion. The discussion centered on making the sputumGram stain an even more valuable clinical tool by reportingmore clinically useful information than currently reported inmost laboratories; see Table 1 for examples. Because suchguided reporting carries some risk of misdirecting providers,guideline development aimed at supporting laboratories inconsistent, evidence-based reporting schemes would be valu-able.

QUANTITATIVE CULTURES

The microbiological assessment of pulmonary samples ob-tained during bronchoscopic procedures is complicated notonly by the diversity of patients who undergo such proceduresand the variety of specimen types obtained but also by thepresence 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 countsof �105/ml of a major isolate suggest a potential pathogen.Bronchial brushings are placed in 1 ml of saline, and thenquantitative culture is performed by plating10 �l of the mate-rial. Counts of �103 CFU/ml correlate with disease in sus-pected pneumonia (20).

Discussion. Although quantitative culture standardizes thelaboratory assessment of these valuable, complex samples, thediscussants emphasized significant problems: (i) the methodsare labor-intensive, (ii) collection methods, including the vol-

* Corresponding author. Mailing address: P&LMS/113, VA Con-necticut 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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ume of saline used in the procedure, are not standardized; and(iii) the time of specimen transport to the laboratory, especiallyfrom referral sites and outpatient procedure centers, fre-quently impacts the subsequent organism load in the sample.Significantly, the studies on which our current quantitativeculture practices are based are approximately 2 decades oldand fail to reflect today’s patient populations, particularly thelarge number of solid organ and bone marrow transplant re-cipients undergoing bronchoscopy. The group recommendedthat new studies assessing the performance and interpretationof quantitative cultures of bronchoscopic specimens, with stan-dardization of both the collection and microbiological analysis,in specific patient populations are necessary to document thevalue of these labor-intensive procedures.

WHAT IS THE ROLE OF THE CLINICALMICROBIOLOGY LAB IN THE

DIAGNOSIS OF AECB?

The chronic bronchitis syndrome consists of impaired exer-cise tolerance, dyspnea on exertion, and chronic productivecough without a specific identified etiology. Patients are usu-ally older with a significant smoking history and may haveother comorbidities. They are at risk for severe pneumonias,ischemic cardiac disease, and other illnesses, some of whosesymptomatologies overlap significantly with acute exacerbationof chronic bronchitis (AECB).

AECB is a poorly understood condition. Clinically, it con-sists of an acute increase in symptoms beyond normal day-to-day variation, typically in one or more of the main symptoms of(i) increased frequency and/or severity of cough, (ii) increasesin volume and/or change in character of sputum production,and (iii) increased dyspnea.

While 70 to 80% of cases of AECB are infection related, itis unclear whether microbiological laboratory diagnosis con-tributes significantly to management of the syndrome. Thelaboratory is rarely informed that a specimen is from a patientwith AECB. Potential bacterial pathogens, such as Haemophi-lus influenzae, Streptococcus pneumoniae, and Moraxella ca-tarrhalis, all of which can contribute to exacerbations, are alsoisolated from patients between exacerbations. Although theseorganisms most likely contribute to the underlying pathology,their isolation from sputum in the setting of an exacerbation isof little or no significance. Similarly, organisms such as Staph-ylococcus aureus, though infrequently isolated, may have littleto do with the exacerbation when found (17). AECB is stronglyassociated with acquisition of new strains of some of these

organisms, but strain typing is currently expensive, slow, andlimited in availability. Antibiotics improve outcomes in AECB,particularly in the sickest patients, but therapy is empiricalrather than pathogen driven. Respiratory viruses, includingrhinovirus, influenza virus, parainfluenza virus, coronavirus,adenovirus, respiratory syncytial virus, and human metapneu-movirus, are frequently associated with AECB; however, ther-apeutic options are limited, with the exception of modestlyeffective therapies for influenza. Thus, the value of viral testingis also dubious. On the face of it, it appears that the laboratoryhas little role in managing chronic obstructive pulmonary dis-ease (COPD), and cost-effective practice would involve limit-ing microbiological workup. The 2007 Global Initiative forChronic Obstructive Lung Disease (GOLD) guidelines and2001 guidelines from the American College of Physicians statethat sputum cultures should not be performed during mostexacerbations of COPD.

Discussion. Some laboratory tests show value in AECB.Testing for respiratory syncytial virus has prognostic signifi-cance, and for patients admitted to the hospital, results of viraldiagnostics have infection control significance. It might be ex-pected that detection of a viral etiology would allow discontin-uation of antibiotics, but critically, the safety of this approachis yet unproven by clinical trials.

A barrier to limiting laboratory use is that the diagnosis ofAECB is rarely straightforward. Other illnesses, includingpneumonia, enter the differential diagnosis. Of patients withCOPD who died within 24 h of admission with a COPD exac-erbation, 28% died of pneumonia (21). So, while microbiolog-ical testing is of limited value in managing AECB, it may stillbe incumbent on the laboratory to test sputa from these pa-tients as a part of a comprehensive evaluation that includesother 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 suchutilization might not come to their attention.

HOW DO WE OPTIMIZE THE MICROBIOLOGICALEVALUATION OF CF PATIENTS WITH

EXACERBATIONS?

In common with COPD patients, patients with cystic fibrosis(CF) have underlying lung disease which typically results incontinual symptoms and important alterations in lung physiol-ogy and microbiota (10). Similarly, both environmental andmicrobial alterations can trigger increased symptoms in pa-tients with CF.

TABLE 1. Interpretive reporting of sputum Gram stains

Descriptive report Interpretive report

2� lancet-shaped Gram-positive cocci in pairs....................................................Consistent with Streptococcus pneumoniae3� Gram-positive cocci in clusters, 2� Gram-positive cocci in

pairs and chains, 2� Gram-negative rods ........................................................Consistent with Staphylococcus aureus and normal respiratory flora3� 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 thatsuggests a specific pathogen or group of pathogens

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A relatively predictable series of pathogens colonize therespiratory tract of CF patients. Nontypeable Haemophilus in-fluenzae and Staphylococcus aureus are typically found early inlife, with S. aureus being one of the most common bacterialpathogens recovered from the respiratory tract of persons withCF (14). On the other hand, the prevalence of infection causedby Pseudomonas aeruginosa varies significantly by age, rangingfrom about 25% for children age 5 years and younger to 80%for adults 25 to 34 years of age (6). Other bacterial opportun-ists include Stenotrophomonas maltophilia, Achromobacter xy-losoxidans, Burkholderia cepacia complex, Ralstonia species,Cupriavidus species, Pandoraea species, and nontuberculousmycobacteria (NTM), particularly rapid growers. In additionto bacteria, fungi such as Aspergillus species, Scedosporiumspecies, and Exophiala dermatidis have been implicated ascauses of chronic colonization or infection of the airways (14).A set of evidence-based guidelines for the use of selectivemedia and procedures is available (16). Proper testing of CFspecimens is materials and labor-intensive, with testing per-formed for both symptomatic disease and organism-specificsurveillance.

The role of the microbiology laboratory in management ofCF patients is complicated by the chronic nature of the illnessand by the intricacy of the host-microbe interactions:

● The complex bacterial flora requires selective media andcomplex protocols for isolation of possible pathogens.

● It is rarely possible to eradicate colonizing pathogens suchas Pseudomonas and S. aureus, although early eradicationprotocols for P. aeruginosa are successful in some patients.The goal of therapy is often clinical improvement ratherthan eradication of infection.

● Inhaled therapies that achieve high local antibiotic con-centrations are employed both for chronic suppressionand for treatment of exacerbations.

● Antibiotic pharmacokinetics are altered in patientswith CF.

● Unusual or altered strains of common bacteria (e.g., mu-coid P. aeruginosa [8]) and uncommon pathogens (Burk-holderia cepacia complex [13], rapidly growing mycobac-teria) provide challenges for bacterial identification andfor routine antibiotic susceptibility testing.

Discussion. There was comparatively little discussion on thistopic. Current guidelines, while complex and labor-intensive,are well crafted, evidence based, and effective for the currentstate of our capabilities in clinical microbiology and manage-ment of CF, especially if surveillance cultures can be limited to4/year, as the guidelines recommend. CLSI is currently devel-oping guidelines for antibiotic susceptibility testing of organ-isms isolated from CF patients, which should clarify some ofthe issues, or at least provide standard procedures for furtherassessment.

WHAT IS THE OPTIMUM LABORATORY EVALUATIONOF PATIENTS WITH POSSIBLE PULMONARY

TUBERCULOSIS?

The tubercle bacillus was described by Koch in 1882, but thediagnosis of pulmonary tuberculosis (TB) is still a complex,slow, uncertain, and expensive process. Optimal laboratory

diagnosis requires multiple specimens taken at intervals foracid-fast staining, possibly molecular assays for direct detectionof Mycobacterium tuberculosis, culture in rapid broth and onsolid medium, subsequent identification employing either phe-notypic or molecular methods, and where appropriate, antimi-crobial susceptibility testing.

The canonical “three sputa rule” for ruling out pulmonarytuberculosis has come under fire from at least two directions.Shorter hospital stays, high hospital occupancy rates, and costcontrols drive providers to move patients out of isolationrooms with airborne precautions as rapidly as possible. In-creasingly, accessible molecular diagnostics promise bettersensitivity than acid-fast smear and microscopy in rapid detec-tion of M. tuberculosis, potentially decreasing the number ofspecimens required (2), but at a significant cost and with a stillunproven impact on overall diagnostic yield and public health(15). The latest CDC guidelines (3) recommend that all pa-tients suspected of having pulmonary tuberculosis have at leastone 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 infec-tion status of patients (1, 3, 4). In contrast to the venerabletuberculin skin test (TST), which requires a carefully per-formed injection, a return visit, and a highly subjective readingprocedure, clinicians need to perform only a simple venipunc-ture to collect specimens for IGRA. Conversely, from a labo-ratory perspective, IGRAs are awkward, requiring specializedcollection materials and/or time-limited and often labor-inten-sive specimen processing prior to testing. IGRAs provide someperformance advantages compared to the TST, as there islimited cross-reactivity with nontuberculous mycobacteria andnone with Mycobacterium bovis BCG vaccine strains. SomeIGRAs may be more sensitive than the TST, although the lackof a gold standard for latent TB infection makes this determi-nation difficult (7). The relative sensitivity of IGRAs and theTST in immunocompromised populations and in young chil-dren is still unclear (12). Also, for individual patients, results ofthe TST and the two available IGRAs may disagree, furthercomplicating the interpretation of these tests. Like the NAATsfor pulmonary tuberculosis, IGRAs provide clinical conve-nience and some improvement in performance at significantcost to the laboratory.

Discussion. None of the discussants in the small group andfew in the meeting at large have implemented routine testingby NAAT for all patients with a suspicion of TB, regardless ofthe acid-fast bacillus (AFB) smear result. Only one NAAT isFDA approved for smear-negative specimens, so access toappropriate testing is an issue, but the major concerns voicedwere doubts about the effectiveness of the intervention and thecost to laboratories. Numerous studies have been published onthe ability of nucleic acid amplification (NAA) assays to di-rectly detect M. tuberculosis complex in clinical specimens.However, drawing definitive conclusions regarding the perfor-mance sensitivity, specificity, predictive values, and clinical util-ity of NAA assays from the myriad of studies is a daunting task.It is a given that the most significant advantage of NAA assaysis their rapid turnaround time for the direct detection of M.tuberculosis, which may have important implications for patientmanagement and tuberculosis control. However, assessing theperformance of these tests on the basis of the current literature

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is problematic. More clinical correlative studies are needed sothat a better understanding of when and how to use NAATs inconjunction with available clinical information may becomeevident, such that these assays are utilized in a cost-effectivemanner with optimum patient management. The performancecharacteristics of most NAATs performed on smear-negativespecimens have not been defined and during routine use mightbe expected to be poorer than in carefully controlled andlimited-duration studies due to accumulating contaminationand operator errors. The costs would likely be borne by labo-ratories, unless provider ordering can be efficiently driven byan electronic medical record or similar system; convincing hos-pitals to bear those costs is a daunting task. The cost to findone undiagnosed TB patient in most centers will be extremelyhigh. The public health benefits are predominantly theoretical,on the basis of estimates of the fraction of smear-negativepatients who would spread tuberculosis. The laboratory israrely, if ever, informed of the diagnosis, so it is unclear howtesting would be confined to patients tested for tuberculosis, asopposed to those in whom the likely diagnosis is NTM infec-tion. This guideline will meet continued resistance from thelaboratory community, unless more compelling data are pro-vided, more streamlined and less expensive tests are FDAapproved, ordering and utilization can be controlled, and re-imbursement is provided. None of these conditions currentlyapplies.

IGRAs clearly have a recognized role in detection of tuber-culosis infection in TST-positive, BCG-immunized individuals.However, more clinical data as to their performance are re-quired before conclusions regarding their usefulness in chil-dren and various immunocompromised populations can bemade. From a laboratory perspective, the role of IGRAs willalso require recognition of the increased costs in labor andreagents to the laboratory, as weighed against the ease of usefor clinics and improved compliance with screening and expo-sure management due to elimination of follow-up visits.

SUMMARY OF NEEDS

A number of needs were identified during this session:

● Evidence-based guidelines for interpretive reporting ofthe sputum Gram stain will allow laboratories to provideaccessible, clinically relevant information to guide themanagement of pneumonia patients.

● Updated studies of the utility and performance of quan-titative cultures of lower respiratory tract specimens incurrent patient populations with standardized samplingand analytical methods are required to document thevalue (if any) of these procedures.

● Clinical trials are needed to determine whether patientswith AECB and in whom a viral agent is detected stillbenefit from empirical antibiotic therapy; the importanceof routine diagnosis of respiratory viral infection in thispopulation is currently undocumented.

● Standardized protocols for susceptibility testing in CF pa-tients, currently in development, will allow standardizationof practice and will support further research on the ther-apeutic microbiology of CF.

● Outcome and cost-effectiveness studies of the routine em-ployment of IGRAs for employee health and risk-basedscreening for latent tuberculosis will provide evidence toestablish their appropriate use.

● Outcome studies of the public health impact of routinetesting by NAATs on AFB smear-negative patients fortuberculosis are necessary to document the value, if any,of this extremely expensive, unfunded intervention.

Session discussants: Matthew Binnicker, Paul Bourbeau, KarenCarroll, Christian Coogan, David T. Durack, George Goedesky,Amanda T. Harrington, Nathan A. Ledeboer, Michael Loeffelholz,Linda M. Mann, Alexander J. McAdam, Michael A. Saubolle, SharonShinn, and B. Jane Smith.

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