|Year : 2006 | Volume
| Issue : 1 | Page : 20-24
Role of sputum cultures in diagnosis of respiratory tract infections
Associate Professor (Medicine), Government Medical College, Surat - 395 001., India
A K Bhattacharya
Associate Professor (Medicine), Government Medical College, Surat - 395 001.
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
Bhattacharya A K. Role of sputum cultures in diagnosis of respiratory tract infections. Lung India 2006;23:20-4
Lower respiratory tract infections are quite common in the general population, occuring with increased frequency in older individuals and those with chronic diseases or compromised immune function. Etiologic diagnosis of the responsible pathogen is made by culture of respiratory tract secretions or by isolation of a compatible organism from blood (or pleural fluid) cultures.
While a positive blood or pleural fluid culture definitely identifies the pathogen, an organism growing from a respiratory specimen is not as readily implicated as the etiologic agent. Many organisms may be normal flora or colonizers of the respiratory tract and not responsible for the clinical syndrome. As a result, there is considerable controversy about the diagnostic value of many respiratory specimens. A rationale approach to specimen collection and the interpretation of results must be used if clinically useful information is to be Obtained  .
| Expectorated Sputum|| |
Expectorated sputum is the most common lower respiratory tract specimen received by the microbiology laboratory. It is also often the most problematic to assess. The first, and the most important, step in evaluation is the Gram stain. Gram stains are useful to:
- Assess the suitability of the sputum specimen for further processing and interpretation.
- Predict a likely etiologic agent by identification of a predominant bacterial morphology in an adequate (purulent) specimen.
There have been several guidelines proposed for evaluation of sputum Gram stain quality, using different combinations and cutoffs of squamous epithelial cells (SECs) and/or polymorphonuclear leukocytes (PMNs) per low power field (LPF, 10X objective)  , but none is considered to be clearly superior. Our laboratory rejects all specimens with more than 10 SECs/LPF (gram stain 1), without considering the number of PMNs. Rejection criteria have not been evaluated for Legionella or mycobacteria, and our laboratory accepts sputum specimens for diagnosis of these pathogens without screening for cellular content.
If the specimen is rejected as unsuitable for culture, the clinician is notified, and a repeat specimen is requested. If Gram stains are used for screening, an examination should be performed as soon as possible after a sample arrives in the laboratory to minimize delay in acquiring another sample.
A good quality sputum Gram stain may or may not offer assistance in determining the bacteriologic etiology of pneumonia. Many purulent specimens will have a mixture of different organisms, without a single predominant morphologic type. However, a specimen that does demonstrate a predominant organism can be quite useful (gram stan 2). In fact, it may prove to be the only diagnostic information available, since some fastidious organisms clearly seen on Gram stain may be overgrown by indigenous respiratory flora on solid media (eg, Streptococcus pneumoniae).
Sputum specimens may have less utility in the etiologic diagnosis of atypical pneumonia. In general, patients with these infections have mucoid sputum with abundant PMNs and sparse organisms on Gram stain.
Collection of the expectorated sputum for optimal yield - upto 30 percent of patients with bacterial pneumonia may be unable to produce a sputum speciman, even under optimal conditions , .Approaches used to improve the quality of the specimen obtained include:
- Obtaining the specimen prior to antibiotic treatment
- Rinsing the mouth prior to expectoration
- No food for one to two hours prior to expectoration
- Inoculation of the culture media immediately after the specimen is obtained or immediately after prompt transport to the microbiology laboratory
A poor quality of specimen,
- may only contain saliva or nasal secretions.
- when it is small in quantity (<2ml).
| Gross Findings of the Sputum|| |
I. Estimate daily volume of sputum
A. Small amounts
- Lung abscess
B. Copious amounts (<200 cc/day)
- Bronchopleural fistula
II. Sputum Color
A. Bloody sputum (Hemoptysis) B. Rusty sputum (Prune-juice)
- Pneumococcal pneumonia
C. Purulent sputum (yellow, green, dirty-gray)
- Color alone does not distinguish bacterial infection
III. Sputum Turbidity
A. Frothy sputum (Air bubbles, Hemoglobin)
- Pulmonary edema
B. Foamy, clear material
- Nasal secretions
IV. Sputum Viscosity
A. Bloody gelatinous sputum (Currant-Jelly)
- Klebsiella pneumonia
- Pneumococcal pneumonia
B. Stringy Mucoid Sputum (may also apear frothy)
- Follows asthma exacerbation
C. Cloudy, Mucoid Sputum
- Chronic bronchitis
D. Three layered appearance (Stagnant, Purulent Sputum)
- Lung Abscess
V. Sputum with Feculent Odor
A. Anaerobic infection
When are sputum Gram stains and cultures indicated? - There is considerable controversy about the utility of sputum specimens in community-acquired pneumonia (CAP) (see below). The value in nosocomial pneumonia, especially ventilatorassociated pneumonia (VAP), is more universally acknowledged.
Special stains of respiratory secretions may be performed as clinically indicated. These include:
- Acid fast stains for mycobacteria and nocardia.
- Fluorescent antibody stains for Legionella and respiratory syncytial virus.
- Quellung stains for Streptococcus pneumoniae laboratories whose technologists are trained in this technique).
Bronchitis - Sputum Gram stains and cultures have no role in the management of acute bronchitis in otherwise healthy individuals. Similarly, they are not indicated in the initial management of acute exacerbations of chronic obstructive pulmonary disease.
Community-acquired pneumonia ----- The American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA) have recently published revised guidelines for the management of CAP , . Both sets of guidelines recognize the limitations of sputum Gram stain and culture. However, the ATS recommends obtaining sputum for Gram stain or culture in patients requiring hospitalization when a drug-resistant pathogen or an unusual organism is suspected, and otherwise considers this testing optional for both in-and outpatient management. The IDSA suggests that Gram stain analysis alone as an optional test for patients with CAP who are managed as outpatients, and endorses routine sputum Gram stain and culture for all hospitalized patients with CAP in order to:
- Optimize antibiotic selection, especially for potentially resistant organisms (eg, penicillin resistant S. pneumoniae).
- Permit selection of antibiotics that are cost effective, least likely to induce resistance, and least likely to have adverse reactions.
- Identify pathogens of possible epidemiological significance.
- Eliminate from consideration those potential pathogens that do not appear on a good quality Gram stain or its corresponding culture, especially enteric Gram negative rods and S. aureus, which rarely cause false negative sputum examination.
- Alert physicians to the possibility of an "atypical" pathogen in those patients with clinical evidence of pneumonia and negative stains and cultures.
Nosocomial pneumonia - Sputum Gram stain and culture are indicated for all patients with nosocomial pneumonia. The specimen may be either expectorated sputum or suctioned secretions, depending upon whether the patient is ill enough to require endotracheal intubation.
In evaluating possible nosocomial pneumonia, sputum culture results cannot be interpreted in the absence of clinical data, especially in the intubated patient. Hospitalized patients become colonized rapidly with enteric Gram negative bacilli, with a frequency that is dictated by the severity of their associated conditions and their prior antibiotic exposure. It is often impossible to determine the true clinical significance of these organisms. Purulent sputum and a culture that grows a predominant organism may be indicative of tracheobronchitis, rather than parenchymal infection, in a patient without fever, peripheral leukocytosis, deteriorating oxygenation, change in quantity or character of sputum, or a new infiltrate on chest radiograph.
Sputum obtained via bronchoscopy - Culture results from specimens obtained at bronchoscopy are often considered to be more useful, expecially in establishing an etiologic agent for VAP  . However, intubation or insertion of the bronchoscope introduces upper airway flora into the lower airways, making qualitative cultures routinely positive. Thus, as with culture of endotracheal aspirates, culture of specimens obtained via ordinary bronchoscopic aspiration has high sensitivity and low specificity.
In comparison to qualitative cultures, quantitative cultures of specimens obtained by bronchoalveolar lavage (BAL) or protected specimen brush (PSB) have been reported to be the most discriminating approach to microbiologic diagnosis  . When these techniques are used, it is imperative that specimen handling be optimized, since fastidious organisms may not survive prolonged transport times. In addition, use of a diluent such as bacteriostatic saline in BAL or brush specimens can inhibit the growth of microorganisms. Lidocaine itself has antibacterial properties, and the use of lidocaine with bacteiostatic additives will predictably produce false negative cultures.
| Rejection Criteria for Diagnosis of Tuberculosis|| |
Patients need to be instructed as to the proper method of sputum collection. The material brought up from the lungs after a productive cough constitutes the material desired. A series of at least three single specimens (but usually not more than six) should be collected initially (preferably on different days) from sputum-producing patients. A sterile, wide-mouth specimen container with a tightly fitting screw top lid is adequate. Specimens should be clearly labeled with patient-identifying information and the date of collection. Every effort should be made to prevent the exterior of the container from becoming contaminated during the collection. The container should be placed in a disposable watertight plastic bag before being transported to the laboratory. No fixative or preservation agents should be used.
If there is breach of any of the above, the criteria are not matched and can be rejected.
| Staining and Microscopic Examination|| |
Factors influencing the sensitivity of smears include staining technique, centrifugation speed, reader experience, and the prevalence of tuberculosis disease in the population being tested. Laboratory errors, prolonged specimen decontamination, shortened incubation times or culture, cross-contamination of smears, and water of stains contaminated with acidfast organisms can result in smear-positive, culture negative specimens.
| Sputum Culture|| |
Routine media used for the isolation and identification of respiratory pathogens include blood agar, chocolate agar, and McConkey agar. Blood agar supports the growth of Gram positive cocci and most Gram negative rods, and is especially useful for evaluation of the colony morphology and hemolysis of streptococci. Chocolate agar permits recovery of Haemophilus influenzae d other fastidious organisms that may grow less well on blood agar. McConkey agar is selective for Gram negative bacteria and allows further classification into lactose positive or negative organisms, based upon their ability to ferment lactose.
Interpretation - Culture results are reported in a semiquantatative manner (1+ to 4+ in some laboratories, rare-few-moderate-abundant in others). Most true pathogens are present in at least 3+ (moderate) amounts.
As stated above, sputum cultures may not yield a specific pathogen. Since the oropharynx and upper airway are virtually always colonized with indigenous flora, culture of expectorated sputum often will grow mixed flora, even in the absence of bacterial lower respiratory tract infection [Figure 1]. For some organisms, the concentration in culture is important, and clinical correlations are critical. Some organisms are virtually never pulmonary pathogens, such as coagulase-negative staphylococci, enterococci, Gram positive bacilli other than nocardia, H. parainfluenzae, and streptococci other than, S. pyogenes and S. angiosus. Potential pathogens also can colonize the respiratory tract, especially in patients with chronic illnesses or recent hospitalization. The yield of sputum cultures is further diminished if the patient has received antibiotics prior to producing the specimen, which occurs in approximatley 25 percent of cases  . The greatest problem in this regard is with fastidious pathogens, such as S. pneumoniae and H. influenzae. The experienced technologist can select colonies with morphologies consistent with known respiratory pathogens, but their presence, especially in a mixed specimen, does not equate with causality.
Quantitative sputum cultures - Quantitative thresholds for BAL and PSB cultures have been extrapolated from quantitative cultures of infected lung tissue  . The generally accepted breakpoints for PSB and BAL are 10 3 and 10 4 colony forming units (CFU)/mL, respectively.
Two general approaches to quantitative culture have been established. The simplest is the "calibrated loop" method, analogous to that used for routine urine cultures. A measured amount of specimen, chosen to allow discrimination at the established threshold, is plated onto solid media and colonies are counted after appropriate incubation. The second method is serial dilution which involves preparation of two serial 100fold dilutions from the original specimen, with aliquots of all three samples then plated to solid agar. This latter approach is the most accurate, as it permits the investigator to count colonies over a wider range of initial organism concentrations, but the former is more suitable for routine microbiology laboratories.
Several studies have analyzed the sensitivity and specificity of quantitative cultures in identifying the etiologic agent of nosocomial pneumonia, using postmortem lung biospy cultures and histology as the gold standard. Further complicating this issue are the lack of agreement among pathologists over the histologic diagnosis of pneumonia and the nonspecificity of postmortem lung cultures. One study found variability in the frequency of the diagnosis of pneumonia among four pathologists reviewing histology from postmortem specimens of 39 patients; the application of pre-established histologic criteria resulted in the reclassification from no histologic pneumonia to histologic pneumonia in six patients  .
Utility of quantitative cultures in diagnosing bacterial VAP is still being questioned, and it is not clear that a threshold can be established to optimize both specificity and sensitivity of this approach  . It has been shown that the most useful results are obtained in patients who have not recently received antibiotics or who have not had a recent change in antibiotic regimen. BAL or PSB are expensive and labor-intensive and may best be reserved for such cases  .
However, one randomized, uncontrolled, multicenter trial in patients with suspected VAP, which compared invasive diagnosis (quantitative cultures of samples obtained by PSB or BAL) with strict clinical criteria and ATS practice guidelines alone showed that the invasive management group had  ,
- Fewer pathogens isolated that were resistant to the initial treatment used (1 versus 24 patients).
- Reduced mortality at day 14 (16.2 perent versus 25.8 percent).
- Decreased antibiotic use.
- Decreased mortality at 28 days (by multivariate proportional hazards regression analysis).
Bronchoscopy collection is most useful for the diagnosis of infection due to M. tuberculosis in patients with negative sputum studies, or for Pneumocystis carinii or occasional other fungal or viral pathogens, or in pinpointing a noninfectious etiology such as malignancy 
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