# Presentation: 718774

## Utilization of Gram Stain and Culture Results In Clinical Trials for Lower Respiratory Tract Infections

- Sousan S. Altaie, Ph.D.
- Office of Drug Evaluation IV
- Division of Anti-Infective Drug Products
- altaies@cder.fda.gov

## Current Situation

- Gram stain result is part of inclusion criteria in lower respiratory tract infections.
- The smear should contain < 10 SEC and >25 WBCs PLP ( 100 X magnification )

## Issues to be Addressed

- Why screen Lower Respiratory Tract  specimens before processing for culture?
- What are Gram stain criteria for acceptability of culture results?
- Should Gram stain be part of inclusion criteria?
- How should Gram stain be used in the overall picture?

## Why Screen Lower Respiratory Tract Specimens?

## Why Screen Specimens?

- The upper respiratory tract to the level of larynx is the major source of contamination for most specimens, including expectorated sputum, nasopharyngeal aspirates, and bronchoscopy aspirates such as BAL.

## Why Screen Specimens?

- Gram-stained smears should be prepared for all sputa submitted for bacterial cultures to determine the extent of contamination with saliva and therefore, acceptability of specimen for bacterial culture.

## Background

- Prior to ~ 1971 clinical labs cultured expectorated sputum specimens without considering the quality of the specimen and  reported all the respiratory pathogens isolated.
- Reports of “nonvalue” of sputum culture in diagnosis of pneumonia, especially pneumococcal pneumonia, flooded the literature.

## Background

- “The routine sputum culture for the diagnosis of acute bacterial pneumonia may be a sacred cow.”
- Barret-Conner,  Elizabeth. 1971.  Am. Rev. Respir. Dis. 103:1010-1027

## Background

- There were multiple attempts to improve the quality of the sputum culture by washing, quantitation, and microscopic screening.

## What are the criteria for an acceptable specimen?

## Classification of Gram-stained SpecimensBartlett R.C., 1974, In Medical Microbiology. Wiley J. & Sons, NY

- Neutro

## Relationship Between Specimen Group and Number of Bacterial Isolates Murray P.R., et al., 1975, Mayo Clin. Proc. 50:339-344

- Number of WBCs bore no relationship to the number of isolates.

**Notes:**
All comers in one month, 382 specimens
categorized patients on the basis of microscopic screening
compared culture results with  47 TTA collected during the past 12 months

## Classification of Gram-stained SpecimensGeckler et al., 1977, J. Clin. Microbiol. 6(4):396-399

**Notes:**
Paired specimens(sputum and TTA) from 100 patients with clinical diagnosis of pneumonia
pathogens: S pneumo, H. flu, N. mening, GAS, S aureus, Enteric rods
This is were <25 epithelial cell became a criteria

## Relation Ship Between Specimen Group and Number of PathogensGeckler et al., 1977, J. Clin. Microbiol. 6(4):396-399

**Notes:**
-mean number of pathogen/specimen is less than 1 because some specimens did not grow any pathogens.

-ideally group 5 should only be cultured

## Rejection Criteria for Endotracheal Aspirates from AdultsMorris A.J. et al. J. Clin. Microbiol. 1993, 31(5):1027-1029

## Culture results for 201 ETAs with no organisms seen on Gram stain Morris A.J. et al. J. Clin. Microbiol. 1993, 31(5):1027-1029

**Notes:**
88% were either sterile or grew varying amounts of normal flora

## What are the criteria for an acceptable LRT specimen?

- Respiratory specimens with <10 SEC/LPF from patients with a clinical diagnosis of pneumonia should produce culture results leading to etiology of the infection.

## How should Sputum Gram stain results be used? 

## Sputum Gram Stain Assessment in Septic CAPGleckman R., et al. 1988, J. Clin. Microbiol. 26(5):846-849

-  59 septic patients with valid (>25 WBCs, <10 sec) sputum
- >10 organisms/oil field = significant bacterial count
- 12/59 did not produce helpful results in guiding therapy.
- In remaining 47 patients Gram-stained sputum precisely predicted the blood culture isolate with sensitivity of 85.1%.

**Notes:**
--Prospective study over 4.5 years to determine the ability of sputum Gram stain to predict the cause of CAP
--Blood Culture isolate, rather than sputum culture served as reference standard to provide precise identification of the etiological agent

category A= NOS (1)
category B=mixed flora with  no morphotypes >10/oil field (7)
category C= mixed flora with >2 morphotypes at counts >10/of (4)
category D=one morphotype in <10/of
category E=one morphotype in>10/of with or without an added morphotype in counts<5/of

--category A-C were not assessed because such gram stain results by definition would not assist clinician in appropriate selective therapy. 

## Sputum Gram Stain Assessment in Septic CAPGleckman R., et al. 1988, J. Clin. Microbiol. 26(5):846-849

- If a clinician had been guided by the valid Gram-stained sputum specimen and offered momotherapy, initial treatment would have been ideal for 40 patients, acceptable for 4 patients and inappropriate for 3 patients. Overall  94% of the time appropriate monotherapy could have been selected

## Correlation of Gram stain, blood culture, and sputum culture Gleckman R., et al. 1988, J. Clin. Microbiol. 26(5):846-849

## How should Sputum Gram stain results be used?

- Gram stain results should be used to determine the adequacy of a sputum  specimen to produce an interpretable culture that may lead to the etiology of the LRT infection.

## Should Gram stain be part of inclusion criteria? 

## Should Gram stain be part of inclusion criteria?

- It is important not to overlook the point that pneumonia is a clinical diagnosis.  Microbiology procedures are only of value for attempting to establish an etiologic diagnosis, not for confirming or disproving the presence of pneumonia; the latter is determined on the basis of the patient’s clinical and radiographic findings.

## Interpretation of Sputum Cultures

- Culture results should be interpreted by correlating clinical observations with the results of direct examination, cytology screening, and quantitative or semiquantitative culture, as well as the pathogenic potential of the organism(s) recovered.

## Clinical Microbiology Group

- Albert  Sheldon, Ph.D.
- Frederick  Marsik, Ph.D
- Harold Silver, B.S.
- Peter Dionne, M.S.
- James King, Ph.D.

## Thought of the Day

- “Expectorated sputum represents the time-honored but often most controversial specimen used to identify pathogens in the lower respiratory tract.”
- CUMITECH 7A, American Society for Microbiology