280. Intensive Examination of Sputum for the Presence of Streptococcus pneumoniae (Spn), Including PCR Hybridization of 16S rRNA in Patients with Community-Acquired Pneumonia (CAP)
Session: Poster Abstract Session: Diagnostic Microbiology; Novel Molecular Methods
Thursday, October 3, 2013
Room: The Moscone Center: Poster Hall C
Background: Identification of Spn as a cause of CAP has declined since the preantibiotic era. Current microbiologic techniques may not detect the organism in sputum cultures. Enhanced methods, using traditional microbiologic techniques and/or 16S rRNA hybridization of sputum or culture plates for pneumococcal 16S ribosomal RNA may facilitate diagnosis.

Methods: In Phase I, sputum cultures from patients (pt) with CAP, enriched for those that were positive for Spn, were studied as follows.  100-200 mcl of sputum and a loopful of bacterial colonies from the area of heavy growth on an overnight culture were analyzed to detect 16S rRNA of Spn (Accuprobe; Gen-Probe, San Diego, CA).  In phase II, a more intense search was made of sputum culture plates by obtaining a loop of heavy growth on a blood agar plate and replating on blood agar with a careful search of alpha hemolytic colonies for colonial morphology and optochin susceptibility and also analyzing by Accuprobe. Values >50,000 were regarded as positive and <25,000 as negative. 

Results: In phase I, sputum from 34 patients was studied. Routine sputum culture identified Spn in 8; Accuprobe of 5 of these culture plates (62.5%) documented the presence of Spn (>50,000 counts).  In an additional 2 samples that were culture negative for Spn, Accuprobe on the plate was positive.  Accuprobe directly on sputum was positive in 8 of the 8 that yielded Spn by culture and in an additional 9 that were negative on sputum culture (including 2 that were culture negative but Accuprobe positive on the plate).  In Phase II, sputum from 6 pneumonia patients was studied. Routine culture did not yield Spn. Careful examination and subculture of the blood agar plates identified Spn in 3 (50%).  Accuprobe of 2 of these was positive and yielded a result at the upper limit of negative (24,520 counts) in the third. Accuprobe directly on sputum was not positive in any of these 6 samples.

Conclusion: More intensive study of sputum using classical microbiological techniques and/or molecular probe is likely to identify Spn in a greater proportion of pt with CAP. Additional samples are planned to reach 40 pt in phase II.

Daniel Musher, MD, FIDSA1,2, Charles Stager, PhD1,2 and Carlo Zuno, MD1,2, (1)Michael E. Debakey VA Medical Center, Houston, TX, (2)Baylor College of Medicine, Houston, TX


D. Musher, None

C. Stager, None

C. Zuno, None

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