Accurate antimicrobial susceptibility testing is vital for patient care and surveillance of emerging antimicrobial resistance. The National Committee for Clinical Laboratory Standards (NCCLS) outlines generally agreed upon guidelines for reliable and reproducible results. In January 1997 we surveyed 320 laboratories participating in the New York State Clinical Evaluation Program for General Bacteriology proficiency testing. Our survey addressed compliance with NCCLS susceptibility testing guidelines for bacterial species designated a problem (Staphylococcus aureus and Enterococcusspecies) or fastidious (Streptococcus pneumoniae,Haemophilus influenzae, and Neisseria gonorrhoeae) organism. Specifically, we assessed compliance with guidelines for inoculum preparation, medium choice, number of disks per plate, and incubation conditions for disk diffusion tests. We also included length of incubation for S. aureus andEnterococcus species. We found overall compliance with the five characteristics listed above in 80 of 153 responding laboratories (50.6%) for S. aureus and 72 of 151 (47.7%) laboratories for Enterococcus species. The most common problem was an incubation time shortened to less than 24 h. Overall compliance with the first four characteristics was reported by 92 of 221 (41.6%) laboratories for S. pneumoniae, 49 of 163 (30.1%) laboratories for H. influenzae, and 11 of 77 (14.3%) laboratories for N. gonorrhoeae. Laboratories varied from NCCLS guidelines by placing an excess number of disks per plate. Laboratories also reported using alternative media forEnterococcus species, N. gonorrhoeae, andH. influenzae. This study demonstrates a need for education among clinical laboratories to increase compliance with NCCLS guidelines.

A genomics-based PCR method was developed and used to test specimens from patients involved in a large outbreak of Mycoplasma pneumoniae in a closed religious community in New York State. New P1 adhesin gene primers were designed to bind to 9 of 10 target sequences in the repetitive-element sequences obtained from the whole genome sequence of M. pneumoniae. This PCR method had a sensitivity of 0.006 CFU and a specificity of 100% for M. pneumoniae. The PCR was validated by testing a subset of patient samples by culture and comparing the results to those obtained by PCR. Of the initial 280 samples tested, 73 were positive by PCR and 22 were positive by culture. All samples positive by culture were also positive by PCR. Follow-up testing of selected patients 3 to 6 weeks after antibiotic treatment revealed that eight samples remained positive by PCR and that three samples remained positive by culture. Additionally, no nonspecific PCR inhibition was detected as a result of the specimen type, transport medium, or sample preparation methodology. The study demonstrates that the PCR described here is a rapid, sensitive, and specific method for the identification of M. pneumoniae and was helpful for the detection and monitoring of the outbreak.Mycoplasma pneumoniae is primarily a respiratory pathogen that is responsible for approximately 15 to 20% of all community-acquired pneumonias (8). The clinical presentation of patients with M. pneumoniae infection is not significantly different from that of patients with infections caused by other respiratory pathogens such as Chlamydia pneumoniae (22), so diagnosis of infection relies primarily on laboratory testing. M. pneumoniae is, however, a fastidious organism, requiring a laborious effort and 21 days or more for growth in culture. Likewise, serology is lacking in sensitivity, has questionable specificity (22), and is dependent on specific collection times relative to the onset of illness (5). DNA probes have also been used, but cross-reactivity between M. pneumoniae and Mycoplasma genitalium has been observed, and methods that use these probes also lack sensitivity (4).Numerous PCR approaches have been developed to provide a rapid, sensitive method for the detection of M. pneumoniae. PCR targets have included the P1 adhesin gene (3,4,5,12,17,21), the ATPase operon (11), genomic clones (2, 7), or a combination of these (1). The P1 adhesin gene is responsible for cytadherence, which is necessary for colonization and infection of host cells by M. pneumoniae (22). This gene is also an intriguing target for PCR because of its repetitive nature within the M. pneumoniae genome. Approximately 8% of this genome is composed of repetitive DNA elements with regions homologous to the P1 adhesin gene (10). Use of the entire genomic DNA sequence enabled us to design primers that would theoretically amplify the maximum number of target molecules, resulting in an improved sensitivity of the PCR for M. pneumoniae.A large outbreak of respiratory illness occurred in a closed religious communit...