title: Laboratory implementation of a rapid three-stain technique for detection of microorganisms from lower respiratory specimens.
abstract: A rapid, cost-effective method for the evaluation of lower respiratory specimen has become increasingly important in the diagnosis of pulmonary diseases in immunocompromised patients. In the past, the technically demanding, time-consuming, and expensive Gomori-methenamine-silver (GMS) stain was the principal means for the evaluation of these specimens. In this study, we compared the GMS stain with a new rapid, three-stain protocol for the evaluation of lower respiratory specimens. Lower respiratory specimens were obtained by bronchoalveolar lavage (BAL). Conventional Wright/Giemsa and Gram stains were utilized, as well as a contemporary strain, calcofluor white (CW). A cell count was performed on the BAL specimens, and cytospins were stained by the three stains. The calcofluor white-stained slides were examined with an epi-fluorescent microscope, whereas the other stains were evaluated with a conventional light microscope. Gomorimethenamine-silver (GMS), acid-fast bacillus (AFB), and Papanicolaou (PAP) stains were performed as controls. Thirty-two BAL procedures were performed in 20 (63%) male patients and 12 (37%) female patients. The clinical diagnosis was pneumonia in 31% of the patients, malignant hematologic disease in 28%, acute respiratory distress syndrome (ARDS) in 9%, and acquired immunodeficiency syndrome (AIDS) in 28%. Of these specimens, 78% were adequate for interpretation and 22% were inadequate. Bacteria were found in 50% (16/32) of all BALs, fungi were found in 9% (3/32), and Pneumocystis carinii was found in 9% (3/32). Gram-positive bacteria were most frequently found in patients with pneumonia (80%, 4/5), whereas P. carinii was identified in patients with AIDS. There were no false-positive results. One CW stain was equivocal for P. carinii due to high fluorescent background. Laboratory implementation of the rapid, three-staining technique was accomplished without difficulty in microbiology and hematology laboratory sections. Specimen evaluation with the rapid staining protocol was technically easy to perform; however, experience in ultraviolet fluorescent microscopy was crucial for interpretation of CW stain. All results were available in 2 hr, cost was reduced by 30%, and the assays were available 7 days/week. Further studies are ongoing to substantiate the sensitivity, specificity, and predictive value of this technique, as well as clinical guidelines for its optimal utilization.
authors.affiliation: Department of Pathology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown 26506, USA.
authors.forename: M
authors.initials: M
authors.lastname: Maymind
authors.forename: J G
authors.initials: JG
authors.lastname: Thomas
authors.forename: H L
authors.initials: HL
authors.lastname: Abrons
authors.forename: R S
authors.initials: RS
authors.lastname: Riley
mesh_terms: D000328:Adult; D000368:Aged; D001228:Aspergillosis / Q000175:diagnosis*; D001232:Aspergillus fumigatus / Q000302:isolation & purification*; D018893:Bronchoalveolar Lavage; D002177:Candidiasis / Q000175:diagnosis*; D048429:Cell Size; D002675:Child, Preschool; D005260:Female; D006094:Gram-Positive Bacteria / Q000302:isolation & purification; D006801:Humans; D008297:Male; D008875:Middle Aged; D011020:Pneumonia, Pneumocystis / Q000175:diagnosis* / Q000382:microbiology; D013194:Staining and Labeling