ABSTRACT
We compared the FXG™: RESP (Asp +) real-time PCR assay (Myconostica Ltd) with two microscopic staining methods (direct immunofluorescence [IFA] and calcofluor white) for the detection of Pneumocystis jirovecii in 411 respiratory specimens submitted for P. jirovecii examination. We considered the specimen to be microscopically positive if the organism could be visualized through the use of either IFA or calcofluor white. A second, published real-time PCR assay targeting the cdc2 gene of P. jirovecii was used to adjudicate those specimens that were microscopically negative but Myconostica PCR positive. The Myconostica PCR positive samples were deemed to be true positives if they were concordant with microscopically positive results or if they were positive by the second PCR assay. As a result, the Myconostica PCR assay was found to be more sensitive than the two microscopy methods in detecting P. jirovecii (10.5% true positivity rate by PCR, 8.0% by immunofluorescence, and 7.1% by calcofluor white). The Myconostica PCR assay showed 93.5% sensitivity, 95.1% specificity, 70.5% positive predictive value, and 99.1% negative predictive value. Its high negative predictive value suggests a role of the Myconostica PCR assay in ruling out Pneumocystis pneumonia.
Subject(s)
Benzenesulfonates/metabolism , Fluorescent Antibody Technique, Direct/methods , Microbiological Techniques/methods , Pneumocystis carinii/isolation & purification , Pneumonia, Pneumocystis/diagnosis , Real-Time Polymerase Chain Reaction/methods , Staining and Labeling/methods , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Male , Microscopy/methods , Middle Aged , Mycology/methods , Pneumonia, Pneumocystis/microbiology , Sensitivity and Specificity , Young AdultABSTRACT
Amoebic keratitis causes significant ocular morbidity in contact lens wearers. Current diagnostic methods for amoebic keratitis are insensitive and labor-intensive and have poor turnaround time. We evaluated four laboratory methods for detection of acanthamoebae in clinical specimens. Deidentified, delinked consecutive specimens from patients with suspected amoebic keratitis were assayed for acanthamoebae by direct smear analysis, culture, and PCR using two different primer sets specific for Acanthamoeba ribosomal DNA. The consensus reference standard was considered fulfilled when the results for any two of the four tests were positive, and the outcome measures were sensitivity and specificity. Of 107 specimens assayed over an 18-month period, 20 were positive for acanthamoebae. The sensitivity and specificity of each assay were as follows, respectively: for smear analysis, 55% (95% confidence interval [CI], 33.2 to 76.8%) and 100%; for culture, 73.7% (95% CI, 54.4 to 93.0%) and 100%; for PCR using Nelson primers, 90% (95% CI, 76.9 to 100%) and 90.8% (95% CI, 84.7 to 96.9%); and for PCR using JDP primers, 65% (95% CI, 44.1 to 85.9%) and 100%. Nelson primer PCR demonstrated a single-organism level of analytic sensitivity. The performance characteristics of the assays varied by specimen type, with contact lenses and casings showing the highest rates of detectable acanthamoebae and the highest diagnostic sensitivities for direct smear analysis, culture, and JDP primer PCR, though these results are based on small numbers and should be interpreted cautiously. These findings have important implications for clinicians collecting diagnostic specimens and for diagnostic laboratories, especially in outbreak situations.