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. 2015 Jun 3;2(3):ofv080.
doi: 10.1093/ofid/ofv080. eCollection 2015 Sep.

Use of Oropharyngeal Washes to Diagnose and Genotype Pneumocystis Jirovecii

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Free PMC article

Use of Oropharyngeal Washes to Diagnose and Genotype Pneumocystis Jirovecii

Jonathan J Juliano et al. Open Forum Infect Dis. .
Free PMC article

Abstract

Pneumocystis jirovecii is a symbiotic respiratory fungus that presents in 2 clinical forms: pneumonia in immunocompromised patients or colonization, defined by the presence of the organism without associated clinical symptoms. Currently, diagnosis requires invasive bronchoscopy, which may not be available in some settings and is inappropriate for detecting colonization in healthy individuals. Noninvasive diagnostic techniques and molecular strain typing tools that can be used on these samples are critical for conducting studies to better understand transmission. We evaluated 2 real-time polymerase chain reaction (PCR) assays targeting dihydropteroate synthase and the major surface glycoprotein for detection in 77 oropharyngeal washes (OPWs) from 43 symptomatic human immunodeficiency virus-infected patients who underwent bronchoscopy. We also evaluated the ability of a new microsatellite (MS) genotyping panel to strain type infections from these samples. Each PCR used individually provided a high sensitivity (>80%) for detection of pneumonia but a modest specificity (<70%). When used in combination, specificity was increased to 100% with a drop in sensitivity (74%). Concentration of organisms by PCR in the OPW tended to be lower in colonized individuals compared with those with pneumonia, but differences in concentration could not clearly define colonization in symptomatic individuals. Oropharyngeal wash samples were genotyped using 6 MSs with ≥4 alleles successfully genotyped in the majority of colonized patients and ≥5 alleles in patients with pneumonia. The MS profile was consistent over time within patients with serial OPWs analyzed. Microsatellite genotyping on noninvasive samples may aid in studying the molecular epidemiology of this pathogen without requiring invasive diagnostic techniques.

Keywords: microsatellite; molecular epidemiology; pneumocystis; real-time PCR.

Figures

Figure 1.
Figure 1.
Concentration of Pneumocystis jirovecii in the extracted DNA from oropharyngeal washes (OPWs) of colonized (polymerase chain reaction [PCR] positive/bronchoalveolar lavage [BAL] or induced sputum [IS] negative) versus infected (PCR positive/BAL or IS positive) samples. The figure shows a box and whiskers plot of the concentration of P jirovecii in the extracted DNA from colonized and infected individuals as determined by the dhps real-time PCR. The box represents the 25th to 75th percentile, and the whiskers represent the minimum and maximum values (t test; P > .05). The major surface glycoprotein PCR data were not analyzed in a similar fashion because there were insufficient numbers of PCR-positive samples in colonized patients, and no standard curve was included in the PCR to allow for accurate quantification.
Figure 2.
Figure 2.
Longitudinal analysis of microsatellites (MSs) from oropharyngeal washes (OPWs) from individual patients. The MS genotypes from longitudinal OPW samples collected from individuals are shown. Within A and B, each subpanel represents data about allele sizes detected from 1 MS. The x-axis shows the day of collection during the illness. The y-axis shows the repeat size of the detected alleles. On a given day, each MS can have more than 1 allele detected, representing polyclonal samples. The MS repeat size of the majority allele (largest peak detected) is shown by the red circle. The MS repeat size of minor alleles are shown with blue squares or triangles. A, A relatively simple pattern over a 2-day period in which genotypes are regularly repeated between samples. B, Genotypes from 7 samples collected over a 16-day period. Both infections are polyclonal with at least 3 clones in the infections. However, B shows the dynamic nature of allele genotyping based on timing of sampling.
Figure 3.
Figure 3.
Impact of changing peak calling threshold. A, The microsatellite (MS) genotyping profile for a MS longitudinally in 1 patient using 33% height of the dominant allele as the cutoff for allele calling (the color scheme is the same as in Figure 2). B, The same information with additional minor peaks being called (green circles) using a 10% height cutoff.

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