Figures
- Figure 1–
Pneumocystis jirovecii PCR values compared with direct immunofluorescence (IF) testing in bronchoalveolar lavage fluid. a) Correlation between semiquantitative estimation of P. jirovecii density in direct IF staining and PCR values. Semiquantitative microscopy results are provided (number of cysts/trophozoites per field of vision, magnitude 200×) with the following scores: IF+: few (<1); IF++: many (1–10); and IF+++: abundant (> 10). b) Receiver-operating characteristic curve showing positive PCR value versus definitive Pneumocystis pneumonia (PCP) as defined by positive IF testing. c) P. jirovecii PCR values in cases of positive IF testing (definitive PCP) and negative IF testing. Two cut-offs are shown. At >1,450 pathogens·mL−1, the positive predictive value for positive IF testing (and definitive PCP) is 98.0%. At 1–85 pathogens·mL−1, the negative predictive value is 100% for positive IF testing. At 85–1,450 pathogens·mL−1, PCR is not able to predict IF test results. In this case, additional IF testing is helpful in diagnosing definitive PCP. AUC: area under the curve. *: p<0.05, **: p<0.001 (nonparametric Mann–Whitney U-test).
- Figure 2–
Decision-making algorithms in the diagnosis and therapy of Pneumocystis pneumonia (PCP) in HIV-negative patients with clinically suspected PCP. On the basis of our data, the “immunofluorescence (IF) first” algorithm should be preferred for logistical and economic reasons. IF allows rapid diagnosis of PCP, and in cases of IF negativity, PCR should be performed to exclude PCP. However, the use of PCR as first test is theoretically possible and presented as an alternative. Note that quantitative PCR is not able to differentiate between colonisation and infection: therefore, the PCR test should be performed only in cases of PCP suspicion. Pos: positive; Neg: negative.
- Figure 3–
Definitive clinical judgment according to PCR value with positive Pneumocystis jirovecii PCR in bronchoalveolar lavage fluid (BALF). Definitive Pneumocystis pneumonia (PCP) was defined as positive immunofluorescence (IF) testing (patient group 1; table 1). In group 2, of 171 IF-negative patients, a positive PCR result was detected in 18. With extensive clinical review, these cases were diagnosed either as possible PCP or PCP excluded. No statistical significance was detected for P. jirovecii pathogen density in BALF; therefore, PCR testing in cases of negative IF testing does not allow discrimination between possible PCP and P. jirovecii colonisation (PCP excluded). #: p=0.01; ***: p<0.001 (nonparametric Mann–Whitney U-test).
Tables
- Table 1– Patient characteristics
Characteristics Group 1: IF positive Group 2: IF negative Subjects 71 171 Age yrs 58.92 (17–83)±15.89 56.68 (15–82)±13.55 Male/female 45/26 (63.4) 109/62 (63.7) Immunocompromised 71 (100) 136 (79.5) Autoimmune disease# 18 (25.3) 53 (31.0) Rheumatoid arthritis 4 17 Giant cell arteritis 6 0 ANCA-positive vasculitis 4 2 Systemic sclerosis 0 9 Granulomatosis with polyangiitis 0 6 Other 6 20 Solid organ transplantation# 28 (39.4) 37 (21.6) Liver 1 6 Heart 10 15 Kidney 16 14 Lung 0 5 >1 organ 1 1 Active haemato-oncological disease# 23 (32.4) 41 (24.0) Solid tumour 3 6 Acute myeloid leukaemia 1 9 Acute lymphoblastic leukaemia 1 2 Chronic lymphatic leukaemia 3 4 Hodgkin’s lymphoma 3 2 Non-Hodgkin’s lymphoma 12 14 Multiple myeloma 2 9 Other haematological disease 0 9 Other cause of immunosuppression# 2 (2.8) 5 (2.9) Death 18 (25.4) 16 (9.34) Pneumocystis diagnosis Definitive PCP (positive IF testing) 71 (100) 0 (0) PCR Pneumocystis jirovecii results Positive qualitative PCR 71 (100) 18 (10.5) Quantitative PCR (log10) in case of positivity median (range) 3.8 (1.94–6.17) 2.18 (1.15–4.72) Data are presented as n, median (range)±sd, n (%), unless otherwise stated. IF: immunofluorescence; ANCA: antineutrophil cytoplasmic antibody; PCP: Pneumocystitis pneumonia. #: the most relevant immunosuppression is chosen. For the specified diagnoses, more than one diagnosis per patient is possible.
- Table 2– Results of the diagnostic procedure according to the performance of the currently recommended standard diagnostic approach with immunofluorescence (IF) as first test
First test Second test Results IF PCR Total subjects Definitive PCP (IF test positive) Possible PCP PCP excluded Positive Positive 71 (100) 71 (100) 0 0 Negative 0 0 Negative Positive 18 (10.5) 0 3 (16.6) 15 (83.3) Negative 153 (89.5) 0 1 (0.65)# 152 (99.3) Data are presented as n (%). PCP: Pneumocystitis pneumonia. #: PCP could not be formally ruled out in only one patient with rheumatoid arthritis and lung involvement. The treating physicians prescribed 3-week therapy against Pneumocystis jirovecii, but judged the acute lung disease as “more likely to be caused by autoimmune disease exacerbation rather than PCP” in the discharge letter. Retrospective reviewers unaware of the PCR result agreed with this interpretation.
- Table 3– Results of the diagnostic procedure according to a theoretical approach based primarily on quantitative PCR Pneumocystis jirovecii as the first test#
First test Second test Results PCR P. jirovecii copies·mL−1 IF Total subjects Definitive PCP (IF test positive) Possible PCP PCP excluded >1450 Positive 50 (98.0) 50 (100) 0 0 Negative 1 (2.0) 0 0 1 (100) 85–1450 Positive 21 (70) 21 (100) 0 0 Negative 9 (30) 0 2 (22.2) 7 (77.7) 1–85 Positive 0 0 Negative 8 (100) 0 1 (12.5) 7 (87.5) Negative Positive 0 0 Negative 153 (100) 0 1 (0.65)¶ 152 (99.3) Data are presented as n (%). IF: immunofluorescence; PCP: Pneumocystitis pneumonia. #: definitive conclusions are possible only with high copy numbers (>1,450 copies·mL−1) and negative results. In the range 1–1,450 copies·mL−1, quantitative PCR is not able to differentiate between “possible PCP” and “PCP excluded” and clinical suspicion remains essential. ¶: PCP could not be formally ruled out in only one patient with rheumatoid arthritis and lung involvement. The treating physicians prescribed 3-week therapy against Pneumocystis jirovecii, but judged the acute lung disease as “more likely to be caused by autoimmune disease exacerbation rather than PCP” in the discharge letter. Retrospective reviewers unaware of the PCR result agreed with this interpretation.
Supplementary material
Files in this Data Supplement:
- Supplementary material -
Bronchoalveolar lavage (BAL) processing and polymerase chain reaction (PCR) methods
Direct IF testing
Real-time PCR
Table 1
e-Table 1
- Supplementary material -
