Clinical significance of Pneumocystis jirovecii DNA detection by real-time PCR in hematological patient respiratory specimens.

Dear Editor,

We read with interest a recent paper in this Journal by Luzatti and colleagues, who explored the significance of the presence of Herpes simplex virus (HSV) DNA in lower respiratory tract (LRT) specimens for the diagnosis of HSV pneumonia in immunocompromised patients. The authors underlined the difficulty in gauging the clinical relevance of such a laboratory finding in the absence of histopathological data, as HSV shedding in the LRT may occur in the absence of disease. The interpretation of real-time PCR results for diagnosis of Pneumocystis jirovecii (PJ) pneumonia (PJP) faces an analogous challenge, since the presence of PJ DNA in LRT may reflect colonization (carriage) rather than infection. There is limited information on the clinical value of PJ real-time PCR in diagnosing PJP in patients with hematological diseases;3, 4, 5, 6 this is exceedingly challenging as the sensitivity of direct examination procedures is suboptimal due to low fungal burdens.

Here, we report on our experience on this matter. A total of 219 episodes of pneumonia occurring in 192 consecutive patients with hematological disorders in which PJP was considered in the differential etiological diagnosis were included. Of these, 127 episodes developed in patients undergoing either allogeneic hematopoietic stem cell transplantation-allo-HSCT- (n = 86) or autologous-HSCT (n = 19), and 92 in non-transplant patients (acute leukemia, n = 61; lymphoma, n = 16; chronic leukemia, n = 8; others, n = 2). The patients were attended at the Hospital Clínico Universitario-HCU-(n = 100) or at the Hospital Universitario Politécnico “La Fe” –HLF- (n = 92) between June 2014 and August 2019. No patients in the cohort tested positive for HIV. This study was approved by the respective hospital ethics committee and informed consent was obtained from all patients.

A single specimen per episode was available for diagnosis (BAL fluids, n = 179; sputa, n = 22; TA, n = 17 and bronchial biopsy, n = 1). The RealCycler PJIR kitⓇ (Progenie Molecular, Spain) was used at HCU, and the Pneumocystis jirovecii Real Time PCR Detection (CerTest Biotech; Zaragoza, Spain) was employed at HLF (see footnote in Table 1 ). Both assays target the large sub-unit of ribosomal (mtLSU) RNA gene. Preliminary experiments using 5 BAL specimens indicated that both assays yield comparable PCR cycle thresholds (CTs) (median, 28.2, range, 26.4–32.3 vs. median 27.5; range, 26.3–33.1, respectively; P = 0.89).

Table 1

Characteristics of patients testing positive by real-time PCR for Pneumocystis jirovecii according to their final clinical diagnosis.

Parameter	Pneumocystis jirovecii pneumoniaa		P valueb
	Yes (n = 12)	No (n = 15)
Hematological disease in non-transplant patients
Acute myeloid leukemia	3	3	0.97
Non-Hodgkin's lymphoma	2	1
Acute lymphocytic leukemia	2	2
Chronic lymphocytic leukemia	1	1
Type of hematopoietic stem cell transplantation
Allogeneic	1	7	0.07
Autologous	3	1
Detection/recovery of other microorganisms in respiratory specimens
Yes	3	14	0.001
No	9	1
Specimen type in which Pneumocystis jirovecii was detectedc
Bronchoalveolar fluid	6	12	0.45
Sputa	5	2
Tracheal aspirate	1	1
Antibiotic prophylaxis against Pneumocystis jiroveciid
Yes	0	4	0.03
No	12	11
Treatment with trimethoprim-sulfamethoxazole upon detection of Pneumocystis jirovecii DNA by PCR	12	11	0.73
Corticosteroid use in the previous month to specimen sampling
Yes	2	4	0.66
No	10	11
Oxygen therapy
Yes	9	13	0.63
No	3	2
Exitus
Yes	4e	8	0.44
No	8	7

The probability of Pneumocystis jirovecii (PJ) pneumonia (PJP) for each patient was retrospectively evaluated by an expert committee including infectious diseases and microbiology specialists at both centers, on the basis of (i) documented PJ presence in respiratory specimens by microscopy; (ii) compatibility of clinical signs and symptoms (at least 2 of the following: subtle onset of progressive dyspnea, pyrexia, nonproductive cough, hypoxaemia and chest pain), (iii) compatible (suggestive) radiological findings (chest radiograph and/or high-resolution computed tomographic scan detection of interstitial opacities and/or diffuse infiltration infiltrates); (iv) complete resolution of symptoms after a full course of anti-PJP treatment; (v) absence of alternative diagnosis. The efficacy of therapy was assessed on a daily basis. PJP was ruled out if real-time PCR for PJ tested negative, or if clinical recovery occurred in the absence of PJ-targeted antimicrobial therapy. PJ colonization (carriage) was the most likely possibility when patients did not meet the above criteria and an alternate diagnosis was made.

Frequencies were compared using the χ2 test (Fisher exact test) for categorical variables. Two-sided exact P values were reported and P values ≤ 0.05 were considered statistically significant. The data were analyzed with the SPSS (version 20.0) statistical package.

Respiratory tract specimens were obtained following conventional procedures. Specimens were examined for presence of ascus or trophic forms of PJ by microscopy following blue toluidine, calcofluor white or Grocott's methenamine silver staining. Cytospin preparations were prepared from BAL specimens for direct examination. Sputa and TA samples were mixed v/v with Sputasol (Oxoid, UK) and vortexed for 5 min. All samples were centrifuged at 3000 g for 10 min, and the pellets were resuspended 1/10 in 0.9% NaCl for further processing. For real-time PCR, DNA was extracted from 200 µL of specimens using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) on either QIA Symphony or EZ-1 platforms (Qiagen), following the manufacturer's instructions. At HCU, a commercially-available real-time PCR assay previously evaluated by others, the RealCycler PJIR kitⓇ (Progenie Molecular, Spain), which targets the mitochondrial large sub-unit of ribosomal (mtLSU) RNA gene, was used according to the manufacturer's instructions (http://www.progenie-molecular.com/PJIR-U-IN.pdf). At HLF, the commercially-available Pneumocystis jirovecii Real Time PCR Detection. (CerTest Biotech; Zaragoza, Spain), which also targets the large sub-unit of ribosomal (mtLSU) RNA gene, was employed following the manufacturer instructions (https://www.certest.es/wpontent/uploads/2019/02/VIASURE_Real_Time_PCR_Pneumocystis_jirovecii_sp1.pdf). At both centers PCR were performed in the Applied Biosystems 7500 fast real-time PCR platform (Applied Biosystems, CA, USA). PCR results were reported as positive or negative. For positive samples, threshold cycle (CT) values were also recorded. No standard curve was generated with a positive control for quantitative estimations.

Antimicrobial prophylaxis for PJP was performed with trimethoprim-sulfamethoxazole (TMP/SMX), one double-strength tablet (160 mg TMP/800 mg SMX) given 2 (in allogeneic HSCT patients) or 3 times a week with oral folic acid (15,16). Patients with suspicion of PJP according to the attending physician were treated with TMP/SMX 15–20 mg/kg (TMP) 75–100 mg/kg (SMX) per day for 2–3 weeks.

Antimicrobial prophylaxis against PJ and of PJP occurrence

Anti-PJ prophylaxis was in place in 114 episodes of pneumonia occurring in 85 transplant and 29 non-transplant patients, and not in the remaining 93 episodes (31 transplant and 62 non-transplant patients). This information could not be retrieved for 12 episodes

Anti-PJ prophylaxis was in place in 114 episodes of pneumonia occurring in 85 transplant and 29 non-transplant patients, and not in the remaining 93 episodes (31 transplant and 62 non-transplant patients). This information could not be retrieved for 12 episodes.

In all these cases, death was attributable to PJP.

All specimens tested negative by direct examination for PJ, whereas 27 were positive by real-time PCR (BAL, n = 18; sputa, n = 7, and TA, n = 2); Following stringent clinical, microbiological and imaging criteria (Table 1), PJP was deemed to be the most probable diagnosis in 12 episodes occurring in unique patients. No histopathological confirmation of PJP was available for any patient.

PCR CT values inversely correlate with fungal burden in the sample. which is higher in patients with PJP than in colonized individuals. Here, overall, PJ PCR CTs in specimens from patients with PJP tended to be lower than in PJ carriers (P = 0.39); when only BAL fluid specimens were considered, the difference reached statistical significance (median, 29.0; range, 26.4–34.7 vs. median 34.6; range, 30.0–41.0; P = 0.04). This finding is likely related to use of more standardized procedures for BAL fluid sampling. Receiver operating characteristic (ROC) curve analysis showed that a threshold CT value of 30.0 in BAL specimens displayed a sensitivity of 85.7% (95% CI, 45.0–100%) and a specificity of 80% (95% CI, 40.8–100%) for PJP diagnosis. A number of studies have established different CTs cut-offs for that purpose,6, 7, 8, 9. In our view, however, the variability in the performance of different PCR assays and sampling conditions, heterogeneity of patient populations, and in particular the lack of a PJ international standard material for PCR result normalization precludes defining a consensus universal threshold nowadays.

The absence of anti-PJ prophylaxis, treatment with corticosteroids and serum LDH levels ≥400 U/L have been shown to be associated with PJP. Here, patients not undergoing anti-PJ prophylaxis were more likely to display a clinically significant PJ PCR result (Table 1). In turn, ROC curve analysis indicated that a cut-off LDH value ≥400 U/L had a sensitivity of 81.8% (CI 95%, 59.0–100%) and specificity of 67% (95% CI, 34.0–99.3%) for PJP diagnosis. In univariate regression logistic models, serum LDH values ≥400 U/L were associated with a clinically significant positive PCR PJ result (OR, 9.0; 95% CI, 1.2–63.8; P = 0.02). In contrast, corticosteroid use within the month before sampling was not different between patients with clinically significant PJ detection and PJ carriers (Table 1).

Detection or recovery of other microbial agents (one or more) was documented in 17 of the 27 specimens testing positive by PJ PCR (Table 2 ). In line with a previous report, this microbiological finding was significantly less frequent (P = 0.001) in specimens from patients with PJP than in colonized patients; in fact, microbial co-detection was inversely associated with PJP in univariate logistic regression models (OR, 0.024; 95% CI, 0.002–0.26; P = 0.002).

Table 2

Microbiological findings in patients testing positive for Pneumocystis jirovecii DNA by real-time PCR.

Patient/clinical categorization	Specimen	Microorganisms detected or recovereda
1/PJP	BAL fluid	Respiratory syncitial virus type B/Candida albicans
2/PJP	BAL fluid	Escherichia coli
3/PJP	BAL fluid	Enterococus faecium
4/Carriage	Sputum	Rhinovirus
5/Carriage	BAL fluid	Aspergillus flavus
6/Carriage	BAL fluid	Pseudomonas aeruginosa
7/Carriage	BAL fluid	Coronavirus 229e
8/Carriage	Sputum	Parainfluenza virus type 3
9/Carriage	BAL fluid	E. coli
10/Carriage	BAL fluid	Respiratory syncitial virus type B/Aspergillus fumigatus
11/Carriage	BAL fluid	Parainfluenza virus type 3
12/Carriage	BAL fluid	Pseudomonas aeruginosa/Human metapneumovirus/Aspergillus fumigatus
13/Carriage	BAL fluid	Cytomegalovirus/Aspergillus fumigatus
14/Carriage	BAL fluid	Stenotrophomonas maltophilia/Human metapneumovirus
15/Carriage	BAL fluid	Aspergillus fumigatus
16/Carriage	TA	Parainfluenza virus type 3
17/Carriage	TA	Parainfluenza virus type 3

BAL, bronchoalveolar lavage; PJP, Pneumocysis jirovecii pneumonia; TA, tracheal aspirate.

As per our routine protocol, all specimens were examined by Gram and acid-fast bacilli stain. Samples were also examined for presence of respiratory viruses (RVs) using either the Luminex xTAG RVP Fast assay (Luminex Molecular Diagnostics, Austin, TX,USA) at HCU, or the CLART® PneumoVir assay (Genomica, Coslada, Spain) at both centers, as previously reported. Semiquantitative (sputa) and quantitative (BAL and TA) cultures for bacteria were performed on conventional media: bacterial loads >104 CFU/mL or >105 CFU/ml were deemed to be clinically relevant on BAL fluids and TA samples, respectively. Specimens were cultured on BCYE-alpha agar, BD (Becton Dickinson) MGIT® (Mycobacteria Growth Indicator Tube)/Lowenstein-Jensen agar slants and Sabouraud agar for recovery of Legionella pneumophila, Mycobacterium spp., and other fungal organisms, respectively. The Platelia™ Aspergillus Ag Kit (Bio-Rad, Hercules, CA, USA) was used for quantitation of Aspergillus spp. galactomannan in BAL fluid and serum specimens. All BAL fluid specimens underwent cytomegalovirus (CMV) PCR testing using the RealTime CMV PCR assay (Abbott Molecular) at HCU or the CMV R-GENE® assay (Biomerieux) at HLF, as previously reported.

Strengths of the current study are the following: (i) clinical categorization of PJP was based upon stringent criteria defined by a multidisciplinary team; (ii) only hematological patients were included; (iii) a comprehensive routine investigation of microbial causes of pneumonia other than PJ was conducted; (iv) the experience of two centers was collected. In addition to its retrospective nature, our study also has some limitations: (i) we cannot completely rule out that some patients categorized as being PJ carriers did in fact have PJP, as most of these patients received full courses of TMP/SMX in combination with antimicrobials targeting other microbial agents. The lack of standardized criteria for PJP diagnosis makes clinical misclassification of patients a potential drawback in studies such as ours, particularly when no positive microscopy or histopathology findings are available; (ii) although we evaluated over 200 patients, only 12 presumptively had PJP; (iii) two different commercially-available PCR assays were used across centers. Nevertheless, we found them to yield rather comparable CTs.

In summary, we found that a positive PJ PCR result in respiratory specimens from transplant and non-transplant hematological patients with pneumonia frequently reflects colonization rather than infection; PCR CT values in BAL fluids, serum LDH levels and lack of co-detection of other microorganisms potentially involved may be helpful in clinical categorization in the absence of positive by PJ microcopy results.

CRediT authorship contribution statement

José Luis Piñana: Writing - review & editing, Data curation, Conceptualization. Eliseo Albert: Formal analysis, Data curation. María Dolores Gómez: Writing - review & editing, Formal analysis. Ariadna Pérez: Writing - review & editing, Data curation. Juan Carlos Hernández-Boluda: Writing - review & editing, Data curation. Juan Montoro: Writing - review & editing, Data curation, Data curation. Miguel Salavert: Writing - review & editing, Data curation. Eva María González: Writing - review & editing, Formal analysis. Mar Tormo: Writing - review & editing. Estela Giménez: Writing - review & editing, Formal analysis, Data curation. Marta Villalba: Writing - review & editing, Data curation. Aitana Balaguer-Roselló: Writing - review & editing, Data curation. Rafael Hernani: Writing - review & editing, Data curation. Felipe Bueno: Writing - review & editing, Formal analysis, Data curation. Rafael Borrás: Writing - review & editing, Formal analysis. Jaime Sanz: Writing - review & editing, Data curation, Conceptualization. Carlos Solano: Writing - review & editing, Data curation, Conceptualization. David Navarro: Writing - review & editing, Writing - original draft.