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Quantitative real-time PCR and the (1 → 3)-β-d-glucan assay for differentiation between Pneumocystis jirovecii pneumonia and colonization
ORIGINAL ARTICLE
MYCOLOGY
Quantitative real-time PCR and the (1 fi 3)-b-D-glucan assay for differentiation between Pneumocystis jirovecii pneumonia and colonization Y. Matsumura1, Y. Ito2, Y. Iinuma3, K. Yasuma1, M. Yamamoto1, A. Matsushima1, M. Nagao1, S. Takakura1 and S. Ichiyama1 1) Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, 2) Department of Respiratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, and 3) Department of Infectious Diseases, Kanazawa Medical University, Kanazawa, Japan
Abstract We evaluated whether quantitative PCR (qPCR) and (1 fi 3)-b-D-glucan assays could be used to differentiate Pneumocystis pneumonia (PCP) from Pneumocystis jirovecii colonization in immunocompromised patients with pulmonary infiltrates. A total of 40 bronchoalveolar lavage samples and 107 induced sputum samples from 147 patients who were suspected of having PCP were obtained for PCR detection of P. jirovecii. Diagnoses of definite PCP, probable PCP, pneumonia with P. jirovecii colonization (colonization) and pneumonia without colonization (non-colonization) were made in 11, 42, 15 and 60 patients, respectively. A PCP diagnosis was undetermined in 19 patients. The copy numbers, determined using qPCR, were significantly higher in definite PCP and probable PCP patients than in colonized patients. The area under the receiver-operating characteristic curve (AUC), sensitivity and specificity for discriminating definite PCP from colonization were 0.96, 100.0% and 80.0%, respectively, at a cut-off value of 1300 copies/mL. The values for discriminating probable PCP from colonization were 0.71, 66.7% and 73.3%, respectively, at a cut-off value of 340 copies/mL. b-D-glucan levels were significantly higher in patients with both definite PCP and probable PCP than in colonized patients. The AUC, sensitivity and specificity for discriminating definite PCP were 0.91, 100.0% and 80.0%, respectively, at a cut-off value of 15.6 pg/mL. The values for discriminating probable PCP were 0.78, 76.2% and 73.3%, respectively, at a cut-off value of 6.0 pg/mL. Both qPCR and the b-D-glucan assay displayed high accuracy for discriminating colonization from definite PCP and displayed moderate accuracy for discriminating colonization from probable PCP. Keywords: Colonization, immunocompromised host, Pneumocystis jirovecii pneumonia, real-time PCR, b-D-glucan Original Submission: 22 March 2011; Revised Submission: 6 June 2011; Accepted: 11 June 2011 Editor: E. Roilides Article published online: 20 June 2011 Clin Microbiol Infect 2012; 18: 591–597 10.1111/j.1469-0691.2011.03605.x Corresponding author: S. Ichiyama, Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan E-mail: [email protected]
Introduction Pneumocystis pneumonia (PCP) is one of the most prevalent infections in human immunodeficiency virus (HIV)-infected patients [1]. PCP also occurs in non-HIV immunocompromised patients receiving transplantation, immunosuppressive therapy and antitumour chemotherapeutic agents [2]. The standard method for laboratory diagnosis of PCP is micro-
scopic visualization of Pneumocystis jirovecii in respiratory samples, including bronchoalveolar lavage (BAL) or induced sputum (IS). Non-HIV patients often develop PCP with a lower fungal burden than that of HIV-infected patients [3], and this lower fungal burden can result in false-negative results following microscopic examination [4]. Because of this problem, polymerase chain reaction (PCR), a highly sensitive technique, has been used for diagnosing PCP. However, Pneumocystis colonization, defined as the detection of the organism or its DNA without signs or symptoms of pneumonia, has been reported in several studies [5]. In addition, some patients with pulmonary infiltrates who were PCRpositive for P. jirovecii have been diagnosed as having pneumonia resulting from a different aetiology than PCP, and the
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positive PCR results were considered indicative of colonization with P. jirovecii [4–6]. Although a positive conventional PCR result cannot distinguish colonization from infection, several studies have shown that the copy number of the specific P. jirovecii gene that is measured using quantitative real-time PCR (qPCR) is significantly higher in patients with PCP than in colonized patients [6–12]. Of the various serum markers tested, it has also been reported that the serum (1 fi 3)-b-D-glucan (b-Dglucan) level is the best test for PCP diagnosis [13–16]. To date, however, only one study [17] with a limited number of patients has assessed the b-D-glucan assay for discrimination of PCP from colonization. In this study, we evaluated whether qPCR and the b-Dglucan assay could be used to discriminate PCP from colonization with P. jirovecii in immunocompromised patients with pulmonary infiltrates.
Materials and Methods Patients and clinical samples
A total of 56 BAL and 161 IS samples from 217 patients suspected of PCP were prospectively obtained for Pneumocystis PCR at Kyoto University Hospital, a 1182-bed tertiary care hospital, between January 2008 and December 2010. In cases in which multiple samples were taken from a patient, the sample from the first episode of PCP was studied. Fifty-six patients without new ground-glass opacities by chest computed tomography and 14 immunocompetent patients were excluded. The remaining 147 eligible immunocompromised patients had new ground-glass opacities and clinical presentations such as fever, cough, sputum, dyspnoea or leucocytosis. These patients provided 40 BAL samples and 107 IS samples. The Ethics Committee of Kyoto University Graduate School and the Faculty of Medicine approved this study and waived the need to obtain informed consent from each patient. Diagnosis of PCP
The diagnosis of definite PCP was established by the microscopic identification of P. jirovecii using Gomori methenamine silver staining. A diagnosis of probable PCP was made when the patient did not have microscopically visible P. jirovecii but had a clinical presentation compatible with PCP, including complete resolution of pulmonary infiltrates after a full course of anti-PCP treatment [4]. The remaining non-PCP patients who had positive qPCR results without any signs or symptoms of PCP and who improved without anti-PCP treatment were diagnosed as ‘pneumonia with P. jirovecii colonization’ (colonization) [5]. Non-PCP patients having nei-
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ther observable P. jirovecii nor positive qPCR results were classified as ‘pneumonia without P. jirovecii colonization’ (noncolonization). Patients who had positive PCR results and pulmonary infiltrates that were resolved by a full course of antimicrobial treatment for concomitant organisms with anti-PCP drugs or who did not improve with or without anti-PCP treatment were classified as undetermined because it was difficult to discriminate between probable PCP and colonization for these patients. Clinical data
The clinical information acquired by a medical chart review included age, sex, underlying diseases, immunosuppressive therapies and prophylaxis for PCP during the month prior to sample collection, clinical symptoms, laboratory values, and the final diagnosis made by the physician. Hypoxia was defined as an arterial PaO2 < 70 mmHg on room air or a requirement for supplemental oxygen. Real-time qPCR
Respiratory samples were centrifuged at 1000 · g for 10 min. The pellet was resuspended in 200 lL of the original fluid, and DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany). DNA was eluted in a final volume of 100 lL. Forward (5¢-CATATGATTCTATATTAA TGGATGTGGAGA-3¢) and reverse (5¢-TTTATAGCAG GAATAACTCGAGAAATCT-3¢) primers were designed to amplify a 142-bp segment of the single-copy dihydropteroate synthase gene. Real-time PCR for P. jirovecii was performed in a 25 lL reaction volume containing 0.9 lM of each primer, 0.25 lM TaqMan probe (5¢-FAM-ATATCAATTATCGT CGCCC-MGB-3¢), 1 · TaqMan Gene Expression Master Mix (Applied Biosystems (ABI)), and 5 lL purified DNA. Thermal cycling was performed in an ABI PRISM 7000 Sequence Detection system (ABI) as follows: 2 min at 50C, 10 min at 95C, followed by 50 cycles of 15 s at 95C and 1 min at 60C. Quantification was performed using ABI PRISM software and was based on the extrapolation of data to standard curves, which were generated by the amplification of a tenfold dilution series of a plasmid standard (5–5 · 105 copies/ reaction). To generate the plasmids, the PCR-amplified product was cloned into the pCR-TOPO vector (Invitrogen, Carlsbad, CA, USA) and sequenced. A copy number of less than the detection limit of 5 copies/reaction was considered to be 1 copy/reaction. The quantified copy numbers of Pneumocystis are expressed as copy numbers per 1 mL of sample. b-D-glucan assay
Plasma b-D-glucan was measured with the b-glucan test WAKO (Wako Pure Chemical Industries, Tokyo, Japan).
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Matsumura et al. qPCR and b-D-glucan assays for P. jirovecii pneumonia
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Plasma samples were collected prior to the collection of BAL or IS samples and the initiation of treatment. The assay was performed at our institution on the same day that the plasma was obtained. Statistical analysis
Categorical variables were compared using the Fisher exact test. Continuous variables were compared using the Kruskal– Wallis test or the Mann–Whitney U-test. b-D-glucan values under the detection limit of 4.3 pg/mL were considered to be 4.3 pg/mL. Receiver-operating characteristic (ROC) curves for the b-D-glucan levels and copy numbers were constructed, and their optimal cut-off values were determined with the maximum Youden index. We compared the area under the curve (AUC) of the two ROC curves using the method described by Delong et al. [18]. A p value <0.05 was considered statistically significant. All statistical analyses were carried out using Stata version 11.2 (StataCorp, College Station, TX, USA).
Results A diagnosis of definite PCP, probable PCP, colonization, noncolonization or undetermined was made in 11, 42, 15, 60 and 19 patients, respectively. BAL samples were obtained from 7, 10, 6, 12 and five of these patients, respectively. All definite patients received anti-PCP treatment. The final
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diagnoses of the colonized patients included bacterial pneumonia (n = 4), interstitial pneumonia (n = 3), invasive aspergillosis (n = 2), atypical pneumonia (n = 1), acute respiratory distress syndrome (ARDS) due to sepsis (n = 1), tuberculosis (n = 1), pulmonary embolism (n = 1), cryptogenic organizing pneumonia (n = 1), and pulmonary alveolar proteinosis (n = 1). Among the non-colonized patients, the final diagnoses included interstitial pneumonia (n = 18), bacterial pneumonia (n = 10), ARDS due to sepsis (n = 9), drug-induced pneumonia (n = 5), pulmonary haemorrhage (n = 5), viral pneumonia (n = 3), tuberculosis (n = 2), pulmonary oedema (n = 1), invasive aspergillosis (n = 1), radiation pneumonitis (n = 1), lymphangitis carcinomatosa (n = 1), cryptogenic organizing pneumonia (n = 1), and unknown (n = 3). Clinical features
Table 1 shows the patient demographics and characteristics. Three definite patients and two probable patients had HIV infection, whereas none of the colonized or non-colonized patients did. An analysis of laboratory test results revealed significant differences in thrombocyte, C-reactive protein, blood urea nitrogen and total bilirubin levels between the four groups. Real-time qPCR
The qPCR and b-D-glucan assay results are presented in Fig. 1. All definite patients and 40 of the 42 probable patients were qPCR-positive. Copy numbers were significantly higher
TABLE 1. Patient demographics and clinical characteristics Definite PCP Characteristics Age, years Male sex Underlying disease Collagen vascular disease Solid malignancy Haematological malignancy Organ transplantation HIV infection Corticosteroids Immunosuppressive agents other than corticosteroids Anti-tumour chemotherapy PCP prophylaxis Hypoxia Laboratory findings Neutrophils, /mm3 Lymphocytes, /mm3 Thrombocytes, /mm3 C-reactive protein, mg/dL Albumin, mg/dL Creatinine, mg/dL Blood urea nitrogen, mg/dL Total bilirubin, mg/dL Lactate dehydrogenase, IU/L
Probable PCP
Colonized
Non-colonized
(n = 42)
(n = 15)
(n = 60)
p value
55 (40–65) 8 (73%)
63 (54–69) 23 (55%)
65 (54–71) 7 (47%)
60 (52–70) 35 (58%)
0.52 0.62
6 0 2 0 3 7 4
(55%) (0%) (18%) (0%) (27%) (64%) (36%)
26 (62%) 8 (24%) 7 (17%) 3 (7%) 2 (5%) 31 (74%) 23 (55%)
9 (60%) 2 (13%) 4 (27%) 2 (13%) 0 (0%) 11 (73%) 7 (47%)
30 (50%) 19 (32%) 7 (12%) 8 (13%) 0 (0%) 43 (72%) 23 (38%)
0.69 0.08 0.46 0.58 0.002 0.93 0.39
0 (0%) 0 (0%) 7 (64%)
10 (24%) 2 (5%) 26 (62%)
4 (27%) 2 (3%) 8 (53%)
14 (23%) 12 (20%) 44 (73%)
0.32 0.7 0.40
6200 (2400–9600) 800 (500–1000) 13.8 (12.3–20.9) 3.0 (1.1–6.8) 3.2 (2.6–3.3) 0.6 (0.5–0.7) 15 (10–16) 0.6 (0.4–0.8) 502 (275–598)
6400 (4100–11 100) 700 (400–1100) 20.3 (14.3–30.4) 5.9 (2.7–9.7) 3.1 (2.5–3.4) 0.7 (0.5–1.4) 18 (12–25) 0.5 (0.4–0.7) 330 (260–428)
56 000 (1400–7800) 400 (200–1300) 15.1 (6.3–21.3) 2.4 (0.6–9.7) 2.9 (2.4–3.6) 0.7 (0.5–1.4) 16 (14–23) 0.7 (0.5–0.8) 323 (277–381)
6350 (4225–10 600) 700 (200–1100) 13.1 (4.7–28.7) 8.2 (2.3–15.4) 2.8 (2.3–3.3) 0.8 (0.5–1.4) 23 (15–36) 0.8 (0.5–1.3) 308 (237–500)
0.41 0.77 0.04 0.049 0.70 0.41 0.04 0.049 0.50
(n = 11)
Data are presented as the n (%) or median (interquartile range). Some patients had two or more underlying diseases or conditions. PCP, Pneumocystis pneumonia; HIV, human immunodeficiency virus.
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p = 0.002
(a)
p = 0.02
p = 0.12
p = 0.03
(a)
p = 0.13
0.75 Sensitivity 0.50
6 4
0.25
2
Copy number b-D-glucan
0
Log10 copies (/mL of sample)
8
p = 0.02
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1.00
594
Probable Colonization PCP BAL
Definite PCP
Probable Colonization PCP IS
0.00
0.25
p = 0.001
(b)
0.75
1.00
0
0.25
100
Sensitivity 0.50
200
0.75
300
400
p = 0.25
0.50 1-specificity
1.00
p < 0.001
(b)
b-D glucan (pg/mL)
0.00
Definite PCP
Probable PCP
Colonization
Copy number b-D-glucan
Non-colonization
FIG. 1. P. jirovecii copy numbers (a) and b-D-glucan levels (b) according to patient group. In the box and whisker plots, the boxes contain
0.00
Definite PCP
0.00
0.25
50% of the sample data, with the median value indicated by a horizontal bar. The whiskers contain 1.5 · the interquartile range. (a)
0.50 1-specificity
0.75
1.00
FIG. 2. Receiver-operator characteristic curves of P. jirovecii copy
The copy numbers were significantly higher in the definite PCP
numbers and b- D-glucan levels for the diagnosis of PCP. (a) The def-
group when compared with the colonized group in both BAL and IS
inite PCP group was compared with the colonized group. The
samples. The copy numbers were not significantly different between
arrows indicate cut-off values of 15.6 pg/mL for b-D-glucan level and
the BAL and IS samples in the definite and probable PCP groups
1300 copies/mL for copy number. (b) The probable PCP group was
(p 0.57 and p 0.29), but were significantly different in the colonized
compared with the colonized group. The arrows indicate cut-off val-
group (p 0.02). (b) The b-D-glucan levels were significantly higher in
ues of 6.0 pg/mL for b-D-glucan level and 340 copies/mL for copy
both the definite and probable PCP groups when compared with the
number.
colonized group.
in the definite group (median 460 000 copies/mL, range 1400–15 000 000) than in the colonized group (median 180, range 10–15 000; p < 0.001). This difference was significant for both BAL (p 0.002) and IS (p 0.02) samples. The ROC curve (Fig. 2a) measured an AUC of 0.96, a sensitivity of 100.0% and a specificity of 80.0% for discriminating the definite group from the colonized group, with a cut-off value of 1300 (Table 2). The copy numbers were also significantly higher in the probable group (median 1300, range 10– 7 100 000) than in the colonized group (p 0.001). When
BAL and IS samples were analysed separately, the difference was not statistically significant (p 0.12 for BAL and p 0.13 for IS). The AUC, sensitivity and specificity were reduced to 0.71, 66.7% and 73.3%, respectively, with a cut-off value of 340 (Fig. 2b). The definite group had significantly higher copy numbers than the probable group (p < 0.001). The copy numbers were not significantly different between the BAL and IS samples in the definite and probable groups (p 0.57 and p 0.29, respectively), but they were significantly different in the colonized group (p 0.02). To discriminate the definite and probable groups from the colonized group, ROC analysis
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TABLE 2. Diagnostic performance of P. jirovecii copy numbers and b-D-glucan for PCP detection Test Copy number Definite PCP vs. colonized Probable PCP vs. colonized Definite and probable PCP vs. colonized b-D-glucan Definite PCP vs. colonized Definite PCP vs. non-colonized Definite PCP vs. colonized and non-colonized Probable PCP vs. colonized Probable PCP vs. non-colonized Probable PCP vs. colonized and non-colonized Definite and probable PCP vs. colonized and non-colonized
AUC (95% CI)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
0.96 (0.89–1.00) 0.71 (0.58–0.85) 0.76 (0.65-0.88)
100.0 66.7 73.6
80.0 73.3 73.3
78.6 87.5 90.7
100.0 44.0 44.0
0.91 0.94 0.93 0.78 0.81 0.80 0.83
100.0 100.0 100.0 76.2 76.2 76.2 81.1
80.0 90.0 88.0 73.3 83.3 81.3 81.3
78.6 64.7 55.0 88.9 76.2 69.6 75.4
100.0 100.0 100.0 52.4 83.3 85.9 85.9
(0.79–1.00) (0.88–0.99) (0.88–0.99) (0.65–0.90) (0.72–0.89) (0.72-0.88) (0.76-0.90)
The cut-off values for copy number were 1300 copies/mL (definite PCP) and 340 copies/mL (probable PCP and definite and probable PCP). When the following changes were made to the cut-off values, the PPV was calculated as follows: 340 copies/mL for definite PCP (78.6%), 1300 copies/mL for probable PCP (87.5%), and 1300 copies/mL for definite and probable PCP (91.4%). The cut-off values for b-D-glucan were 15.6 pg/mL (definite PCP) and 6.0 pg/mL (probable PCP and definite and probable PCP). When the following changes were made to the cut-off values, the PPV was calculated as follows: 6.0 pg/mL for definite PCP (vs. colonized, 78.6%; vs. non-colonized, 52.4%; vs. colonized and non-colonized, 44.0%), 15.6 pg/mL for probable PCP (vs. colonized, 89.3%; vs. non-colonized, 80.6%; vs. colonized and non-colonized, 73.5%), and 15.6 pg/mL for definite and probable PCP (80.0%). PCP, Pneumocystis pneumonia; AUC, area under the receiver-operator characteristic curve; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value.
produced an AUC of 0.76, a sensitivity of 73.6% and a specificity of 73.3%, with a cut-off value of 1300. b-D-glucan assay
The b-D-glucan levels were significantly higher in the definite group (median 39.5 pg/mL, range 15.7–352.7) than in the colonized group (median 4.3, range 4.3–61.5, p < 0.001) and the non-colonized group (median 4.3, range 4.3–160.4; p < 0.001, Fig. 1b). The ROC curves (Fig. 2a) produced an AUC of 0.91, a sensitivity of 100.0% and a specificity of 80.0% for discriminating the definite group from the colonized group and values of 0.94, 100% and 90.0%, respectively, for discriminating the definite group from the non-colonised group, with a cut-off value of 15.6 (Table 2). Similar to the results obtained for the definite group, the b-D-glucan levels were significantly higher in the probable group (median 23.1, range 4.3–1007) than in the colonized (p 0.001) and non-colonized groups (p < 0.001). The ROC curves (Fig. 2b) produced an AUC of 0.78, a sensitivity of 76.2% and a specificity of 73.3% for discriminating the probable group from the colonized group, and values of 0.81, 76.2% and 83.3%, respectively, for discriminating the probable group from the non-colonized group, with a cut-off value of 6.0. The b-D-glucan levels were similar between the definite and probable groups (p 0.25) and between the colonized and non-colonized groups (p 0.62). To discriminate the definite and probable groups from the colonized and non-colonized groups, ROC analysis produced an AUC of 0.83, a sensitivity of 81.1% and a specificity of 81.3%, with a cut-off value of 6.0. Among the four patients in the colonized group with a positive b-D-glucan level (>6.0 pg/mL), two had probable invasive pulmonary aspergillosis, whereas the others did not have any detectable fungal infections. Among the ten patients
with positive b-D-glucan levels in the non-colonized group, one had candidaemia, one had cryptococcaemia, one received haemodialysis, and four received albumin or intravenous immunoglobulin therapy. The others had no detectable fungal infections, bacteraemia or treatment with amoxicillinclavulanate. The AUC analyses showed that the qPCR and b-D-glucan assays had excellent and comparable diagnostic performances for discriminating the definite group from the colonized group (p = 0.38, Fig. 2a). When the probable group was compared with the colonized group, the diagnostic performances of the two assays were also comparable (p 0.40, Fig. 2b).
Discussion Previous studies assessing qPCR for the diagnosis of PCP have targeted various genes and determined various cut-off values for the discrimination of PCP from colonization [7– 12]. However, the sensitivities (84–100%) and specificities (93–100%) of the qPCR assays were sufficient, provided that positive microscopy results were also obtained [7–9]. We used the dihydropteroate synthase gene as a PCR target because it is a single-copy gene in the P. jirovecii genome [19], which allowed us to show that the copy numbers of the definite PCP patients were significantly higher than the copy numbers of the colonized patients, with an excellent AUC, sensitivity and specificity. Previous studies have reported a wide range of qPCR cut-off values that do not effectively discriminate PCP from colonization, and that approximately one-third to one-half of PCP patients have a
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copy number in this range [7–10]. In this study, three of 11 definite PCP patients had a copy number in this ‘grey zone’ between definite PCP and colonization. Although the reference standard for the diagnosis of PCP is microscopic visualization of P. jirovecii, probable or presumptive PCP has been defined as compatible with clinical conditions, and treatment for PCP has commenced without microbiological findings in previous studies [4,6,10]. In this study, we demonstrated that the qPCR results from the probable PCP patients were significantly higher than those from the colonized patients but were significantly lower than those from the definite PCP patients. As a result, the AUC comparing probable PCP with colonized patients was reduced from 0.96 to 0.71, and the range of copy numbers fully overlapped. Several studies have shown that b-D-glucan assays have 92–100% sensitivity and 86–94% specificity for the diagnosis of definite PCP when compared with controls with or without pneumonia [16,20–22]. Damiani et al. [17] examined six definite PCP patients and eight colonized patients and found 100.0% sensitivity and 75.0% specificity. Our study also demonstrated a sensitivity of 100.0% and specificities of 80.0% and 90.0% in the discrimination of definite PCP from nonPCP with or without colonization, respectively. Del Bono et al. [23] have reported that b-D-glucan was a reliable marker for diagnosing presumptive PCP. However, they did not perform microscopy or PCR, and the colonization status of their controls was unknown. In contrast, almost all of our probable PCP patients had positive PCR results, and our controls were separated into colonized and non-colonized groups. We found similarly low levels of b-D-glucan in both control groups. The b-D-glucan assay may be superior to qPCR for the diagnosis of PCP in terms of the difficulties in collecting respiratory samples, such as the invasiveness of the procedure and the variability of sample quality. However, the b-D-glucan assay is not specific for Pneumocystis and falsepositive results have been reported [24,25]. This study had some limitations. First, we combined the data from the BAL and IS samples to evaluate the qPCR. However, when the BAL and IS samples were analysed separately, the copy numbers between definite PCP and colonized patients remained significantly different. The copy numbers were higher in probable PCP patients than in colonized patients, but the difference was not statistically significant due to the small number of patients. Second, we had a small number of definite PCP patients in this cohort. This small number was probably because BAL samples were only obtained from a quarter of all patients, and we used only Gomori methenamine silver staining to detect P. jirovecii microscopically; repeated assessments using other stains or
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more sensitive fluorescent antibody stains were not performed [26]. In conclusion, we found that a b-D-glucan assay and qPCR assay both had high accuracies in discriminating definite PCP from pneumonia with P. jirovecii colonization. Both the b-Dglucan and the qPCR assays also had moderate accuracies in discriminating probable PCP from colonization. These results suggest that patients who have a clinical presentation and radiological findings compatible with PCP and an elevated bD-glucan level or an appropriate qPCR copy number may be categorized as probable PCP patients.
Author Contributions YM participated in the design of the study, reviewed the medical records, performed the qPCR studies, performed the statistical analysis, and drafted the manuscript. Y. Ito participated in the design of the study, reviewed the medical records, interpreted the data and revised the manuscript. Y. Iinuma and SI participated in manuscript preparation. KY, MY, AM, MN and ST participated in the qPCR studies.
Transparency Declaration This work was not specially funded. Some of the results were generated during routine diagnostic activities. No commercial relationships or potential conflicts of interest exist.
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8. Huggett JF, Taylor MS, Kocjan G et al. Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients. Thorax 2008; 63: 154–159. 9. Alanio A, Desoubeaux G, Sarfati C et al. Real-time PCR assay-based strategy for differentiation between active Peumocystis jirovecii pneumonia and colonization in immunocompromised patients. Clin Microbiol Infect 2011 [Epub ahead of print]. 10. Flori P, Bellete B, Durand F et al. Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens. J Med Microbiol 2004; 53: 603–607. 11. Larsen HH, Huang L, Kovacs JA et al. A prospective, blinded study of quantitative touch-down polymerase chain reaction using oral-wash samples for diagnosis of Pneumocystis pneumonia in HIV-infected patients. J Infect Dis 2004; 189: 1679–1683. 12. Fillaux J, Malvy S, Alvarez M et al. Accuracy of a routine real-time PCR assay for the diagnosis of Pneumocystis jirovecii pneumonia. J Microbiol Methods 2008; 75: 258–261. 13. Iikuni N, Kitahama M, Ohta S, Okamoto H, Kamatani N, Nishinarita M. Evaluation of Pneumocystis pneumonia infection risk factors in patients with connective tissue disease. Mod Rheumatol 2006; 16: 282–288. 14. de Boer MG, Gelinck LB, van Zelst BD et al. b-D-glucan and s-adenosylmethionine serum levels for the diagnosis of Pneumocystis pneumonia in HIV-negative patients: a prospective study. J Infect 2011; 62: 93–100. 15. Nakamura H, Tateyama M, Tasato D et al. Clinical utility of serum beta-D-glucan and kl-6 levels in Pneumocystis jirovecii pneumonia. Intern Med 2009; 48: 195–202. 16. Tasaka S, Hasegawa N, Kobayashi S et al. Serum indicators for the diagnosis of Pneumocystis pneumonia. Chest 2007; 131: 1173–1180.
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MYCOLOGY
Quantitative real-time PCR and the (1 fi 3)-b-D-glucan assay for differentiation between Pneumocystis jirovecii pneumonia and colonization Y. Matsumura1, Y. Ito2, Y. Iinuma3, K. Yasuma1, M. Yamamoto1, A. Matsushima1, M. Nagao1, S. Takakura1 and S. Ichiyama1 1) Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, 2) Department of Respiratory Medicine, Kyoto University Graduate School of Medicine, Kyoto, and 3) Department of Infectious Diseases, Kanazawa Medical University, Kanazawa, Japan
Abstract We evaluated whether quantitative PCR (qPCR) and (1 fi 3)-b-D-glucan assays could be used to differentiate Pneumocystis pneumonia (PCP) from Pneumocystis jirovecii colonization in immunocompromised patients with pulmonary infiltrates. A total of 40 bronchoalveolar lavage samples and 107 induced sputum samples from 147 patients who were suspected of having PCP were obtained for PCR detection of P. jirovecii. Diagnoses of definite PCP, probable PCP, pneumonia with P. jirovecii colonization (colonization) and pneumonia without colonization (non-colonization) were made in 11, 42, 15 and 60 patients, respectively. A PCP diagnosis was undetermined in 19 patients. The copy numbers, determined using qPCR, were significantly higher in definite PCP and probable PCP patients than in colonized patients. The area under the receiver-operating characteristic curve (AUC), sensitivity and specificity for discriminating definite PCP from colonization were 0.96, 100.0% and 80.0%, respectively, at a cut-off value of 1300 copies/mL. The values for discriminating probable PCP from colonization were 0.71, 66.7% and 73.3%, respectively, at a cut-off value of 340 copies/mL. b-D-glucan levels were significantly higher in patients with both definite PCP and probable PCP than in colonized patients. The AUC, sensitivity and specificity for discriminating definite PCP were 0.91, 100.0% and 80.0%, respectively, at a cut-off value of 15.6 pg/mL. The values for discriminating probable PCP were 0.78, 76.2% and 73.3%, respectively, at a cut-off value of 6.0 pg/mL. Both qPCR and the b-D-glucan assay displayed high accuracy for discriminating colonization from definite PCP and displayed moderate accuracy for discriminating colonization from probable PCP. Keywords: Colonization, immunocompromised host, Pneumocystis jirovecii pneumonia, real-time PCR, b-D-glucan Original Submission: 22 March 2011; Revised Submission: 6 June 2011; Accepted: 11 June 2011 Editor: E. Roilides Article published online: 20 June 2011 Clin Microbiol Infect 2012; 18: 591–597 10.1111/j.1469-0691.2011.03605.x Corresponding author: S. Ichiyama, Department of Clinical Laboratory Medicine, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan E-mail: [email protected]
Introduction Pneumocystis pneumonia (PCP) is one of the most prevalent infections in human immunodeficiency virus (HIV)-infected patients [1]. PCP also occurs in non-HIV immunocompromised patients receiving transplantation, immunosuppressive therapy and antitumour chemotherapeutic agents [2]. The standard method for laboratory diagnosis of PCP is micro-
scopic visualization of Pneumocystis jirovecii in respiratory samples, including bronchoalveolar lavage (BAL) or induced sputum (IS). Non-HIV patients often develop PCP with a lower fungal burden than that of HIV-infected patients [3], and this lower fungal burden can result in false-negative results following microscopic examination [4]. Because of this problem, polymerase chain reaction (PCR), a highly sensitive technique, has been used for diagnosing PCP. However, Pneumocystis colonization, defined as the detection of the organism or its DNA without signs or symptoms of pneumonia, has been reported in several studies [5]. In addition, some patients with pulmonary infiltrates who were PCRpositive for P. jirovecii have been diagnosed as having pneumonia resulting from a different aetiology than PCP, and the
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positive PCR results were considered indicative of colonization with P. jirovecii [4–6]. Although a positive conventional PCR result cannot distinguish colonization from infection, several studies have shown that the copy number of the specific P. jirovecii gene that is measured using quantitative real-time PCR (qPCR) is significantly higher in patients with PCP than in colonized patients [6–12]. Of the various serum markers tested, it has also been reported that the serum (1 fi 3)-b-D-glucan (b-Dglucan) level is the best test for PCP diagnosis [13–16]. To date, however, only one study [17] with a limited number of patients has assessed the b-D-glucan assay for discrimination of PCP from colonization. In this study, we evaluated whether qPCR and the b-Dglucan assay could be used to discriminate PCP from colonization with P. jirovecii in immunocompromised patients with pulmonary infiltrates.
Materials and Methods Patients and clinical samples
A total of 56 BAL and 161 IS samples from 217 patients suspected of PCP were prospectively obtained for Pneumocystis PCR at Kyoto University Hospital, a 1182-bed tertiary care hospital, between January 2008 and December 2010. In cases in which multiple samples were taken from a patient, the sample from the first episode of PCP was studied. Fifty-six patients without new ground-glass opacities by chest computed tomography and 14 immunocompetent patients were excluded. The remaining 147 eligible immunocompromised patients had new ground-glass opacities and clinical presentations such as fever, cough, sputum, dyspnoea or leucocytosis. These patients provided 40 BAL samples and 107 IS samples. The Ethics Committee of Kyoto University Graduate School and the Faculty of Medicine approved this study and waived the need to obtain informed consent from each patient. Diagnosis of PCP
The diagnosis of definite PCP was established by the microscopic identification of P. jirovecii using Gomori methenamine silver staining. A diagnosis of probable PCP was made when the patient did not have microscopically visible P. jirovecii but had a clinical presentation compatible with PCP, including complete resolution of pulmonary infiltrates after a full course of anti-PCP treatment [4]. The remaining non-PCP patients who had positive qPCR results without any signs or symptoms of PCP and who improved without anti-PCP treatment were diagnosed as ‘pneumonia with P. jirovecii colonization’ (colonization) [5]. Non-PCP patients having nei-
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ther observable P. jirovecii nor positive qPCR results were classified as ‘pneumonia without P. jirovecii colonization’ (noncolonization). Patients who had positive PCR results and pulmonary infiltrates that were resolved by a full course of antimicrobial treatment for concomitant organisms with anti-PCP drugs or who did not improve with or without anti-PCP treatment were classified as undetermined because it was difficult to discriminate between probable PCP and colonization for these patients. Clinical data
The clinical information acquired by a medical chart review included age, sex, underlying diseases, immunosuppressive therapies and prophylaxis for PCP during the month prior to sample collection, clinical symptoms, laboratory values, and the final diagnosis made by the physician. Hypoxia was defined as an arterial PaO2 < 70 mmHg on room air or a requirement for supplemental oxygen. Real-time qPCR
Respiratory samples were centrifuged at 1000 · g for 10 min. The pellet was resuspended in 200 lL of the original fluid, and DNA was extracted using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany). DNA was eluted in a final volume of 100 lL. Forward (5¢-CATATGATTCTATATTAA TGGATGTGGAGA-3¢) and reverse (5¢-TTTATAGCAG GAATAACTCGAGAAATCT-3¢) primers were designed to amplify a 142-bp segment of the single-copy dihydropteroate synthase gene. Real-time PCR for P. jirovecii was performed in a 25 lL reaction volume containing 0.9 lM of each primer, 0.25 lM TaqMan probe (5¢-FAM-ATATCAATTATCGT CGCCC-MGB-3¢), 1 · TaqMan Gene Expression Master Mix (Applied Biosystems (ABI)), and 5 lL purified DNA. Thermal cycling was performed in an ABI PRISM 7000 Sequence Detection system (ABI) as follows: 2 min at 50C, 10 min at 95C, followed by 50 cycles of 15 s at 95C and 1 min at 60C. Quantification was performed using ABI PRISM software and was based on the extrapolation of data to standard curves, which were generated by the amplification of a tenfold dilution series of a plasmid standard (5–5 · 105 copies/ reaction). To generate the plasmids, the PCR-amplified product was cloned into the pCR-TOPO vector (Invitrogen, Carlsbad, CA, USA) and sequenced. A copy number of less than the detection limit of 5 copies/reaction was considered to be 1 copy/reaction. The quantified copy numbers of Pneumocystis are expressed as copy numbers per 1 mL of sample. b-D-glucan assay
Plasma b-D-glucan was measured with the b-glucan test WAKO (Wako Pure Chemical Industries, Tokyo, Japan).
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Plasma samples were collected prior to the collection of BAL or IS samples and the initiation of treatment. The assay was performed at our institution on the same day that the plasma was obtained. Statistical analysis
Categorical variables were compared using the Fisher exact test. Continuous variables were compared using the Kruskal– Wallis test or the Mann–Whitney U-test. b-D-glucan values under the detection limit of 4.3 pg/mL were considered to be 4.3 pg/mL. Receiver-operating characteristic (ROC) curves for the b-D-glucan levels and copy numbers were constructed, and their optimal cut-off values were determined with the maximum Youden index. We compared the area under the curve (AUC) of the two ROC curves using the method described by Delong et al. [18]. A p value <0.05 was considered statistically significant. All statistical analyses were carried out using Stata version 11.2 (StataCorp, College Station, TX, USA).
Results A diagnosis of definite PCP, probable PCP, colonization, noncolonization or undetermined was made in 11, 42, 15, 60 and 19 patients, respectively. BAL samples were obtained from 7, 10, 6, 12 and five of these patients, respectively. All definite patients received anti-PCP treatment. The final
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diagnoses of the colonized patients included bacterial pneumonia (n = 4), interstitial pneumonia (n = 3), invasive aspergillosis (n = 2), atypical pneumonia (n = 1), acute respiratory distress syndrome (ARDS) due to sepsis (n = 1), tuberculosis (n = 1), pulmonary embolism (n = 1), cryptogenic organizing pneumonia (n = 1), and pulmonary alveolar proteinosis (n = 1). Among the non-colonized patients, the final diagnoses included interstitial pneumonia (n = 18), bacterial pneumonia (n = 10), ARDS due to sepsis (n = 9), drug-induced pneumonia (n = 5), pulmonary haemorrhage (n = 5), viral pneumonia (n = 3), tuberculosis (n = 2), pulmonary oedema (n = 1), invasive aspergillosis (n = 1), radiation pneumonitis (n = 1), lymphangitis carcinomatosa (n = 1), cryptogenic organizing pneumonia (n = 1), and unknown (n = 3). Clinical features
Table 1 shows the patient demographics and characteristics. Three definite patients and two probable patients had HIV infection, whereas none of the colonized or non-colonized patients did. An analysis of laboratory test results revealed significant differences in thrombocyte, C-reactive protein, blood urea nitrogen and total bilirubin levels between the four groups. Real-time qPCR
The qPCR and b-D-glucan assay results are presented in Fig. 1. All definite patients and 40 of the 42 probable patients were qPCR-positive. Copy numbers were significantly higher
TABLE 1. Patient demographics and clinical characteristics Definite PCP Characteristics Age, years Male sex Underlying disease Collagen vascular disease Solid malignancy Haematological malignancy Organ transplantation HIV infection Corticosteroids Immunosuppressive agents other than corticosteroids Anti-tumour chemotherapy PCP prophylaxis Hypoxia Laboratory findings Neutrophils, /mm3 Lymphocytes, /mm3 Thrombocytes, /mm3 C-reactive protein, mg/dL Albumin, mg/dL Creatinine, mg/dL Blood urea nitrogen, mg/dL Total bilirubin, mg/dL Lactate dehydrogenase, IU/L
Probable PCP
Colonized
Non-colonized
(n = 42)
(n = 15)
(n = 60)
p value
55 (40–65) 8 (73%)
63 (54–69) 23 (55%)
65 (54–71) 7 (47%)
60 (52–70) 35 (58%)
0.52 0.62
6 0 2 0 3 7 4
(55%) (0%) (18%) (0%) (27%) (64%) (36%)
26 (62%) 8 (24%) 7 (17%) 3 (7%) 2 (5%) 31 (74%) 23 (55%)
9 (60%) 2 (13%) 4 (27%) 2 (13%) 0 (0%) 11 (73%) 7 (47%)
30 (50%) 19 (32%) 7 (12%) 8 (13%) 0 (0%) 43 (72%) 23 (38%)
0.69 0.08 0.46 0.58 0.002 0.93 0.39
0 (0%) 0 (0%) 7 (64%)
10 (24%) 2 (5%) 26 (62%)
4 (27%) 2 (3%) 8 (53%)
14 (23%) 12 (20%) 44 (73%)
0.32 0.7 0.40
6200 (2400–9600) 800 (500–1000) 13.8 (12.3–20.9) 3.0 (1.1–6.8) 3.2 (2.6–3.3) 0.6 (0.5–0.7) 15 (10–16) 0.6 (0.4–0.8) 502 (275–598)
6400 (4100–11 100) 700 (400–1100) 20.3 (14.3–30.4) 5.9 (2.7–9.7) 3.1 (2.5–3.4) 0.7 (0.5–1.4) 18 (12–25) 0.5 (0.4–0.7) 330 (260–428)
56 000 (1400–7800) 400 (200–1300) 15.1 (6.3–21.3) 2.4 (0.6–9.7) 2.9 (2.4–3.6) 0.7 (0.5–1.4) 16 (14–23) 0.7 (0.5–0.8) 323 (277–381)
6350 (4225–10 600) 700 (200–1100) 13.1 (4.7–28.7) 8.2 (2.3–15.4) 2.8 (2.3–3.3) 0.8 (0.5–1.4) 23 (15–36) 0.8 (0.5–1.3) 308 (237–500)
0.41 0.77 0.04 0.049 0.70 0.41 0.04 0.049 0.50
(n = 11)
Data are presented as the n (%) or median (interquartile range). Some patients had two or more underlying diseases or conditions. PCP, Pneumocystis pneumonia; HIV, human immunodeficiency virus.
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p = 0.002
(a)
p = 0.02
p = 0.12
p = 0.03
(a)
p = 0.13
0.75 Sensitivity 0.50
6 4
0.25
2
Copy number b-D-glucan
0
Log10 copies (/mL of sample)
8
p = 0.02
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1.00
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Probable Colonization PCP BAL
Definite PCP
Probable Colonization PCP IS
0.00
0.25
p = 0.001
(b)
0.75
1.00
0
0.25
100
Sensitivity 0.50
200
0.75
300
400
p = 0.25
0.50 1-specificity
1.00
p < 0.001
(b)
b-D glucan (pg/mL)
0.00
Definite PCP
Probable PCP
Colonization
Copy number b-D-glucan
Non-colonization
FIG. 1. P. jirovecii copy numbers (a) and b-D-glucan levels (b) according to patient group. In the box and whisker plots, the boxes contain
0.00
Definite PCP
0.00
0.25
50% of the sample data, with the median value indicated by a horizontal bar. The whiskers contain 1.5 · the interquartile range. (a)
0.50 1-specificity
0.75
1.00
FIG. 2. Receiver-operator characteristic curves of P. jirovecii copy
The copy numbers were significantly higher in the definite PCP
numbers and b- D-glucan levels for the diagnosis of PCP. (a) The def-
group when compared with the colonized group in both BAL and IS
inite PCP group was compared with the colonized group. The
samples. The copy numbers were not significantly different between
arrows indicate cut-off values of 15.6 pg/mL for b-D-glucan level and
the BAL and IS samples in the definite and probable PCP groups
1300 copies/mL for copy number. (b) The probable PCP group was
(p 0.57 and p 0.29), but were significantly different in the colonized
compared with the colonized group. The arrows indicate cut-off val-
group (p 0.02). (b) The b-D-glucan levels were significantly higher in
ues of 6.0 pg/mL for b-D-glucan level and 340 copies/mL for copy
both the definite and probable PCP groups when compared with the
number.
colonized group.
in the definite group (median 460 000 copies/mL, range 1400–15 000 000) than in the colonized group (median 180, range 10–15 000; p < 0.001). This difference was significant for both BAL (p 0.002) and IS (p 0.02) samples. The ROC curve (Fig. 2a) measured an AUC of 0.96, a sensitivity of 100.0% and a specificity of 80.0% for discriminating the definite group from the colonized group, with a cut-off value of 1300 (Table 2). The copy numbers were also significantly higher in the probable group (median 1300, range 10– 7 100 000) than in the colonized group (p 0.001). When
BAL and IS samples were analysed separately, the difference was not statistically significant (p 0.12 for BAL and p 0.13 for IS). The AUC, sensitivity and specificity were reduced to 0.71, 66.7% and 73.3%, respectively, with a cut-off value of 340 (Fig. 2b). The definite group had significantly higher copy numbers than the probable group (p < 0.001). The copy numbers were not significantly different between the BAL and IS samples in the definite and probable groups (p 0.57 and p 0.29, respectively), but they were significantly different in the colonized group (p 0.02). To discriminate the definite and probable groups from the colonized group, ROC analysis
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TABLE 2. Diagnostic performance of P. jirovecii copy numbers and b-D-glucan for PCP detection Test Copy number Definite PCP vs. colonized Probable PCP vs. colonized Definite and probable PCP vs. colonized b-D-glucan Definite PCP vs. colonized Definite PCP vs. non-colonized Definite PCP vs. colonized and non-colonized Probable PCP vs. colonized Probable PCP vs. non-colonized Probable PCP vs. colonized and non-colonized Definite and probable PCP vs. colonized and non-colonized
AUC (95% CI)
Sensitivity (%)
Specificity (%)
PPV (%)
NPV (%)
0.96 (0.89–1.00) 0.71 (0.58–0.85) 0.76 (0.65-0.88)
100.0 66.7 73.6
80.0 73.3 73.3
78.6 87.5 90.7
100.0 44.0 44.0
0.91 0.94 0.93 0.78 0.81 0.80 0.83
100.0 100.0 100.0 76.2 76.2 76.2 81.1
80.0 90.0 88.0 73.3 83.3 81.3 81.3
78.6 64.7 55.0 88.9 76.2 69.6 75.4
100.0 100.0 100.0 52.4 83.3 85.9 85.9
(0.79–1.00) (0.88–0.99) (0.88–0.99) (0.65–0.90) (0.72–0.89) (0.72-0.88) (0.76-0.90)
The cut-off values for copy number were 1300 copies/mL (definite PCP) and 340 copies/mL (probable PCP and definite and probable PCP). When the following changes were made to the cut-off values, the PPV was calculated as follows: 340 copies/mL for definite PCP (78.6%), 1300 copies/mL for probable PCP (87.5%), and 1300 copies/mL for definite and probable PCP (91.4%). The cut-off values for b-D-glucan were 15.6 pg/mL (definite PCP) and 6.0 pg/mL (probable PCP and definite and probable PCP). When the following changes were made to the cut-off values, the PPV was calculated as follows: 6.0 pg/mL for definite PCP (vs. colonized, 78.6%; vs. non-colonized, 52.4%; vs. colonized and non-colonized, 44.0%), 15.6 pg/mL for probable PCP (vs. colonized, 89.3%; vs. non-colonized, 80.6%; vs. colonized and non-colonized, 73.5%), and 15.6 pg/mL for definite and probable PCP (80.0%). PCP, Pneumocystis pneumonia; AUC, area under the receiver-operator characteristic curve; CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value.
produced an AUC of 0.76, a sensitivity of 73.6% and a specificity of 73.3%, with a cut-off value of 1300. b-D-glucan assay
The b-D-glucan levels were significantly higher in the definite group (median 39.5 pg/mL, range 15.7–352.7) than in the colonized group (median 4.3, range 4.3–61.5, p < 0.001) and the non-colonized group (median 4.3, range 4.3–160.4; p < 0.001, Fig. 1b). The ROC curves (Fig. 2a) produced an AUC of 0.91, a sensitivity of 100.0% and a specificity of 80.0% for discriminating the definite group from the colonized group and values of 0.94, 100% and 90.0%, respectively, for discriminating the definite group from the non-colonised group, with a cut-off value of 15.6 (Table 2). Similar to the results obtained for the definite group, the b-D-glucan levels were significantly higher in the probable group (median 23.1, range 4.3–1007) than in the colonized (p 0.001) and non-colonized groups (p < 0.001). The ROC curves (Fig. 2b) produced an AUC of 0.78, a sensitivity of 76.2% and a specificity of 73.3% for discriminating the probable group from the colonized group, and values of 0.81, 76.2% and 83.3%, respectively, for discriminating the probable group from the non-colonized group, with a cut-off value of 6.0. The b-D-glucan levels were similar between the definite and probable groups (p 0.25) and between the colonized and non-colonized groups (p 0.62). To discriminate the definite and probable groups from the colonized and non-colonized groups, ROC analysis produced an AUC of 0.83, a sensitivity of 81.1% and a specificity of 81.3%, with a cut-off value of 6.0. Among the four patients in the colonized group with a positive b-D-glucan level (>6.0 pg/mL), two had probable invasive pulmonary aspergillosis, whereas the others did not have any detectable fungal infections. Among the ten patients
with positive b-D-glucan levels in the non-colonized group, one had candidaemia, one had cryptococcaemia, one received haemodialysis, and four received albumin or intravenous immunoglobulin therapy. The others had no detectable fungal infections, bacteraemia or treatment with amoxicillinclavulanate. The AUC analyses showed that the qPCR and b-D-glucan assays had excellent and comparable diagnostic performances for discriminating the definite group from the colonized group (p = 0.38, Fig. 2a). When the probable group was compared with the colonized group, the diagnostic performances of the two assays were also comparable (p 0.40, Fig. 2b).
Discussion Previous studies assessing qPCR for the diagnosis of PCP have targeted various genes and determined various cut-off values for the discrimination of PCP from colonization [7– 12]. However, the sensitivities (84–100%) and specificities (93–100%) of the qPCR assays were sufficient, provided that positive microscopy results were also obtained [7–9]. We used the dihydropteroate synthase gene as a PCR target because it is a single-copy gene in the P. jirovecii genome [19], which allowed us to show that the copy numbers of the definite PCP patients were significantly higher than the copy numbers of the colonized patients, with an excellent AUC, sensitivity and specificity. Previous studies have reported a wide range of qPCR cut-off values that do not effectively discriminate PCP from colonization, and that approximately one-third to one-half of PCP patients have a
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copy number in this range [7–10]. In this study, three of 11 definite PCP patients had a copy number in this ‘grey zone’ between definite PCP and colonization. Although the reference standard for the diagnosis of PCP is microscopic visualization of P. jirovecii, probable or presumptive PCP has been defined as compatible with clinical conditions, and treatment for PCP has commenced without microbiological findings in previous studies [4,6,10]. In this study, we demonstrated that the qPCR results from the probable PCP patients were significantly higher than those from the colonized patients but were significantly lower than those from the definite PCP patients. As a result, the AUC comparing probable PCP with colonized patients was reduced from 0.96 to 0.71, and the range of copy numbers fully overlapped. Several studies have shown that b-D-glucan assays have 92–100% sensitivity and 86–94% specificity for the diagnosis of definite PCP when compared with controls with or without pneumonia [16,20–22]. Damiani et al. [17] examined six definite PCP patients and eight colonized patients and found 100.0% sensitivity and 75.0% specificity. Our study also demonstrated a sensitivity of 100.0% and specificities of 80.0% and 90.0% in the discrimination of definite PCP from nonPCP with or without colonization, respectively. Del Bono et al. [23] have reported that b-D-glucan was a reliable marker for diagnosing presumptive PCP. However, they did not perform microscopy or PCR, and the colonization status of their controls was unknown. In contrast, almost all of our probable PCP patients had positive PCR results, and our controls were separated into colonized and non-colonized groups. We found similarly low levels of b-D-glucan in both control groups. The b-D-glucan assay may be superior to qPCR for the diagnosis of PCP in terms of the difficulties in collecting respiratory samples, such as the invasiveness of the procedure and the variability of sample quality. However, the b-D-glucan assay is not specific for Pneumocystis and falsepositive results have been reported [24,25]. This study had some limitations. First, we combined the data from the BAL and IS samples to evaluate the qPCR. However, when the BAL and IS samples were analysed separately, the copy numbers between definite PCP and colonized patients remained significantly different. The copy numbers were higher in probable PCP patients than in colonized patients, but the difference was not statistically significant due to the small number of patients. Second, we had a small number of definite PCP patients in this cohort. This small number was probably because BAL samples were only obtained from a quarter of all patients, and we used only Gomori methenamine silver staining to detect P. jirovecii microscopically; repeated assessments using other stains or
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more sensitive fluorescent antibody stains were not performed [26]. In conclusion, we found that a b-D-glucan assay and qPCR assay both had high accuracies in discriminating definite PCP from pneumonia with P. jirovecii colonization. Both the b-Dglucan and the qPCR assays also had moderate accuracies in discriminating probable PCP from colonization. These results suggest that patients who have a clinical presentation and radiological findings compatible with PCP and an elevated bD-glucan level or an appropriate qPCR copy number may be categorized as probable PCP patients.
Author Contributions YM participated in the design of the study, reviewed the medical records, performed the qPCR studies, performed the statistical analysis, and drafted the manuscript. Y. Ito participated in the design of the study, reviewed the medical records, interpreted the data and revised the manuscript. Y. Iinuma and SI participated in manuscript preparation. KY, MY, AM, MN and ST participated in the qPCR studies.
Transparency Declaration This work was not specially funded. Some of the results were generated during routine diagnostic activities. No commercial relationships or potential conflicts of interest exist.
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ª2011 The Authors Clinical Microbiology and Infection ª2011 European Society of Clinical Microbiology and Infectious Diseases, CMI, 18, 591–597
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Matsumura et al. qPCR and b-D-glucan assays for P. jirovecii pneumonia
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ª2011 The Authors Clinical Microbiology and Infection ª2011 European Society of Clinical Microbiology and Infectious Diseases, CMI, 18, 591–597