Real Time Quantitative PCR Assay for <i>Pneumocystis jirovecii</i> Detection

Meliani, Leila; Develoux, M.; Marteau-Miltgen, M; Magne, Denis; Barbu, Véronique; Poirot, Jean-Louis; Roux, Patricia

doi:10.1111/j.1550-7408.2003.tb00670.x

Cited by 38 publications

(24 citation statements)

References 3 publications

(3 reference statements)

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“…We used a previously described protocol of qPCR targeting the mitochondrial large subunit DNA locus (23). Briefly, DNA was extracted from 500 l of BAL fluid using the Qiagen DNA blood minikit (Qiagen) and automated purification on a QiaSymphony SP system.…”

Section: Methodsmentioning

confidence: 99%

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Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii

et al. 2015

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Quantitative PCR (qPCR) is now a key diagnostic tool forPneumocystispneumonia. However, cutoffs to distinguish between infected and colonized patients according to their HIV status have not yet been determined. According to clinical, radiological, and biological data, we retrospectively classified bronchoalveolar lavage (BAL) samples subjected to qPCR over a 3-year period into four categories, i.e., definite PCP, probable PCP,Pneumocystiscolonization, and no infection. Fungal burden was then analyzed according to the HIV status of the patients. Among 1,212 episodes of pneumonia screened in immunocompromised patients, 52 and 27 HIV-positive patients were diagnosed with a definite and probable PCP, whereas 4 and 22 HIV-negative patients had definite and probable PCP, respectively. Among patients with definite or a probable PCP, HIV-negative patients had a significantly lower burden than HIV-positive patients (P< 10−4). In both groups, the median fungal burden was significantly higher in patients with definite PCP than in colonized patients. A single cutoff at 1.5 × 104copies/ml allowed to differentiate colonized and infected HIV-positive patients with 100% sensitivity and specificity. In HIV-negative patients, cutoff values of 2.87 × 104and 3.39 × 103copies/ml resulted in 100% specificity and sensitivity, respectively. Using cutoffs determined for the whole population would have led us to set aside the diagnosis of PCP in 9 HIV-negative patients with definite or probable PCP. qPCR appeared to be the most sensitive test to detectPneumocystisin BAL samples. However, because of lower inocula in HIV-negative patients, different cutoffs must be used according to the HIV status to differentiate between colonized and infected patients.

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Section: Methodsmentioning

confidence: 99%

“…A run was validated only if the qPCR efficiency was between 98 and 105%. The analytic sensitivity (lower limit of P. jirovecii DNA detection) of our method had been calculated at 180 copies of the targetml in a previous study (23).…”

Section: Methodsmentioning

confidence: 99%

Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii

et al. 2015

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“…P. jirovecii DNA was quantified in the 46 BAL fluid samples in which the fungus was previously detected using a real-time qPCR assay targeting the mtLSU rRNA gene of P. jirovecii, as described elsewhere (29). Specificity of the two primers and the probe for P. jirovecii DNA was checked using Blast software.…”

Section: Methodsmentioning

confidence: 99%

Combined Quantification of Pulmonary Pneumocystis jirovecii DNA and Serum (1→3)-β- d -Glucan for Differential Diagnosis of Pneumocystis Pneumonia and Pneumocystis Colonization

et al. 2013

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This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1¡3)-␤-D-glucan (BG) level detection to distinguish Pneumocystis pneumonia (PCP) from pulmonary colonization with P. jirovecii. Forty-six patients for whom P. jirovecii was initially detected in BAL fluid samples were retrospectively enrolled. Based on clinical data and results of P. jirovecii detection, 17 and 29 patients were diagnosed with PCP and colonization, respectively. BAL fluid samples were reassayed using a qPCR assay targeting the mitochondrial large subunit rRNA gene. qPCR results and serum BG levels (from a Fungitell kit) were analyzed conjointly. P. jirovecii DNA copy numbers were significantly higher in the PCP group than in the colonization group (1.3 ؋ 10 7 versus 3.4 ؋ 10 3 copies/l, P < 0.05). A lower cutoff value (1.6 ؋ 10 3 copies/l) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 ؋ 10 4 copies/ l) achieving 100% specificity were determined. Applying these two values, 13/17 PCP patients and 19/29 colonized patients were correctly assigned to their patient groups. For the remaining 14 patients with P. jirovecii DNA copy numbers between the cutoff values, PCP and colonization could not be distinguished on the basis of qPCR results. Four of these patients who were initially assigned to the PCP group presented BG levels of >100 pg/ml. The other 10 patients, who were initially assigned to the colonization group, presented BG levels of <100 pg/ml. These results suggest that the combination of the qPCR assay, applying cutoff values of 1.6 ؋ 10 3 and 2 ؋ 10 4 copies/l, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses.

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“…Before extraction, all samples were centrifuged at 3,000 rpm for 5 min. The qPCR described by Meliani et al was performed (54). The amplified target was the mitochondria1 large subunit (mtLSU) rRNA gene.…”

Section: Samples and Patientsmentioning

confidence: 99%

Detection of Pneumocystis jirovecii by Quantitative PCR To Differentiate Colonization and Pneumonia in Immunocompromised HIV-Positive and HIV-Negative Patients

et al. 2016

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dPneumocystis jirovecii pneumonia (PCP) is an acute and life-threatening lung disease caused by the fungus Pneumocystis jirovecii. The presentation of PCP in HIV-positive patients is well-known and consists of a triad of dyspnea, fever, and cough, whereas the presentation of PCP in HIV-negative patients is atypical and consists of a sudden outbreak, O 2 desaturation, and a rapid lethal outcome without therapy. Despite the availability of direct and indirect identification methods, the diagnosis of PCP remains difficult. The cycle threshold (C T ) values obtained by quantitative PCR (qPCR) allow estimation of the fungal burden. The more elevated that the fungal burden is, the higher the probability that the diagnosis is pneumonia. The purposes of the present study were to evaluate the C T values to differentiate colonization and pneumonia in a population of immunocompromised patients overall and patients stratified on the basis of their HIV infection status. Testing of bronchoalveolar lavage (BAL) fluid samples from the whole population of qPCR-positive patients showed a mean C T value for patients with PCP of 28 (95% confidence interval [CI], 26 to 30) and a mean C T value for colonized patients of 35 (95% CI, 34 to 36) (P < 10 ؊3 ). For the subgroup of HIV-positive patients, we demonstrated that a C T value below 27 excluded colonization and a C T value above 30 excluded PCP with a specificity of 100% and a sensitivity of 80%, respectively. In the subgroup of HIV-negative patients, we demonstrated that a C T value below 31 excluded colonization and a C T value above 35 excluded PCP with a specificity of 80% and a sensitivity of 80%, respectively. Thus, qPCR of BAL fluid samples is an important tool for the differentiation of colonization and pneumonia in P. jirovecii-infected immunocompromised patients and patients stratified on the basis of HIV infection status with different C T values.

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Real Time Quantitative PCR Assay for Pneumocystis jirovecii Detection

Cited by 38 publications

References 3 publications

Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii

Impact of HIV Infection Status on Interpretation of Quantitative PCR for Detection of Pneumocystis jirovecii

Combined Quantification of Pulmonary Pneumocystis jirovecii DNA and Serum (1→3)-β- d -Glucan for Differential Diagnosis of Pneumocystis Pneumonia and Pneumocystis Colonization

Detection of Pneumocystis jirovecii by Quantitative PCR To Differentiate Colonization and Pneumonia in Immunocompromised HIV-Positive and HIV-Negative Patients

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