A highly sensitive novel PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveloar lavage specimens from immunocompromised patients

Tia, Taweesak; Putaporntip, Chaturong; Kosuwin, Rattiporn; Kongpolprom, Napplika; Kawkitinarong, Kamon; Jongwutiwes, Somchai

doi:10.1111/j.1469-0691.2011.03656.x

Cited by 33 publications

(31 citation statements)

References 21 publications

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“…Indeed, Pneumocystis jirovecii cannot be cultured, and its detection is based on staining methods using respiratory specimens, which suffer from low sensitivity (214). Consequently, many studies have evaluated the performance of PCR on respiratory specimens for PCP diagnosis, and the results seem promising, with sensitivity values as high as 100% (91,(215)(216)(217)(218)(219)(220)(221)(222)(223)(224). However, in the case of PCP, colonization is a significant issue, with rates as high as 22% in high-risk populations (225), and more importantly, since DNA is fairly stable, it is difficult to distinguish between active and previous infections (226).…”

Section: Pcrmentioning

confidence: 99%

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

et al. 2014

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SUMMARY Invasive fungal infections constitute a serious threat to an ever-growing population of immunocompromised individuals and other individuals at risk. Traditional diagnostic methods, such as histopathology and culture, which are still considered the gold standards, have low sensitivity, which underscores the need for the development of new means of detecting fungal infectious agents. Indeed, novel serologic and molecular techniques have been developed and are currently under clinical evaluation. Tests like the galactomannan antigen test for aspergillosis and the β-glucan test for invasive Candida spp. and molds, as well as other antigen and antibody tests, for Cryptococcus spp., Pneumocystis spp., and dimorphic fungi, have already been established as important diagnostic approaches and are implemented in routine clinical practice. On the other hand, PCR and other molecular approaches, such as matrix-assisted laser desorption ionization (MALDI) and fluorescence in situ hybridization (FISH), have proved promising in clinical trials but still need to undergo standardization before their clinical use can become widespread. The purpose of this review is to highlight the different diagnostic approaches that are currently utilized or under development for invasive fungal infections and to identify their performance characteristics and the challenges associated with their use.

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Section: Pcrmentioning

confidence: 99%

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

et al. 2014

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“…BAL örnekleri 1500 rpm'de 10 dakika santrifüj edildikten sonra elde edilen pelletin 200 μl'si DNA ekstraksiyonu için kullanıldı 18 . DNA ekstraksiyon kiti (Macherey-Nagel, Almanya) ile üretici fi rma prosedürlerine göre elde edilen DNA örnekleri, amplifi kasyon işlemi yapılıncaya kadar -20°C'de saklandı.…”

Section: Dna Ekstraksiyonu Ve İki Turlu Pcr Yöntemiunclassified

“…Birinci PCR döngüsünde pAZ102-E (5'-GATGGCTGTTTCCAAGCCCA-3') ve pAZ102-H (5'GTGTACGTTGCAAAGTA CTC-3') primerleri; ikinci PCR döngüsünde ise pAZ102-X (5'-GTGAAATACAAATCGGACTA GG-3') ve pAZ102-Y (5'-TCACTTAATATTA-ATTGGGGAGC-3) primerleri kullanıldı 18 . Her bir reaksiyon için 2.5 μl 10x reaksiyon tamponu, 2.5 μl MgCl 2 (25 mM stok), 2.5 μl dNTP (2 mM stok), 1 μl Taq DNA polimeraz (1 U/μl stok), 0.75 μl primer (10 μM stok) ve 1 μl DNA örneği eklendikten sonra steril distile su ile son hacim 25 μl'ye tamamlandı.…”

Section: Dna Ekstraksiyonu Ve İki Turlu Pcr Yöntemiunclassified

“…Her bir reaksiyon için 2.5 μl 10x reaksiyon tamponu, 2.5 μl MgCl 2 (25 mM stok), 2.5 μl dNTP (2 mM stok), 1 μl Taq DNA polimeraz (1 U/μl stok), 0.75 μl primer (10 μM stok) ve 1 μl DNA örneği eklendikten sonra steril distile su ile son hacim 25 μl'ye tamamlandı. Amplifi kasyon, 94°C'de 5 dakikalık ön denatüras-yonun ardından 40 döngü; 94°C'de 1 dakika, 56°C'de 1 dakika, 72°C'de 1.5 dakika ve son uzama basamağı için 72°C'de 5 dakika olacak şekilde düzenlendi 18 . Her iki döngü için aynı miktar ve süreler uygulandı.…”

Section: Dna Ekstraksiyonu Ve İki Turlu Pcr Yöntemiunclassified

“…Reaksiyon sonunda 10 μl PCR ürünü, %1.5'lik agaroz jelde 100 volt altında 40 dakika elektroforeze tabi tutulduktan sonra 1 μg/ml etidyum bromür ile boyanarak ultraviole ışık altında görüntülendi. Birinci PCR döngüsü sonunda 346 baz çifti (bç), nPCR sonunda ise 267 bç uzunluğunda amplikon saptanması durumunda örnekler pozitif olarak değerlendirildi 18 . Tüm PCR testleri her bir örnek için üç kez tekrar edildi.…”

Section: Dna Ekstraksiyonu Ve İki Turlu Pcr Yöntemiunclassified

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Investigation of Pneumocystis jirovecii Pneumonia and Colonization in Iatrogenically Immunosuppressed and Immunocompetent Patients

Özkoç

Delıbaş²

2015

Mikrobiyol Bul

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ÖZPneumocystis pnömonisi (PCP), immün sistem bozukluğu olan hastalarda ölümcül seyredebilen bir akciğer enfeksiyonudur. Bununla birlikte Pneumocystis jirovecii'nin sağlıklı kişilerde ve diğer kronik akciğer hastalarında kolonize olabildiği bilinmektedir. Bu çalışmada, immün sistemi normal ve iyatrojenik olarak baskılanmış hastaların, PCP ve P.jirovecii kolonizasyonu yönünden değerlendirilmesi amaçlanmıştır. Çalışmaya, Ocak 2011-Nisan 2014 tarihleri arasında farklı pulmoner semptomları nedeniyle bronkoskopi yapılan toplam 92 hasta (66 erkek, 26 kadın; yaş aralığı: 18-93 yıl, ortanca: 58.5) dahil edilmiştir. Hastaların 65'i immünosüpresif ilaç (38'i anti-kanser, 15'i anti-rejeksiyon/immünomodülatör ve 12'si kortikosteroid) tedavisi alan, 27'si ise almayan olgulardır. Hastalara ait bronkoalveolar lavaj (BAL) sıvısı örnekleri P.jirovecii ribozomal RNA büyük alt ünitesini kodlayan mitokondriyal geni (mtLSUrRNA) çoğal-tan iki turlu (nested) PCR (nPCR) yöntemi ile değerlendirilmiş; ayrıca tüm örnekler Giemsa ve Gomori'nin metenamin gümüş (GMG) boyama yöntemiyle boyanarak incelenmiştir. Çalışmada, nPCR ile 92 BAL örneğinin 31 (%33.7)'inde P.jirovecii DNA'sı saptanmıştır. Birinci PCR aşamasında, immünosüpresif altı (%6.4) hasta pozitif iken, nPCR ile immünosüpresif 65 hastanın 26 (%40)'sı ve immünokompetan 27 hastanın beşi (%18.5) pozitif bulunmuştur. nPCR ile pozitif saptanan 31 örneğin sadece beşinde (%16.1) Giemsa ve GMG boyaları ile P.jirovecii kist ve trofozoitleri gözlenmiştir. nPCR pozitif olguların immüno-süpresif olma olasılığı, nPCR negatif hastalara göre istatistiksel olarak anlamlı düzeyde yüksek bulunmuş (χ²= 3.940; p= 0.047); bu farkın organ transplant alıcıları ve anti-rejeksiyon/immünomodülatör ilaç tedavisi alan hastalarda daha belirgin olduğu (sırasıyla, χ²= 6.715, p= 0.01; χ²= 5.550, p= 0.018) izlenmiştir. nPCR pozitif saptanan hastalar klinik, laboratuvar ve radyolojik olarak incelendiğinde; immünosüpresif grupta yer alan beş (2 böbrek transplantı, 1 kemik iliği transplantı, 1 akciğer kanseri ve 1 interstisyel Geliş Tarihi

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Diagnostic value of bronchoscopy in patients with hematologic malignancy and pulmonary infiltrates

et al. 2014

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Pulmonary infections are a major cause of morbidity and mortality in patients with hematologic malignancy. Bronchoscopy is at present still the traditional first investigation in immunosuppressed patients that have developed pulmonary infiltrates. There is limited data available on the validity of fiberoptic bronchoscopy (FOB) with bronchoalveolar lavage (BAL) to determine the etiology of pulmonary infiltrates with concurrent hematologic malignancy. We retrospectively analyzed the microbiological results of 206 bronchoscopic examinations and treatment changes used in 187 patients with hematologic malignancy and pulmonary infiltrates. Bacteria, fungi, and viruses were found in 85 (41.3 %), 49 (23.8 %), and 55 (28.6 %) of cases, respectively, and overall yield of bronchoscopy was 65.0 %. We compared the microbiological findings with respect to neutropenia, hematopoietic stem cell transplantation (HSCT) status, and the type of malignancy. There were significantly more bacterial and viral infections detected in post-HSCT patients, and more viruses were detected in patients without neutropenia. Galactomannan (GM) was measured in 149 BAL samples. With a GM index threshold of ≥0.5, the sensitivity, specificity, and positive and negative predictive values (PPV and NPV, respectively) of the BAL GM assay were 93.94 %, 86.21 %, 65.96 %, and 98.04 %, respectively. Treatment was modified in 62 cases (30.1 %). There was no significant relationship of treatment modification with the underlying disease, HSCT, or neutropenia. Bronchoscopy with BAL is a valuable diagnostic tool to determine the etiology and appropriate treatment in patients with hematologic malignancy and pulmonary infiltrates. A BAL GM test is recommended when invasive pulmonary aspergillosis is suspected.

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A highly sensitive novel PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveloar lavage specimens from immunocompromised patients

Cited by 33 publications

References 21 publications

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

Investigation of Pneumocystis jirovecii Pneumonia and Colonization in Iatrogenically Immunosuppressed and Immunocompetent Patients

Diagnostic value of bronchoscopy in patients with hematologic malignancy and pulmonary infiltrates

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