The clinical utility of polymerase chain reaction for the detection of Pneumocystis Carinii using a clinical diagnosis as the gold standard
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Gold standard (test)
Clinical Diagnosis
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Quantitative Polymerase Chain Reaction in Diagnosis of Pneumocystis Pneumonia , Quantitative Polymerase Chain Reaction in Diagnosis of Pneumocystis Pneumonia , کتابخانه دیجیتال دانشگاه علوم پزشکی اصفهان
Pneumocystis pneumonia
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カリニ肺炎は日和見感染症の一つで, ニューモシスティス-カリニの感染・増殖によりおきる. この疾患は免疫不全患者に発症しやすく, AIDS患者の死因の第一位を占めている. ニューモシスティス-カリニは真核微生物であるが, AIDSの流行による発症急増まで基礎的な研究がほとんど行われなかった. このカリニ肺炎パラサイトの分類に関しては, これまで原虫説と真菌説とが対峙し結論に至っていなかった. われわれはニューモシスティス-カリニから5SリボゾームRNAを分離し, その塩基配列を決定することによって, 分子系統学的にこれが原虫と菌類の中間種であることを明らかにした. さらにこの分析結果を利用して, 5SリボゾームRNA遺伝子のPCR増幅法による実用的なニューモシスティス-カリニ遺伝子診断法を臨床的に確立した. すでに広範な臨床応用が国内で実施され, その有効性が実証された. このPCR診断は患者の治療効果の判定や, 副作用の回避・投薬プロトコル作成にも顕著な効果を示した.
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Two amplification steps were made to detect Pneumocystis carinii DNA by polymerase chain reaction (PCR). pAZ102-E and pAZ102-H (standard PCR), pAZ102-L2 (sense), and pAZ102-E (antisense) (two-step PCR) were used as primers. The amplification products were analyzed by ethidium bromide. After the two-step PCR, ethidium bromide detected all samples positive by oligohybridization after one amplification step. Our two-step PCR is a rapid, cost-effective, and clinically suitable method for the detection of P. carinii infection.
Ethidium bromide
Taq polymerase
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Abstract Background Pneumocystis jirovecii pneumonia (PJP) affects immunocompromised patients and contributes significantly to mortality. Outcomes depend on early treatment, making timely and accurate diagnosis critical. Typically, PJP diagnosis is through identification of trophozoite or cyst forms in bronchoalveolar lavage (BAL) fluid or sputum, a labor-intensive and insensitive process. Options for more accessible and sensitive molecular detection are limited. It is known that patients may be colonized, which can cast doubt on the clinical significance of low levels of DNA amplification in qualitative result reporting. In this study, we describe a real time (rt) PCR assay utilizing analyte specific reagent primers targeting the mtLSU gene of P. jirovecii and correlate amplification with morphological PJP identification. Methods IUHPL Clinical Microbiology assessed sputum or BAL fluid from 109 patients with clinical concern for PJP microscopically via fungal stains (GMS, calcofluor white). Comparative rtPCR was conducted as follows. First, 2µL of residual specimen or control were mixed with an 8µL combination of rtPCR mastermix, control DNA, and primer pairs (Simplexa). No nucleic acid extraction was performed. Real time PCR was executed and analyzed on the LIAISON MDX (DiaSorin) platform. Qualitative amplification results and cycle threshold (CT) values were correlated with microscopic methods to establish performance. Chart review was performed to assess the clinical impact of this assay. Results P. jirovecii was microscopically detected in 26% (29/109) of samples, while 31.1% (34/109) exhibited amplification by rtPCR. Agreement between the two methods was 95.4%; rtPCR demonstrated 100% sensitivity and 93.8% specificity in comparison. Conclusion Our results indicate that this assay has exceptional negative predictive value (100%), and therefore may be valuable as a screening test. Considering this data alone, the positive predictive value is lower (85.3%). Further examination of the data, however, revealed that 80% (4/5) of discrepant results demonstrated CT values of >34, while the highest CT for a microscopically positive sample was 31.2. Further clinical correlation may establish a CT cutoff that will reduce false positive and potentially clinically insignificant cases. Disclosures All Authors: No reported disclosures
Primer (cosmetics)
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Background The definitive diagnosis of Pneumocystis pneumonia requires direct visualization of the organism; in recent years, polymerase chain reaction (PCR) assays, although not standardized, have become widely used diagnostic tools. A concern is that newer, more sensitive PCR assays will not differentiate colonization from infection. We evaluated the clinical implications of 2 different PCR assays for the diagnosis of Pneumocystis pneumonia. Methods Adult patients with a bronchoalveolar lavage or sputum sample positive for Pneumocystis by PCR from February 2014 to February 2018 were studied. Samples were tested with end-point (EP) PCR before February 2017 and with real-time (RT) PCR after February 2017. A strict case definition based on host factors, clinical presentation, radiological and pathological findings, and response to therapy was used to categorize cases as proven, probable, possible, and no Pneumocystis pneumonia. Positive EP-PCR and RT-PCR results were deemed to be true positives or false positives based on this categorization of patients. Results Of 1693 patients tested, 153 (9%) had a positive PCR test result, and 118 met the case definitions for proven (n = 2), probable (n = 70), and possible (n = 46) Pneumocystis pneumonia., Among 35 patients with a positive PCR test result who did not the meet criteria for Pneumocystis infection and who had a verified alternative diagnosis, 8 (23%) were false positive by EP-PCR and 27 (77%) by RT-PCR ( P < 0.0001). Among the 27 patients who did not have host factors that would classically place them at risk of Pneumocystis infection, RT-PCR gave significantly more false-positive results (n = 12; 44%) than did EP-PCR (n = 2; 7%; P < 0.001). Conclusions Reverse transcription PCR gave significantly more positive results than did EP-PCR in patients who likely were colonized only and did not have Pneumocystis infection. Host risk factors, clinical presentation, and radiological findings should be considered before ordering PCR testing for Pneumocystis .
Pneumocystis pneumonia
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Although polymerase chain reaction (PCR) has shown promise in diagnosing Pneumocystis jirovecii pneumonia (PCP), no clinically validated real-time
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