Revathy Mani, Therese Kulandai Lily, Bagyalakshmi Radhakishnan, Chandrasekar Chokaliingam, Kumar Suria, Madhavan Hajib N
L & T Microbiology Research Center, Vision Research Foundation, Kamal Nayan Bajaj Research Centre, Chennai, India.
Indian J Med Res. 2014 Sep;140(3):406-13.
BACKGROUND & OBJECTIVES: As there are no standard laboratory techniques for the rapid detection of Pneumocystis jirovecii in India, this study was undertaken to evaluate and establish an optimal and rapid technique for the detection of P. jirovecii by comparing three different techniques - staining technique, application of a real time polymerase chain reaction (RT-PCR) targeting kex 1 gene and application of nested PCR targeting mitochondrial large subunit (mtLSU) gene for rapid detection of P. jirovecii in HIV positive patients.
One hundred and fifty sputum specimens from HIV positive (n = 75) and HIV negative (n = 75) patients were subjected to three different techniques -KOH/Calcoflour and Grocott methanamine silver staining (GMS), RT-PCR targeting kex1 gene, PCR targeting mtLSU region followed by DNA sequencing and BLAST analysis.
Among the 75 HIV positive patients, P. jirovecii was detected in 19 (25.33%) patients by the staining techniques, and in 23 (30.65%) patients each by PCR targeting mtLSU region and by RT- PCR targeting kex1 gene of P. jirovecii. PCR based DNA sequencing targeting mtLSU region revealed 97-100 per cent sequence homology with P. jirovecii sequences in GenBank.
INTERPRETATION & CONCLUSIONS: Of the three techniques for detection of P. jirovecii evaluated in this study, false negativity was found to be more in staining technique and it also required high technical expertise to interpret the result. Both nested PCR and RT-PCR were reliable and equally sensitive, in rapid detection of P. jirovecii, but RT-PCR technique also generated the copy numbers for knowing the severity of infection.
由于印度尚无用于快速检测耶氏肺孢子菌的标准实验室技术，本研究旨在通过比较三种不同技术——染色技术、针对kex 1基因的实时聚合酶链反应（RT-PCR）以及针对线粒体大亚基（mtLSU）基因的巢式PCR，来评估并建立一种用于检测耶氏肺孢子菌的最佳快速技术，以实现对HIV阳性患者耶氏肺孢子菌的快速检测。
对150份来自HIV阳性（n = 75）和HIV阴性（n = 75）患者的痰液标本采用三种不同技术进行检测——氢氧化钾/荧光增白剂和Grocott甲胺银染色（GMS）、针对kex1基因的RT-PCR、针对mtLSU区域的PCR，随后进行DNA测序和BLAST分析。
在75例HIV阳性患者中，通过染色技术检测到19例（25.33%）耶氏肺孢子菌感染，通过针对耶氏肺孢子菌mtLSU区域的PCR和针对kex1基因的RT-PCR分别检测到23例（30.65%）。基于mtLSU区域的PCR DNA测序显示与GenBank中耶氏肺孢子菌序列的同源性为97%-100%。
在本研究评估的三种耶氏肺孢子菌检测技术中，染色技术的假阴性率更高，且结果判读需要较高的技术专业知识。巢式PCR和RT-PCR在耶氏肺孢子菌的快速检测中均可靠且同样敏感，但RT-PCR技术还能生成拷贝数用于了解感染的严重程度。
