Graves D C, Chary-Reddy S, Becker-Hapak M
Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Mol Cell Probes. 1997 Feb;11(1):1-9. doi: 10.1006/mcpr.1996.0070.
A hybridization assay for the detection of Pneumocystis carinii was developed using a repetitive DNA fragment of P.c. hominis. The assay was specific as different micro-organisms typically found in the respiratory tract, normal human lung DNA (A 549 cell line) and normal rat lung DNA did not react with the repetitive probe. In a slot blot (SB) hybridization assay, the repetitive probe was able to detect as few as 100 P.c. hominis organisms with no false-positives. The results of the SB hybridization assay were compared with an immunofluorescence (IFA) assay for the detection of P.c. hominis in 84 induced sputum (IS) samples obtained from 52 human immunodeficiency virus (HIV)-seropositive patients, 22 HIV-seronegative patients and 10 healthy individuals. Samples from 24 patients clinically diagnosed with P. carinii pneumonia (PCP) were positive for P.c. hominis by both assays. In addition, the SB assay detected P.c. hominis in 14 patients (10 HIV-positive and four HIV-negative) who were negative by IFA. All 14 samples showed a positive PCR signal for the P.c. hominis dihydrofolate reductase gene, further confirming the presence of P.c. hominis in these specimens. Twelve of these patients had a clinical course highly suggestive of PCP and were either on P. carinii prophylaxis or P. carinii chemotherapy. The other two samples were from HIV-positive patients who had respiratory illness due to causes other than P.c. hominis (disseminated histoplasmosis and fatal Bordetella pneumonia). Detection of P.c. hominis in these samples suggests that these patients may have subclinical colonization by P.c. hominis. Furthermore, P.c. hominis was detected in all 12 sequential IS samples from six AIDS patients who had primary episodes of PCR using the SB assay, while P.c. hominis was detected only in eight samples by IFA (66.6%). All six patients developed recurrent PCP within 6 months from the time the assays were performed, further illustrating the potential of the SB hybridization assay in monitoring PCP recurrence. Thus, the ability of the SB hybridization assay to detect a low parasite load suggests that this assay may become an important supplemental tool, along with current cytological methods, for detecting P.c. hominis in patient populations with lower burdens of the organism and in identifying asymptomatic carriers of the parasite in healthy and immunosuppressed individuals.
利用人肺孢子虫的重复DNA片段开发了一种用于检测卡氏肺孢子虫的杂交试验。该试验具有特异性，因为呼吸道中常见的不同微生物、正常人肺DNA（A549细胞系）和正常大鼠肺DNA均不与该重复探针发生反应。在狭缝印迹（SB）杂交试验中，该重复探针能够检测低至100个人肺孢子虫生物体，且无假阳性。将SB杂交试验的结果与免疫荧光（IFA）试验的结果进行比较，以检测从52例人类免疫缺陷病毒（HIV）血清阳性患者、22例HIV血清阴性患者和10名健康个体获得的84份诱导痰（IS）样本中的人肺孢子虫。24例临床诊断为卡氏肺孢子虫肺炎（PCP）的患者样本通过两种试验检测人肺孢子虫均呈阳性。此外，SB试验在14例IFA检测为阴性的患者（10例HIV阳性和4例HIV阴性）中检测到人肺孢子虫。所有14份样本的人肺孢子虫二氢叶酸还原酶基因PCR信号均为阳性，进一步证实了这些标本中存在人肺孢子虫。其中12例患者的临床病程高度提示PCP，且正在接受卡氏肺孢子虫预防或卡氏肺孢子虫化疗。另外两份样本来自HIV阳性患者，其呼吸道疾病由人肺孢子虫以外的原因引起（播散性组织胞浆菌病和致命的博德特氏菌肺炎）。在这些样本中检测到人肺孢子虫表明这些患者可能有人肺孢子虫的亚临床定植。此外，使用SB试验在6例有PCR初发的艾滋病患者的所有12份连续IS样本中检测到人肺孢子虫，而通过IFA仅在8份样本中检测到人肺孢子虫（66.6%）。所有6例患者在进行试验后的6个月内均出现复发性PCP，进一步说明了SB杂交试验在监测PCP复发方面的潜力。因此，SB杂交试验检测低寄生虫载量的能力表明，该试验可能会成为一种重要的补充工具，与目前的细胞学方法一起，用于检测寄生虫负荷较低的患者群体中的人肺孢子虫，并识别健康个体和免疫抑制个体中的寄生虫无症状携带者。
