Perandin F, Manca N, Galati L, Piccolo G, Calderaro A, Viani I, Ricci L, Dettori G, Chezzi C, Turano A
Dipartimento di Diagnostica, Università degli Studi di Brescia, Spedali Civili, Italia.
New Microbiol. 2001 Jan;24(1):69-76.
A PCR method involving a genus-specific oligonucleotides set and Southern blot hybridization with four species-specific probes to P. falciparum, P. vivax, P. malariae and P. ovale was evaluated for the detection of malaria parasites in blood samples from 101 patients with clinically suspect malaria infection imported to Italy. Plasmodium falciparum was the main species detected. As determined by microscopy, 53 (52.4%) patients had malaria and of these: 40 (75.5%) were infected with P. falciparum; 7 (13.2%) with P. vivax; 1 (1.9%) with P. ovale; 3 (5.7%) with P. malariae; 1 (1.9%) with P. vivax or P. ovale; and 1 (1.9%) with P. falciparum or P. vivax. Ninety-seven out 101 blood samples were submitted to ParaSight-F test which showed a sensitivity of 94.73%, and a specificity of 93.22%, as compared to microscopy. The PCR assay using the genus-specific oligonucleotide primer set (pg-PCR) was able to detect 53 (52.4%) infections and showed a sensitivity of 100% and a specificity of 100%, when compared to microscopy. The parasite species were identified by Southern blot hybridization using species-specific probes and 40 (75.5%) samples were P. falciparum positive, 5 (9.4%) P. vivax positive, 4 (7.5%) P. ovale positive, and 2 (3.8%) P. malariae positive. When the Southern blot results were compared to those of blood-film diagnosis, we observed some disagreement. In particular, compared to Southern blot, microscopy underestimated P. ovale infection; blood film analysis recognised only 1 P. ovale sample, whereas Southern blot recognised 4 P. ovale positive samples (by microscopy, 2 of these were detected as P. vivax, 1 as P. ovale or P. vivax, and the other as P. falciparum or P. vivax). Southern blot hybridization was unable to identify one P. falciparum and one P. vivax positive case detected by microscopy. We also plan to use a reference nested-PCR assay to clarify the disagreement observed between microscopy and Southern blot hybridization.
评估了一种聚合酶链反应（PCR）方法，该方法涉及一组属特异性寡核苷酸，并与针对恶性疟原虫、间日疟原虫、三日疟原虫和卵形疟原虫的四种种特异性探针进行Southern印迹杂交，用于检测101例输入意大利的临床疑似疟疾感染患者血样中的疟原虫。恶性疟原虫是检测到的主要虫种。通过显微镜检查确定，53例（52.4%）患者患有疟疾，其中：40例（75.5%）感染恶性疟原虫；7例（13.2%）感染间日疟原虫；1例（1.9%）感染卵形疟原虫；3例（5.7%）感染三日疟原虫；1例（1.9%）感染间日疟原虫或卵形疟原虫；1例（1.9%）感染恶性疟原虫或间日疟原虫。101份血样中的97份进行了ParaSight-F检测，与显微镜检查相比，其灵敏度为94.73%，特异性为93.22%。使用属特异性寡核苷酸引物组的PCR检测（pg-PCR）能够检测到53例（52.4%）感染，与显微镜检查相比，其灵敏度和特异性均为100%。通过使用种特异性探针的Southern印迹杂交鉴定寄生虫种类，40例（75.5%）样本恶性疟原虫呈阳性，5例（9.4%）间日疟原虫呈阳性，4例（7.5%）卵形疟原虫呈阳性，2例（3.8%）三日疟原虫呈阳性。当将Southern印迹结果与血涂片诊断结果进行比较时，我们观察到一些不一致之处。特别是，与Southern印迹相比，显微镜检查低估了卵形疟原虫感染；血涂片分析仅识别出1例卵形疟原虫样本，而Southern印迹识别出4例卵形疟原虫阳性样本（通过显微镜检查，其中2例被检测为间日疟原虫，1例被检测为卵形疟原虫或间日疟原虫，另1例被检测为恶性疟原虫或间日疟原虫）。Southern印迹杂交无法识别显微镜检查检测到的1例恶性疟原虫阳性病例和1例间日疟原虫阳性病例。我们还计划使用参考巢式PCR检测来澄清显微镜检查和Southern印迹杂交之间观察到的不一致之处。
