Kammalac Ngouana Thierry, Drakulovski Pascal, Krasteva Donika, Kouanfack Charles, Reynes Jacques, Delaporte Eric, Boyom Fabrice Fekam, Mallié Michèle, Bertout Sebastien
Clinical Biology Laboratory, Yaoundé Central Hospital, Yaoundé, Cameroon.
IRD UMI 233 TransVIHMI - UM INSERM U1175 'TransVIHMI' Laboratoire de Parasitologie et Mycologie Médicale, UFR Pharmacie, Université de Montpellier, Montpellier, France.
J Med Microbiol. 2016 Jul;65(7):579-589. doi: 10.1099/jmm.0.000265. Epub 2016 Apr 20.
Cryptococcal meningitis is a dreadful opportunistic fungal infection amongst human immunodeficiency virus (HIV)-infected patients. One complication in the management of the disease is the possible infection of a patient by two or more different strains of Cryptococcus neoformans. This study investigated the intra-individual genetic diversity and antifungal susceptibility of C. neoformans isolates from Yaoundé (Cameroon) HIV-infected patients with cryptococcal meningitis. Twenty-five clinical isolates were obtained during a prospective study. Five colonies were randomly collected from each initial sample. The 150 isolates obtained (125 colonies and 25 initial samples) were submitted to serotyping by multiplex PCR. Genotyping analyses were achieved using RFLP, and minisatellite- and microsatellite-length polymorphism. The antifungal susceptibility testing was carried out using a Sensititre YeastOne kit. Seven antifungals were tested: itraconazole, fluconazole, amphotericin B, ketoconazole, 5-fluorocytosine, posaconazole and voriconazole. The 150 isolates were identified as C. neoformans serotype A and genotype VNI. The microsatellite and minisatellite sequence analyses generated 15 genotypes. Six out of 25 (24 %) patients were found to be infected by two different genotypes. Antifungal susceptibility showed several profiles: posaconazole (0.015-0.25 µg ml-1), amphotericin B (0.06-1 µg ml-1), fluconazole (0.5-16 µg ml-1), itraconazole (0.008-0.12 µg ml-1), ketoconazole (0.008-0.12 µg ml-1), 5-fluorocytosine (0.25-16 µg ml-1) and voriconazole (0.008-0.12 µg ml-1). It was noted that isolates from the same patient might present different susceptibility profiles to an antifungal drug with differences of more than four dilutions. The results achieved highlighted the possible presence of isolates with different genotypes in a patient with dissimilar antifungal susceptibility profiles during a single episode of cryptococcal meningitis.
隐球菌性脑膜炎是人类免疫缺陷病毒（HIV）感染患者中一种可怕的机会性真菌感染。该疾病治疗中的一个并发症是患者可能被两种或更多不同菌株的新型隐球菌感染。本研究调查了来自雅温得（喀麦隆）患有隐球菌性脑膜炎的HIV感染患者的新型隐球菌分离株的个体内遗传多样性和抗真菌药敏性。在一项前瞻性研究中获得了25株临床分离株。从每个初始样本中随机收集5个菌落。对获得的150株分离株（125个菌落和25个初始样本）进行多重PCR血清分型。使用限制性片段长度多态性（RFLP）、小卫星和微卫星长度多态性进行基因分型分析。使用Sensititre YeastOne试剂盒进行抗真菌药敏试验。测试了七种抗真菌药物：伊曲康唑、氟康唑、两性霉素B、酮康唑、5-氟胞嘧啶、泊沙康唑和伏立康唑。150株分离株被鉴定为新型隐球菌血清型A和基因型VNI。微卫星和小卫星序列分析产生了15种基因型。25名患者中有6名（24%）被发现感染了两种不同的基因型。抗真菌药敏性显示出多种情况：泊沙康唑（0.015 - 0.25 µg/ml）、两性霉素B（0.06 - 1 µg/ml）、氟康唑（0.5 - 16 µg/ml）、伊曲康唑（0.008 - 0.12 µg/ml）、酮康唑（0.008 - 0.12 µg/ml）、5-氟胞嘧啶（0.25 - 16 µg/ml）和伏立康唑（0.008 - 0.12 µg/ml）。值得注意的是，来自同一患者的分离株对一种抗真菌药物可能呈现不同的药敏情况，差异超过四个稀释度。所取得的结果突出表明，在隐球菌性脑膜炎的单次发作期间，患者体内可能存在具有不同基因型且抗真菌药敏情况不同的分离株。
