In vitro and in vivo activity of a possible novel antifungal small molecule against Candida albicans

Nosocomial infections by fungi are important causes of morbidity and mortality, and the adhesion capacity of yeast on abiotic and biotic surfaces has been considered an important step in this process. Als3 proteins are widely studied for their ability to allow Candida albicans to bind to various surfaces. The objective of the present study was to verify, with more details, the action of F2768-0318 in relation to its antifungal activity as well as its ability to act on C. albicans virulence factors related to adhesion and biofilm formation in vitro and in vivo by inhibiting the Als3 protein. F2768-0318 was assessed in tests of biofilm formation and adhesion on abiotic surfaces (polystyrene plates) and adherence on biotic surfaces, including human endocervical (HeLa) cells, human umbilical vein endothelial cells (HUVECs), and fresh buccal epithelial cells (BEC). Our results showed F2768-0318 was useful in reducing the adhesion and biofilm formation of C. albicans on abiotic surfaces, indicating the possibility of treating hospital materials and preventing biofilm formation on these types of equipment. Further studies are still needed, including optimization of the molecule to allow this molecule to be effective on other types of surfaces, such as human cells.     

Isavuconazole is highly active in vitro against Candida species isolates but shows trailing effect

ObjectiveIsavuconazole is a triazole previously shown to have potent in vitro activity against Aspergillus spp., Mucorales, and Candida spp. Unlike for other azoles, it is unclear if isavuconazole may induce a trailing effect. We studied isavuconazole MICs for a large collection of Candida isolates from blood samples and determined the extent of the trailing effect when using the EUCAST Edef 7.3.1 method.Methods761 molecularly identified Candida isolates from blood samples of 742 patients admitted to the hospital (January 2007 to September 2017) were evaluated and further tested for in vitro susceptibility to isavuconazole following the EUCAST E.Def 7.3.1 test method.ResultsC. albicans showed the highest susceptibility, followed by C. parapsilosis and C. tropicalis (geometric mean MIC 0.003 vs 0.005/0.006, respectively; P < 0.001). In contrast, C. glabrata, and C. krusei had significantly higher MIC values (geometric mean MIC 0.094 vs 0.093, respectively). Isavuconazole MIC distributions were not truncated at the lowest concentration tested, except for C. albicans. Overall, the mean percentage of trailing was 12.9% but differences among species were observed: C. glabrata, C. albicans, and C. tropicalis exhibited higher trailing in comparison to C. parapsilosis and non-Candida yeasts (P < 0.001). The percentage of non-wild-type C. albicans (considering the heavy trailer isolates as wild-type), C. parapsilosis and C. glabrata isolates were 0.56% (2/355), 1.5% (3/200), and 4.65% (4/86), respectively.ConclusionsIsavuconazole showed high in vitro activity against Candida spp., particularly against C. albicans. Trailing effect is commonly observed with isavuconazole, particularly with C. glabrata.     

对流感病毒神经氨酸酶N1亚型抗体筛选体系的评价

目的 比较真核表达、活病毒感染、酵母展示系统用于流感病毒神经氨酸酶(NA) N1亚型抗体筛选的差别,寻找合适的抗体筛选系统.方法 将pVRC-VN NA-HAtag质粒转染入293T,通过酵母展示获得重组NA.通过反向遗传学获得重组病毒A/Sichuan/1/2009.用荧光发光法检测NA性能,用流式细胞仪检测NA与5种候选抗体结合情况.结果 真核表达、酵母展示系统均可以使NA在表面展示,且具有与活病毒NA相似的特性.在候选的5种抗体中,真核表达以及病毒的NA可与抗体1、5结合,病毒的NA还能与抗体4结合;而酵母展示系统的NA与5种抗体均不能结合.结论 真核表达、真病毒感染系统适合NA抗体的筛选,酵母展示系统表达的NA其表面抗体识别的表位有差异.     

酵母双杂交技术筛选α干扰素结合蛋白的基因

目的 筛选人肝细胞cDNA文库中与α干扰素（IFNα）蛋白具有相互作用的蛋白基因。方法 用聚合酶链反应（PCR）扩增IFNα基因，连接入酵母表达载体pGBKT7中构建诱饵质粒，转染酵母细胞AH109，Western blot证明IFNα蛋白能够在AH109中表达。然后将AH109与转染了人肝cDNA文库质粒pACT2的酵母细胞Y187进行配合，在营养缺陷型培养基和X-α-半乳糖（X-α-gal）上进行双重筛选阳性菌落，提取质粒后转化DH5α大肠埃希菌并经氨苄西林抗性筛选，提取单克隆菌落质粒，酶切答定正确者进行测序，再行生物信息学分析。结果 成功克隆化IFNα基因并在酵母细胞中表达，应用酵母双杂交筛选出阳性菌落34个，经生物信息学分析，排除读码框架不正确者，最后得到8种已知基因：玻璃体连接蛋白、纤维蛋白原α多肽、人类免疫缺陷病毒（HIV）1Tat相互作用蛋白2、精氨酸酶、NADH脱氢酶1β亚复合物、转铁蛋白受体2α、酒精脱氢酶IBβ多肽、肝细胞癌蛋白（HCC-1）；2种染色体基因：人染色体17，克隆RP11-35083，人染色体10，克隆RP11-35101。结论 成功克隆出IFNα基因，并从肝细胞cDNA文库中筛选出8种能与IFNα具有结合作用的蛋白基因。     

重症监护病房患者酵母样真菌感染调查分析

目的：了解症监护监房（ICU）患者酵母样真菌感染的病原学变化及其临床意义。方法：用常规方法和miniVITAL血培养仪进行培养，用常规方法及ID32C或ATBExpression鉴定。结果：酵母样真菌感染的患者144例，分离真菌152株，内有念珠菌（假丝酵母菌）133株，占总数的87．5％，念珠菌中白色珠菌33株，占21．7％，白色念珠菌以外的其他酵母样菌119株占78．3％，结论：ICU患者大量使用广谱抗生素或长期留置导管等均为酵母样真菌医院感染的危险因素。     

木糖醇对口腔假丝酵母菌体外增殖及产酸的影响

目的初步研究木糖醇对口腔假丝酵母菌体外增殖及产酸的影响,寻求新的预防口腔假丝酵母菌感染的方法。方法对100例戴义齿患者的唾液样本进行假丝酵母菌的培养鉴定及分离纯化后,观察4种常见致病性假丝酵母菌(白假丝酵母菌、热带假丝酵母菌、光滑假丝酵母菌和克柔假丝酵母菌)在不同比例木糖醇替代葡萄糖(葡萄糖∶木糖醇比例分别为4∶1、3∶2、2∶3、1∶4和0∶5)沙氏培养液中的增殖和产酸情况,了解木糖醇对不同假丝酵母菌生长增殖的抑制作用;并在有氧和缺氧条件下,比较白假丝酵母菌在含不同浓度(0%、5%、10%、15%、20%)的木糖醇培养液中培养6、12、24、48和72h后木糖醇对白假丝酵母菌增殖与产酸抑制作用的差异。结果与含葡萄糖和木糖醇的培养液相比,仅含木糖醇的培养液中,4种假丝酵母菌的D600值均降低,而pH值均升高(P<0.05)。与不含木糖醇的培养液相比,在有氧条件下,除含5%木糖醇48h的pH值和D600值、10%木糖醇48h的D600值、5%木糖醇72h的D600值变化不显著外,其余各时间点含不同浓度木糖醇的白假丝酵母菌培养液D600值均降低,pH值均升高(P<0.05),而在缺氧条件下,各时间点的白假丝酵母菌培养液D600值均升高,pH值均降低(P<0.05)。结论木糖醇对4种常见致病性假丝酵母菌的增殖和产酸都有明显的抑制作用,提示木糖醇作为糖替代品和致病性假丝酵母菌抑制剂,在口腔疾病的预防与保健中将发挥重要作用。     

HIV Antibodies: Further Questions and a Plea for Clarification

Summary

The existence of specific antibody/protein reactions is the crucial assumption underlying proof of HIV isolation, proof of HIV infection and the causative role of HIV in AIDS. However, since

1. antibodies which react with the ‘HIV’ proteins arise following allogenic stimuli in non-HIV-infected animals and humans, as well as in mice and humans with autoimmune disorders; antibodies to antigens from both mycobacteria and yeasts cross-react with HIV env and gag proteins;

2. individuals belonging to the AIDS risk groups are subjected to allogenic stimuli and have high levels of autoimmune antibodies, while the vast majority of patients in the AIDS risk groups are infected with either or both mycobacteria or yeasts;

the evidence for the existence of HIV and its putative role in AIDS must be reappraised.     

Superficial mycoses at the Hospital do Servidor Público Municipal de S?o Paulo between 2005 and 2011

BACKGROUND

Superficial mycoses are fungal infections limited to the outermost layers of the skin. Dermatophytic filamentous fungi and yeasts are the major causative agents of these mycoses. Dermatophytosis is one of the clinical conditions caused by fungal infections most commonly found in dermatological practice. Thus, knowledge of the ecology of dermatophytes provides a better understanding of the natural history of dermatophytosis.

OBJECTIVE

This study aimed to investigate epidemiological and mycological features of superficial mycoses diagnosed from 2005 to 2011 in the Dermatology Clinic of the Hospital do Servidor Público Municipal de São Paulo, Brazil.

METHOD

This retrospective study was conducted in the Laboratory of Medical Mycology at the Dermatology Clinic of the Hospital do Servidor Público Municipal de São Paulo. Mycological examinations of 9042 patients with clinical suspicion of superficial mycoses performed between 2005 and 2011 were reviewed.

RESULTS

Of 9042 direct microscopic examinations, 2626 (29%) were positive for dermatophytes, 205 (2.3%) were positive for Malassezia, 191 (2.1%) were positive for other types of yeast, 48 (0.5%) were positive for bacteria, and 5972 (66%) were negative. Mean age of patients was 48 years, 6920 (77%) patients were female and 2112 (23%) were male.

CONCLUSION

The biota consisted of six dermatophyte species: T. rubrum, T. mentagrophytes, M. gypseum, T. tonsurans, E. floccosum, and M. canis. The most common site of involvement was the nail and foot in adults and scalp in children, with a female predominance. Both Candida and Malassezia were more prevalent in adult women, the former most commonly affecting the interdigital region and nails and the latter the chest and neck.     

Thermotolerance of Histoplasma capsulatum at 40 °C predominates among clinical isolates from different Latin American regions

The yeast phase of 22 Histoplasma capsulatum clinical isolates from Mexico, Argentina, Colombia, and Guatemala and three reference strains, one from Panama and two from the United States of America (USA), were screened for thermosensitivity characteristics using different analyses. Growth curves at 0, 3, 6, 12, 24, and 30 h of incubation at 37 and 40 °C, the growth inhibition percentage at 40 °C, and the doubling time at 37 and 40 °C were determined for all yeasts studied. Most of the isolates examined exhibited thermotolerant phenotypes at 40 °C, whereas a thermosensitive phenotype at 40 °C was only detected in the Downs reference strain from the USA. Growth inhibition values lower than 33.8% supported the predominance of the thermotolerant phenotype at 40 °C. The doubling time means found for the different isolates were 5.14 h ± 1.47 h at 37 °C and 5.55 h ± 1.87 h at 40 °C. This is the first report to underscore the predominance of thermotolerant and delayed doubling time phenotypes in H. capsulatum clinical isolates from different regions of Latin America.