分子生物学技术在侵袭性真菌感染诊疗中的应用进展

侵袭性真菌感染近年来呈上升趋势,临床分离真菌的构成比有所变化,耐药菌株的增加亦给临床的诊断、治疗和预防带来一定困难。因此,建立快速、准确的分子生物学鉴定方法,对侵袭性真菌感染的早期诊断有着重要意义。该文就分子生物学技术在侵袭性真菌感染早期诊疗中的应用研究作一综述。     

侵袭性和非侵袭性肺炎链球菌耐药率的比较分析

目的研究侵袭性和非侵袭性肺炎链球菌的耐药谱的差异,指导合理应用抗生素及感染管理。方法回顾性统计分析2009至2011年来天台县人民医院就诊患者分离肺炎链球菌的标本来源及耐药性,比较侵袭性和非侵袭性肺炎链球菌耐药率之间的差异。结果共分离出肺炎链球菌642株,痰液中分离出584株,非痰液中分离出58株,其中血液中分离出32株,脑脊液中分离出20株,其他分离出6株,所有肺炎链球菌对万古霉素和利奈唑胺均敏感,对青霉素、红霉素、克林霉素、四环素及复方新诺明耐药严重,对左氧氟沙星、氯霉素比较敏感;侵袭性分离株对青霉素、红霉素、克林霉素、左氧氟沙星、四环素及氯霉素的耐药率显著高于非侵袭性肺炎链球菌。结论该院分离的肺炎链球菌主要来自痰液标本,耐青霉素肺炎链球菌的检出率高,大环内酯类耐药严重,存在一定比例的侵袭性感染,非侵袭菌株与侵袭性菌株耐药谱之间存在一定差异,临床治疗应该区别对待,系统的监测细菌耐药性,合理选择抗菌药物。     

改变医疗的新一代诊断、评价技术

新的医疗技术普及的关键在于诊断、评价技术。尤其是可对活体内分子的行为进行无侵袭性监视的分子成像技术格外引人注目。企业的参与日趋活跃，技术的焦点也逐渐明朗。[编者按]     

侵袭性真菌感染早期诊断技术的研究进展

侵袭性真菌感染（invasive fungal infections,IFIs）是真菌入侵人体导致血流、各脏器或全身播散的严重感染,以念珠菌为主的酵母样真菌和曲霉为主的丝状真菌最常见。近年来IFIs发病率及死亡率在全球范围内有显著上升趋势,严重威胁着人类的健康。早期快速的诊断方法现己成为真菌感染研究领域的热点和难点,对于患者及时治疗和死亡率的降低有十分重要的意义。本文旨对目前侵袭性真菌早期相关诊断技术以及临床研究的问题和现状予以总结,同时预测该领域未来的发展趋势。     

侵袭性真菌病的诊断：现状与展望

近二十年来,医学科学很多领域都取得重大进步。但全球范围内,侵袭性真菌病的发病率及死亡率却仍明显上升,严重威胁人类健康。侵袭性真菌病发病隐匿、临床表现不典型、治疗手段有限、病死率与致残率高,早期、特异的诊断对于改善预后意义重大。目前,以培养、病理为代表的形态学诊断方法虽有局限,但仍是侵袭性真菌病诊断的金标准;以G试验、GM试验、高分辨率CT为代表的新兴血清学及影像学诊断方法值得在临床大力推广;而以PCR技术为基础的核酸诊断技术方法前景光明,但其临床应用之路却仍任重而道远。联合使用并不断改良现有培养、病理等形态学诊断方法、血清学方法及先进影像学技术是提高侵袭性真菌病诊断水平的现实最佳途径。     

临床侵袭性真菌病实验室诊断学术研讨会征文通知

当前侵袭性真菌感染在临床上的危害日益增大,其诊断和治疗存在严峻挑战,由中华医学会检验分会临床微生物学组主办,卫生部主管《医学参考报》检验医学频道和北京协和医院检验科临床微生物专业组联合承办的此次会议将邀请国内外知名临床侵袭性真菌病学专家就临床侵袭性真菌病实验室诊断技术进行深入研讨。会议将围绕以下重点议题展开：（1）临床常见侵袭性真菌的分离培养及形态学鉴定技术。（2）分子生物学诊断技术研究进展。（3）侵袭性真菌体外药敏试验方法。（4）侵袭性真菌耐药监测及耐药机制的研究。     

艾滋病合并侵袭性真菌感染35例临床分析

目的 分析艾滋病(AIDS)合并侵袭性真菌感染的发生情况及临床特点,为其诊治提供参考。方法 回顾性分析85例AIDS患者的临床资料,总结侵袭性真菌感染的发病情况及临床特点。结果 85例AIDS患者继发侵袭性真菌感染35例,感染率为41．2％,感染部位以消化道为主,占44．4％,致病菌以白念珠菌(白假丝酵母)为主,占57．4％,侵袭性真菌感染病例主要发生于CD4~+T淋巴细胞计数＜100个/μl的患者,占71．8％。35例患者中27例治愈,8例死亡。结论 侵袭性真菌感染是AIDS患者主要的机会性感染之一,其发生与CD4~+T淋巴细胞计数密切相关,临床上以消化道真菌感染多见,侵袭性真菌感染的病死率高。     

重视侵袭性真菌病的诊治

随着免疫抑制剂、广谱抗菌素、器官移植和侵袭性操作应用的增加,侵袭性真菌病的发生率也相应地增加。临床医生的误诊误治,更加重了这种倾向。要纠正这种倾向,必须提高对侵袭性真菌病的认识水平。分清真菌相关的概念,了解微生物学特性,熟悉流行病学现状和趋势,重视早期诊断尤其是疑似线索,对实现真正意义上的分层治疗是非常重要的。     

侵袭性真菌感染的流行病学和诊断研究进展

伴随着侵袭性真菌感染(Invasive fungal infection,IFI)的迅猛增长,逐渐认识到对许多真菌诊断的重要性,本文就侵袭性真菌感染的流行概况、危险因素以及传统和新近发展的诊断方法作一综述。     

实时荧光定量PCR在侵袭性曲霉病诊断中的研究进展

实时荧光定量PCR自20世纪90年代初起被用于侵袭性曲霉病的诊断研究,具有较好的灵敏度和特异度,但由于技术操作多种多样,尚未统一,该技术仍未正式列入诊断标准.近年来,随着研究增加,对该技术操作的标准化研究取得了明显的进展.现从技术问题,包括标本种类、DNA提取方式、靶序列的选择、国际标准化研究进程以及该技术在侵袭性曲霉病的诊断价值和应用两个层面进行综述.     

Oocyte Scoring Enhances Embryo-Scoring in Predicting Pregnancy Chances with IVF Where It Counts Most

Context

Our center’s quality improvement optimization process on many occasions anecdotally suggested that oocyte assessments might enhance embryo assessment in predicting pregnancy chances with in vitro fertilization (IVF).

Objective

To prospectively compare a morphologic oocyte grading system to standard day-3 morphologic embryo assessment.

Design, Setting, Patients

We prospectively investigated in a private academically-affiliated infertility center 94 consecutive IVF cycles based on 6 criteria for oocyte quality: morphology, cytoplasm, perivitelline space (PVS), zona pellucida (ZP), polar body (PB) and oocyte size, each assigned a value of -1 (worst), 0 (average) or +1 (best), so establishing an average total oocyte score (TOS). Embryo assessment utilized grade and cell numbers of each embryo on day-3 after oocyte retrieval. Clinical pregnancy was defined by presence of at least one intrauterine gestational sac.

Interventions

Standard IVF cycles in infertile women.

Main Outcome Measures

Predictability of pregnancy based on oocyte and embryo-grading systems.

Results

Average age for all patients was 36.5 ± 7.3 years; mean oocyte yield was 7.97± 5.76; Patient specific total oocyte score (PTOS) was -1.05 ± 2.24. PTOS, adjusted for patient age, was directly related to odds of increased embryo cell numbers (OR 1.12, P = 0.025), embryo grade (OR 1.19, P < 0.001) and clinical pregnancy [OR 1.58 (95%CI 1.23 to 2.02), P < 0.001]. Restricting the analysis to day three embryos of high quality (8-cell/ good grades), TOS was still predictive of clinical pregnancy (OR 2.08 (95%CI 1.26 to 3.44, P = 0.004). Among the 69 patients with embryos of Grade 4 or better available for transfer 23 achieved Clinical Pregnancy. When the analysis was restricted to the 69 transfers with good quality embryos (≥ Grade 4) the Oocyte Scoring System (TOS) (AUC±SE 0.863±0.044, oocyte score) provided significantly greater predictive value for clinical pregnancy compared to the embryo grade alone (AUC 0.646 ± 0.072, embryo grade) p = 0.015.

Conclusions

Oocyte-scoring, thus, provides useful clinical information especially in good prognosis patients with large numbers of high quality embryos. This finding appears of particular importance at a time when many IVF centers are committing sizable investments to closed incubation systems with time-lapse photography, which are exclusively meant to define embryo morphology.     

Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics

The assessment of oocyte quality in human in vitro fertilization (IVF) is getting increasing attention from embryologists. Oocyte selection and the identification of the best oocytes, in fact, would help to limit embryo overproduction and to improve the results of oocyte cryostorage programs. Follicular fluid (FF) is easily available during oocyte pick-up and theorically represents an optimal source on non-invasive biochemical predictors of oocyte quality. Unfortunately, however, the studies aiming to find a good molecular predictor of oocyte quality in FF were not able to identify substances that could be used as reliable markers of oocyte competence to fertilization, embryo development and pregnancy. In the last years, a well definite trend toward passing from the research of single molecular markers to more complex techniques that study all metabolites of FF has been observed. The metabolomic approach is a powerful tool to study biochemical predictors of oocyte quality in FF, but its application in this area is still at the beginning. This review provides an overview of the current knowledge about the biochemical predictors of oocyte quality in FF, describing both the results coming from studies on single biochemical markers and those deriving from the most recent studies of metabolomics     

Effect of vitrification on human oocyte maturation rate during in vitro maturation procedure: A systematic review and meta-analysis

Combination of in vitro maturation (IVM) and cryopreservation offers new opportunities for women with contraindication in ovarian stimulation, and females who desire to postpone the childbearing due to different problems. There are still controversies regarding IVM procedure and its impact on oocytes fertilization capability. This systematic review and meta-analysis were conducted to evaluate the impact of vitrification on human oocyte maturation rate during IVM procedure. In this review, we searched Medline, Embase, Scopus and ISI web of science to identify English-language studies. The last search was implemented on 3 February 2018. The original articles which assessed maturation rate after vitrification of MI or GV oocytes were included. Animal trials and the studies that performed cryopreservation using slow-freeze method were excluded. Bias and quality assessments were performed. 2476 articles were screened primarily. After duplication removing and the application of inclusion and exclusion criteria, 14 studies included for the analysis. All studies compared maturation rate between the oocytes that were vitrified at the GV or MI stage before maturation and oocytes which were matured in vitro without vitrification. Meta-analysis showed that oocyte vitrification at GV stage had a significant negative impact on maturation rate (RR = 0.76, 95% CI: 0.66–0.88); I² = 85.2%; P = 0.000). Finally, based on our results, oocyte vitrification decreases the maturation rate by 24%.     

Noninvasive bovine oocyte quality assessment: possibilities of a single oocyte culture

Although bovine embryos are routinely produced in vitro for several decades, there still exists a critical need for techniques to accurately predict the oocyte's developmental competence in a noninvasive way, before the in vitro embryo production procedure. In this review, several noninvasive methods to evaluate oocyte quality are discussed, such as morphological assessment of the cumulus oocyte complex and the use of brilliant cresyl blue. Because an individual oocyte and embryo culture method can possibly generate additional insights into the factors that determine oocyte quality, the second part of this review summarizes the state of the art of bovine single oocyte culture. The optimization of individual in vitro embryo production can obviously accelerate the quest for better noninvasive oocyte quality markers, because more information about the oocyte's requirements and intrinsic quality will be revealed. Although each step of in vitro culture has to be re-examined in light of the hampered production of single embryos, the reward at the end will be substantial. Individual scored oocytes will be traceable along the in vitro embryo production procedure and the final blastocyst outcome can be linked to the original oocyte quality and follicular environment without the bias caused by simultaneously developing embryos.     

Epigenetic changes associated with oocyte aging

It is well established that the decline in female reproductive outcomes is related to postovulatory aging of oocytes and advanced maternal age. Poor oocyte quality is correlated with compromised genetic integrity and epigenetic changes during the oocyte aging process. Here, we review the epigenetic alterations, mainly focused on DNA methylation, histone acetylation and methylation associated with postovulatory oocyte aging as well as advanced maternal age. Furthermore, we address the underlying epigenetic mechanisms that contribute to the decline in oocyte quality during oocyte aging.     

Maternal diabetes and oocyte quality

Maternal diabetes has been demonstrated to adversely affect preimplantation embryo development and pregnancy outcomes. Emerging evidence has implicated that these effects are associated with compromised oocyte competence. Several developmental defects during oocyte maturation in diabetic mice have been reported over past decades. Most recently, we further identified the structural, spatial and metabolic dysfunction of mitochondria in oocytes from diabetic mice, suggesting the impaired oocyte quality. These defects in the oocyte may be maternally transmitted to the embryo and then manifested later as developmental abnormalities in preimplantation embryo, congenital malformations, and even metabolic disease in the offspring. In this paper, we briefly review the effects of maternal diabetes on oocyte quality, with a particular emphasis on the mitochondrial dysfunction. The possible connection between dysfunctional oocyte mitochondria and reproductive failure of diabetic females, and the mechanism(s) by which maternal diabetes exerts its effects on the oocyte are also discussed.