The contribution of galactomannan detection in the diagnosis of invasive aspergillosis in bone marrow transplant recipients

Until recently, accurate microbiological diagnosis of invasive aspergillosis (IA) was seldom established in HSCT recipients. Blood samples are rarely positive for Aspergillus species, the reliability of the cultures depends of the specimen (if taken from a normally sterile site or not) and biopsy samples require invasive procedures, rarely recommended in patients with severe thrombocytopenia. Implementing the international consensus defining the microbiological criteria for the diagnosis of Aspergillus infection, we retrospectively evaluated the role of serum galactomannan (GM) detection by EIA to diagnose IA among HSCT patients with proven invasive fungal infection (IFI) and the impact of serum storage in GM concentrations. The EIA assay allowed categorizing as “probable” 5 of the 10 cases of “possible” aspergillosis (50%). Considering a lower cut-off level for the reaction (1.0), 80% of the cases could be categorized as “probable” aspergillosis. Positive or undetermined results were detected one to 4 months before the diagnosis of IA in eight of the 11 patients (72.7%) with proven IFI. Retesting the stored samples after a second storage for four years, we could observe lower reactivity in 20% of the samples. The detection of galactomannan by the EIA test represents a major advance in the diagnosis of IA in HSCT recipients at high risk of IA. A better understanding of the kinetics of the GM in different clinical situations is necessary to maximize the benefit of the test in Aspergillus surveillance.     

国产血清半乳甘露聚糖检测试剂对侵袭性肺曲霉菌病的诊断价值评估

目的：评估国产血清半乳甘露聚糖（Galactomannan,GM）检测试剂对侵袭性肺曲霉菌病的诊断价值。方法根据血液病/恶性肿瘤患者侵袭性真菌病的诊断标准与治疗原则（第四次修订版）[1]收集临床确诊侵袭性肺曲霉菌病（inva-sive pulmonary aspergillosis,IPA）、临床诊断IPA、拟诊IPA、排除IPA四组病例。采用天津贻诺琦公司酶联免疫吸附法（ELISA）试剂检测纳入的86例患者血清标本的GM浓度,分析其敏感性、特异性、阳性预测值（PPV）、阴性预测值（NPV）。结果86例病例中,临床诊断27例、拟诊12例、排除47例。在3种不同的阳性判断标准下,敏感性：9444%、9630%、6296%;特异性：5625%、4576%、6441%;PPV：4474%、4483%、4474%;NPV：9643%、9643%、7917%。统计学分析证实标准1（即血清GM值〉095μg/L为阳性,〈075μg/L为阴性,075~095μg/L为灰区,未将灰区加入计算）在3种判断标准中最优,故选择其为最终判断标准。结论该血清GM检测试剂盒诊断性能较好,可以用于侵袭性肺曲霉菌病的辅助诊断。     

Towards a molecular diagnosis of invasive aspergillosis and disseminated candidosis

A lot of in-house polymerase chain reaction assays have been reported for diagnosis of invasive aspergillosis and disseminated candidosis. Encouraging results have been published to anticipate the diagnosis over the conventional microbiological methods. However, the absence of standardized methods has led to diverging results. As a consequence, these tests are not recognized as consensual diagnostic criteria, in contrast with some antigenemia detection kits. The major breakthrough for improving the results of these methods is the emergence of real-time technologies. This markedly improves the reliability of the PCR results by dramatically decreasing the risk of false positive results due to PCR products carryover. Moreover, using the quantitative results provided by this technique, this allows to rapidly compare the efficiency of primers, probes, and DNA extraction methods. Therefore, the hope is to identify the more specific and sensitive parameters to implement comparative studies. Automated DNA extraction should also be useful to achieve this goal. Whatever sophisticated technology is used, we still have to define the meaning of detecting nucleic acids in a given clinical sample. This seems simple in normally sterile anatomical sites but less obvious for example in respiratory specimens for invasive aspergillosis or in blood for candidosis in heavily colonized patients. Additional studies of the kinetics of fungal DNA are needed. The development of real-time technology should improve our knowledge in order to give the clinicians informative clues for making a decision.     

侵袭性曲霉菌病13例临床特点分析

目的分析侵袭性肺曲霉病(IPA)的临床特点,提高对本病的认识。方法对13例确诊的IPA患者的临床资料进行回顾性分析。结果 IPA患者中存在基础疾病或危险因素者占69.2%(9/13);临床表现包括咳嗽、黄痰(13/13)、高热(11/13)、胸闷或呼吸困难(7/13)和咯血(2/13)等;92.3%(12/13)患者有高白细胞血症,WBC大多为(15~30)×109;10例G试验检测的患者中有9例出现阳性;影像学检查:肺内多发空洞及"空气新月征"9例(69.2%),"晕轮征"2例(15.4%),支气管炎表现6例(46.2%),胸腔积液3例(23.1%),颅内播散病灶1例;气管镜检查以粘膜水肿、脓痰、管腔坏死物为主;9例患者痰培养或肺泡灌洗液培养阳性(占69.2%),所有患者病理检查均见曲霉菌丝。所有患者均接受抗真菌药物治疗,4例好转出院,9例恶化放弃治疗或死亡。结论 IPA是临床严峻挑战;掌握该病的临床特点、实验室检查和胸部影像特征,及时的气管镜、组织病理检查有助于IPA确诊。临床医师应高度重视曲霉病。     

Usefulness of galactomannan detection in the diagnosis and follow-up of hematological patients with invasive aspergillosis

The usefulness of galactomannan detection using the Platelia Aspergillus test for the diagnosis of invasive aspergillosis was studied in 849 sera from 54 hematological patients with prolonged neutropenia, which were classified according to the risk for invasive aspergillosis. Three patients developed a proven invasive aspergillosis, one a probable invasive aspergillosis and 17 patients a possible invasive aspergillosis. Thirty-three patients showed no evidence of invasive aspergillosis. All patients with proven invasive aspergillosis had a high risk for invasive aspergillosis, while the one having probable invasive aspergillosis had intermediate risk. Detection of galactomannan in this study showed a sensitivity of 66.7% for patients with proven invasive aspergillosis and 50% for patients with proven and probable invasive aspergillosis. The specificity was 98% or higher in all groups studied. The predictive positive and negative values for patients with proven invasive aspergillosis were 66.7% and 98%, respectively. A rise in the concentration of galactomannan was observed in patients who failed to respond to the antifungal treatment. Galactomannan antigenemia preceded post-mortem histological diagnosis of invasive aspergillosis in two patients by 17 and 81 days, respectively. In conclusion, detection of galactomannan by the Platelia Aspergillus test allows for a specific and relatively sensitive diagnosis of invasive aspergillosis in hematological patients with a high and intermediate risk for invasive aspergillosis.     

Local retrospective analysis of galactomannan cut-off values in bronchoalveolar lavage fluids for diagnosis of invasive aspergillosis

Galactomannan antigen (GM) testing has been used for decades to screen immunocompromised patients for invasive aspergillosis (IA). Recent publications suggested that using a higher cut-off value than 0.5 in bronchoalveolar lavage fluid (BALF) could be more discriminant for hematology patients. We retrospectively analyzed the values of GM in BALF over 7 years (from 2010 to 2016). Performance indicators of the GM in BALF, according to three different cut-off values (0.5, 0.8, 1.5), were calculated using Stata 14.1. IA classification for hematology patients was based on European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria, as defined in 2008. A number of 716 GM were performed on BALF from 2010 to 2016 (597 patients) and 66 were positive (>?0.5). Among these 597 patients, 27 IA were diagnosed, 13 with a positive GM in BALF, 9 with a negative GM in BALF, and 5 unclassified IA (ICU patients). The analysis of performance indicators, based on our local data, did not demonstrate any significant difference using a higher cut-off value of GM in BALF. This result may be explained by the local recruitment of patients and by pre-analytic variations during BALF realization.     

米卡芬净治疗重症侵袭性真菌感染40例的临床分析

目的评价米卡芬净治疗重症侵袭性真菌感染患者的疗效和安全性。方法采用回顾性分析2007年12月~2009年6月大连医科大学附属二院ICU病房40例侵袭性真菌感染患者应用米卡芬净治疗的临床资料。结果40例患者中,痊愈10例(25%),显效18例(45%),总有效率为70%,真菌清除率为60.8%,不良反应少,所有患者均耐受治疗。结论米卡芬净治疗重症侵袭性真菌感染安全、有效。     

Development of a chromosomal DNA probe for the laboratory diagnosis of aspergillosis

Chromosomal DNA was extracted from clinical isolates of Aspergillus fumigatus of human and animal origin using the protoplast lysate method. The probe was developed by the nick translation of the chromosomal DNA genome fraction with p³² as the radiolabel. Hybridization of the probe with endonuclease-cleaved DNA of the same species resulted in a pattern of recognition sites specific for the species. The latter was not seen in other species encountered in clinical specimens. Trials were carried out on sputum experimentally inoculated with the fungus where crude DNA was directly extracted, treated with the endonuclease and hybridized with the probe. The efficacy of the probe was as good with the crude as the purified DNA. The specificity of the probe was determined by testing it against single and mixed DNA populations extracted from different species of several fungal and bacterial genera isolated from and/or known to occur in clinical specimens of respiratory infection origin. The sensitivity of the probe was assessed by detecting a DNA concentration in the specimen equivalent to 3 C.F.U.This research project was supported by the Mayo Visiting Clinician Program, Mayo Medical Center, Rochester, MN 55905, USA.     

Value of Aspergillus galactomannan antigen detection in the diagnosis and follow-up of invasive aspergillosis in hematological patients

Serum galactomannan detection is considered to be a useful test for early diagnosis and follow-up of invasive aspergillosis. From February to September 2002, adult patients hospitalized in our Hematology Unit for receiving intensive chemotherapy and/or hematopoietic stem cell transplant were prospectively studied. We analyzed a total of 760 samples obtained from 100 patients. Eleven patients (11%) having a positive result (OD index >1.5 ng/ml) in two consecutive Platelia Aspergillus tests were considered galactomannan-positive cases. On the other hand, 12 patients (12%) were diagnosed of proven or probable invasive aspergillosis. Sensitivity (66.6%), specificity (95.5%), positive predictive value (72.7%) and negative predictive value (96.7%) were comparable to those of larger series. Galactomannan positivity allowed also to anticipate invasive aspergillosis diagnosis (from two to 17 days before radiographic findings and from two to 15 days before mycological culture). Moreover, kinetics of antigenemia could be useful for assessing therapeutic response. Once accepted galactomannan test as a diagnostic criterium for invasive aspergillosis knowing potential causes of false positive results is of paramount importance.     

侵袭性真菌感染治疗新进展

随着免疫受损人群的增多,近年来侵袭性真菌感染的发生率逐渐升高,由此导致的致病率也逐年上升.如何及时诊断并有效的治疗侵袭性真菌感染已成为临床上面临的挑战之一.该文就侵袭性真菌感染的流行病学、病因学及现有的治疗策略等方面进行综述.     

实体器官移植术后隐球菌感染诊治的研究进展

隐球菌是实体器官移植术后最常见的致病性真菌之一,主要通过呼吸道入侵机体并播散至全身,尤嗜中枢神经系统。隐球菌感染如不及时治疗,病死率极高。实体器官移植术后隐球菌感染的主要危险因素包括术前发热、术后免疫抑制剂和抗生素使用、术后导管留置时间、术后感染及大剂量糖皮质激素使用等。其临床症状缺少特异性,早期诊断较困难。实体器官移植术后抗隐球菌治疗主要包括两性霉素B、氟胞嘧啶、氟康唑等,具体方案通常视患者免疫状态和器官功能而定。本文综述了实体器官移植术后隐球菌病诊断和治疗方面的研究进展。     

两性霉素B对小儿白血病化疗后肺部侵袭性真菌感染的疗效研究

目的探讨两性霉素B对小儿白血病化疗后肺部侵袭性真菌感染（PIFI）的疗效。方法选取沈阳军区总医院于2012年4月至2014年8月收治的54例白血病化疗后PIFI患儿为研究对象,随机分成A、B两组,每组27例。B组采用米卡芬净,A组采用两性霉素B。对比两组临床疗效、G试验结果、肺部CT改变情况及不良反应发生率。结果 （1）A组治疗有效率为66.67%,明显优于B组的44.44%,且治疗后A组死亡6例（22.22%）,低于B组的13例（48.15%）,组间对比差异有统计学意义（P〈0.05）;（2）所有受试患儿均行动态G试验检测,发现阳性者43例（79.63%）,A组22例、B组21例（P〉0.05）。其中37例（86.05%）治疗后转阴,A组20例,B组17例（P〉0.05）;治疗前,54例患儿高分辨CT影像显示其肺部出现片状或结节状高密度影,呈现明显新月形或空洞改变;（3）治疗后,两组患者均无严重腹泻、头晕、头痛等不良反应发生（P〉0.05）;A组3例因耐受不足停药,B组4例因肾功能改变而停止治疗,不良反应发生对比差异无统计学意义（P〉0.05）。结论对白血病化疗后出现PIFI症状的患者给予两性霉素B进行治疗,疗效显著,安全可靠,值得临床推广。     

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.     

Evaluation of three serological tests for detection of anti-candidal antibodies in diagnosis of invasive candidiasis

Immunoblot detection of antibody against 47 KD cytoplasmic antigen ofCandida albicans was evaluated in diagnosis of invasive candidiasis and compared to whole cell agglutination and gel diffusion tests for detection of anticandidal antibody in 64 patients. The patients included 17 with culture proved candidemia, 34 with significant candiduria (more than 10,000 colony forming units per ml of urine) and 13 with nonsignificant candiduria. Antibody against 47 KD antigen was found to be the best indicator for diagnosis of invasive candidiasis even in patients with malignancy. The sensitivity of this procedure was 82.4%, specificity 86.7%, positive predictive value 77.8%, negative predictive value 89.7% and efficacy 85.1%. The gel diffusion procedure lacked in sensitivity whereas whole cell agglutination lacked in specificity. Detection of antibody against 47 KD antigen proved to be a valuable adjunct in the diagnosis of invasive candidiasis.     

Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice

The bone marrow is believed to play important roles during fracture healing such as providing progenitor cells for inflammation, matrix remodeling, and cartilage and bone formation. Given the complex nature of bone repair, it remains difficult to distinguish the contributions of various cell types. Here we describe a mouse model based on bone marrow transplantation and genetic labeling to track cells originating from bone marrow during fracture healing. Following lethal irradiation and engraftment of bone marrow expressing the LacZ transgene constitutively, wild type mice underwent tibial fracture. Donor bone marrow-derived cells, which originated from the hematopoietic compartment, did not participate in the chondrogenic and osteogenic lineages during fracture healing. Instead, the donor bone marrow contributed to inflammatory and bone resorbing cells. This model can be exploited in the future to investigate the role of inflammation and matrix remodeling during bone repair, independent from osteogenesis and chondrogenesis.     

The effects of staphylococcal protein A on human lymphokine-activated killer cell induction

Summary A murine IgG2b monoclonal antibody directed to the constant part of the human / T cell receptor (BMA031) was investigated in a pilot study as an initial treatment for acute graft-versus-host disease (aGvHD) after allogeneic bone marrow transplantation. The treatment protocol consisted of 5 mg BMA031 on 5 consecutive days with continuation of the prophylactic baseline immuno suppression using cyclosporin. Seven patients with grades II–III acute graft-versus-host disease were entered on the protocol and six patients completed the full treatment course. Mild to moderate acute adverse reactions to the first BMA031 infusion occurred in three patients. A nearly complete decline of circulating T lymphocytes was observed during BMA031 therapy, but the T cells returned to pretreatment values within 1 week after the last infusion. Serum pharmacokinetics of free antibody best fitted to a two-compartment open model with a mean initial half-life of 6 h and an estimated mean terminal half-life of 40 h. One patient developed antimurine antibodies of the IgM subclass. In five patients a complete and sustained resolution of all disease manifestations was attained, while in one patient a temporary response of skin involvement with aGvHD was noted. These results indicate that BMA031 can be safely administered as initial treatment of aGvHD. The therapeutic responses observed warrant its further clinical evaluation in this setting.     

Effect of repeated allogeneic bone marrow mononuclear cell transplantation on brain injury following transient focal cerebral ischemia in rats

Aims

Transplantation of bone marrow mononuclear cells (BMMCs) exerts neuroprotection against cerebral ischemia. We examined the therapeutic timepoint of allogeneic BMMC transplantation in a rat model of focal cerebral ischemia, and determined the effects of repeated transplantation outside the therapeutic window.

Main methods

Male Sprague–Dawley rats were subjected to 90 minute focal cerebral ischemia, followed by intravenous administration of 1 × 10⁷ allogeneic BMMCs or vehicle at 0, 3 or 6 h after reperfusion or 2 × 10⁷ BMMCs 6 h after reperfusion. Other rats administered 1 × 10⁷ BMMCs at 6 h after reperfusion received additional BMMC transplantation or vehicle 9 h after reperfusion. Infarct volumes, neurological deficit scores and immunohistochemistry were evaluated 24 or 72 h after reperfusion.

Key findings

Infarct volumes at 24 h were significantly decreased in transplantation rats at 0 and 3 h, but not at 6 h, after reperfusion, compared to vehicle-treatment. Even high dose BMMC transplantation at 6 h after reperfusion was ineffective. Repeated BMMC transplantation at 6 and 9 h after reperfusion reduced infarct volumes and significantly improved neurological deficit scores at 24 and 72 h. Immunohistochemistry showed repeated BMMC transplantation reduced ionized calcium-binding adapter molecule 1, 4-hydroxy-2-nonenal and 8-hydroxydeoxyguanosine expression at 24 and 72 h after reperfusion.

Significance

Intravenous allogeneic BMMCs were neuroprotective following transient focal cerebral ischemia, and the therapeutic time window of BMMC transplantation was > 3 h and < 6 h after reperfusion in this model. Repeated transplantation at 6 and 9 h after reperfusion suppressed inflammation and oxidative stress in ischemic brains, resulting in improved neuroprotection.     

Mapping of susceptibility and protective loci for acute GVHD in unrelated HLA-matched bone marrow transplantation donors and recipients using 155 microsatellite markers on chromosome 22

Despite matching donors and recipients for the human leukocyte antigens (HLAs) expressed by the major histocompatibility genomic region of the short arm of chromosome 6, several recipients still develop acute graft-versus-host disease (aGVHD) after bone marrow transplantation (BMT). This is possibly due to non-HLA gene polymorphisms, such as minor histocompatibility antigens (mHas) and genes coding for cytokines. However, a detailed genetic background for aGVHD has not yet been established. To find novel susceptibility and/or protective loci for aGVHD, a whole genome-wide association study of donors and recipients needs to be performed. As the first step to such a study, we retrospectively analyzed polymorphisms of 155 microsatellite markers spread across the long arm of chromosome 22 in 70 pairs of HLA-matched unrelated BMT donors and recipients. We performed individual typing and then compared the markers’ allele frequencies (1) between all the aGVHD (grades III and IV GVHD) and GVHD-free (grade 0 GVHD) groups in donors and recipients and (2) between the aGVHD and aGVHD-free groups in donor/recipient pairs that were matched and mismatched for the microsatellite marker’s allele. Screening of the microsatellite markers revealed five loci with a significant difference between the aGVHD and GVHD-free groups and revealed eight loci on chromosome 22, where the microsatellite allele mismatched markers were associated with aGVHD. This screening analysis suggests that several aGVHD-associated susceptible and protective loci exist on chromosome 22, which may encompass novel gene regions that need to be elucidated for their role in aGVHD.