Identification and antifungal susceptibility profiles of Kodamaea ohmeri based on a seven-year multicenter surveillance study
《七年间多中心监测研究中 Kodamaea ohmeri 的鉴定及抗真菌敏感性谱型分析》

Introduction  介绍

Invasive fungal disease (IFD) is an important cause of morbidity and mortality in hospitalized patients.¹ The China Hospital Invasive Fungal Surveillance Net (CHIF-NET) program was the first, and currently the largest, national surveillance program established to provide updated information on the epidemiology of invasive fungal infections in mainland China. It was initiated in 2009, and by the seventh surveillance year (2016), as many as 73 hospitals from 30 of the 34 provinces in China had participated, enabling collection of over 8,000 yeast isolates. Although Candida species remain the most common fungal pathogens worldwide, recent reports have highlighted the emergence of infections caused by less-common pathogenic yeasts.^2,3 One such emerging pathogen is Kodamaea ohmeri.
侵袭性真菌病（IFD）是住院患者发病率和死亡率的重要原因。 ¹ 中国医院侵袭性真菌监测网（CHIF-NET）项目是中国第一个，也是目前最大的全国性监测项目，旨在提供中国大陆侵袭性真菌感染流行病学的最新信息。该项目始于 2009 年，到第七次监测年（2016 年），已有来自中国 34 个省份中的 30 个省份的 73 家医院参与，收集了超过 8000 株酵母菌分离株。尽管 Candida 种属仍然是全球最常见的真菌病原体，但最近的报告强调了由较少常见的病原酵母菌引起的感染的出现。 ^{2 3} 其中一种新兴病原菌是 Kodamaea ohmeri。

K. ohmeri, previously known as Pichia ohmeri and Yamadazyma ohmeri, is an ascosporogenous yeast, and a teleomorph of Candida guilliermondii var. membranaefaciens, which has been commonly used in the food industry for the fermentation of pickles, rinds, and other fruit.⁴ Now, the genus Kodamaea is divided into 5 species (K. anthrophila, K. kakaduensis, K. laetipori, K. nitidulidarum, K. ohmeri) and only K. ohmeri shows pathogenicity in humans.⁴ Since the first case report of sepsis due to K. ohmeri,⁵ several case reports of various IFDs, including sepsis or fungemia,^6,7 catheter-related bloodstream infection,^8,9 peritonitis,¹⁰ and endocarditis,^11,12 with high mortality due to K. ohmeri have gradually accumulated. Moreover, nosocomial outbreaks of K. ohmeri infection in the pediatric ward have also been reported.¹³ All this evidence suggests that K. ohmeri should be added to the growing list of opportunistic fungal pathogens in humans, and calls for early recognition and appropriate treatment.
K. ohmeri，之前被称为 Pichia ohmeri 和 Yamadazyma ohmeri，是一种有性孢子酵母，是 Candida guilliermondii var. membranaefaciens 的同源真菌，常用于食品工业中的泡菜、果皮及其他水果的发酵。 ⁴ 现在，Kodamaea 属分为 5 个种（K. anthrophila、K. kakaduensis、K. laetipori、K. nitidulidarum、K. ohmeri），只有 K. ohmeri 在人类中表现出致病性。 ⁴ 自首次报道由于 K. ohmeri 引起的败血症以来， ⁵ 关于各种侵袭性真菌病（包括败血症或菌血症， ^{6 7} 导管相关血流感染， ^{8 9} 腹膜炎， ¹⁰ 和心内膜炎）， ^{11 12} 由于 K. ohmeri 导致的高死亡率病例逐渐累积。此外，K. ohmeri 在儿科病房引起的医院暴发感染也有所报道。 ¹³ 所有这些证据表明，K. ohmeri 应该被添加到人类机会性真菌病原体的不断增加的名单中，并呼吁早期识别和适当治疗。

Despite the increasing clinical significance in IFD, there are limited data on the occurrence and distribution of K. ohmeri globally. Here, we studied the epidemiology and antifungal susceptibility patterns of K. ohmeri clinical isolates based on the multicenter surveillance program-CHIF-NET in China over seven years.
尽管 K. ohmeri 在侵袭性真菌感染（IFD）中的临床意义日益增加，但全球范围内关于 K. ohmeri 的流行病学和抗真菌药物敏感性模式的数据有限。在此，我们基于中国多中心监测项目-CHIF-NET，对过去七年中的 K. ohmeri 临床分离株的流行病学和抗真菌药物敏感性模式进行了研究。

Materials and methods  材料与方法

Results  结果

Detailed information of the study isolates is summarized in Table 1. The 62 isolates were collected from 62 patients at 24 hospitals located in 14 provinces across China over seven years. Thirty-four (34/62, 54.8%) of the strains were isolated in CHIF-NET year 2016, ten of which were isolated from one single hospital (Figure 1). The majority of the isolates were from patients admitted to surgical department (38.7%), medical department (32.3%), and intensive care unit (ICU) (14.5%), followed by pediatrics (6.5%), emergency (3.2%), organ transplantation (1.6%), dermatology (1.6%), and rehabilitation (1.6%). Among various specimen types, more than half of the isolates (54.8%) were recovered from blood, followed by catheter (16.1%), wound (8.1%), ascitic fluid (8.1%), drainage fluid (6.5%), broncho-alveolar lavage fluid (3.2%), pleural effusion (1.6%), and cerebrospinal fluid (1.6%) (Table 1).
该研究分离株的详细信息总结见 Table 1 。62 株分离株来自中国 14 个省份 24 家医院的 62 名患者，历时七年。其中 34 株（34/62，54.8%）菌株在 CHIF-NET 2016 年分离，其中有 10 株来自单一医院（ Figure 1 ）。大多数分离株来自外科病房（38.7%）、内科病房（32.3%）和重症监护室（ICU，14.5%），其次是儿科（6.5%）、急诊科（3.2%）、器官移植科（1.6%）、皮肤科（1.6%）和康复科（1.6%）。在各种标本类型中，超过一半的分离株（54.8%）来自血液，其次是导管（16.1%）、伤口（8.1%）、腹水（8.1%）、引流液（6.5%）、支气管肺泡灌洗液（3.2%）、胸腔积液（1.6%）和脑脊液（1.6%）（ Table 1 ）。

Discussion  讨论

For decades, K. ohmeri has been recognized as a fungal contaminant but not as a human pathogen. Systemic infections due to K. ohmeri have generally been considered to be rare. Consequently, little attention has been paid to this insignificant yeast until 1998 when the first case of fungemia caused by K. ohmeri was described.⁵ Most published studies to date on K. ohmeri infections are sporadic cases commonly seen in Asian countries like Korea,^20,21 Japan,^8,22 and India.^23–26 The infections were reported more often in children than in adults, and almost all patients had one or more underlying conditions alongside immunodeficiency.⁸ Mortality rates due to K. ohmeri invasive infections have been reported to be as high as 50%.^13,20 The largest cluster of K. ohmeri infection reported was set in a single hospital in North India, presenting as 38 fungemia cases, 78.9% of which were isolated from neonates in intensive care units.²⁶ Several surveillance studies in Spain, Malaysia, and Tunisia have also reported the isolation of K. ohmeri but with limited numbers.^27–29 So far, our study presents as the first, largest, and multicenter epidemiological study of K. ohmeri clinical isolates causing IFDs in China.
几十年来，K. ohmeri 被认为是一种真菌污染菌，而不是人类病原体。K. ohmeri 引起的全身性感染通常被认为较为罕见。因此，直到 1998 年首次报道了由 K. ohmeri 引起的血流感染病例之前，人们对这种微不足道的酵母菌的关注很少。 ⁵ 目前为止，关于 K. ohmeri 感染的大多数已发表研究都是零星病例，常见于韩国、 ^{20 21} 日本、 ^{8 22} 印度等亚洲国家。 ^{23 – 26} 感染在儿童中比在成人中更为常见，几乎所有的患者都伴有免疫缺陷或其他基础疾病。 ⁸ K. ohmeri 引起的侵袭性感染的死亡率据报道高达 50%。 ^{13 20} 在北印度的一家医院中报告的最大 K. ohmeri 感染群集包括 38 例血流感染病例，其中 78.9% 的病例来自重症监护室的新生儿。 ²⁶ 西班牙、马来西亚和突尼斯的几项监测研究也报告了 K. ohmeri 的分离，但数量有限。 到目前为止，本研究是首个、最大规模且多中心的中国临床分离株 K. ohmeri 导致侵袭性真菌病的流行病学研究。

During the 7-year surveillance, a total of 62 K. ohmeri isolates from cases of IFDs were collected and re-identified by 26S rDNA sequencing at the Central hospital, PUMCH. However, according to the original results submitted by the participating local hospitals, less than half of the isolates were correctly identified as K. ohmeri using different methods. The CHROMagar Candida chromogenic growth medium, which was developed based on the characteristic color change of the colonies, is an extremely useful tool in the clinical lab to assist in routine identification of common Candida species. However, this medium failed to correctly identify all the 16 K. ohmeri isolates reported by local hospitals in this study. It is known that K. ohmeri colonies can undergo a unique color change from pink to blue when grown on CHROMagar medium, and this phenomenon takes at least 2–3 days to form pink-blue colonies. Moreover, a full week may be required to obtain complete blue color colony development.²⁰ Thus it is not surprising to note that 9, 4, and 3 isolates amongst the 16 misidentified isolates were incorrectly identified as C. albicans, C. glabrata, and C. tropicalis, respectively, which may be a result of determining the color of the isolates either too early or too late by lab staff. This identification confusion has been previously reported.²⁶ Actually, this is how the largest cluster of K. ohmeri infection was discovered, in which 38 (25.7%) of 148 previously identified C. tropicalis isolates were re-identified as K. ohmeri by genotypic characterisation.²⁶ Therefore, while CHROMagar is a useful and simple identification tool, careful and patient observation is necessary for correct identification of K. ohmeri.
在 7 年的监测期间，中央医院 PUMCH 通过 26S rDNA 测序共收集并重新鉴定出 62 株 K. ohmeri 分离株，用于侵袭性真菌病（IFD）病例。然而，根据参与的当地医院提交的原始结果，使用不同方法鉴定的分离株中，只有不到一半被正确鉴定为 K. ohmeri。基于菌落颜色变化特征开发的 CHROMagar Candida 选择性培养基，在临床实验室中是一个非常有用的工具，用于常规鉴定常见念珠菌属物种。然而，该培养基未能正确鉴定本研究中当地医院报告的 16 株 K. ohmeri 分离株。已知 K. ohmeri 在 CHROMagar 培养基上生长时，菌落会经历从粉红色到蓝色的独特颜色变化，这一现象至少需要 2-3 天才能形成粉蓝菌落。此外，完全蓝色菌落的形成可能需要一周时间。因此，16 株被错误鉴定的分离株中有 9 株、4 株和 3 株分别被错误地鉴定为 C. albicans、C. glabrata 和 C. 热带 isolates，这可能是由于实验室工作人员过早或过晚确定菌株颜色所致。这种鉴定混淆之前已有报道。实际上，正是通过这种方式发现了最大的 K. ohmeri 感染集群，在其中，148 株之前鉴定为 C. tropicalis 的菌株中有 38 株（25.7%）被基因型鉴定重新鉴定为 K. ohmeri。因此，虽然 CHROMagar 是一种有用的简单鉴定工具，但正确鉴定 K. ohmeri 需要仔细和耐心的观察。 ²⁶ 实际上，正是通过这种方式发现了最大的 K. ohmeri 感染集群，在其中，148 株之前鉴定为 C. tropicalis 的菌株中有 38 株（25.7%）被基因型鉴定重新鉴定为 K. ohmeri。 ²⁶

Several other fungal identification methods with various levels of accuracy for specific organisms are used by many local hospitals in China. Among 16 isolates identified by API 20C system, seven (43.8%) were misidentified as C. glabrata (n=4), C. lusitaniae (n=1), C. albicans (n=1), and C. guilliermondii (n=1). The Vitek 2 Compact system correctly identified 70% (14/20) of isolates, with two misidentified as C. guilliermondii, two as C. famata, and one of each as C. pelliculosa and C. rugosa. Both the API 20C and Vitek 2 Compact systems have been shown to yield false-positive results, identifying C. haemulonii or C. parapsilosis as K. ohmeri.^20,30 It is also known that C. auris and C. haemulonii are closely related and cannot be distinguished with conventional identification methods.³¹ Due to the emerging role of C. auris as a multidrug-resistant fungal pathogen with high morbidity and mortality, early accurate identification of this organism is crucial for patient management.³² In this case, the use of the faster molecular diagnostic tools for the proper identification of fungal pathogens is strongly recommended. The other four methods including ATB32C, BD Phoenix100, RapID™ YEAST PLUS, and Vitek MS, which were used for fungal identification by a small number of hospitals, yielded a wide variety of results which were difficult to generalize.
在中国的许多当地医院中，使用了多种不同准确度的其他真菌鉴定方法。在使用 API 20C 系统鉴定的 16 个菌株中，有 7 个（43.8%）被误鉴定为 C. glabrata（4 个）、C. lusitaniae（1 个）、C. albicans（1 个）和 C. guilliermondii（1 个）。Vitek 2 Compact 系统正确鉴定了 70%（14/20）的菌株，其中有 2 个被误鉴定为 C. guilliermondii，2 个被误鉴定为 C. famata，1 个被误鉴定为 C. pelliculosa，1 个被误鉴定为 C. rugosa。API 20C 系统和 Vitek 2 Compact 系统均被证实会产生假阳性结果，将 C. haemulonii 或 C. parapsilosis 误鉴定为 K. ohmeri。 ^{20 30} 已知 C. auris 和 C. haemulonii 关系密切，常规鉴定方法无法区分两者。 ³¹ 鉴于 C. auris 作为一种多重耐药真菌病原体，具有高发病率和高死亡率的新兴作用，早期准确鉴定该菌种对于患者的管理至关重要。 ³² 因此，强烈建议使用更快的分子诊断工具来正确鉴定真菌病原体。 其他四种方法，包括 ATB32C、BD Phoenix100、RapID™ YEAST PLUS 和 Vitek MS，仅少数医院使用，结果多样，难以概括。

Nevertheless, the Vitek MS system performed the best among all the methods with 100% accuracy, albeit only two isolates identified using this method. Therefore, we performed a parallel study for identification of all the 62 K. ohmeri isolates using three commonly used phenotypic methods in our lab. To the best of our knowledge, this is the first evaluation study on the performance of three phenotypic methods for the identification of K. ohmeri. In general, the Vitek 2 Compact system (82.3% vs 70%) and Vitek MS (98.4% vs 100%) showed similar results with those submitted by the local hospitals. The Bruker system demonstrated a difference in the identification accuracy of K. ohmeri based on the sample preparation method. The identification accuracy obtained using the protein extraction method (96.8%) was comparable to that of Vitek MS, while the direct transfer method was only comparable to that of Vitek 2 Compact only when the genus identification cut-off value (>1.7) was adopted (83.9%). While MALDI-TOF MS is increasingly being used to identify Candida species in clinical laboratories, only one study has previously reported on the use of Bruker system for identification of K. ohmeri.⁸ In that study, the protein extraction procedure was used, which yielded a high confidence score although only one isolate was tested.⁸ These data suggest that both Vitek MS and Bruker system with protein extraction method for sample preparation can be used as a fast and accurate tool for K. ohmeri identification.
尽管 Vite 克 MS 系统在所有方法中表现最佳，准确率为 100%，但仅识别出两个菌株。因此，我们对实验室常用的三种表型方法进行了并行研究，以识别所有 62 株 K. ohmeri 菌株。据我们所知，这是首次评估三种表型方法用于识别 K. ohmeri 性能的研究。总体而言，Vitek 2 Compact 系统（82.3% vs 70%）和 Vitek MS（98.4% vs 100%）的识别结果与当地医院提交的结果相似。Bruker 系统在基于样本制备方法识别 K. ohmeri 的准确性上存在差异。使用蛋白质提取方法（96.8%）的识别准确率与 Vitek MS 相当，而直接转移方法仅在采用属识别阈值（>1.7）时（83.9%）与 Vitek 2 Compact 相当。虽然 MALDI-TOF MS 在临床实验室中越来越多地用于鉴定念珠菌属，但仅有一项研究报道了使用 Bruker 系统识别 K. ohmeri 的情况。 ohmeri. ⁸ 在该研究中，使用了蛋白质提取程序，尽管只测试了一个分离株，但获得了高置信度评分。 ⁸ 这些数据表明，无论是使用 Vitek MS 系统还是 Bruker 系统结合蛋白质提取方法进行样品准备，都可以作为快速而准确的 K. ohmeri 鉴定工具。

For the first time, we also compared the in-vitro susceptibilities of 8 antifungal agents using standard BMD and the commercial SYY against 62 K. ohmeri isolates. The commercial SYY system is an adapted microbroth susceptibility testing system based on the M27-A3 standard for yeasts, which has been widely used in antifungal susceptibility testing of yeasts and is now being evaluated in molds.^33,34 Excellent EAs have been reported in triazoles and echinocandins against Candida spp., ranging from 92.3% to 100%.^35,36 Surprisingly, significant differences in EAs against K. ohmeri isolates were observed, especially for itraconazole presenting as 22.6%. We excluded experimental errors by repeating both procedures, and the results were confirmed. We tried to find a possible explanation for this finding, but it proved difficult as there is limited data reported in literature on this aspect. Most of the published literature concerning K. ohmeri infections only used one method, either the commercial SYY or the standard BMD, to determine the MIC values. Only one previous study used BMD to confirm the MIC for micafungin using a colorimetric method for a single K. ohmeri isolate causing fungemia and found a 640-fold difference in the MIC value between the two methods.²² Despite the scarce evidence, this should serve as a reminder that validation of the antifungal susceptibility test by standard BMD for rare yeasts like K. ohmeri is important as susceptibility results often play an important role in the choice of antifungal agent to administer.
首次比较了 8 种抗真菌药物（包括标准 BMD 和商业 SYY）对 62 株 K. ohmeri 分离株的体外敏感性。商业 SYY 系统是基于 M27-A3 标准的改良微量肉汤稀释法，广泛用于酵母菌的抗真菌敏感性测试，目前也在评估其在霉菌中的应用。 ^{33 34} 在三唑类和棘白菌素类药物中，对念珠菌属的优秀抗菌活性（EAs）报道范围为 92.3%至 100%。 ^{35 36} 然而，令人惊讶的是，对 K. ohmeri 分离株的 EAs 存在显著差异，尤其是伊曲康唑的 EAs 仅为 22.6%。我们通过重复实验排除了实验误差，结果得到了确认。我们试图寻找这一发现的可能解释，但发现文献中关于这一方面的数据有限。大多数关于 K. ohmeri 感染的研究仅使用一种方法，要么是商业 SYY，要么是标准 BMD 来确定 MIC 值。仅有一项先前的研究使用 BMD 方法，通过比色法确认米卡芬净的 MIC 值，针对单株 K. ohmeri 导致菌血症的分离株，并发现两种方法之间的 MIC 值相差 640 倍。尽管证据有限，这仍应作为提醒，对于如 K. ohmeri 这样的罕见酵母菌，通过标准 BMD 验证抗真菌敏感性试验非常重要，因为敏感性结果往往在选择抗真菌药物方面起着重要作用。

Since no CBP or ECV has been established to date for K. ohmeri, we could not compare the susceptibility or resistance patterns of our isolates with those reported in literature. However, based on MIC distribution of susceptibility results by BMD, our findings are in agreement with previous results showing that K. ohmeri strains have low MICs to all antifungal agents tested except for fluconazole, with MIC₅₀ and MIC₉₀ as high as 4 and 8 μg/mL, respectively.²⁶ Twenty-four of the tested isolates exhibited MICs of ≥8 μg/mL, one of which had the highest MIC of >256 μg/mL. Compared to the large cluster from India,²⁶ the present isolates had similar MIC_90s for amphotericin B, itraconazole and posaconazole, while a two-fold increase in MIC₉₀ was observed for voriconazole and caspofungin, and a four-fold increase for fluconazole. Previous studies concluded that amphotericin B or echinocandin should be considered to be a good antifungal choice for treatment of K. ohmeri infections.^8,37 Considering that antifungal treatment should be adjusted according to susceptibility results of the clinical isolates, based on the MIC results of our isolates, all the eight antifungal agents tested except fluconazole, may be successful in treating most of the K. ohmeri infections according to the in vitro susceptibility results.
由于目前尚未建立 K. ohmeri 的 CBP 或 ECV，我们无法将我们的分离株的敏感性或耐药模式与文献中的报告进行比较。然而，根据 BMD 方法得出的敏感性结果的 MIC 分布，我们的发现与之前的研究结果一致，即 K. ohmeri 菌株对所有抗真菌剂的 MIC 值较低，除了氟康唑外，其余抗真菌剂的 MIC ₅₀ 和 MIC ₉₀ 分别为 4 μg/mL 和 8 μg/mL。 ²⁶ 在测试的 24 个分离株中，有 24 个分离株的 MIC 值≥8 μg/mL，其中一个分离株的 MIC 值最高为>256 μg/mL。与印度的大群落相比， ²⁶ 目前的分离株对两性霉素 B、伊曲康唑和泊沙康唑的 MIC _90s 相似，而对伏立康唑和卡泊芬净的 MIC ₉₀ 分别增加了两倍和四倍，对氟康唑的 MIC 增加了四倍。先前的研究得出结论，两性霉素 B 或棘白菌素应被视为治疗 K. ohmeri 感染的良好抗真菌选择。 考虑到抗真菌治疗应根据临床分离株的敏感性结果进行调整，根据我们分离株的 MIC 结果，在体外敏感性试验中，除了氟康唑外，其余八种抗真菌药物可能对大多数 K. ohmeri 感染有效。

In conclusion, this is the first systemic study regarding the epidemiology, identification, and antifungal susceptibility profiles of K. ohmeri isolates in China. Our study emphasizes the need for accurate identification of clinical K. ohmeri isolates as an emerging human pathogen in China and the importance of validation of antifungal susceptibility results by standard BMD.
总之，这是首次对中国地区 K. ohmeri 分离株的流行病学、鉴定及抗真菌敏感性谱系进行系统研究。本研究强调了准确鉴定临床 K. ohmeri 分离株作为中国新兴的人类病原体的重要性，并强调了使用标准 BMD 验证抗真菌敏感性结果的重要性。

Disclosure  披露

All authors report no conflicts of interest in this work.
所有作者声明在本研究中不存在利益冲突。