洁净室滋生霉菌，如何调查？如何控制？如何消毒？

The MoldyNightmare: Questions and Answers

霉菌的噩梦：问题和答案

以下问答来自 PDA 药物微生物学会议的早餐会议“霉菌污染和修复——发霉的噩梦”。后发表在PDA电子期刊，The Moldy Nightmare: Questions and Answers, Part 1，作者真菌学家Ziva Abraham，在微生物和质量保证方面拥有超过35年的学术，研究，和工作经验。

Question

问题

If you see low-level recovery (1 or 2 CFU) on an airlock gowning bench every three months or so, with no other recoveries in the airlock, would you suspect inadequate cleaning practices?

如果每三个月左右在更衣间更衣长凳上检测到低水平霉菌（1 或 2 CFU），而更衣间中没有检测到其他微生物，你会怀疑是清洁措施不足吗？

Answer

回答

Low recovery in gowning areas is a common phenomenon, as this is the entry point into the controlled areas from the uncontrolled areas. Having said that, it is important to have a science-based cleaning program to address all mold that can be tracked through foot-borne traffic. The cleaning and disinfection program should include use of a general-purpose disinfectant with a surfactant that helps break surface tension and aids in cleaning as well as periodic use of a sporicidal agent to address bacterial and fungal spores.

更衣区域的低回收是一种普遍现象，因为这是从非受控制区域进入受控区域的入口。话虽如此，制定一个基于科学的清洁程序来解决可以通过人员带入的所有霉菌非常重要。清洁和消毒程序应包括使用带有表面活性剂的广谱消毒剂以帮助打破表面张力并有助于清洁，以及定期使用杀孢子剂来解决细菌和真菌芽孢。

Question

问题

If mold is found only in air sampling (not on surface), what should be the action plan?

如果仅在空气采样中发现霉菌（而不是在表面），应该采取什么行动计划？

Answer

回答

Mold recovery in air samples may be due to multiple reasons. Surface contaminants can become airborne, so the source could be foot- or wheel-borne contamination brought in. Inadequate storage of monitoring equipment and inadequate wipe-down procedures can be another reason. Monitoring equipment without a HEPA-filtered exhaust is known to be a contamination source. Growth of mold in walls after leaks, compromised HEPA filters, or mold growing on seals can cause airborne mold recovery. It is also important to track the mold to its source and map the transport into the area where it was recovered. Often dead spaces (e.g., in cleanrooms where the air is not cleared due to the location of HEPA filters), returns, and cleanroom and barrier system integration may allow the contaminants to linger for a long time.

空气样品中的霉菌可能由多种原因造成。表面污染物可能会在空气中传播，因此可能来源于足底或轮子上的污染物。监测设备保存不当和擦拭程序不当可能是另一个原因。众所周知，排气没有HEPA过滤的监测设备是污染源。HEPA过滤器受损泄漏后墙壁上的霉菌生长，或密封件上的霉菌生长会导致空气中的霉菌。追踪霉菌的来源及其传播途径也很重要。通常，死角（例如，洁净室中局部区域由于HEPA过滤器的位置布局而空气未被清除），回风以及洁净室和屏障系统的相互作用可能会使污染物长时间徘徊。

If the mold is found in filling areas, especially RABs, the area between the HEPAs without a diffuser membrane is hard to clean and can hold contaminants that can become airborne.

如果在灌装区域（尤其是RAB）中发现霉菌，则HEPA之间没有散流板的区域很难清洁，并且可能会容纳可能通过空气传播的污染物。

Finally, depending upon the mold species recovered, especially the deuteromycotous fungi, which proliferate very fast, an abiotic factor such as moisture, carbon source, mineral oil, etc., should be looked at.

最后，根据回收的霉菌种类，特别是增殖非常快的半知菌类真菌，应考虑水分，碳源，矿物油等非生物因素。

In summary, the investigation should be based on the genus recovered and the source, and the remedial action should be based on the above-mentioned points.

综上所述，调查应以所检测到的霉菌的属和来源为依据，补救措施应以上述几点为依据。

Once the source has been determined, increasing use of sporicidal agent, especially where the mold source is found, and trending monitoring data to assess the effectiveness of remedial measures is recommended. If a decision for using fogging is made, as the root cause is undetermined, the chemistry of the fog, the fog size, and the number of foggers to be used to cover the area in question should be considered. The smaller the fog particle size, the more buoyant, allowing it to stay in the air for longer periods of time.

一旦确定了霉菌来源，建议增加杀孢子剂的使用，特别是在发现霉菌源的情况下，并建议使用趋势监测数据来评估补救措施的有效性。如果决定使用雾化（消毒），由于根本原因尚未确定，应考虑（消毒剂）雾的化学性质、（消毒剂）雾的大小以及用于覆盖相关区域的雾化器数量。雾粒径越小，浮力越大，使其在空气中停留的时间更长。

Question

问题

What mold genus should be included in disinfection efficacy studies?

消毒功效研究中应包括哪些霉菌属？

Answer

回答

It is beneficial to use a USP-recommendedAspergillus strain along with the one or two predominant mold isolates recovered from the environment or product testing. Per the recent European standard, EN 13697, it is recommended to use the mature Aspergillus spores for disinfectant qualification.

使用USP推荐的曲霉菌菌株以及从环境或产品测试中回收的一种或两种主要霉菌分离株是有益的。根据最近的欧洲标准EN 13697，建议使用成熟的曲霉菌孢子进行消毒剂确认。

Question

问题

Why do some molds fail during disinfectant qualification studies?

为什么有些霉菌在消毒剂确认研究中会失效？

Answer

回答

Disinfectant label claim testing is performed using colorless deuteromycotous fungiAspergillus and Trichophyton. Disinfectants with fungicidal claims, while using this testing method, may not be able to kill some colored Deuteromycota, most Ascomycota, and some Zygomycota.

消毒剂标签声明测试是使用无色半知菌类霉菌曲霉菌和毛癣菌进行的。使用这种测试方法时，具有杀真菌声明的消毒剂可能无法杀死一些有色的半知菌类霉菌，大多数子囊菌和一些接合菌。

It is recommended to understand the structures of the mold recovered in the cleanroom against those tested for fungicidal label claim. In some cases, increasing the contact time may help with the required kill, while in other cases prevention is the best strategy.

建议了解在洁净室中检测到的霉菌的结构，以对照那些测试的杀真菌标签声明。在某些情况下，增加接触时间可能有助于所需的杀灭，而在其他情况下，预防是最好的策略。

Question

问题

Is rotation of cleaning agent essential? Is it true that microbes grow resistant to the cleaning agent?

清洁剂的轮换是否必不可少？微生物对清洁剂产生耐受性是真的吗？

Answer

回答

There is no documented evidence of resistance, but to address all types of bacterial and mold contamination a rotation program utilizing a general-purpose disinfectant with surfactant, as well as a sporicidal agent, is effective at eliminating vegetative forms and spores.

没有书面的耐受性证据，但为了解决所有类型的细菌和霉菌污染，使用带有表面活性剂的广谱消毒剂以及杀孢子剂的轮换程序可有效消除繁殖体和芽孢。

Question

问题

Can you comment on the degree of disinfectant resistance between fungi and bacterial spores?

能评论一下真菌和细菌芽孢的消毒剂耐受性吗？

Answer

回答

Among the bacteria, the spore formers are harder to eliminate. For example,Bacillus cereus has the least kill even with sporicidal agents. It is hard for the disinfectant to penetrate the spore structure. As for ascomycotous mold, whose sexual spores are protected by two layers as compared to one layer in bacterial spores, it is harder to eliminate these fungi. Hence, preventing the entry of hard-to-kill bacterial spores and ascomycotous fungi that are soil or cellulous material-borne respectively is a winning strategy.

在细菌中，芽孢更难消除。例如，即使使用杀孢子剂，对蜡样芽孢杆菌的杀伤力也最低。消毒剂很难穿透芽孢结构。至于子囊菌霉菌，其有性芽孢受到两层保护，而细菌芽孢中有一层，因此更难消除这些真菌。因此，防止土壤或纤维材料携带的难以杀死的细菌芽孢和子囊菌真菌进入是一种成功的策略。

Identification

鉴别

Question

问题

Should mold colonies be read on the fifth or seventh day of incubation?

应该在培养的第5天还是第7天读取霉菌菌落？

Answer

回答

Many deuteromycotous mold such as Aspergillus, Penicillium, Paecilomyces, etc., are fast growers. By day 5, many of these fast-growing mold may sporulate and create additional colonies or cover the entire plate in a fungal mat. Both these scenarios will lead to false positive results.

许多半知菌类的霉菌，如曲霉、青霉、拟青霉等，都是快速生长的。到第 5 天，许多这些快速生长的霉菌可能会形成孢子并产生额外的菌落或以真菌垫覆盖整个碟子。这两种情况都会导致假阳性结果。

It is best to count colonies on day 3 and check on day 5. This way the day 3 read will allow counting the colonies of fast-growing mold and day 5 read will allow counting colonies of slower growing mold.

最好在第 3 天计数菌落，并在第 5 天进行复查。这样，第 3 天的读数将允许对快速生长的霉菌的菌落进行计数，而第 5 天的读数将允许对生长较慢的霉菌的菌落进行计数。

Question

问题

What are the best methods/technologies to definitively identify fungi?

正确鉴定真菌的最佳方法/技术是什么？

Answer

回答

A large and diverse library in an identification system/identification service is the key to correct identification and reduced chances of unidentified isolates.

鉴定系统中庞大而多样的数据库是正确鉴定和减少无法鉴定机会的关键。

Question

问题

When building a mold identification SOP, if you want to include a requirement to save identified contaminants in a library, how would you recommend storing them?

在构建霉菌鉴别 SOP 时，如果想要求将已鉴别的污染物保存在库中，建议如何保存它们？

Answer

回答

Mold can be cryopreserved or lyophilized. There are services available for both these methods of preservation. Cryopreservation can also be done inhouse using liquid media and 10-15% glycerol. If cryopreserved in-house, procedures must be established to perform periodic checks for purity of the cultures.

霉菌可以冷冻保存或冻干。这两种方法可选用外部服务。企业内部也可以使用液体培养基和10-15% 甘油进行冻存。如果在企业内部冷冻保存，则必须建立程序以定期检查培养物的纯度。

Question

问题

You mentioned earlier that Mold identification should be confirmed by looking under microscope. Do you think this is necessary even with sequence-based identifications?

您之前提到，霉菌鉴定应通过在显微镜下观察来确认。即使对于基于序列的鉴定，这也是必要的吗？

Answer

回答

Sequence-based identification is ideal, microscopy and microscopy techniques give an inclination of the mold type and its probable source so that while waiting for the identification results, investigation and remedial actions can be initiated.

基于序列的鉴定是理想的，显微镜和显微镜技术可以确定霉菌类型及其可能的来源，以便在等待鉴定结果的同时，可以启动调查和补救措施。

Question

问题

Often mold identification results are noted as “mycelia sterilia” or “unidentified.” What is the reason behind these results?

霉菌鉴别结果通常被标记为“菌丝体”或“未鉴定”。这些结果背后的原因是什么？

Answer

回答

“Mycelia sterilia” are rare in nature. Such results are normally generated by a laboratory when they cannot get the mold to sporulate while using conventional identification methods.

“菌丝体” 在自然界中很罕见。当实验室在使用常规鉴定方法无法使霉菌孢子形成时，通常会生成此类结果。

Unidentified mold as a result from any conventional method used can be attributed to no sporulation. Non mycologists who do not comprehend the growth requirements of the mold in question may not be able to provide nutritional growth media for sporulation purposes.

由于使用任何常规方法而导致的未识别霉菌的结果，可归因于没有孢子形成。不了解相关霉菌生长要求的非真菌学家可能无法为孢子形成提供营养生长培养基。

On the other hand, an unidentified mold result from a phenotypic, proteotypic, or genotypic ID system alludes to some key factors such as the mold not catalogued in the system’s library, mixed culture or laboratory error when preparing the sample to run on the system.

另一方面，由表型、蛋白型或基因型 ID 系统产生的未识别霉菌暗示了一些关键因素，例如霉菌未在系统库中编目、混合培养或在制备样品以在系统上运行时出现实验室错误。

Question

问题

How do you categorize mold into weak or strong? is there a list that can be referred to?

如何将霉菌分为弱霉菌或强霉菌？有没有可以参考的列表？

Answer

回答

Colorless deuteromycotous fungi are weaker than colored deuteromycotous fungi and ascomycotous fungi. Among the zygomycotous fungi, the asexual spores are weaker than the sexually produced zygospores.

无色半知菌类真菌比有色半知菌类真菌和子囊菌弱。在接合菌真菌中，无性孢子比有性产生的接合孢子弱。

Specific Molds

特定霉菌

Question

问题

From the IDs obtained, how does one understand the sexual stage of the mold?

如何从获得的ID中了解霉菌的性阶段？

Answer

回答

If the ID system and the fungal identification service provider keeps up with the taxonomy and nomenclature changes, the result will be reported in the sexual stage of the fungus. It is up to the microbiologist to research the asexual stage to assess patient risk. Note that the sexual stage is hard to kill and asexual stage is most infectious.

如果鉴别系统和真菌鉴定服务提供商跟上分类法和命名法的变化，结果将在真菌的性阶段中报告。微生物学家负责研究无性阶段以评估患者风险。请注意，性阶段很难杀死，而无性阶段最具传播性。

Question

问题

What is the likelihood that these molds will reproduce sexually in our facilities, as the conditions are extreme for mold? Is it more likely we will only see the asexual spores?

由于霉菌的条件非常极端，这些霉菌在我们的设施中有性繁殖的可能性有多大？我们更有可能只看到无性孢子吗？

Answer

回答

There is a likelihood of all mold that have an anamorphic and teleomorphic phase to be present at both stages in the cleanroom. Depending upon the identification system or methodology used, the result may be reported in the asexual or sexual name of the mold. The asexual stage is the one that is the most infectious while the sexual stage is harder to eliminate. It is advised that no matter the identification result, the laboratory should research if the mold has both phases and address remediation as well as conduct a pathogen specific assessment accordingly.

这两个阶段的霉菌都有可能出现在洁净室。根据所使用的鉴定系统或方法，结果可能会以霉菌的无性或有性名称报告。无性阶段是最具传播性的阶段，而有性阶段更难消除。建议无论鉴定结果如何，实验室都应研究霉菌是否同时具有两个阶段并解决补救措施，并相应地进行微生物特异性评估。

Question

问题

Do you have experience withTalaromyces ruber? What is the best way to get rid of them from the incubators and cleanrooms?

你有红色篮状菌的经验吗？从培养箱和洁净室中清除它们的最佳方法是什么？

Answer

回答

Talaromyces ruber is described as a sexual state of Penicillium rubrum that produces soft walled ascomata covered with interwoven hyphae. Penicillum is the asexual phase while Talaromyces is the sexual phase; if the sexual stage is not eliminated, it will produce asexual spores and the cycle of asexual and sexual reproduction will continue. As incubators are ideal breeding ground for mold, it is recommended to disinfect the incubator using a sporicidal agent followed by a wipedown on frequent basis. Water pans or humidity generating tanks should also be cleaned frequently.

红色篮状菌被描述为红色青霉的有性状态，产生覆盖着交织菌丝的软壁子囊。青霉菌是无性期，而 篮状菌 是有性期;如果不消除有性阶段，就会产生无性孢子，无性繁殖的循环会继续。由于培养箱是霉菌滋生的理想场所，因此建议使用杀孢子剂对培养箱进行消毒，然后经常擦拭。水盘或产生湿气的水箱也应经常清洁。

Question

问题

If theT. ruber is still recovered after the VHP, what other things can we try to eliminate it if we recover it in our samples in Grade C clean room areas? We recovered it mostly during operational environmental monitoring.

如果在 VHP 后仍然检测到红色篮状菌，我们在 C 级洁净室区域的样品中检测到它，我们还可以尝试哪些其他方法来消除它？我们主要在环境监测中检测到它。

Answer

回答

Talaromyces ruberis described as a sexual state of Penicillium rubrum that produces soft walled ascomata covered with interwoven hyphae. It would be beneficial to understand where exactly the mold is found, and if it is found in the same general area. Fogging is as successful as the effectiveness of the fog chemistry to kill the organism, the capability of the fog to reach the source of the contamination, and the buoyancy the fog is. Bouyancy of the fog is a key parameter as the fog should stay long enough in the air and should reach all areas in the room.

红色篮状菌被描述为红色青霉的有性状态，产生覆盖着交织菌丝的软壁子囊。了解霉菌的确切位置以及是否在同一区域发现霉菌将是有益的。雾化的成功与雾化学杀死生物体的有效性、雾到达污染源的能力以及雾的浮力相关。雾的浮力是一个关键参数，因为雾应该在空气中停留足够长的时间，并且应该到达房间的所有区域。

It is recommended to trend the contaminant and try and find its route of entry as well as use the trend to understand if it is localized within the cleanroom, from where it is proliferating and spreading.

建议对污染物进行趋势分析，尝试找到其进入路线，并利用趋势来了解它是否局限在洁净室内，在哪里繁殖和扩散的？

Question

问题

How to get rid ofCladosporium?

如何清除枝孢霉菌？

Answer

回答

Cladosporium likes the lower temperatures of 20 to 25°C. Beyond being brought into the cleanroom via foot and wheel borne traffic, it proliferates in niches where there is moisture and lower temperatures. That being said, it can also grow in warmer temperatures as well.

Auditing cold rooms and refrigerators is a good start. It is also recommended to inspect labels of vials stored in coldrooms as paper and glue are used as a carbon source by most mold. Having an informational monitoring program for coldrooms and a routine cleaning and disinfection program may reduce the occurrence of this mold. Cladosporium sporulation method makes it very proliferous, it sporulates in branching chains of conidia which are very small and disseminate easily.

枝孢霉菌喜欢 20 至 25°C 的低温。 除了通过人员脚步和推车轮子带入洁净室外，它还会在潮湿和温度较低的壁龛中繁殖。话虽如此，它也可以在温暖的温度下生长。审查冷藏室和冰箱是一个好的开始。还建议检查冷藏室中储存的小瓶的标签，因为大多数霉菌都使用纸张和粘胶剂作为碳源。制定冷藏室信息监测计划以及常规清洁和消毒计划可以减少这种霉菌的发生。枝孢菌孢子形成方法使其迅速增殖，它在分生孢子的分支链中形成孢子，这些分生孢子链非常小，很容易传播。

Question

问题

Cladosporium species found in air samples, does it point to any source?

在空气采样中发现的枝孢霉菌属物种，它可能有哪种来源吗？

Answer

回答

Any contamination on the floor can become airborne in a cleanroom due to air patterns and movement. IfCladosporium is not recovered on the floors at all it is beneficial to look at the abiotic factors that help it proliferate. Cladosporium is prevalent in coldrooms and refrigerators as it prefers the colder temperatures. It would be beneficial to audit these sources.

由于气流和移动，地板上的任何污染物都可能在洁净室中传播到空气中。如果在地板上没有检测到枝孢霉菌，那么研究帮助它增殖的非生物因素是有益的。枝孢菌普遍存在于冷藏室和冰箱中，因为它喜欢较冷的温度。审查这些来源将是有益的。

It is recommended to trend the most common sampling points where Cladosporium is recovered and look for abiotic sources or airflow patterns which may explain this phenomenon.

建议对发现枝孢霉菌的最常见采样点进行趋势分析，并寻找可能解释这种现象的非生物来源或气流模式。

Cladosporium creates branching chains of conidia, in short many more spores than most fungi, hence it is a very common mold recovered in cleanrooms.

枝孢霉菌产生分生孢子的分支链，简而言之，孢子比大多数真菌多得多，因此它是在洁净室中发现的非常常见的霉菌。