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The MoldyNightmare: Questions and Answers

霉菌的噩夢：問題和答案

 

以下問答來自 PDA 藥物微生物學會議的早餐會議“霉菌污染和修復——發霉的噩夢”。后發表在PDA電子期刊，The Moldy Nightmare: Questions and Answers, Part 1，作者真菌學家Ziva Abraham，在微生物和質量保證方面擁有超過35年的學術，研究，和工作經驗。

 

Question

 

問題

 

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?

 

如果每三個月左右在更衣間更衣長凳上檢測到低水平霉菌（1 或 2 CFU），而更衣間中沒有檢測到其他微生物，你會懷疑是清潔措施不足嗎？

 

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.

 

更衣區域的低回收是一種普遍現象，因為這是從非受控制區域進入受控區域的入口。話雖如此，制定一個基于科學的清潔程序來解決可以通過人員帶入的所有霉菌非常重要。清潔和消毒程序應包括使用帶有表面活性劑的廣譜消毒劑以幫助打破表面張力并有助于清潔，以及定期使用殺孢子劑來解決細菌和真菌芽孢。

 

Question

 

問題

 

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

 

如果僅在空氣采樣中發現霉菌（而不是在表面），應該采取什么行動計劃？

 

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.

 

空氣樣品中的霉菌可能由多種原因造成。表面污染物可能會在空氣中傳播，因此可能來源于足底或輪子上的污染物。監測設備保存不當和擦拭程序不當可能是另一個原因。眾所周知，排氣沒有HEPA過濾的監測設備是污染源。HEPA過濾器受損泄漏后墻壁上的霉菌生長，或密封件上的霉菌生長會導致空氣中的霉菌。追蹤霉菌的來源及其傳播途徑也很重要。通常，死角（例如，潔凈室中局部區域由于HEPA過濾器的位置布局而空氣未被清除），回風以及潔凈室和屏障系統的相互作用可能會使污染物長時間徘徊。

 

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.

 

如果在灌裝區域（尤其是RAB）中發現霉菌，則HEPA之間沒有散流板的區域很難清潔，并且可能會容納可能通過空氣傳播的污染物。

 

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，根據回收的霉菌種類，特別是增殖非常快的半知菌類真菌，應考慮水分，碳源，礦物油等非生物因素。

 

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.

 

綜上所述，調查應以所檢測到的霉菌的屬和來源為依據，補救措施應以上述幾點為依據。

 

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.

 

一旦確定了霉菌來源，建議增加殺孢子劑的使用，特別是在發現霉菌源的情況下，并建議使用趨勢監測數據來評估補救措施的有效性。如果決定使用霧化（消毒），由于根本原因尚未確定，應考慮（消毒劑）霧的化學性質、（消毒劑）霧的大小以及用于覆蓋相關區域的霧化器數量。霧粒徑越小，浮力越大，使其在空氣中停留的時間更長。

 

Question

 

問題

 

What mold genus should be included in disinfection efficacy studies?

 

消毒功效研究中應包括哪些霉菌屬？

 

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推薦的曲霉菌菌株以及從環境或產品測試中回收的一種或兩種主要霉菌分離株是有益的。根據最近的歐洲標準EN 13697，建議使用成熟的曲霉菌孢子進行消毒劑確認。

 

Question

 

問題

 

Why do some molds fail during disinfectant qualification studies?

 

為什么有些霉菌在消毒劑確認研究中會失效？

 

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.

 

消毒劑標簽聲明測試是使用無色半知菌類霉菌曲霉菌和毛癬菌進行的。使用這種測試方法時，具有殺真菌聲明的消毒劑可能無法殺死一些有色的半知菌類霉菌，大多數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.

 

建議了解在潔凈室中檢測到的霉菌的結構，以對照那些測試的殺真菌標簽聲明。在某些情況下，增加接觸時間可能有助于所需的殺滅，而在其他情況下，預防是最好的策略。

 

Question

 

問題

 

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

 

清潔劑的輪換是否必不可少？微生物對清潔劑產生耐受性是真的嗎？

 

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.

 

沒有書面的耐受性證據，但為了解決所有類型的細菌和霉菌污染，使用帶有表面活性劑的廣譜消毒劑以及殺孢子劑的輪換程序可有效消除繁殖體和芽孢。

 

Question

 

問題

 

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

 

能評論一下真菌和細菌芽孢的消毒劑耐受性嗎？

 

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.

 

在細菌中，芽孢更難消除。例如，即使使用殺孢子劑，對蠟樣芽孢桿菌的殺傷力也最低。消毒劑很難穿透芽孢結構。至于子囊菌霉菌，其有性芽孢受到兩層保護，而細菌芽孢中有一層，因此更難消除這些真菌。因此，防止土壤或纖維材料攜帶的難以殺死的細菌芽孢和子囊菌真菌進入是一種成功的策略。

 

Identification

 

鑒別

 

Question

 

問題

 

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

 

應該在培養的第5天還是第7天讀取霉菌菌落？

 

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.

 

許多半知菌類的霉菌，如曲霉、青霉、擬青霉等，都是快速生長的。到第 5 天，許多這些快速生長的霉菌可能會形成孢子并產生額外的菌落或以真菌墊覆蓋整個碟子。這兩種情況都會導致假陽性結果。

 

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 天計數菌落，并在第 5 天進行復查。這樣，第 3 天的讀數將允許對快速生長的霉菌的菌落進行計數，而第 5 天的讀數將允許對生長較慢的霉菌的菌落進行計數。

 

Question

 

問題

 

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

 

正確鑒定真菌的最佳方法/技術是什么？

 

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.

 

鑒定系統中龐大而多樣的數據庫是正確鑒定和減少無法鑒定機會的關鍵。

 

 

Question

 

問題

 

 

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?

 

在構建霉菌鑒別 SOP 時，如果想要求將已鑒別的污染物保存在庫中，建議如何保存它們？

 

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.

 

霉菌可以冷凍保存或凍干。這兩種方法可選用外部服務。企業內部也可以使用液體培養基和10-15% 甘油進行凍存。如果在企業內部冷凍保存，則必須建立程序以定期檢查培養物的純度。

 

Question

 

問題

 

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，霉菌鑒定應通過在顯微鏡下觀察來確認。即使對于基于序列的鑒定，這也是必要的嗎？

 

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.

 

基于序列的鑒定是理想的，顯微鏡和顯微鏡技術可以確定霉菌類型及其可能的來源，以便在等待鑒定結果的同時，可以啟動調查和補救措施。

 

Question

 

問題

 

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

 

霉菌鑒別結果通常被標記為“菌絲體”或“未鑒定”。這些結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.

 

“菌絲體” 在自然界中很罕見。當實驗室在使用常規鑒定方法無法使霉菌孢子形成時，通常會生成此類結果。

 

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.

 

由于使用任何常規方法而導致的未識別霉菌的結果，可歸因于沒有孢子形成。不了解相關霉菌生長要求的非真菌學家可能無法為孢子形成提供營養生長培養基。

 

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 系統產生的未識別霉菌暗示了一些關鍵因素，例如霉菌未在系統庫中編目、混合培養或在制備樣品以在系統上運行時出現實驗室錯誤。

 

Question

 

問題

 

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

 

如何將霉菌分為弱霉菌或強霉菌？有沒有可以參考的列表？

 

 

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.

 

無色半知菌類真菌比有色半知菌類真菌和子囊菌弱。在接合菌真菌中，無性孢子比有性產生的接合孢子弱。

 

Specific Molds

 

特定霉菌

 

Question

 

問題

 

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

 

如何從獲得的ID中了解霉菌的性階段？

 

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.

 

如果鑒別系統和真菌鑒定服務提供商跟上分類法和命名法的變化，結果將在真菌的性階段中報告。微生物學家負責研究無性階段以評估患者風險。請注意，性階段很難殺死，而無性階段最具傳播性。

 

Question

 

問題

 

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?

 

由于霉菌的條件非常極端，這些霉菌在我們的設施中有性繁殖的可能性有多大？我們更有可能只看到無性孢子嗎？

 

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.

 

這兩個階段的霉菌都有可能出現在潔凈室。根據所使用的鑒定系統或方法，結果可能會以霉菌的無性或有性名稱報告。無性階段是最具傳播性的階段，而有性階段更難消除。建議無論鑒定結果如何，實驗室都應研究霉菌是否同時具有兩個階段并解決補救措施，并相應地進行微生物特異性評估。

 

Question

 

問題

 

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

 

你有紅色籃狀菌的經驗嗎？從培養箱和潔凈室中清除它們的最佳方法是什么？

 

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.

 

紅色籃狀菌被描述為紅色青霉的有性狀態，產生覆蓋著交織菌絲的軟壁子囊。青霉菌是無性期，而 籃狀菌 是有性期;如果不消除有性階段，就會產生無性孢子，無性繁殖的循環會繼續。由于培養箱是霉菌滋生的理想場所，因此建議使用殺孢子劑對培養箱進行消毒，然后經常擦拭。水盤或產生濕氣的水箱也應經常清潔。

 

Question

 

問題

 

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 后仍然檢測到紅色籃狀菌，我們在 C 級潔凈室區域的樣品中檢測到它，我們還可以嘗試哪些其他方法來消除它？我們主要在環境監測中檢測到它。

 

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.

 

紅色籃狀菌被描述為紅色青霉的有性狀態，產生覆蓋著交織菌絲的軟壁子囊。了解霉菌的確切位置以及是否在同一區域發現霉菌將是有益的。霧化的成功與霧化學殺死生物體的有效性、霧到達污染源的能力以及霧的浮力相關。霧的浮力是一個關鍵參數，因為霧應該在空氣中停留足夠長的時間，并且應該到達房間的所有區域。

 

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.

 

建議對污染物進行趨勢分析，嘗試找到其進入路線，并利用趨勢來了解它是否局限在潔凈室內，在哪里繁殖和擴散的？

 

Question

 

問題

 

How to get rid of Cladosporium?

 

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.

 

枝孢霉菌喜歡 20 至 25°C 的低溫。 除了通過人員腳步和推車輪子帶入潔凈室外，它還會在潮濕和溫度較低的壁龕中繁殖。話雖如此，它也可以在溫暖的溫度下生長。審查冷藏室和冰箱是一個好的開始。還建議檢查冷藏室中儲存的小瓶的標簽，因為大多數霉菌都使用紙張和粘膠劑作為碳源。制定冷藏室信息監測計劃以及常規清潔和消毒計劃可以減少這種霉菌的發生。枝孢菌孢子形成方法使其迅速增殖，它在分生孢子的分支鏈中形成孢子，這些分生孢子鏈非常小，很容易傳播。

 

Question

 

問題

 

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

 

在空氣采樣中發現的枝孢霉菌屬物種，它可能有哪種來源嗎？

 

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.

 

由于氣流和移動，地板上的任何污染物都可能在潔凈室中傳播到空氣中。如果在地板上沒有檢測到枝孢霉菌，那么研究幫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，因為它喜歡較冷的溫度。審查這些來源將是有益的。

 

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.

 

建議對發現枝孢霉菌的最常見采樣點進行趨勢分析，并尋找可能解釋這種現象的非生物來源或氣流模式。

 

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

 

枝孢霉菌產生分生孢子的分支鏈，簡而言之，孢子比大多數真菌多得多，因此它是在潔凈室中發現的非常常見的霉菌。