样本处理时间对猫犬小孢子菌培养性能的影响

王帆

The influence of sample processing time on the performance of Microsporum canis cultures in cats

样本处理时间对猫犬小孢子菌培养性能的影响

作者：Aline E. Santana, Fabio P. Sellera, Kilder D. Filgueira，Carlos P. Taborda and Archivaldo Reche-Junior

翻译：赵胜楠

Background – Fungal culture is widely used as a diagnostic tool for detecting dermatophytosis. However, the presence of fungal contaminants can influence the culture’s performance and compromise the diagnosis.

Objective – To verify whether the sample processing time can affect the performance of fungal culture for the diagnosis of Microsporum canis infection in cats.

Animals – Forty Persian cats.

Methods and materials – Hair and scale samples were collected by combing the coat using a 5 x 5 cm sterile polyester carpet. The carpets were assigned randomly to four groups based on time point of processing samples after collection (i.e. used for culture on a selective agar medium for dermatophytes): Group 1: 8 h (n = 10); Group 2: 24 h (n = 10); Group 3: 48 h (n = 10); and Group 4: 72 h (n = 10). Cultures were compared regarding the degree of fungal invasion by either M. canis or nondermatophytic contaminant moulds (NDM).

Results – Processing samples after 24 h of storage resulted in increased isolation rates of NDM and decreased isolation rates of M. canis. Samples processed after 48 h and 72 h presented more than half of the plates with a high degree of fungal contamination (i.e. NDM occupying ≥50% of the total fungal mass). However, samples processed after 8 h and 24 h presented a lower degree (P < 0.05) of NDM plate invasion and higher recovery rates of M. canis when compared to samples processed after 48 h and 72 h.

Conclusions and clinical importance – Delayed processing time is closely associated with the overgrowth of contaminants and with lower recovery rates of M. canis.

背景 – 真菌培养被广泛用作检测皮肤癣菌病的诊断工具。然而, 真菌污染物的存在可能影响培养基的表现并影响诊断。

目的 – 验证样本处理时间是否影响用来诊断猫的犬小孢子菌感染的真菌培养基的表现。

动物 – 40只波斯猫。

方法和材料 – 使用5x5cm无菌聚酯材料采集毛发和皮屑样本。根据采集后处理样本的时间点 (即用于皮肤癣菌选择性琼脂培养基培养) , 将样本随机分为4组: 第1组, 8 h(n = 10); 第2组, 24 h(n = 10); 第3组, 48 h(n = 10);第4组, 72 h(n = 10)。比较犬小孢子菌或非皮肤癣菌的污染霉菌(NDM) 在培养基中的真菌侵袭程度。

结果 – 储存 > 24 h后处理样本导致NDM分离率增加, 而犬小孢子菌分离率降低。48h和72h后处理的样本显示超过一半的平皿真菌污染程度高 (即NDM≥50%的真菌总质量) 。然而, 与48h和72h后处理的样本相比, 8h和24h后处理的样本呈现较低程度(P < 0.05)的NDM平皿侵袭和较高的犬小孢子菌生长率。

结论和临床重要性 – 处理时间延迟与污染菌过度生长和犬小孢子菌生长率较低密切相关。

Introduction

介绍

Dermatophytosis is a contagious and zoonotic dermatomycosis of cats. Currently, several diagnostic techniques are available to confirm clinical suspicion, and the fungal culture remains one of the most employed diagnostic tools. Additionally, culture allows the identification of fungal species involved and helps to monitor the response to antifungal therapy.

皮肤癣菌病是猫的一种接触性、人畜共患的皮肤真菌病。目前，有几种诊断技术可用于证实临床怀疑，真菌培养仍然是使用最多的诊断工具之一。此外，培养可鉴定涉及的真菌种属，并有助于监测对抗真菌治疗的反应。

The accuracy of fungal culture can be influenced by several factors, such as sampling method and culture media, as well as proper inoculation on the agar plates.Although no previous studies have specifically reported the influence of the sample processing time on the performance of the dermatophyte cultures, a general recommendation is that clinical samples ideally should be processed immediately after sampling to prevent over growth of contaminants. The presence of fungal or bacterial contaminants can lead to a misleading result by suppressing the growth of or masking the presence of the targeted pathogen.Consequently, this kind of contamination can substantially affect the final diagnosis and compromise treatment.

真菌培养的准确性可受多种因素影响，包括采样方法、培养基选择，以及在琼脂平皿上合适的接种技术。尽管之前没有研究专门报告样本处理时间对皮肤癣菌培养性能的影响，但一般建议是理想情况下的临床样本应在采样后立即处理，以防止污染物过度生长。真菌或细菌污染物的存在可能通过抑制目标病原体的生长或掩盖其存在而导致误导性结果。因此，这种污染会严重影响最终诊断和治疗。

In this study, we performed an investigation to verify whether the sample processing time can affect the performance of fungal culture for the diagnosis of Microsporum canis infection in cats.

在这项研究中，我们进行了一项调查以验证样品处理时间能否影响真菌培养诊断猫犬小孢子菌感染的性能。

Methods and materials

方法和材料

This study was approved by the Institutional Animal Care and Use Committee of our Institute. The owners signed an informed consent before inclusion.

本研究获得了本研究所动物护理和使用委员会的批准。入选前主人签署了知情同意书。

Animals

动物

The study included 40 Persian cats (22 males, 18 females) ranging in age from three to 10 years, coming from the same cattery. All cats presented multifocal alopecia and scaling. The animals had no prior history of treatment with antifungals in the 20 days before sample collection.

该研究包括40只波斯猫（22只雄性，18只雌性），年龄范围为3-10岁，来自同一猫舍。所有猫均出现多灶性脱毛和皮屑。在样本采集前20天内，动物无抗真菌药既往治疗史。

Skin and hair sampling

皮肤和毛发采样

Hair and scales were collected by combing the coat with a 5cm x 5cm sterile polyester carpet square according to the modified Mariat et al collection method.

根据改良的Mariat等人的采集方法，用5cm x 5cm的正方形无菌聚酯材料梳理被毛收集毛发和皮屑。

Before use, the carpets were cut in squares, individually wrapped and autoclaved for 20min at 122°C. Then, the collection was performed by gently rubbing the cats for approximately 2min from the head and neck to the dorsum, trunk, ventrum, limbs and tail. After collection, all of the carpet squares were individually wrapped in the original aluminum foil, placed in individual plastic bags, and submitted to a mycology reference laboratory. The samples were kept at room temperature until being processed.

使用前，将材料剪成正方形，单独包裹并在122 ℃下高压灭菌20 min。然后，从猫的头颈部至背部、躯干、腹侧、四肢和尾部轻轻摩擦约2 min进行采集。采集后，将所有方形材料单独包裹在原始铝箔中，置于单个塑料袋中，并提交至真菌学参考实验室。样本在室温下保存直至处理。

Fungal culture

真菌培养

The carpet samples were randomly assigned to four groups (10 for each group). Samples were processed after 8h (Group 1), 24h(Group 2), 48h (Group 3) and 72h (Group 4) of storage. All carpets were inoculated on agarose plates (Mycosel Agar, Becton Dickinson; Sparks, MD, USA) by gently pressing once onto the plate surface for 10 s. Fungal cultures were incubated at 25°C for 15 days after which plate photographs were taken. Identification of dermatophytes and nondermatophytic contaminant moulds (NDM) was performed using morphological keys (colony colour and texture, type of conidia).

将材料样本随机分为4组（每组10只）。在储存8 h（组1）、24 h（组2）、48 h（组3）和72 h（组4）后处理样本。将所有地毯接种于琼脂平皿上，在平皿表面轻轻按压10 s。真菌培养物在25 ℃下培养15天，然后对平皿进行拍照。使用形态学关键词（菌落颜色和质地、分生孢子类型）鉴定皮肤癣菌和非皮肤癣菌污染霉菌(NDM)。

Comparison of groups

组间比较

Groups were compared using the following parameters: (i) number of plates with M. canis; (ii) number of plates with NDM; (iii) degree of M. canis invasion; and (iv) degree of NDM invasion.

使用以下参数比较各组：(i)含有犬小孢子菌的平皿数量；(ii)含有NDM的平皿数量；(iii)犬小孢子菌侵袭程度；和(iv)NDM侵袭程度。

The fungal invasion by either M. canis or NDM was calculated using the open-source IMAGEJ software (National Institutes of Health; Bethesda, MD, USA; https://imagej.nih.gov/ij/). Briefly, the program analysed all of the fungal plate images and quantified the pixels corresponding to the relative surface occupied by M. canis and NDM.

使用开源的IMAGEJ软件（美国国立卫生研究院；Bethesda,MD,USA；https://imagej.nih.gov/ij/）计算了犬小分生孢子菌和NDM的真菌侵袭情况。简而言之，该程序分析了所有真菌平皿图像，并量化了与犬小孢子菌和NDM占据的相对表面相对应的像素。

The degree of fungal contamination and the impact of NDM on ease of visualising M. canis colonies were rated using a modified semi-quantitative scoring system proposed previously.This uses four degrees of contamination as follows: Low, <10% NDM contribution to the fungal invasion of the plate, very easy visualisation of M. canis colonies; Moderate, 20–40% NDM, easy visualisation; High, 50–60% NDM, difficult visualisation; Very high, 80-90% NDM, very difficult visualisation.

使用先前提出的改良半定量评分系统对真菌污染程度和NDM对犬小孢子菌菌落可视化难易度的影响进行评级。这使用了四种污染程度，如下所示：低，< 10%NDM对平皿真菌侵袭的作用，非常容易观察犬小孢子菌菌落；中等，20-40%NDM，容易观察；高，50-60%NDM，难以观察；非常高，80-90%NDM，非常难以观察。

Statistical analysis

统计学分析

Statistical analysis was performed with PRISM v8 (Graphpad Inc.; San Diego, CA, USA) and a P-value of <0.05 was considered statistically significant. Kruskal–Wallis test with Dunn’s multiple comparison post-test was used to compare fungal invasion of the plate by M. canis and NDM. Descriptive statistics (median and range) also were used to summarise the data.

统计分析使用PRISM v8（Graphpad Inc.；San Diego，CA，USA），P值 < 0.05被视为具有统计学意义。采用Kruskal—Wallis检验与Dunn‘s多重比较试验后比较犬小孢子菌和NDM对平皿的真菌侵袭情况。描述性统计量（中位数和范围）也被用来总结数据。

Results

结果

Fungal culture

真菌培养

A total of 40 fungal cultures were performed. Microsporum canis was found in 100% of cultures analysed. Fungal contamination was identified in 35 of 40 (87.5%)plates with different degrees of invasion. Examples of culture plates with contaminant moulds are shown in Figure 1. The results of fungal invasion by either M. canis or NDM are summarised in Table 1. Individual plate results can be found in Table S1 and individual images are presented in Supporting information Figure S1.

共进行40次真菌培养。在研究分析的培养基中，犬小孢子菌检出率100%。40个不同侵袭程度的平皿中有35个(87.5%)鉴定出真菌污染。含有污染霉菌的培养板示例见图1。犬小孢子菌或NDM对真菌侵袭的结果总结见表1。单个平皿结果见表S1，单个图像见支持信息图S1。

When comparing the contribution to the fungal invasion of the plate by M. canis, we found a significantly higher plate invasion in Group 1 when compared to groups 2, 3 and 4; (all P < 0.05). No difference was found between plates of groups 3 and 4 (P > 0.05).

当比较犬小孢子菌对平皿真菌侵袭的作用时，我们发现第1组的平皿侵袭显著高于第2、3和4组；（所有P < 0.05）。组3和组4平皿之间未发现差异(P > 0.05)。

Regarding the presence of fungal contaminants (NDM), a lower plate invasion was found in Group 1 when compared to groups 2, 3 and 4; (all P < 0.05). A lower NDM invasion was found in Group 2 when compared to Group 3 (P < 0.05). No difference was found between groups 3 and 4 (P > 0.05). NDM isolated were Alternaria sp., Aspergillus sp. and Cladosporium sp. Interestingly, Cladosporium sp., a melanised fungi, was found only in Group 4.

关于真菌污染物(NDM)的存在，与组2、3和4相比，组1的平皿侵袭较低；（所有P < 0.05）。与第3组相比，第2组的NDM侵袭较低(P < 0.05)。组3和组4之间未发现差异(P > 0.05)。分离的NDM为链格孢属、曲霉菌属和枝孢属。有趣的是，仅在第4组中发现了一种黑化真菌枝孢霉属。

Group 1 displayed a greater ease of visualisation of M. canis colonies than the other groups as a result of the low degree of fungal contamination. However, more than half of the plates from groups 3 and 4 presented a high degree of fungal contamination, with difficult visualisation of M. canis colonies.

由于真菌污染程度较低，第1组的犬小孢子菌菌落可视化程度高于其他组。然而，组3和组4中超过一半的平皿存在高度真菌污染，难以观察到犬小孢子菌菌落。

Discussion

讨论

In this study, we investigated whether sample processing time can influence the performance of M. canis culture. Our findings demonstrated that delayed processing time is associated with the overgrowth of fungal contaminants and with lower rates of M. canis on agarose plates. At least half of the samples processed after 8 h resulted in pure culture of M. canis (not showing the presence of fungal contaminants). However, more than half of samples processed after 48 h and 72 h showed plates with a high degree of fungal contamination (i.e. NDM occupying ≥50% of the total fungal mass). Significantly higher recovery rates of M. canis were found in cultures of samples processed after 8h and 24h. Overall, these results indicate that delayed processing of samples may affect the performance of M. canis as a consequence of contaminant overgrowth.

在本研究中，我们研究了样本处理时间能否影响犬小孢子菌培养的性能。我们的发现表明，延迟处理时间与真菌污染物的过度生长和在琼脂平皿上犬小孢子菌的检出率低有关。8 h后处理的样本中至少有一半得到犬小孢子菌的纯培养物（未显示存在真菌污染物）。然而，在48h和72h后处理的样本中超过一半显示出具有高度真菌污染的平皿（即NDM占总真菌量的≥50%）。在8h和24h后处理的样本培养物中发现犬小孢子菌的检出率显著提高。总之，这些结果表明，由于污染物过度生长，样本处理延迟可能影响犬小孢子菌的生长。

Despite the fact that all Petri dishes contained solid growth media supplemented with cycloheximide, an inhibitor of a wide range of nondermatophyte moulds and yeasts, contaminant moulds were found in 35 (87.5%) plates. These findings are consistent with previous studies that showed that NDM can grow in selective media containing cycloheximide.

尽管所有培养皿均含有添加放线菌酮的固体生长培养基，一种广泛的非皮肤癣菌霉菌和酵母菌抑制剂，但在35个(87.5%)平皿中发现了污染霉菌。这些发现与之前的研究一致，表明NDM可以在含有放线菌酮的选择性培养基中生长。

The NDM fungal contaminant genera included Alternaria, Aspergillus and Cladosporium. Although these are considered to be environmental fungi, a recent study based on next-generation sequencing demonstrated that these genera could be found colonising the skin of cats. Moreover, previous studies that investigated feline skin microbial communities using culture-dependent methods also isolated these genera. Thus, it can be hypothesised that the NDM isolated in this study did not come from the environment and, rather, from the skin of the sampled animals. Additionally, all clinical samples remained individually wrapped until they were processed into culture media to minimise the risk of contamination by environmental micro-organisms. Interestingly, the genus Cladosporium was found only in samples processed over 72h. Nine of 10 plates of this group exhibited this melanised fungus. We propose that this fungus already was colonising the skin microbiota and its overgrowth led to the suppression of other fungi; Cladosporium is characterised by rapid growth with easy spread over plates that can suppress slow-growing fungi such as M. canis.

NDM真菌污染物属包括链格孢霉属、曲霉菌属和枝孢霉属。尽管这些被认为是环境真菌，但最近一项基于新一代测序的研究证明，这些种属可以定殖在猫的皮肤上。此外，之前利用培养依赖的方法调查猫皮肤微生物群落的研究也分离出了这些属。因此，可以假设，本研究中分离的NDM不是来自环境，而是来自采样动物的皮肤。此外，所有临床样本均保持单独包装，直至接种到培养基，以将环境微生物污染的风险降至最低。有趣的是，枝孢霉属仅在处理超过72h的样本中发现。该组10个平皿中有9个平皿显示出这种黑化真菌。我们认为这种真菌已经在皮肤微生物群中定殖，其过度生长导致了其他真菌的抑制；枝孢霉的特征是快速生长，容易在平皿上扩散，可以抑制缓慢生长的真菌，如犬小孢子菌。

Fungal contamination also was identified in sample plates processed after 8h and 24h, yet the degree of NDM contamination did not compromise the identification of M. canis. Thus, the high yield of M. canis found in these plates led to a proper visualisation and identification of this dermatophyte species. These findings might be valuable to veterinary clinicians that need to ship samples of suspected cases of dermatophytosis to off-site testing laboratories.

在8h和24h后处理的样本平皿中也发现了真菌污染，但NDM污染的程度不影响犬小孢子菌的鉴别。因此，在这些平皿中发现的犬小孢子菌的检出率高使得该皮肤癣菌种属的适当可视化和鉴别。这些发现可能对需要将皮肤癣菌病疑似病例样本运送至非现场检测实验室的兽医临床医生有价值。

It is accepted currently that dermatophytes can remain viable within skin samples for a long time,1、yet the present study points out that delayed processing of samples may make the isolation and identification of M. canis challenging, especially for nonexperts. In particular, prolonged storage can favour the development of NDM that may suppress the growth of M. canis. It follows that dermatological samples, while not as perishable as other biological samples (e.g. blood, urine), should be processed within a brief time window when performing cultures for M. canis. The last consideration is that re-culturing generally is recommended in the case of heavily contaminated plates, which delays the diagnostic workflow further.

目前公认皮肤癣菌可以在皮肤样本内长期存活，然而本研究指出，样本的延迟处理可能使犬小孢子菌的分离和鉴定具有挑战性，尤其是对于非专家。特别是，长期储存有利于NDM的发展，可能抑制犬小孢子菌的生长。因此，皮肤病学样本虽然不像其他生物样本（例如血液、尿液）那样容易腐烂，但在进行犬小孢子菌培养时，应在短时间内进行处理。最后一个考虑是，对于严重污染的平皿，通常建议进行再培养，这会进一步延迟诊断工作流程。

A limitation of the present study is that samples were collected from different animals, which could harbour different fungal loads of both M. canis and NDM. Although we attempted to reduce this bias by selecting cats from the same cattery and with a similar lesion pattern, we recognise that further studies will be necessary to validate our findings. It also would be interesting to verify whether our conclusions apply to other types of samplings (e.g. skin scrapings, toothbrush technique) and infections by other dermatophyte species.

本研究的局限性在于样本采集自不同的动物，其可能含有不同的犬小孢子菌和NDM真菌量。尽管我们试图通过选择来自同一猫舍、具有相似病变模式的猫来减少这种偏倚，但我们认识到有必要进行进一步的研究来验证我们的发现。验证我们的结论是否适用于其他类型的采样（例如，皮肤刮片、牙刷技术）和其他皮肤癣菌属感染也很有意义。

In conclusion, to the best of our knowledge, this is the first study to evaluate the influence of sample processing time on the performance of M. canis cultures in cats. The results reported here suggest that sample processing time is an important parameter and should be considered to optimise the recovery of M. canis on culture plates.

总之，据我们所知，这是首次评估样本处理时间对猫的犬小孢子菌培养性能影响的研究。本文报告的结果表明，为了优化培养皿上犬小孢子菌的检出率，应关注样本处理时间这一重要参数。

Figure 1. Examples of dermatophyte cultures obtained after 15 days of incubation, showing different degrees of nondermatophytic mould (NDM) contamination.

图1：培养15天后的皮肤癣菌培养基示例，可见不同程度的非皮肤癣菌污染物（NDM）生长。

(a) 8 h after collection, fungal culture presents a low degree of contamination (NDM occupying <10% of overall fungal mass). (b) 24 h after collection, moderate degree of contamination (NDM 20–40% of overall fungal mass). (c) 48 h after collection, very high degree of contamination (NDM 80–90% of overall fungal mass). (d) 72 h after collection, very high degree of contamination (NDM 80–90% of overall fungal mass). Fungal identification: BLACK * = NDM; RED * = Microsporum canis; GREEN * = melanised NDM.

（a）采样后8h组，真菌培养基污染程度低（NDM占所有真菌量＜10%）。
（b）采样后24h组，真菌培养基污染程度中等（NDM占所有真菌量20-40%）。

（c）采样后48h组，真菌培养基污染程度非常高（NDM占所有真菌量80-90%）。

（d）采样后72h组，真菌培养基污染程度非常高（NDM占所有真菌量80-90%）。

真菌鉴定：黑*=NDM；红*=犬小孢子菌；绿*=黑化NDM

青春年少

郑江涛