Histopathological diagnosis of onychomycosis by periodic acid-Schiff-stained nail clippings

BACKGROUND: The current laboratory methods for diagnosing fungal infections of the nails are the potassium hydroxide (KOH) scraping technique and fungal culture. However, due to the long incubation period required for fungal culture and the reported rate of approximately 30% false negative results observed when using these methods, a quick and highly specific screening test for diagnosing onychomycosis is urgently needed. OBJECTIVES: In a prospective study, to compare the traditional mycological diagnostics using culture medium and KOH preparation with the histopathological diagnosis of onychomycosis by periodic acid-Schiff (PAS)-stained nail clippings. METHODS: Material from 387 nails of 350 patients suspected of having onychomycosis was obtained and a KOH stain as well as two fungal cultures (Kimmig agar with and without cycloheximide) were prepared. In addition, the same specimen was histopathologically examined (PAS stain). RESULTS: Culture medium and KOH preparation respectively revealed 100 and 156 cases of onychomycosis, as compared with 182 cases by histological examination. Histological examination gave a significantly higher rate of positive results (P < 0.05). Considering the total number of positive results given by at least one of the three methods (total = 438), histological evaluation was found to give the highest rate of successful recognition of mycotic infection (41.6%). CONCLUSIONS: The histopathological evaluation of PAS-stained nail clippings is very quick and easy to perform, and will increase the frequency of diagnosing onychomycotic disease above that achieved by culture and KOH preparation alone. However, because information concerning the vitality of the fungi and accurate identification of the specific pathogen is not available through this investigation alone, mycological culture continues to remain the indisputable 'gold standard' of mycological diagnostics.     

Onycomycosis due to artificial nails

Background  The use of artificial nails (ANs) as part of nail-care cosmetics is very popular. Several side effects and complications, such as contact dermatitis and bacterial and fungal infections, have been reported in patients using ANs.
Objective  The purpose of this study was to identify the fungal pathogens in nail abnormalities appearing in patients with ANs.
Methods  We evaluated 68 patients suffering from nail changes and paronychia, which appear after removal of ANs. Mycological samples were obtained from two sites: distal parts of the involved nail and the proximal nail fold. KOH examination and fungal culture were used for detection and identification of fungal infection.
Results  Mycological results from the distal part of the nail showed positive KOH test in 57 cases (83.8%), and culture was positive in 67 patients (98.5%). Mycological results obtained from the proximal nail fold showed positive KOH test in 36 patients (52.9%); in 36 of the cases, culture was positive. Candida spp. were the most common pathogen. Both KOH and culture results were significantly better while sampling from the distal part of the nail compared with sampling from the proximal nail fold ( P =  0.0001).
Conclusion  Onychomycosis was found to be very common in nail changes due to ANs, leading to an increased risk of transmitting microbial infections. Therefore, health care personnel and workers in the food industry should avoid using ANs.     

Antifungal susceptibility patterns of yeasts and filamentous fungi isolated from nail infection

Background Onychomycosis is the nail infection caused by a wide spectrum of fungi species, including yeasts, dermatophytes and filamentous fungi non‐dermatophytes (FFND). This fungal infection represents an important medical problem because it involves the patient′s life quality. Objective The aim was to isolate and identify the fungal agents of onychomycosis, and to determine the in vitro susceptibility to antifungal agents. Methods During the period of March 2008 to March 2009, 114 patients clinically suspected of having onychomycosis were examined. Demographic data, mainly age and gender were obtained from each patient. The nail samples collected (136) were submitted to direct examination with potassium hydroxide 20% and grown on Sabouraud dextrose agar. The in vitro antifungal susceptibility testing was performed according to the method of broth microdilution, recommended by the Clinical Laboratory Standards Institute (CLSI). Results Onychomycosis was observed in 95 (83.3%) patients, including 16 men (16.8%) and 79 women (83.2%), with mean age of 48.1 years. Candida parapsilosis, Trichophyton rubrum and Fusarium spp were the fungi most frequently isolated. The most of the isolated yeasts showed susceptibility to antifungal agents studied. Among filamentous fungi, high MIC values to itraconazole were found for T. rubrum and T. mentagrophytes, while Fusarium spp showed decreased susceptibility to itraconazole and voriconazole. Conclusion C. parapsilosis was the most common fungal species isolated from patients with onychomycosis. The different response obtained by in vitro susceptibility testing to drugs shows the importance of these methods to assist clinicians in choosing the best therapeutic option.     

Evaluation of a newly-developed immunochromatography strip test for diagnosing dermatophytosis

Background Traditionally, dermatophytosis, a common disease affecting millions of people world‐wide, has been diagnosed by direct microscopy and fungal culture. The immunochromatography (ICG) strip test was recently developed. Methods We compared the performance of the ICG strip test for the detection of dermatophytes in samples from human skin and nails with direct microscopy. The 160 samples, consisting of 88 skin and 72 nail specimens, were subjected to direct microscopy study using a 20% KOH solution and to examination with the ICG strip test. Of 160 samples, 18 were examined by fungal culture using Sabouraud dextrose agar medium. Results We found that the overall sensitivity and specificity of the ICG test were 83.5% and 66.7%; they were 82.1% and 76.2% for the 88 skin and 85.4% and 58.3% for the 72 nail specimens, respectively. Conclusions Our findings indicate that the efficacy of the ICG test is comparable to direct microscopy for the detection of dermatophytes. Performance of the assay was easy, and results were available quickly. We suggest that it is an effective tool for dermatophytosis screening.     

Superficial mycoses in immunodepressed patients (AIDS)

HIV infection has the capacity to distort the epidemiology and clinical course of infectious diseases, producing atypical manifestations and changing diagnoses. Superficial fungal infections are frequent in HIV-positive/AIDS patients and are no exception. These infections are frequently different in immunodepressed patients (AIDS), with a modified course or exacerbations. This chapter discusses the diagnosis and treatment of superficial mycoses in HIV patients, including cutaneous alterations caused by Candida, dematiaceous fungi agents of phaeohyphomycosis, Malassezia spp, dermatophyte, and filamentous nondermatophyte fungi.     

Fusarium Solani: a causative agent of skin and nail infections

Fusarium spp are non-dermatophytic hyaline moulds found as saprophytes and plant pathogens. Human infections are probably a result of various precipitating predisposing factors of impaired immune status. Immunocompetent individuals of late are also vulnerable to various unassuming saprophytic and plant pathogens. To stress the need to identify correctly and institute appropriate antifungal therapy in newly emerging human fungal infectious agents. Repeated mycological sampling of the skin and nails of the suspected fungal infection were processed as per the standard format including direct microscopy and fungal culture on Sabouraud's dextrose agar. The fungus was isolated as Fusarium solani. Fusarium is an important plant pathogen and soil saprophyte. Infection is acquired by direct inoculation or inhalation of spores. It is associated with a variety of diseases like keratitis, onychomycosis, eumycetoma, skin lesions and disseminated diseases.     

Great toenail onychomycosis caused by Syncephalastrum racemosum

Nondermatophyte molds are fungi found in soil and decaying plant debris and are generally considered to be uncommon or secondary pathogens of diseased nails. Prevalence rates of onychomycoses caused by nondermatophyte molds range between 1.45 percent and 17.60 percent. The most common nondermatophyte molds associated with nail disease are Scopulariopsis, Scytalidium, Fusarium, Aspergillus and Onychocola canadensis. Syncephalastrum racemosum, a nondermatophyte mold, belongs to the class Zygomycetae. Only one well-documented case of human disease attributed to this organism has been described. We describe a 45-year-old man with culture proven toenail onychomycosis due to Syncephalstrum racemosum.     

念珠菌性包皮龟头炎的菌种分布及其药敏试验分析

目的：了解念珠菌性包皮龟头炎的致病菌种及其对抗真菌药物的敏感性。方法：分别采用沙保罗琼脂平板、法国生物梅里埃公司的酵母样真菌鉴定卡（ID 32C）、酵母样真菌药敏卡（ATB FUNGUS3）对包皮龟头分泌物进行念珠菌的培养、鉴定和药敏试验。结果：1227例患者共培养出356株念珠菌,阳性率为29．0％。其中,白色念珠菌297株（83．5％）、光滑念珠菌41株（11．5％）、热带念珠菌14珠（3．9％）,其它念珠菌4株（1．1％）。白色念珠菌和光滑念珠菌对5-氟胞嘧啶、两性霉素B、氟康唑、伊曲康唑、伏立康唑的敏感率分别为98％、100％、97．3％、61．3％、100％和95．1％、100％、19．5％、12．2％、95．1％。结论：白色念珠菌是念珠菌性包皮龟头炎的主要致病菌种,不同菌种对药物的敏感性有所不同。     

APPLICATION OF POLYMERASE CHAIN REACTION (PCR) AND PCR BASED RESTRICTION FRAGMENT LENGTH POLYMORPHISM FOR DETECTION AND IDENTIFICATION OF DERMATOPHYTES FROM DERMATOLOGICAL SPECIMENS

Objective:

To develop and optimize polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) targeting 18S rDNA and internal transcribed spacer (ITS) region of fungi for rapid detection and identification of dermatophytes.

Materials and Methods:

Two PCR-RFLP methods targeting 18S rDNA and ITS regions of fungi were optimized using standard and laboratory isolates of dermatophytes and other fungi. Sixty-eight dermatological clinical specimens (nail clippings (56), material obtained from blisters (8), hair root (2), scraping from scaly plaque of foot (1) and skin scraping (1) collected by the dermatologist were subjected to both the optimized PCR-RFLP and conventional mycological (smear and culture) methods.

Results:

PCRs targeting 18S rDNA and the ITS region were sensitive to detect 10 picograms and 1 femtogram of T. rubrum DNA, respectively. PCR targeting 18S rDNA was specific for dermatophytes and subsequent RFLP identified them to species level. PCR-RFLP targeting the ITS region differentiated dermatophytes from other fungi with identification to species level. Among the 68 clinical specimens tested, both PCR-RFLP methods revealed the presence of dermatophytes in 27 cases (39.7%), whereas culture revealed the same only in 2 cases (7.40%), increasing the clinical sensitivity by 32.3%. Among 20 smear positive specimens, both PCR-RFLP methods detected dermatophytes in 12 (17.6%). Both the methods detected the presence of dermatophytes in 13 (19.11%) smear and culture negative specimens, increasing the clinical sensitivity by 36.1%.

Conclusion:

PCR-RFLP methods targeting 18S rDNA and the ITS regions of fungi were specific and highly sensitive for detection and speciation of dermatophytes.     

Onychomycosis infections in the Middle Black Sea Region, Turkey

Background  Onychomycosis, a fungal infection of the nail, is caused by dermatophytes, yeasts, and nondermatophyte molds. The causative pathogen and incidence of onychomycosis depend on age, gender, geographic and climatic conditions, living habits, and immune status of the host.
Aim  To determine the incidence and etiologic agents of onychomycosis in the Middle Black Sea Region, Turkey.
Methods  Two hundred and seventy-six specimens were collected from patients with suspected onychomycosis during January 2004 to May 2008.
Results  Culture positivity was obtained in 240 of the 276 samples. Dermatophytes were isolated in 225 samples. The most common causative agent of onychomycosis was Trichophyton rubrum (91) (38%), followed by Trichophyton mentagrophytes (49) (20.4%), Epidermophyton floccosum (41) (17%), and Trichophyton verrucosum (34) (14.2%). Seven isolates were identified as yeasts (2.9%). Nondermatophyte molds were isolated from eight samples (3.3%).
Conclusions  This survey reveals that the etiologic agents of onychomycosis in our area show large discrepancies from those in other regions of Turkey and Europe.     

Epidemiologic and clinicomycologic profile of onychomycosis from north India

Background  Onychomycosis is an important public health problem because of the increase in immunosuppressive states. Large-scale studies in India are scarce, and so the baseline incidence of onychomycosis is not firmly established.
Methods  Three hundred and two clinically suspected cases of onychomycosis were included in this study. Nail samples were collected for direct microscopic examination and culture. Clinical patterns and associated relevant factors were noted according to a predetermined protocol.
Results  The associated predisposing conditions included diabetes mellitus (3.9%) and systemic lupus erythematosus (2.3%). Distal and lateral subungual onychomycosis was the most common clinical pattern (62%), followed by total dystrophic onychomycosis (20.2%). The most common fungal isolates were dermatophytes (49.5%), followed by Candida spp. (40.4%) and nondermatophyte molds (10.1%). Of the dermatophytes, Trichophyton rubrum (47%) was the most common isolate, followed by Trichophyton tonsurans (20.4%). Of the Candida spp., Candida albicans was the most common (60%).
Conclusions  Until recently, yeasts and nondermatophyte molds were regarded as contaminants, but their emergence as a significant cause of onychomycosis in immunocompromised patients calls for mycologic diagnosis and antifungal susceptibility testing in onychomycosis. The recognition of the changing patterns of onychomycosis will aid in the therapeutic approach and the implementation of control measures.     

Tinea capitis in early infancy treated with itraconazole: a pilot study

Background  Tinea capitis is the most common fungal infection of the scalp in childhood, but a very rare disorder in the first year of life.
Objective  To evaluate the efficacy, tolerability and safety of itraconazole in 7 children aged between 3 and 46 weeks (median: 36 weeks) suffering from tinea capitis caused by Microsporum canis .
Methods  Prospective case note study. In all patients KOH testing and fungal cultivation on Sabouraud dextrose agar were performed.
Results  7 patients (5 girls and 2 boys) were included in the period between 2001 and 2008. The causative etiologic agent was Microsporum canis in all children. The patients received itraconazole 5mg/kg bodyweight daily for 3 to 6 weeks with no clinically side effects being noted. In all patients clinical and mycological cure could be achieved.
Conclusion  Itraconazole proved to be a safe and effective treatment option for Microsporum canis induced tinea capitis in children in their first year of life.     

Evaluation of the association of superficial dermatophytosis and athletic activities with special reference to its prevention and control

Objective This study was conducted to evaluate the association of superficial mycosis and athletic activities with special references to its prevention and control in Tehran. Participating in various kinds of sports can lead to direct and indirect exposures to and transmission of micro‐organisms between athletes and also passive observers. Methods A retrospective study of superficial fungal infections in athletes was carried out during the period of March 2002 to December 2006 on 656 mycological proven cases of dermatophytosis found in athletes in Tehran. Mycologic examination consisted of culturing of pathologic material followed by direct microscopic observation. Mycologic cultures were carried out on Sabouraud Chloramphenicol Agar, Sabouraud Chloramphenicol and Cyclohexamide Agar, and Dermatophyte Agar incubated at 25 °C for at least 28 d. Diagnosis was based on macroscopic and microscopic characteristics of the colonies. Results A total of 1075 athletes, from 201 institutions, suspected of cutaneous fungal infections were examined and 656 (61%) were found to be positive for fungal infections. The fungal isolates comprised Trichophyton tonsurans (56%), Epidermophyton floccosum (11.8%), Trichophyton mentagrophytes (8.9%), Trichophyton rubrum (8.3%), Trichophyton verrucosum (3.9%), Trichophyton violaceum (3.3%), Microsporum canis (2.5%), and Malassezia spp. (5.3%). The distribution of lesions on the body in decreasing order was as follows: trunk, groin, hair and scalp, sole, toe webs, finger nails, and toe nails. Fungal infections were more commonly seen in wrestlers and individuals that participate in aerobics. Conclusion The results suggest that athletic activity seems to be a predisposing factor, especially for fungal infections. Guidelines are provided regarding measures to prevent transmission of infectious diseases in athletic settings, including hygiene, infection control practices, and education of officials, coaches, trainers, and sports participants.     

PCR-RFLP技术快速鉴定八种致病丝状病原真菌的实验研究

目的 建立能快速鉴定深部丝状真菌感染病原菌的PCR-RFLP方法。方法 用真菌通用引物扩增烟曲霉、黄曲霉、土曲霉、黑曲霉、杂色曲霉、构巢曲霉、尖端赛多孢和串珠镰刀菌的ITS区,分别用HhaⅠ、HaeⅢ、HinfⅠ、TaqⅠ和MspⅠ 5种限制性核酸内切酶对PCR产物进行酶切,建立以PCR为基础的RFLP方法,然后对22株临床株和2株环境分离株进行PCR-RFLP图谱分析。结果 对PCR产物进行RFLP分析可以准确鉴定8种深部致病丝状真菌,从DNA提取到酶切分析可以在1个工作日完成。22株临床株和2株环境分离株PCR-RFLP鉴定结果与传统的形态学鉴定结果一致。结论 PCR-RFLP技术是一种能够快速鉴定丝状真菌的有效方法。     

Onychomycosis in Tunis area: epidemiological and mycological data]

BACKGROUND: Onychomycosis is by far the most frequent cause of nail disease. We describe epidemiological and mycological features observed in the Tunis area in Tunisia. MATERIAL AND METHODS: Data were collected from 292 nail samples performed in 255 patients with suspected onychomycosis. RESULTS: Request for samples were made late, on the average 48 months after development of nail disorders. Most of the patients were women (63.5%). One hundred ninety-six samples were positive (67%), 130 from toe nails and 66 from finger nails. Simultaneous infections of both finger and toe nails were found in 22 cases. Associated onychomycosis and skin mycosis was found preferentially in feet onychomycosis. The sensitivities of direct examination and culture depended on the site of the onychomycosis. Cultures were more sensitive for hands where yeasts, particularly Candida albicans, predominated, but the direct examination was more sensitive for feet where dermatophytes, particularly Trichophyton rubrum, predominated. CONCLUSION: Mycological examination is compulsory for confirmation of onychomycosis. It is also recommended before initiating a costly long-term treatment.     

Filamentous fungi and yeasts on mattresses covered with different encasings.

Besides mites, filamentous fungi and yeasts play an important role as domestic allergens. Among different allergen avoidance strategies the efficacy of synthetic mattress encasings has been demonstrated for the reduction of house dust mites. Whether these synthetic encasings are also able to reduce the growth of fungi on the mattress under domestic conditions has not been assessed so far. To determine if the fungal growth on mattresses can be reduced by the use of synthetic encasings we assessed the fungal colonisation of mattresses covered either by conventional cotton encasings or by polyurethane encasings impermeable to particles > 3 mum. Within a 12-month period dust samples were obtained from the mattresses. Fungal quantities were measured by counting colonies on agar plates incubated at 20 degrees C and 37 degrees C. The counts of fungi were significantly higher on mattresses with cotton encasings. Penicillium spp. and Aspergillus spp. were isolated most frequently. Therefore the application of synthetic encasings with similar properties to the encasings used in this investigation is recommended as part of an allergen avoidance strategy for patients sensitised to fungal allergens.     

Dermatophyte identification in skin and hair samples using a simple and reliable nested polymerase chain reaction assay

Summary Background Dermatophyte identification in tinea capitis is essential for choosing the appropriate treatment and in tinea infections to identify the possible source. The failure of fungi to grow in cultures frequently occurs, especially in cases of previous antifungal therapy. Objectives To develop a rapid polymerase chain reaction (PCR) sequencing assay for dermatophyte identification in tinea capitis and tinea corporis. Material and methods Fungal DNA was extracted from hair and skin samples that were confirmed to be positive by direct mycological examination. Dermatophytes were identified by the sequence of a 28S ribosomal DNA subunit amplicon generated by nested PCR. Results Nested PCR was found to be necessary to obtain amplicons in substantial amounts for dermatophyte identification by sequencing. The results agreed with those of classical mycological identification in 14 of 23, 6 of 10, and 20 of 23 cases of tinea capitis, tinea corporis and tinea pedis, respectively, from which a dermatophyte was obtained in culture. In seven of the 56 cases, another dermatophyte was identified, revealing previous misidentification. A dermatophyte was identified in 12 of 18, three of five, and four of nine cases of tinea capitis, tinea corporis and tinea pedis, respectively, in cases in which no dermatophyte grew in culture. Conclusions Although the gold standard dermatophyte identification from clinical samples remains fungal cultures, the assay developed in the present study is especially suitable for tinea capitis. Improved sensitivity for the identification of dermatophyte species was obtained as it is possible to identify the dermatophyte when the fungus fails to grow in cultures.     

改良皮肤癣菌培养基在甲真菌病诊断中的应用

目的 采用改良皮肤癣菌试验培养基（改良DTM）检测甲真菌病临床标本，并与皮肤癣菌试验培养基（DTM）比较，以评价其临床实用性。方法 收集临床拟诊甲真菌病的标本，分别接种于改良DTM、DTM、含放线菌酮及氯霉素的沙氏培养基（SCCA）和含氯霉素的沙氏培养基（SCA）；记录菌株的开始生长时间、培养基开始变色时间及开始变色时菌落的直径。以专业真菌实验室的鉴定结果为金标准，将DTM和改良DTM的结果与之比较。结果 ①改良DTM、DTM、SCCA的分离率、菌种的生长速度及形态无显著差异。②所有分离的皮肤癣菌均能使两种显色培养基变色，改良DTM的开始变色时间（5.83 ± 0.39 d）早于DTM（7.32 ± 0.41 d），两组比较，t = 2.63，P = 0.01。③大多数分离的非皮肤癣菌也能使两种培养基变色，以开始变色时菌落的直径大小（≥5 mm）为非皮肤癣菌和皮肤癣菌的鉴别点，与金标准有高度的一致性。结论 DTM和改良DTM比传统的鉴定方法结果报告时间提前1周左右，改良培养基的配方较DTM经济，肉眼观察其开始变色时间早于DTM，值得进一步的深入研究。     

New criteria for the laboratory diagnosis of nondermatophyte moulds in onychomycosis

Background  Nondermatophyte moulds (NDM) may be found as aetiological agents or as contaminants in onychomycosis. The classic and most used criteria for the diagnosis of NDM are those established by English in 1976.
Objectives  The aim of this article is to re-evaluate the laboratory criteria for the diagnosis of NDM in onychomycosis.
Patients and methods  Patients with suspected NDM of the nail underwent five consecutive examinations by both KOH and mycological culture; at the first visit, three samples from the affected nail were taken and were examined separately. Later those patients underwent four consecutive examinations; during this stage only a single sample for both KOH and culture was taken. We compared the culture results obtained from the three nail samples obtained at the first visit with the results from the four consecutive visits.
Results  We noted a clear trend showing that as the number of positive cultures increases (one to three cultures) during the first examination, the percentage of subsequent positive cultures, taken during the four consecutive visits, also increased.
Conclusions  We suggest that when NDM infection is found in the first culture, the patient should be re-examined in a subsequent visit in which three separate samples are taken from the affected nail. If NDM is confirmed in all three cultures, the diagnosis of NDM is established. Treatment should be recommended in patients who show positive results in all three cultures.     

Molecular detection of dermatophytes and nondermatophytes in onychomycosis by nested polymerase chain reaction based on 28S ribosomal RNA gene sequences

Background  Onychomycosis is often caused by dermatophytes, but the role of nondermatophytes is underestimated due to the difficulty of identifying them by conventional direct microscopy and culture.
Objectives  This study aims to detect nondermatophytes, as well as dermatophytes, in the nail samples of patients with onychomycosis using a polymerase chain reaction (PCR)-based culture-independent method.
Materials and methods  The nested PCR assay targeting the sequence of the 28S ribosomal RNA gene was used to amplify fungal DNAs from 50 microscopy-positive nail specimens. Newly designed primer sets for dermatophyte universal, Trichophyton rubrum , T. mentagrophytes , Aspergillus spp., Scopulariopsis brevicaulis , Fusarium solani , F. oxysporum , F. verticillioides , Candida albicans and C. tropicalis were used after confirmation of their specificity.
Results  Forty-seven cases (94%) were positive for fungal DNA, among which dermatophytes were detected in 39 cases (83·0%): T. rubrum in 35 cases (74·5%) and T. mentagrophytes in eight cases (17·0%). Surprisingly, nondermatophytes were detected in 18 cases (38·3%), both dermatophytes and nondermatophytes in 10 cases (21·3%) and nondermatophytes alone in eight cases (17·0%). Aspergillus spp. alone was observed in five cases (10·6%).
Conclusions  This study indicates that most of the affected nail plates of patients with onychomycosis were positive for specific fungal DNAs, and suggests that nondermatophytes detected at high rates may be involved in the pathogenesis of onychomycosis.