Scholarly article on topic 'Cases of Tinea capitis due to pale isolates of Trichophyton violaceum (Trichophyton glabrum) in South-East Europe. A challenge to the clinical laboratory'

Cases of Tinea capitis due to pale isolates of Trichophyton violaceum (Trichophyton glabrum) in South-East Europe. A challenge to the clinical laboratory Academic research paper on "Clinical medicine"

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{" Trichophyton violaceum " / " Trichophyton glabrum " / " Tinea capitis " / "South-East Europe"}

Abstract of research paper on Clinical medicine, author of scientific article — Manthoula Valari, Ageliki Stathi, Theoni Petropoulou, Talia Kakourou, Anastasia Pangali, et al.

Abstract Two recent indigenous cases of tinea capitis in children due to pale isolates of Trichophyton violaceum are reported herein for the first time from South-East Europe (Greece). Pale isolates of Trichophyton violaceum, reported in the past as Trichophyton glabrum, are thus far sporadically reported only from African or Asian countries. The cases reported herein raise the awareness of its existence in the community, assigning special importance to its accurate identification in the clinical laboratory.

Academic research paper on topic "Cases of Tinea capitis due to pale isolates of Trichophyton violaceum (Trichophyton glabrum) in South-East Europe. A challenge to the clinical laboratory"

Medical Mycology Case Reports 1 (2012) 66-68

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Cases of Tinea capitis due to pale isolates of Trichophyton violaceum (Trichophyton glabrum) in South-East Europe. A challenge to the clinical laboratory

Manthoula Valaria, Ageliki Stathib, Theoni Petropoulou c, Talia Kakourou c, Anastasia Pangalib, Michael Arabatzis d,e'*

a Pediatric Dermatology Department, Aghia Sophia Children's Hospital, Athens, Greece b Microbiology Laboratory, Aghia Sophia Children's Hospital, Athens, Greece

c First Department of Pediatrics, University of Athens, Aghia Sophia Children's Hospital, Athens, Greece

d First Department of Dermatology-Venereology, Medical School, Aristotle University, Thessaloniki, Greece (Lecturer Elect), Athens, Greece e Mycology Laboratory (K.A.70/3/6915), Department of Microbiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece

ARTICLE INFO

ABSTRACT

Article history: Two recent indigenous cases of tinea capitis in children due to pale isolates of Trichophyton violaceum

Received 14July 2012 are reported herein for the first time from South-East Europe (Greece). Pale isolates of Trichophyton

Received in revised form violaceum, reported in the past as Trichophyton glabrum, are thus far sporadically reported only from

7 Augus' 2012 African or Asian countries. The cases reported herein raise the awareness of its existence in the

Accepted 20 August 2012 community, assigning special importance to its accurate identification in the clinical laboratory.

© 2012 International Society for Human and Animal Mycology. Published by Elsevier B.V. All rights Keywords: reserved.

Trichophyton violaceum Trichophyton glabrum Tinea capitis South-East Europe

1. Introduction

Changes in dermatophyte epidemiology in Europe should be continuously monitored and reported, especially in view of the increased recent immigration from endemic areas for rare anthro-pophilic species. Anthropophilic dermatophytes such as Trichophyton soudanense, Trichophyton tonsurans, Trichophyton violaceum and Microsporum audouinii have recently sporadically emerged in several countries such as Belgium, Denmark, Germany, France, Spain, or UK [1-3].

Pale isolates of the anthrophilic dermatophyte Trichophyton violaceum, reported in the past under the synonym Trichophyton glabrum, constitute an unusual variant, thus far recently reported only from east/north African (Ethiopia, Morocco) or east/central Asian countries (Japan, Uzbekistan) [4-7].

Two recent cases of tinea capitis in children due to pale isolates of T. violaceum are reported here for the first time from Greece. The cases are also the first recently reported from Europe.

* Corresponding author at: Mycology Laboratory (K.A.70/3/6915), Department of Microbiology, Medical School, National and Kapodistrian University of Athens, Athens, Greece. Tel.: + 30 210 7462146; fax: + 30 210 7462147. E-mail address: marabatz@med.uoa.gr (M. Arabatzis).

The cases extend the known geographical distribution of this difficult to identify dermatophyte and raise the awareness of its existence in the community, assigning special importance to its accurate identification in the clinical laboratory.

2. Cases

2.1. Case 1

A four year-old boy presented to the outpatient clinics of our hospital (July 2009, day 0) with progressive scaly lesions on the crown area of the scalp of 3 weeks duration (day -21). The boy had an unremarkable medical history. He reportedly had recreational contact with horses and no other family member was similarly affected. He had been born to Albanian parents and periodically travelled to Albania. On examination, a mildly inflammatory, sole circular area with profuse accompanying scaling but no alopecia was observed on the crown area of the scalp. The hairs were easy to detach when pulled. Differential diagnosis at the time included T. capitis, seborrhoeic dermatitis and psoriasis.

Direct microscopic examination of lesional hair fragments and skin scale material with 20% KOH revealed dermatophyte spores

2211-7539/$-see front matter © 2012 International Society for Human and Animal Mycology. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.mmcr.2012.08.003

inside hairs in an endothrix invasion pattern and hyphae in the skin scales. Cultures of hair and skin clinical material in Dermatophyte Test Medium (DTM) and in standard Sabouraud dextrose agar supplemented with chloramphenicol (0.05 g L_1) and cyclo-heximide (0.5 g L_ 1) at 27 °C, yielded multiple, slowly growing, pale in colour colonies. Primary and secondary cultures in DTM turned the medium red, indicating the possible presence of a dermatophyte. The isolate subsequently formed plane, glabrous, whitish to lightly buff colonies in malt extra (MEA) and potato dextrose (PDA) agars (Fig. 1), with a maximum diameter of 0.5 cm in 7 day at 25 °C. Tested for thiamine dependence in Trichophyton agars 1 (no thiamine) and 4 (thiamine), it was strongly stimulated by the vitamin. Microscopically in MEA and PDA, it showed hyaline true hyphae with no other dermatophyte structures such as micro-, macro- or chlamydoconidia at both 25 °C and 37 °C. Bacterial contamination of the strain was ruled out by subcultur-ing in blood agar at 37 °C. The above morphology and culture characteristics of the isolate were consistent with the presence of either a dermatophyte such as T. verrucosum or pale T. violaceum or a non-sporulating contaminant with the ability to turn DTM red. The isolate was identified as T. violaceum by sequencing the internal transcribed spacer (ITS) region, using the general fungal primer pair 1TS1/1TS5 [8]. The amplified ITS sequence (GenBank accession no. JQ322677) showed 99% sequence homology to the GenBank archived T. violaceum (BLAST; /http://www.ncbi.nlm. nih.gov>). To definitely confirm the identification, the derived ITS sequence was aligned with the GenBank dermatophyte reference T. rubrum complex homologue sequences [9] and phylogenetic analysis was derived via the Neighbour-Joining method in the MEGA v.4 software [10,11]. The analysis placed the strain in the T. violaceum clade, with the highest homology seen with the T. glabrum Genbank reference sequence (accession no. AJ270796, deposited by Graser et al.) [9]. The strain was deposited to the University of Athens/ Hellenic Collection of Pathogenic Fungi (UOA/HCPF) as no. 11910.

Subsequently, the boy was dispatched with a prescription of oral fluconazole 100 mg o.d. (equivalent to 6 mg Kg_ 1) and ketoconazole 2% shampoo, both for six weeks. He was lost to the follow-up until December 2009 (day +161), when he was seen in the outpatient clinics with gradual worsening of his lesion. On examination, the previous scaly and inflammatory area on the crown area of the scalp had enlarged and hair loss had appeared. Additionally, broken hairs but no ''black dots'' were visible. Cultures grew again a pale T. violaceum and oral terbina-fine 125 mg o.d. in combination with ketoconazole 2% shampoo was administered for three weeks. Fungal cultures of material from the scalp of the patients' family members were negative.

Clinical and mycological cure was documented at the end of the treatment period (day +185) and after one year (day + 521).

As no break points have been set for dermatophytes, antifungal susceptibility was retrospectively tested to record potential resistance for the isolate to the major antifungals, following the Clinical Laboratory Standards Institute guidelines M38-A2 [12]. Hence, the recorded minimum inhibitory concentration (MIC) for the strain were: amphotericin B, 4 mg/ml; griseofulvin, 2 mg/ml; fluconazole, 8 mg/ml; itraconazole, 0.25 mg/ml; posaconazole, 0.5 mg/ml; voriconazole 0.25 mg/ml; amorolfine 16 mg/ml and terbinafine, 0.064 mg/ml.

2.2. Case 2

The second case was a 9 year-old girl presented (March 2010, day 0) with scaling of the palms and of the crown area of the scalp (with no alopecia or broken hairs), of three weeks duration (day -21). The patient had a history of haemoglobin H disease for which she had successfully undergone hematopoietic stem cell transplantation at 4.5 years of age. From the age of six she had venesections for lowering the total iron load. She was generally healthy and her psycho-physical development was normal. The patient belonged to the Romany ethnic group and had never travelled outside Greece. Similarly to our first case, she had no contact with animals and her family members were unaffected.

Direct microscopy of clinical material from both palms and scalp was positive for dermatophyte spores or hyphae and cultures from both sites grew a pale T. violaceum strain. The strain's amplified ITS sequence (GenBank accession no. JQ322678) showed 100% homology to the 1TS sequence of the first case T. violaceum isolate. The strain was deposited to the University of Athens/ Hellenic Collection of Pathogenic Fungi as UOA/HCPF no. 13250. The retrospectively recorded minimum inhibitory concentration (MIC) for the strain UOA/HCPF no. 13250 were: amphotericin B, 8; griseofulvin, 4 mg/ml; fluconazole, 32 mg/ml; itraconazole, 1 mg/ml; posaconazole, 1 mg/ml; voriconazole 0.5 mg/ml; amorolfine 16 mg/ml and terbinafine, 0.064 mg/ml.

The patient was treated with itraconazole 65 mg b.i.d. in oral liquid form (equivalent to 5 mg Kg_ 1 per day) until mycological cure was observed at five months of treatment (day +175). Clinical cure was observed after the first month of treatment for the palm lesions (day + 55) and after the second for the scalp ones (day + 85). Liver SGOT and SGPT enzyme levels remained within normal range during this period. The patient remained free of lesions 16 months after the end of therapy (day + 505).

Fig. 1. Whitish to lightly buff cultures of the pale T. violaceum isolate UOA/HCPF 13250 at 25 °C in (a) malt extract agar and (b) potato dextrose agar, after 2 weeks.

3. Discussion

The cases reported herein document the existence of the pale T. violaceum variant for the first time in Greece. Pale isolates have never been reported before from Greece (or, indeed, from any other European country in the last two decades), although T. violaceum was common in the past and it has been recently reported sporadically from Greeks, Roma people or Albanian and African immigrants [13]. It is unclear if the Greek isolates represent a recent introduction, a recent local development or they were simply unrecognised until now. The fact that the patients described here belong to the known endemic ethnic groups for T. violaceum in Greece, additionally points towards the autochthonous origin of the Greek pale isolates. Certainly, it seems that the pale variant is not related to a specific geographical area as, until now, it has been reported from the main T. violaceum endemic areas in North/East Africa and central Asia.

1n the clinical laboratory, the unusual, white or yellowish, pale T. violaceum isolates are especially important as their lack of the normal violaceous colour characteristic of T. violaceum is not easily pointing to the correct identification. They could be easily discarded as contaminants, especially in the hectic everyday conditions of a clinical laboratory. Besides the obvious clinical and epidemiological reasons for not missing the correct identification in the clinical laboratory, an extra reason is to avoid recurrent family or institutional T. violaceum outbreaks [14]. In this context, 1TS sequencing can confirm the correct identification of the isolates.

Clinically, it seems that both classic T. violaceum and its pale variant cause indistinguishable infections. 1nterestingly, the study isolates presented high M1C values to fluconazole, an attribute possibly related to the failure of the fluconazole treatment in our first case. The clinical significance of high dermatophyte M1Cs is not entirely clear as correlation studies between antifungal susceptibilities and clinical outcome has not been conclusively performed in dermatophytoses.

1n conclusion, the present cases represent the first instance of infections caused by the pale variant of T. violaceum in Greece. The cases extend the known geographical distribution of the variant and raises the awareness for this difficult to identify dermatophyte.

Conflict of interest

There are none.

Acknowledgements

The 1nternational Society for Human and Animal Mycology (1SHAM) for encouraging dermatophyte research. Afroditi Milioni and Stavroula Kritikou for technical support. Galderma France, for providing the amorolfine compound. SARG K.A.70/3/6915, National and Kapodistrian University of Athens, Greece, for funding the investigative aspects of this work.

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