Isolation of Opportunistic Fungi from Dermatophytic Samples
N. El Khizzi,
A.M. Al Rasheed,
H.H. Al Ajlan
This study was carried out for three years during November 2004 to November 2007. The aim of this study was to isolate the fungal flora from skin and nails samples which are usually present in the household environment. This study excludes the recoganized dermotophytes like Trichophyton, Microsporum and Epidermatophyton. Candida species were not included. Out of a total five hundred samples of skin and nail received during this study period, yielded 19 fungal species from skin and nails belonging to fungal genera Alternaria, Aspergillus, Bipolaris, Cladosporium, Exophiala, Fusarium, Graphium, Malassezia, Prototheca, Rhizopus, Rhodotorula, Trichosporon and Ulocladium. Aspergillus was the leading genus represented by six species. Previously well known methodology for isolation of dermatophytes was followed here except that Sabouraud dextrose agar with three antibiotics viz., Ciprofloxacin, vancomycin and gentamycin sulphate (0.05 g L-1 each) was used here to suppressed the growth of bacteria hence enhanced the isolation of molds. Only KOH positive cases are included here. Although, these fungi may not cause infections as true dermatophytes but these fungi should also be considered for tropical antifungal treatment to avoid further spreading of these fungi as a precaution.
Received: July 27, 2010;
Accepted: October 01, 2010;
Published: March 28, 2011
Molds or non-dermatophytic filamentous fungi (generally belong to Hyphomycetes
or Fungi Imperfectii) are quite often suspected as pathogen by public than by
the medical care community but during past few decades these molds getting increasing
attention by medical mycologist due to increased in number of skin and nail
infections in humans and animals by these so called environmental fungi or laboratory
contaminant (Evans and Richardson, 1989) because these
fungi produced lesions that are similar to those caused by dermatophytes (Kwon-Chung
and Bennet, 1992). Like dermatophytes, these fungi also have the capability
of utilizing keratin in-vitro and produce proteolytic enzymes
including keratinase. The outbreak of cutaneous infection is triggered by increasing
in number of immunodeficient population (Vennewald and Wlollina,
The problem of increased fungal infection could also be attributed to modern
living conditions with closed house environment, use of wall to wall carpets
which provide a very suitable environment to these molds for growth. All these
molds which causes cutaneous, sub-cutaneous, allergic or even systemic mycoses
are common inhabitant of household environment (Bokhary
and Parvez, 1995; Bakhali and Parvez, 1999).
Alternaria species have been reported to cause cutaneous infections
in both immunocompetent and immunocompromised patients (Sood
et al., 2007; Robertshaw and Higgins, 2005;
Romano et al., 2005; Mayser
et al., 2004). Aspergillus species are well recognized as
a causal organisms of pulmonary aspergillosis in immunocompromised patients
but cutaneus aspergillosis occurs less frequent and thus remain poorly described
as primary or secondary infections although there are some reports of Aspergillus
causing cutaneous infections in immunocompromised hosts (Romano
and Miracco, 2003) but numerous reports have described primary or secondary
cutaneous aspergillosis in an array of non-HIV infected immunodeficient patients,
including burn victims, neonatus, individual with the cancer, bone marrow and
solid organ transplant patients (Riddel et al., 2004;
Talbot et al., 2002). Although, in healthy hosts
cutaneous aspergillosis can also develop in surgical wounds by traumatic inoculation
or by exposure to high spore counts (Romano and Miracco,
2003). Initial lesions of cutaneous aspergillosis appears as macules, nodules,
plaques or popules. In the case of neonatus, lesions occurs with prulent discharge
or pustules (Fleming et al., 2002; Restrepo
et al., 2004). In the case of catheter, infection begins from arm
board or occlusive tape and resulting in hemorrhage bulla type infection. Infection
in the case of intravenous catheter puncture, typically begins with erythma
and induration at the skin puncture site and progress to necrosis that extends
radially from initial focus. High fever, a change in the character of the wound
surface, swelling induration and tenderness are the symptoms of primary cutaneous
aspergillosis arising in a wound. Erythematous macules or papules that evolved
to hemorrhage bullae or ulceration nodules are the symptoms for secondary cutaneous
aspergillosis, secondary lesion can resemble ecthyma gangrenosum, traditionally
caused by Pseudomonas aeruginosa (Warnok, 2006; Groll
and Walsh, 2001).
Cutaneous infections caused by Bipolaris species and Cladosporium
species (Cladophialophora) are mainly restricted to wound infections
in the case of immunocompromised patients but could also cause infections in
immunocompetant hosts (Fleming et al., 2002).
Exophiala could cause cutaneous or sub-cutaneous infections while Graphium
(Pseudoallescheria boydii or Scedesporium apiospermum) could also
cause deep mycoses (Riddel et al., 2004; Talbot
et al., 2002). Prototheca although not a fungus but an alga
which lacks chlorophyll is well known to cause cutaneous mycoses but respond
well to antifungals (Kantrow and Boyd, 2003; Zaitz
et al., 2006). Rhizopus and Ulocladium could cause
skin infections specially in immunocompromised patients and children (Oh
and Notrica, 2002).
Trichosporan is a yeast fungus which is well known to cause infections
in humans and animals, starting from hairs to skin to deep mycoses like endocarditis
and meningitis (Kwon-Chung and Bennet, 1992; Bassetti
et al., 2004; Pini et al., 2005;
Ramos et al., 2004). Fusarium species now well recognized
as causal organism of skin and nails (Kwon-Chung and Bennett,
The aim of this study to focus on isolation of non-dermatophytic (molds) flora from dermatophytic samples (skin and nails), which are usually inhabitant of household environment, laboratory contaminants or so called environmental fungi but these fungi are also the causal organisms of superficial mycoses to deep mycoses and could not be ignored just as an environmental fungi.
MATERIALS AND METHODS
Samples of skin scrapings and nail clippings were collected from Dermatology and Out-patient clinics. A total number of 500 samples (400 skin scraping and 100 nails scraping) were collected during the study period between Nov. 2004 to Nov. 2007.
Isolation of fungi was carried out according to Standard Operating Procedure
for Medical Mycology (Parvez, 2010; Evans
and Richardson, 1989).
Medium for isolation: Sabouraud Dextrose Agar and Dermatophyte Medium (Oxoid Ltd. London) were use for isolation of these fungi. Three antibiotics, ciprofloxacin, vancomycin and gentamycin sulphate (0.05 g L-1 each) were added to Sabouraud dextrose agar. Potato dextrose agar (Oxoid Ltd. London) was also used but without the addition of antibiotics.
Isolation of fungi from nails: Samples of nails clippings was at first dipped into sterile water into a sterile petri dish for 10-15 min for softening of nail. Then small pieces were cut by using sterile scalpel and 5-10 pieces were put on the surface of medium plates. These plates were then incubated at 30°C for further study.
Isolation of fungi from scraping: Scarping samples were directly cultured by putting scraping pieces over the agar plates. These plates then incubated at 30°C for further study.
Microscopic examination of samples: Microscopic examination was done by using KOH (20% KOH + 15% glycerol) mount. Parker blue-black ink was also added when required.
Identification of fungi: Identification of isolated fungi was carried
out according to the following literature: (Evans and Richardson,
1989; Ellis, 1971, 1976; Raper
and Fennell, 1965; Zycha et al., 1969; Howard,
1983; Nelson et al., 1983), while the identification
of Rhodotorula muciliginosa and Prototheca wickerhamii were done
by API 20C system (BioMerieux Vitek, Inc., France).
Isolated fungi were sub-cultured and maintained on Sabouraud Dextrose Agar slants in tissue culture bottles.
RESULTS AND DISCUSSION
Non dermatophytic fungi isolated form dermatophytic samples during the period between November 2004 and November 2007 are summarized in Table 1. True dermatophytes like Trichophyton, Microsporum, Epidermatophyton and also Candida species which were isolated during the study period but not included here. Three yeast fungi Malassezia furfur, Rhodatorula muciliginosa and Trichosporon beigellii, are included here. Prototheca wickerhamii which is not a fungus but an alga devoid of chlorophyll is also included in the Table 1. Out of 400 skin scraping samples 30 samples (7.5%) were found positive and out of 100 nails samples, 9 samples (9%) were found positive. A total number of 19 fungal species were isolated from scraping and nail samples. Seventeen fungal species were isolated from scraping while only 5 species could be isolated from nail samples. Aspergillus was the predominant genus and represented by 5 species followed by Alternaria (3 species). All other fungi were represented by one species each.
Alternaria alternata, Fusarium solani and Exophiala jeanselmei were the only fungi which were isolated from both type of samples. Aspergillus fumigatus and A. niger were represented by highest number of isolates (4 each) followed by A. flavus (3 isolates), Alternaria alternata, Fusarium solani, Prototheca wickerhamii and Ulocladium chartarum (2 isolates each). All other species was represented by one isolate each in the case of skin scraping. While in the case of nails, Fusarium solani represented by highest no. of isolates (4 isolates) followed by Alternaria (2 isolates), Cladosporium, Exophiala and Rhodotorula represented by one isolates each.
Molds that isolated from different type of skin samples are summarized in Table 2, highest number of isolates were yielded from scalp (11 isolates) followed by underarm, neck, chest region (7 isolates), leg (5 isolates) and thigh (4 isolates). Scraping samples from palm and face yielded two isolates each.
|| Isolation of fungi from skin and nail samples (n = 400 from
skin and n = 100 for nail)
||Isolation of molds from different type of skin samples
Table 3 gave the details of positive cases according to age
group and male and female ratio of positive cases. The highest number of positive
cases were found among the age group of 50 years and older persons in both type
|| Number of positive cases according to age group and also
male and female ratio
In the case of skin scraping, the age group of 11-30 years yielded lowest number
of isolates while age group 31-50 was the second highest in the number of isolates.
In the case of female, the ratio of the number of positive cases were almost
double as compared to male.
Dermatophytes like Trichophyton, Microsporum and Epidermatophyton
which are traditionally only accepted causal organism of superficial cutaneous
mycoses among medical community but the mold flora which has the ability to
utilize keratin and therefore could cause superficial cutaneous infection have
usually been ignored. The mold flora isolated here like Alternaria, Aspergillus,
Bipolaris, Cladosporium, Exophiala, Fusarium, Rhizopus
and Ulocladium are usually common inhabitant of household environment
(Niedoszytko et al., 2007; Bokhary
and Parvez, 1995; Bakhali and Parvez, 1999). But
the serious problem is that, unlike true dermatophytes, the pathogenicity of
these mold flora are not restricted to skin or nail infections (Niedoszytko
et al., 2007). Alternaria species could cause sub-cutaneous
phaeohyphomycosis both in immunodeficient and immunocompetent patients (Sood
et al., 2007; Robertshaw and Higgins, 2005).
Aspergillus species could pose more problems as not only causing cutaneous
infection but also sub-cutaneous nodules, posing problem of infections in neonate,
cancer patients, patients on catheter, transplant patients and a leading agent
of fungal sinusitis (Romano and Miracco, 2003; Warnok,
2006; Restrepo et al., 2004). The fungal
species reported here as causative agent of cutaneous mycoses but they are also
reported to cause serious and fatal deep mycoses (Warnok,
2006; Groll and Walsh, 2001).
The isolation of mold from cutaneous sample should not be overlooked as they
cause almost similar lesion that are produced by true dermatophytes (Kwon-Chung
and Bennet, 1992). Secondly, they could infect other parts of the body especially
in the case of any sort of immunodeficiency or wound or trauma.
Isolation of such a number of opportunistic mold from dermatophytic samples which could cause fatal deep mycoses and difficult to cure. Therefore, it is suggested that along with dermatophytic samples, a brief clinical history of patient should also be noted like patient having some sort of immunodeficiency symptoms, diabetes, trauma, wound, use of long term antibiotics, any kind of transplant etc. These will help in co-relating fungal isolation and infection.
Although, these fungi may not cause infections as true dermatophytes but these fungi could grow initially on dead skin cells on scalp and other parts of the body which could cause allergenic type itching or rashes. Hence resulting in scraping of the site by the person which may leads to a deeper infection or spreading of these fungi to other parts of the body. Therefore, we recommend that if these fungi isolated from dermatophytic samples, these fungi should also be considered for tropical antifungal treatment to avoid further spreading of these fungi as a precaution.
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