Onychomycosis in diabetic patients in Fako Division of Cameroon: prevalence, causative agents, associated factors and antifungal sensitivity patterns

Study design and settings

We conducted a cross-sectional descriptive and analytical hospital-based study from May to August 2014 at the Diabetic Clinics of the Regional hospital of Buea (RHB) and the Regional hospital of Limbe (RHL) found in Buea and Limbe respectively. Buea and Limbe constitute the two major cities of Fako division. These institutions serve as secondary referral hospitals for other hospitals and health institutions- public, faith based and private-in the region and beyond. They also serves as teaching hospitals to several university related health institutions within the region and beyond. These hospitals receive diabetic patients with both Types I and II diabetes of all ages every day of the week but specific days of the week are set as clinic days with specialist consultations and patient education being carried out.

Participants and sampling

The study participants constituted attendants of the diabetic clinics of the RHB and RHL. To be eligible, participants had to be diabetic of age 21 years or more. All participants were examined by a dermatologist for signs of onychomycosis. Participants with gestational diabetes, two foot amputation and those who had been on antifungal treatment during the preceding four weeks were excluded. Convenient and consecutive sampling was used to select participants once they agreed to participate.

Data collection, variables and measurement

A structured questionnaire (see Additional file 1: Questionnaire) was used to collect data from each participant. This questionnaire was used to assess demographic associated factors (age, gender, diabetic clinic attended), behavioural factors (occupation, level of education, hobbies, foot wear and nail cutting habits), and clinical factors (duration of diabetes, presence of foot ulcer, other co-morbid disease, amputation and structural deformity, family history of onychomycosis, trauma, clinical manifestation of onychomycosis). Data collection was carried out by the investigator (a trained microbiologist) and this minimised potential bias.

Specimen collection

Nail specimens were collected from participants with suspected nail lesions. Severity of nail damage was classified as mild (<25% involvement or <4 nails involved), moderate (26–74% involvement or 5–8 nails involved) and severe (≥75% involvement or ≥9 nails involved). Where one or more of the toenails appeared clinically abnormal, then the two toenails that are clinically most likely to have onychomycosis were sampled. To obtain nail specimens from dystrophic nails, cleansing of the nail area with 90% alcohol was carried out to remove contaminants such as bacteria, any discoloured, dystrophic or brittle parts of the nail and affected nail was cut superficially as far back as possible using a No. 22 surgical blade including any crumbly material. In case of superficial involvement (as in white superficial onychomycosis) nail scrapings were collected and the free edge of the nail plate was also used for investigation. A separate sample was collected if there appeared to be fingernail involvement as well as dermatophyte infection on another part of the body of any patient. Nail specimens were scraped directly onto black carbon papers, which made it easier to see how much material had been collected, carefully folded and placed inside white envelopes and sealed providing ideal conditions for transportation to the laboratory. The envelopes containing samples were carefully labeled with patient code, age, sex, date, place and time of collection. Analysis was carried out within 24 h of specimen collection.

Sample processing

Five to six small fragments (1–2 mm) or scrape material from nails was placed on a glass slide containing a drop of 10% Potassium Hydroxide and glycerol (10% KOH + glycerol), covered with a cover-slip, gently heated by passing through a flame 2 or 3 times (not allowing solution to boil) and put aside to digest for at least 30 min at room temperature, then examined with ×10 and ×40 objectives for the presence of septate fungal hyphae. Negative specimens were kept and re-examined the next day to avoid reporting false-negative results because of delayed clearance and as need arose some slides were kept till culture results became available. The glycerol prevented the preparation from drying off and made the slides still readable after several days.

Approximately 20 representative small nail fragments from each participant irrespective of their microscopy results were scattered on Sabouraud Dextrose Agar (SDA) plates supplemented with chloramphenicol. The plates were sealed with proprietary tape to prevent air-borne contamination in the laboratory and incubated at room temperature (27 °C) for 7–14 days while visually monitoring of plate was done on a daily basis for fast growing fungi. Pure isolates were obtained by sub-culturing on new SDA plates and colonies growing out of the inoculation area were regarded as contaminants.

Identification of fungus grown on culture plates was based on the colony morphology, reverse pigmentation on SDA and the observation of sporulating fungus with Lactophenol cotton blue stain. Identification to species level was done using the cellotape flag method and the slide culture procedure as described elsewhere [8, 9].

Susceptibility testing was carried out using the Kirby–Bauer disk diffusion method endorsed by the Clinical Laboratory Standards Institute (CLSI) [10] on dried SDA plates. Briefly, a tiny portion of the fungal colony was emulsified in 4 ml of sterile physiological saline, mixed and the turbidity of the suspension compared with 0.5 McFarland standard. A sterile swab was used to streak the suspension on SDA plates in three directions, rotating the plate approximately 60° to ensure even distribution. The isolates were tested against five antifungal agents including Amphotericin B (20 mcg, Biogram™), Ketoconazole (10 mcg Biogram™), Miconazole (50 mcg, Bioanalyse^®), Griseofulvin (10 mcg, Bioanalyse^®) and Itraconazole (50 mcg Bioanalyse^®). After 24–48 h incubation at 27 °C, the diameter of the zones of inhibition were measured and interpreted according to the CLSI criteria [10].

Data management and analysis

Questionnaire data was systematically checked for errors during data entry. All data obtained from the questionnaires were keyed into an Excel spread sheet (Microsoft Excel 2007 software) and cross checked for errors. It was then analyzed using the statistical package for social sciences (SPSS) version 20. To assess demographic, clinical and behavioral factors associated with onychomycosis, the logistic regression analysis was used to identify associated factors such as age, sex, duration and type of diabetes, presence of co-morbid diseases and family history of onychomycosis, occupation, footwear, habits of nail cutting. Odds ratios were used to report the association between onychomycosis and these associated factors. All statistical tests used were two-tailed, and values of P < 0.05 were considered statistically significant.

The strobe guideline for reporting observational studies was used in writing this manuscript [11].