Real-time PCR: A rapid and sensitive method for diagnosis of dermatophyte induced onychomycosis, a comparative study

Peer review under responsibility of Alexandria University Faculty of Medicine.

Available online 29 August 2015

Figures & data

Figure 2 Results of nested PCR of clinical specimens from patients with onychomycosis. Lane M 100-bp DNA ladder (molecular mass markers); lane 1,2,3, nested-PCR-positive specimens (288 bp); lane 4,5 nested-PCR-negative specimens.

Figure 3 Melting curve analysis of PCR product of pan dermatophyte gene (melts at 81.9 °C).

Figure 1 (a) Slide culture of T. mentagrophytes. (b) Culture of T. mentagrophytes on SDA (microscopic). (c) Slide culture of T. verrucosum. (d) Culture of T. verrucosum on SDA. (e) Slide culture of T. rubrum. (f) Culture of T. rubrum on SDA.

Figure 4 Receiver operating characteristic (ROC) curve for direct KOH microscopy, nested PCR and RT-PCR against the gold standard test (fungal culture) for the diagnosis of onychomycosis (N = 80). KOH microscopy: n = 66 positive and n = 14 negative; Area Under the Curve (AUC) = 0.48, P < 0.001 Nested PCR: n = 52 positive and n = 28 negative; AUC = 0.57, P < 0.001. RT-PCR: n = 58 positive and n = 22 negative; AUC = 0.67, P < 0.001.

Table 1 Socio-demographic characteristics of the study population.

Characteristics		N = 80
Age	Mean ± SD; (range)	43.52 ± 15.04; (22–77)
Sex	Male/female; (% male)	50/30; (62.50)
Residence	Urban/rural; (% urban)	42/38; (52.50)

Table 5 Analysis for results of direct KOH microscopy, nested PCR and RT-PCR against the reference test (fungal culture).

	Direct KOH microscopy	Nested PCR	RT-PCR
Sensitivity	80.00%	73.3%	93.30%
Specificity	16.00%	40.00%	40.00%
Positive predictive value	36.40%	42.30%	48.30%
Negative predictive value	57.10%	71.40%	90.90%
Area under the curve (AUC)	0.48	0.58	0.67

Table 2 Patients with suspected cases of onychomycosis.

Disease		N = 80
Trauma	Yes/no; (% yes)	10/70; (12.50)
DM	Yes/no; (% yes)	22/58; (27.50)
Anemia	Yes/no; (% yes)	28/52; (35.00)
PVD	Yes/no; (% yes)	16/64; (20.00)
Associated nail disease	No disease; (%)	42; (52.50)
	Paronychia; (%)	24; (30.00)
	Nail pitting; (%)	14; (17.50)
Treatment	Yes/no; (% yes)	30/50; (37.50)

DM: diabetes mellitus, PVD: peripheral vascular disease.

Table 3 Clinical features of patients with suspected cases of onychomycosis.

Fingers (46) = 57.5%	Toes (34) = 42.5%	Total (80) = 100%
Distal = 8	Distal = 4	12; (15.00)
Distolateral = 8	Distolateral = 10	18; (22.50)
Proximal = 7	Proximal = 11	18; (22.50)
Proximal & distolateral = 1	Proximal & distolateral = 1	2; (2.50)
Total dystrophic = 22	Total dystrophic = 8	30; (37.50)
Clinical features		N = 80
Nail shape	Distal; (%)	12; (15.00)
	Distolateral; (%)	18; (22.50)
	Proximal; (%)	18; (22.50)
	Proximal & distolateral; (%)	2; (2.50)
	Total dystrophic; (%)	30; (37.50)
Number of nails affected	One or more fingers; (%)	46; (57.50)
	One or more toes; (%)	34; (42.50)

Table 4 Comparison of different methods used for diagnosis of onychomycosis among the study group.

Test		N = 80
Direct KOH microscopy	Yes/no; (% yes)	66/14; (82.50)
Fungal culture	Negative; (%)	42; (52.50)
	Non-dermatophyte; (%)	8; (10.00)
	Dermatophyte; (%)	30; (37.50)
Nested PCR	Yes/no; (% yes)	52/28; (65.00)
RT-PCR	Yes/no; (% yes)	58/22; (72.50)