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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 8  |  Issue : 1  |  Page : 47-52

Arbitrarily primed polymerase chain reaction-based identification of dystrophic onychomycosis


1 Centre for Interdisciplinary Biomedical Research, Adesh University, Bathinda, Punjab, India
2 Department of Dermatology, Adesh Institute of Medical Sciences and Research, Bathinda, Punjab, India

Date of Submission09-Mar-2020
Date of Decision28-Apr-2020
Date of Acceptance01-May-2020
Date of Web Publication20-Jun-2020

Correspondence Address:
Dr. Prabhjot Kaur Gill
Centre for Interdisciplinary Biomedical Research, Adesh University, Bathinda - 151 101, Punjab
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/amhs.amhs_31_20

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  Abstract 


Background and Objective: Onychomycosis is a fungal infection of the nails; when caused by dermatophytes, it is called as Tinea unguium. The aim was to explicate the utility of arbitrarily primed polymerase chain reaction (AP-PCR) to augment the early and accurate clinical diagnosis of dystrophic onychomycosis. Materials and Methods: The collected nail samples were divided into three portions. The first portion was explored for direct microscopic examination, the second portion was used for culturing of the nail samples on sabouraud dextrose agar media, and the third portion was used for DNA extraction followed by AP-PCR identification of dermatophytes. Results: All 48 samples with dystrophic onychomycosis were diagnosed by three methods, namely, 20% Potassium hydroxide microscopy, culture growth, and AP-PCR. With all the methods, Trichophyton rubrum was found as major causative agents for dermatophyte nail infection (60%), followed by Trichophyton mentagrophytes (23% cases). AP-PCR is a convenient method to achieve a higher dermatophyte identification rate with lesser time and shows complete concordance with conventional culture for two Trichophyton species. Conclusion: AP-PCR is a rapid, specific, and sensitive procedure for the diagnosis of nail fungal infection.

Keywords: Arbitrarily primed polymerase chain reaction, dermatophytes, dystrophy, fungal infection, onychomycosis


How to cite this article:
Kaur S, Gupta A, Gill PK. Arbitrarily primed polymerase chain reaction-based identification of dystrophic onychomycosis. Arch Med Health Sci 2020;8:47-52

How to cite this URL:
Kaur S, Gupta A, Gill PK. Arbitrarily primed polymerase chain reaction-based identification of dystrophic onychomycosis. Arch Med Health Sci [serial online] 2020 [cited 2020 Oct 30];8:47-52. Available from: https://www.amhsjournal.org/text.asp?2020/8/1/47/287356




  Introduction Top


Onychomycosis refers to fungal infection of the nail unit; when caused by dermatophytes, it is known as Tinea unguium. Onychomycosis is a fungal infection of nail, which is caused by T. unguium, specifically a dermatophytic invasion of the nail plate. The high prevalence and incidence of dermatophyte make them more important public health problem, while infections are not life-threatening.[1] Therefore, nail infection is a cosmetic problem with serious physical, psychological morbidity that can lead to self-consciousness and impairment of daily functioning.[2] Laboratory investigation is necessary to accurately differentiate between fungal infections and other skin diseases before starting treatment. Onychomycosis fungal groups are dermatophyte, which includes Trichophyton rubrum and Trichophyton mentagrophytes.[3] Nail discoloration and other onychodystrophy should be differentiated from onychomycosis, which is classified as distal lateral subungual onychomycosis, white superficial onychomycosis, proximal subungual onychomycosis, candidal onychomycosis (CO), and total dystrophic onychomycosis.[4] CO lacks gross distortion and accumulated detritus and mainly affects fingernails. Dermatophytoses of the fingers and toe nails are specifically difficult to suppress with drug treatment as compared to other sites of the body, because of the hard, protective nail plate, steady growth of the nail, and relatively less penetration of the chemotherapeutic agents.[5] About 1.5%–6% of cases in onychomycosis, mostly seen in toenails of elderly individuals with a history of trauma and possibly due to poor peripheral circulation. However, the prevalence of onychomycosis is increasing because of many reasons such as increasing population which increases chronic health problems, trauma, age, gender, and increasing use of swimming pools.[6] The treatments of onychomycosis are generally very expensive and long with potential side effects. Therefore, proper diagnosis of disease is very important. The laboratory diagnosis of clinical specimen for onychomycosis is done on the basis of direct microscopic and culturing on sabouraud dextrose agar (SDA) media followed by morphological identification of dermatophytes. The microscopic examination is a rapid method, but these diagnostic methods have lack of specificity and sensitivity with false negative results up to 15% and genus or species identification is not possible.[7] The culture morphological and microscopic examination of colonies is specific for species identification, but it is time-consuming due to slow growth, which takes 10–15 days. Consequently, crucial advancements are required for the diagnosis of dermatophytes. In the last few years, many polymerase chain reaction (PCR) assays have been developed for direct detection of fungi in the nail samples using specific primers which includes nontranscribed spacer regions (NTS), chitin synthase gene (CHS), metalloprotease gene, tubulin gene, real-time PCR, and nested PCR with different sensitivity and specificity.[7],[8],[9] Arbitrarily primed PCR (AP-PCR) is a rapid method for the diagnosis of specific fungal species and more acceptable for its accuracy and speed.[10] Therefore, in the present study, AP-PCR assays have been used for the detection of fungi in 48 nail samples using four random primers, which identified various dermatophyte species rapidly and economically. The present research also compared the result of AP-PCR with microscopy and culture method-based identification.


  Materials and Methods Top


The present research was carried out in the Centre for Interdisciplinary Biomedical Research, Adesh University, Bathinda, in the period of June 2017–December 2018. The sample was collected from the Outpatient Department of Dermatology, Adesh Hospital, Bathinda. Written informed consent was obtained from the patients according to their language before participation. Only 48 patients were reported in the present investigation with clinical symptoms and signs of dystrophic onychomycosis such as discoloration, thickening, dryness of nail, and nail separated from the nail bud. The full medical history was also taken which further includes gender, age, occupation, and any allergy. History included the duration of the onychomycosis, past history included history of any other microbial infection, diabetes mellitus, any other disease or specific habit. General examination of infected site like diabetic feet, predisposing factor, Tinea pedis were done.

Collection of nail samples

Clinical presentation of end-stage nail disease was identified by the clinicians and reported as dystrophic onychomycosis and therefore included in the study. Prior to sampling, the infected nails were cleaned with a 70% ethanol solution. Approximately 25–30 mg digital nail scrapings were taken with a sterile surgical blade or presterilized nail clippers and were collected in the sterilized Eppendorf tubes.

Methods for investigation

The collected specimen was divided into three portions. The first portion was explored for direct microscopic examination, the second portion was used for culturing of the nail samples, and the third portion was used for DNA extraction directly from the nail samples.

Direct microscopic examination

The sample was used for direct microscopic examination using 20% KOH and examined under ×40 and ×100 (oil immersion) objectives (Olympus CX31RTSF). The nail sample was placed on the clean slide after teasing with forceps added few drops of 20% KOH, and after placing the coverslip, the sample was pressed properly and examined under low- and high-power objectives. The detailed examinations were done for fungal hyphae, spores, and budding cells.

Culturing of nail samples

SDA media with gentamycin (20 mg/ml) was used for the growth of dermatophytes from nail and incubated at 37°C for 7–15 days. After the growth of the fungi, the cultures were identified through macroscopically and microscopically. Macroscopically, it was identified on the basis of colony morphology, size, shape, pigment production, and consistency both front and reverse sides of Petri plates. The microscopic examination using done lactophenol cotton blue tease mount and fungal cultures were identified on the basis of hyphae, fruiting bodies, and spores.

Molecular detection of dermatophytes

Fungal DNA was extracted from the nail samples by crushing the nail sample in mortar pestle followed by incubation of nail fragment with the addition of digestion buffer, proteinase K, and 10% SDS, for 2–3 h in water bath at 65°C in 15 ml falcons with intermittent shaking. After incubation, the samples were vortex vigorously followed by the addition of CTAB/NaCl and incubated at 56°C in water bath for about 1 h. After that, added equal amount of chloroform and isoamyl alcohol in 24:1 followed by centrifugation at 7000 rpm for 10 min at 25°C and removed the supernatant in another tubes and added phenol–chloroform–isoamyl alcohol (PCI-25:24:1). The samples were centrifuged again for 10 min and removed the supernatant in fresh tube followed by the addition of chilled isopropanol and incubated for 30 min at −20°C. The tubes were centrifuged at 7000 rpm for 10 min at 4°C, the supernatant were discarded, and the pellets were washed with 70% ethanol twice. The DNA pellets were air-dried and suspended in 50 μl of sterilized MilliQ water.

Arbitrarily primed polymerase chain reaction amplification

Arbitrarily primed PCR was conducted for all isolates with each primer separately. Amplification reaction was performed with total reaction mixture of 20 μl in which DNA 3 μl, master mix 10 μl, and 2 μl random primers, i.e., OPAA11 (5'-ACCCGACCTG-3'), OPU15 (5'-ACGGGCCAGT-3'), OPAA17 (5'-GAGCCCG ACT-3'), and OPD18 (5'-GAGAGCCAAC-3') and 5 μl MilliQ water. The PCR cycling was 32 cycles of 94°C for 30s, 36°C for 45s, and 72°C for 90s. After PCR amplification, the samples were electrophoresis in 1.2% agarose gel in 1X TAE buffer and stained by ethidium bromide and visualized in the Biorad gel doc EZ imager. The different molecular weight bands appeared on the gel were interpreted with previous reported data by Zarrin et al.[10] The DNA isolated from the nail clippings and from the pure fungal cultures was amplified with the random primers and banding patterns were compared.

Ethics approval

The study was carried out after the approval of the Institutional Research Committee and Ethical Committee of the Adesh University, Bathinda (AU/ECFM/38/2018). Written consent was taken from the participants.

Statistical analysis

All the necessary analyses were done using MS-Excel 2007. The means of patient demographics were interpreted among subgroups. Chi-square test was used for categorical variables. The differences between experimental groups were analyzed using two-tailed unpaired t-test. Two-sided P≤ 0.5 was considered statistically significant.


  Results Top


Only 48 patients with dystrophic fungal nail infection were reported in a 1½ year in the outpatient department at a tertiary care hospital. Out of 30 samples, 12 toenail and 18 fingernail samples were collected from female patients. Whereas, only 18 samples from males with 11 toenail and 7 fingernail samples were collected [Table 1]. All the 48 nail samples proceeded for the dermatophytes identification by three methods; with 20% KOH direct microscopy-35 cases showed positive results, while the culturing of nails on SDA media were positive in 42 cases, whereas with AP-PCR 44 cases were positive and 4 cases were negative [Table 2]. The AP-PCR showed the significantly (P 0.00) highest sensitivity (90.47%) as compared to KOH (70.45%) and culture method (87.50%). Whereas, AP-PCR specificity (89.65%) comparable to KOH (92%) and culture method (83%) in the dystrophic nail infection [Table 2]. With all the methods, T. rubrum was found as a major causative agent for dermatophyte nail infection [Figure 1] and [Figure 2] and represented 60% of cases in the present region, followed by T. mentagrophytes in 23% of cases [Figure 2] and [Figure 3]. On the contrary, 4% nondermatophyte infection was detected and major fungal was found to be Aspergillus flavus on the SDS media, whereas PCR showed negative results for nondermatophytes infections [Table 2]. Therefore, AP-PCR allowed more rapid and specific identification ≤2 days in comparison with growth of fungi on SDA media ≤28 days.
Table 1: Dystrophic onychomycosis infestation on finger and toe nails

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Table 2: Association of dystrophic onychomycosis with sociodemographic characteristics

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Figure 1: (a) Patient with dystrophic onychomycosis; (b) Trichophyton rubrum on the sabouraud dextrose agar media; (c) cherry red pigment on reverse side of plate; (d) microscopic appearance of Trichophyton rubrum spores

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Figure 2: Identification of onychomycosis with arbitrarily primed polymerase chain reaction and polymerase chain reaction products of two dermatophytes are shown. Lane M, Molecular weight marker; Lane 1-4, Primers OPAA11 (350, 750 bp), OPU15 (250, 400, 450, 600 bp) OPAA17 (300, 400, 500, 650, 900 bp) OPD18 (300, 400, 750, 1000 bp) respectively for Trichophyton mentagrophytes; Lane 5-8, OPAA11 (350, 450, 600 bp), OPU15 (250, 350, 550, 750, 800 bp), OPAA17 (250, 450, 700, 800, 1200 bp), OPD18 (350, 400, 700, 800, 900, 1300 bp), respectively, for Trichophyton rubrum

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Figure 3: (a) Patient with dystrophic Onychomycosis; (b) Trichophyton mentagrophytes on the sabouraud dextrose agar media; (c) microscopic appearance of Trichophyton mentagrophytes spores

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Arbitrarily primed polymerase chain reaction-based Identification

The PCR-based identification of 48 isolates was done by comparing the molecular size of the DNA bands with four random primers. In T. mentagrophytes, primer OPAA11 showed only two bands such as 350 and 750 bp, while in T. rubrum, primer revealed three bands such as 350, 450, and 600 bp. In primer OPU15, T. mentagrophytes exhibited four bands such as 250, 400, 450, and 600 bp, whereas in T. rubrum, OPU15 showed five bands such as 250, 350, 550, 750, and 800 bp. With the use of primer OPAA17, T. mentagrophytes exhibited 300, 400, 500, 650, and 900 bp, while in T. rubrum revealed 250, 450, 700, 800, and 1200 bp bands. The primer OPD18 with AP-PCR showed bands from 300, 400, 750, and 1000 bp in T. mentagrophytes, while in T. rubrum, it showed 350, 400, 700, 800, 900, and 1300 bp bands [Figure 2]. The direct DNA isolated from the nail samples and DNA-isolated pure culture of dermatophytes yielded similar banding pattern after amplification. Therefore, DNA isolated directly from the nail samples provided quick and promising results and explored as diagnostic method.


  Discussion Top


In the current study, AP-PCR was introduced as molecular diagnostic for dystrophic onychomycosis as compared to KOH and culture method, because AP-PCR method is cost-effective as compared to the real-time PCR and nested PCR. Previously, Zarrin et al.[10] also reported four random primers such as OPAA11, OPU15, OPAA17, and OPD18 for the identification of dermatophyte species with agreement of the present study, while the Girgis et al.[11] used only one primer OPAA17. Similarly, Watanabe and Ishida [12] reported that PCR identification method was achieved higher (100%) and rapid identification rate as compared to cultural methods (69.8%), but showed complete concordance for the occurrence of T. rubrum and T. mentagrophytes by both methods. Spiliopoulou et al.[13] also reported that T. rubrum is the major pathogen for nail infection followed by T. mentagrophytes. On the contrary, in some studies, T. mentagrophytes reported as the most prevalent dermatophytes,[2],[14] which may be due to the different geographic regions. All the samples never produce T. mentagrophytes and T. rubrum together, proposed that mixed infection is unusual [Figure 1] and [Figure 3].

In the existing investigation, 94% of patients reported from rural area with agriculture occupation and underprivileged population [Table 2]. The infection site of patients was mostly finger nails (52.08%) as compared to toe nails (47.5%), which was contradicted with the recently reported research, in which the author found 90% of the toenail infection and 50% of finger nail onychomycosis.[15]

In the current study, the incidence of dystrophic onychomycosis was distributed among farmers, homemakers, students, etc., but overall, more female patients (62.5%) reported for the dystrophy than male patients (37.5%) [Table 2]. All the reported females with dystrophy were homemakers with age ranges from 22 to 65 years. On the basis of gender, the prevalence of onychomycosis reported different. Bitew and Wolde [16] and Lubis et al.[3] reported that females were more prone to onychomycosis than males, while others demonstrated that males are more affected to fungal nail infection than females.[12],[17] In the present study, females were highly prone to fungal infection, maybe because of wet hands as whole day busy in household works such as cooking, washing, and cleaning. In addition, some female patients reported that the infection increases and become painful with the usage of detergent and soap, which further accelerated the infection. Highest visiting OPD of dystrophic onychomycosis (25%–33%) was seen in patients between 20 and 40 years and lowest prevalence (6%) in patients older than 60 years [Table 2]. Because of elderly peoples were least bothered about the nail infections therefore avoid the medication. Loo [18] stated that the prevalence of onychomycosis was nearly 20% in patients aged >60 years. Similarly, Araiza-Santibánez et al.[19] conducted retrospective study in elderly patients and found 90% nail disorders was onychomycosis, many of these patients was suffering with hypertension or diabetes mellitus, which leads to complicated treatment outcomes and affected the quality of life of elderly patients. Therefore, elderly patients should awarded adequate health education and monitoring, which will improve the quality of life. In the current study, patients with onychomycosis are not willing to take the medical treatment till it become severe/dystrophic, consequently in the worldwide difference in the prevalence of onychomycosis are found. The fungi known to cause onychomycosis varies among geographic areas, primarily due to differences in climate. In the current comparative study, AP-PCR showed the highest sensitivity (88.57%) and specificity (89.23%) and the probability of positive predictive value was 86.36%, which suggested that the method has a high strength to determine the true positive results [Table 2]. A study by Lubis et al.[3] reported 85.71% sensitivity of PCR-restriction fragment length polymorphism (PCR-RFLP) method and specificity value 28.57%. Mehlig et al.[20] reported the MycoDerm Biotype Diagnostic kit for 21 fungi belonging dermatomycoses in Europe and evaluated sensitivity (87.30%), specificity (94.3%), and positive predictive values of 87.3%.

Mechanism of infection and risk factors for onychomycosis

The dermatophyte produce keratinases when warm and moist environment of shoe and microtraumatic pressure on the nail break the hyponychial seal and dermatophyte diffuse into nail bed, that start the infection into toes or fingers which spread gradually from the hyperkeratotic sole to the distal hyponychia space of micro-traumatized nail units. The dermatophyte infects the nail bed when the distal nail hyponychium is breached and it spread as onycholysis and subungual hyperkeratosis.[21],[22] Risk factors include age, diabetes mellitus, psoriasis, diminished blood circulation, slow-growing nails, immunodeficiency and living with family members that have onychomycosis, heavy perspiration, humid or moist work environment, wearing artificial nails, wearing socks and leather shoes, previous injury or infection to the skin or nail, occupation (working with chemical like pesticides), detergents, prolonged exposure to pathogenic fungi, suboptimal immune function, inability to trim the toenails increases the risk of onychomycosis, T. pedis.[7],[15],[23]


  Conclusion Top


The current study concluded that clinical examination is not adequate for the diagnosis of onychomycosis, whereas AP-PCR is a significant diagnostic tool, which can be explored as an alternative to diagnose onychomycosis. AP-PCR achieved a higher dermatophyte identification rate with rapid results and showed complete concordance with conventional methods for two Trichophyton species. Therefore, further study is underway for the surveillance of onychomycosis in the present region, so that accurate prevalence of different types of onychomycosis on the basis of gender and age will be reported and also validate the AP-PCR method.

Acknowledgment

The authors are thankful to Honorable Chancellor, Vice-Chancellor of Adesh University and Chairperson, Centre for Interdisciplinary Biomedical Research, Adesh University, for providing necessary lab facilities.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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