mycoses
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Morphopathological aspects of healthy nails and nails affected by onychomycosis Olga Zaikovska,1 Mara Pilmane2 and Janis Kisis3 1 3
RSU Doctoral Department, Riga Stradins University, Riga, Latvia, 2Institute of Anatomy and Anthropology, Riga Stradins University, Riga, Latvia and Department of Infectology and Dermatology, Riga Stradins University, Riga, Latvia
Summary
Patients of onychomycosis are common in the dermatology practice. Contemporary morphology creates opportunities to study the functional units of the nail when such infections occur from morphopathological point of view. There were 22 nails biopsies from onychomycosis patients taken for the research of morphopathological changes in the thickened nail plate affected by onychomycosis. Samples of cadaverous’ nails were used as a control material. The material was stained with haematoxylin and eosin and immunohistochemical methods. Terminal deoxynucleotidyl transferase dUTP nick end labelling reaction and periodic acid-Schiff reaction were also performed. We found patchy hypertrophy in the granulose layer of the epidermis, with focal acanthosis. In the horn layer, we identified nests of parakeratosis of various sizes, with incorporations of homogenous and eosinophil masses. We found high levels of interleukin 6 and interleukin 10 positive cells in the nail bed and in the bloodstream. Interleukin 1, however, was not a part of any of the functional units of any of the nails. Significant amount of fibres containing human beta defensin-2 were found in the bed and plate of the nail. Therefore one can conclude that as regards the nails affected by onychomycosis, the most effective morphopathogenical processes include cytokine and defensin excretion occurrence in the nail bed.
Key words: Onychomycosis, nail biopsy, antimicrobial peptides, defensin 2, interleukins.
Introduction Onychomycosis is a chronic infection that is difficult to treat; it leads to gradual destruction of the nail plate. During the age of antibiotics and preservatives, damaged nails have become a riddle for diagnosis and therapy. The disease is associated with certain risk groups, various cosmetic and psychological issues and frequent
Correspondence: O. Zaikovska, K aßu l 25, LV-1058, Rıga, Latvia. Tel.: +371 26548019. Fax: +371 67220080. E-mail: [email protected] Submitted for publication 7 January 2014 Revised 8 February 2014 Accepted for publication 23 February 2014
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57, 531–536
recrudescence and therapy that is not always successful. There are data on a range of certain pathogens of the onychomycosis in several countries on the basis of results of the culture of the pathogen material. Until now, there have not been any extensive studies carried out in Latvia that would include and distinguish the spread and the range of the pathogens of onychomycosis. According to data provided by Estonia, only 69% of the patients with clinically detected onychomycosis were diagnosed also by mycological tests. Each patient is usually diagnosed with on average 5.4 nails affected by onychomycosis. The main symptoms of onychomycosis are as follows: change in the nail colour, hyperkeratosis and fragility of the nail. Most often the pathogen of the disease is Trichophyton rubrum.1 Onychomycosis of the feet occurs seven times more often than the onychomycosis of the hands. Incidence
doi:10.1111/myc.12191
O. Zaikovska et al.
of onychomycosis increases with the age as the growth rate of nails and blood circulation in the distal parts of the extremities change.2 Development of onychomycosis is determined by several predisposed factors. The main factors mentioned in the German guidelines of onychomycosis are as follows: angiopathy, peripheral neuropathy, wearing of tight footwear, deformation of feet, repeated injuries and other metabolic changes.3 Nowicki together with co-authors refers also to immunosuppression and psoriasis.4 In daily practice onychomycosis is diagnosed by microscopy and culture. The latest data show that biopsy of the sick nail is the most sensitive diagnostic method of onychomycosis – its sensitivity is 80%.5 Both histology and microscopy together are more sensitive than the culture method (53%), however, histology and microscopy together provide the most precise diagnostics of onychomycosis.6 To demonstrate fungi in histological sections two staining methods can be used: the periodic acid-Schiff (PAS) reaction that stains fungi deeply red, and the methylamine silver nitrate method that stains fungi black. Fungi stain positively with the PAS reaction due to the presence of cellulose and chitin in the cell walls – two substances rich in neutral polysaccharides. In the absence of apparent fungi, the histological picture of fungal infections is not possible to diagnose. Depending on the degree of reaction of the tissue to the presence of fungi, the histological features of an acute, a sub-acute or a chronic dermatitis can be seen.7 However, nail biopsy is not used in daily practice for diagnostics of onychomycosis as it is complicated to obtain the nail material, the procedure requires additional skills, presence of staff and patient’s compliance. Moreover, patients do not like the method as it requires period of rehabilitation that usually includes also restrictions of sport activities and tenderness after the procedure. Therefore, there is not much information on nail histology and its peculiarities in the literature. However, it is suggested to use nail biopsy in situations when it is not possible to differentiate between nails affected by onychomycosis and other diseases affecting nails (psoriasis, lichen planus, longitudinal melanonychia, trachyonychia, tumours).8,9 Using the modern morphology possibilities, the nail unit affected by onychomycosis will be studied from several points of view: blood supply, innervation, apoptosis process, inflammatory cytokines and release of antimicrobial peptides.
532
Material and methods Material
To evaluate morphological and immunohistochemical picture of onychomycosis, 5 mm punch biopsies from a nail unit were taken. The study included 22 patients corresponding to preestablished selection criteria to the exclusion of a variety of possible factors influencing the results (a previous drug therapy, diseases affecting nail condition). Eligible patients were aged from 31 to 81 years with clinically and microscopic/ culture confirmed diagnosis of onychomycosis. Samples of the cadaverous’ nails were used as control material (n = 5). The sample material did not have any clinical indications of onychomycosis, inflamed chronic skin diseases clinically and in anamnesis, with no antibiotic treatment within the last 2 weeks. Samples were obtained not later than 12 h after death. The study was approved by the Ethical Committee at Riga Stradins University, permit issued on 26.01.2012. Method
• Five millimetre punch nail unit biopsies were fixed in Stefanini’s solution, dehydrated and embedded in paraffin. Four-micrometre-thick sections were prepared from each tissue specimen and stained routinely with haematoxylin and eosin. PAS reaction was also performed. • Immunohistochemical method. Tissue was determined for the human beta defensin-2 (hBD-2) (VJU01, 1 : 100; R&D Systems, Minneapolis, MN, USA), human 1 alpha interleukin (Il-1) (SC-9983, 1 : 50; Santa Cruz Biotechnology, Dallas, TX, USA), interleukin 6 (IL-6) (NYRhIL6, 1 : 50; Santa Cruz Biotechnology, Dallas, TX, USA) and interleukin 10 (IL-10) (AB34843, 1 : 400, Abcam, Cambridge, England), protein gene product (PGP 9.5) (Z511601, 1 : 600; DAKO, Glostrup, Denmark), vascular endothelial growth factor (SC-7269, 1 : 50; Santa Cruz, Dallas, TX, USA), matrix metalloproteinase 2 (MMP-2) (AF902, 1 : 100; R&D Systems, ASV, Minneapolis, MN, USA). Apoptosis was made visible on the material applying terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method (11684817910, 1 : 10; Roche, Mannheim, Germany). • For visual illustration of our findings, we used Leica DC 300F digital camera and image processing and analysis software Image Pro Plus (Media Cybernetics, © 2014 Blackwell Verlag GmbH Mycoses, 2014, 57, 531–536
Onychomycosis affected nail
Inc., Rockville, MD, USA). The intensity of immunostaining was graded semiquantitatively. Few positive structures in the visual field were labelled with +, moderate number of positive structures in the visual field was labelled with ++, numerous positive structures in the visual field were labelled with +++ and abundance of positive structures in the visual field was marked with ++++.10 • For statistical analysis the Mann–Whitney U-test and SPSS17 (SPSS Inc. Chicago, IL, USA) was used.
Results We found patchy hypertrophy in the granulose layer of the epidermis and focal acanthosis in all onychomycosis cases. In stratum corneum, parakeratosis and incorporated eosinophil homogenous masses were observed. There was oedema, diffuse lymphocytic infiltration observed in derma in few cases. Small amount of VEGF was detected in the endotheliocytes of the onychomycosis affected nail unit connective tissue part, in healthy nails, however, VEGF was not found (stat. significance 0.025). Performing PGP 9.5 reaction there was a small amount of nerve fibre detected in nails affected by onychomycosis both in connective tissues and nail bed. The same was observed in the control group patients. Statistically substantial differences with regard to the presence of PGP 9.5 in the affected and the healthy nails were not found. Significant amount of MMP-2 was detected in connective tissue, nail plate and nail bed affected by onychomycosis (Fig. 1). However, in healthy nails it was not found. The statistically significant difference in the
affected and the healthy nails was found in the nail plate (0.016) and nail connective tissue (0.000). Explicit apoptosis processes in the bed of the nail affected by disease (Fig. 2) were found performing immunohistochemical TUNEL reaction. Statistically significant difference between the affected and the healthy nail is 0.001. Numerous hBD-2 containing fibres were found in the nail bed (Fig. 3), but moderate number of hBD-2 positive cells was found in underlying connective tissue and nail plate of onychomycosis affected nails. Statistically significant difference between all three groups – nail bed (0.000), nail plate (0.029) and nail connective tissue (0.002) was detected comparing the affected and the healthy nail.
Figure 2 Abundance of apoptotic cell in nail bed. TUNEL, IMH,
9250.
Figure 1 High expression of MMP-2 in connective tissue and
Figure 3 High expression of HBD-2 in nail bed and underlying
vessels. MMP-2, IMH, 9100.
connective tissue. HBD-2 IMH, 9200.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57, 531–536
533
O. Zaikovska et al.
muscles of blood vessels (Fig. 4), however, only few positive cells were found in plate structure. This is also validated by the statistical analysis – statistical significance only between nail bed (0.001) and nail connective tissue (0.006) detected. Moderate IL-10 containing structures were found in nail bed, but there were few positive cells in connective tissue and plate structure. Nevertheless, statistical analysis displays a significant difference with regards to IL – it shows 10 times higher presence in affected nails, respectively – in nail bed (0.035) and plate (0.007). Overview of all staining method semiquantitative and statistical results is available in Table 1.
Figure 4 Abundance of IL-6 containing endothelial cells and
smooth muscle in blood vessels in nail bed. IL-6 IMH, 9200.
Few IL-1 positive structures were detected in six preparations, particularly in the nail bed. Numerous IL-6 positive cells were found in nail bed and smooth
Discussion The nail apparatus consists or four main elements: the nail plate, the matrix, the nail folds (proximal, distal and lateral) and nail bed with connective tissue beneath it.
Table 1 Overview of all staining results. Onychomysosis affected nails Method PGP 9,5 Mann–Whitney Significance VEGF Mann–Whitney Significance IL-1 Mann–Whitney Significance IL-6 Mann–Whitney Significance IL-10 Mann–Whitney Significance MMP-2 Mann–Whitney Significance TUNEL (AI) Mann–Whitney Significance hBD-2 Mann–Whitney Significance
U
Nail bed
Connective tissue
Nail plate
Nail bed
Connective tissue
Nail plate
+ 32,500 0.086
+ 42,500 0.228 + 22,500 0.025 0/+ 18,000 0.006 +/++ 15,500 0.006 + 28,000 0.54 +/++ 2,500 0.000
0 44,000 0.185
+/++
+
0/+
U
U
U
U
U
U
U
Control group nails
0/+ 10,000 0.001 ++/+++ 11,000 0.001 +/++ 22,000 0.35 +/++ 54,000 0.420 49 10,000 0.001 ++/+++ 4,000 0.000
0
0/+ 46,500 0.342 + 45,500 0.266 + 15,500 0.007 + 24,000 0.016
+/++
0/+
0/+
+
0/+
0/+
++
+
0/+
+
0
0/+
0/+
0/+
18
+/++ 10,000 0.002
+ 23,500 0.029
0/+
Designation of semiquantative method: 0 negative reaction; 0/+ positive few elements in some preparations; + few positive structures in the visual field; ++ moderate number of positive structures in visual field; +++ numerous positive structures in the visual field; ++++ abundance of positive structures in the visual field. Mann–Whitney U-test – displays the ranking differences between the control group and studied group, that is, the lower the Mann– Whitney U-test, the higher the ranking difference. Significance – if significance is
Diagnosis,Therapy and Prophylaxis of Fungal Diseases
Original article
Morphopathological aspects of healthy nails and nails affected by onychomycosis Olga Zaikovska,1 Mara Pilmane2 and Janis Kisis3 1 3
RSU Doctoral Department, Riga Stradins University, Riga, Latvia, 2Institute of Anatomy and Anthropology, Riga Stradins University, Riga, Latvia and Department of Infectology and Dermatology, Riga Stradins University, Riga, Latvia
Summary
Patients of onychomycosis are common in the dermatology practice. Contemporary morphology creates opportunities to study the functional units of the nail when such infections occur from morphopathological point of view. There were 22 nails biopsies from onychomycosis patients taken for the research of morphopathological changes in the thickened nail plate affected by onychomycosis. Samples of cadaverous’ nails were used as a control material. The material was stained with haematoxylin and eosin and immunohistochemical methods. Terminal deoxynucleotidyl transferase dUTP nick end labelling reaction and periodic acid-Schiff reaction were also performed. We found patchy hypertrophy in the granulose layer of the epidermis, with focal acanthosis. In the horn layer, we identified nests of parakeratosis of various sizes, with incorporations of homogenous and eosinophil masses. We found high levels of interleukin 6 and interleukin 10 positive cells in the nail bed and in the bloodstream. Interleukin 1, however, was not a part of any of the functional units of any of the nails. Significant amount of fibres containing human beta defensin-2 were found in the bed and plate of the nail. Therefore one can conclude that as regards the nails affected by onychomycosis, the most effective morphopathogenical processes include cytokine and defensin excretion occurrence in the nail bed.
Key words: Onychomycosis, nail biopsy, antimicrobial peptides, defensin 2, interleukins.
Introduction Onychomycosis is a chronic infection that is difficult to treat; it leads to gradual destruction of the nail plate. During the age of antibiotics and preservatives, damaged nails have become a riddle for diagnosis and therapy. The disease is associated with certain risk groups, various cosmetic and psychological issues and frequent
Correspondence: O. Zaikovska, K aßu l 25, LV-1058, Rıga, Latvia. Tel.: +371 26548019. Fax: +371 67220080. E-mail: [email protected] Submitted for publication 7 January 2014 Revised 8 February 2014 Accepted for publication 23 February 2014
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57, 531–536
recrudescence and therapy that is not always successful. There are data on a range of certain pathogens of the onychomycosis in several countries on the basis of results of the culture of the pathogen material. Until now, there have not been any extensive studies carried out in Latvia that would include and distinguish the spread and the range of the pathogens of onychomycosis. According to data provided by Estonia, only 69% of the patients with clinically detected onychomycosis were diagnosed also by mycological tests. Each patient is usually diagnosed with on average 5.4 nails affected by onychomycosis. The main symptoms of onychomycosis are as follows: change in the nail colour, hyperkeratosis and fragility of the nail. Most often the pathogen of the disease is Trichophyton rubrum.1 Onychomycosis of the feet occurs seven times more often than the onychomycosis of the hands. Incidence
doi:10.1111/myc.12191
O. Zaikovska et al.
of onychomycosis increases with the age as the growth rate of nails and blood circulation in the distal parts of the extremities change.2 Development of onychomycosis is determined by several predisposed factors. The main factors mentioned in the German guidelines of onychomycosis are as follows: angiopathy, peripheral neuropathy, wearing of tight footwear, deformation of feet, repeated injuries and other metabolic changes.3 Nowicki together with co-authors refers also to immunosuppression and psoriasis.4 In daily practice onychomycosis is diagnosed by microscopy and culture. The latest data show that biopsy of the sick nail is the most sensitive diagnostic method of onychomycosis – its sensitivity is 80%.5 Both histology and microscopy together are more sensitive than the culture method (53%), however, histology and microscopy together provide the most precise diagnostics of onychomycosis.6 To demonstrate fungi in histological sections two staining methods can be used: the periodic acid-Schiff (PAS) reaction that stains fungi deeply red, and the methylamine silver nitrate method that stains fungi black. Fungi stain positively with the PAS reaction due to the presence of cellulose and chitin in the cell walls – two substances rich in neutral polysaccharides. In the absence of apparent fungi, the histological picture of fungal infections is not possible to diagnose. Depending on the degree of reaction of the tissue to the presence of fungi, the histological features of an acute, a sub-acute or a chronic dermatitis can be seen.7 However, nail biopsy is not used in daily practice for diagnostics of onychomycosis as it is complicated to obtain the nail material, the procedure requires additional skills, presence of staff and patient’s compliance. Moreover, patients do not like the method as it requires period of rehabilitation that usually includes also restrictions of sport activities and tenderness after the procedure. Therefore, there is not much information on nail histology and its peculiarities in the literature. However, it is suggested to use nail biopsy in situations when it is not possible to differentiate between nails affected by onychomycosis and other diseases affecting nails (psoriasis, lichen planus, longitudinal melanonychia, trachyonychia, tumours).8,9 Using the modern morphology possibilities, the nail unit affected by onychomycosis will be studied from several points of view: blood supply, innervation, apoptosis process, inflammatory cytokines and release of antimicrobial peptides.
532
Material and methods Material
To evaluate morphological and immunohistochemical picture of onychomycosis, 5 mm punch biopsies from a nail unit were taken. The study included 22 patients corresponding to preestablished selection criteria to the exclusion of a variety of possible factors influencing the results (a previous drug therapy, diseases affecting nail condition). Eligible patients were aged from 31 to 81 years with clinically and microscopic/ culture confirmed diagnosis of onychomycosis. Samples of the cadaverous’ nails were used as control material (n = 5). The sample material did not have any clinical indications of onychomycosis, inflamed chronic skin diseases clinically and in anamnesis, with no antibiotic treatment within the last 2 weeks. Samples were obtained not later than 12 h after death. The study was approved by the Ethical Committee at Riga Stradins University, permit issued on 26.01.2012. Method
• Five millimetre punch nail unit biopsies were fixed in Stefanini’s solution, dehydrated and embedded in paraffin. Four-micrometre-thick sections were prepared from each tissue specimen and stained routinely with haematoxylin and eosin. PAS reaction was also performed. • Immunohistochemical method. Tissue was determined for the human beta defensin-2 (hBD-2) (VJU01, 1 : 100; R&D Systems, Minneapolis, MN, USA), human 1 alpha interleukin (Il-1) (SC-9983, 1 : 50; Santa Cruz Biotechnology, Dallas, TX, USA), interleukin 6 (IL-6) (NYRhIL6, 1 : 50; Santa Cruz Biotechnology, Dallas, TX, USA) and interleukin 10 (IL-10) (AB34843, 1 : 400, Abcam, Cambridge, England), protein gene product (PGP 9.5) (Z511601, 1 : 600; DAKO, Glostrup, Denmark), vascular endothelial growth factor (SC-7269, 1 : 50; Santa Cruz, Dallas, TX, USA), matrix metalloproteinase 2 (MMP-2) (AF902, 1 : 100; R&D Systems, ASV, Minneapolis, MN, USA). Apoptosis was made visible on the material applying terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) method (11684817910, 1 : 10; Roche, Mannheim, Germany). • For visual illustration of our findings, we used Leica DC 300F digital camera and image processing and analysis software Image Pro Plus (Media Cybernetics, © 2014 Blackwell Verlag GmbH Mycoses, 2014, 57, 531–536
Onychomycosis affected nail
Inc., Rockville, MD, USA). The intensity of immunostaining was graded semiquantitatively. Few positive structures in the visual field were labelled with +, moderate number of positive structures in the visual field was labelled with ++, numerous positive structures in the visual field were labelled with +++ and abundance of positive structures in the visual field was marked with ++++.10 • For statistical analysis the Mann–Whitney U-test and SPSS17 (SPSS Inc. Chicago, IL, USA) was used.
Results We found patchy hypertrophy in the granulose layer of the epidermis and focal acanthosis in all onychomycosis cases. In stratum corneum, parakeratosis and incorporated eosinophil homogenous masses were observed. There was oedema, diffuse lymphocytic infiltration observed in derma in few cases. Small amount of VEGF was detected in the endotheliocytes of the onychomycosis affected nail unit connective tissue part, in healthy nails, however, VEGF was not found (stat. significance 0.025). Performing PGP 9.5 reaction there was a small amount of nerve fibre detected in nails affected by onychomycosis both in connective tissues and nail bed. The same was observed in the control group patients. Statistically substantial differences with regard to the presence of PGP 9.5 in the affected and the healthy nails were not found. Significant amount of MMP-2 was detected in connective tissue, nail plate and nail bed affected by onychomycosis (Fig. 1). However, in healthy nails it was not found. The statistically significant difference in the
affected and the healthy nails was found in the nail plate (0.016) and nail connective tissue (0.000). Explicit apoptosis processes in the bed of the nail affected by disease (Fig. 2) were found performing immunohistochemical TUNEL reaction. Statistically significant difference between the affected and the healthy nail is 0.001. Numerous hBD-2 containing fibres were found in the nail bed (Fig. 3), but moderate number of hBD-2 positive cells was found in underlying connective tissue and nail plate of onychomycosis affected nails. Statistically significant difference between all three groups – nail bed (0.000), nail plate (0.029) and nail connective tissue (0.002) was detected comparing the affected and the healthy nail.
Figure 2 Abundance of apoptotic cell in nail bed. TUNEL, IMH,
9250.
Figure 1 High expression of MMP-2 in connective tissue and
Figure 3 High expression of HBD-2 in nail bed and underlying
vessels. MMP-2, IMH, 9100.
connective tissue. HBD-2 IMH, 9200.
© 2014 Blackwell Verlag GmbH Mycoses, 2014, 57, 531–536
533
O. Zaikovska et al.
muscles of blood vessels (Fig. 4), however, only few positive cells were found in plate structure. This is also validated by the statistical analysis – statistical significance only between nail bed (0.001) and nail connective tissue (0.006) detected. Moderate IL-10 containing structures were found in nail bed, but there were few positive cells in connective tissue and plate structure. Nevertheless, statistical analysis displays a significant difference with regards to IL – it shows 10 times higher presence in affected nails, respectively – in nail bed (0.035) and plate (0.007). Overview of all staining method semiquantitative and statistical results is available in Table 1.
Figure 4 Abundance of IL-6 containing endothelial cells and
smooth muscle in blood vessels in nail bed. IL-6 IMH, 9200.
Few IL-1 positive structures were detected in six preparations, particularly in the nail bed. Numerous IL-6 positive cells were found in nail bed and smooth
Discussion The nail apparatus consists or four main elements: the nail plate, the matrix, the nail folds (proximal, distal and lateral) and nail bed with connective tissue beneath it.
Table 1 Overview of all staining results. Onychomysosis affected nails Method PGP 9,5 Mann–Whitney Significance VEGF Mann–Whitney Significance IL-1 Mann–Whitney Significance IL-6 Mann–Whitney Significance IL-10 Mann–Whitney Significance MMP-2 Mann–Whitney Significance TUNEL (AI) Mann–Whitney Significance hBD-2 Mann–Whitney Significance
U
Nail bed
Connective tissue
Nail plate
Nail bed
Connective tissue
Nail plate
+ 32,500 0.086
+ 42,500 0.228 + 22,500 0.025 0/+ 18,000 0.006 +/++ 15,500 0.006 + 28,000 0.54 +/++ 2,500 0.000
0 44,000 0.185
+/++
+
0/+
U
U
U
U
U
U
U
Control group nails
0/+ 10,000 0.001 ++/+++ 11,000 0.001 +/++ 22,000 0.35 +/++ 54,000 0.420 49 10,000 0.001 ++/+++ 4,000 0.000
0
0/+ 46,500 0.342 + 45,500 0.266 + 15,500 0.007 + 24,000 0.016
+/++
0/+
0/+
+
0/+
0/+
++
+
0/+
+
0
0/+
0/+
0/+
18
+/++ 10,000 0.002
+ 23,500 0.029
0/+
Designation of semiquantative method: 0 negative reaction; 0/+ positive few elements in some preparations; + few positive structures in the visual field; ++ moderate number of positive structures in visual field; +++ numerous positive structures in the visual field; ++++ abundance of positive structures in the visual field. Mann–Whitney U-test – displays the ranking differences between the control group and studied group, that is, the lower the Mann– Whitney U-test, the higher the ranking difference. Significance – if significance is
