studentjournalofmedicine

Modulation of Intestinal Inflammation by Yeasts and Cell Wall Extracts: Strain Dependence and Unexpected Anti-Inflammatory Role of Glucan Fractions

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Jagadeesh Bayry, Editor

Abstract

Yeasts and their glycan components can have a beneficial or adverse effect on intestinal inflammation. Previous research has shown that the presence of Saccharomyces cerevisiae var. boulardii (Sb) reduces intestinal inflammation and colonization by Candida albicans. The aim of this study was to identify dietary yeasts, which have comparable effects to the anti-C. albicans and anti-inflammatory properties of Sb and to assess the capabilities of yeast cell wall components to modulate intestinal inflammation. Mice received a single oral challenge of C. albicans and were then given 1.5% dextran-sulphate-sodium (DSS) for 2 weeks followed by a 3-day restitution period. S. cerevisiae strains (Sb, Sc1 to Sc4), as well as mannoprotein (MP) and β-glucan crude fractions prepared from Sc2 and highly purified β-glucans prepared from C. albicans were used in this curative model, starting 3 days after C. albicans challenge. Mice were assessed for the clinical, histological and inflammatory responses related to DSS administration. Strain Sc1-1 gave the same level of protection against C. albicans as Sb when assessed by mortality, clinical scores, colonization levels, reduction of TNFα and increase in IL-10 transcription. When Sc1-1 was compared with the other S. cerevisiae strains, the preparation process had a strong influence on biological activity. Interestingly, some S. cerevisiae strains dramatically increased mortality and clinical scores. Strain Sc4 and MP fraction favoured C. albicans colonization and inflammation, whereas β-glucan fraction was protective against both. Surprisingly, purified β-glucans from C. albicans had the same protective effect. Thus, some yeasts appear to be strong modulators of intestinal inflammation. These effects are dependent on the strain, species, preparation process and cell wall fraction. It was striking that β-glucan fractions or pure β-glucans from C. albicans displayed the most potent anti-inflammatory effect in the DSS model.

Introduction

Probiotics are a popular alternative to antibiotics [1]. The positive effects of probiotics on humans and animals result either from a direct nutritional effect or a health effect, with probiotics acting as bioregulators of the intestinal microflora and reinforcing the host’s natural defences [2].

Saccharomyces cerevisiae var. boulardii (Sb) is described as a biotherapeutic agent in the clinical literature and is reported to be efficacious in the prevention of antibiotic-associated diarrhoea and colitis in humans [3], [4]. Orally administered Sb demonstrated clinical and experimental effectiveness in gastrointestinal diseases through modulation of host cell signalling pathways implicated in the pro-inflammatory response such as IL-1β and TNF-α. Sb exerts a trophic effect that restores intestinal homeostasis and activates expression of peroxisome proliferator–activated receptor-gamma which protects against gut inflammation [5].

It has recently been reported that Sb decreases inflammation and intestinal colonization by C. albicans in a BALB/c mouse model of colitis induced by dextran-sulphate-sodium (DSS) [6]. Interestingly, parallel studies indicated that Sb reduces C. albicans adhesion to human intestinal cell lines and decreases pro-inflammatory cytokine mRNA levels in response to C. albicans infection [7], [8].

Sb is generally administered as a lyophilized powder [9] and its use as a food additive has only been reported in a number of cases such as in the fermentation of raw vegetable materials [10] and incorporation into commercial yoghurts [11].Taxonomic studies indicate that Sb should be considered as a Saccharomyces cerevisiae strain [12], [13]. This leads to the question “do other strains of S. cerevisiae also possess probiotic properties?” [14].

In the present study, low doses of DSS were administered to mice for 2 weeks to induce colonic inflammation and promote the establishment of C. albicans colonization, followed by a 3-day restitution period. Either S. cerevisiae strains or glycan fractions were then administered daily by oral gavage for 2 weeks, starting 3 days after the C. albicans challenge, in order to assess their curative effects on both colonic inflammation and acceleration of colonic epithelial restoration. Using the DSS mouse model each dietary yeast was found to have its own effect on colitis and C. albicans colonization. The impact of orally administered glycan fractions extracted from S. cerevisiae that may reverse the adverse effects of DSS and C. albicans, and the biological activity of soluble β-glucan isolated from the C. albicans cell wall, were then investigated in this DSS mouse model.

Results

Comparison of the probiotic potential of S. cerevisiae var. boulardii and S. cerevisiae 1-1 strain

S. cerevisiae 1-1 strain (Sc1-1) was selected from our collection as having previously exhibited a probiotic effect. This yeast is prepared as an active dry yeast so that it can react quickly to its environment (Table 1). As Sc1-1 was comparable in vitro to Sb in terms of decreasing growth and germ tube formation by C. albicans (data not shown), the aim was to compare the Sc1-1 and Sb strains for their ability to reduce C. albicans colonization and intestinal inflammation.

Table 1

Yeast strains used in the investigation.

The model used is summarized in Fig. 1A and can be defined as “curative”. In this model, a single dose of C. albicans was administered to mice receiving DSS. Either the Sb or Sc1-1 strain was given 3 days later when C. albicans colonization was established. Low mortality was observed in mice that received DSS or DSS+C. albicans whereas none of the mice given the same regimen plus S. cerevisiae died (Fig. 1B).

Figure 1

Comparison of the probiotic potential of S. cerevisiae var. boulardii and S. cerevisiae 1-1 strain.

Concerning C. albicans colonization in mice that received DSS (Fig. 1C), the number of colony-forming units (CFUs) in stools gradually increased from the day of C. albicans administration. From S. cerevisiae administration on day 4 to the endpoint, a dramatic reduction in number of C. albicans CFUs was observed. No difference was observed between the two S. cerevisiae strains in their activity on C. albicans clearance (Fig. 1C).

The cumulated clinical and histological scores (Fig. 1D–E) were higher in mice that received DSS or DSS+C. albicans whereas they were reduced significantly by the administration of either Sb or Sc1-1 (Fig. 1D–E). Both strains were equally effective in reducing intestinal inflammation assessed by these parameters (Fig. 2).

Figure 2

Histological analysis of DSS-induced colitis in mice.

To analyze the possible mediators involved in the reduction of the inflammatory response to DSS and C. albicans in the colon following S. cerevisiae administration, we focused on levels of TNF-α and IL-10 mRNA as representative pro- and anti-inflammatory cytokines. As shown in Fig. 1F–G, administration of either Sb or Sc1-1 was associated with a significant reduction in TNF-α expression and increase in IL-10 production. No difference was observed between the two S. cerevisiae strains in their ability to redirect the inflammatory response.

Analysis of the anti-C. albicans and anti-inflammatory properties of other S. cerevisiae strains with different preparation processes

As both Sc1-1 and Sb strains displayed identical beneficial effects in this specific model the Sc1-1 strain was used as the reference strain for probiotic activity. In this part of the study, the influence of yeast preparation procedure and other yeast strains was investigated.

From an initial screening involving 10 strains or preparation procedures, five representative examples of strain activities were selected and compared to Sc1-1. These consisted of strains Sc1-2, Sc2, Sc3 and Sc4.

Sc1-1 showed important differences in mortality (Fig. 3A), ability to reduce C. albicans colonization (Fig. 3B) and a decrease in both histological and clinical scores (Fig. 3C–D). The Sc1-1 strain gave excellent results for all parameters, as did Sc3, which is used for its probiotic activities (Fig. 4) Interestingly, some of the beneficial effects induced by Sc1-1 were abolished when this strain was prepared as an instant dry yeast (Table 1). The most striking results were observed with the Sc4 strain, which did not display any particular effect on C. albicans colonization over 2 days compared to the other S. cerevisiae strains. However, the mice were extremely constipated, clinically inflamed and highly colonized with C. albicans starting from day 7. From this point on, we discontinued collecting faeces as the mice were becoming extremely ill. This yeast, which presents optimal growth at low temperatures, dramatically exacerbated both the clinical and histological scores and was associated with high mortality (around 80%). We also found that both C. albicans and Sc4 increased the staging of colitis in mice (Fig. 4). These high clinical and histological scores were associated with high numbers of C. albicans CFUs in different parts of the gut from moribund mice (data not shown).

Figure 3

Assessment of biological effect of S. cerevisiae 1-1 versus other S. cerevisiae strains.
Figure 4

Histological analysis of DSS-induced colitis in mice.

Finally, Sc2, another industrial strain used for the production of yeast proteins, displayed an “intermediate” behaviour with reduced beneficial effects and slight worsening of mortality and clinical scores.

Identification of the cell wall fractions supporting the beneficial and adverse effects on C. albicans colonization and inflammation

For this purpose, Sc2 was used as this strain can be easily induced to undergo autolysis in order to prepare cell wall extracts including mannoprotein (MP) and β-glucan fractions (Fig. 5).

Figure 5

Schematic diagram of glycan preparation from S. cerevisiae.

In contrast to living cells, none of the cell wall extracts induced mortality (Fig. 6A). However, the changes in body weight and clinical scores were worsened considerably by MP fractions while these parameters were ameliorated by β-glucan fractions (Fig. 6B, F). Notably, the MP fraction as well as the Sc2 strain induced an important loss in body weight up to day 9 that was correlated with its incapacity to control C. albicans colonization (Fig. 6C). By contrast, administration of β-glucan fraction maintained normal body weight, reduced inflammation scores and promoted C. albicans clearance (Fig. 6A–F). Although the body weight of mice receiving MP fractions started to increase from day 10, the clinical activity score was higher than that of mice receiving β-glucan fractions (Fig. 6E).

Figure 6

Effect of glycan fractions derived from S. cerevisiae on Candida DSS-treated mice.

Activity of the homologous C. albicans oligoglucoside fraction in the DSS mouse model

As the glucoprotein fraction from S. cerevisiae unexpectedly displayed a protective effect in this model, it was decided to investigate both the structure and biological activity of the β-glucan fraction from C. albicans. The harsh whole cell extraction procedure leads to fraction-1, known as yeast ghosts (Fig. 7). Fraction-1 (F1) was analyzed by fluorescence microscopy with various fluorescent probes specific for cell wall glycans in comparison to zymosan, which is widely used for β-glucan immunological studies (Fig. 8). Both zymosan and F1 were labelled with monoclonal antibody (mAb) 2G8 specific for β-1,3 glucans and WGA which binds to chitin (Fig. 8). In contrast to zymosan, which was stained with both Concanavalin A (ConA) and GNL, no mannose residue signals were observed for F1. Immunofluorescent staining with antibodies to β-mannose, liable to be synthesized by C. albicans, was also negative with F1 (data not shown). Thus, yeast ghosts have no mannose residues in their cell wall (Fig. 8).

Figure 7

Schematic diagram of β-glucan preparation from C. albicans.
Figure 8

Immunofluorescence staining of C. albicans ghost cells and zymosan with various fluorescent probes specific for yeast cell wall glycans.

MALDI-MS analysis of the soluble fraction derived from F1 (F2) established that it consisted of a highly polydisperse hexose polymer consisting of 3–27 hexose residues (Fig. 9A). According to its reactivity with anti β-1,3 glucans, this component was susceptible to zymolyase digestion which produced a set of small water soluble fragments (2–5 Glc), as demonstrated by thin-layer chromatography (data not shown) and MALDI-MS analyses (Fig. 9B). After purification of this fraction by reverse phase and adsorption chromatography, its structure was established by NMR as a mixture of β1,3-substituted glucan oligomers with free reducing ends. Furthermore, these oligomers were shown to be partially substituted by a random single β1,6 glucopyranose residue (Fig. 9C).

Figure 9

Structural analysis of glycan fraction.

Mortality, weight loss, clinical activity, histological score and C. albicans colonization

After structure characterization of the β-oligoglucoside fraction (F2) extracted from C. albicans, the biological activity of F2 was tested in the DSS mouse model. After C. albicans challenge, mice were given F2 (1 mg/day) from day3 up to the endpoint (Fig. 1A). F2 administration significantly prevented mouse mortality due to either DSS or DSS+C. albicans (Fig. 10A). In contrast to DSS and DSS+C. albicans mice, which developed severe colitis and lost body weight, F2 administration reversed the adverse effect of colitis and the mice showed a significant amelioration of both the clinical inflammation score and body weight (Fig. 10B–D). Histological examination of colon sections from mice receiving either DSS or DSS+C. albicans showed important colonic inflammation which was associated with mucosal cell loss, crypt damage, mucosal ulceration and accompanying submucosal oedema (Fig. 10C–D). F2 administration significantly reduced colonic inflammation due to either DSS or DSS+C. albicans (Fig. 10C–D). C. albicans colonization in DSS-treated mice showed a steady increase as assessed by the number of CFUs in faeces which was consistent with the high load of C. albicans recovered from the stomach, ileum and colon of this group of mice at the endpoint of the experiments (Fig. 10E–F).

Figure 10

Effect of β-oligoglucoside fractions derived from C. albicans on Candida DSS-treated mice.

In contrast to the higher numbers of C. albicans CFUs recovered from different compartments of the gut in DSS-treated mice, oral administration of F2 decreased the number of C. albicans CFUs recovered from stools and all gut segments of DSS-treated mice (Fig. 10E–F). Furthermore, the reduction in clinical and histological scores was consistent with these low numbers of C. albicans CFUs in different parts of the gut (Fig. 11 and Table 2).

Figure 11

Summary of the effects of S. cerevisiae strains or glycan fractions on Candida DSS-treated mice.
Table 2

Effect of different strains and cell wall extracts on C. albicans colonization and inflammation in the curative C. albicans DSS model.

Discussion

Excessive use of antifungal agents has been implicated in the emergence of antifungal resistance in C. albicans and constitutes a serious clinical problem in hospitals by affecting the natural balance of the intestinal microflora in these individuals [15]. The non-pathogenic yeast, Sb, which is widely prescribed for the treatment of antibiotic-induced gastrointestinal disorders and Clostridium difficile-associated enteropathies, has been shown to be an alternative approach to counterbalance the equilibrium of the intestinal microflora and modulate the innate immune defence [16], [17]. Orally administered Sb was successful in both the treatment of inflammatory bowel disease (IBD) and the elimination of C. albicans colonization [7], [8], [18][21].

Recently, it has been shown that Sb decreases both C. albicans colonization and intestinal inflammation in a mouse model of DSS-induced colitis [6]. Following this study, S. cerevisiae strains, MP and β-glucan fractions were screened in a mouse model of DSS-induced colitis. As Sb is considered taxonomically to be a strain of S. cerevisiae [12], [13], strain Sc1-1 was compared to Sb in the DSS model. Sc1-1 is a gastro-resistant strain that reacts rapidly to its environment and is widely used in the food industry. Incidentally, it was observed that both Sc1-1 and Sb strains reduced C. albicans filamentation in vitro and C. albicans adhesion to plastic-plate wells (data not shown). In the present study, we did not chose a prophylactic but a curative model in which the animals develop colitis with histological features that are similar to those seen in patients with IBD before starting their treatment.

In this model low doses of DSS were used in order to establish C. albicans colonization, followed by S. cerevisiae or yeast extracts administration to assess their effects on the inflamed colon and colonic epithelium restitution.

Two weeks of DSS administration were scheduled to induce moderate colonic inflammation in mice, with low mortality rates. A recent study by Samonis et al. showed that mice receiving a high daily oral dose of C. albicans (around 108 CFU/day) for 2 weeks did not respond to Sb treatment [22]. In our model, a single inoculum of C. albicans was used and Candida colonization was maintained naturally in the mouse gastrointestinal tract by the DSS-induced colitis since a high C. albicans dose could dramatically hide the beneficial effect of Sb. In the present study, and similar to the Sb strain, Sc1-1 decreased both C. albicans colonization and intestinal inflammation in terms of clinical and histological score and mortality. Another notable finding was the acceleration of colonic epithelium restoration in mice treated with these dietary yeasts leading to the absence of submucosal oedema and epithelial erosion. Mechanistically, a recent report on intestinal inflammation showed that Sb secretes motogenic factors that enhance intestinal epithelial cell restitution [23].

Regarding the RT-PCR results, both Sb and Sc1-1 reduced the expression levels of pro-inflammatory cytokine TNF-α mRNA in the colonic mucosa with subsequent enhancement of IL-10 mRNA expression that inhibits intestinal injury [24]. Additionally, different pro-inflammatory cytokines were investigated in this set of experiments and were consistent with TNF-α expression. Further investigation is required to determine the role of Th17/Treg responses in different sets of experiments [25], [26]. A recent study in patients with IBD showed that Sb reduced TNF-α production and significantly inhibited T-cell proliferation induced by intestinal inflammation [19]. Generally, the biological activities of S. cerevisiae in gastrointestinal inflammatory conditions are mediated through modulation of host pro-inflammatory responses not only by the whole yeast, but also by secreted factors able to interfere with host signalling molecules that control inflammation at different levels such as NF-κB [27], [28]. Sb produces a soluble anti-inflammatory factor that inhibits NF-κB activation and attenuates pro-inflammatory signalling in host cells. In addition, Sb stimulates IL-10 secretion from intraepithelial lymphocytes infected by C. albicans and Escherichia coli [29]. As Sc1-1 was shown to be comparable to Sb and presents the same beneficial features against C. albicans and intestinal inflammation, Sc1-1 was considered as the reference strain in the DSS model. To assess if the observed anti-inflammatory properties were strain-dependent, other S. cerevisiae strains were selected deliberately for their high phenotypic diversity. The possible influence of yeast preparation process on anti-C. albicans activity was also studied. Surprisingly, some strains had a dramatic effect in the DSS mouse model and the process of yeast preparation also had an influence on the yeast’s biological properties [30]. Each strain selected in this study was well characterized in vitro in terms of cell growth, osmostress, fermentation, viability and metabolites. However, different factors could influence the biological activity of the strains when introduced by gavage in the DSS mouse model: (i) the resistance of the cell wall related to the yeast preparation process [30]; (ii) the viability of the strain in the stomach, ileum and colon; (iii) its interaction with the microflora and intestinal mucosa [31]; and (iv) its ability to produce soluble anti-inflammatory factors in the milieu triggering expression of mediators in the intestinal epithelium and cells of monocyte lineage present in the submucosae [27]. Altogether, each strain has its own unique properties and supports specific activities within the host. The in vitro findings, together with the results for all S. cerevisiae strains analyzed in this study, suggest that Sc1-1 has beneficial biological activities reversing all aspects of colitis, including histological damage, diarrhoea and mucosal levels of the pro-inflammatory mediator TNF-α.

The cell wall is an essential structural component of yeast cells playing a central role in the interaction of yeasts with their environment. Unfortunately, the biological activities of S. cerevisiae cell wall components are still unclear in terms of C. albicans colonization and intestinal inflammation. Two components (MP and β-glucans) produced industrially were explored in our experimental model. With MP fraction administration, C. albicans colonization was not consistent with intestinal inflammation parameters, suggesting that MP fractions have differential effects on C. albicans colonization and intestinal inflammation. In contrast to MP fractions, GP fraction administration decreased the number of C. albicans CFUs concomitantly to all intestinal inflammation parameters.

Both of these components are known to be potent immunological activators, but their mechanisms of action are different and controversial [32][34]. As an example, both MP and β-glucans act positively on tumour cells and several microbial infections [34], [35]. Conversely, administration of β-glucans derived from C. albicans has been shown to exacerbate arthritis in mice [36]. Structurally, MP have extensive N-and O-linked mannosylation which serve as ligands for galectin-3 (Gal-3), mannose receptor and DC-SIGN on macrophages and dendritic cells [37]. Different MP express β-Man epitopes, which have been identified as the principal ligand for Gal-3 [38]. In a previous study using the DSS model with C. albicans, Gal-3 knock-out mice were less affected by intestinal inflammation and C. albicans colonization than wild-type animals [39]. Recently, it was shown that C. glabrata deficient in β-Man was less virulent in DSS-treated mice as revealed by low clinical and histological scores and reduction of C. glabrata colonization [40]. β-glucans have affinities towards different receptors such as CD11b/CD18 [41], located on neutrophils, or Dectin-1 on macrophages [42]. This results in β-glucan activation of cytokine production and in turn activation of adaptive immunity. Thus, β-glucans attenuate the impact of colitis compared to MP [43].

As our results also showed a beneficial effect of β-glucans on inflammation/colonization, insoluble ghost yeast cells derived from C. albicans containing β-glucans were prepared and compared to zymosan which is widely used in β-glucan studies where many investigators refer to it as β-glucan [44], [45]. Zymosan stimulates the production and activity of pro-inflammatory cytokines [45]. Additionally, when chemically characterized zymosan containing only β-(1–3)-glucans was added to macrophage cells, the production of IL-10, reactive oxygen species (ROS) and TNF-α increased in a dose-dependent way [46]. Bonifazi et al. demonstrated the capacity of zymosan to activate both inflammatory and tolerogenic dendritic cells (DCs) leading to the triggering of both Th17 and Treg cells in vivo [47]. Our observations showed that zymosan contains both mannans and β-glucans exposed together on the cell wall surface in comparison to C. albicans ghosts that contain only β-glucans. This evidence prevented us from further studies on zymosan. Different observations showed that the biological activities of soluble β-glucans differ from those of cell-associated β-glucans [32], [48], [49]. Ishibashi et al. showed that insoluble cell wall β-glucans induced intensive inflammatory and immunomodulating activities compared to soluble β-glucans [49]. Following the β-glucan analysis, the chemical structure of the soluble β-glucan fraction derived from C. albicans ghosts was characterized and its biological activities were tested in the DSS mouse model. Interestingly, orally administered β-glucans from C. albicans decreased intestinal inflammation and C. albicans colonization.Several reports show that β-glucan enhances the immune response and improves the clearance of pathogenic bacteria in animal models [50][52]; this supports our findings that smaller oligoglucosides derived from C. albicans showed beneficial activities against C. albicans and these results are comparable to β-glucans derived from S. cerevisiae. However, it may also be hypothesized that these individual oligoglucosides could block receptors such as dectin-1 and CD11b/CD18 and prevent multivalent binding necessary for strong triggering of the inflammatory responses [53]. Besides the importance of yeast molecules sensing for immune response, a third player may also possibly act in the general interplay. This is the mouse microbiota. Oligosaccharides are well known prebiotics active on the intestinal flora [54], [55], and although such a role has not been investigated for C. albicans derived oligoglucosides it cannot be ruled out. Altogether, these results demonstrate that oligoglucosides behave differently from the original C. albicans whole yeast cells in the DSS mouse model.

In summary, Sc1-1 was found to be comparable to Sb and had beneficial biological activities against C. albicans and intestinal inflammation. Clinical trials are currently being conducted with Sc1-1 and promising results have been seen in patients with IBD. In the second part of this study, we focused on cell wall components involved in direct contact with the host and demonstrated that, in contrast to MP, β-glucan fractions from either S. cerevisiae or C. albicans have a more potent anti-inflammatory effect against colonic colitis induced by DSS in mice. In conclusion, this study generated some progress in deciphering the nature of the yeast molecular components differentially favouring inflammation and/or C. albicans clearance. Future studies will include experiments on oligosaccharide administration to mice in order to determine how these glycans stimulate the growth of beneficial bacteria in the gut and boost the immune system providing therapeutic perspectives for digestive disorders and life-threatening fungal infections of endogenous origin.

Materials and Methods

Yeast strains

The yeast strains used in this study are shown in Table 1.

Preparation of β-glucan fractions from yeasts

The composition in dry matter of spray-dried S. cerevisiae Sc2 cell wall fractions is shown in Table 3 and the preparation procedure for MP and β-glucan fractions from the cell wall of the same strain is shown in Fig. 1. The fractionation and digestion procedure for extraction of the β-glucan fraction from C. albicans is summarized in Fig. 2. Briefly, the cell pellet of C. albicans (50 g wet weight) was incubated twice in 200 ml of 1 M NaOH at 70°C for 30 min. After washing with distilled water, the supernatant was removed and the pellet was oxidized with 100 mM NaIO4 (Sigma-Aldrich, France) at room temperature for 24 h in the dark [56]. After completion of the reaction, excess periodate was destroyed by adding ethylene glycol. After washing several times with water, the pellet was reduced with 1 M NaBH4 (Sigma Aldrich, France) at room temperature. The reaction was terminated by lowering the pH to 5 by the addition of acetic acid. After washing several times with water, the insoluble fraction was then lyophilized to produce fraction-1. Fraction-1 was treated with zymolyase 20T (0.2 mg/mL, Immuno™; ICN Biomedicals Inc.) at 37°C for 3 h. Zymolyase inactivation was performed at 70°C for 5 min. After centrifugation, the supernatant was dialyzed against distilled water. The dialyzed solution was loaded onto a Sep-Pak C18 column (Alltech) equilibrated with 0.1% TFA (trifluoroacetic acid). Eluate-1 was evaporated and the resulting oligoglucosaccharides were dissolved in distilled water and further purified on a carbograph column (Alltech carbograph SPE column). Eluate-2 from the carbograph column was lyophilized to produce fraction-2 (F2). Fluorescence microscopy was performed to assess surface oligomannose expression on fraction-1 in comparison to zymosan (Sigma-Aldrich). Fraction-1 and zymosan suspensions deposited on slides were incubated with either monoclonal antibody (mAb) 2G8 specific for β-1,3 glucans [57], [58], or wheat germ agglutinin(WGA), which binds to chitin [59], or Concanavalin A (ConA) or Galanthus nivalis lectin (GNL) or DAPI, as described previously [6], [40]. For animal experimentation, fraction-2 was suspended in water and divided into 200 µL aliquots (each aliquot of 200 µL contained 1 mg of β-glucans).

Table 3

Composition in dry matter of spray-dried Sc2 cell wall fractions.

Structural analysis of β-glucans

NMR experiments were performed at 300 K using a Bruker AvanceII 900 MHz spectrometer equipped with a 5 mm triple-resonance cryoprobe. Prior to NMR spectroscopic analyses in deuterium, oligosaccharides were repeatedly exchanged in 2H2O (99.97% 2H, Euriso-top; Saint-Aubin, France) with intermediate freeze-drying and finally dissolved in 2H2O and transferred into Shigemi (Allison Park, USA) tubes. Chemical shifts (ppm) were calibrated taking the methyl group from internal acetone at δ1H 2.225 and δ13C 31.55 ppm. MALDI-TOF mass spectra were acquired on a Voyager Elite DE-STR mass spectrometer (Perspective Biosystems, Framingham, MA). Prior to analysis, samples were prepared by mixing 1 µL of oligosaccharide solution (1–5 pmol) with 1 µL of 2,5 dihydroxybenzoic acid matrix solution (10 mg/mL in CH3OH/H2O, 50[ratio]50, vol/vol) directly on the target. Between 50 and 100 scans were averaged for each spectrum.

Animals

Six- to 8-week-old female BALB/c mice were used. All mice were maintained by Charles River Laboratories (France). Four sets of experiments were performed independently and each experiment was divided into control groups (eight mice/cage), including assessment of the effect of DSS alone, and experimental groups (10 mice/cage).

Ethics statement

All mouse experiments were performed according to protocols approved by the Subcommittee on Research Animal Care of the Regional Hospital Centre of Lille, France, and in accordance with the European legal and institutional guidelines (86/609/CEE) for the care and use of laboratory animals.

Inoculum preparation and induction of colitis

Each animal was inoculated on day 1 by oral gavage with 200 µL of phosphate-buffered saline (PBS) containing 107 live C. albicans cells. Mice were given 1.5% DSS (MW 36–50 kDa; MP Biomedicals, LLC, Germany) in drinking water from day 1 to day 14 to induce intestinal inflammation. Three days after C. albicans oral challenge, mice were administered by oral gavage with a single-daily dose of either 107 lyophilized S. cerevisiae strains or 1 mg of β-glucan fraction for 2 weeks. Lyophilized S. cerevisiae strains were rehydrated for 30 min in PBS at 37°C before administering to the mice [60]. The presence of yeasts in the intestinal tract was followed daily by performing plate counts of faeces (approximately 0.1 g/sample) collected from each animal [39]. The faecal samples were suspended in 1 mL saline, ground in a glass tissue homogenizer and plated onto Candi-Select medium (Bio-Rad Laboratories, Marnes la Coquette, France). This chromogenic medium is designed for the isolation of yeasts from clinical specimens and is intended to differentiate medically important yeast species depending on the colour of the colonies [61]. Colonies of C. albicans were counted after 48 h incubation at 37°C. The results were noted as colony forming units (CFUs)/µg of faeces.

Presence of C. albicans colonization in the gastrointestinal tract

To check for C. albicans colonization, the animals were sacrificed and the gastrointestinal tract was removed and separated into the stomach, ileum and colon. The tissues were cut longitudinally. After removal of intestinal contents, the tissues were washed several times in PBS to minimize surface contamination from organisms present in the lumen [62]. Serial dilutions of homogenates were performed. The results were noted as C. albicans CFUs/mg of tissue.

Assessment of clinical parameters

The mortality rate of DSS-treated mice was determined daily and a colon biopsy was taken immediately after death for histological analysis. Total body weight was measured daily. The data are expressed as mean percent change from starting body weight. Daily clinical activity score ranging from 0 to 8 was calculated as described elsewhere [39], [63].

Determination of histological score

Rings of the transverse part of the colon were fixed overnight in 4% paraformaldehyde-acid and embedded in paraffin for histological analysis. Cross-sections (4 µm thick) were stained with haematoxylin-eosin (Sigma-Aldrich, France). Histological scores were evaluated by two independent, blinded investigators who observed two sections per mouse at magnifications of ×10 and ×100. The scores were determined in accordance with Siegmund et al. [63] and the sections were evaluated for the following two subscores: (i) a score for the presence and confluence of inflammatory cells, including neutrophils, in the lamina propria and submucosa or transmural extension; and (ii) a score for epithelial damage, focal lymphoepithelial lesions, mucosal erosion and/or ulceration and extension to the bowel wall. The two subscores were added together and the combined histological score ranged from 0 (no changes) to 6 (extensive cell infiltration and tissue damage).

Real-time mRNA quantification

Total RNA was isolated from colon samples using a NucleoSpin RNA II kit (Macherey-Nagel, France) following the manufacturer’s instructions, with 20–50 units of DNase I (RNase-free) at 37°C for 30 min to avoid contamination with genomic DNA. RNA quantification was performed by spectrophotometry (Nanodrop; Nyxor Biotech, France). Reverse transcription of mRNA was carried out in a final volume of 26 µL from 1 µg total RNA using 300 U M-MLV reverse transcriptase (Invitrogen, France) according to the manufacturer’s instructions with 500 ng oligo(dT) 12–18 and 50 U ribonuclease inhibitor (RNase-Out, Promega). PCR was performed using an ABI 7000 prism sequence detection system (Applied Biosystems, France) with SYBR green (Applied Biosystems, France). Amplification was carried out in a total volume of 25 µL containing 0.5 µL of each primer [6], [39] and 1 µL of cDNA prepared as described above. SYBR green dye intensity was analyzed using Abiprism 7000 SDS software (Applera Corp.). All results were normalized to the housekeeping gene β-actin.

Statistical analysis

Data are expressed as the mean ± SE of five mice in each group. All comparisons were analyzed by the Mann-Whitney U test. Statistical analyses were performed using the StatView™ 4.5 statistical program (SAS Institute Inc., Meylan, France). Differences were considered significant when the P value was <0.05.

Acknowledgments

The authors thank Nadine FRANÇOIS, Caroline DUBUQUOY, Emilie GANTIER, and Edmone ERDUAL for their excellent technical assistance and Val HOPWOOD for editing the manuscript.

Funding Statement

This work was funded by the program LEVACI issued from the French Government research plan FUI 5th AAP (DGE-Lille University contract number 082906131, European funds FEDER and local funds from the Région Nord-Pas de Calais, Lille Métropole Communauté Urbaine). LEVACI partners belong to research clusters Nutrition-Santé-Longévité and Végépolys. This work was also funded by the FP7 Health 260338 “ALLFUN” project “Fungi in the setting of inflammation, allergy and auto-immune diseases: translating basic science into clinical practices.” The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Herpes Zoster Vaccine Effectiveness against Incident Herpes Zoster and Post-herpetic Neuralgia in an Older US Population: A Cohort Study

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Stephan Harbarth, Academic Editor
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Abstract

Background

Herpes zoster is common and has serious consequences, notably post-herpetic neuralgia (PHN). Vaccine efficacy against incident zoster and PHN has been demonstrated in clinical trials, but effectiveness has not been studied in unselected general populations unrestricted by region, full health insurance coverage, or immune status. Our objective was to assess zoster vaccine effectiveness (VE) against incident zoster and PHN in a general population-based setting.

Methods and Findings

A cohort study of 766,330 fully eligible individuals aged ≥65 years was undertaken in a 5% random sample of Medicare who received and did not receive zoster vaccination between 1st January 2007 and 31st December 2009.

Incidence rates and hazard ratios for zoster and PHN were determined in vaccinated and unvaccinated individuals. Analyses were adjusted for age, gender, race, low income, immunosuppression, and important comorbidities associated with zoster, and then stratified by immunosuppression status. Adjusted hazard ratios were estimated using time-updated Cox proportional hazards models.

Vaccine uptake was low (3.9%) particularly among black people (0.3%) and those with evidence of low income (0.6%). 13,112 US Medicare beneficiaries developed incident zoster; the overall zoster incidence rate was 10.0 (9.8–10.2) per 1,000 person-years in the unvaccinated group and 5.4 (95% CI 4.6–6.4) per 1,000 person-years in vaccinees, giving an adjusted VE against incident zoster of 0.48 (95% CI 0.39–0.56). In immunosuppressed individuals, VE against zoster was 0.37 (95% CI 0.06–0.58). VE against PHN was 0.59 (95% CI 0.21–0.79).

Conclusions

Vaccine uptake was low with variation in specific patient groups. In a general population cohort of older individuals, zoster vaccination was associated with reduction in incident zoster, including among those with immunosuppression. Importantly, this study demonstrates that zoster vaccination is associated with a reduction in PHN.

Please see later in the article for the Editors’ Summary

Introduction

Herpes zoster is a significant public health problem affecting 1 million individuals in the US per year and associated with important sequelae [1],[2]. Herpes zoster occurs following reactivation of latent varicella zoster virus (VZV) infection and presents with a painful vesicular rash, which frequently in older individuals leads to prolonged pain, post-herpetic neuralgia (PHN), with a major impact on quality of life [2]. Vaccine efficacy has been shown in trials [3],[4]; in a selected insured population [5]; and among people with any of five specific immune-mediated diseases [6] but not among an unselected population in a clinical setting. Zhang et al. demonstrated that despite Advisory Committee for Immunization Practices (ACIP) recommendations, individuals with immunosuppression received the live herpes zoster vaccine in clinical practice [7]. The lack of adherence to ACIP recommendations on vaccination is not entirely surprising given that individuals with immunosuppression are not only at increased risk of incident herpes zoster but also at significantly increased risk of herpes zoster complications, in particular prolonged, severe PHN [8],[9]. Previous research has suggested that the varicella vaccine may be efficacious and safe in people with immunosuppressive disorders [10][12]. Similar evidence about vaccine effectiveness (VE) is lacking in relation to the zoster vaccine in individuals with serious immune suppression, beyond effectiveness among those with the selected immune-mediated disorders examined to date.

Important outstanding research questions with great relevance to policy include VE in unselected population-based elderly US populations; this includes effectiveness against PHN, which has not been assessed in routine practice. The report by Zhang et al. also highlights the additional importance of studying further VE in those with immunosuppression [6]. This is the first study to the best of our knowledge to assess the effectiveness of herpes zoster vaccine against both incident herpes zoster and PHN in an unselected older population including those with immunosuppression.

Methods

Ethics

Ethics approval was obtained from Centers for Medicare & Medicaid Services (CMS) (data use agreement 21520) and the Ethics committee of the London School of Hygiene and Tropical Medicine. Any data cell containing fewer than 11 beneficiaries have not been shown as per the CMS Data Use Agreement.

Data Source

Medicare is a US administrative claims program mainly for individuals aged >65 y covering 15% of the US population. There are 44 million beneficiaries, of which more than half the individuals are aged 65–75 y.[13] This study was based on the 5% random Medicare Standard Analytic Files (SAF) including Denominator, Inpatient hospital discharge records (MedPAR), Physician/Supplier (Carrier) and Outpatient files from January 1st 2007 to December 31st 2009 obtained from the CMS.

Study Population

Study participants were aged 65 y or greater with at least 12 mo continuous enrolment in Medicare parts A (which covers inpatient care) and B (physician services and facility charges) and at least 6 mo continuous enrolment in part D (drug benefits) of Medicare. The start of follow-up was the first date an individual fulfilled all the eligibility criteria with an additional 12-mo baseline pre-study observation period added to ensure observation of incident rather than prevalent zoster. End of follow-up was defined as the earliest of end of eligibility, date of death, development of herpes zoster, or the end of the study period. Individuals enrolled in health maintenance organizations or Medicare Advantage plans were excluded from the study as their records are not processed by CMS, hence information on clinical events is not available. Individuals with episodes of herpes zoster in the first year pre-study observation period were excluded from analysis to exclude prevalent cases. Additionally, individuals who received the herpes zoster vaccine during the baseline pre-study observation period were excluded from analysis (Figure 1).

Figure 1

Flow chart of analysis cohort.

Exposure

Herpes zoster vaccine was identified based on the presence of Current Procedural Terminology (CPT) code 90736. Additionally, specific National Drug Codes (NDCs) for herpes zoster vaccine were identified. A definite administration date was considered present if a CPT code 90741 or Healthcare Common Procedural Coding system (HCPCS) code G0377 was present within 7 d of vaccine purchase; otherwise the date of recording of the NDC code for herpes zoster vaccine was considered to be the administration date.

Outcomes

Incident herpes zoster cases were identified as those with both the presence of a specific International Classification of Diseases, Ninth Revision, Clinical Modification (ICD-9-CM) diagnostic code for herpes zoster, excluding those with specific ICD-9-CM codes for PHN, and the use of antivirals, including acyclovir, famciclovir, or valacyclovir, within 7 d either before or after the diagnostic code for herpes zoster [6]. Cases were identified from outpatient, inpatient, or health care provider (carrier) files. The approach of using both the presence of diagnostic codes and receipt of antivirals was proposed by Zhang et al. to increase the positive predictive value of a herpes zoster diagnosis in an administrative data source [6]. Incident herpes zoster was defined as an episode of herpes zoster without any evidence of herpes zoster or PHN for at least 1 y previously. A sensitivity analysis was undertaken defining incident herpes zoster as the presence of an ICD-9 code for herpes zoster irrespective of receipt of antiviral therapy. PHN was identified using a modification of the method proposed by Klompas et al. for administrative sources [14]. On the basis of this method, PHN was identified as those with a first episode of zoster with a further zoster diagnostic code after 90 d with a relevant prescription for analgesia, anticonvulsant, or antidepressant therapy on the same day as the recorded consultation. The presence of codes for non-specific neuralgia or for neurological complications of zoster after 90 d was also consistent with PHN. The PHN analysis was repeated after 30 d using the same diagnostic criteria.

Covariates

Current age was determined using date of birth identified from the Medicare beneficiary file; age was categorized into 5-y age bands as a time-varying covariate. Age at the start of the study was also modelled as a continuous variable in a sensitivity analysis. Race was identified from the Medicare SAF Denominator files, derived from the Social Security Administration’s master beneficiary record (designated via self-report) and categorized into white, black, and other (not including those with missing data on race). It is critical to study race in any study of zoster epidemiology as incidence rates in individuals with black skin are significantly lower than in white individuals [15]. “State buy -in” at any point during follow-up was assessed as a marker for low income. “State buy-in” reflects that the state pays Medicare premiums for an individual who is eligible due to low income. It can be assumed that people with state buy-in have resources that are less than twice that of the Supplemental Security Income threshold; hence “state buy-in” can be used a proxy for low income. We also determined the proportions of individuals receiving herpes zoster vaccination and developing incident herpes zoster by state and used these data to define quintiles of states defined by each of these two variables.

Immunosuppression status was identified as a time-varying covariate. Once individuals developed leukaemia, lymphoma, or HIV, as determined by the presence of two diagnostic ICD-9-CM codes on different days within outpatient, inpatient, or provider files, they were defined as being immunosuppressed from that point forward. These specific disorders were selected as these are specific ACIP contraindications for the zoster vaccine [7].

Immunosuppressive medications were identified from the part D drug files and an individual was defined as being immunosuppressed for 6 mo following the prescription of any immunosuppressive medication. If a patient received a further script during that period, they remained immunosuppressed. Other comorbidities including immune-mediated disorders or others previously identified as being associated with increased risks of zoster such as chronic obstructive pulmonary disease (COPD), diabetes, and systemic lupus erythematosis (SLE) were identified from medical records by the presence of two ICD-9-CM codes on different days in the outpatient or carrier (provider) files or one or more codes from inpatient records. Individuals with autoimmune disorders such as SLE were considered immunocompetent unless they received immunosuppressive therapy; these disorders are not ACIP contraindications to vaccination.

Statistical Analysis

Characteristics associated with receipt of the herpes zoster vaccine were explored by examining proportions of person-years of follow-up contributed by demographic and clinical attributes. Incidence rates for herpes zoster and PHN overall and by population characteristics were determined by identifying the number of events divided by person-years of follow-up. Cox regression was used to derive hazard ratios for herpes zoster and PHN in the vaccinated compared with the unvaccinated, adjusting for relevant confounders identified from the previous literature [3],[15], including age, gender, race, low income, immunosuppression, and comorbidities associated with herpes zoster, with age and immunosuppression being included in the analysis as time-varying covariates. Other comorbidities including COPD were treated as binary variables. State quintiles of vaccination and zoster incidence were added to the model to determine if they were confounders. Checks for collinearity were undertaken by assessing variance inflation factors for independent variables. Interaction terms were explored for associations between vaccination and age group and gender. VE was calculated as (1 – the adjusted hazard ratio). Stratified analysis of VE by immune status was adjusted for demographic characteristics with a sensitivity analysis adjusting for other comorbidities. A further sensitivity analysis was undertaken assessing VE against PHN using logistic regression amongst those with zoster with at least 6 mo of follow-up following zoster. All analyses were undertaken using STATA (version 11.0).

Results

Vaccination Rates

Of the 766,330 eligible participants, 29,785 (3.9% of people; 2.1% of person-time) had herpes zoster vaccination during the study period. Vaccination rates were lower in the oldest age group (1.5% in those aged 80 y or greater), in black individuals (0.3% compared to 2.4% in white individuals), and lower in those with evidence of low income (Table 1)—0.6% in those with evidence of low income were vaccinated as compared to 2.6% in individuals with no evidence of low income. 140,925 individuals were immunosuppressed at some point during follow-up and 4,469 of these individuals were immunosuppressed at the time of herpes zoster vaccination.

Table 1

Person-years by vaccination status and characteristics.

Herpes zoster Incidence Rates

Incidence rates for herpes zoster using the antiviral definition were higher in older age groups, in women, in those with any immunosuppression (adjusted hazard ratio 1.80 [95% CI 1.70–1.90]) and in those with specified immune-mediated disorders, including inflammatory bowel disease and SLE, and other disorders such as chronic kidney disease and COPD (Table 2). Lower incidence rates were seen in people who reported being black (adjusted hazard ratio 0.51 ([95% CI 0.47–0.56]) and those with any evidence of low income (adjusted hazard ratio 0.86 [95% CI 0.82–0.90]).

Table 2

Incidence rates for herpes zoster by disease definition and patient characteristics.

Herpes zoster Vaccine Effectiveness

Overall, 154 vaccinees experienced incident herpes zoster episodes (defined using the specific antiviral definition) during 28,291 person-years of follow-up compared to 12,958 events in 1,291,829 person-years of follow-up in those not vaccinated, giving an incidence rate of herpes zoster in vaccinees of 5.4 (95% CI 4.6–6.4) per 1,000 person-years compared to 10.0 (95% CI 9.8–10.2) per 1,000 person-years in those not vaccinated. All variance inflation factors were less than 1.2, suggesting collinearity was not a major issue, and no significant interactions were detected. The overall vaccine effectiveness (VE) for herpes zoster in vaccinees adjusted for age, gender, race, immunosuppression, low income, and comorbidity was 0.48 (95% CI 0.39–0.56) (Table 3). Incorporating age at the start of the study as a continuous variable did not change study findings: adjusted VE, 0.48 (95% CI 0.40–0.56). The median time to vaccine failure was 168 d. In immunocompromised vaccinees, there were 24 events in 1,981 person-years of follow-up, giving an adjusted VE of 0.37 (95% CI 0.06–0.58). Adjusting for state quintiles of either proportions receiving herpes zoster vaccination or proportions with incident herpes zoster did not modify study findings (adjusted VE 0.48 [95% CI 0.30–0.56] and 0.48 [95% CI 0.30–0.56, respectively]). Proportions vaccinated per state varied from 0.05% to 11.02% (Figure 2) and proportions of individuals developing incident herpes zoster during follow-up per state varied from 0% to 7%. At 90 d or greater following zoster, the adjusted VE was 0.59 (95% CI 0.21–0.79) for PHN in vaccinees compared to those not vaccinated, after adjusting for age, gender, race, and other comorbidities (numbers suppressed to remain compliant with CMS’s small-sized cell privacy policy). At 30 d or longer following zoster, 16 vaccinees developed PHN during 71,457 person-years of follow-up compared to 1,665 events during 2,563,404 person-years of follow-up in those not vaccinated, giving an adjusted VE of 0.62 (95% CI 0.37–0.77) for PHN, after adjusting for age, gender, race, immunosuppression status, and other comorbidities (Table 4). Results of the logistic regression analysis amongst those with zoster showed materially similar estimates of protection against PHN, albeit with wider confidence intervals (adjusted VE 0.64 [95% CI 0.11–0.85]). Lower VE against incident herpes zoster and PHN was seen when using the general rather than the specific disease definition.

Figure 2

Percentage vaccinated by state.
Table 3

Zoster vaccine effectiveness against incident herpes zoster by characteristics and disease definition.
Table 4

Zoster vaccine effectiveness against PHN by characteristics and disease definition.

Discussion

This is the first population-based study, to the best of our knowledge, to demonstrate the effectiveness of herpes zoster vaccination against PHN in a routine population setting. Study findings are consistent with efficacy data from the Shingles Prevention Study (SPS) randomised controlled trial (RCT) [3]. This work complements the findings of previous observational studies that have shown effectiveness of herpes zoster vaccination in immunocompetent insured individuals in southern California and in older individuals with selected immune-mediated diseases, as this study is the first study to the best of our knowledge to determine effectiveness against incident herpes zoster in a population-based cohort of older individuals across the US, not restricted by geographic region, immune status, or insurance status [5],[6].

Low uptake of herpes zoster vaccination (4%) was seen overall with variations in uptake by age, race, and low income levels. Overall VE of 48% was demonstrated against incident herpes zoster, 62% against PHN after 30 d and 59% against PHN after 90 d. In immunosuppressed individuals, VE against incident herpes zoster was 37%.

In the SPS RCT, Oxman et al. demonstrated herpes zoster vaccine efficacy against incident herpes zoster (51%) and PHN (67%) in 38,546 immunocompetent individuals (19,270 of whom were vaccinated) aged 60 y or greater with no history of herpes zoster [3]. Subsequent to this study, Schmader et al. performed an RCT in 22,439 immunocompetent individuals aged 50–59 y in North America and Europe, demonstrating vaccine efficacy of 69.8% for incident herpes zoster; efficacy against PHN was not determined [4]. Our findings are closer to those of the SPS study with similar estimates and confidence intervals for VE against incident zoster, which likely relates to the older age of participants in our study population. Our findings for VE against PHN were similar to those observed in the SPS trial [3]. RCTs typically have excellent internal validity, but post-licensure observational studies are necessary to inform generalisability of research findings.

Our study also confirms the results of the study by Tseng et al., which demonstrated the effectiveness of the herpes zoster vaccine against herpes zoster incidence in 75,761 immunocompetent vaccinees aged 60 y or greater matched (1[ratio]3) to unvaccinated members; all study participants were fully insured individuals in Kaiser Permanente Southern California [5]. The authors reported VE of 55% against incident herpes zoster, which is very similar to our estimates, despite differences in the study population. Zhang et al. assessed herpes zoster VE in 463,541 Medicare beneficiaries aged 60 y or greater with a restricted range of immune-mediated diseases and 18,683 vaccinees and found an adjusted hazard ratio of 0.61 (95% CI 0.52–0.71) overall [6]. The lower VE in this population likely reflects their underlying immunosuppression. The authors did not observe an increase in risk of herpes zoster following vaccination in those with immune-mediated disease, nor did they detect any cases of herpes zoster in individuals vaccinated while on biologic therapy.

Medicare is an administrative data source so some misclassification of exposures and outcomes is possible. However, this misclassification is likely to be random leading to the possibility of bias towards the null. Despite taking steps to guard against misclassification of the PHN by using the method proposed by Klompas et al. [14] for administrative data sources, the incidence of PHN in this study is lower than in the SPS [3]—1.38 per 1,000 person-years in the placebo group compared to 0.65 per 1,000 in the unvaccinated in our study—which might suggest some misclassification. In a recent study combining administrative data with medical record review, 3.9% of those with zoster developed PHN, which is lower than the 6.7% of people observed in this study [16]. There is a possibility that we underestimated herpes zoster vaccine uptake if individuals paid for their own vaccination; however, given that all of these individuals have part D Medicare (drug benefit) coverage, individual payment is unlikely because of the cost of the vaccine (US$159 for a single dose, not including administration costs). As these are observational data, the exposure—herpes zoster vaccination—was not randomly allocated. Our study demonstrates that vaccine uptake was not random and was likely to have been influenced by the demographic characteristics of beneficiaries. As data on exposures and outcomes were not collected for research purposes, there are unmeasured potential confounders including smoking and obesity, which are not routinely recorded in an administrative data source, despite the availability of diagnostic codes. Previous studies have not suggested that either of these covariates are major risk factors for the development of incident herpes zoster or PHN and, therefore, they are unlikely to confound the associations [15]. The study period was relatively short; the first 12 mo following eligibility was excluded to enable study of incident rather than prevalent herpes zoster. This limited duration results in the inability to study long-term vaccine effects but does not impact the study of VE. Additionally, while VE was assessed in individuals with immunosuppression, assessment of adverse effects or vaccine safety was not the hypothesis under study. The number of vaccinated immunosuppressed individuals in the study was modest resulting in a lack of precision of the estimate of VE in this group.

Our study is a large population-based cohort; the size of the cohort gives sufficient statistical power to study VE against herpes zoster and PHN and results are estimated relatively precisely; our findings would be unlikely if there was no effect in the population. In addition, Medicare beneficiaries are reasonably representative of the general US elderly population, with 98% of Americans aged 65 y or greater being enrolled in Medicare in 2009, increasing the generalisability of our findings [13]. Medicare datasets have high quality data available on demographic details of beneficiaries and clinical encounters, including prescription data. A strict definition for herpes zoster was used and therefore misclassification of incident herpes zoster is not likely, although it is not possible to completely exclude misclassification [16],[17]. The higher VE when using the specific definition could reflect some misclassification of zoster using the general definition. Alternatively, those with zoster who did not receive antivirals might include a large proportion with very mild disease; in the SPS, the zoster vaccine was shown to have higher efficacy against zoster with appreciable acute morbidity than against any zoster [3]. If those not receiving antivirals had milder incident zoster, this could lead to over-estimation of VE while providing reasonable estimates for significant zoster episodes. In this study VE was determined after adjusting for a wide range of confounders, including demographic details, immunosuppression, and immune-mediated diseases; despite the large size of this dataset, in some instances adjusting for confounding led to wide confidence intervals, for example when assessing VE in immunocompromised vaccinees.

Conclusions

Herpes zoster vaccination was associated with a significant reduction in incident herpes zoster and PHN in routine clinical use. This study also supports effectiveness of the vaccine against incident herpes zoster in immunosuppressed individuals, although the number of immunosuppressed individuals was small, resulting in lack of precision in the estimate. Given that these individuals are at greatest risk of both herpes zoster and complications, this may have important implications for policy. The findings are relevant beyond US medical practice, being of major importance to the many countries, including the UK, that are actively considering introducing the zoster vaccine into routine practice in the near future.

Despite strong evidence supporting its effectiveness, clinical use remains disappointingly low with particularly low vaccination rates in particular patient groups. This study shows that herpes zoster vaccination is associated with a reduction in PHN in routine clinical use. As PHN is the major complication of herpes zoster and is associated with highly significant morbidity and adverse impacts on quality of life, substantial efforts are needed to increase vaccine use in routine care of elderly individuals.

Acknowledgments

We thank Krishnan Bhaskaran for advice on statistical analysis and Ole Hoffstad for assistance creating Figure 2.

Abbreviations

ACIP
Advisory Committee for Immunization Practices
CMS
Centers for Medicare & Medicaid Services
COPD
chronic obstructive pulmonary disease
PHN
post-herpetic neuralgia
RCT
randomised controlled trial
SLE
systemic lupus erythematosis
SPS
Shingles Prevention Study
VE
vaccine effectiveness

Funding Statement

This research was funded by an NIHR Clinician Scientist award from the National Institute for Health Research, UK Department of Health, awarded to SML. The funders of the study had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the UK Department of Health.

References

1. Yawn BP, Saddier P, Wollan PC, St Sauver JL, Kurland MJ, et al. (2007) A population-based study of the incidence and complication rates of herpes zoster before zoster vaccine introduction. Mayo Clin Proc 82: 1341–1349. [PubMed]
2. Johnson RW, Bouhassira D, Kassianos G, Leplège A, Schmader KE, et al. (2010) The impact of herpes zoster and post-herpetic neuralgia on quality-of-life. BMC Med 8: 37. [PMC free article] [PubMed]
3. Oxman M, Levin M, Johnson G, Schmader K, Straus S, et al. (2005) A vaccine to prevent herpes zoster and postherpetic neuralgia in older adults. N Engl J Med 352: 2271–2284. [PubMed]
4. Schmader KE, Levin MJ, Gnann JW, McNeil SA, Vesikari T, et al. (2012) Efficacy, safety, and tolerability of herpes zoster vaccine in persons aged 50–59 years. Clin Infect Dis 54: 922–928. [PubMed]
5. Tseng HF, Smith N, Harpaz R, Bialek SR, Sy LS, et al. (2011) Herpes zoster vaccine in older adults and the risk of subsequent herpes zoster disease. JAMA 305: 160–166. [PubMed]
6. Zhang J, Xie F, Delzell E, Chen L, Winthrop KL, et al. (2012) Association between vaccination for herpes zoster and risk of herpes zoster infection among older patients with selected immune-mediated diseases. JAMA 308: 43–49. [PMC free article] [PubMed]
7. Harpaz R, Ortega-Sanchez I, Seward J (2008) Prevention of herpes zoster: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 57: 1–30; quiz CE32–34. [PubMed]
8. Gebo K, Kalyani R, Moore R, Polydefkis M (2005) The incidence of, risk factors for, and sequelae of herpes zoster among HIV patients in the highly active antiretroviral therapy era. J Acquir Immune Defic Syndr 40: 169–174. [PubMed]
9. Strangfeld A, Listing J, Herzer P, Liebhaber A, Rockwitz K, et al. (2009) Risk of herpes zoster in patients with rheumatoid arthritis treated with anti-TNF-alpha agents. JAMA 301: 737–744. [PubMed]
10. Gourishankar S, McDermid J, Jhangri G, Preiksaitis J (2004) Herpes zoster infection following solid organ transplantation: incidence, risk factors and outcomes in the current immunosuppressive era. Am J Transplant 4: 108–115. [PubMed]
11. Son M, Shapiro ED, LaRussa P, Neu N, Michalik DE, et al. (2010) Effectiveness of varicella vaccine in children infected with HIV. J Infect Dis 201: 1806–1810. [PMC free article] [PubMed]
12. Gershon AA, Levin MJ, Weinberg A, Song LY, LaRussa PS, et al. (2009) A phase I–II study of live attenuated varicella-zoster virus vaccine to boost immunity in human immunodeficiency virus-infected children with previous varicella. Pediatr Infect Dis J 28: 653–655. [PMC free article] [PubMed]
13. Center for Medicare and Medicaid Services (2009) Center for Medicare and Medicaid Services 2009 data compendium. Baltimore (Maryland): Center for Medicare and Medicaid Services.
14. Klompas M, Kulldorff M, Vilk Y, Bialek SR, Harpaz R (2011) Herpes zoster and postherpetic neuralgia surveillance using structured electronic data. Mayo Clin Proc 86: 1146–1153. [PMC free article] [PubMed]
15. Thomas S, Hall A (2004) What does epidemiology tell us about risk factors for herpes zoster? Lancet Infect Dis 4: 26–33. [PubMed]
16. Yawn BP, Wollan P, St Sauver J (2011) Comparing shingles incidence and complication rates from medical record review and administrative database estimates: how close are they? Am J Epidemiol 174: 1054–1061. [PMC free article] [PubMed]
17. Donahue J, Choo P, Manson J, Platt R (1995) The incidence of herpes zoster. Arch Intern Med 155: 1605–1609. [PubMed]

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studentjournalofmedicine

A strange case of ingrown toenail treated with phenol

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Abstract

We experienced a strange case of ingrown toenail, which had developed as a huge mass and enveloped the nail of the left first toe. The patient had self-treated his ingrown toenail for a period of one year with an ointment available over the counter. However, the granulation tissue on both sides of the nail had increased gradually and advanced over the nail plate in the medial direction. Finally, the granulation tissue on both sides had adhered to the nail and epithelial cells advanced over the granulation tissue completely. During surgery, the epithelized granulation tissue was excised at the bilateral terminal base point, and the posterior nail fold and the nail matrix were cauterized completely with phenol. Eighteen months after the operation there was no recurrence of the ingrown toenail.

INTRODUCTION

The ingrown toenail is a common problem which occurs mostly in the first toe and causes a high amount of morbidity in affected patients. In this report we document a strange case of ingrown toenail of the first toe, which had developed as a huge mass and enveloped the nail. We describe here the cauterization of the nail matrix with phenol after surgical resection of the tumor, a treatment that had good results.

CASE REPORT

A 56-year-old man came to our office complaining of pain, offensive smelling discharge and disappearance of the nail of the right great toe. Physical examination showed epithelized mass on the nail plate and fistulation from the proximal nail fold to the tip of the toe, epithelization did not occur on the nail plate side of the mass (Figure 1). The clinical diagnosis of this tumor was epithelized granulation tissue caused by ingrown toenail, however squamous cell carcinoma was not denied completely.

Figure 1

The first toe of a 56-year-old man. Photograph shows epithelized granulation tissue on the nail plate and fistulation from the proximal nail fold to the tip of the toe

The operative treatment involved anesthetizing the toe with a digital nerve block using 1% lignocaine. The tumor mass was excised at the bilateral terminal base point, and was sent to pathology for an immediate diagnosis. The pathological finding was granulation tissue with partial scar formation, and the external surface of the tumor was covered with epithelial cells. Next, the nail plate was incised longitudinally from top to the root at a width of approximately 5mm. The posterior nail fold and the nail matrix were cauterized completely with an 88% phenol-immersed cotton-tipped applicator for five minutes. The excessive skin and soft tissue of the tip of the toe were excised and trimmed (Figure 2).

Figure 2

Bilateral chemical matricectomy of the matrix was done with phenol after resection of the tumor

Eighteen months after the operation, there is no recurrence of the ingrown toenail (Figure 3).

Figure 3

Eighteen months after the operation there was no recurrence of the ingrown toenail

DISCUSSION

Ingrown toenail deformity is a common nail pathology that causes intractable pain and discomfort, hindering normal walking and markedly decreasing the quality of life of patients. Ingrown toenails could be a cause of granulation tissue of the lateral nail fold of the finger or toe (1). However, an ingrown toenail creating such a huge mass on the nail plate, as in this case, is very rare. In the literature, to our knowledge, there have been no reports of cases similar to our patient.

In the case presented here, the patient had treated his ingrown toenail himself for one year with an ointment purchased over the counter before he came to our office. However the granulation tissue of both sides of the nail had increased gradually and advanced on the nail plate in the medial direction. Finally, the granulation tissue from both sides adhered and epithelial cells advanced over the granulation tissue completely. We believe such pathogenesis of this tumor is very uncommon.

Conservative and surgical forms of therapy for ingrown nails have been used, however, there is no common or unique form of treatment (26). In recent years, matrix phenolization of the nail bed has been used increasingly, and has been reported to give less discomfort and lower recurrence rates (7,8). The procedure of phenolization is easy to perform and does not require specialized equipment (9). In our reported case, after resection of the epithelized granuloma, both sides of the posterior nail fold and the nail matrix were cauterized completely with phenol. After this simple treatment, there was no recurrence of the ingrown toenail. The only disadvantage of the treatment was the smallness of the toenail, however, the patient did not complain of this aesthetic disadvantage due to the advantages of freedom from pain and the bad odour.

REFERENCES

1. Chapeskie H. Ingrown toenail or overgrown toe skin? Can Fam Physician. 2008; 54:1561–1562. [PMC free article] [PubMed]
2. Moriue T, Yoneda K, Moriue J, et al. A simple therapeutic strategy with super elastic wire for ingrown toenails. Dermatol Surg. 2008; 1729–1732. [PubMed]
3. Arai H, Arai T, nakajima H, et al. Formable acrylic treatment for ingrowing nail with gutter splint and sculptured nail. Int J Dermatol. 2004; 43: 759–765. [PubMed]
4. Matumoto K, Hashimoto I, Nakanishi H, et al. Resin splint as a new conservative treatment for ingrown toenails. J Medical Invest. 2010; 57:321–325. [PubMed]
5. Nazari S. A simple and practical method in treatment of ingrown nails: splinting by flexible tube. J Eur Acad Dermatol Venereol. 2006; 20: 1302–1306. [PubMed]
6. Rounding C, Hulm S. Surgical treatment for ingrown toenails. Cochrane Database Syst Rev. 2005; 2; CD001514. [PubMed]
7. Di Chiaccihio N, Belda W, Jr, Di Chiacchio NG, et al. Nail matrix phenolization for treatment of ingrowing nail: technique report and recurrence rate of 267 surgeries. Dermatol Surg. 2010; 36: 534–537. [PubMed]
8. Tatlican S, Yamangokturk B, Eren C, et al. Comparison of phenol applications of different durations for the cauterization of germinal matrix: an efficicacy and safety study. Acta Orthop Traumatol Turc. 2009; 43: 298–302. [PubMed]
9. Kimata Y, Uetake M, Tsukada S, et al. Follow-up study of patients treated for ingrown nails with the nail matrix phenolization method. Plast Reconstr Surg. 1995; 95: 719–724. [PubMed]

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studentjournalofmedicine

Tinea Incognito in Korea and Its Risk Factors: Nine-Year Multicenter Survey

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Copyright © 2013 The Korean Academy of Medical Sciences.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Tinea incognito (TI) is a dermatophytic infection which has lost its typical clinical appearance because of improper use of steroids or calcineurin inhibitors. The incidence of TI is increasing nowadays. We conducted retrospective review on 283 patients with TI from 25 dermatology training hospitals in Korea from 2002-2010 to investigate the demographical, clinical, and mycological characteristics of TI, and to determine the associated risk factors. More than half (59.3%) patients were previously treated by non-dermatologists or self-treated. The mean duration of TI was 15.0 ± 25.3 months. The most common clinical manifestations were eczema-like lesion, psoriasis-like, and lupus erythematosus-like lesion. The trunk and face were frequently involved, and 91 patients (32.2%) also had coexisting fungal infections. Among 67 isolated strains, Trichophyton rubrum was the most frequently detected (73.1%). This is the largest study of TI reported to date and the first investigational report concerning TI in Korea. We suggest that doctors should consider TI when a patient has intractable eczema-like lesions accompanied by tinea pedis/unguium. Furthermore, there should be a policy change, which would make over-the-counter high-potency topical steroids less accessible in some countries, including Korea.

Keywords: Calcineurin Inhibitor, Dermatophytoses, Korea, Steroid, Tinea, Tinea Incognito

INTRODUCTION

Tinea incognito (TI) is the term given to a dermatophyte infection, which has been modified in appearance by improper use of steroids or calcineurin inhibitors (1, 2). Since it was first described by Ive and Marks in 1968 (3), a few case reports and a number of review articles have been published on TI in English journals (1, 2, 4-6). Though typical dermatophytic infection on most skin surfaces except scalp, volar areas and nails usually present as annular lesions with erythematous scaly border and central clearing. Therefore, dermatophytic infection may be confused with other skin disorders such as granuloma annulare, discoid lupus erythematosus, pityriasis rosea, erythema annulare centrifugum, erythema migrans, or other dermatological lesions (7). Most textbooks or review articles state that mycological confirmation with laboratory testing before the start of antifungal therapy is recommended because the clinical diagnosis of fungal presence could be inaccurate (8). However, this is not easy to do in practice because of time pressure, inadequate access to equipment, lack of experience, low reproducibility, and so on (8, 9). Misdiagnosed dermatophytic disorders could be treated improperly with steroids. As some high-potency topical steroids are easily accessible as over-the-counter (OTC) products and non-dermatologists can also prescribe topical steroids freely without any fungal examination, the incidence of TI seems to be gradually increasing in Korea (10). However, there has been no published large-scale study on TI in Korea, as yet. For this reason, we investigated the demographics and past medical histories of TI patients, and clinical and mycological characteristics of TI in Korea.

MATERIALS AND METHODS

Patients

In the period from 2002 to 2010, cases of TI were collected retrospectively from dermatologic departments of 25 general hospitals in Korea, which had a dermatology training program. We defined 4 criteria for diagnosis of TI: 1) loss of typical clinical appearance of tinea (Fig. 1A); 2) previous history of corticosteroid (topical or systemic) or calcineurin inhibitor application to present lesions; 3) positive for at least 1 mycological evaluation (KOH examination, mycologic culture, or skin biopsy with DPAS stain); and 4) improvement after antifungal treatment. To be enrolled in this study, all patients had to satisfy all the 4 diagnostic criteria listed above. Only the cases confined to dorsum of hands and feet were included to avoid confusion with tinea pedis and tinea manus.

Fig. 1

Various features of tinea incognito (A-G). Vitiligo-like (A; pre-treatment, B; after 4 weeks of application of topical pimecrolimus, C; 6 weeks after topical antifungal treatment), contact dermatitis-like (D), nonspecific eczema-like (E), seborrheic dermatitis-like

Ethics statement

The study protocol was approved by Institutional Review Boards or Ethics Committees of Pusan National University Hospital and informed consent was obtained.

Demographics, past histories, and clinical characteristics

Clinical data including charts and clinical photos from 25 hospitals were systematically and retrospectively reviewed. Demographic information included age, gender, coexisting diseases, and other dermatologic diseases. Past medical histories included the duration of TI, how the patients obtained the topical steroid or calcineurin inhibitor, and treatment modality. After dividing the patients into 3 groups (dermatologist-treated, non-dermatologist-treated, and self-treated TI groups), the duration of TI and treatment modality were compared among the 3 groups. Regarding the clinical characteristics of TI, the distribution, the most likely clinical feature, and coexisting fungal infections were investigated.

Mycological data

The KOH examination (20% potassium hydroxide) was performed to check for the presence of fungi. Mycological culture was performed on Sabouraud dextrose agar with chloramphenicol and cycloheximide. After incubation at 25℃ for at least 3 weeks, dermatophytes were identified by means of gross morphology, light microscopy, and/or biopsy with PAS stain (11).

Statistical analysis

Pearson’s chi-square test was used to compare the frequency of treatment modalities and one-way ANOVA was used to compare the duration of TI among dermatologist-treated, non-dermatologist-treated, and self-treated TI patients group. A P value of less than 0.05 was considered statistically significant.

RESULTS

Demographics

After thorough review, 283 patients fulfilled the diagnostic criteria of TI in this study. The mean age was 44.0 ± 22.5 yr (range 3-94) and 125 patients (44.3%) were female. Table 1 shows the age distribution of TI patients with a slightly lower frequency of patients with TI under 10 and over 80 yr old. Sixty-five patients (23.0%) had coexisting diseases at first clinic visit such as hypertension in 37 (13.1%), diabetes in 23 (8.1%), and hepatitis in 7 (2.5%). Five patients had underlying malignancy (1.8%), 2 patients suffered from angina, and 2 patients had asthma. In addition, 1 patient had adrenal insufficiency, 1 patient had myasthenia gravis, 1 had depression, and 1 had epilepsy. Sixteen patients (5.7%) had coexisting dermatologic diseases including 5 patients with atopic dermatitis (1.8%), 4 patients with psoriasis (1.4%), 3 with systemic lupus erythematosus (1.1%), and 2 with seborrheic dermatitis (0.7%). There was 1 patient with rosacea, and 1 patient with bullous pemphigoid.

Table 1

Demographics and past histories of 283 cases of tinea incognito in Korea during 2002-2010

Past medical histories

The mean duration of TI in the study patients was 15.0±25.3 months. While mean duration of self-treated TI patients was 9.0±11.1 months, that of TI patients treated by dermatologists and non-dermatologists was 16.4±25.8 and 15.7±28.1 months, respectively. There was no statistical significance among the 3 groups (P = 0.234) (Table 2).

Table 2

Mean duration of the disease and previous treatment modalities according to past physician’s specialty

Before coming to the teaching hospital, 40.6% of TI patients received treatment from a dermatologist, 43.8% from non-dermatologists, and another 15.5% were self-treated. While all of self-treated patients used topical steroids only, people treated by dermatologists or non-dermatologists used various treatment modalities such as topical/systemic steroids, topical/systemic antibiotics, topical calcineurin inhibitor, steroid intralesional injection, or a combination of aforementioned agents. Overall, most of TI patients were treated with topical steroids only (86.9%), and other treatment modalities included topical and systemic steroids (6.4%), topical steroid and topical calcineurin inhibitor (1.4%), and topical calcineurin inhibitor (0.7%), etc. There were no significant differences in treatment modalities according to past physician’s specialty (p > 0.05).

Clinical characteristics

Overall, the trunk (30.4%) is the most commonly affected area of TI followed by the face (24.4%), foot (13.8%), multiple involvements (13.8%), the groin (9.9%), and hand (7.8%) (Table 3). The clinical features were variable, but regardless of distribution, over more than three-quarters of all study patients showed eczema-like (82.0%) lesions which included nonspecific eczema, contact dermatitis, seborrheic dermatitis, and atopic dermatitis. Less often, TI mimicked psoriasis (6.0%), lupus erythematosus (2.5%), impetigo (1.4%), urticaria (1.2%), folliculitis (0.7%), and other dermatological lesions (Table 3). According to the anatomical distribution, facial TI presented as eczema-like (76.8%), lupus erythematosus-like (8.7%), impetigo-like (2.9%), and vitiligo-like (2.9%) lesions. Trunk TI presented as eczema-like (79.1%) and psoriasis-like (10.5%) lesions, and almost all of groin, hand, and foot TI resembled eczema. When TI involved multiple sites, it appeared similar to eczema (69.2%), psoriasis (15.4%), folliculitis (2.6%), and other dermatological lesions (Table 3). In children, TI was most likely to be found in the facial area (11.6%), and the trunk (11.6%), and least likely to be found in the groin (3.6%).

Table 3

Clinical and mycological characteristics of 283 cases of tinea incognito in Korea during 2002-2010

In 91 cases (32.2%), other fungal diseases such as tinea pedis (42.9%), tinea unguium (31.9%), tinea pedis et unguium, or tinea unguium/tinea corporis (25.3%) were diagnosed apart from TI sites (Table 3). According to anatomical distribution, TI of the trunk, groin, or hand was commonly seen with tinea pedis (>50.0%), TI involving foot or multiple areas usually accompanied tinea unguium, and facial TI was strongly associated with tinea pedis et unguium.

Mycological data

Direct microscopic examination was performed in all cases and 260 cases (91.9%) were positive. Of 49 biopsied specimens, 42 (85.7%) showed fungal hyphae and/or spores by D-PAS stain. Sixty-seven cases (23.7%) were cultured in our study and Trichophyton rubrum was the most frequently detected dermatophyte (49/67, 73.%), regardless of TI distribution. Trichophyton mentagrophytes (6/67, 9.0%) and Microsporum canis (6/67, 9.0%) were the second-most frequently detected causative agents, and T. tonsurans, T. verrucosum and M. gypseum were also isolated in a few cases. While only 1 or 2 species of dermatophytes were found in groin, hand, and foot TI, various kinds of fungi were identified in face or trunk TI (Table 3).

DISCUSSION

Tinea incognito had been defined as tinea modified by the improper use of systemic or topical corticosteroids. However, as the use of topical calcineurin inhibitors has been increasing gradually in many dermatologic diseases such as atopic dermatitis, seborrheic dermatitis, intertriginous psoriasis, contact dermatitis and other dermatological lesions (12), the number of cases of modified tinea has also increased (7, 13, 14). Thus, we propose that TI be defined as certain dermatophytoses which have lost their usual clinical manifestation because of erroneous use of systemic/topical corticosteroids or topical calcineurin inhibitor, as in 1 recent article (2). In addition, we think that TI, which involves the hand or the foot, should be confined to the dorsal surface, because tinea pedis and tinea manus cannot be definitively differentiated from TI involving the palm or the sole. It has been suggested that the use of immunosuppressants decreases the fungus-induced local inflammation, and this may allow the fungus to grow slowly with less erythema or scaling causing a “modification” of the typical manifestation of tinea (7).

While TI seems to be common in dermatology practices currently, only a few numbers of large scale studies have been reported (1, 2, 4). These studies were done in Italy, Spain, and Iran. Our study in Korea was designed to be the largest scale study on TI. While previous case reports of TI in Korean literature (10, 15-21) (Table 4) showed female predominance, this study showed relatively equal gender distribution and relatively uniform age distribution (mean: 44.0±22.5 yr) were found except for patients over 80 yr. A recent article regarding TI in Italy (1) also reported equal gender distribution and similar mean age (42 yr), and another article in Iran (4) also revealed equal gender distribution with slightly younger mean age (32.6 yr). Based on these data, we can postulate that TI is common in middle-aged persons with little difference in gender. Moreover, 65 (23.0%) patients in our study had coexisting non-dermatologic diseases such as hypertension, diabetes, hepatitis, malignancy, and so on, and 16 (5.7%) patients had coexisting dermatologic disorders requiring systemic steroids or other immunosuppressants. This was lower than the previous Italian report in that 40% of patients with TI had non-dermatological pathologies which required treatment with systemic steroids (1). Though the percentage of the patients in this study who received immunosuppressive therapy was lower than in the Italian report, the possibility of TI should be kept in mind whenever the patient with skin lesions is on immunosuppressant medications.

Table 4

Previous reports of tinea incognito in Korean literatures

There have been no published data regarding TI according to past treating physician’s specialty or treatment modalities, as yet. Based on our study, over half of the patients were either treated by non-dermatologists (124/283, 43.8%) or self-treated (44/283, 15.5%). TI was thought to be associated with easy access to high-potency OTC topical steroids such as betamethasone valerate by patients and with lack of understanding of tinea by non-dermatologists. Therefore, there should be swift policy changes to limit OTC access of high-potency steroids to patients in Korea. This would limit inappropriate tinea treatment by patients. Furthermore, to reduce the number of cases of TI caused by non-dermatologists, education regarding skin diseases including fungal infections could be provided by Korean Dermatologic Associations.

Surprisingly, about 40% of the patients were treated by dermatologists in this study. Even though tinea can mimic many other skin disorders and there could be selection bias, this ratio seems to be too high. This may mean a lack of mycological evaluation and carelessness of dermatologists when diagnosing tinea infection. It is important for dermatologists to consider fungal infection in the differential diagnosis of skin disorders, and increase the use of laboratory tests for mycological evaluation. In practice, the medical cost for mycological examinations is very low in Korea. However, the patient load is high and doctors are pressed for time. This could be the prime reason for misdiagnosis of fungal infections (22). Therefore, we think that if physicians were better paid for mycological evaluations there might be more active mycological examinations and fewer misdiagnoses of fungal infections.

On the aspect of distribution, the trunk was the most commonly involved site of TI and the face was another commonly involved area, as reported in other original articles (1, 4) and previous Korean literatures. Another recent study regarding 54 childhood TI cases also reported similar results with highest incidence in the trunk and face (2), and our study backed it up with the same results. From these findings, we can postulate that the most common sites of TI are trunk and face regardless of age.

The clinical features of TI were reported to be variable, and the most prevalent features seen are eczema-like disorders such as nonspecific eczema, contact dermatitis, and atopic dermatitis (1, 4, 7), and previous Korean reports about TI also in accordance with it. Similarly, 232 TI patients in this study showed quite various clinical features such as eczema-like, psoriasis-like, lupus erythematosus-like, and etc. Specifically, nearly all cases of hand and foot TI showed eczema-like features. Therefore, when dealing with recalcitrant eczematous lesions on the hand or foot, mycological examination should always be considered. Compared to TI of groin, and hand and foot, where the eczema-like features were quite high, TI of the face, trunk, or multiple areas showed more variable features. Therefore, not only eczema-like lesions but also other recalcitrant skin manifestations resembling psoriasis, lupus erythematosus, impetigo, urticaria, etc., should also be carefully evaluated to rule out TI especially when skin diseases involve the face, the trunk, or multiple areas (Table 5).

Table 5

Suggested risk factors of tinea incognito

Moreover, one-third of our study population (32.2%) had combined fungal diseases, which involved distant areas from present TI, and most of them had dermatophytic infection on their feet regardless of affected areas, including tinea pedis and tinea unguium. Therefore, in patients with refractory skin disease especially resembling eczema and also with concomitant tinea pedis or tinea unguium, TI should be ruled out because these coexisting fungal infections could be an autoinoculation source of superficial dermatophytic infection in another body part at any time.

As many previous studies confirmed (1, 23-25), Trichophyton rubrum (T. rubrum) was also the most frequently identified dermatophyte. T. rubrum is one of the anthropophilic dermatophytes and the most common pathogen in tinea corporis, tinea cruris, tinea manus, tinea pedis, and tinea unguium (26). Since TI affecting the trunk, groin, hands, and feet accounted for almost 60% of T1 in our study, it would not be surprising that T. rubrum was the most commonly isolated dermatophyte. Moreover, the high prevalence of combined tinea pedis and tinea unguium might also have contributed to the high isolation rate of T. rubrum. Anthropophilic dermatophytes have adapted to humans and elicit a mild to non-inflammatory host response unlike zoophilic and geophilic infections (26). This mild inflammatory response might be the cause of the long duration of TI because topical corticosteroids or topical calcineurin inhibitor could alleviate the inflammation, which could be the main mechanism of disguising the typical manifestation of tinea.

In summary, our research was the largest study of TI in Korea to date. We investigated the characteristics of TI according to the primary physician’s specialty though the clinical and mycological results were similar to previous studies. From this study, we can suggest that long-lasting erythematous scaly skin lesions unresponsive to steroids or calcineurin inhibitor as the most important risk factors of TI. Not only truncal or facial involvement, but also combined tinea pedis/unguium or the history of immunosuppressant treatment could also be a good clue in diagnostic approach of TI. Moreover, we can suggest several things to reduce the incidence of TI based on our results. First, reform of OTC sales system of high-potency topical steroids is needed so that they are not as easily available to the public in some countries including Korea. Second, non-dermatologists need to be informed and educated that superficial dermatophytic infection could appear in a variety of forms. Care by experienced dermatologists could also be needed especially when dealing with long-lasting erythematous scaly skin lesions, which have proven to be unresponsive to steroids or calcineurin inhibitor treatment. Finally, we recommend dermatologists not to neglect TI as a possibility in cases of recalcitrant variable skin lesions, not hesitating to do active mycological examinations, which would give them some critical clues in diagnosis of TI, and doing careful clinical examinations when finding combined tinea pedis or tinea unguium.

ACKNOWLEDGMENTS

The authors have no conflicts of interest to disclose.

Footnotes

This work was subsidized by a research grant from the Janssen Award in 2010.

References

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Articles from Journal of Korean Medical Science are provided here courtesy of Korean Academy of Medical Sciences
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Characteristics of Androgenetic Alopecia in Asian

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Won-Soo Lee, M.D., Ph.D.corresponding author and Hae-Jin Lee, M.D.
Copyright © 2012 The Korean Dermatological Association and The Korean Society for Investigative Dermatology
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Androgenetic alopecia (AGA), or pattern hair loss, is a common disorder in Asian men and women, with a reported incidence of up to 73% among general population. There are several descriptions regarding the characteristics of AGA in patients of European descent. Asian patients with AGA have different types of hair loss and family histories from Europeans, which may affect treatment response. Therefore, in this review, prevalence, hair loss patterns, familial factors, androgen receptor gene polymorphisms of Asian AGA patients, and management based on algorithmic guidelines for AGA are discussed. This review may be useful for dermatologists in clinical practice for diagnosing and designing management approaches for Asian patients with AGA.

Keywords: Androgenetic alopecia, Asians

INTRODUCTION

The term “androgenetic alopecia (AGA)” was introduced by Orentreich1 in 1960, but the same condition in men has also been termed male pattern alopecia, common baldness, male pattern baldness, and male pattern hair loss (MPHL). Androgen dependence and hereditary factors are less obvious in affected women than in affected men, thus the term pattern hair loss, which is a much broader concept, is preferred for women. AGA is the most common type of alopecia that occurs after puberty in both sexes. Patients typically present with progressive thinning and shortening of hair in affected areas. AGA is clearly a stressful experience for both sexes, but it may be substantially more distressing for women. In this review, Asian characteristics of prevalence, hair loss patterns, familial factors, androgen receptor (AR) gene polymorphisms, and management of AGA will be discussed.

PREVALENCE OF AGA IN ASIAN

There are population differences in the prevalence and types of AGA. In individuals of European descent, the prevalence of AGA has been well documented by Hamilton2 and Norwood3. A study of European-American men in the USA revealed a predominance for frontal baldness (Type A variant in Norwood-Hamilton classification) in 12% and a Type III or worse pattern in 16% of males aged 18~29 years old, which increased progressively to 53% in those aged 40~49 years old4. A study of 20~50 year old Norwegian men most commonly reported Type I (31%), followed by Type II (26%) and Type V or worse (20%)5.

AGA is also a common disorder in Asian people. Takashima et al.6 and Kakizo7 studied AGA in Japanese and found that it was minimal before the age of 40 and that, although the incidence increased with age, it remained lower than in Europeans. Japanese men develop AGA approximately one decade later than Europeans, and the prevalence is 1.4-fold lower in each decade of life6. In Korean men, the prevalence of AGA (Norwood III or above) at all ages was 14.1% and increased steadily with advancing age, but remained lower than that of Europeans: 2.3% in the third decade, 4.0% in the fourth decade, 10.8% in the fifth decade, 24.5% in the sixth decade, 34.3% in the seventh decade and 46.9% over 70 years. Type III vertex involvement was the most common type in the third decade to the seventh decade in Korean men; over 70 years, type VI was most common. A ‘female pattern’ was observed in 11.1% of cases8. In Korean women, the prevalence of AGA (Ludwig I or above) at all ages was 5.6%, and also increased steadily with advancing age: 0.2% in the third decade, 2.3% in the fourth decade, 3.8% in the fifth decade, 7.4% in the sixth decade, 11.7% in the seventh decade and 24.7% over age 70 years. Type I was the most common type up to the sixth decade; over 60 years, Type I and II were similar in prevalence. Type III was not observed8.

The age specific prevalence of AGA in Taiwanese men was compatible to that among Korean men but was lower than that among individuals of European descent9. Smoking status, current amount of cigarette smoking, and smoking intensity were statistically significant factors for AGA after controlling for age and family history9. A population-based cross sectional study was carried out in 7,056 subjects (3,519 men and 3,537 women) in Shanghai, China10. The prevalence of AGA in Chinese men was 19.9%, and the prevalence of female pattern hair loss (FPHL) in men was 0.1%. The most common type of hair loss in men was type III vertex (3.5%). The prevalence of AGA in Chinese men was lower than in European men but was similar to that in Korean men. However, over the age of 60 it approached the prevalence in European men and was higher than in Korean men. The prevalence of AGA in Chinese women was 3.1%, while MPHL in Chinese women was found in those aged over 50 years (0.4%), and the most common type was Ludwig type I (1.4%). Interestingly, the prevalence of AGA in Chinese women was lower than that in Korean women and European women, and type I was the most common type10.

In Singapore, Tang et al.11 reported a prevalence of 63% for Norwood type I to VII. The prevalence increased with age from 32% among young adults aged 17 to 26 years to almost 100% for those in their eighties. In Thailand, Pathomvanich et al.12 conducted a randomized study including 1,124 Asian men (local Thai and Chinese) between the ages of 18 and 90. The prevalence of baldness was reported as 38.52%; this figure approached that of Europeans, rather than the one fourth to one third reported in previous studies of Asians6. The prevalence increased with age, affecting 11% of young adults aged over 20 years and reaching 61.78% at 70 years of age. There are, however, two limitations of this survey. First, the small number of men included over 80 years of age (31 men) might have affected the results when compared to the Norwood study of the same age group. In addition, there were two Asian subgroups involved in this study, Thai and Chinese.

According to these studies mentioned, the prevalence of AGA in Chinese and Korean men was similar to, but significantly lower than, the prevalence in Thailand. The highest prevalence among the Asian groups studied was the 63% observed in Singapore; this discrepancy may be attributed to the diverse populations residing in the country or the inclusion of the almost normal Norwood type I in the Singapore study. In contrast, type II was the most common pattern among Indian males until the sixth decade, followed by type III Vertex after the sixth decade. The type A variant was only seen in 1% of Indian males, and FPHL was only observed in 0.2% of Indian males. These results suggest a less extensive balding pattern in Indian male population than in other Asian populations13.

In summary, Asian men with AGA have different characteristics from those of men of European descent. There are similar increases in prevalence with age among all the Asian groups studied. This high prevalence in older men suggests that this form of hair loss may be a normal consequence of aging. However, particularly in younger men, hair loss can have significant psychosocial manifestations, and can in turn have a significant economic impacts on household health expenditures14. The wide variation in prevalence rates in the current Asian studies would require a more standardized protocol.

AR GENE POLYMORPHISM IN AGA

Balding scalps are characterized by high levels of the potent androgen dihydrotestosterone (DHT) and increased expression of the androgen receptor gene. Most AGA patients have an androgen dependent trait, although it is thought to be under the control of multiple genes, such as genes for the AR, insulin-like growth factor-1, and DHT regulations15,16. The human AR gene is on the X chromosome at Xq11-12. The AR is a structurally conserved member of the nuclear receptor superfamily. The amino terminal domain is required for transcriptional activation and contains a region of polyglutamine that is encoded by CAG trinucleotide repeats. In humans, the number of CAG repeats is polymorphic. Expansion of CAG repeats in the AR has clinical implications for human disease17. A low number of CAG repeats in the AR gene implies increased risk factors for coronary heart disease18 and prostate cancer19. In recent studies, neurotrophic factors, especially brain-derived nerve factor, were found to have potential importance mediating the effects of androgens on hair follicles, serving as negative regulatory control signals20. These findings suggest that other regulatory signals may affect the pathogenesis of AGA, as well as AR gene polymorphisms.

The ubiquity of the AR gene Stu I restriction site, and the higher incidence of shorter triplet repeat haplotypes in bald men, suggests that these markers are very close to a functional variant that is a necessary component of the polygenic determination of male pattern baldness. A meta-analysis study by Zhuo et al.21 suggests that the G allele of the AR Stu I polymorphism might be a potential risk factor for AGA, especially in subjects of European descent. Functional mutations in or near the AR gene may explain the high reported levels of expression of this gene in the balding scalp22. Shorter CAG repeat lengths may be associated with the development of androgen mediated skin disorders such as AGA, hirsutism, and acne in men and women23. These findings suggest that the CAG repeat length in AR may affect androgen mediated gene expression in hair follicles and sebaceous glands in men and women with androgenic skin disorders14,23. Interestingly, when the number of triplet repeats (CAG+GGC) was plotted against degree of symptom improvement after treatment with finasteride, a broad correlation between these variables was observed24. The smaller the repeat number, the greater the improvement with finasteride. The group of patients with shorter repeat regions in the AR gene responded better to finasteride than did those with longer repeat regions, although patients with shorter repeats tended to have severe initial symptoms. The determination of such polymorphisms is thought to be useful in drug choice for AGA patients24.

Jung et al.25 compared CAG repeat numbers within the AR genes of 64 male Korean AGA patients with those of 40 normal male controls in a preliminary study. There was no significant difference in the number of CAG repeats between the Korean AGA patients and controls. There were no correlations between CAG repeat numbers and age of onset or severity of AGA in Korean AGA patients. These results suggests that AR receptor CAG polymorphisms in the Korean male population might not play a major role in AGA susceptibility. Nevertheless, a more extensive study to clarify whether there are real population-based differences in AR gene polymorphisms is needed.

Recently, advanced genetic studies of AGA have been published. Hayes et al.26 reported that the gene locus of E211A is significantly lower in proportion in the vertex and vertex balding group and frontal balding group compared with the no balding group. Therefore, the AR-E211 A allele, in linkage with the functional repeat sequences, is associated with a lower risk of metastatic prostate cancer and a lower risk of alopecia. Moreover, a study using genome-wide linkage study revealed a locus associated with AGA on chromosome 3q2627.

Hillmer et al.28 investigated the signatures of genetic variants of AR and their relationships to the AGA risk haplotype. Haplotype homozygosity suggested that the AGA risk haplotype was driven to high frequency by positive selection in Europeans, although a low meiotic recombination rate contributed to high haplotype homozygosity. Further, they detected high levels of population differentiation and a series of fixed derived alleles along an extended region centromeric to AR in the Asian HapMap sample.

RACIAL DIFFERENCES IN PATTERNS OF HAIR LOSS IN AGA AND THE BASP CLASSIFICATION FOR AGA

Asian men with AGA display different characteristics compared with men of other ethnicity. However, there is an increase in prevalence with age among all the Asian groups studied similar to that observed in other ethnic population8-12,29. The reason for this increase rate in the prevalence of AGA compared to Europeans remains unknown, but a transition toward a more Western diet and lifestyle may play a role.

There are also populational differences in the patterns of hair loss in AGA. In a previous study, FPHL was observed in 11.1% of Korean males with AGA8. In a Chinese study, MPHL was found in 13 of 108 (12%) women with AGA, all of whom were over 50 years of age10. In an Indian study, although it was possible to classify 80% of cases of AGA and II (28%) and III (15%) were the most common types of AGA, 27 patients of 150 male subjects (18%) did not fit into specific patterns according to the Norwood Hamilton classification30. In addition, the type ‘a’ variant was noted in 20% of patients, clearly indicating the limitations of the existing classifications. There is considerable overlap in types IV, V and VI in the Norwood classification, with the ‘a’ variants further confusing the picture30.

Various classification methods have been proposed for describing AGA. In 1950, Beek31 published a classification system, based on 1,000 males of European descent, which used two evolutionary aspects: frontal and frontovertical baldness. In the following year, the first systematic classification of AGA was established by Hamilton2, who sub-classified patterns of baldness based on frontoparietal, frontal recession, and vertex thinning, then evaluated a large group of men and women for the presence of specific patterns of hair loss from the prenatal period through the tenth decade of life. In 1975, Norwood3 refined Hamilton’s classification by emphasizing temporofrontal or vertex only subcategories of hair loss into seven types with a type A variant and reported the incidence of male pattern baldness at various ages in 1,000 adult male subjects of European descent. An additional pattern was introduced as the Norwood-Hamilton classification in a clinical trial of finasteride in MPHL32. Olsen33,34 proposed assigning separate designations (temporal, frontal, mid and vertex) to areas of the scalp that bald at different rates in different individuals with MPHL. Ludwig35 presented quite a different picture of hair loss in women from that described by Hamilton2. He emphasized preservation of the frontal fringe despite progressive centrifugal loss over the top of the scalp and arbitrarily designated three gradations of hair loss. Olsen36 proposed that frontal accentuation (or the ”Christmas tree” pattern) should be considered an additional pattern of hair loss in women, which helps to distinguish AGA from potential hair loss mimicries in women. Presently, the Norwood-Hamilton classification32 for MPHL and the Ludwig classification35 for female AGA are the most commonly used classification methods for assessing AGA worldwide.

Each of these existing classifications has substantial limitations in clinical setting. The Norwood-Hamilton classification (Fig. 1)3 is very detailed and is less stepwise classifications in its descriptions, making it difficult to memorize for common use. It does not list some non-typical types of baldness, such as FPHL in men. Additionally, many women with MPHL cannot be classified using the Ludwig classification system (Fig. 2)35. For most of these classification systems, clinicians must use different classification systems for each gender in order to correctly classify patterns34.

Fig. 1

The Norwood-Hamilton classification of male balding defines two major patterns and several less common types. Thinning starts in both temples as well as the crown/vertex and slowly progresses to encompass the entire top of the scalp (cited from Ref. 3
Fig. 2

The Ludwig pattern of hair loss (3-point). There are three main classes, each with increasing hair loss (cited from Ref. 35).

Thus, a more widely accepted, accurate, and stepwise method for classifying AGA would be of great benefit. Lee et al.37 devised a new classification system, named the Basic and Specific (BASP) classification (Fig. 3), which is comprehensive and systematic regardless of population or gender. The BASP classification37 was based on observed patterns of hair loss. The basic (BA) types represent the shape of the anterior hairline, and the specific types (SP) represent the density of hair on distinct areas (frontal and vertex). There are four basic types (L, M, C, and U) and two specific types (F and V). The final type is assigned according to a combination of the assigned BA and SP types.

Fig. 3

The BASP classification system includes four basic types (L, M, C, and U) and two specific types (V and F). The basic types represent the shape of the anterior hairline, and the specific types represent the density of hair on specific areas (frontal and

A total of 2,213 Korean subjects, comprised of 1,768 males and 445 females, were classified according to the BASP classification37. According to the severity of the phenotype, both the basic and specific types were subclassified into subtypes in order to generate a more stepwise and systematic classification. It is possible to describe patterns of hair loss in detail using the BASP method, and, thus estimate the further extent of hair loss and therapeutic response to a certain therapy. For both sexes, the majority of patients enrolled in the study were in the third and fourth decades of life (65.1% of males and 56.68% of females). In males, the older as well as the younger group were more likely to have little recession of the frontal hairline (classified as type M1-2) and diffuse thinning over the top of the scalp (type F1-2). The women in the study developed typical female AGA.

In men, regardless of age, 1,434 of the 1,768 males were classified as type M, accounting for 81.1% of cases. Among the subtypes and according to the severity of baldness, the majority of subjects below 50 years of age were classified as type M1, whereas most subjects over the age of 50 were classified as type M2. The incidence of Type L (9.3%) tended to decrease with age, but those of types C (5.8%) and U (3.8%) tended to increase. In women, type L showed the highest frequency in all age groups, accounting for 210 (47.2%) of 445 female subjects. Regardless of age, types M, C, and U were the next most common in order, observed in 121 women (27.2%), 111 women (25.0%), and 3 women (0.6%) of the 445 subjects, respectively. Type C0 was the second most common subtype in female subjects between the second and fourth decade of life, and its incidence decreased with age. In men, type F, which is identical to FPHL in the Ludwig classification, was observed in 42.4% (749/1,768) of male subjects, and type V was observed in 19.8% (350/1768). The grades of both types increased slightly with age. In women, type F was observed in 70.6% (314/445) of female subjects with AGA.

The BASP classification is a stepwise, systematic, and universal classification system for AGA, regardless of race or sex. It is an easily available comprehensive classification system. The BASP classification may prove particularly useful in communicating the exact amount and distribution of hair loss in those with AGA37. For these reasons, we use the basic and specific (BASP) classification in this review.

FAMILIAL FACTORS IN AGA

Family history plays an important role in the onset of AGA, which is believed to be influenced by genetic factors. However, the exact mode of inheritance has not been well characterized. Although there are some reports regarding the prevalence of AGA in male paternal family members, reports regarding the maternal side are rare. The AGA prevalence in male family members of patients (30.3%) was higher than those of controls (8.5%)38. AGA prevalence on the paternal side was greater than on the maternal side. However, no differences were found between paternal and maternal AGA prevalence, analyzed according to the age of onset and severity of AGA. These results suggest that AGA expression might be influenced by familial AGA prevalence and that paternal AGA prevalence has a greater effect in general on AGA expression than maternal AGA prevalence38.

In another Korean study, a family history of baldness was present in 48.5% of men and 45.2% of women with AGA8. In a Chinese study, a family history of AGA was present in 55.8% of men and 32.4% of women with AGA10. In contrast to the Korean study, the proportion of Chinese men with a positive family history was higher, suggesting that genetic background is important for determining the prevalence of AGA in Chinese men, and confirming that subjects with positive family histories are at greater risk of developing severe AGA9. The proportion of Chinese women with positive family histories was lower, further indicating that AGA is a polygenetic hereditary disease. In a Singaporean study, a positive family history of AGA was recorded in 58.9% (151/256) of subjects. Male patients tend to be more likely to have a father or male sibling with a similar problem, whereas female patients tend to be more likely to have a mother or female sibling with AGA39. In an Indian study of 150 subjects, positive family histories were found in 127 (85%) of subjects, paternal in 101 (67%), maternal in seven (5%) and both in 19 (13%). In 23 (15%) patients, no family history could be elicited.

In a Taiwanese study, an association was detected between moderate or severe AGA and family history of AGA from paternal relatives, whereas there was no corresponding association with maternal relatives9. Moderate or severe AGA was associated with a family history of AGA among first degree and second degree relatives but not among third degree relatives after adjusting for age. In addition, family histories of AGA among paternal relatives were predictive of moderate or severe AGA after adjusting for age. These findings do not support an association between moderate or severe AGA and a family history of AGA among maternal relatives. Moreover, a family history of AGA is associated with the risk of early onset AGA. This implies that those with a family history of AGA may have a higher risk of early onset AGA and a higher risk of developing severe AGA. Most importantly, early onset AGA showed a dose dependent association with AGA grade after adjusting for age and family history. From a clinical point of view, this suggests that patients with early onset AGA should receive early advice to prevent further deterioration9. These results of various epidemiologic familial studies suggest that AGA expression is influenced by familial AGA prevalence and, particularly, that paternal AGA prevalence has more effect on AGA expression than maternal AGA prevalence.

A recent study by Lee et al.40 using the BASP classification revealed that familial factors affecting the morphology of AGA in Asians differ between males and females, and for each BASP subtype. Parental influences on anterior hairline shape in men were predominantly from the paternal side, whereas these effects were less notable in women. In patients without family histories of AGA, a higher frequency of early-onset AGA than late-onset AGA was identified in men but not in women. Basic types of hair loss had a higher degree of heritability from the paternal side of the family, regardless of the specific type. This study provides detailed information indicating that each hair loss pattern according to the BASP classification has different familial factors in Asians40. Therefore, we can provide appropriate information to patients if we obtain careful personal and familial histories of AGA.

MANAGEMENT OF AGA IN ASIAN

General consideration

AGA is often related to poor self-image and low selfrespect. The problem must be viewed in perspective; an emphatic approach is important, as different people are affected in various ways when they lose hair. Patients should avoid hair-care products likely to injure the scalp and/or hair. Patients should maintain adequate diet, especially with adequate protein. The National Institutes of Health of the United States recommended daily allowance for protein is 0.8 g/kg41. Topical medications act only where the medication is applied; therefore, the whole area at risk of hair loss (the top of the scalp) should be treated with a given topical agent. If possible, any drugs that could negatively affect hair growth should be stopped and alternative substitutes used. Any underlying scalp disorders, such as seborrheic dermatitis or scalp psoriasis, should be treated as these conditions can affect the ability to use topical treatments for hair loss without irritation.

The typical man with MPHL who seeks treatment has significant concerns about the condition and has already engaged in considerable efforts to obtain information and at times even resorted to self-medication. Individualized consideration of attitudes, concerns, self-treating efforts, and expectations is crucial for effective management of men seeking medical treatment for MPHL42. Research has shown that most men and women who have unwanted hair loss have distressing experiences that diminish their body image43. Because of the psychological impact of hair loss, patients may seek inappropriate and unproven therapies. However, they must also appreciate the real goals and true limitations of each form of therapy. It is important that misconceptions should also be corrected. Some patients mistakenly think that their hormone levels are too high. Others erroneously place too many restrictions on their hair and grooming (e.g., hair styling, teasing, hair spray, washing frequency, hair color or permanents)44.

Knowledge and understanding of the genetic and physiological basis of AGA may help allay misconceptions and anxiety about its occurrence, and indirectly influence patient willingness to seek treatment for this condition45. Clinicians should follow the progress of their patients periodically to identify problems, utilizing photographic records of treatment results.

Medical treatment

A reduction in hair loss is usually seen after 3~6 months of medical treatment, and visible hair regrowth is observed after 6~12 months. Continuous treatment is needed to ensure sustained benefits. Unfortunately, available medical treatments are not curative. Ensuring that patients understand the limitations of these treatments is an important aspect of the management of AGA. Patients should be counseled that treatment for AGA will not restore hair growth to its prepubertal density and that the main aim is to prevent further progression of hair loss. Currently there are two agents, topical minoxidil and oral finasteride (only for males), approved by the United States Food and Drug Association (FDA) for the treatment of AGA.

1) Male

(1) Topical minoxidil solution

Topical minoxidil solution is administered at a dosage of 1 ml twice daily. Its mechanism of action is unknown. However, the main benefit appears to be a prolongation of the anagen phase and hair shaft diameter, irrespective of the underlying cause of baldness. It is well established that 5% minoxidil is more effective than 2% or 3% solution. Patients should be warned that during the initial 2~8 weeks, a temporary telogen effluvium may occur in some patients, which is self-limiting and subsides when subsequent anagen regrowth begins, and should not be a cause for treatment cessation46.

A recent advancement in the use of minoxidil as a hair loss treatment is the development of a 5% topical foam. Placebo controlled, double-blind trials have demonstrated that the hydroalcoholic foam is efficacious, safe, and well accepted cosmetically by patients14.

(2) Oral finasteride

Oral finasteride, a potent type II 5α-reductase inhibitor, should be administered at a daily dosage of 1 mg. In clinical trials over a 2-year period in men aged 18~41 years, the number of responding hairs was established after 1 year and continued treatment increased the length, diameter, and pigmentation of these hairs so that the coverage of the scalp increased over time. On stopping finasteride, the balding process resumed. An extension of the above study to 5 years showed that finasteride 1 mg/day was well tolerated, and led to durable improvements in scalp hair growth47.

Finasteride is generally well tolerated, side effects are typically mild and do not require discontinuation of therapy. Rare side effects may include some loss of libido and erectile function. At present, there is no proven benefit for finasteride in women. A placebo-controlled study in postmenopausal women with AGA given finasteride 1 mg/day over 1 year showed no significant benefit47.

2) Female

(1) Topical minoxidil solution

Topical minoxidil solution is administered at a dosage of 1 ml twice daily. The 5% solution was compared with the 2% solution in 2 studies involving 493 women. On the basis of hair-count data, the 5% solution was not significantly more effective than the 2% solution48. Patients should be warned that in the initial 2~8 weeks, a temporary telogen effluvium may occur in some patients, which is self-limiting and subsides when subsequent anagen regrowth begins, and should not be a cause for treatment cessation46. Side effects include hypertrichosis which occurs in 6% of women using 2% minoxidil, and 14% among those using the 5% solution46. This occurs on the face and resolves within 1~6 months after drug discontinuation. However, hypertrichosis diminishes or disappears after about 1 year, even with continued use of minoxidil.

(2) Oral antiandrogens

Cyproterone acetate, spironolactone and flutamide can be used as alternatives to minoxidil, but most of the antiandrogen therapies have not been rigorously studied in FPHL49. In general, better results are seen in women with hyperandrogenism. Side effects are generally greater with cyproterone acetate and spironolactone41.

Surgical management

Despite advances in medical therapy, hair transplantation remains the only means of permanent hair restoration in cases of severe AGA. It is contraindicated in patients with systemic diseases such as hypertension, cardiac disease, and diabetes mellitus, all of which must be controlled before hair transplantation. Local diseases such as cutaneous lupus erythematosus, morphea, alopecia areata, and scalp folliculitis must be quiescent for at least 6 months before hair transplantation. Complications of hair transplantation include ingrown hairs and foreign body reactions, infections, cobblestoning, graft depression, epidermal cysts, bleeding, headaches, scarring (keloid and hypertrophic scars), poor hair growth, arteriovenous fistula, osteomyelitis, wound dehiscence, telogen effluvium, accelerated hair loss, delayed temporary marked thinning, curly, lusterless hair, chronic mild folliculitis, and patient dissatisfaction.

After 4~6 months, the skin surfaces of the grafts have usually blended in perfectly with the surrounding scalp. In some patients, the grafts may be a shade lighter in color until they are “aged” by sun exposure50-52.

Other alternative medical therapies

Dutasteride is a dual type I and type II 5a-reductase inhibitor. In clinical trials, oral dutasteride showed significantly greater efficacy than placebo according to phototrichometric hair count, subject self-assessment, and investigator and panel photographic assessment53. Dutasteride is generally well tolerated, with rare side effects that may include some loss of libido and erectile function. Dutasteride is only approved by the Korean FDA for the treatment of AGA.

Topical alfatradiol may be an alternative, though reports of its efficacy have variable results to treat AGA54. Under the influence of 17alpha-estradiol (alfatradiol), an increased conversion of testosterone to 17beta-estradiol and androstendione to estrone improves hair growth55. Topical alfatradiol is available in Europe, South America, and Korea. Kim et al.56 reported single center, open-label, non-comparative, phase IV study of the efficacy and safety of alfatradiol (17α-estradiol) solution on female pattern hair loss in Korean women. Hair counts and diameter from baseline to 4 and 8 months after treatment were significantly increased in treated patients.

Bimatoprost and latanoprost, which are prostaglandin (PG) analogues, demonstrate stimulatory effects on hair growth of eyebrows and eyelashes and pigmentation in a high numbers of patients57. Currently bimatoprost is approved as eyelash growth enhancer. It might be used for the treatment of AGA off-label. The expressions of PG receptors were examined in mouse skin hair follicles, and mRNA was identified in dermal papilla and outer root sheath follicular structures during the anagen phase. In addition, other studies have demonstrated the ability of PG to stimulate movement from telogen to anagen in mice.

Ketoconazole might also be used for the treatment of AGA. The mechanism of ketaconazole is unknown, but may involve inhibition of inflammation, or anti-androgenic properties58. There is some evidence, both in humans and in rodents, that this agent may stimulate hair growth14. Prostaglandin analogues and ketoconazole are not approved for AGA treatment and further studies are needed to investigate the therapeutic effect on AGA.

Other devices and non-medical aesthetic aids

Devices can be used as alternative tools for the treatment of AGA. Laser hair comb (Low-level laser therapy)59,60 and Fractional photothermolysis laser61 have been tried to treat AGA. However, these treatments cannot be substituted for the medical and surgical approaches previously mentioned.

Non-medical approaches can provide cosmetic relief to both men and women with thinning hair, if medical treatments are not indicated, not effective, or not desired by the patient. Non-medical aesthetic aids include wigs, hairpieces, hair extensions, and topical powder makeup. They can also be used as adjuvant tools to medical or surgical treatments41.

CONCLUSION

Although the clinical aspects of AGA are recognized in both men and women and the role of DHT is well documented, much remains to be determined regarding the most appropriate treatments for AGA based on genetics and pathophysiology. AGA is a disconcerting experience for both sexes, but it may be substantially more distressing for women. Therefore, dermatologists should take into account the psychological well-being of patients with AGA, which can lead to the choice of an appropriate treatment. Moreover, most of the previously published studies of AGA were conducted among only patients of European descent. There are effective treatments, medical or surgical, currently available for some men and women with AGA. Compared to other populations, Asian patients with AGA have different types of hair loss and family histories, which may influence treatment response. This review of AGA in Asians may be practical for informing dermatologists regarding their approaches to understand, diagnose and treat Asian patients with AGA in clinical practice.

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