The prevalence of unsuspected onychomycosis and its causative organisms in a multicentre Canadian sample of 30 000 patients visiting physicians' offices.

BACKGROUND: Onychomycosis is difficult to treat and a concern for many patients. Prevalence estimates of onychomycosis in North American clinic samples have been higher than what has been reported for general populations. OBJECTIVE: A large, multicentre study was conducted to estimate the prevalence of toenail onychomycosis in the Canadian population. METHODS: Patients were recruited from the offices of three dermatologists and one family physician in Ontario, Canada. Nail samples for mycological testing were obtained from normal and abnormal-looking nails. This sample of 32 193 patients includes our previous published study of 15 000 patients. RESULTS: Abnormal nails were observed in 4350 patients. Of these, the prevalence of culture-confirmed toenail onychomycosis was estimated to be 6.7% (95% CI, 6.41-6.96%). Following sex and age adjustments for the general population, the estimated prevalence of toenail onychomycosis in Canada was 6.4% (95% CI, 6.12%-6.65%). The distribution of fungal organisms in culture-confirmed onychomycosis was 71.9% dermatophytes, 20.4% non-dermatophyte moulds and 7.6% yeasts. Toenail onychomycosis was four times more prevalent in those over the age of 60 years than below the age of 60 years. CONCLUSION: The present data highlights that onychomycosis may be a growing medical concern among ageing patients.

Nivolumab for Metastatic Melanoma.

Melanoma is an aggressive skin cancer with a generally poor prognosis at Stage III-IV disease. Traditionally, metastatic melanoma was treated by surgical resection, when possible, and with systemic chemotherapy. New developments in molecular biology have led to the identification of immune checkpoints which are exploited by malignant cells, allowing them to go undetected by the immune system. Nivolumab (Opdivo®) is a human monoclonal antibody which prevents immune inhibition by interacting with PD-1 on tumor cells; thus, increasing tumor-specific T cell proliferation. Nivolumab has demonstrated efficacy superior to that of standard chemotherapy and relative safety in clinical trials. Indeed, the outcomes for patients with advanced melanoma are being improved by novel biologic agents such as nivolumab.

An open comparative study of nail drilling as adjunctive treatment for toenail onychomycosis (.).

Introduction Novel treatment regimens are being developed to improve drug penetration through the nail plate. This study investigated the efficacy of nail drilling regimens for the treatment of onychomycosis. Methods Participants were assigned to holes with combination (oral plus topical terbinafine) therapy (Group 1), holes with topical terbinafine (Group 2) or topical terbinafine only (Group 3). Measurement of clear nail and mycology was performed at baseline and at weeks 4, 10, 16, 22 and 28. Mixed linear models were used to compare mean percent clear nail. Mycological cure rates were also tabulated for each group. Tolerability and adverse events were documented. Results Ninety-eight participants were enrolled (106 nails). Both groups with holes had significantly higher percentage of clear nail compared with topical terbinafine alone. Although no significant difference between the two groups where holes were drilled in the nail plate, Group 1 demonstrated improvement over Group 3 earlier than Group 2 (visit 2 versus visit 4). Group 1 also had the highest mycological cure rates. Conclusion Treatment with holes plus topical terbinafine produces significantly greater improvement in toenails' appearance and higher mycological cure rates compared to treating the dorsal aspect of the nail plate with topical terbinafine alone.

Ivermectin 1% cream for rosacea.

The etiology of papulopustular rosacea (PPR) is not well understood yet appears to involve both the innate and adaptive immune response in addition to possible infestation with Demodex mites. Current treatments for PPR consist mainly of antibiotics. Ivermectin cream 1%, a new topical treatment for PPR, possesses both anti-inflammatory and anti-parasitic properties. After 12 weeks of treatment, subjects treated with ivermectin cream 1% had significantly greater reductions in PPR symptoms and enhanced diseaserelated quality of life improvements compared to subjects who received vehicle. Furthermore, PPR symptoms continued to improve with prolonged treatment (40 weeks). Ivermectin cream 1% offers a multi-pronged approach to combat the complex pathophysiology of rosacea.

The prevalence of culture-confirmed toenail onychomycosis in at-risk patient populations.

Onychomycosis is a fungal infection of the nail and is the most common nail affliction in the general population. Certain patient populations are at greater risk of infection and the prevalence of onychomycosis reported in the literature has yet to be summarized across these at-risk groups. We performed a systematic review of the literature and calculated pooled prevalence estimates of onychomycosis in at-risk patient populations. The prevalence of dermatophyte toenail onychomycosis was as follows: general population 3.22% (3.07, 3.38), children 0.14% (0.11, 0.18), the elderly 10.28% (8.63, 12.18), diabetic patients 8.75% (7.48, 10.21), psoriatic patients 10.22% (8.61, 12.09), HIV positive patients 10.40% (8.02, 13.38), dialysis patients 11.93% (7.11, 19.35) and renal transplant patients 5.17% (1.77, 14.14). Dialysis patients had the highest prevalence of onychomycosis caused by dermatophytes, elderly individuals had the highest prevalence of onychomycosis caused by yeasts (6.07%; 95% CI = 3.58, 10.11) and psoriatic patients had the highest prevalence of onychomycosis caused by non-dermatophyte moulds (2.49%; 95% CI = 1.74, 3.55). An increased prevalence of onychomycosis in certain patient populations may be attributed to impaired immunity, reduced peripheral circulation and alterations to the nail plate which render these patients more susceptible to infection.

When is onychomycosis onychomycosis? A cross-sectional study of fungi in normal-appearing nails.

BACKGROUND: The presence of fungal organisms in healthy-looking toenails has previously been reported in individuals with a known dermatophyte infection and in those with onycholysis, but has not been extensively studied in individuals who do not present with foot pathology. OBJECTIVES: To determine the prevalence of fungal organisms in the toenails and on the soles of normal-appearing feet. METHODS: Adults who visited a dermatology clinic between June 2012 and February 2013 for concerns unrelated to fungal infection of the nails and feet participated in this study. Participants' feet were clinically examined, and skin and nail samples were collected and sent for potassium hydroxide (KOH) light microscopy and culture. RESULTS: Five hundred and eighty-five individuals with normal-appearing feet and toenails participated in this study. Fungal organisms were detected in 9·2%, 3·9% and 3·1% of participants' toenails by KOH, culture, and a combination of KOH and culture, respectively, while fungal organisms were present on the soles of the feet of 7·0%, 2·9% and 1·4% of participants by KOH, culture and both these methods combined, respectively. A significant association between the presence of fungal organisms in toenails and on the soles of the feet was found (P < 0·01). CONCLUSIONS: The presence of fungal organisms in the nail, even in the absence of clinical signs, may be termed 'subclinical' onychomycosis. The normal-appearing nail plate may act as a reservoir for infectious dermatophyte and nondermatophyte organisms. When left unimpeded by the host's immune system, these organisms are inclined to proliferate to produce clinically apparent disease.

High throughput HLA genotyping using 454 sequencing and the Fluidigm Access Array™ System for simplified amplicon library preparation.

The human leukocyte antigen (HLA) class I and class II loci are the most polymorphic genes in the human genome; distinguishing the thousands of HLA alleles is challenging. Next generation sequencing of exonic amplicons with the 454 genome sequence (GS) FLX System and Conexio Assign ATF 454 software provides high resolution, high throughput HLA genotyping for eight class I and class II loci. HLA typing of potential donors for unrelated bone marrow donor registries typically uses a subset of these loci at high sample throughput and low cost per sample. The Fluidigm Access Array System enables the incorporation of 48 different multiplex identifiers (MIDs) corresponding to 48 genomic DNA samples with up to 48 different primer pairs in a microfluidic device generating 2304 parallel polymerase chain reactions (PCRs). Minimal volumes of reagents are used. During genomic PCR, in this 4-primer system, the outer set of primers containing the MID and the 454 adaptor sequences are incorporated into an amplicon generated by the inner HLA target-specific primers each containing a common sequence tag at the 5' end of the forward and reverse primers. Pools of the resulting amplicons are used for emulsion PCR and clonal sequencing on the 454 Life Sciences GS FLX System, followed by genotyping with Conexio software. We have genotyped 192 samples with 100% concordance to known genotypes using 8 primer pairs (covering exons 2 and 3 of HLA-A, B and C, and exon 2 of DRB1, 3/4/5 and DQB1) and 96 MIDs in a single GS FLX run. An average of 166 reads per amplicon was obtained. We have also genotyped 96 samples at high resolution (14 primer pairs covering exons 2, 3, and 4 of the class I loci and exons 2 of DRB1, 3/4/5, DQA1, DQB1, DPB1, and exon 3 of DQB1), recovering an average of 173 sequence reads per amplicon.

Prodigious substrate specificity of AAC(6')-APH(2"), an aminoglycoside antibiotic resistance determinant in enterococci and staphylococci.

BACKGROUND: High-level gentamicin resistance in enterococci and staphylococci is conferred by AAC(6')-APH(2"), an enzyme with 6'-N-acetyltransferase and 2"-O-phosphotransferase activities. The presence of this enzyme in pathogenic gram-positive bacteria prevents the successful use of gentamicin C and most other aminoglycosides as therapeutic agents. RESULTS: In an effort to understand the mechanism of aminoglycoside modification, we expressed AAC(6')-APH(2") in Bacillus subtilis. The purified enzyme is monomeric with a molecular mass of 57 kDa and displays both the expected aminoglycoside N-acetyltransferase and O-phosphotransferase activities. Structure-function analysis with various aminoglycosides substrates reveals an enzyme with broad specificity in both enzymatic activities, accounting for AAC(6')-APH(2")'s dramatic negative impact on clinical aminoglycoside therapy. Both lividomycin A and paromomycin, aminoglycosides lacking a 6'-amino group, were acetylated by AAC(6')-APH(2"). The infrared spectrum of the product of paromomycin acetylation yielded a signal consistent with O-acetylation. Mass spectral and nuclear magnetic resonance analysis of the products of neomycin phosphorylation indicated that phosphoryl transfer occurred primarily at the 3'-OH of the 6-aminohexose ring A, and that some diphosphorylated material was also present with phosphates at the 3'-OH and the 3"'-OH of ring D, both unprecedented observations for this enzyme. Furthermore, the phosphorylation site of lividomycin A was determined to be the 5"-OH of the pentose ring C. CONCLUSIONS: The bifunctional AAC(6')-APH(2") has the capacity to inactivate virtually all clinically important aminoglycosides through N- and O-acetylation and phosphorylation of hydroxyl groups. The extremely broad substrate specificity of this enzyme will impact on future development of aminoglycosides and presents a significant challenge for antibiotic design.

Aminoglycoside antibiotic phosphotransferases are also serine protein kinases.

BACKGROUND: Bacterial resistance to aminoglycoside antibiotics occurs primarily through the expression of modifying enzymes that covalently alter the drugs by O-phosphorylation, O-adenylation or N-acetylation. Aminoglycoside phosphotransferases (APHs) catalyze the ATP-dependent phosphorylation of these antibiotics. Two particular enzymes in this class, APH(3')-IIIa and AAC(6')-APH(2"), are produced in gram-positive cocci and have been shown to phosphorylate aminoglycosides on their 3' and 2" hydroxyl groups, respectively. The three-dimensional structure of APH (3')-IIIa is strikingly similar to those of eukaryotic protein kinases (EPKs), and the observation, reported previously, that APH(3')-IIIa and AAC(6')-APH(2") are effectively inhibited by EPK inhibitors suggested the possibility that these aminoglycoside kinases might phosphorylate EPK substrates. RESULTS: Our data demonstrate unequivocally that APHs can phosphorylate several EPK substrates and that this phosphorylation occurs exclusively on serine residues. Phosphorylation of Ser/Thr protein kinase substrates by APHs was considerably slower than phosphorylation of aminoglycosides under identical assay conditions, which is consistent with the primary biological roles of the enzymes. CONCLUSIONS: These results demonstrate a functional relationship between aminoglycoside and protein kinases, expanding on our previous observations of similarities in protein structure, enzyme mechanism and sensitivity to inhibitors, and suggest an evolutionary link between APHs and EPKs.

Inhibition of aminoglycoside antibiotic resistance enzymes by protein kinase inhibitors.

Bacterial resistance to the aminoglycoside antibiotics is manifested primarily through the expression of enzymes which covalently modify these drugs. One important mechanism of aminoglycoside modification is through ATP-dependent O-phosphorylation, catalyzed by a family of aminoglycoside kinases. The structure of one of these kinases, APH(3')-IIIa has recently been determined by x-ray crystallography, and the general fold is strikingly similar to eukaryotic protein kinases (Hon, W. C., McKay, G. A., Thompson, P. R., Sweet, R. M., Yang, D. S. C., Wright, G. D., and Berghuis, A. M. (1997) Cell 89, 887-895). Based on this similarity, we have examined the effect of known inhibitors of eukaryotic protein kinases on two aminoglycoside kinases, APH(3')-IIIa and the enzyme AAC(6')-APH(2") which also exhibits acetyl-CoA-dependent aminoglycoside modification activity. We report that several known protein kinase inhibitors are also good inhibitors of aminoglycoside kinases. Compounds belonging to the isoquinolinesulfonamide group are especially effective in this regard, giving competitive inhibition in the micromolar range with respect to ATP and noncompetitive inhibition versus the aminoglycoside substrate. This study provides the basis for future aminoglycoside kinase inhibitor design and for the development of compounds which could reverse antibiotic resistance in the clinic.

Alginate Encapsulation of the White Rot Fungus Phanerochaetechrysosporium

In the laboratory, the white rot fungus Phanerochaetechrysosporium degrades numerous organic pollutants. Lack of aslow-release delivery system to toxic waste sites, for this and other fungi,however, constitutes an important barrier to practical implementation. Inthis study, the use of calcium alginate as an encapsulant for mycelia wasinvestigated; samples were in the form of pellets 1-3 mm in diameter. Whenrefrigerated, alginate-embedded mycelia of P. chrysosporium wereviable for one year, both with and without nutrient supplementation. At roomtemperature, in the absence of nutrient supplementation, viability decreasedsharply within 2 months. Addition of sawdust or corncob grits extended theviability of alginate-embedded mycelia; nevertheless, after 9 months onlyabout 20% of the pellets stored at room temperature yielded fungalgrowth. Spores of P. chrysosporium, embedded in alginate pelletstogether with corncob grits, gave 75% viability after 9 months ofstorage at room temperature. Alginate-embedded mycelia were used in Petriplate toxicity tests with 2,4,6-trinitrotoluene (TNT) and gave more rapid andreproducible results than tests performed with mycelial plugs. Theseexperiments demonstrated the feasibility of encapsulating P.chrysosporium in calcium alginate pellets, thus providing a potentialmethod of delivering white rot fungi to toxic waste sites, as well as fordeveloping a system of standardized toxicity testing in plate assays.

Differences in sexual behavior among HIV discordant and concordant gay men in primary relationships.

We investigated differences in unprotected anal intercourse among gay men in HIV concordant and discordant primary relationships. Individuals were recruited in 1992 from household- and bar-based samples of gay/bisexual men in Portland, Oregon, and Tucson, Arizona. Respondents were men who reported that they were in primary relationships of > or = 1 month and who reported their own and their partner's HIV status (n = 785). Comparisons were made between three groups: (a) HIV + respondents/HIV + partners; (b) HIV- respondents/HIV- partners; and (c) respondents whose partner's HIV status was different from their own (discordant), on sexual behavior, psychosocial, and demographic variables. Men in HIV concordant relationships reported significantly higher rates of unprotected anal intercourse (54% for +2 and 48% for --) than discordant couples (17%). HIV- men in concordant relationships were more likely to be monogamous (58%) and younger (22% < or = 25 years) than the other two groups. There were no significant differences among the groups regarding the kind of sexual behaviors they engaged in with nonprimary partners. The substantially lower rate of unprotected anal intercourse among men in discordant relationships compared to men in concordant relationships suggests that individuals and couples make judgments about sex and behavior based on knowledge of one's own and one's partner's HIV status.

HIV risk among Latino gay men in the Southwestern United States.

Using two different methods-bar and community household sampling-159 Latino gay men were recruited in the city of Tucson, Arizona. In addition to demographics, the study questionnaire assessed participants' sexual activity during the last 30 days with primary and nonprimary partners, condom use in the last year, and eight psychosocial constructs that have been predictive of HIV risk in different studies of (mostly white) gay/bisexual men. Questionnaires were available only in English; this Latino sample is thus likely to overrepresent highly acculturated, English-speaking men. Results show that 22% of the sample engaged in unprotected anal intercourse with nonmonogamous partners during the last 30 days; 51% of the sample reported at least one instance of unprotected anal intercourse during the last year. Of those men who practiced any anal intercourse during the last 30 days, 67% practiced unprotected anal intercourse with primary partners and 44% practiced unprotected intercourse with casual partners. Thus, the majority of Latino gay men who practiced anal intercourse in the month prior to the interview were not using condoms. Men who practiced unprotected intercourse with nonmonogamous partners reported lower annual incomes and were less educated. Two cognitive variables (behavioral intentions and perceptions of self-efficacy and self-control) and two behavioral variables (sex under the influence of alcohol and/or drugs and sex in public environments) emerged as the most important correlates of HIV risk.

N-acetylcysteine inhibits germination of conidia and growth of Aspergillus spp. and Fusarium spp.

N-Acetylcysteine inhibited hyphal growth and germination of conidia of Aspergillus spp. and Fusarium spp. N-Acetylcysteine inhibited conidial germination as well as or better than L-cysteine. Cysteine-related compounds may provide a potential therapeutic strategy against agriculturally and medically important fungal pathogens.

Evaluation of an HIV risk reduction intervention among African-American homosexual and bisexual men.

OBJECTIVE: To provide the first data which evaluates an HIV risk reduction intervention designed to reduce HIV high-risk sexual behavior in African-American homosexual and bisexual men. SUBJECTS: Participants (n = 318) were recruited from bars, bathhouses, and erotic bookstores, and through homosexual African-American organizations, street out-reach, media advertisements, and personal referrals of individuals aware of the study. METHODS: Participants were randomized into a single or triple session experimental group or a wait-list control group. Both experimental interventions included AIDS risk education, cognitive-behavioral self-management training, assertion training, and attempts to develop self-identity and social support. Data collection involved assessments of self-reported changes in sexual behavior at 12- and 18-month follow-up. RESULTS: Participants in the triple session intervention greatly reduced their frequency of unprotected anal intercourse (from 46 to 20%) at the 12-month follow-up evaluation and (from 45% to 20%) at the 18-month follow-up evaluation. However, levels of risky behavior for the control group remained constant (from 26 to 23% and from 24 to 18%) at 12- and 18-month follow-up evaluations, respectively. In addition, levels of risky behavior for the single session intervention decreased only slightly (from 47 to 38% and from 50 to 38%) at the 12- and 18-month follow-up evaluations, respectively. CONCLUSIONS: Results were interpreted to demonstrate the superiority of a triple session over a single session intervention in reducing risky sexual behavior in this cohort.

Decisions to get HIV tested and to accept antiretroviral therapies among gay/bisexual men: implications for secondary prevention efforts.

The objective of this study was to report prevalence rates of adherence by HIV-seropositive individuals to medical recommendations for the treatment of HIV infection, a behavioral pattern referred to as AIDS secondary prevention. We report cross-sectional data (n = 2,593) from two household-based and two bar-based samples of gay/bisexual men, gathered in 1992 in Tucson, Arizona, and Portland, Oregon. The main outcome variables were prevalence of HIV antibody testing and adherence to recommended secondary prevention behaviors to prevent onset of AIDS symptoms. Approximately one-third of the gay/bisexual men in these samples do not know their current HIV status. Of the gay/bisexual men who do know that they are HIV-seropositive, approximately three-fourths adhere to each of the secondary prevention recommendations, as appropriate to their stage of disease progression. In a multivariate logistic model, three variables distinguished between HIV-seropositive men who did and did not adhere: perceived antiviral treatment norms (OR = 1.4, CI = 1.1-1.7), perceived efficacy of secondary prevention treatments (OR = 1.4, CI = 1.1-1.7), and quality of the relationship with one's health-care provider (OR = 2.5, CI = 1.6-4.0). These findings indicate that efforts to support AIDS secondary prevention behaviors can occur not only through health education to change the perceptions of at-risk communities about the options available to delay the onset of opportunistic infections among HIV-seropositive individuals but also by enhancing effective doctor/patient communication.