Sensitivity of different DNA extraction methods and PCR to detect resistance in patients with leprosy stratified by the bacilloscopic index.

INTRODUCTION: Antimicrobial resistance in leprosy is an emerging problem, and the quantitative impact of low bacilloscopic indexes (BIs) on the sensitivity of molecular tests is unknown. We aimed to evaluate the sensitivity of gene sequencing for the detection of mutations related to antimicrobial resistance in Mycobacterium leprae in patients with low BIs using an analytical model. METHODS: Patients with leprosy were included and divided into two groups depending on their BIs (≥ 2+ and < 2+). The sensitivities of the two DNA extraction methods were compared after amplifying and sequencing the repetitive element (RLEP), folP1, rpoB and gyrA in M. leprae. RESULTS: We included 56 patients with leprosy: 35 had BIs less than 2+ (22 had negative slit-skin smear [SSS] results) and 21 patients had BIs greater than or equal to 2+. The sensitivity of the amplification of the RLEP target and the gene sequencing of folP1, rpoB and gyrA was 50 to 70% lower in patients with a BI less than 2+ and was significantly reduced in patients with lower BIs for all targets (p < 0.001). One patient had a mutation in the folP1 gene, and 14 patients had mutations in the gyrA gene, but no mutations related to antimicrobial resistance were found. CONCLUSIONS: We can conclude that the sensitivity of molecular tests is directly related to the BI, but these tests can still detect up to 20% of the targets in patients with BIs < 2+. New strategies to improve the sensitivity for detecting antimicrobial resistance in leprosy patients and reasonable clinical criteria for follow-up and the introduction of alternative treatments must be developed.

Sensitivity of different DNA extraction methods and PCR to detect resistance in patients with leprosy stratified by the bacilloscopic index

ABSTRACT Introduction: Antimicrobial resistance in leprosy is an emerging problem, and the quantitative impact of low bacilloscopic indexes (BIs) on the sensitivity of molecular tests is unknown. We aimed to evaluate the sensitivity of gene sequencing for the detection of mutations related to antimicrobial resistance in Mycobacterium leprae in patients with low BIs using an analytical model. Methods: Patients with leprosy were included and divided into two groups depending on their BIs (≥ 2+ and < 2+). The sensitivities of the two DNA extraction methods were compared after amplifying and sequencing the repetitive element (RLEP), folP1, rpoB and gyrA in M. leprae. Results: We included 56 patients with leprosy: 35 had BIs less than 2+ (22 had negative slit-skin smear [SSS] results) and 21 patients had BIs greater than or equal to 2+. The sensitivity of the amplification of the RLEP target and the gene sequencing of folP1, rpoB and gyrA was 50 to 70% lower in patients with a BI less than 2+ and was significantly reduced in patients with lower BIs for all targets (p < 0.001). One patient had a mutation in the folP1 gene, and 14 patients had mutations in the gyrA gene, but no mutations related to antimicrobial resistance were found. Conclusions: We can conclude that the sensitivity of molecular tests is directly related to the BI, but these tests can still detect up to 20% of the targets in patients with BIs < 2+. New strategies to improve the sensitivity for detecting antimicrobial resistance in leprosy patients and reasonable clinical criteria for follow-up and the introduction of alternative treatments must be developed.

The interference of polypharmacy and the importance of clinical pharmacy advice in the treatment of leprosy: a case-control study.

INTRODUCTION: Although supervised doses are essential for reducing leprosy treatment failure, the impact of specific drug interactions has rarely been assessed. This study aimed to estimate the risk of leprosy treatment suspension in patients receiving polypharmacy. METHODS We performed this case-control study in which the primary outcome was defined as the need to discontinue multibacillary leprosy treatment for at least one supervised dose, and the main risk factor was the detection of polypharmacy. Multivariate analysis by logistic regression was used for calculating odds ratio (OR). RESULTS: This study included 103 patients, of whom 43 needed to discontinue leprosy treatment (hemolysis = 26, hepatitis = 2, hemolysis associated with hepatitis = 6, and suspected treatment resistance = 9) and the rest did not. The severity of drug interactions had no effect on treatment discontinuation. Patients who used five or more drugs in addition to leprosy treatment had almost a 4-fold greater risk of treatment suspension (OR, 3.88; 95% confidence interval: 1.79-9.12; p < 0.001). The number of drugs used also positively influenced the occurrence of hemolysis (p < 0.001). No patient presented evidence of molecular resistance to rifampicin, dapsone, or ofloxacin treatment, as evidenced by genetic sequencing detection of rpoB, folp1, and gyrA mutations. CONCLUSIONS: Polypharmacy has deleterious effects on the already difficult-to-adhere-to treatment of leprosy and polypharmacy induces hemolysis. Additional measures must be taken to avoid the undesirable effects of inadequate polypharmacy.

The interference of polypharmacy and the importance of clinical pharmacy advice in the treatment of leprosy: a case-control study

Abstract INTRODUCTION: Although supervised doses are essential for reducing leprosy treatment failure, the impact of specific drug interactions has rarely been assessed. This study aimed to estimate the risk of leprosy treatment suspension in patients receiving polypharmacy. METHODS We performed this case-control study in which the primary outcome was defined as the need to discontinue multibacillary leprosy treatment for at least one supervised dose, and the main risk factor was the detection of polypharmacy. Multivariate analysis by logistic regression was used for calculating odds ratio (OR). RESULTS: This study included 103 patients, of whom 43 needed to discontinue leprosy treatment (hemolysis = 26, hepatitis = 2, hemolysis associated with hepatitis = 6, and suspected treatment resistance = 9) and the rest did not. The severity of drug interactions had no effect on treatment discontinuation. Patients who used five or more drugs in addition to leprosy treatment had almost a 4-fold greater risk of treatment suspension (OR, 3.88; 95% confidence interval: 1.79-9.12; p < 0.001). The number of drugs used also positively influenced the occurrence of hemolysis (p < 0.001). No patient presented evidence of molecular resistance to rifampicin, dapsone, or ofloxacin treatment, as evidenced by genetic sequencing detection of rpoB, folp1, and gyrA mutations. CONCLUSIONS: Polypharmacy has deleterious effects on the already difficult-to-adhere-to treatment of leprosy and polypharmacy induces hemolysis. Additional measures must be taken to avoid the undesirable effects of inadequate polypharmacy.

Coati (Nasua nasua) attacks on humans: case report.

Coatis [including Nasua nasua, the ring-tailed coati], are medium-sized mammals widely distributed in the Americas. They are social animals, whose normal diet includes insects, fruits, and small vertebrates, and rarely prey on larger sized animals. There are, to our knowledge, no reports in the medical literature of attacks on humans. This report describes a coati attack on 2 children in their home. The children sustained deep scratches and bites. The animal may have injured the humans in a defensive strike, but motivation for attack was uncertain. Coati attacks may occur in places where there is interaction between these mammals and humans.

Familial primary localized cutaneous amyloidosis in Brazil.

BACKGROUND: Macular and lichen amyloidosis are clinical variants of primary localized cutaneous amyloidosis (PLCA). Most cases are sporadic, but approximately 10% of cases may be familial. To our knowledge, the clinicopathologic and molecular features of such pedigrees, however, have not been studied in detail. OBSERVATIONS: We assessed 2 Brazilian families with either lichen-type (family 1 had 14 affected subjects) or macular-type (family 2 had 7 affected subjects) PLCA. Typically, in both pedigrees, the onset of symptoms was around puberty, and pruritus usually began on the lower legs. Findings from lesional skin biopsy samples from both families showed thioflavin T-positive material in the papillary dermis, which was more prominent in the lichen phenotype in family 1. Spontaneous improvement occurred in 3 subjects (from both families) after age 25 years. All affected individuals in family 1 had a heterozygous missense mutation in the OSMR gene (p.I691T), but no pathogenic mutation in OSMR was found in family 2. CONCLUSIONS: Familial PLCA shows autosomal dominant inheritance, but there is clinical and genetic heterogeneity and variable clinical penetrance. Demonstration of mutations in the OSMR gene provides new insight into mechanisms of itch and apoptosis in human skin.

Oncostatin M receptor-beta mutations underlie familial primary localized cutaneous amyloidosis.

Familial primary localized cutaneous amyloidosis (FPLCA) is an autosomal-dominant disorder associated with chronic skin itching and deposition of epidermal keratin filament-associated amyloid material in the dermis. FPLCA has been mapped to 5p13.1-q11.2, and by candidate gene analysis, we identified missense mutations in the OSMR gene, encoding oncostatin M-specific receptor beta (OSMRbeta), in three families. OSMRbeta is a component of the oncostatin M (OSM) type II receptor and the interleukin (IL)-31 receptor, and cultured FPLCA keratinocytes showed reduced activation of Jak/STAT, MAPK, and PI3K/Akt pathways after OSM or IL-31 cytokine stimulation. The pathogenic amino acid substitutions are located within the extracellular fibronectin type III-like (FNIII) domains, regions critical for receptor dimerization and function. OSM and IL-31 signaling have been implicated in keratinocyte cell proliferation, differentiation, apoptosis, and inflammation, but our OSMR data in individuals with FPLCA represent the first human germline mutations in this cytokine receptor complex and provide new insight into mechanisms of skin itching.