Genotyping of Borrelia from formalin-fixed paraffin-embedded skin biopsies of cutaneous borreliosis and tick bite reactions by assays targeting the intergenic spacer region, ospA and ospC genes.

BACKGROUND: Lyme borreliosis has a broad spectrum of clinical presentations involving the skin, joints and nervous system. The variable manifestations have been attributed to different Borrelia genospecies but genotyping required culture or fresh tissue. However, in dermatology practice, formalin-fixed paraffin-embedded biopsies are used for dermatopathological examination. Polymerase chain reaction (PCR) for Borrelia burgdorferi sensu latu has been established on such specimens, but studies attempting genotyping of subspecies or strains are lacking. OBJECTIVES: To adapt PCR assays for genotyping of Borrelia using paraffin-embedded biopsies, to identify Borrelia genospecies and to compare clinicopathological features of different genospecies. METHODS: Eighty-two paraffin-embedded biopsies from 68 patients, with erythema migrans, acrodermatitis chronica atrophicans, lymphocytoma cutis or tick bite reactions, were studied with assays targeting the intergenic spacer (IGS), ospA and ospC, followed by sequencing and phylogenetic analysis. Clinicopathological data were analysed comparing different Borrelia genospecies. RESULTS: Genotyping by IGS, ospA and ospC was successful in 85% of patients (91% B. afzelii, 7% B. garinii, 2% B. bavariensis). ospA serotyping identified type 2 (90%), type 3 (8%) and type 4 (2%). ospC-PCR was positive in 40% of the patients revealing 12 different groups, noninvasive forms being seen only in tick bite reactions and erythema migrans. No major clinicopathological differences could be identified between the genospecies, but neural inflammation and arthralgia were seen more often in lesions caused by invasive ospC strains. CONCLUSIONS: Genotyping of Borrelia can be easily implemented in a routine dermatopathology setting, especially as a fast method to confirm early cutaneous borreliosis. Genotyping could also enable earlier treatment of patients infected with invasive strains.

A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy.

PURPOSE: To identify the biochemical and molecular genetic defect in a 16-year-old patient presenting with apical hypertrophic cardiomyopathy and neuropathy suspected for a mitochondrial disorder. METHODS: Measurement of the mitochondrial energy-generating system (MEGS) capacity in muscle and enzyme analysis in muscle and fibroblasts were performed. Relevant parts of the mitochondrial DNA were analysed by sequencing. Transmitochondrial cybrids were obtained by fusion of 143B206 TK(-) rho zero cells with patient-derived enucleated fibroblasts. Immunoblotting techniques were applied to study the complex V assembly. RESULTS: A homoplasmic nonsense mutation m.8529G-->A (p.Trp55X) was found in the mitochondrial ATP8 gene in the patient's fibroblasts and muscle tissue. Reduced complex V activity was measured in the patient's fibroblasts and muscle tissue, and was confirmed in cybrid clones containing patient-derived mitochondrial DNA. Immunoblotting after blue native polyacrylamide gel electrophoresis showed a lack of holocomplex V and increased amounts of mitochondrial ATP synthase subcomplexes. An in-gel activity assay of ATP hydrolysis showed activity of free F(1)-ATPase in the patient's muscle tissue and in the cybrid clones. CONCLUSION: We describe the first pathogenic mutation in the mitochondrial ATP8 gene, resulting in an improper assembly and reduced activity of the complex V holoenzyme.

Measurement of peritoneal fluid handling in children on continuous ambulatory peritoneal dialysis using autologous hemoglobin.

OBJECTIVE: Previous measurements of peritoneal fluid handling in children treated by continuous ambulatory peritoneal dialysis (CAPD) were performed with human albumin as a fluid marker. A major disadvantage of this substance is that endogenous patient albumin enters the peritoneal cavity during the dwell period. For this reason peritoneal fluid kinetics were measured in a group of children on CAPD, using autologous hemoglobin as a volume marker. DESIGN: Autologous hemoglobin was added to dialysate containing 1.36% glucose as a volume marker. Marker clearance (MC), which is presently the best available approximation of lymphatic absorption in the clinical setting, and transcapillary ultrafiltration (TCUF) were measured during a 4-hour dwell. SETTING: University hospital. PATIENTS: Children on CAPD (N = 9), with a median age of 8.1 years (range 2.1-3.2 years). RESULTS: MC was 521 +/- 166 mL/4 hour/1.73 m2, which is high compared to the literature data on adult CAPD patients. TCUF was 519 +/- 92 mL/4 hour/1.73 m2, which is similar to data concerning adult patients. TCUF reached no maximum during the 4-hour dwell, and the deviation of the TCUF curve from linear was markedly less than usually seen in adult patients. CONCLUSIONS: MC in children treated with CAPD is higher when compared to the literature data on adults. Difficulties to achieve sufficient ultrafiltration in children could be caused by relatively small differences between MC and TCUF from the beginning to the end of the dwell.

Familial selective IgA deficiency with circulating anti-IgA antibodies: a distinct group of patients?

Two families were investigated in which the mothers had selective IgA deficiency and circulating class-specific anti-IgA antibodies. Both gave birth to two children who were found to be IgA deficient. Three of these children developed anti-IgA antibodies before puberty. In vitro immunoglobulin production studies performed in the children of both families revealed an IgA B cell defect combined with IgA-specific excessive T suppressor function in all four. The mechanisms by which transplacental passage of maternal anti-IgA antibodies could have interfered with the developing IgA system in the offspring are discussed.