A battery of tandem mass spectrometry assays with stable isotope-dilution for the quantification of 15 anti-tuberculosis drugs and two metabolites in patients with susceptible-, multidrug-resistant- and extensively drug-resistant tuberculosis.

OBJECTIVE: Anti-tuberculosis (antiTB) drugs are characterized by an important inter-interindividual pharmacokinetic variability poorly predictable from individual patients' characteristics. Therapeutic drug monitoring (TDM) may therefore be beneficial for patients with Mycobacterium tuberculosis infection, especially for the management of multidrug/extensively drug resistant- (MDR/XDR)-TB. Our objective was to develop robust HPLC-MS/MS methods for plasma quantification of 15 antiTB drugs and 2 metabolites, namely rifampicin, isoniazid plus N-acetyl-isoniazid, pyrazinamide, ethambutol (the conventional quadritherapy for susceptible TB) as well as combination of agents against MDR/XDR-TB: bedaquiline, clofazimine, delamanid and its metabolite M1, levofloxacin, linezolid, moxifloxacin, pretomanid, rifabutin, rifapentine, sutezolid, and cycloserine. METHODS: Plasma protein precipitation was used for all analytes except cycloserine, which was analyzed separately after derivatization with benzoyl chloride. AntiTB quadritherapy drugs (Pool1) were separated by Hydrophilic Interaction Liquid Chromatography (column Xbridge BEH Amide, 2.1 × 150 mm, 2.5 µm, Waters®) while MDR/XDR-TB agents (Pool 2) and cycloserine (as benzoyl derivative) were analyzed by reverse phase chromatography on a column XSelect HSS T3, 2.1 × 75 mm, 3.5 µm (Waters®). All runs last <7 min. Quantification was performed by selected reaction monitoring electrospray tandem mass spectrometry, using stable isotopically labelled internal standards. RESULTS: The method covers the clinically relevant plasma levels and was extensively validated based on FDA recommendations, with intra- and inter-assay precision (CV) < 15% over the validated ranges. Application of the method is illustrated by examples of TDM for two patients treated for drug-susceptible- and MDR-TB. CONCLUSION: Such convenient extraction methods and the use of stable isotope-labelled drugs as internal standards provide an accurate and precise quantification of plasma concentrations of all major clinically-used antiTB drugs regimens and is optimally suited for clinically efficient TDM against tuberculosis.

Cigarette smoking and culture conversion in patients with susceptible and M/XDR-TB.

BACKGROUND: Tuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Active cigarette smoking may have a significant impact on treatment responses to anti-tuberculosis treatment. OBJECTIVE: To ascertain the effect of smoking on Mycobacterium tuberculosis sputum culture conversion rates following treatment initiation in patients with susceptible, multidrug-resistant and extensively drug-resistant TB (M/XDR-TB). METHOD: Sputum cultures of smoking and non-smoking patients with pulmonary TB (PTB) treated at a referral centre in Germany were evaluated. RESULTS: Between January 2012 and March 2017, 247 patients with PTB treated at the Medical Clinic of Research Center Borstel, Borstel, Germany, were included in the study. Of 247 patients, 65 (26.3%) were infected with multidrug-resistant strains of M. tuberculosis (MDR-TB). Sputum culture examinations were performed on a weekly basis. Active smoking (n = 111; time to culture conversion [TCC] 50.7 days, interquartile range [IQR] 26.5-73.0) and former smoking (n = 72; TCC 43.1 days, IQR 19.8-56.0) significantly delayed culture conversion rates (P < 0.001) when compared with never smoking (n = 64; TCC 33.2 days, IQR 8.0-50.3). Delay in TCC among smoking, non-MDR-TB patients (n = 138; TCC 47.3 days, IQR 19.0-89.0) was comparable with non-smoking, MDR-TB patients (n = 20; TCC 53.0 days, IQR 18.0-71.0). The shortest TCC was observed in non-smoking, non-MDR-TB patients (n = 44; TCC 33.0 days, IQR 10.0-48.5), whereas the longest was seen in smoking, MDR-TB patients (n = 45; TCC 60.7 days, IQR 33.3-89.0); P < 0.001). CONCLUSION: Active cigarette smoking and, to a lesser extent, former cigarette smoking, substantially delayed culture conversion in PTB.

A six-year experience with anal cytology in women with HPV in the lower genital tract: utility, limitations, and clinical correlation.

This study assessed the utility and limitations of anal cytology as a screening method for women infected with human papilloma virus (HPV) in the lower genital tract. Furthermore, this study aimed to establish risk factors for pathological anal cytology/biopsy findings, the prevalence of anatomopathological lesions associated with positive anal brushings, and the frequency of concomitant lesions of the lower genital tract. A cross-sectional, retrospective, descriptive study in 207 women with HPV-associated lesions of the lower genital tract and 25 women with immunosuppression was carried out. Anal cytology, high resolution anoscopy, and biopsy of suspicious lesions were performed. In total, 232 anal brushings were performed: 184 (79.3%) were negative, 24 (10.34%) showed atypical squamous cells of undeterminated significance, 18 (7.7%) showed low-grade squamous intraepithelial lesions, and 6 (2.6%) showed high-grade squamous intraepithelial lesion. Cytohistological correlation was obtained for 70 cases. The sensitivity of anal cytology in detecting intraepithelial lesions was 70%, whereas the specificity was 93%. The sensitivity of the method for detecting high-grade lesions (84%) was higher, than that for detecting low-grade lesions (66%). The most frequently associated pathology was vulvar lesion. It is important to perform anal brushings in women who have had lower genital tract biopsies for HPV-associated lesions due to the high prevalence of anal lesions in such patients. Anal cytology is useful for detecting high-grade lesions but the sensitivity for detecting low-grade lesions is low. It is of the utmost importance to perform high-resolution anoscopy and biopsy in women with suspicious lesions in order to confirm the pathology.

Mutación del gen k-ras en tumores colorrectales: asociación con variantes histológicas / K-ras mutation in colorectal cancer: association with histologic types

Antecedentes: Cerca del 40% de los tumores colorrectales presentan metástasis en el trascurso de su evolución. La sobrevida de los pacientes con cáncer colorrectal ha aumentado en las últimas décadas de algunos meses a mas de 22 meses, gracias a la administración de diferentes drogas quimioterápicas. El desafio actual consiste en seleccionar los pacientes que se beneficiaran de estas terapéuticas. El gen K-ras constituye un marcador biológico de respuesta al tratamiento con anticuerpos monoclonales. No se han descripto asociaciones histológicas con la presencia de mutación del gen K-ras. Objetivo: Analizar la frecuencia de mutaciones del K-ras en los tumores colorrectales. Determinar la relación entre la mutación del K-ras y las variables histopatológicas. Lugar de aplicación: Hospital Universitario. Diseño: Estudio observacional. Población: Pacientes tratados por cáncer colorrectal con estudio de mutación de K-ras. Método: Presentación de trabajo. Resultados: Entre abriI de 2009 y marzo de 2011, estudiamos 145 pacientes tratados por cáncer colorrectal, en los que se determinó el estado del gen K-ras. Encontramos un K-ras no mutado en 106 pacientes (73%) y un K-ras mutado en 39 pacientes (27%). En el análisis de localización tumoral, estadios TNM y sus subcategorías, y mutaciones del K-ras, no hubo diferencias en cuanto a la distribución de mutaciones del K-ras.Analizamos las variables histológicas en 43 pacientes. Encontramos una asociación significativa entre la presencia de infiltración linfocitaria intratumoral de tipo leve a moderada y la presencia de mutaciones del K-ras (p=0,01). Conclusiones: La mutación del K-ras es frecuente en los tumores colorrectales. Algunas variables histológicas orientarían el muestreo genético. A medida que se introduzcan en la práctica habitual fármacos, como el cetuximab, la determinación de estos y otros parámetros moleculares deberá realizarse de rutina.

Mutación del gen k-ras en tumores colorrectales: asociación con variantes histológicas / K-ras mutation in colorectal cancer: association with histologic types

Antecedentes: Cerca del 40% de los tumores colorrectales presentan metástasis en el trascurso de su evolución. La sobrevida de los pacientes con cáncer colorrectal ha aumentado en las últimas décadas de algunos meses a mas de 22 meses, gracias a la administración de diferentes drogas quimioterápicas. El desafio actual consiste en seleccionar los pacientes que se beneficiaran de estas terapéuticas. El gen K-ras constituye un marcador biológico de respuesta al tratamiento con anticuerpos monoclonales. No se han descripto asociaciones histológicas con la presencia de mutación del gen K-ras. Objetivo: Analizar la frecuencia de mutaciones del K-ras en los tumores colorrectales. Determinar la relación entre la mutación del K-ras y las variables histopatológicas. Lugar de aplicación: Hospital Universitario. Diseño: Estudio observacional. Población: Pacientes tratados por cáncer colorrectal con estudio de mutación de K-ras. Método: Presentación de trabajo. Resultados: Entre abriI de 2009 y marzo de 2011, estudiamos 145 pacientes tratados por cáncer colorrectal, en los que se determinó el estado del gen K-ras. Encontramos un K-ras no mutado en 106 pacientes (73%) y un K-ras mutado en 39 pacientes (27%). En el análisis de localización tumoral, estadios TNM y sus subcategorías, y mutaciones del K-ras, no hubo diferencias en cuanto a la distribución de mutaciones del K-ras.Analizamos las variables histológicas en 43 pacientes. Encontramos una asociación significativa entre la presencia de infiltración linfocitaria intratumoral de tipo leve a moderada y la presencia de mutaciones del K-ras (p=0,01). Conclusiones: La mutación del K-ras es frecuente en los tumores colorrectales. Algunas variables histológicas orientarían el muestreo genético. A medida que se introduzcan en la práctica habitual fármacos, como el cetuximab, la determinación de estos y otros parámetros moleculares deberá realizarse de rutina.(AU)

Adhesión a dentina radicular tratada con arginina utilizando sistemas de auto-grabado / Adhesion to arginine-treated root dentin using self-etching bonding systems

El objetivo de este estudio fue evaluar, comparativamente, la resistencia de la unión adhesiva de resinas compuestas a dentina radicular, previamente tratada con arginina (AR) y otras técnicas, utilizando un sistema de auto-grabado (SAG). Se seleccionaron ocho terceros molares libres de caries y de reciente extracción, de los cuales se utilizó la dentina radicular. Se asignaron cuatro grupos de acuerdo al tratamiento realizado: A) AA (acondicionamiento ácido) + AR; B) AA; C) AA + piedra pómez (PP) (control); D) PP (control absoluto). Seguidamente, las superficies fueron tratadas con un SAG y cargadas con un composite. Las probetas fueron sometidas a cargas traccionales utilizando una máquina Instron. A partir de los valores registrados, se obtuvieron los resultados de resistencia de la unión adhesiva, que fueron analizados mediante análisis de varianza de 1 vía y p rueba de comparaciones múltiples de Bonferroni. Se encontraron diferencias estadísticamente significativas (P<0,05) entre los grupos C y B y entre los grupos A y B, concluyendo que AR no interfirió en la resistencia adhesiva en relación a los grupos control.

Adhesión a dentina radicular tratada con arginina utilizando sistemas de auto-grabado / Adhesion to arginine-treated root dentin using self-etching bonding systems

El objetivo de este estudio fue evaluar, comparativamente, la resistencia de la unión adhesiva de resinas compuestas a dentina radicular, previamente tratada con arginina (AR) y otras técnicas, utilizando un sistema de auto-grabado (SAG). Se seleccionaron ocho terceros molares libres de caries y de reciente extracción, de los cuales se utilizó la dentina radicular. Se asignaron cuatro grupos de acuerdo al tratamiento realizado: A) AA (acondicionamiento ácido) + AR; B) AA; C) AA + piedra pómez (PP) (control); D) PP (control absoluto). Seguidamente, las superficies fueron tratadas con un SAG y cargadas con un composite. Las probetas fueron sometidas a cargas traccionales utilizando una máquina Instron. A partir de los valores registrados, se obtuvieron los resultados de resistencia de la unión adhesiva, que fueron analizados mediante análisis de varianza de 1 vía y p rueba de comparaciones múltiples de Bonferroni. Se encontraron diferencias estadísticamente significativas (P<0,05) entre los grupos C y B y entre los grupos A y B, concluyendo que AR no interfirió en la resistencia adhesiva en relación a los grupos control.(AU)

Synthesis and immunochemical characterization of neoglycoproteins containing epitopes of the inner core region of Pseudomonas aeruginosa RNA group I lipopolysaccharide.

The disaccharide allyl 2-acetamido-2-deoxy-alpha-D-galactopyranosyl-(1-->3)-7-O-carbamoyl-L-glycero-alpha-D-manno-heptopyranoside 5 (GalNAc-cmHep-allyl) was synthesized starting from 1 and 2. Compound 5, cmHep-allyl and the disaccharide cmHep-(1-->3)-Hep-allyl were converted into cysteamine-spacered derivatives and conjugated to bovine serum albumin (BSA) to yield the neoglycoconjugates 7--9, respectively. These conjugates were used to immunize mice and to prepare monoclonal antibodies (mAbs) which were characterized in comparison to mAbs obtained after immunization with heat-killed Pseudomonas aeruginosa strain H4. Two antibodies obtained after immunization with the neoglycoconjugates bound strongly to cmHep-BSA and with lower affinity to cmHep-Hep-BSA but did not bind to GalNAc-cmHep-BSA or to H4 LPS. Another antibody obtained after immunization with heat-killed bacteria bound to LPS and GalNAc-cmHep-BSA but not to cmHep-Hep-BSA or cmHep-BSA

Sistema bact-alert de hemocultivos: evaluación de los subcultivos terminales / Bact-Alert system for hemocultures: evaluation of terminal subcultures

Few laboratory microbiological procedures are as important as the isolation of microorganisms from blood. To evaluate the usefulness of the terminal subcultures, 5669 blood cultures giving negative results after 7 days of incubation in the Bact/Alert System (Organon Teknika) were studied. Bottles were distributed as follows: 1562 adult aerobic bottles, 119 adult anaerobic bottles, 3960 pediatric bottles and 28 FAN bottles. From 5669 blood cultures, 10 subcultures that yielded growth had not been detected by the system. These included 5 adult aerobic bottles and 5 pediatric bottles, 7 of these microorganisms were considered contaminants according to clinical data (2 Micrococcus spp, 1 staphylococci coagulase negative, 1 Burkholderia cepacia, 1 Peptoestreptococcus spp, 1 Corynebacterium spp, 1 Scedosporium spp) while the other 3 were considered true bacteremia (1 Pseudomonas aeruginosa, 1 Proteus mirabilis, 1 Streptococcus sanguis), although no one made any change in treatment on the basis of the previous isolation. Based on these results the routinary utilization of terminal subcultures is not advisable and should be used only for special cases or a second system of blood culture should be added according to clinical or epidemiological data.

Sistema bact-alert de hemocultivos: evaluación de los subcultivos terminales / Bact-Alert system for hemocultures: evaluation of terminal subcultures

Few laboratory microbiological procedures are as important as the isolation of microorganisms from blood. To evaluate the usefulness of the terminal subcultures, 5669 blood cultures giving negative results after 7 days of incubation in the Bact/Alert System (Organon Teknika) were studied. Bottles were distributed as follows: 1562 adult aerobic bottles, 119 adult anaerobic bottles, 3960 pediatric bottles and 28 FAN bottles. From 5669 blood cultures, 10 subcultures that yielded growth had not been detected by the system. These included 5 adult aerobic bottles and 5 pediatric bottles, 7 of these microorganisms were considered contaminants according to clinical data (2 Micrococcus spp, 1 staphylococci coagulase negative, 1 Burkholderia cepacia, 1 Peptoestreptococcus spp, 1 Corynebacterium spp, 1 Scedosporium spp) while the other 3 were considered true bacteremia (1 Pseudomonas aeruginosa, 1 Proteus mirabilis, 1 Streptococcus sanguis), although no one made any change in treatment on the basis of the previous isolation. Based on these results the routinary utilization of terminal subcultures is not advisable and should be used only for special cases or a second system of blood culture should be added according to clinical or epidemiological data.(AU)

[Bact-Alert system for hemocultures: evaluation of terminal subcultures]. / Sistema bact-alert de hemocultivos: evaluación de los subcultivos terminales.

Few laboratory microbiological procedures are as important as the isolation of microorganisms from blood. To evaluate the usefulness of the terminal subcultures, 5669 blood cultures giving negative results after 7 days of incubation in the Bact/Alert System (Organon Teknika) were studied. Bottles were distributed as follows: 1562 adult aerobic bottles, 119 adult anaerobic bottles, 3960 pediatric bottles and 28 FAN bottles. From 5669 blood cultures, 10 subcultures that yielded growth had not been detected by the system. These included 5 adult aerobic bottles and 5 pediatric bottles, 7 of these microorganisms were considered contaminants according to clinical data (2 Micrococcus spp, 1 staphylococci coagulase negative, 1 Burkholderia cepacia, 1 Peptoestreptococcus spp, 1 Corynebacterium spp, 1 Scedosporium spp) while the other 3 were considered true bacteremia (1 Pseudomonas aeruginosa, 1 Proteus mirabilis, 1 Streptococcus sanguis), although no one made any change in treatment on the basis of the previous isolation. Based on these results the routinary utilization of terminal subcultures is not advisable and should be used only for special cases or a second system of blood culture should be added according to clinical or epidemiological data.

Sistema bact-alert de hemocultivos: evaluación de los subcultivos terminales. / [Bact-Alert system for hemocultures: evaluation of terminal subcultures]

Few laboratory microbiological procedures are as important as the isolation of microorganisms from blood. To evaluate the usefulness of the terminal subcultures, 5669 blood cultures giving negative results after 7 days of incubation in the Bact/Alert System (Organon Teknika) were studied. Bottles were distributed as follows: 1562 adult aerobic bottles, 119 adult anaerobic bottles, 3960 pediatric bottles and 28 FAN bottles. From 5669 blood cultures, 10 subcultures that yielded growth had not been detected by the system. These included 5 adult aerobic bottles and 5 pediatric bottles, 7 of these microorganisms were considered contaminants according to clinical data (2 Micrococcus spp, 1 staphylococci coagulase negative, 1 Burkholderia cepacia, 1 Peptoestreptococcus spp, 1 Corynebacterium spp, 1 Scedosporium spp) while the other 3 were considered true bacteremia (1 Pseudomonas aeruginosa, 1 Proteus mirabilis, 1 Streptococcus sanguis), although no one made any change in treatment on the basis of the previous isolation. Based on these results the routinary utilization of terminal subcultures is not advisable and should be used only for special cases or a second system of blood culture should be added according to clinical or epidemiological data.

Elucidation of the structure of an alanine-lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4.

Lipopolysaccharide (LPS) of Pseudomonas aeruginosa rough mutant H4 was isolated by hot water/phenol extraction followed by a modified phenol/chloroform/petroleum ether procedure. Upon SDS/PAGE, the LPS showed a strong major band corresponding to the expected rough-type LPS. Additional faint high molecular-mass bands revealed that the O-chain was present, indicating that the H4 mutant is genetically unstable. Mild acid hydrolysis of the LPS removed lipid A and released a phosphorylated core oligosaccharide that was purified by gel-permeation chromatography and high-performance anion-exchange liquid chromatography. The oligosaccharide contained two residues of L-glycero-D-manno-heptose (Hep) and one residue each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and GalNAc. Upon matrix-assisted laser desorption/ionization mass spectroscopy in the negative ion mode, the main fraction expressed a peak for the molecular ion [M-H]- at m/z 1106.41, which was compatible with a carbamoylated, trisphosphorylated tetrasaccharide. The structure was further investigated using one- and two-dimensional homonuclear and heteronuclear correlated NMR spectroscopy at pD 3 and, after borohydride reduction, at pD 9. The NMR data of the two phosphorylated tetrasaccharides recorded at different pD allowed determination of the positions of the three phosphate (P) groups and the carbamoyl group (Cm) thus establishing the following structure of the core oligosaccharide: [equation: see text] Two unusual structural features in the core oligosaccharide of P. aeruginosa were identified for the first time, i.e. the replacement of an amide-linked alanyl group in GalN with an acetyl group and the phosphorylation at position 6 of HepII.

[Approach to repeated pregnancy of women with uterine malformation (author's transl)]. / Unser geburtshilfliches Verhalten bei erneuter Schwangerschaft und Fehlbildungen des Uterus.

Twelve cases of uterine malformation are described in this paper. Each of them had a previous history of a full-term pregnancy. In nine cases earlier diagnosis had been based on histerosalpingography. Caesarean section had to be performed on five cases for abnormal presentations, while seven deliveries had been normal. Diagnosis was made for three cases more recently during various gynaecological procedures.