Molecular characterization of multiple-drug-resistant Mycobacterium tuberculosis isolates from northwestern Russia and analysis of rifampin resistance using RNA/RNA mismatch analysis as compared to the line probe assay and sequencing of the rpoB gene

This investigation evaluated the potential of RNA/RNA mismatch analysis for the detection of rifampin resistance among 38 multiple-drug-resistant (MDR) isolates of Mycobacterium tuberculosis from northwestern Russia. The results obtained were compared with a commercialized line probe assay and rpoB sequencing, and the genetic diversity of the isolates was also investigated in parallel using spoligotyping and variable number of tandem DNA repeats (VNTR). The mismatch analysis revealed 3 distinct RNA cleavage profiles permitting the subdivision of the strains into mutation groups 1 to 3, the most common being group 1 (28 of 38 isolates) that contained a majority of strains with a TCG531>TTG (Ser->Leu) mutation, followed by group 2 (6 of 38 isolates) characterized by different mutations in the codon CAC526 (His), and group 3 (4 of 38 isolates), all characterized by a GAC516(Asp) mutation. Spoligotyping revealed the Beijing type to be the most prevalent among mismatch group 1 (24 out of 28 strains), suggesting that the most frequent rpoB mutation among the Beijing family in our setting was TCG531 >TTG (Ser->Leu). All the Beijing type isolates were also characterized by a unique VNTR pattern made up of exact tandem repeats (ETR)-A to E of 42435. We conclude that the Beijing genotype constitutes the major family of MDR-TB isolates currently circulating in northwestern Russia, and that the in-house RNA/RNA mismatch analysis may be successfully used for rapid and reliable diagnosis of rifampin-resistant tuberculosis in this setting.     

Global distribution of Mycobacterium tuberculosis spoligotypes

We present a short summary of recent observations on the global distribution of the major clades of the Mycobacterium tuberculosis complex, the causative agent of tuberculosis. This global distribution was defined by data-mining of an international spoligotyping database, SpolDB3. This database contains 11708 patterns from as many clinical isolates originating from more than 90 countries. The 11708 spoligotypes were clustered into 813 shared types. A total of 1300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.     

Use of spoligotyping to study the evolution of the direct repeat locus by IS6110 transposition in Mycobacterium tuberculosis

Based on the variability of 43 spacers within the direct repeat (DR) locus of Mycobacterium tuberculosis complex organisms, spoligotyping is a rapid method that aids in the study of the epidemiology of tuberculosis. It was recently hypothesized that despite its presence in the DR locus, spacer 31 could not be amplified in M. tuberculosis clinical isolates belonging to spoligotype 50 due to the insertion of an extra copy of IS6110 between spacers 31 and 32 that could lead to an asymmetrical split of the primer targets (I. Filliol, C. Sola, and N. Rastogi, J. Clin. Microbiol. 38:1231--1234, 2000). In the present investigation, previous observations were extended to 25 clinical isolates of type 50 showing that the primer set IS6-DRb that selectively amplified the left and central DR regions was indeed able to demonstrate the presence of spacer 31. IS6110-restriction fragment length polymorphism (RFLP) and DR-RFLP showed that type 50 isolates were characterized by the presence of two copies of IS6110 associated with the DR locus and an additional double IS6110 band of 1.4 kb. The primer set IS3-IS6 was then used to selectively amplify a 750-bp inter-IS6110 fragment within the DR locus. The sequencing of the central DR region corroborated our previous findings and showed that the absence of spacer 31 among the type 50 isolates was due to the asymmetric insertion of an extra copy of IS6110 between spacers 31 and 32, leading to an unequal split of the DRa-DRb target into two portions, of 6 and 30 bp, respectively. These results show that the DR locus constitutes an ideal IS6110 preferential locus (ipl), permitting the insertion of two or more copies of IS6110, and provide new clues for epidemiological and phylogenetic interpretation of changes in IS6110-RFLP and spoligotyping profiles.     

Delayed retroperitoneal haematoma after failed lumbar plexus block

A 72-yr-old patient was to undergo a left lumbar plexus blockby the posterior approach to achieve postoperative analgesiaafter hip replacement. The block failed after three unsuccessfulattempts to identify nerve structures and a fascia iliaca compartmentblock was performed. Postoperatively the patient received enoxaparinand then phenylindanedione for thromboprophylaxis. She was re-admitted2 weeks after surgery because of a lower limb motor deficitand a left retroperitoneal haematoma requiring blood transfusion.Clinicians need to be aware of this potential complication oflumbar plexus block in patients receiving thrombphylaxis.     

Molecular characterization and biodiversity of Mycobacterium tuberculosis in the Antilles-Guiana region and comparative analysis in a metropolitan region, Aquitaine

Tuberculosis is a highly contagious infectious disease in recrudescence whose epidemiologic monitoring is reinforced by molecular biology. In this context, we were particularly interested in the cases of tuberculosis of French West Indies and French Guiana (FWI-FG). This study covered a period of two years (1997 and 1998) and focused on the demographical and epidemiological characteristics of the cases diagnosed by an analysis of their genotypes. Our results were confronted with a French metropolitan area (Aquitaine) with similar demographic background. Moreover, Aquitaine area has privileged links with FWI-FG region and also has a similar network for monitoring tuberculosis as ours. So we used a PCR method called spoligotyping as a first line method to optimize the alternative IS6110-RFLP method which remains cumbersome. A total of 105 strains of FWI-FG and 172 strains of Aquitaine were typed by spoligotyping and by the standard IS6110-RFLP method. The results of the first grouping by spoligotyping were analyzed in comparison with IS6110-RFLP. The results obtained showed a rate of recent transmission of tuberculosis being 34.3% in FWI-FG and 10.5% in Aquitaine. These observations underlined a high degree of polymorphism in the Aquitaine region as compared to the FWI-FG region. Thanks to the various profiles obtained by spoligotyping, we could study their distribution in the three areas and highlight common types like type 53, 50 and 42 and types found locally like the types 33 and 14 found respectively in Aquitaine and FWI as well as endemic types like type 76 found only in FG. These results are discussed in the context of the evolution of clinical isolates of tubercle bacilli with time.     

Role of embB codon 306 mutations in Mycobacterium tuberculosis revisited: a novel association with broad drug resistance and IS6110 clustering rather than ethambutol resistance

Mutations at position 306 of embB (embB306) have been proposed as a marker for ethambutol resistance in Mycobacterium tuberculosis; however, recent reports of embB306 mutations in ethambutol-susceptible isolates caused us to question the biological role of this mutation. We tested 1,020 clinical M. tuberculosis isolates with different drug susceptibility patterns and of different geographical origins for associations between embB306 mutations, drug resistance patterns, and major genetic group. One hundred isolates (10%) contained a mutation in embB306; however, only 55 of these mutants were ethambutol resistant. Mutations in embB306 could not be uniquely associated with any particular type of drug resistance and were found in all three major genetic groups. A striking association was observed between these mutations and resistance to any drug (P < 0.001), and the association between embB306 mutations and resistance to increasing numbers of drugs was highly significant (P < 0.001 for trend). We examined the association between embB306 mutations and IS6110 clustering (as a proxy for transmission) among all drug-resistant isolates. Mutations in embB306 were significantly associated with clustering by univariate analysis (odds ratio, 2.44; P = 0.004). In a multivariate model that also included mutations in katG315, katG463, gyrA95, and kasA269, only mutations in embB306 (odds ratio, 2.14; P = 0.008) and katG315 (odds ratio, 1.99; P = 0.015) were found to be independently associated with clustering. In conclusion, embB306 mutations do not cause classical ethambutol resistance but may predispose M. tuberculosis isolates to the development of resistance to increasing numbers of antibiotics and may increase the ability of drug-resistant isolates to be transmitted between subjects.     

Alternative Method of Oral Dosing for Rats

Oral administration of drugs to laboratory rodents typically is achieved by using the gavage technique. Although highly effective, this method occasionally can cause esophageal injury as well as restraint-associated distress, particularly with repeated use. The aim of this study was to assess an alternative oral dosing method that could reduce the distress and morbidity associated with standard gavage techniques. The palatability and pharmacokinetic profile of 2 medicines approved for the treatment of Alzheimer disease, donepezil and galantamine, were investigated in male Lister hooded rats by using a syringe-feeding method and compared with results from traditional gavage administration. In addition, the stimulant nicotine was tested by using the syringe-feeding method in a separate series of experiments. Animals reliably learned to drink voluntarily from the syringe, and latency to drink decreased rapidly. The addition of donepezil, galantamine, or nicotine to sucrose had no apparent effect on the palatability of the solution, although nicotine produced aversive effects that inhibited subsequent voluntary intake. Oral bioavailability was improved by using syringe feeding with donepezil but not galantamine. Both drugs improved cognitive performance in the novel object recognition test, with similar behavioral profiles between the 2 methods of administration. Our results suggest that the syringe-feeding technique is an effective alternative oral dosing method in rats.Abbreviation: NOR, novel object recognitionOral administration of substances is a common procedure in scientific experiments using laboratory animals and typically is achieved in conscious animals by using the intragastric gavage technique. Gavage is the introduction of a solution into the stomach by means of a tube and is used clinically and for research. In laboratory animals, dosage by gavage involves removing the animal from its cage, manually restraining it, inserting a small-diameter tube into the esophagus, and delivering the drug directly into the stomach by means of a syringe. Although highly effective, care must be taken to ensure that the tube or needle does not enter the trachea or damage the esophagus or stomach. In one study,⁷ a 32% mortality rate was attributable to asphyxia caused by impacted food and bedding material in the oropharynx of gavaged rats; granulomatous inflammation caused by the gavaging procedure appeared to be the source of the impaction. To ensure competent administration of the test substance, the animal must be restrained manually, which can distress the animal. A study using implanted telemetry transponders to investigate the intensity and duration of the stress caused by oral gavage demonstrated that the acute effect of the procedure on laboratory rats can last for 30 to 60 min afterward.⁴Over the years, the gavage procedure has been refined to reduce morbidity and mortality in laboratory animals through the use of flexible cannulas, but no procedure as yet has been found to effectively replace this method.^13,17 Several investigators have investigated novel means of oral drug delivery in rats. For instance, one group developed a technique for oral drug administration to rats by using premixed drug–chocolate pellets.⁸ Results from this technique demonstrated appropriate levels of drug absorption as indicated by effects of the test drugs in an in vivo model. Although effective, this method has several limitations. First, the theobromine and caffeine contents in chocolate may proscribe its use for oral drug administration in drug discovery and efficacy studies. Second, the drug must be stable, mix easily, and remain active in chocolate and be adequately palatable in dry form mixed with chocolate.⁸ Other investigators introduced a novel method of oral drug administration in a study in which a mixture of 5% sucrose solution and a neuroleptic drug (clozapine, haloperidol, or diazepam) was given to the test animals by using a syringe.¹⁴ Behavioral effects associated with these drugs then were investigated. All animals adapted to daily drug administration without any difficulties and displayed minimal effects on wellbeing, as reflected by voluntary participation in both drug administration and behavioral testing. This alternative method of oral administration was sufficient to cause changes in behavioral parameters previously reported by using traditional methods of drug administration.^6,14 However, the study did not compare the novel method with traditional methods of oral drug administration nor report on pharmokinetic exposure after syringe delivery.The aim of the present study was to investigate the palatability and pharmacokinetic profile of donepezil and galantamine, which are approved for the treatment of Alzheimer disease, by dosing male Lister hooded rats with the syringe method and comparing subsequent results with those from the traditional administration method of orogastric gavage. In a separate series of experiments, we also evaluated the stimulant nicotine (frequently used as a positive control in many cognitive behavioral tests) by using both dosing methods. We then investigated the efficacy of syringe feeding of donepezil and galantamine in enhancing cognitive performance in the novel object recognition (NOR) test and compared the results with those after gavage dosing. The NOR assessment is a simple test of recognition memory that exploits the tendency or preference of rats to explore a novel rather than a familiar object in their environment.^1,5 The assay is known to be particularly sensitive to both animal handling and drug administration that may induce distress, making this test an excellent cognitive model for evaluating the efficacy of the 2 methods of oral administration under study.     

Histocytopathological diagnosis of Rosai–Dorfman disease: Case report

Sinus histiocytosis with massive lymphadenopathy (Rosai–Dorfman disease) being a rare benign proliferative self‐limiting disease of the cells of macrophage‐histiocyte family is of unknown etiology and presents with massive lymphadenopathy. We are hereby reporting a case of RDD presenting with massive bilateral cervical and submandibular lymphadenopathy, diagnosed by histocytopathology.