Independent inter and intra-observer agreement of the Schizas's classification of degenerative lumbar stenosis: Comparison among three levels of surgical training. / [Artículo traducido] Acuerdo inter- e intraobservador independiente de la clasificación de Schizas de estenosis lumbar degenerativa. Comparación entre tres niveles de entrenamiento quirúrgico.

INTRODUCTION AND OBJECTIVES: Lumbar spinal stenosis is a common age-related condition that affects the quality of life. Multiple classifications have been developed to quantify the severity of stenosis affecting comparison between studies and homogenous communication among surgeons and researchers. Even though this classification has not shown a direct clinical correlation, Schizas's classification appears to be a simple method to assess stenosis. Our objective was to evaluate the inter and intraobserver independent agreement of the Schizas's classification to assess stenosis severity. Additionally, we aimed to compare agreement among three levels of training in spine surgery. MATERIALS AND METHODS: An independent inter and intra observer agreement was conducted among junior, senior orthopedic residents and attending spine surgeons. Ninety lumbar levels from 30 patients were evaluated by 16 observers. Weighted kappa agreement was used. RESULTS: Overall interobserver and intraobserver agreement was of 0.57 (95% CI=0.52-0.63) and 0.69 (0.55-0.79), respectively. Interobserver agreement according to level of training yielded values of 0.53 (0.46-0.60) for junior residents, 0.61 (0.54-0.67) for senior residents and 0.67 (0.59-0.74) for attendings. Intraobserver agreement was of 0.54 (0.48-0.60) for junior, 0.60 (0.55-0.66) for senior and 0.66 (0.60-0.72) for attendings. CONCLUSION: The Schizas's classification showed moderate interobserver and substantial intraobserver agreement. Among attending surgeons, substantial inter and intraobserver agreement was observed. The classification allowed acceptable communication among trained spine surgeons.

Independent inter and intra-observer agreement of the Schizas's classification of degenerative lumbar stenosis: Comparison among three levels of surgical training.

INTRODUCTION AND OBJECTIVES: Lumbar spinal stenosis is a common age-related condition that affects the quality of life. Multiple classifications have been developed to quantify the severity of stenosis affecting comparison between studies and homogenous communication among surgeons and researchers. Even though this classification has not shown a direct clinical correlation, Schizas's classification appears to be a simple method to assess stenosis. Our objective was to evaluate the inter and intraobserver independent agreement of the Schizas's classification to assess stenosis severity. Additionally, we aimed to compare agreement among three levels of training in spine surgery. MATERIALS AND METHODS: An independent inter and intra observer agreement was conducted among junior, senior orthopedic residents and attending spine surgeons. Ninety lumbar levels from 30 patients were evaluated by 16 observers. Weighted kappa agreement was used. RESULTS: Overall interobserver and intraobserver agreement was of 0.57 (95% CI=0.52-0.63) and 0.69 (0.55-0.79), respectively. Interobserver agreement according to level of training yielded values of 0.53 (0.46-0.60) for junior residents, 0.61 (0.54-0.67) for senior residents and 0.67 (0.59-0.74) for attendings. Intraobserver agreement was of 0.54 (0.48-0.60) for junior, 0.60 (0.55-0.66) for senior and 0.66 (0.60-0.72) for attendings. CONCLUSION: The Schizas's classification showed moderate interobserver and substantial intraobserver agreement. Among attending surgeons, substantial inter and intraobserver agreement was observed. The classification allowed acceptable communication among trained spine surgeons.

Wound-related complications in the surgical treatment of vertebral metastatic disease - A case series analysis. / [Artículo traducido] Complicaciones relacionadas con la herida en el tratamiento quirúrgico de la enfermedad metastásica vertebral: análisis de una serie de casos.

INTRODUCTION AND OBJECTIVES: Spinal metastases (SM) account for 5-30% of patients with cancer, causing pain, deformity and/or neurological deficit. Postoperative complications are a concerning subject and wound-related complications (WRC) may delay adjuvant treatment. The objective of this study was to analyze the incidence of WRC in patients with SM that underwent surgical treatment as well as possible risk factors related to the occurrence of complications. MATERIALS AND METHODS: Patients with SM operated between 2011 and 2021 were analyzed. Demographics characteristics, primary tumor, general and neurological status, Tokuhashi score, type of surgical treatment, surgical length, preoperative serum albumin and hemoglobin, pre and postoperative adjuvant treatment were analyzed. The incidence and risk factors of WRC -surgical site infection, hematoma, and/or dehiscence- at 90 days was evaluated. Patients were classified in two groups according to the absence/presence of WRC. RESULTS: 198 patients (121 males and 77 females) with an average age of 65 years (range 54-73 years) were analyzed. WRC were observed in 44 patients (22%). On multivariable analysis, significant predictors for developing WRC were low Tokuhashi score (OR=7.89, 95% CI=1.37-45.35, p=.021), prostate cancer as primary tumor (6.73, 1.14-39.65, p=.035), and preoperative serum albumin level ≤3.5g/dL (2.31, 1.02-5.22, p=.044). There was no difference between groups on 90 days survival rate (p=.714). CONCLUSIONS: In our series, the incidence of WRC was 22%, main risk factors for complications were low Tokuhashi score, lower preoperative serum albumin, and prostate cancer. Finally, short-term survival rate was not affected by the occurrence of WRC.

Wound-related complications in the surgical treatment of vertebral metastatic disease - A case series analysis.

INTRODUCTION AND OBJECTIVES: Spinal metastases (SM) account for 5-30% of patients with cancer, causing pain, deformity and/or neurological deficit. Postoperative complications are a concerning subject and wound-related complications (WRC) may delay adjuvant treatment. The objective of this study was to analyze the incidence of WRC in patients with SM that underwent surgical treatment as well as possible risk factors related to the occurrence of complications. MATERIALS AND METHODS: Patients with SM operated between 2011 and 2021 were analyzed. Demographics characteristics, primary tumor, general and neurological status, Tokuhashi score, type of surgical treatment, surgical length, preoperative serum albumin and hemoglobin, pre and postoperative adjuvant treatment were analyzed. The incidence and risk factors of WRC - surgical site infection, hematoma, and/or dehiscence - at 90 days was evaluated. Patients were classified in two groups according to the absence/presence of WRC. RESULTS: 198 patients (121 males and 77 females) with an average age of 65 years (range 54-73 years) were analyzed. WRC were observed in 44 patients (22%). On multivariable analysis, significant predictors for developing WRC were low Tokuhashi score (OR=7.89, 95% CI=1.37-45.35, p=0.021), prostate cancer as primary tumor (6.73, 1.14-39.65, p=0.035), and preoperative serum albumin level ≤3.5g/dL (2.31, 1.02-5.22, p=0.044). There was no difference between groups on 90 days survival rate (p=0.714). CONCLUSIONS: In our series, the incidence of WRC was 22%, main risk factors for complications were low Tokuhashi score, lower preoperative serum albumin, and prostate cancer. Finally, short-term survival rate was not affected by the occurrence of WRC.

Successful Allogenic Stem Cell Transplantation in Patients with Inherited CARD9 Deficiency.

Autosomal recessive (AR) CARD9 (caspase recruitment domain-containing protein 9) deficiency underlies invasive infections by fungi of the ascomycete phylum in previously healthy individuals at almost any age. Although CARD9 is expressed mostly by myeloid cells, the cellular basis of fungal infections in patients with inherited CARD9 deficiency is unclear. Therapy for fungal infections is challenging, with at least 20% premature mortality. We report two unrelated patients from Brazil and Morocco with AR CARD9 deficiency, both successfully treated with hematopoietic stem cell transplantation (HSCT). From childhood onward, the patients had invasive dermatophytic disease, which persisted or recurred despite multiple courses of antifungal treatment. Sanger sequencing identified homozygous missense CARD9 variants at the same residue, c.302G>T (p.R101L) in the Brazilian patient and c.301C>T (p.R101C) in the Moroccan patient. At the ages of 25 and 44 years, respectively, they received a HSCT. The first patient received a HLA-matched HSCT from his CARD9-mutated heterozygous sister. There was 100% donor chimerism at D + 100. The other patient received a T cell-depleted haploidentical HSCT from his CARD9-mutated heterozygous brother. A second HSCT from the same donor was performed due to severe amegakaryocytic thrombocytopenia despite achieving full donor chimerism (100%). At last follow-up, more than 3 years after HSCT, both patients have achieved complete clinical remission and stopped antifungal therapy. HSCT might be a life-saving therapeutic option in patients with AR CARD9 deficiency. This observation strongly suggests that the pathogenesis of fungal infections in these patients is largely due to the disruption of leukocyte-mediated CARD9 immunity.

Molecular epidemiology of hospital-onset methicillin-resistant Staphylococcus aureus infections in Southern Chile.

Methicillin-resistant Staphylococcus aureus (MRSA) is a pathogen of public health importance. In Chile, the Cordobes/Chilean clone was the predominant healthcare-associated MRSA (HA-MRSA) clone in 1998. Since then, the molecular epidemiological surveillance of MRSA has not been performed in Southern Chile. We aimed to investigate the molecular epidemiology of HA-MRSA infections in Southern Chile to identify the MRSA clones involved, and their evolutionary relationships with epidemic international MRSA lineages. A total of 303 single inpatient isolates of S. aureus were collected in the Valdivia County Hospital (2007-2008), revealing 33% (100 MRSA/303) prevalence for HA-MRSA infections. The SCCmec types I and IV were identified in 97% and 3% of HA-MRSA, respectively. All isolates lacked the pvl genes. A random sample (n = 29) of all MRSA was studied by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), SCCmec subtyping, agr and spa typing, and virulence genes profiling. PFGE analysis revealed the predominance (89%, 26/29) of pulsotype A and three additional pulsotypes, designated H1, I33, and G1. Pulsotype A (ST5-SCCmecI-spa-t149) is clonally related to the Cordobes/Chilean clone. Pulsotype H1 (ST5-SCCmecIVNT-spa-t002) is genetically related to the Pediatric clone (ST5-SCCmecIV). Pulsotype I33 (ST5-SCCmecIVc-spa-t002) is clonally related by PFGE to the community-associated MRSA (CA-MRSA) clone spread in Argentina, I-ST5-IVa-PVL(+). The G1 pulsotype (ST8-SCCmecIVc-spa-t024) is clonally related to the epidemic USA300 CA-MRSA. Here, we demonstrate the stability of the Cordobes/Chilean clone over time as the major HA-MRSA clone in Southern Chile. The identification of two CA-MRSA clones might suggest that these clones have entered into the healthcare setting from the community. These results emphasize the importance of the local surveillance of MRSA infections in the community and hospital settings.

Strategies to develop the nursing process and nursing care plans in the health system in Bolivia.

BACKGROUND: Care plans provide guidelines to offer all patients the same quality assistance. Care plans constitute a base where every stage of the nursing process can be recorded, which will save time when handling documentation. Bolivian nurses are very keen to participate in this global trend in order to grasp the opportunity offered by the international cooperation programmes carried out between Spain and South America. AIMS: To identify obstacles and enablers encountered when implementing the nursing process and to set clear strategies and actions so as to develop the nursing process and care plans in Santa Cruz department (Bolivia) both in hospitals and universities. METHODS: The participatory action research was conducted between the Autonomous University of Gabriel René Moreno (Bolivia) and the University of Almería (Spain). The procedure for data collection included meetings with key informants, interviews, observation and workshops. Data were analysed using the constant comparison and categorized by common themes. RESULTS: Lack of time and instruments, shortage of resources and the need for continuous training came up as obstacles, whereas enablers were the nurses' involvement, the university-hospital coordination and the support given by institutions. Actions taken were the creation of a care plan unit, the implementation of a training programme, design of instruments and meetings held with authorities and institutional representatives. CONCLUSIONS: Conducting action research schemes within international cooperation programmes signifies an excellent opportunity for the nursing system in less-developed countries to be included in international plans with the aim of standardizing the nursing practice.

High frequency of Panton-Valentine leukocidin genes in invasive methicillin-susceptible Staphylococcus aureus strains and the relationship with methicillin-resistant Staphylococcus aureus in Córdoba, Argentina.

In the study presented here, the genetic characteristics of methicillin-susceptible Staphylococcus aureus (MSSA) strains isolated from patients attending hospitals in the city of Córdoba, Argentina, during 1999-2002 were evaluated to determine their genetic relationship with methicillin-resistant S. aureus (MRSA) clones as part of an effort to control the potential emergence of new epidemic MRSA strains. The results showed there is a high frequency of MSSA strains carrying Panton-Valentine leukocidin genes in invasive infections in Córdoba, Argentina, particularly in those occurring in hospital settings. Panton-Valentine leukocidin genes were found in the genomic background of one clone (ST30-N pulsotype) belonging to a successful internationally distributed MSSA lineage (clonal complex 30), which is closely related to the EMRSA-16 pandemic clone. These genes were also detected in the ancestral clone (ST5-M pulsotype) of the most prevalent MRSA epidemic clone causing healthcare-associated infections in this region, known as the Cordobes/Chilean clone. The molecular characterization of circulating MSSA strains, including the detection of Panton-Valentine leukocidin genes, is thus a useful marker for investigating the evolving epidemiology of hospital- and community-acquired MRSA clones.

[Molecular epidemiology of tuberculosis in Guadeloupe from 1994 to 2000]. / Epidémiologie moléculaire de la tuberculose en Guadeloupe de 1994 à 2000.

In Guadeloupe, the incidence of tuberculosis decreased between 1994 and 2000. The rate of resistance to at least one antibiotic remained constant at 11%, whereas the rate of multiple-drug resistance increased from 0.9 to 2.4% in 2000. The proportion of patients of foreign origin (mainly from Haiti and the Dominican Republic) increased whereas the number of French patients decreased. These results show that the epidemiology of tuberculosis in Guadeloupe is similar to industrialized countries as older people, foreigners from countries where TB is endemic, and HIV+ patients are at a higher risk to declare tuberculosis disease. Molecular typing realized by spoligotyping showed the importance of previous successive colonizations and migrations as characterized by the presence of major phylogenetic families originating essentially from Northern Europe (Haarlem), Latin America and Mediterranean (LAM) and from Anglo-Saxon countries (X). The sub-typing of clustered strains by IS6110-RFLP and by a PCR method based on the variable number of tandem DNA repeats (VNTR), highlighted 29 clusters, corresponding to 44.8% of clustered strains, and allowed to estimate the rate of recent transmission at 32.2%. The epidemiologic data associated with fingerprinting results underlined the importance of reactivation cases among older people, a significant number of imported TB cases without evident links, and casual contacts.

Virulence factors of non-O1 non-O139 Vibrio cholerae isolated in Córdoba, Argentina / Factores de virulencia de Vibrio cholerae no-O1 no-O139 aislados en Córdoba, Argentina

V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no-clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and “in vivo” studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.

En este trabajo se analizó la presencia y expresión de genes de virulencia en V. cholerae no-O1 no-O139 de origen clínico y ambiental, aislados en Córdoba, Argentina. La mayoría de las cepas estudiadas contiene los genes toxR y hlyA, pero no ctxA, zot, ace, tcpA y stn. Se analizó la actividad hemolítica y citotóxica de estas cepas en los sobrenadantes de cultivo, así como su potencial enterotóxico en ensayos de asa ileal ligada de conejo. Además, los aislamientos fueron comparados por sus perfiles genéticos en PFGE. Las cepas del medio ambiente mostraron variación en su fenotipo de virulencia y no mostraron relación clonal. Las cepas clínicas fueron muy enterotóxicas, hemolíticas, proteolíticas y mostraron perfiles indistinguibles de PFGE, aunque mostraron diferencias en su actividad citotóxica. En este trabajo se describen por primera vez, utilizando ensayos de cultivo celular e “in vivo”, propiedades de virulencia de V. cholerae no-O1 no-O139 aislados en Argentina.

Virulence factors of non-O1 non-O139 Vibrio cholerae isolated in Córdoba, Argentina.

V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and "in vivo" studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.

Virulence factors of non-O1 non-O139 Vibrio cholerae isolated in Córdoba, Argentina.

V. cholerae non-O1 non-O139 serogroups isolated from clinical and environmental sources in Córdoba, Argentina, were analyzed for the presence and expression of virulence genes. Most of the strains studied contained the genes toxR and hlyA, but lacked ctxA, zot, ace, tcpA and stn. The culture supernatants were tested for hemolytic and cytotoxic activity. The enterotoxic potential of the strains was studied in a rabbit ileal loop assay and their genetic profiles were compared by PFGE. The environmental strains varied in their virulence phenotype and showed no clonal relationships. The clinical strains were highly enterotoxic, hemolytic, proteolytic and showed indistinguishable PFGE profiles, although they differed in their cytotoxic activity. This is the first description, using cell culture and [quot ]in vivo[quot ] studies, of the virulence properties of non-O1 non-O139 V. cholerae from Argentina.

[Molecular characterization and biodiversity of Mycobacterium tuberculosis in the Antilles-Guiana region and comparative analysis in a metropolitan region, Aquitaine]. / Caractérisation moléculaire et biodiversité de Mycobacterium tuberculosis dans la région Antilles-Guyane et analyse comparative à une région métropolitaine, l'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.

[Epidemiological study of tuberculosis in the area of Angers, France, as studied by 3 PCR-based fingerprinting methods]. / Epidémiologie moléculaire de la tuberculose dans l'agglomération angevine étudiée par une stratégie d'association de 3 méthodes de génotypage par PCR.

The new genotyping methods efficiently complement classical epidemiological investigation in order to attempt a global approach to TB control. In the present work, we have studied the genomic diversity of Mycobacterium tuberculosis isolated during the year 1998 within the district of Angers, France (260,000 inhabitants distributed in 29 districts), in order to identify recent transmission events and any related risk factors. The methods used included "spacer oligonucleotide typing" or spoligotyping, "variable number of DNA tandem repeats" or VNTR, and "double repetitive element PCR" or DRE-PCR. The resulting spoligotyping and VNTR results were also feeded to international databases and compared with >10,000 isolates for spoligotyping and 500 isolates for VNTR, representative of about 60 countries. The results obtained underlined that most of the TB cases in our setting probably reflected reactivation cases, as clustered cases indicative of potential events of recent transmission were rare. Furthermore, interrogation of international databases showed that most of the isolates from the Angers region belonged to major conserved families of TB isolates representative of Europe, with only rare cases of Asian origin, or those previously reported in specific epidemies reported from elsewhere.

A data-mining approach to spacer oligonucleotide typing of Mycobacterium tuberculosis.

MOTIVATION: The Direct Repeat (DR) locus of Mycobacterium tuberculosis is a suitable model to study (i) molecular epidemiology and (ii) the evolutionary genetics of tuberculosis. This is achieved by a DNA analysis technique (genotyping), called sp acer oligo nucleotide typing (spoligotyping ). In this paper, we investigated data analysis methods to discover intelligible knowledge rules from spoligotyping, that has not yet been applied on such representation. This processing was achieved by applying the C4.5 induction algorithm and knowledge rules were produced. Finally, a Prototype Selection (PS) procedure was applied to eliminate noisy data. This both simplified decision rules, as well as the number of spacers to be tested to solve classification tasks. In the second part of this paper, the contribution of 25 new additional spacers and the knowledge rules inferred were studied from a machine learning point of view. From a statistical point of view, the correlations between spacers were analyzed and suggested that both negative and positive ones may be related to potential structural constraints within the DR locus that may shape its evolution directly or indirectly. RESULTS: By generating knowledge rules induced from decision trees, it was shown that not only the expert knowledge may be modeled but also improved and simplified to solve automatic classification tasks on unknown patterns. A practical consequence of this study may be a simplification of the spoligotyping technique, resulting in a reduction of the experimental constraints and an increase in the number of samples processed.

Mycobacterium tuberculosis phylogeny reconstruction based on combined numerical analysis with IS1081, IS6110, VNTR, and DR-based spoligotyping suggests the existence of two new phylogeographical clades.

This paper deals with phylogenetic relationships among a set of 90 clinical strains representative of the worldwide diversity of the Mycobacterium tuberculosis complex (Kremer et al. 1999) using eight independent genetic markers: IS6110, IS1081, the direct repeat (DR) locus, and five variable number of tandem DNA repeat loci (VNTR). In a preliminary experiment, phylogenetic trees based on single markers were constructed that led to the detection of some similarities between the VNTR-based and the spoligotyping-based phylogenetic trees. In the second step, a more global phenetic approach based on pairwise comparison of strains within each typing system was used, followed by calculations of mean genetic distances based on all the eight loci and the use of the neighbor-joining algorithm for tree reconstruction. This analysis confirmed our preliminary observations and suggested the existence of at least two new phylogeographical clades of M. tuberculosis, one defined as the "East African-Indian family" (EA-I), which may find its origin on the African or Asian continents, and the other as the "Latin American and Mediterranean" (LA-M) family. The existence of these two families was also validated by an independent phylogenetic analysis of spoligotyping on a larger set of shared types (n = 252) and further corroborated by VNTR and katG-gyrA results. The potential origin of these families of bacilli is discussed based on cattle domestication and human migration history. In conclusion, the information contained in insertion sequence and repetitive DNAs may serve as a model for the phylogenetic reconstruction of the M. tuberculosis complex.

Rapid differentiation of "Mycobacterium canettii" from other Mycobacterium tuberculosis complex organisms by PCR-restriction analysis of the hsp65 gene.

A total of 102 isolates of the Mycobacterium tuberculosis complex, including available "M. canettii" isolates, were studied by PCR-restriction analysis of a 441-bp fragment of the hsp65 gene. PRA upon HhaI enzyme digestion (GCGC) allowed easy differentiation of "M. canettii" from other members of the M. tuberculosis complex (three bands of 260, 105, and 60 bp for "M. canetti," compared to four bands of 185, 105, 75, and 60 bp for other members of the M. tuberculosis complex). Sequencing of the 441-bp hsp65 fragment of "M. canettii" isolates showed the disappearance of an HhaI site at position 235 due to a C-to-T transition that corresponded to position 631 of the homologous hsp65 gene of M. tuberculosis H37Rv. Considering that "M. canettii" may also exist as a stable rough morphotype, we suggest that the true number of "M. canettii" isolates may be underestimated in clinical microbiology laboratories.

Spoligotype database of Mycobacterium tuberculosis: biogeographic distribution of shared types and epidemiologic and phylogenetic perspectives.

We give an update on the worldwide spoligotype database, which now contains 3,319 spoligotype patterns of Mycobacterium tuberculosis in 47 countries, with 259 shared types, i.e., identical spoligotypes shared by two or more patient isolates. The 259 shared types contained a total of 2,779 (84%) of all the isolates. Seven major genetic groups represented 37% of all clustered isolates. Two types (119 and 137) were found almost exclusively in the USA and accounted for 9% of clustered isolates. The remaining 1,517 isolates were scattered into 252 different spoligotypes. This database constitutes a tool for pattern comparison of M. tuberculosis clinical isolates for global epidemiologic studies and phylogenetic purposes.

Genetic diversity of Mycobacterium tuberculosis in Sicily based on spoligotyping and variable number of tandem DNA repeats and comparison with a spoligotyping database for population-based analysis.

In a previous study, we proposed to associate spoligotyping and typing with the variable number of tandem DNA repeats (VNTR) as an alternative strategy to IS6110-restriction fragment length polymorphism (RFLP) for molecular epidemiological studies on tuberculosis. The aim of the present study was to further evaluate this PCR-based typing strategy and to describe the population structure of Mycobacterium tuberculosis in another insular setting, Sicily. A collection of 106 DNA samples from M. tuberculosis patient isolates was characterized by spoligotyping and VNTR typing. All isolates were independently genotyped by the standard IS6110-RFLP method, and clustering results between the three methods were compared. The totals for the clustered isolates were, respectively, 15, 60, and 82% by IS6110-RFLP, spoligotyping, and VNTR typing. The most frequent spoligotype included type 42 that missed spacers 21 to 24 and spacers 33 to 36 and derived types 33, 213, and 273 that, together represented as much as 26% of all isolates, whereas the Haarlem clade of strains (types 47 and 50, VNTR allele 32333) accounted for 9% of the total strains. The combination of spoligotyping and VNTR typing results reduced the number of clusters to 43% but remained superior to the level of IS6110-RFLP clustering (ca. 15%). All but one IS6110-defined cluster were identified by the combination of spoligotyping and VNTR clustering results, whereas 9 of 15 spoligotyping-defined clusters could be further subdivided by IS6110-RFLP. Reinterpretation of previous IS6110-RFLP results in the light of spoligotyping-VNTR typing results allowed us to detect an additional cluster that was previously missed. Although less discriminative than IS6110-RFLP, our results suggest that the use of the combination of spoligotyping and VNTR typing is a good screening strategy for detecting epidemiological links for the study of tuberculosis epidemiology at the molecular level.