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1.  The use of microbead-based spoligotyping for Mycobacterium tuberculosis complex to evaluate the quality of the conventional method: Providing guidelines for Quality Assurance when working on membranes 
BMC Infectious Diseases  2011;11:110.
Background
The classical spoligotyping technique, relying on membrane reverse line-blot hybridization of the spacers of the Mycobacterium tuberculosis CRISPR locus, is used world-wide (598 references in Pubmed on April 8th, 2011). However, until now no inter-laboratory quality control study had been undertaken to validate this technique. We analyzed the quality of membrane-based spoligotyping by comparing it to the recently introduced and highly robust microbead-based spoligotyping. Nine hundred and twenty-seven isolates were analyzed totaling 39,861 data points. Samples were received from 11 international laboratories with a worldwide distribution.
Methods
The high-throughput microbead-based Spoligotyping was performed on CTAB and thermolyzate DNA extracted from isolated Mycobacterium tuberculosis complex (MTC) strains coming from the genotyping participating centers. Information regarding how the classical Spoligotyping method was performed by center was available. Genotype discriminatory analyses were carried out by comparing the spoligotypes obtained by both methods. The non parametric U-Mann Whitney homogeneity test and the Spearman rank correlation test were performed to validate the observed results.
Results
Seven out of the 11 laboratories (63 %), perfectly typed more than 90% of isolates, 3 scored between 80-90% and a single center was under 80% reaching 51% concordance only. However, this was mainly due to discordance in a single spacer, likely having a non-functional probe on the membrane used. The centers using thermolyzate DNA performed as well as centers using the more extended CTAB extraction procedure. Few centers shared the same problematic spacers and these problematic spacers were scattered over the whole CRISPR locus (Mostly spacers 15, 14, 18, 37, 39, 40).
Conclusions
We confirm that classical spoligotyping is a robust method with generally a high reliability in most centers. The applied DNA extraction procedure (CTAB or thermolyzate) did not affect the results in this study. However performance was center-dependent, suggesting that training is a key component in quality assurance of spoligotyping. Overall, no particular spacer yielded a higher degree of deviating results, suggesting that errors occur randomly either in the process of re-using membranes, or during the reading of the results and transferring of data from the film to a digital file. Last, the performance of the microbead-based method was excellent as previously shown by Cowan et al. (J. Clin. Microbiol. 2004) and Zhang et al. (J. Med. Microbiol. 2009) and demonstrated the proper detection of spacer 15 that is known to occasionally give weak signals in the classical spoligotyping.
doi:10.1186/1471-2334-11-110
PMCID: PMC3107175  PMID: 21527037
2.  Variation in Gamma Interferon Responses to Different Infecting Strains of Mycobacterium tuberculosis in Acid-Fast Bacillus Smear-Positive Patients and Household Contacts in Antananarivo, Madagascar▿  
The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop tuberculosis (TB), though many may become latently infected. More precise measurement of the human immune response to M. tuberculosis infection may help us understand this difference and potentially identify those subjects most at risk of developing active disease. Gamma interferon (IFN-γ) production has been widely used as a proxy marker to study infection and to examine the human immune response to specific M. tuberculosis antigens. It has been suggested that genetically distinct M. tuberculosis strains may invoke different immune responses, although how these differences influence the immune responses and clinical outcome in human tuberculosis is still poorly understood. We therefore evaluated the antigen-specific IFN-γ production responses in peripheral blood mononuclear cells from two cohorts of subjects recruited in Antananarivo, Madagascar, from 2004 to 2006 and examined the influence of the infecting M. tuberculosis strains on this response. The cohorts were sputum-positive index cases and their household contacts. Clinical strains isolated from the TB patients were typed by spoligotyping. Comparison of the IFN-γ responses with the spoligotype of the infecting clinical strains showed that “modern” M. tuberculosis strains, like Beijing and Central Asian (CAS) strains, tended to induce lower IFN-γ responses than “ancient” strains, like East African-Indian (EAI) strains, in index cases and their household contacts. These results suggest that new strains may have evolved to induce a host response different from that of ancient strains. These findings could have important implications in the development of therapeutic and diagnostic strategies.
doi:10.1128/CVI.00049-10
PMCID: PMC2897260  PMID: 20463103
3.  Snapshot of Moving and Expanding Clones of Mycobacterium tuberculosis and Their Global Distribution Assessed by Spoligotyping in an International Study†  
Journal of Clinical Microbiology  2003;41(5):1963-1970.
The present update on the global distribution of Mycobacterium tuberculosis complex spoligotypes provides both the octal and binary descriptions of the spoligotypes for M. tuberculosis complex, including Mycobacterium bovis, from >90 countries (13,008 patterns grouped into 813 shared types containing 11,708 isolates and 1,300 orphan patterns). A number of potential indices were developed to summarize the information on the biogeographical specificity of a given shared type, as well as its geographical spreading (matching code and spreading index, respectively). To facilitate the analysis of hundreds of spoligotypes each made up of a binary succession of 43 bits of information, a number of major and minor visual rules were also defined. A total of six major rules (A to F) with the precise description of the extra missing spacers (minor rules) were used to define 36 major clades (or families) of M. tuberculosis. Some major clades identified were the East African-Indian (EAI) clade, the Beijing clade, the Haarlem clade, the Latin American and Mediterranean (LAM) clade, the Central Asian (CAS) clade, a European clade of IS6110 low banders (X; highly prevalent in the United States and United Kingdom), and a widespread yet poorly defined clade (T). When the visual rules defined above were used for an automated labeling of the 813 shared types to define nine superfamilies of strains (Mycobacterium africanum, Beijing, M. bovis, EAI, CAS, T, Haarlem, X, and LAM), 96.9% of the shared types received a label, showing the potential for automated labeling of M. tuberculosis families in well-defined phylogeographical families. Intercontinental matches of shared types among eight continents and subcontinents (Africa, North America, Central America, South America, Europe, the Middle East and Central Asia, and the Far East) are analyzed and discussed.
doi:10.1128/JCM.41.5.1963-1970.2003
PMCID: PMC154710  PMID: 12734235
4.  Extrapulmonary and Pulmonary Tuberculosis in Antananarivo (Madagascar): High Clustering Rate in Female Patients 
Journal of Clinical Microbiology  2002;40(11):3964-3969.
Antananarivo, the capital city of Madagascar, has an endemic focus of tuberculosis (TB). We specifically studied patients with extrapulmonary TB (EPTB) and grouped patients according to infected body site. The strains were characterized by IS6110 fingerprinting and compared with those isolated from patients with pulmonary TB (PTB) during the same period in order to determine the possible association between the genotype and the clinical expression of TB. A total of 316 TB patients were included in this study: 151 individuals with EPTB, 10 with both PTB and EPTB, and 155 with PTB alone. Pleural TB was the major EPTB localization (77%) and was found more often in older patients, while PTB or EPTB in which the localization was other than pleural (other EPTB) was found in younger patients. The male-to-female ratio was slightly higher in pleural TB patients (3.06:1) than in patients with other EPTB (1.35:1). There was no significant difference in the BCG status among patients with PTB, pleural TB, and other EPTB. Analysis of IS6110 patterns showed that 167 patients (52.8%) were assigned to 37 clusters of 2 to 34 patients. Analysis of the IS6110 clusters and the IS6110 families did not show any association with a particular clinical expression of the disease. Patients with PTB or other EPTB were more likely to have strains with one IS6110 copy than patients with pleural TB. The clustering rate was found to be significantly higher in female patients (62%) than in male patients (48%) (P = 0.029), suggesting that Malagasy women were more likely to progress to disease after infection than men.
doi:10.1128/JCM.40.11.3964-3969.2002
PMCID: PMC139634  PMID: 12409359
5.  Global Distribution of Mycobacterium tuberculosis Spoligotypes 
Emerging Infectious Diseases  2002;8(11):1347-1349.
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 11,708 patterns from as many clinical isolates originating from more than 90 countries. The 11,708 spoligotypes were clustered into 813 shared types. A total of 1,300 orphan patterns (clinical isolates showing a unique spoligotype) were also detected.
doi:10.3201/eid0811.020125
PMCID: PMC2738532  PMID: 12453368
Mycobacterium tuberculosis; spoligotyping
6.  Predominance of Serotype-Specific Mucosal Antibody Response in Shigella flexneri-Infected Humans Living in an Area of Endemicity 
Infection and Immunity  2001;69(9):5230-5234.
The mucosal humoral immune response elicited following Shigella flexneri infection in patients living in Antananarivo districts (Madagascar Island) was evaluated by measuring the gut-derived, circulating immunoglobulin A (IgA) antibody-secreting cells (ASC) specific for the major bacterial antigen lipopolysaccharide (LPS). Fifty, 34, 11, and 5% of the S. flexneri-positive patients were infected with serotypes 2a, 1a, 4a, and 3a, respectively. The total number of IgA ASC in infected patients increased significantly, compared to the number in healthy controls, early after the onset of disease. The number of anti-homologous LPS IgA ASC varied among individuals and peaked between days 5 and 10 after the onset of the disease. In the S. flexneri 1a- and 2a-infected patients, the level of IgA ASC cross-reactivity to heterologous S. flexneri serotypes was weak. These data indicate that S. flexneri 2a and 1a are the predominant strains responsible for shigellosis in this area of endemicity and that the anti-LPS antibody response following natural infection is mainly directed against serotype-specific determinants.
doi:10.1128/IAI.69.9.5230-5234.2001
PMCID: PMC98630  PMID: 11500390
7.  A Combination of Two Genetic Markers Is Sufficient for Restriction Fragment Length Polymorphism Typing of Mycobacterium tuberculosis Complex in Areas with a High Incidence of Tuberculosis 
Journal of Clinical Microbiology  2001;39(4):1530-1535.
The incidence of tuberculosis (TB) in Madagascar is 150 cases per 100,000 people. Because of this endemicity, we studied the genetic diversity of Mycobacterium tuberculosis strains isolated in four big cities in 1994 to 1995 with the aim of monitoring TB transmission. Isolates from 316 cases of pulmonary TB (PTM+) were typed by Southern hybridization with genetic markers IS6110 and DR. Of the 316 PTM+ strains, 66 (20.8%) had a single IS6110 band and were differentiated by the DR marker into 33 profiles. Using both markers, 37.7% (119) of the patients were clustered, a proportion similar to that in countries with a high prevalence of TB. There was no significant difference between clustered and nonclustered patients in age, sex, Mycobacterium bovis BCG status, and drug susceptibility of strains. Clustering was significantly greater in the capital, Antananarivo, than in the other cities, suggesting a higher rate of transmission. However, most of the patients in clusters were living in different areas, and, within a distance of 0.7 km, we did not find epidemiologically unrelated strains with the same restriction fragment length polymorphism profile. Despite an apparently low polymorphism, genetic markers such as IS6110 are potentially valuable for monitoring TB transmission. However, the high proportion of Malagasy isolates with a single IS6110 copy makes this marker alone unsuitable for typing. Additional markers such as DR are necessary for the differentiation of the isolates and for epidemiological surveys.
doi:10.1128/JCM.39.4.1530-1535.2001
PMCID: PMC87965  PMID: 11283082

Results 1-7 (7)