Dipstick test for rapid diagnosis of Shigella dysenteriae 1 in bacterial cultures and its potential use on stool samples.

BACKGROUND: We describe a test for rapid detection of S. dysenteriae 1 in bacterial cultures and in stools, at the bedside of patients. METHODOLOGY/PRINCIPAL FINDINGS: The test is based on the detection of S. dysenteriae 1 lipopolysaccharide (LPS) using serotype 1-specific monoclonal antibodies coupled to gold particles and displayed on a one-step immunochromatographic dipstick. A concentration as low as 15 ng/ml of LPS was detected in distilled water and in reconstituted stools in 10 minutes. In distilled water and in reconstituted stools, an unequivocal positive reaction was obtained with 1.6×106 CFU/ml and 4.9×106 CFU/ml of S. dysenteriae 1, respectively. Optimal conditions to read the test have been determined to limit the risk of ambiguous results due to appearance of a faint yellow test band in some negative samples. The specificity was 100% when tested with a battery of Shigella and unrelated strains in culture. When tested on 328 clinical samples in India, Vietnam, Senegal and France by laboratory technicians and in Democratic Republic of Congo by a field technician, the specificity (312/316) was 98.7% (95% CI:96.6-99.6%) and the sensitivity (11/12) was 91.7% (95% CI:59.8-99.6%). Stool cultures and the immunochromatographic test showed concordant results in 98.4 % of cases (323/328) in comparative studies. Positive and negative predictive values were 73.3% (95% CI:44.8-91.1%) and 99.7% (95% CI:98-100%). CONCLUSION: The initial findings presented here for a simple dipstick-based test to diagnose S. dysenteriae 1 demonstrates its promising potential to become a powerful tool for case management and epidemiological surveys.

Sequence analysis of the gene encoding H antigen in Escherichia coli isolated from food in Morocco.

In order to develop other molecular method useful for typing of motile and non motile Escherichia coli strains, a total of 207 strains of E. coli (133 reference strains, 74 food strains) were characterized by analysis of sequences of their amplified flagellin-encoding (fliC) gene products. The collection of reference strains was used for database building of fliC gene sequences. Application of this identification system to 74 E. coli food isolates revealed a reproducible and clear cut classification with very good correlation to results obtained by HhaI restriction of the amplified flagellin gene. The proposed determination of fliC sequences variations should be helpful for epidemiological studies.

Microbial quality control of raw ground beef and fresh sausage in Casablanca (Morocco).

In this study, samples of raw ground beef (n = 150) and fresh sausage (n = 100) were collected randomly from butcheries, supermarkets, and fast-food shops, in Casablanca, Morocco. Two types of meat product samples were considered, one with spices (n = 115) and other without spices (n = 135). All the samples were analyzed for the presence of the following bacteria: Escherichia coli, Staphylococcus, Clostridium perfringens, Salmonella, and Listeria monocytogenes. E. coli strains were further typed by pulsed-field gel electrophoresis (PFGE), Operon O, and characterized for virulence genes by polymerase chain reaction (PCR). Results indicated that counts of E. coli, coagulase-positive Staphylococcus, and C. perfringens were 17%, 9.6%, and 8.7% in samples without spices, respectively; and 23.5%, 23.7%, and 29.6% in samples with spices, respectively. Two pathogenic genes, LT and EAST, were identified separately in four strains of E. coli. Salmonella and L. monocytogenes were isolated in 2.8% and 3.2% of the total samples, respectively.

Macrolide-resistant Shigella sonnei.

Shigella sonnei UCN59, isolated during an outbreak of S. sonnei in January 2007, was resistant to azithromycin (MIC 64 mg/L). The isolate contained a plasmid-borne mph(A) gene encoding a macrolide 2 -phosphotransferase that inactivates macrolides. Emergence of the mph(A) gene in S. sonnei may limit usefulness of azithromycin for treatment of shigellosis.

Surveillance of hemolytic uremic syndrome in children less than 15 years of age, a system to monitor O157 and non-O157 Shiga toxin-producing Escherichia coli infections in France, 1996-2006.

BACKGROUND: Since the 1980s, Shiga toxin-producing Escherichia coli (STEC), especially E. coli O157:H7, has been an important cause of food borne disease in industrial countries. In France, as there was no routine screening for STEC in clinical laboratories, enhanced surveillance of hemolytic uremic syndrome (HUS) in children less than 15 years of age was established in 1996 to monitor trends in the incidence of STEC infections. METHODS: The surveillance system was based on a voluntary national network of pediatricians of 31 pediatric nephrology units in public hospitals. RESULTS: From 1996 to 2006, the mean annual incidence of HUS was 0.71 cases per 100,000 children less than 15 years of age and 1.87 cases per 100,000 children less than 5 years of age. STEC infections were confirmed in 66% of patients; STEC O157 was the most common serogroup identified in STEC-related HUS (83%). In this 11-year period, 96% of HUS cases were sporadic and only 2 outbreaks caused by STEC O157 and by a dual infection of STEC O26 and O80 were detected. CONCLUSIONS: An evaluation of the surveillance of pediatric HUS showed that it is a simple and useful system for monitoring trends in STEC infections in France. It provides the information needed to measure the impact of new and changing vehicles of STEC transmission, and evaluate the effectiveness of prevention measures.

Massively parallel pathogen identification using high-density microarrays.

Identification of microbial pathogens in clinical specimens is still performed by phenotypic methods that are often slow and cumbersome, despite the availability of more comprehensive genotyping technologies. We present an approach based on whole-genome amplification and resequencing microarrays for unbiased pathogen detection. This 10 h process identifies a broad spectrum of bacterial and viral species and predicts antibiotic resistance and pathogenicity and virulence profiles. We successfully identify a variety of bacteria and viruses, both in isolation and in complex mixtures, and the high specificity of the microarray distinguishes between different pathogens that cause diseases with overlapping symptoms. The resequencing approach also allows identification of organisms whose sequences are not tiled on the array, greatly expanding the repertoire of identifiable organisms and their variants. We identify organisms by hybridization of their DNA in as little as 1-4 h. Using this method, we identified Monkeypox virus and drug-resistant Staphylococcus aureus in a skin lesion taken from a child suspected of an orthopoxvirus infection, despite poor transport conditions of the sample, and a vast excess of human DNA. Our results suggest this technology could be applied in a clinical setting to test for numerous pathogens in a rapid, sensitive and unbiased manner.

Long-term population-based genotyping study of Mycobacterium tuberculosis complex isolates in the French departments of the Americas.

The three French overseas departments of the Americas are characterized both by insular (Guadeloupe and Martinique) and continental (French Guiana) settings with a tuberculosis case detection rate that varies from less than 10 per 100,000 per year in insular areas to an estimated incidence of more than 55 per 100,000 in French Guiana. Under a long-term genotyping program, more than three-fourths of all the Mycobacterium tuberculosis isolates (n = 744) received from the three settings were fingerprinted over a 10-year period (1994 to 2003) by spoligotyping and variable number of tandem DNA repeats (VNTRs) in order to understand the current trends in their detection rates, drug resistance, and groups and subpopulations at risk of contracting the disease and to pinpoint the circulating phylogeographical clades of the bacilli. The major difference in the study populations was the nationality of the patients, with a high percentage of immigrants from high-incidence neighboring countries in French Guiana and a low but increasing percentage in the French Caribbean. The rate of recent transmission was calculated to be 49.3% in French Guiana, compared to 27.2% and 16.9% in Guadeloupe and Martinique, respectively. At the phylogeographic level, 77.9% of the isolates studied belonged to four major clades (Haarlem, Latin-American and Mediterranean, T, and X) which are already reported from neighboring Caribbean islands in an international database and may underline potential interregional transmission events.

Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set.

We analyzed a global collection of Mycobacterium tuberculosis strains using 212 single nucleotide polymorphism (SNP) markers. SNP nucleotide diversity was high (average across all SNPs, 0.19), and 96% of the SNP locus pairs were in complete linkage disequilibrium. Cluster analyses identified six deeply branching, phylogenetically distinct SNP cluster groups (SCGs) and five subgroups. The SCGs were strongly associated with the geographical origin of the M. tuberculosis samples and the birthplace of the human hosts. The most ancestral cluster (SCG-1) predominated in patients from the Indian subcontinent, while SCG-1 and another ancestral cluster (SCG-2) predominated in patients from East Asia, suggesting that M. tuberculosis first arose in the Indian subcontinent and spread worldwide through East Asia. Restricted SCG diversity and the prevalence of less ancestral SCGs in indigenous populations in Uganda and Mexico suggested a more recent introduction of M. tuberculosis into these regions. The East African Indian and Beijing spoligotypes were concordant with SCG-1 and SCG-2, respectively; X and Central Asian spoligotypes were also associated with one SCG or subgroup combination. Other clades had less consistent associations with SCGs. Mycobacterial interspersed repetitive unit (MIRU) analysis provided less robust phylogenetic information, and only 6 of the 12 MIRU microsatellite loci were highly differentiated between SCGs as measured by GST. Finally, an algorithm was devised to identify two minimal sets of either 45 or 6 SNPs that could be used in future investigations to enable global collaborations for studies on evolution, strain differentiation, and biological differences of M. tuberculosis.

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.

Spoligotyping of Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in Mumbai, India.

Tuberculosis remains a major health problem in India, with 2 million new cases and 421,000 deaths each year. In this paper, we describe the spoligotyping results of 216 Mycobacterium tuberculosis culture isolates from patients with pulmonary tuberculosis in Mumbai, India. As spoligotyping data from India have rarely been described until now, and as there is limited information on the major circulating clades of M. tuberculosis, the data obtained were also compared to an international spoligotype database (SpolDB4) that contained patterns from 22,546 isolates from more than 100 countries. Eighty-four (39%) of the isolates were definitively marked as orphan strains, indicating the paucity of such data from India. The remaining 132 isolates clustered among 59 shared types; among these, 42 shared types were already present in the database, 17 were newly created, and 5 of them were specifically reported from Mumbai. A total of 9 major types in this study clustered 32% of the isolates. At the phylogenetic level, 30% of the isolates belonged to the Central Asian families CAS1 and CAS2, of the major genetic group (MGG) 1, 29% to MGG 2 and 3 families (spacers 33-36 missing) and 17% to the ancestral East African Indian (EAI) family. Finally, nearly 10% of the isolates belonged to the W-Beijing family in a broad sense, also in the MGG 1 group. In conclusion, historic clones of the MGG 1 group of M. tuberculosis are responsible for roughly 60% of all tuberculosis cases in Mumbai. Together with the fact that organisms presumably of European descent (such as the Haarlem family) were only rarely found, our observations suggest that tuberculosis in Mumbai, India is essentially caused by historical clones of tubercle bacilli undergoing active circulation due to uncontrolled demography, high prevalence of the disease, and a paucity of resources.

Phylogenetic reconstruction of Mycobacterium tuberculosis within four settings of the Caribbean region: tree comparative analyse and first appraisal on their phylogeography.

In order to compare phylogenetic methods and to reconstruct the evolutionary history of the tubercle bacilli, a set of macro-array-based genotyping data of Mycobacterium tuberculosis clinical isolates (called spoligotyping for spacer oligonucleotide typing, which assays the variability of the Direct Repeat -DR- locus), was analyzed in four settings of the Caribbean region (Guadeloupe, Martinique, Cuba and Haiti). A set of 47 alleles, split into 26 shared and 21 unique alleles) representative of 321 individual M. tuberculosis clinical isolates from patients residing in the above regions was studied. The following methods (and software in brackets) were investigated: numerical taxonomy distance methods (TAXOTRON), maximum parsimony procedure (PAUP), median-joining networks (NETWORK), and nested clade analysis (GEODIS). Results using these methods were analyzed, compared and discussed. The latter method (GEODIS) was investigated in detail by introducing geographical data together with genetic variability results to detect a link between population structure and population history, and to test the null hypothesis of no association between geography and genotypes. Irrespective of the methods used, our findings demonstrate that a core structure of four families (or clades) of M. tuberculosis strains is highly prevalent within the islands studied, indirectly reflecting passed colonization history of these different settings. Specificity of M. tuberculosis genotypes in each of the islands is discussed in the light of their respective colonial and contemporary histories.

Genotyping of the Mycobacterium tuberculosis complex using MIRUs: association with VNTR and spoligotyping for molecular epidemiology and evolutionary genetics.

The recent introduction of molecular methods has gained increased acceptance as a powerful tool for epidemiology and phylogeny of tuberculosis (TB). In this investigation, the efficiency of molecular typing using mycobacterial interspersed repetitive units (MIRUs) was assessed on a set of 116 Mycobacterium tuberculosis complex clinical isolates from 11 different geographic origins. The results obtained were compared with spoligotyping and variable number of tandem DNA repeats (VNTRs) typing data. Eighty-nine different MIRU profiles were obtained on the sample studied. Spoligotyping- or VNTR-defined clusters were split into subclusters by MIRU typing. Conversely, almost all of the clinical isolates clustered by MIRUs were shown to belong to spoligotyping-based defined clusters. The calculation of the discriminative power by the Hunter-Gaston index (HGI) for VNTR, spoligotyping and MIRU typing gave the values of, respectively, 0.959, 0.965 and 0.988, showing the high discriminative power of the MIRUs. The allelic diversity of the sample was calculated for each of the MIRU-VNTR loci; five MIRU loci (MIRU nos. 10, 23, 26, 31 and 40) were "highly discriminant", four (MIRU nos. 4, 16, 24 and 39) were "moderately discriminant", and three (MIRU nos. 2, 20 and 27) were "poorly discriminant". Among the three complementary VNTRs (exact tandem repeats ETR-A, ETR-B and ETR-C), ETR-A was the most discriminant locus. A combined numerical analysis of spoligotyping, VNTR and MIRU typing results partly corroborated a recently hypothesized evolutionary scenario for the M. tuberculosis complex. M. canettii would be the first branch to have diverged from a common M. tuberculosis complex ancestor. The East-African Indian (EAI) clade could be the first family to have diverged thereafter. A third branching separated a M. africanum-M. bovis clade, followed by a node separating Beijing versus non-Beijing M. tuberculosis. The Beijing clade was distinct from the Central Asian 1 (CAS1) family. Among non-Beijing strains, branches such as the Latin-American and Mediterranean (LAM), X and Haarlem clades diverged later. In conclusion, the results obtained show the congruence between clades defined by spoligotyping, and MIRU-VNTR, and underline the potential of these methods for M. tuberculosis phylogeny reconstruction. We also conclude that MIRU typing is a very promising method that may be used in a "two PCR-based" genotyping strategy, in conjunction to conventional epidemiological investigations.

Snapshot of moving and expanding clones of Mycobacterium tuberculosis and their global distribution assessed by spoligotyping in an international study.

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.

Characterization of Finnish Mycobacterium tuberculosis isolates by spoligotyping.

The molecular epidemiology of tuberculosis (TB) in Finland was studied by spoligotyping 380 Mycobacterium tuberculosis isolates. The isolates were obtained during a 1-year study period from July 2000 to June 2001 and represented 90% of new M. tuberculosis findings by culture in the whole country during the study period. The spoligotyping results were compared to the World Spoligotyping Database of the Institut Pasteur de Guadeloupe, which contains data from >14,000 M. tuberculosis isolates obtained worldwide. A total of 138 different spoligotypes were identified among the 380 M. tuberculosis isolates. Thirty-eight (10%) isolates had unique spoligotypes, while 342 (90%) isolates belonged to 100 shared types. The four most common spoligotypes caused approximately one-third of the Finnish TB cases. Forty-seven of the 138 (34.1%) spoligotypes and 61 (16.1%) of the 380 M. tuberculosis isolates had spoligotypes that had not been previously reported. Only four (1.1%) patients were infected with an isolate belonging to the Beijing genotype. The characterization of Finnish M. tuberculosis isolates by spoligotyping shows that ubiquitous spoligotypes were common, but many spoligotypes specific to Finland were also found. However, Beijing family isolates were rarely encountered, although this spoligotype is predominant in our eastern and southern neighbors.

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.

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.

Spoligotyping and IS6110-RFLP typing of Mycobacterium tuberculosis from French Guiana: a comparison of results with international databases underlines interregional transmission from neighboring countries.

In this investigation, 94 clinical isolates of Mycobacterium tuberculosis obtained over a 3-year period (1996-1998) from French Guiana were characterized by spoligotyping and IS6110-RFLP and the patterns obtained were compared with genotypes representing the worldwide diversity in an international spoligotyping database (n = 4269) and a IS6110-RFLP database (n = 4189). All the clustered isolates giving < or = 6 copies of IS6110 were further typed using the double-repetitive element (DRE)-PCR. The results obtained underlined the highly diverse nature of the M. tuberculosis population in French Guiana with potential links to neighboring countries within the Americas. It may be hypothesized that the genetic heterogeneity of tubercle bacilli in French Guiana is linked to the high number of imported cases of tuberculosis, that may account for as high as 68% of all tuberculosis cases. Although an epidemiological investigation based on direct interrogation of patients was not performed, available medical records suggested that the clustering of isolates was mostly linked to the following risk factors: pulmonary tuberculosis, smear-positive samples, foreign-born nationals and/or immigrants, and a high rate of HIV-TB coinfection. Thus the persisting foci of endemic disease and increased active transmission due to high population flux and HIV coinfection may be largely responsible for the relatively high incidence of tuberculosis in French Guiana.