Biomedical subjects

Dick van Soolingen

Publications and source records attributed to Dick van Soolingen.

At least 19 recordsLinked to original sources

PubMed2006

Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable-number tandem repeat typing of Mycobacterium tuberculosis.

Molecular typing based on 12 loci containing variable numbers of tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTRs) has been adopted in combination with spoligotyping as the basis for large-scale, high-throughput genotyping of Mycobacterium tuberculosis. However, even the combination of these two methods is still less discriminatory than IS6110 fingerprinting. Here, we define an optimized set of MIRU-VNTR loci with a significantly higher discriminatory power. The resolution and the stability/robustness of 29 loci were analyzed, using a total of 824 tubercle bacillus isolates, including representatives of the main lineages identified worldwide so far. Five loci were excluded for lack of robustness and/or stability in serial isolates or isolates from epidemiologically linked patients. The use of the 24 remaining loci increased the number of types by 40%--and by 23% in combination with spoligotyping--among isolates from cosmopolitan origins, compared to those obtained with the original set of 12 loci. Consequently, the clustering rate was decreased by fourfold--by threefold in combination with spoligotyping--under the same conditions. A discriminatory subset of 15 loci with the highest evolutionary rates was then defined that concentrated 96% of the total resolution obtained with the full 24-locus set. Its predictive value for evaluating M. tuberculosis transmission was found to be equal to that of IS6110 restriction fragment length polymorphism typing, as shown in a companion population-based study. This 15-locus system is therefore proposed as the new standard for routine epidemiological discrimination of M. tuberculosis isolates and the 24-locus system as a high-resolution tool for phylogenetic studies.

Bacterial Typing Techniques↗

PubMed2006

Antituberculosis drug resistance in the south of Vietnam: prevalence and trends.

BACKGROUND: There is limited evidence that the DOTS (directly observed therapy, short course) strategy for tuberculosis (TB) control can contain the emergence and spread of drug resistance in the absence of second-line treatment. We compared drug-resistance levels between 1996 and 2001 in the south of Vietnam, an area with a well-functioning DOTS program. METHODS: Sputum specimens were collected from consecutively diagnosed patients with smear-positive TB at 40 randomly selected public TB clinics. Mycobacterium tuberculosis isolates were tested for susceptibility to first-line drugs. RESULTS: Among 888 new patients in 2001, resistance to any drug was observed in 238 (26.3%), resistance to isoniazid was observed in 154 (16.6%), resistance to rifampin was observed in 22 (2.0%), resistance to ethambutol was observed in 12 (1.1%), resistance to streptomycin was observed in 173 (19.4%), and resistance to both isoniazid and rifampicin (multidrug resistance [MDR]) was observed in 20 (1.8% [95% confidence interval, 1.0%-3.3%]). Among 136 previously treated patients in 2001, any resistance was observed in 89 (62.9%), and MDR was observed in 35 (23.2%). The prevalence of any drug resistance and of streptomycin resistance among new patients had decreased significantly (P<.01) since 1996; there was no increase in the prevalence of MDR. CONCLUSION: The prevalence of drug resistance decreased despite high initial levels of resistance to isoniazid and streptomycin and despite the absence of second-line treatment. Therefore, a DOTS program can contain drug-resistant TB in this setting.

Adolescent↗

PubMed2006

Beijing genotype of Mycobacterium tuberculosis is significantly associated with human immunodeficiency virus infection and multidrug resistance in cases of tuberculous meningitis.

Multidrug-resistant tuberculous meningitis is fatal without rapid diagnosis and use of second-line therapy. It is more common in human immunodeficiency virus (HIV)-positive patients. Beijing genotype strains of Mycobacterium tuberculosis are associated with drug resistance, particularly multidrug resistance, and their prevalence is increasing worldwide. The prevalence of Beijing genotype strains among Mycobacterium tuberculosis isolates from the cerebrospinal fluid of HIV-positive (n = 35) and HIV-negative (n = 187) patients in Ho Chi Minh City was determined. The Beijing genotype was significantly associated with HIV status (odds ratio [OR] = 2.95 [95% confidence interval {CI}, 1.38 to 6.44]; P = 0.016), resistance to any drug (OR = 3.34 [95% CI, 1.87 to 5.95]; P < 0.001) and multidrug resistance (Fisher's exact test; P = 0.001). The association of the Beijing genotype with drug resistance was independent of HIV status. This is the first report of Beijing genotype association with HIV status, which may be an association unique to tuberculous meningitis.

Adolescent↗

PubMed2006

Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology.

BACKGROUND: The Direct Repeat locus of the Mycobacterium tuberculosis complex (MTC) is a member of the CRISPR (Clustered regularly interspaced short palindromic repeats) sequences family. Spoligotyping is the widely used PCR-based reverse-hybridization blotting technique that assays the genetic diversity of this locus and is useful both for clinical laboratory, molecular epidemiology, evolutionary and population genetics. It is easy, robust, cheap, and produces highly diverse portable numerical results, as the result of the combination of (1) Unique Events Polymorphism (UEP) (2) Insertion-Sequence-mediated genetic recombination. Genetic convergence, although rare, was also previously demonstrated. Three previous international spoligotype databases had partly revealed the global and local geographical structures of MTC bacilli populations, however, there was a need for the release of a new, more representative and extended, international spoligotyping database. RESULTS: The fourth international spoligotyping database, SpolDB4, describes 1939 shared-types (STs) representative of a total of 39,295 strains from 122 countries, which are tentatively classified into 62 clades/lineages using a mixed expert-based and bioinformatical approach. The SpolDB4 update adds 26 new potentially phylogeographically-specific MTC genotype families. It provides a clearer picture of the current MTC genomes diversity as well as on the relationships between the genetic attributes investigated (spoligotypes) and the infra-species classification and evolutionary history of the species. Indeed, an independent Naïve-Bayes mixture-model analysis has validated main of the previous supervised SpolDB3 classification results, confirming the usefulness of both supervised and unsupervised models as an approach to understand MTC population structure. Updated results on the epidemiological status of spoligotypes, as well as genetic prevalence maps on six main lineages are also shown. Our results suggests the existence of fine geographical genetic clines within MTC populations, that could mirror the passed and present Homo sapiens sapiens demographical and mycobacterial co-evolutionary history whose structure could be further reconstructed and modelled, thereby providing a large-scale conceptual framework of the global TB Epidemiologic Network. CONCLUSION: Our results broaden the knowledge of the global phylogeography of the MTC complex. SpolDB4 should be a very useful tool to better define the identity of a given MTC clinical isolate, and to better analyze the links between its current spreading and previous evolutionary history. The building and mining of extended MTC polymorphic genetic databases is in progress.

Computational Biology↗

PubMed2006

Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex.

In a previous report, we described a PCR protocol for the differentiation of the various species of the Mycobacterium tuberculosis complex (MTC) on the basis of genomic deletions (R. C. Huard, L. C. de Oliveira Lazzarini, W. R. Butler, D. van Soolingen, and J. L. Ho, J. Clin. Microbiol. 41:1637-1650, 2003). That report also provided a broad cross-comparison of several previously identified, phylogenetically relevant, long-sequence and single-nucleotide polymorphisms (LSPs and SNPs, respectively). In the present companion report, we expand upon the previous work (i) by continuing the evaluation of known MTC phylogenetic markers in a larger collection of tubercle bacilli (n = 125), (ii) by evaluating additional recently reported MTC species-specific and interspecific polymorphisms, and (iii) by describing the identification and distribution of a number of novel LSPs and SNPs. Notably, new genomic deletions were found in various Mycobacterium tuberculosis strains, new species-specific SNPs were identified for "Mycobacterium canettii," Mycobacterium microti, and Mycobacterium pinnipedii, and, for the first time, intraspecific single-nucleotide DNA differences were discovered for the dassie bacillus, the oryx bacillus, and the two Mycobacterium africanum subtype I variants. Surprisingly, coincident polymorphisms linked one M. africanum subtype I genotype with the dassie bacillus and M. microti with M. pinnipedii, thereby suggesting closer evolutionary ties within each pair of species than had been previously thought. Overall, the presented data add to the genetic definitions of several MTC organisms as well as fine-tune current models for the evolutionary history of the MTC.

Animals↗

PubMed2006

Isolation of the genome sequence strain Mycobacterium avium 104 from multiple patients over a 17-year period.

The genome sequence strain 104 of the opportunistic pathogen Mycobacterium avium was isolated from an adult AIDS patient in Southern California in 1983. Isolates of non-paratuberculosis M. avium from 207 other patients in Southern California and elsewhere were examined for genotypic identity to strain 104. This process was facilitated by the use of a novel two-step approach. In the first step, all 208 strains in the sample were subjected to a high-throughput, large sequence polymorphism (LSP)-based genotyping test, in which DNA from each strain was tested by PCR for the presence or absence of 4 hypervariable genomic regions. Nineteen isolates exhibited an LSP type that resembled that of strain 104. This subset of 19 isolates was then subjected to high-resolution repetitive sequence-based PCR typing, which identified 10 isolates within the subset that were genotypically identical to strain 104. These isolates came from 10 different patients at 5 clinical sites in the western United States, and they were isolated over a 17-year time span. Therefore, the sequenced genome of M. avium strain 104 has been associated with disease in multiple patients in the western United States. Although M. avium is known for its genetic plasticity, these observations also show that strains of the pathogen can be genotypically stable over extended time periods.

Bacterial Typing Techniques↗

PubMed2005

Ecotypes of the Mycobacterium tuberculosis complex.

A phylogeny of the Mycobacterium tuberculosis complex has recently shown that the animal-adapted strains are found in a single lineage marked by the deletion of chromosomal region 9 (RD9) [Brosch et al., 2002. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc. Natl Acad. Sci. USA 99 (6), 3684-3689]. We have obtained the spoligotype patterns of the RD9 deleted strains used to generate this new evolutionary scenario and we show that the presence of spoligotype spacers 3, 9, 16, 39, and 40-43 is phylogenetically informative in this lineage. We have used the phylogenetically informative spoligotype spacers to screen a database of spoligotype patterns and have identified further members of a group of strains apparently host-adapted to antelopes. The presence of the spoligotype spacers is congruent with the phylogeny generated by chromosomal deletions, suggesting that recombination is rare or absent between strains of this lineage. The phylogenetically informative spacers, in concert with the previously identified single nucleotide mutations and chromosomal deletions, can be used to identify a series of clades in the RD9 deleted lineage each with a separate host preference. Finally, we discuss the application of the ecotype concept to this series of clades and suggest that the M. tuberculosis complex may best be described as a series of host-adapted ecotypes.

Animals↗

PubMed2005

Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guérin mutants that secrete listeriolysin.

The tuberculosis vaccine Mycobacterium bovis bacille Calmette-Guérin (BCG) was equipped with the membrane-perforating listeriolysin (Hly) of Listeria monocytogenes, which was shown to improve protection against Mycobacterium tuberculosis. Following aerosol challenge, the Hly-secreting recombinant BCG (hly+ rBCG) vaccine was shown to protect significantly better against aerosol infection with M. tuberculosis than did the parental BCG strain. The isogenic, urease C-deficient hly+ rBCG (DeltaureC hly+ rBCG) vaccine, providing an intraphagosomal pH closer to the acidic pH optimum for Hly activity, exhibited still higher vaccine efficacy than parental BCG. DeltaureC hly+ rBCG also induced profound protection against a member of the M. tuberculosis Beijing/W genotype family while parental BCG failed to do so consistently. Hly not only promoted antigen translocation into the cytoplasm but also apoptosis of infected macrophages. We concluded that superior vaccine efficacy of DeltaureC hly+ rBCG as compared with parental BCG is primarily based on improved cross-priming, which causes enhanced T cell-mediated immunity.

Adult↗

PubMed2005

Protection of macaques against Mycobacterium tuberculosis infection by a subunit vaccine based on a fusion protein of antigen 85B and ESAT-6.

Various new tuberculosis (TB) vaccine candidates in combination with new delivery systems, including subunit vaccines, are currently being evaluated by a number of laboratories. One vaccine candidate that has shown promising protective capacity in mice and guinea pigs is a fusion of Ag85B and ESAT-6. In this study, we have investigated the efficacy of this Ag85B-ESAT-6 fusion protein vaccine in a non-human primate model for TB. Vaccination of cynomolgus monkeys with the Ag85B-ESAT-6 fusion protein in two different adjuvant (DDA/MPL, AS02A) resulted in a reduction in bacterial number and/or lung pathology in animals challenged with Mycobacterium tuberculosis. Vaccination prevented an increase in C-reactive protein serum levels, general activation of CD4 and CD8 subsets and boosted development of humoral and cellular immune responses to a spectrum of mycobacterial antigens on exposure to M. tuberculosis infection. We show, in two independent experiments, that vaccination of primates with Ag85B-ESAT-6 induces protective immune responses, suggesting that Ag85B-ESAT-6 is a strong candidate for further clinical evaluation. As far as we are aware this is the first report of protection in primates with a subunit vaccine.

Acyltransferases↗

PubMed2005

ESAT-6 and CFP-10 in clinical versus environmental isolates of Mycobacterium kansasii.

Mycobacterium kansasii consists of 5 genetically distinct groups, of which 2 are associated with human disease. Determinants of the differences in virulence are unknown. Potential genes of interest are esat-6 and cfp-10, which are associated with virulence of Mycobacterium tuberculosis and Mycobacterium bovis but are lacking in bacille Calmette-Guérin and in most environmental mycobacteria (M. kansasii is an exception). We investigated esat-6 and cfp-10 genes in 22 clinical and 14 environmental isolates of M. kansasii. Both were present in all isolates; each genetic group had its own characteristic Southern-blot pattern corresponding to a highly conserved fingerprint pattern. Nucleotide sequences of the genes differed 12.6% and 10.1%, respectively, from the M. tuberculosis homologues, but the deduced amino acid sequences were <5% different. In vitro, clinical and environmental genotypes of M. kansasii expressed CFP-10 and ESAT-6. Thus, virulence of M. kansasii is not directly related to esat-6 and cfp-10 genes or gene expression.

Amino Acid Sequence↗

PubMed2005

Antimycobacterial agents differ with respect to their bacteriostatic versus bactericidal activities in relation to time of exposure, mycobacterial growth phase, and their use in combination.

A number of antimycobacterial agents were evaluated with respect to their bacteriostatic activity (growth inhibition) versus the bactericidal activity against a clinical isolate of Mycobacterium avium (Mycobacterium avium complex [MAC] strain 101) in relation to the time of exposure and the growth phase of the mycobacteria. In terms of growth inhibition the MAC in the active phase of growth was susceptible to clarithromycin, ethambutol, rifampin, amikacin, and the quinolones moxifloxacin, ciprofloxacin, and sparfloxacin. In terms of bactericidal activity in relation to the time of exposure these agents differed substantially with respect to the killing rate. An initial high killing capacity at low concentration was observed for amikacin, which in this respect was superior to the other agents. The bactericidal activity of clarithromycin and ethambutol was only seen at relatively high concentrations and increased with time. Killing by rifampin was concentration dependent as well as time dependent. The bactericidal activity of moxifloxacin was marginally dependent on the concentration or the time of exposure. The activity of clarithromycin in combination with ethambutol was not significantly enhanced compared to single-agent exposure. Only an additive effect was observed. The addition of rifampin or moxifloxacin as a third agent only marginally effected increased killing of MAC. However, by addition of amikacin the activity of the clarithromycin-ethambutol combination was significantly improved. The combination of amikacin and amoxicillin-clavulanic acid exhibited synergistic antimycobacterial activity. Towards MAC at low growth rates, only the quinolones exhibited a bactericidal effect.

Amikacin↗

PubMed2005

Utility of mycobacterial interspersed repetitive unit typing for differentiating multidrug-resistant Mycobacterium tuberculosis isolates of the Beijing family.

Mycobacterial interspersed repetitive unit (MIRU) typing has been found to allow rapid, reliable, high-throughput genotyping of Mycobacterium tuberculosis and may represent a feasible approach to study global M. tuberculosis molecular epidemiology. To evaluate the use of MIRU typing in discriminating drug-resistant M. tuberculosis strains of the Beijing genotype family, 102 multidrug-resistant (MDR) clinical isolates and 253 randomly selected non-MDR isolates collected from 2000 to 2003 in Hong Kong were subjected to 12-locus MIRU typing, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. Spoligotyping showed that 243 (68.5%) of 355 isolates belonged to Beijing family genotype. MIRU typing showed lower discrimination in differentiating between the Beijing family strains (Hunter-Gaston discriminative index [HGI] of 0.8827) compared with the IS6110 RFLP method (HGI = 0.9979). For non-Beijing strains, MIRU typing provided discrimination (HGI = 0.9929) comparable to that of the RFLP method (HGI = 0.9961). There was no remarkable difference in discrimination power between the two methods in differentiating both within and between MDR and non-MDR strains of M. tuberculosis. Dendrograms constructed with the MIRU typing data showed a clear segregation between the Beijing and non-Beijing genotype. Addition of RFLP to MIRU typing offered a higher discrimination ability (92.6%) than did addition of MIRU typing to RFLP (40.0%). This supported the potential use of this method to analyze the global genetic diversity of MDR M. tuberculosis strains that may be at different levels of evolutionary divergence.

Bacterial Typing Techniques↗

PubMed2005

Use of variable-number tandem-repeat typing to differentiate Mycobacterium tuberculosis Beijing family isolates from Hong Kong and comparison with IS6110 restriction fragment length polymorphism typing and spoligotyping.

The Mycobacterium tuberculosis Beijing family isolates may cause more than a quarter of all tuberculosis cases worldwide, are emerging in some areas, and are often associated with drug resistance. Early recognition of transmission of this genotype is therefore important. To evaluate the usefulness of variable-number tandem-repeat (VNTR) typing to discriminate and recognize strains of the Beijing family, M. tuberculosis isolates from Hong Kong were subjected to VNTR analysis, spoligotyping, and IS6110 restriction fragment length polymorphism (RFLP) typing. The allelic diversity of the 14 VNTR loci included in the analysis varied from 0 to 0.618 among Beijing strains. The discriminatory power of VNTR analysis was slightly lower than that of IS6110 RFLP. Our analysis shows that VNTR typing, which has many practical advantages over RFLP typing, can be used for epidemiological studies of Beijing strains. However, VNTR-defined clusters should be subtyped with IS6110 RFLP for maximal resolution.

Bacterial Typing Techniques↗

PubMed2005

Fast ligation-mediated PCR, a fast and reliable method for IS6110-based typing of Mycobacterium tuberculosis complex.

IS6110 restriction fragment length polymorphism (RFLP) analysis is the most widely applied method for strain differentiation of Mycobacterium tuberculosis complex. We have previously described mixed-linker PCR, an IS6110-based PCR method that favorably compared with other typing methods for M. tuberculosis complex according to reproducibility and ability to differentiate between strains. Here we report the further development of this method, called fast ligation-mediated PCR (FLiP), which allows analysis of strains within one working day and starting from less than 1 ng of mycobacterial DNA or a crude cell lysate. Blinded analysis of a standard set of 131 M. tuberculosis complex and nontuberculous isolates showed the ability to differentiate 81 types among 90 M. tuberculosis complex isolates with 84 different IS6110 RFLP fingerprint patterns and detected 97% of the 31 duplicate samples. We suggest that FLiP can serve to rapidly detect chains of transmission prior to starting high-throughput analysis or standard IS6110 RFLP. It may as well serve as a secondary typing technique for other, non-IS6110-based methods.

Bacterial Typing Techniques↗

PubMed2005

Discriminatory power and reproducibility of novel DNA typing methods for Mycobacterium tuberculosis complex strains.

In recent years various novel DNA typing methods have been developed which are faster and easier to perform than the current internationally standardized IS6110 restriction fragment length polymorphism typing method. However, there has been no overview of the utility of these novel typing methods, and it is largely unknown how they compare to previously published methods. In this study, the discriminative power and reproducibility of nine recently described PCR-based typing methods for Mycobacterium tuberculosis were investigated using the strain collection of the interlaboratory study of Kremer et al. This strain collection contains 90 M. tuberculosis complex and 10 non-M. tuberculosis complex mycobacterial strains, as well as 31 duplicated DNA samples to assess reproducibility. The highest reproducibility was found with variable numbers of tandem repeat typing using mycobacterial interspersed repetitive units (MIRU VNTR) and fast ligation-mediated PCR (FLiP), followed by second-generation spoligotyping, ligation-mediated PCR (LM-PCR), VNTR typing using five repeat loci identified at the Queens University of Belfast (QUB VNTR), and the Amadio speciation PCR. Poor reproducibility was associated with fluorescent amplified fragment length polymorphism typing, which was performed in three different laboratories. The methods were ordered from highest discrimination to lowest by the Hunter-Gaston discriminative index as follows: QUB VNTR typing, MIRU VNTR typing, FLiP, LM-PCR, and spoligotyping. We conclude that both VNTR typing methods and FLiP typing are rapid, highly reliable, and discriminative epidemiological typing methods for M. tuberculosis and that VNTR typing is the epidemiological typing method of choice for the near future.

Bacterial Typing Techniques↗

PubMed2005

Isolation of nontuberculous mycobacteria in Zambia: eight case reports.

The isolation of nontuberculous mycobacteria (NTM) raises the question of their clinical significance, especially in an African setting. We found a high percentage of NTM isolated from various specimens, including ones that are normally sterile, among 213 patients in Zambia. Because tuberculosis can affect all parts of the body, we decided to include patients who had signs and symptoms in any part of the body for more than 2 weeks. Most patients had tractus respiratorius (80%) and tractus digestivus (10%) symptoms. During three consecutive days, sputum was collected and two separate sputum specimens were cultured for mycobacteria. Depending on the clinical picture, pleural effusion, ascites, abscess material, or enlarged lymph nodes were also cultured for mycobacteria. A specimen from one sterile body site was collected from 25 patients (60% human immunodeficiency virus [HIV] positive). NTM were isolated from 8 of these 25 specimens. Mycobacterium lentiflavum was isolated from four patients, and Mycobacterium goodii was isolated from one patient. In order to exclude the possibility of laboratory cross-contamination, a novel amplified fragment length polymorphism DNA typing method for M. lentiflavum was developed. Genetic variation was detected, rendering the likelihood of laboratory contamination unlikely. Clinically relevant infection due to NTM occurs in both HIV-positive and HIV-negative patients in Zambia, and their clinical impact seems to be underestimated. This is the first report of M. lentiflavum and M. goodii infections in Africa.

AIDS-Related Opportunistic Infections↗

PubMed2005

Targeted hybridization of IS6110 fingerprints identifies the W-Beijing Mycobacterium tuberculosis strains among clinical isolates.

Targeted IS6110-based RFLP genotyping can be applied to rapidly identify specific groups of biomedically/epidemiologically relevant Mycobacterium tuberculosis clinical isolates. One such group is the W-Beijing strain family (also known as Beijing/W), implicated in significant nosocomial and community outbreaks worldwide. Using previously defined criteria, we developed a simple and accurate method to identify members of the W-Beijing family, based on rehybridization of Southern blot membranes used previously in routine IS6110 DNA fingerprint analysis. The hybridization probe constructed ("W-Beijing polyprobe") contains the PCR-amplified fragments specific for three M. tuberculosis chromosomal loci used for the identification of W-Beijing strains. The targets include the dnaA-dnaN and NTF regions and the direct repeat locus. A total of 526 selected clinical isolates (representative of 253 different IS6110-defined strain types) were analyzed using the W-Beijing polyprobe. A total of 148 isolates from this collection were found to be members of the W-Beijing phylogenetic lineage, comprising 106 strains from the W-Beijing family (46 clusters) and 42 related isolates. Rehybridization results were confirmed by computer-assisted analysis. The sensitivity and specificity of this method were estimated at 98.7% and 99.7%, respectively. This study demonstrates that the W-Beijing polyprobe can accurately and reliably discriminate members of the W-Beijing phylogenetic lineage and the W-Beijing family of M. tuberculosis strains.

Base Sequence↗

PubMed2005

Genomic polymorphisms for Mycobacterium avium subsp. paratuberculosis diagnostics.

Mycobacterium avium subsp. paratuberculosis is an emerging pathogen of mammals and is being actively investigated as a possible zoonotic agent. The lack of reliable diagnostic assays has hampered rational assessment of the prevalence of this organism in humans and animals. We have used a comparative genomic approach to reveal genomic differences between M. avium subsp. paratuberculosis and its close relative M. avium subsp. avium, a highly prevalent environmental organism. From computational and DNA microarray-based study of two prototype strains, M. avium subsp. avium strain 104 and M. avium subsp. paratuberculosis strain K10, we have uncovered two types of large sequence polymorphisms (LSPs): those present in the former but missing in the latter (LSP(A)s) and those only present in the latter (LSP(P)s). We examined the distribution of 3 LSP(A)s and 17 LSP(P)s across a panel of 383 M. avium complex isolates in order to determine their potential utility for the development of accurate diagnostic tests. Our results show that the absence of LSP(A)8 is 100% specific for the identification of M. avium subsp. paratuberculosis. Of the 17 LSP(P)s, 10 regions were not specific for M. avium subsp. paratuberculosis while 7 were shown to be highly specific (>98%) and, in some cases, highly sensitive as well (up to 95%). These data highlight the need to evaluate these regions across a diverse panel of clinical and environmental isolates and indicate the LSPs best suited for M. avium subsp. paratuberculosis diagnostics.

Genome, Bacterial↗