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K Kremer

Publications and source records attributed to K Kremer.

At least 37 records · Page 2Linked to original sources
PubMed2001

Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units.

Large-scale genotyping of Mycobacterium tuberculosis is especially challenging, as the current typing methods are labor-intensive and the results are difficult to compare among laboratories. Here, automated typing based on variable-number tandem repeats (VNTRs) of genetic elements named mycobacterial interspersed repetitive units (MIRUs) in 12 mammalian minisatellite-like loci of M. tuberculosis is presented. This system combines analysis of multiplex PCRs on a fluorescence-based DNA analyzer with computerized automation of the genotyping. Analysis of a blinded reference set of 90 strains from 38 countries (K. Kremer et al., J. Clin. Microbiol. 37:2607-2618, 1999) demonstrated that it is 100% reproducible, sensitive, and specific for M. tuberculosis complex isolates, a performance that has not been achieved by any other typing method tested in the same conditions. MIRU-VNTRs can be used for analysis of the global genetic diversity of M. tuberculosis complex strains at different levels of evolutionary divergence. To fully exploit the portability of this typing system, a website was set up for the analysis of M. tuberculosis MIRU-VNTR genotypes via the Internet. This opens the way for global epidemiological surveillance of tuberculosis and should lead to novel insights into the evolutionary and population genetics of this major pathogen.

Automation↗
PubMed2001

Family outbreaks of tuberculosis in South Moravia, a low incidence area of the Czech Republic.

OBJECTIVES: To identify suspected family outbreaks of tuberculosis in South Moravia, an area with the lowest notification rates in the Czech Republic. METHOD: The insertion sequence IS6110-based RFLP analysis of Mycobacterium tuberculosis was applied in isolates collected from 17 indigenous excretors giving the same family name and/or domicile. The fingerprints were matched with a database of 184 RFLP profiles of Czech M. tuberculosis isolates originating from remote localities. RESULTS: The RFLP analysis revealed uniform fingerprints in each of six indigenous outbreaks consisting of two to six family members or relatives. In three of them no matching case has been found in the Czech fingerprint database. The remaining three clusters showed identical patterns with RFLP profiles of 13 Czech M. tuberculosis strains originating from remote areas: five were from patients living in the immediate vicinity and eight were from distant regions. CONCLUSION: Different clustered genotypes of M. tuberculosis were identified in epidemiologically linked family outbreaks of tuberculosis, half of them indigenous and half matching fingerprints of strains from remote areas. It seems that family outbreaks may contribute to the continued occurrence of tuberculosis in South Moravia, which reports the lowest notification values in the Czech Republic.

Adolescent↗
PubMed2000

Strong attraction between charged spheres due to metastable ionized states

We report a mechanism which can lead to long-range attractions between like-charged spherical macroions, stemming from the existence of metastable ionized states. We show that the ground state of a single highly charged colloid plus a few excess counterions is overcharged. For the case of two highly charged macroions in their neutralizing divalent counterion solution we demonstrate that, in the regime of strong Coulomb coupling, the counterion clouds are very likely to be unevenly distributed, leading to one overcharged and one undercharged macroion. This long-living metastable configuration in turn leads to a long-range Coulomb attraction.

Journal Article↗
PubMed2000

Self-similar chain conformations in polymer gels

We use molecular dynamics simulations to study the swelling of randomly end-cross-linked polymer networks in good solvent conditions. We find that the equilibrium degree of swelling saturates at Q(eq) approximately N(3/5)(e) for mean strand lengths &Nmacr;(s) exceeding the melt entanglement length N(e). The internal structure of the network strands in the swollen state is characterized by a new exponent nu = 0.72+/-0.02. Our findings can be rationalized by a Flory argument for a self-similar structure of mutually interpenetrating network strands, agree partially with the classical Flory-Rehner theory, and are in contradiction to de Gennes' c(*)-theorem.

Journal Article↗
PubMed2000

Usefulness of IS6110-restriction fragment length polymorphism typing of Brazilian strains of Mycobacterium tuberculosis and comparison with an international fingerprint database.

Strains of Mycobacterium tuberculosis isolated from 219 different tuberculosis patients, 115 from patients residing in Rio de Janeiro, 79 from Rio Grande do Sul and the remaining from other regions of the country, were analyzed by IS6110-restriction fragment length polymorphism fingerprinting. The IS6110-DNA patterns from these strains were highly polymorphic: 174 different patterns were observed and 25 patterns were shared by 70 isolates (32%). Most strains (93.4%) had multicopy patterns and only 17% of clustered strains had less than six IS6110 copies. Strain clustering was significantly higher for isolates from Rio Grande do Sul (36.7%) in comparison with strains from Rio de Janeiro (22.6%), but only when using high stringency during cluster analysis. Upon screening of an international database containing 3,970 fingerprints of M. tuberculosis strains, 15% of the patterns of Brazilian strains (21% of the strains) were identical to a fingerprint of an isolate from another country and one particular eight-band pattern forming the largest Brazilian cluster was detected in seven additional countries, suggesting that international transmission of tuberculosis from and to Brazil could be occurring frequently. Alternatively,preferential use of certain IS6110 integration sites could also be important in high-copy number strains, having important consequences for the use of databases for epidemiological studies on a large scale.

Bacterial Typing Techniques↗
PubMed2000

IS6110-RFLP and spoligotyping of Mycobacterium tuberculosis isolates in Iran.

To evaluate and compare the usefulness of IS6110-restriction fragment length polymorphism (RFLP) and spoligotyping in the epidemiology of tuberculosis in Iran, Mycobacterium tuberculosis complex strains, isolated in 2 different areas of Iran, were subjected to RFLP and spoligotyping. The average number of IS6110 copies per strain was 11 and ranged from 5 to 18 among the M. tuberculosis strains. In total, among the 62 isolates, 56 different patterns were observed. 50 strains had unique RFLP patterns (89%) and 12 (11%) revealed patterns that were found among at least 1 other isolate. Spoligotyping of 97 isolates resulted in 42 different patterns, of which 72% were found in 15 clusters. 14 (29%) out of 48 investigated isolates were resistant to 1 or more antituberculosis drugs and 57% of the resistant isolates were isolated from Afghan immigrants. Ten percent of the isolates represented the Beijing genotype, including 4 of the 14 (36%) resistant strains. Three of these resistant Beijing strains were isolated from Afghan patients. IS6110-RFLP typing could be useful for studying the epidemiology of tuberculosis in Iran. IS6110 patterns were polymorphic and the average IS6110 copy number was high.

Cluster Analysis↗
PubMed2000

Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria.

The direct repeat region in Mycobacterium tuberculosis complex strains is composed of multiple direct variant repeats (DVRs), each of which is composed of a 36-bp direct repeat (DR) plus a nonrepetitive spacer sequence of similar size. It has been shown previously that clinical isolates show extensive polymorphism in the DR region by the variable presence of DVRs, and this polymorphism has been used in the epidemiology of tuberculosis. In an attempt to better understand the evolutionary scenario leading to polymorphic DR loci and to improve strain differentiation by spoligotyping, we characterized and compared the DNA sequences of the complete DR region and its flanking DNA of M. tuberculosis complex strains. We identified 94 different spacer sequences among 26 M. tuberculosis complex strains. No sequence homology was found between any of these spacers and M. tuberculosis DNA outside of the DR region or with any other known bacterial sequence. Although strains differed extensively in the presence or absence of DVRs, the order of the spacers in the DR locus was found to be well conserved. The data strongly suggest that the polymorphism in clinical isolates is the result of successive deletions of single discrete DVRs or of multiple contiguous DVRs from a primordial DR region containing many more DVRs than seen in present day isolates and that virtually no scrambling of DVRs took place during evolution. Because the majority of the novel spacer sequences identified in this study were confined to isolates of the rare Mycobacterium canettii taxon, the use of the novel spacers in spoligotyping led only to a slight improvement of strain differentiation by spoligotyping.

Animals↗
PubMed2000

Genetic heterogeneity in Mycobacterium tuberculosis isolates reflected in IS6110 restriction fragment length polymorphism patterns as low-intensity bands.

Mycobacterium tuberculosis isolates with identical IS6110 restriction fragment length polymorphism (RFLP) patterns are considered to originate from the same ancestral strain and thus to reflect ongoing transmission. In this study, we investigated 1,277 IS6110 RFLP patterns for the presence of multiple low-intensity bands (LIBs), which may indicate infections with multiple M. tuberculosis strains. We did not find any multiple LIBs, suggesting that multiple infections are rare in the Netherlands. However, we did observe a few LIBs in 94 patterns (7.4%) and examined the nature of this phenomenon. With single-colony cultures it was found that LIBs mostly represent mixed bacterial populations with slightly different RFLP patterns. Mixtures were expressed in RFLP patterns as LIBs when 10 to 30% of the DNA analyzed originated from a bacterial population with another RFLP pattern. Presumably, a part of the LIBs did not represent mixed bacterial populations, as in some clusters all strains exhibited LIBs in their RFLP patterns. The occurrence of LIBs was associated with increased age in patients. This may reflect either a gradual change of the bacterial population in the human body over time or IS6110-mediated genetic adaptation of M. tuberculosis to changes in the environmental conditions during the dormant state or reactivation thereafter.

Cluster Analysis↗
PubMed2000

The first occurrence of a multi-drug resistant tuberculosis epidemic in the Czech Republic caused by genetically closely related Mycobacterium tuberculosis strains.

DNA fingerprinting based on the detection of the insertion sequence IS6110 in Pvull restriction fragments was applied to M. tuberculosis isolates originating in the first microepidemic of multidrug resistant tuberculosis recorded in the Czech Republic. Their disseminators were 21 individuals living in--or roaming between three distant areas. The age of 17 males ranged from 36 to 64 years (average 45 years) and of 4 females aged from 38 to 52 years. The index person was most probably a former male prisoner, aged 49 years, who disseminated multidrug resistant M. tuberculosis over a period of 28 months. In ten of the patients the following risk factors for tuberculosis were found: imprisonment, homelessness, immigration and previous stay in asylum--or in a psychiatric ward. In six cases, M. kansasii infection preceded tuberculosis. Four out of the 21 patients died. The RFLP analysis separated the patients into two distinct groups: group A comprising 14 members of which M. tuberculosis strains were isolated with six IS6110 copies, whereas the isolates of seven individuals of the group B, the RFLP profile displayed highly similar RFLP patterns compared to the isolates of group A, but with two additional IS6110 copies. In one patient, both A and B patterns were found: the first one in a M. tuberculosis strain isolated in 1993 and the second one in the isolate isolated two years later. Both the appearance of pattern B among the isolates of a part of patients and the switch from A to B pattern in one of patients can be plausibly explained by the unstability of DNA genotypes caused by transposition of IS6110 elements.

Adult↗
PubMed1999

Perforating appendicitis: is it a separate disease? Acute Abdominal Pain Study Group.

OBJECTIVE: To find out whether perforated and unperforated appendicitis are separate diseases and can be distinguished clinically. DESIGN: Prospective multicentre study. SETTING: 11 departments of surgery in Germany and Austria. SUBJECTS: 519 patients over 6 years old who had histologically confirmed acute appendicitis between October 1994 and March 1996. MAIN OUTCOME MEASURES: Differences in history, clinical findings, lab results, clinical course and outcome. RESULTS: 92 of the 519 patients (18%) had perforated appendicitis. The following variables were shown by univariate analysis to be significantly more common in the group with perforated appendicitis: rigidity, reduced abdominal wall movement, abdominal distension, reduced bowel sounds (all p<0.001), pale skin (p<0.005), generalised abdominal tenderness, severe abdominal tenderness (both p<0.01), WCC > or =10(9)/L (p<0.05). By multivariate analysis the following variables were significantly more common in the group with perforated appendicitis: age over 50 years (p<0.0001); change in bowel habit and rigidity of the abdominal wall (both p = 0.001); generalised tenderness (p<0.01); male sex (p<0.01); and distended abdomen (p<0.05). Rectal examination failed to make the distinction. CONCLUSIONS: Perforated and unperforated appendicitis behave clinically like two different diseases. They can in most cases reliably be distinguished using clinical criteria alone. Although greater diagnostic accuracy may result in a higher rate of perforation, close observation and timely intervention will only marginally affect the outcome.

Adult↗
PubMed1999

Molecular epidemiology of tuberculosis in the Netherlands: a nationwide study from 1993 through 1997.

To disclose risk factors for active tuberculosis transmission in the Netherlands, restriction fragment length polymorphism (RFLP) patterns of 78% of the Mycobacterium tuberculosis isolates, from the period 1993-1997, were analyzed. Of the respective 4266 cases, 46% were found in clusters of isolates with identical RFLPs, and 35% were attributed to active transmission. The clustering percentage increased strongly with the number of isolates; taking this into account, fewer cases were clustered than has been reported in other studies. Contact investigations in the five largest clusters of 23-47 patients suggested epidemiological linkage between cases. Of patients identified through contact tracing, 91% were clustered. Demographic risk factors for active transmission of tuberculosis included male sex, urban residence, Dutch and Surinamese nationality, and long-term residence in the Netherlands. Human immunodeficiency virus infection was not an independent risk factor for active transmission. Isoniazid-resistant strains were relatively less frequently clustered, suggesting that these generated fewer secondary cases.

Adult↗
PubMed1999

Usefulness of spoligotyping To discriminate IS6110 low-copy-number Mycobacterium tuberculosis complex strains cultured in Denmark.

Mycobacterium tuberculosis complex strains cultured in Denmark have been analyzed by IS6110 restriction fragment length polymorphism (RFLP) on a routine basis from 1992 and onwards. Due to the influx of immigrants with tuberculosis, the number of strains harboring only one to five copies of IS6110 has increased steadily. Since the discriminatory power of IS6110 fingerprinting for such strains is poor, we have performed additional genotyping of all low-copy-number strains by the recently described PCR-based method known as spoligotyping. A total of 311 clinical strains were typed: 14 Mycobacterium bovis BCG, 48 M. bovis, and 249 M. tuberculosis strains. Spoligotyping correctly differentiated M. bovis and M. bovis BCG from M. tuberculosis strains, but it did not differentiate M. bovis from M. bovis BCG. All M. bovis BCG strains exhibited identical spoligotype patterns. The discriminatory power of spoligotyping of low-copy-number M. tuberculosis strains was higher than that of IS6110 fingerprinting. Based on RFLP typing solely, 83% of the low-copy-number M. tuberculosis strains were found to form part of a cluster, and 75% were found to form a cluster on the basis of spoligotyping. When the two techniques were combined, the amount of clustering decreased to 55%. The combination of these two techniques might be valuable in studying the epidemiology of M. tuberculosis strains harboring few copies of the IS6110 element.

DNA Fingerprinting↗
PubMed1999

Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility.

In this study, the currently known typing methods for Mycobacterium tuberculosis isolates were evaluated with regard to reproducibility, discrimination, and specificity. Therefore, 90 M. tuberculosis complex strains, originating from 38 countries, were tested in five restriction fragment length polymorphism (RFLP) typing methods and in seven PCR-based assays. In all methods, one or more repetitive DNA elements were targeted. The strain typing and the DNA fingerprint analysis were performed in the laboratory most experienced in the respective method. To examine intralaboratory reproducibility, blinded duplicate samples were included. The specificities of the various methods were tested by inclusion of 10 non-M. tuberculosis complex strains. All five RFLP typing methods were highly reproducible. The reliability of the PCR-based methods was highest for the mixed-linker PCR, followed by variable numbers of tandem repeat (VNTR) typing and spoligotyping. In contrast, the double repetitive element PCR (DRE-PCR), IS6110 inverse PCR, IS6110 ampliprinting, and arbitrarily primed PCR (APPCR) typing were found to be poorly reproducible. The 90 strains were best discriminated by IS6110 RFLP typing, yielding 84 different banding patterns, followed by mixed-linker PCR (81 patterns), APPCR (71 patterns), RFLP using the polymorphic GC-rich sequence as a probe (70 patterns), DRE-PCR (63 patterns), spoligotyping (61 patterns), and VNTR typing (56 patterns). We conclude that for epidemiological investigations, strain differentiation by IS6110 RFLP or mixed-linker PCR are the methods of choice. A strong association was found between the results of different genetic markers, indicating a clonal population structure of M. tuberculosis strains. Several separate genotype families within the M. tuberculosis complex could be recognized on the basis of the genetic markers used.

Bacterial Typing Techniques↗
PubMed1999

A multidrug-resistant tuberculosis microepidemic caused by genetically closely related Mycobacterium tuberculosis strains.

IS6110 DNA fingerprinting was used to characterize an outbreak of multidrug-resistant tuberculosis in 21 individuals (17 males and 4 females) living in or roaming among four distantly separated areas in the Czech Republic. The restriction fragment length polymorphism (RFLP) analysis separated the collected Mycobacterium tuberculosis strains into group A, including 14 patients with six IS6110 copies, and group B, with 7 patients displaying highly similar RFLP patterns but with two additional IS6110 bands. A switch from pattern A to pattern B was observed in one patient, and the subsequent detection of subclone B in seven more individuals has been explained by the instability of DNA genotypes caused by transposition of IS6110 elements.

Czech Republic↗
PubMed1999

[A micro-epidemic of tuberculosis in the South Moravia Region].

In the South Moravian region which is the area with the lowest prevalence of bacillary tuberculosis in the Czech Republic (6.6 per 100,000 population in 1996) in 1993-1996 a total of six local and family microepidemics of tuberculosis were detected. For their identification the RFLP fingerprinting method was used based on evidence of repeated sequence of IS6110 in the chromosomal DNA of the examined strains of Mycobacterium tuberculosis predigested with restrictive enzyme PvuII. The profiles of fingerprints of each microepidemic were included by means of a computer programme into the hierarchy of the fingerprint dendrogram of 184 strains of M. tuberculosis which made it possible to identify possible identical profiles of strains from patients from remote places in the Czech Republic. In three family microepidemics involving always two members no identical fingerprint profiles of other Czech strains of M. tuberculosis were revealed. To the fourth cluster formed by six members of one family an identical RFLP profile of a female patient living in a nearby locality was added. In another microepidemic recorded in three brothers identical fingerprints were found another four patients from the South Moravian region and in one from the Central Bohemian region. The last cluster of two brothers was surprisingly enlarged by six identical RFLP profiles of patients from the West Bohemian region and one from Prague. These findings suggest that in areas with a low prevalence tuberculosis persists more frequently than in areas with a high prevalence as familial or local microepidemics.

Adolescent↗
PubMed1998

[Connection between 2 tuberculosis outbreaks demonstrated after 8 years by DNA-fingerprinting of the causative mycobacteria].

OBJECTIVE: To determine the possible connection between two outbreaks of tuberculosis at an 8-year interval by DNA fingerprinting of the causative mycobacteria. DESIGN: Descriptive. SETTING: Regional Public Health Services Zeeland and Noord-Holland, the Netherlands. METHOD: The source patients of the tuberculosis explosions in an island in the province of Zeeland (28 persons infected) and an island of the Noord-Holland area (36 persons infected) were identified in 1986 and 1994 respectively. In 1994. spoligotyping was performed on dead bacteria from the suspected source from 1986. to analyse the chain of transmission. RESULTS: Identical spoligotyping patterns of the source patients proved the transmission of a Mycobacterium tuberculosis strain from a father to his son around 1986. Lack of a positive Mantoux test in the son prohibited prevention of the 1994 explosion. CONCLUSION: Thanks to the applicability of spoligotyping on non-vital mycobacteria this DNA method contributed retrospectively to demonstration of a connection between two out-breaks of tuberculosis at an 8-year interval.

Adolescent↗
PubMed1998

Diagnosis of Mycobacterium microti infections among humans by using novel genetic markers.

As a result of DNA typing of Mycobacterium microti isolates from animals in the United Kingdom and The Netherlands, we diagnosed four human M. microti infections. These are the first M. microti infections among humans to be reported. Three of the patients were immunocompromised and suffered from generalized forms of tuberculosis. The fourth patient was a 34-year-old immunocompetent male with a persistent cough and undefined X-ray abnormalities. Two of the M. microti infections were recognized by their IS6110 restriction fragment length polymorphism (RFLP) patterns, which showed a high degree of similarity with those of M. microti strains isolated from a pig and a ferret in The Netherlands. The two other human M. microti infections were recognized by using the recently developed DNA fingerprinting method, "spoligotyping," directly on clinical material. All M. microti isolates from the United Kingdom and The Netherlands were found to contain an exceptionally short genomic direct repeat region, resulting in identical two-spacer sequence reactions in spoligotyping. In contrast, the highly similar IS6110 RFLP patterns of the vole strains from the United Kingdom differed considerably from the RFLPs of all M. microti strains isolated in The Netherlands, suggesting that geographic isolation led to divergent strains in the United Kingdom and on the continent.

Adult↗