Computer analysis of IS6110 RFLP patterns of Mycobacterium tuberculosis.
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Biomedical subjects
Publications and source records attributed to K Kremer.
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SETTING: Mycobacterium avium complex (MAC) includes major acquired immune deficiency syndrome (AIDS)-associated pathogens. Formerly, MAC serotyping was used for epidemiological purposes. Recently, restriction fragment length polymorphism (RFLP) typing has become available. OBJECTIVE: Examination of the usefulness of insertion sequence IS1245 in RFLP typing of MAC isolates and the association with IS901 RFLP. DESIGN: Ninety-four serovar reference strains were compared with 144 clinical and animal MAC isolates in RFLP typing. RESULTS: All but four strains containing M. avium-specific-rRNA possessed IS1245. Most human isolates showed polymorphic multiband IS1245 patterns, which were associated with serovars 4, 6 and 8. Sequential clinical isolates obtained at up to five years' distance displayed indistinguishable/closely related patterns. Eleven M. paratuberculosis isolates showed indistinguishable six-band patterns. All 29 MAC isolates from 23 bird species, 7/23 from mammals and 1/81 clinical isolates showed an IS1245 three-band pattern, associated with serovars 1, 2 and 3. All these IS1245 'bird' type strains showed closely related IS901 RFLPs. Only three IS1245 'non-bird' type strains contained IS901, but exhibited completely different RFLP patterns. CONCLUSION: IS1245-RFLP typing is useful for the classification of M. avium and epidemiology of most human isolates. The highly conserved IS901 and IS1245 RFLPs among 'bird' type isolates provide proof that these strains constitute a separate taxon within the MAC.
SETTING: Molecular typing has become an important tool for examining the extent of active transmission of tuberculosis. OBJECTIVES: To examine transmission of tuberculosis in Cuba using IS6110 restriction fragment length polymorphism (RFLP) typing and to evaluate the utility of spoligotyping. DESIGN: One hundred and sixty Mycobacterium tuberculosis strains isolated over a one year period in Cuba were subjected to RFLP and spoligotyping. RESULTS: Forty-eight percent of the isolates were found in 19 clusters of strains with identical RFLP patterns. In general, cluster sizes were limited, except for two large institutional outbreaks. Age was strongly inversely correlated to clustering. Most streptomycin-resistant isolates were found in clusters. Fifteen spoligotype clusters comprised 78% of the isolates. Significantly different IS6110 RFLP types subdivided 11 spoligotype clusters, whereas none of the IS6110 clusters were subdivided by spoligotyping. CONCLUSIONS: Considering the short study period, 48% clustering is high, indicating that recent transmission plays an important role in Cuba. Although resistance is still a minor problem, transmission of streptomycin-resistant strains occurs. The high polymorphism observed with IS6110 RFLP indicates that this marker is useful for future molecular epidemiological studies in Cuba. Spoligotyping appeared less suitable for population-based studies.
The results of rectal digital examinations performed on 477 patients upon admission with histopathologically proven acute appendicitis from a total of 2280 patients with acute abdominal pain were analyzed. Although 13.7% of the patients experienced pain on the right side and 7.4% pain in the pouch of Douglas during rectal examination, none of the rectal examination parameters was statistically significant for the diagnosis of acute appendicitis. There are well established and statistically significant clinical indications, such as guarding, rigidity, rebound tenderness or abdominal distention that actually make the unpleasant rectal-digital examination superfluous for patients with suspected appendicitis.
Large epidemiological studies recently published suggest that perforating appendicitis may represent a separate disease entity. To find out whether perforating and nonperforating appendicitis may be distinguished by clinical features (history and examination, lab tests) data from 519 cases of acute appendicitis (17.7% perforations) were analysed, and significant differences were recorded in various aspects of history and clinical examination. It was concluded that perforating and nonperforating appendicitis may clinically be distinguished reliably in the majority of cases.
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Restriction endonuclease PvuII plays a central role in restriction fragment length polymorphism analysis of Mycobacterium tuberculosis complex isolates with IS6110 as a genetic marker. We have investigated the basis for an apparent dichotomy in PvuII restriction fragment pattersn observed among strains of the M. tuberculosis complex. The chromosomal regions of two modified PvuII restriction sites, located upstream of the katG gene and downstream of an IS1081 insertion sequence, were studied in more detail. An identical 10-bp DNA sequence (CAGCTGGAGC) containing a PvuII site was found in both regions, and site-directed mutagenesis analysis revealed that this sequence was a target for modification. Strain-specific modification of PvuII sites was identified in DNA from over 80% of the nearly 800 isolates examined. Furthermore, the proportion of modifying and nonmodifying strains differs significantly from country to country.
The epidemiological relationship between tuberculosis cases in a prison and between cases within five families was investigated. Therefore, the isolated Mycobacterium tuberculosis strains were subjected to restriction fragment length polymorphism (RFLP) analysis using insertion sequence IS6110 as a probe. In case of 11 patients, the expected links of transmission were confirmed by RFLP typing. In contrast, in case of 4 patients the conclusion of classical contact tracing were not in agreement with the DNA fingerprinting results. These findings reinforce the usefulness of this recently developed technique as an additional tool in contact tracing. The IS6110DNA fingerprints of all strains investigated consisted of 7 to 13 bands and showed a high degree of polymorphism. Comparison of these fingerprints with those recorded in the Czech Republic previously, revealed the presence of a predominant DNA fingerprint type, without a known connection between the cases. Furthermore, other patterns found in the present study showed a high degree of similarity with the previously obtained fingerprints. Part of the patients were sampled twice. All of these double isolates showed identical fingerprints, confirming the previously described stability of IS6110DNA fingerprints. In contrast, from one couple of strains, isolated from husband and wife both suffering from tuberculosis, a slight change in one of the two patterns was observed. The patterns shared 10 band positions, confirming the expected relationship between these cases, but one of the patterns contained one additional band.
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Histological sections of formalin fixed, paraffin-embedded tissue comprising 195 specimens of human lung carcinomas were Feulgen stained. The nuclei of the histomorphological images were segmented using an automated image analyzing system, and the attributed minimum spanning trees (MST) were calculated. Features related to the DNA-content of the nuclei (integrated optical density (IOD), IOD-entropy, S-phase related fraction, percentage > 5C, etc.), and structural parameters (minimum distance between tumor cell nuclei, minimum distance between tumor cells and neighboring lymphocytes, MST-entropy, MST-current of entropy (entropiefluss), distance between neighboring proliferating tumor cells, etc.) were measured. The following results were obtained: the measured IOD and MST features showed significant differences between the primary carcinomas and metastatic carcinomas in the intrapulmonary lymph nodes. The survival of patients was remarkably improved if the carcinomas displayed a low S-phase related fraction, a low percentage of tumor cells > 5C, a low number of stem lines, and a low MST-current of entropy.
The clinical significance of the entropy of the integrated optical density E (IOD) and the corresponding entropiefluss EF (IOD) is discussed as well as the structural entropy E (MST) and its entropiefluss EF (MST). The E (IOD) is based upon the formula given by Stenkvist and Strande. It describes the statistical entropy of the DNA distribution of tumor cell nuclei; the E (MST) is based upon differences in IOD and distance between neighboring tumor cell nuclei and describes the deviation of tumor structure from a "biological constant" structure. The entropiefluss measures the current of entropy or irreversibly created heat which has to be removed through the surface of the tumor. It is a universally valid description of a thermodynamically open system and measures the distance of such a system from its equilibrium stage. The clinical significance of these parameters is tested on 241 specimens of human lung carcinomas and intrapulmonary metastases of extrapulmonary malignancies. The surgical specimens were fixed with buffered formalin. Histological sections cut from paraffin-embedded tissue were FEULGEN stained. The nuclei of the histomorphological images were segmented using an automated image analyzing system, and the attributed minimum spanning trees (MST) were calculated. The following results were obtained: The measured E (IOD) and E (MST) showed significant differences in the primary carcinomas compared to those measured in intrapulmonary lymph node metastases or intrapulmonary metastases. The survival of patients was remarkably improved if the carcinomas displayed a low E (MST), EF (IOD), and EF (MST).
Histologic sections of formalin-fixed, paraffin-embedded tissue consisting of 53 surgical specimens, 48 biopsy specimens and 29 intrapulmonary lymph nodes of human non-small cell lung carcinoma were Feulgen stained. The biopsy specimens were obtained before and the surgical specimens after induction chemotherapy (down staging). The integrated optical density (IOD) of tumor cell nuclei was measured using an automated image analysis system, and the attributed minimum spanning trees (MSTs) were computed. The measured features included IOD (DNA content of the nuclei), IOD entropy, S-phase-related fraction (SRF), percentage of tumor cells with an IOD > 5C (P5C), standard deviation of IOD and structural parameters (minimum distance between neighboring tumor cell nuclei, minimum distance between tumor cell nuclei and neighboring lymphocytes, MST entropy, minimum distance between neighboring proliferating tumor cell nuclei, etc.). The measured IOD and MST features showed significant differences in the biopsy specimens as compared to those measured in the surgically excised carcinomas and those growing in the intrapulmonary lymph nodes (lymph node metastases). After down staging, the SRF was significantly diminished, and the percentage of cells with an IOD > 5C increased (P < .05). The survival of patients was remarkably improved if both the carcinomas before and after induction chemotherapy displayed a low SRF, a low number of stemlines and low MST entropy.
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