Fixed versus live endothelial cells: The effect of glutaraldehyde fixation manifested by characteristic bands on the Raman spectra of cells.

This work shows an impact of glutaraldehyde (GA) fixation on endothelial cells. Raman spectroscopy imaging was used as a method to monitor biochemical content of the cells due to GA fixation since this is an approach frequently used for studying cells by means of Raman imaging. To get a deeper insight into the changes and to understand them better the measurements of live and fixed cells were performed using two lasers, i.e. 488 and 532 nm. It has been demonstrated that GA fixation affects lipids, proteins, nucleic acid and carbohydrates to small extent. The application of 488 nm laser line seems to be more efficient for live cells due to the small impact of cytochrome resonance on Raman spectra, however 532 nm line is more beneficial for fixed cells due to higher quantum efficiency of the detector, thus leading to higher intensity of Raman bands. Generally, the changes due to fixation are not pronounced but cannot be ignored and the knowledge about them can help in a proper interpretation of data collected for fixed versus live cells.

Impact of Donor Risk Index on Risk of Hepatic Artery Thrombosis in Patients After Orthotopic Liver Transplantation.

BACKGROUND: Hepatic artery thrombosis (HAT) is one of the most severe complications after liver transplantation (LT). HAT can lead to early graft loss and retransplantation or death of the recipient. METHODS: This retrospective cohort study was conducted using data from patients treated between January 2008 and December 2013 in the Department of General, Transplant and Liver Surgery at the Medical University of Warsaw. A total of 750 patients underwent LT over this period. RESULTS: HAT occurred in 27 patients (2.1%). The median DRI was 1.414 (IQR 1.103-1.578) points and median donor age was 47 (IQR 33-56) years. The optimal cut-off value of DRI in predicting HAT was ≥1.328 points. The cutoff point was characterized by sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 88.0%, 41.3%, 5.5% and 98.9%, respectively (AUC = 0.605, 95% CI 0.477-0.733). A DRI ≥1.328 was a significant risk factor for HAT (OR = 5.16, 95% confidence interval [CI] 1.529-17.48, P = .008). The optimal cutoff point for donor age was 50 years and was characterized by sensitivity, specificity, PPV, and NPV of 66.7%, 55.8%, 5.3%, and 97.8%, respectively. Donor age ≥50 years (OR = 2.53, 95% CI 1.123-5.714, P = .025) was a significant risk factor for HAT. CONCLUSION: DRI is a clinically relevant factor that allows estimating the risk of HAT after liver transplantation from a deceased donor. To reduce the incidence of this complication, the allocation of organs taken from donors at DRI exceeding 1.328 for recipients without other HAT risk factors should be considered.

Outcomes of Patients With Poorly Differentiated Hepatocellular Carcinoma After Liver Transplantation.

BACKGROUND: Liver transplantation (LT) outcomes for patients with poorly differentiated (G3) hepatocellular carcinoma (HCC) are unsatisfactory. The aim of this study was to evaluate outcomes in patients with poorly differentiated HCC undergoing LT. PATIENTS AND METHODS: There were 192 HCC patients after LT in the Department of General, Transplant and Liver Surgery, Medical University of Warsaw, between January 2001 and April 2014. The study group comprised 24 patients with poorly differentiated tumors. RESULTS: Disease-free survival (DFS) for all patients was 49.5% at 5 years. The 5-year DFS for patients who met the Milan criteria (n = 9, 88.9%) was significantly better compared to those who did not (n = 15, 28.0%, P = .025). Multivariable analysis revealed that only the largest tumor diameter (P = .014) and α-fetoprotein (AFP) concentration (P = .001) were independent risk factors for DFS. The optimal cut-off AFP and tumor size that could distinguish patients with the highest risk were ≥500 ng/mL and ≥3.5 cm, respectively. DFS for patients with AFP <500 ng/mL and tumor size <3.5 cm was 100% after 2.8 years, and for those with ≥500 ng/mL or tumor size ≥3.5 cm was 46.9% after 5 years. However, the DFS for patients with AFP ≥500 ng/mL and tumor size ≥3.5 cm was only 12.5% after 4.7 years (P = .002). CONCLUSIONS: Outcomes of patients with poorly differentiated HCC treated with LT can be characterized with acceptable survival when applying criteria based on tumor size <3.5 cm and AFP <500 ng/mL.

Prognostic Factors and Outcomes of Patients After Liver Retransplantation.

BACKGROUND: Despite great progress and improvement in results of orthotopic liver transplantation (OLTx), 10%-20% of patients still require retransplantation (re-OLTx). The aim of the study was to present long-term results of liver retransplantation and to determine the factors influencing outcomes. PATIENTS AND METHODS: From December 1994 to July 2014, a total of 1461 liver transplantations were performed in the Department of General, Transplant and Liver Surgery of Medical University of Warsaw. There were 92 retransplantations (6.3%), including 40 early re-OLTx (up to 30 days). The most common indication for re-OLTx were vascular complications (41/92, 44.6%). Influence of clinical variables on short- and long-term outcomes was analyzed. RESULTS: Postoperative mortality was 30.4% (28/92). One-year, 3-year and 5-year survival for all patients was 59.8%, 56.5% and 54.1%, respectively. The best results were achieved in patients undergoing retransplantation due to chronic rejection and biliary complications, whose 5-year survival rates were 75.0% and 72.9% respectively. There was no difference in long-term survival after early and late retransplantations (60.9% and 49.3%, respectively; P = .158). Multivariable analysis revealed factors associated with longer survival of patients, namely, higher preoperative hemoglobin concentration (P = .001), increased blood transfusions (P = .048), and decreased fresh frozen plasma transfusions (P = .004). CONCLUSIONS: Liver retransplantation is a method providing satisfactory outcomes in selected patients. The perioperative period has a major impact on patient outcome.

Identification of Lactobacillus strains with probiotic features from the bottlenose dolphin (Tursiops truncatus).

AIMS: In order to develop complementary health management strategies for marine mammals, we used culture-based and culture-independent approaches to identify gastrointestinal lactobacilli of the common bottlenose dolphin, Tursiops truncatus. METHODS AND RESULTS: We screened 307 bacterial isolates from oral and rectal swabs, milk and gastric fluid, collected from 38 dolphins in the U.S. Navy Marine Mammal Program, for potentially beneficial features. We focused our search on lactobacilli and evaluated their ability to modulate TNF secretion by host cells and inhibit growth of pathogens. We recovered Lactobacillus salivarius strains which secreted factors that stimulated TNF production by human monocytoid cells. These Lact. salivarius isolates inhibited growth of selected marine mammal and human bacterial pathogens. In addition, we identified a novel Lactobacillus species by culture and direct sequencing with 96·3% 16S rDNA sequence similarity to Lactobacillus ceti. CONCLUSIONS: Dolphin-derived Lact. salivarius isolates possess features making them candidate probiotics for clinical studies in marine mammals. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to isolate lactobacilli from dolphins, including a novel Lactobacillus species and a new strain of Lact. salivarius, with potential for veterinary probiotic applications. The isolation and identification of novel Lactobacillus spp. and other indigenous microbes from bottlenose dolphins will enable the study of the biology of symbiotic members of the dolphin microbiota and facilitate the understanding of the microbiomes of these unique animals.

[The microbiotics of the human body]. / De microbiota van het menselijk lichaam.

--Our bodies are home to complex microbial communities. --In most samples, a greater microbial diversity is revealed by using culture-independent, molecular techniques than by conventional methods. --The composition of the human-associated microbiota differs in each individual, and at each anatomical site within an individual. --The intestinal colonization of newborn infants seems to be driven by environmental factors and random processes, rather than by the composition of the parent's microbial communities. --A set of fraternal twins showed almost identical microbial communities. --Investigating the composition of the human-associated microbiota will enable us to better understand the role of commensals in health and disease.

Somatic APC mosaicism: an underestimated cause of polyposis coli.

BACKGROUND: The patient with 10 or more adenomas in the colon poses a diagnostic challenge. Beside germline mutations in the APC and MUTYH genes, only four cases of mosaic APC mutations have been reported. AIM: Given the relatively high frequency of de novo APC mutations in familial adenomatous polyposis (FAP), an investigation was carried out into whether the proportion of somatic mosaic APC mutations is currently underestimated. METHODS: Between 1 January 1994 and 31 December 2005 germline mutation analysis was performed in 599 consecutive index patients with polyposis coli referred for diagnostic APC scanning using a combination of denaturing gradient gel electrophoresis (DGGE) and protein truncation test (PTT). Variants were analysed by direct sequencing with primers flanking those used for DGGE and PTT, and quantified using pyrosequencing. RESULTS: Scrutinizing the molecular genetic results and family data of 242 index patients with pathogenic APC mutations led to the identification of 10 mosaic cases (4%). C>T transitions were observed in CGA sites in four of the 10 cases with somatic mosaicism, which is significantly more than 26 of the 232 non-mosaic cases (p = 0.02). Phenotypes of patients with somatic mosaicism ranged from an attenuated form of polyposis coli to florid polyposis with major extracolonic manifestations. CONCLUSIONS: Mosaicism occurs in a significant number of APC mutations and it is estimated that one-fifth of the de novo cases of FAP are mosaic. Clinically, the severity of manifestations in offspring and the recurrence risk for siblings of apparently sporadic polyposis patients may be underestimated due to parental APC mosaicism.

[Increased sensitivity for detection of mutations in exon 15 of the APC gene in patients with familial adenomatous polyposis]. / Zvýsení citlivosti pro detekci mutací v 15. exonu APC genu u pacientu s familiární adenomatózní polypózou.

BACKGROUND: Protein Truncation Test (PTT) was used to detect mutations in exon 15 of the APC gene in patients with Familial Adenomatous Polyposis. This method is limited by its ability to detect polypetide chains up to a certain minimum length. The aim of this study was to increase the sensitivity of detection of mutations in this region by using the technique of Denaturing Gradient Gel Electrophoresis (DGGE). METHODS AND RESULTS: Study were performed on 122 patients without detected mutations in the APC gene. The patients were divided into two independent groups 15A and 15A+15B (with 51 and 71 patients respectively). All the patients were tested with the DGGE and the positive findings were confirmed with sequencing. No mutation was detected in the group 15A (0%). In group 15A+15B one (1.4%) polymorphism and four (5.63%) patients with nonsense mutations were detected. CONCLUSIONS: DGGE is an effective method for detecting mutations in the first part of exon 15 of APC gene. It allows detecting any change in DNA strand. DGGE complements PTT in scanning of the whole exon 15 of APC gene.

Management of an outbreak of Enterobacter cloacae in a neonatal unit using simple preventive measures.

Enterobacter cloacae is becoming an increasingly important nosocomial pathogen. Outbreaks of E. cloacae in intensive care units and burns units have been described frequently. In December 1999, a neonate with line sepsis was transferred from a university hospital to the neonatal unit of the Diakonessen Hospital. Blood culture yielded E. cloacae. An outbreak of E. cloacae was occurring in the university hospital at that time. In February 2000, a second neonate in our hospital developed line sepsis caused by E. cloacae. Direct measures taken included cohorting of infected children, disinfection of incubators, thermometers and wards, and screening patients. Of nine neonates, seven were colonized with E. cloacae. Despite these measures, the outbreak continued. Forty-one patients were screened; 15 were colonized. Environmental searches yielded E. cloacae in a sink and on two thermometers. Sixteen isolates were typed by arbitrarily primed PCR using four primers. All the patient isolates and the two isolates from thermometers were identical. The strain isolated from the sink was unrelated. Amplified fragment length polymorphism typing showed that the outbreak clone was identical to that in the university hospital. After the introduction of disposable thermometer covers, E. cloacae colonization slowly decreased.

Atypical HNPCC owing to MSH6 germline mutations: analysis of a large Dutch pedigree.

Hereditary non-polyposis colorectal cancer (HNPCC) is the most common genetic susceptibility syndrome for colorectal cancer. HNPCC is most frequently caused by germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1. Recently, mutations in another MMR gene, MSH6 (also known as GTBP), have also been shown to result in HNPCC. Preliminary data indicate that the phenotype related to MSH6 mutations may differ from the classical HNPCC caused by defects in MSH2 and MLH1. Here, we describe an extended Dutch HNPCC family not fulfilling the Amsterdam criteria II and resulting from a MSH6 mutation. Overall, the penetrance of colorectal cancer appears to be significantly decreased (p<0.001) among the MSH6 mutation carriers in this family when compared with MSH2 and MLH1 carriers (32% by the age of 80 v >80%). Endometrial cancer is a frequent manifestation among female carriers (six out of 13 malignant tumours). Transitional cell carcinoma of the urinary tract is also relatively common in both male and female carriers (10% of the carriers). Moreover, the mean age of onset of both colorectal cancer (MSH6 v MSH2/MLH1 = 55 years v 44/41 years) and endometrial carcinomas (MSH6 v MSH2/MLH1 = 55 years v 49/48 years) is delayed. As previously reported, we confirm that the pattern of microsatellite instability, in combination with immunohistochemical analysis, can predict the presence of a MSH6 germline defect. The detailed characterisation of the clinical phenotype of this kindred contributes to the establishment of genotype-phenotype correlations in HNPCC owing to mutations in specific mismatch repair genes.

The evolution of epidemic Vibrio cholerae strains.

The emergence of the novel Vibrio cholerae strain, O139 Bengal, which caused a large epidemic in Southeast Asia, underlines the adaptability of pathogenic microorganisms. Recent studies reveal that horizontal transfer of cell-wall polysaccharide genes played a central role in the emergence of this strain and that its genesis may not be as unique as initially believed.

[Severe watery diarrhea caused by Vibrio cholerae in lambs]. / Ernstige waterige diarree door Vibrio cholerae bij lammeren.

An outbreak of watery diarrhoea in lambs is described. Seventeen lambs died within 24 hours after the start of the diarrhoea. At necropsy Vibrio cholerae was isolated from the organs and intestines of three lambs. The strains did not react with O1 or O139 antisera, the strains responsible for cholera epidemics among humans. It is concluded that the diarrhoea in the lambs was caused by V. cholerae non-01/non-139. This microorganism had not been described before in lambs in the Netherlands.

Evidence for subtelomeric exchange of 3.3 kb tandemly repeated units between chromosomes 4q35 and 10q26: implications for genetic counselling and etiology of FSHD1.

Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy, clinically characterized by asymmetric weakness of muscles in the face, shoulder girdle and upper arm. Deletion of an integral number of 3.3 kb repeated units within a highly polymorphic EcoRI fragment at chromosome 4q35, generating a relatively short EcoRI fragment (< 35 kb), has been shown to cause FSHD1. Probe p13E-11 detects these short fragments in FSHD1 patients, and has therefore been used for diagnostic DNA analysis. However, the reliability of this analysis has been hampered by cross-hybridization of p13E-11 to chromosome 10q26-linked EcoRI fragments of comparable size, which also contain a variable number of 3.3 kb repeated units. Recently, a BinI restriction site was identified within each of the repeated units derived from chromosome 10q26, which enables differentiation of the two polymorphic p13E-11 loci in most cases without haplotype analysis. Remarkably, applying the differential analysis to screen DNA of 160 Dutch cases referred to us for FSHD1 diagnosis, we obtained evidence for subtelomeric exchange of 3.3 kb repeated units between chromosomes 4q35 and 10q26 in affected and unaffected individuals. Subsequently, analysis of 50 unrelated control samples indicated such exchange between chromosomes 4q35 and 10q26 in at least 20% of the population. These subtelomeric rearrangements have generated a novel interchromosomal polymorphism, which has implications for the specificity and sensitivity of the differential restriction analysis for diagnostic purposes. Moreover, the high frequency of the interchromosomal exchanges of 3.3 kb repeated units suggests that they probably do not contain (part of) the FSHD1 gene, and supports position effect variegation as the most likely mechanism for FSHD1.

DNA fingerprinting of Vibrio cholerae strains with a novel insertion sequence element: a tool to identify epidemic strains.

A novel Vibrio cholerae insertion sequence element, designated IS1004, was characterized and used for DNA fingerprinting of Vibrio spp. IS1004 comprises 628 bp and contains an open reading frame whose product shows a large degree of sequence identity with the IS200-encoded transposase. IS1004 was present in one to eight copies in most of the V. cholerae strains analyzed. The IS1004-generated fingerprints of epidemic V. cholerae strains with serotype O1 were closely related, although it was possible to distinguish between the two biotypes, classical and El Tor. Non-O1 serotype strains generally showed heterogeneous patterns unrelated to those of the epidemic O1 strains. Several strains were observed with identical or related fingerprint patterns but expressed different serotypes. Conversely, strains with different fingerprint patterns but identical serotypes were also found. These observations indicate that the gene clusters coding for distinct O antigens may be transferred horizontally between V. cholerae strains. Two examples of non-O1 strains with a fingerprint resembling that of epidemic O1 strains were found; they were the O139 Bengal strain and an O37 strain. The O139 Bengal strain is closely related to the El Tor biotype. The O37 strain was responsible for a large cholera outbreak in Sudan in 1968 and was classified as a noncholera vibrio. Our study, however, shows that the O37 Sudan strain is genetically closely related to classical O1 strains. Similar to O139 Bengal, O37 Sudan lacked most of the O1 antigen cluster but did contain flanking genes. Thus, O37 Sudan represents a second example of an epidemic V. cholerae strain carrying non-O1 antigens. This study underlines the importance of genotypic methods for the differentiation of V. cholerae strains and for recognition of strains with epidemic potential.

Genetic organization and functional analysis of the otn DNA essential for cell-wall polysaccharide synthesis in Vibrio cholerae O139.

In 1992 a new Vibrio cholerae strain, designated V. cholerae O139 Bengal, emerged which has been responsible for large outbreaks of cholera in India and Bangladesh. Previously, we have shown that this strain arose from a V. cholerae O1 strain by the acquisition of novel DNA. Sequence analysis revealed that the novel DNA is flanked by two genes, rfaD and rfbQRS, which are also found in O1 strains. The mosaic structure of rfaDVCO139 indicated that it was one of the regions involved in recombination between donor and acceptor DNA. However, sequence divergence between the O1 and O139 rfbQRS genes indicated that the second recombination site between donor and O1-acceptor DNA is probably located downstream of rfbDVCO139. The DNA region between rfaDVCO139 and rfbQRSVCO139, designated otn, contained seven open reading frames (ORFs). Two ORFs, otnA and otnB, showed homology with genes involved in cell-wall polysaccharide synthesis. Mutations in otnA and otnB indicated that they are required for capsule synthesis but not lipopolysaccharide synthesis. The otn DNA is also found in V. cholerae O69 and O141 strains, and the organization of this DNA was essentially identical to that in the O139 strain. However, sequence divergence of the otnAB genes indicated that the O139 otn DNA region was not derived from the O69 or O141 strains. No antigenic relationship was found between the different V. cholerae serotypes carrying otn DNA, so the genes determining the antigenic specificity of the O antigen or capsule must be located outside the otn DNA. The O139 otn DNA contained a JUMPstart sequence, which is associated with polysaccharide-synthetic genes in several bacterial species. Furthermore, a repeat motif was observed in extragenic regions. A number of observations suggest that these sequences may facilitate gene flow between V. cholerae strains and the assembly of clusters of functionally related genes.

Genesis of the novel epidemic Vibrio cholerae O139 strain: evidence for horizontal transfer of genes involved in polysaccharide synthesis.

Only Vibrio cholerae strains of serotype O1 are known to cause epidemics, while non-O1 strains are associated with sporadic cases of cholera. It was therefore unexpected that the recent cholera epidemic in Asia was caused by a non-O1 strain with the serotype O139. We provide evidence that O139 arose from a strain closely related to the causative agent of the present cholera pandemic, V. cholerae O1 El Tor, by acquisition of novel DNA which was inserted into, and replaced part of, the O antigen gene cluster of the recipient strain. Part of the novel DNA was sequenced and two open reading frames (otnA and otnB) were observed, the products of which showed homology to proteins involved in capsule and O antigen synthesis, respectively. This suggests that the otnAB DNA determines the distinct antigenic properties of the O139 cell surface. The otnAB DNA was not detected in O1 strains, but was present in two non-O1 V. cholerae strains with serotypes O69 and O141. In the O69 and O139 strains the otnAB genes were located proximate to the putative insertion sequence (IS) element rfbQRS, which is associated with O antigen synthesis genes in O1 strains, and may have played a role in the insertion of the otnAB DNA in the recipient chromosome. Our results suggest that the O139 strain arose by horizontal gene transfer between a non-O1 and an O1 strain. The acquired DNA has altered the antigenic properties of the recipient O1 strain, providing a selective advantage in a region where a large part of the population is immune to O1 strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction and analysis of a Vibrio cholerae delta-aminolevulinic acid auxotroph which confers protective immunity in a rabbit model.

Vibrio cholerae CVD101 is a very effective live vaccine. Although this strain does not produce active cholera toxin because of a mutation in the gene for the cholera toxin A subunit, it still shows residual pathogenicity. To attenuate CVD101 further, we set out to isolate derivatives of CVD101 which were limited in their ability to proliferate in vivo. Two delta-aminolevulinic acid auxotrophs of CVD101, designated V286 and V287, were isolated by transposon mutagenesis and penicillin enrichment. Southern blotting revealed that the mutants differed with respect to the location of the transposon insertion. Under aerobic conditions, in the absence of delta-aminolevulinic acid, both mutants showed diminished growth compared with CVD101. The growth of V286 was most severely affected. Microaerophilic growth of both mutants was less affected. Competition experiments with a rabbit model showed that strain V286 was found in numbers 10(3)- to 10(4)-fold lower than its parental strain. This observation indicates that strain V286 is impaired in its ability to colonize the rabbit intestine. It also supports an important role for aerobic growth in the colonization of the intestine by V. cholerae. Vaccination of rabbits with a single dose of strain V286 resulted in full protection against challenge with a virulent strain. Strain V286 was not shed from rabbits in a cultivatable form. Our results suggest that delta-aminolevulinic acid auxotrophy can attenuate V. cholerae by limiting its ability to colonize without affecting its capacity to induce protective immunity. Furthermore, this type of mutation may prevent the spread of V. cholerae vaccine strains in the environment.

Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains.

Most strains of the Mycobacterium tuberculosis complex carry multiple copies of an IS3-like element, and these strains are highly polymorphic with regard to the site of integration in the chromosome. In contrast, Mycobacterium bovis BCG contains a single copy of the insertion element, and in all strains this copy is integrated at the same site in the chromosome. In this study, we determined the sequence of the single-copy insertion element from M. bovis BCG, IS987, and its flanking regions. The analysis of IS987 revealed that this element was virtually identical to the sequence of IS986 from M. tuberculosis. IS987 is located in a region containing direct repeats (DRs). The cloned flanking regions contained 20 virtually identical DRs of 36 bp, each separated by 35 to 41 bp of spacer DNA. Analysis of chromosomal DNA by the polymerase chain reaction revealed the presence of a cluster of 49 DRs, and IS987 is inserted in the 30th DR. Furthermore, the DR sequences were found to occur only in species of the M. tuberculosis complex and not in nine other mycobacterial species tested. Analysis of 14 M. tuberculosis strains revealed the presence of one insertion sequence element in the DR-containing region of eight strains, two insertion sequence elements were located in the DR region of five strains, and one strain did not contain an insertion sequence element in this region. Additionally, the DR-containing regions of these 14 M. tuberculosis strains were polymorphic in length and composition. We conclude that the DR cluster is a specific, hot-spot region for integration of insertion elements in the chromosome of M. tuberculosis complex strains.

Specific detection of Mycobacterium tuberculosis complex strains by polymerase chain reaction.

During the screening of a Mycobacterium tuberculosis lambda gt-11 gene library with monoclonal antibodies, we detected a recombinant clone, lambda PH7311, which contained a mycobacterial DNA insert that hybridized specifically with DNA of M. tuberculosis complex strains. Part of this insert was sequenced and used for the development of an M. tuberculosis complex-specific polymerase chain reaction (PCR). Only strains belonging to species of the M. tuberculosis complex group contained an amplifiable fragment of 158 base pairs (bp). This fragment was absent in all strains tested belonging to 15 other mycobacterial species. After amplification by PCR and dot blot hybridization with a digoxigenin-labeled oligonucleotide, the limit of detection of purified genomic M. tuberculosis DNA amounted to a quantity corresponding to 20 bacterial cells. By this technique about 10(3) M. tuberculosis bacteria were detectable in sputum. Using PCR, we were also able to detect M. tuberculosis cells in clinical material such as pleural fluid, bronchial washings, and biopsies, and these results were comparable with those obtained by classical bacterial culture. Of 34 M. tuberculosis strains, 5 did not carry the amplifiable 158-bp fragment, which occurs usually as a single copy in the chromosome. Evidence is presented that the 158-bp fragment is located near a repeated sequence in the chromosome. We presume that strains which did not carry the 158-bp fragment have lost a chromosomal segment by a genetic rearrangement induced by the repetitive DNA element.