PCR and DNA sequencing in establishing the aetiology of bacterial infections in children

The results of partial polymerase chain reaction (PCR) amplification of the bacterial 16S rRNA gene and subsequent DNA sequencing of clinical samples from children are described. In 13 out of 62 samples, DNA from bacteria likely to be the cause of infection was identified. In the vast majority (11/13) of samples with significant pathogen culture the specimen had been negative. Antibiotics had been given in all cases except for three prior to sampling. PCR and subsequent DNA sequencing is a valuable supplementary tool for establishing the cause of bacterial infections in children when culture is negative.     

Single-Molecule Long-Read 16S Sequencing To Characterize the Lung Microbiome from Mechanically Ventilated Patients with Suspected Pneumonia

In critically ill patients, the development of pneumonia results in significant morbidity and mortality and additional health care costs. The accurate and rapid identification of the microbial pathogens in patients with pulmonary infections might lead to targeted antimicrobial therapy with potentially fewer adverse effects and lower costs. Major advances in next-generation sequencing (NGS) allow culture-independent identification of pathogens. The present study used NGS of essentially full-length PCR-amplified 16S ribosomal DNA from the bronchial aspirates of intubated patients with suspected pneumonia. The results from 61 patients demonstrated that sufficient DNA was obtained from 72% of samples, 44% of which (27 samples) yielded PCR amplimers suitable for NGS. Out of the 27 sequenced samples, only 20 had bacterial culture growth, while the microbiological and NGS identification of bacteria coincided in 17 (85%) of these samples. Despite the lack of bacterial growth in 7 samples that yielded amplimers and were sequenced, the NGS identified a number of bacterial species in these samples. Overall, a significant diversity of bacterial species was identified from the same genus as the predominant cultured pathogens. The numbers of NGS-identifiable bacterial genera were consistently higher than identified by standard microbiological methods. As technical advances reduce the processing and sequencing times, NGS-based methods will ultimately be able to provide clinicians with rapid, precise, culture-independent identification of bacterial, fungal, and viral pathogens and their antimicrobial sensitivity profiles.     

Direct 16S rRNA Gene Sequencing from Clinical Specimens,with Special Focus on Polybacterial Samples and Interpretation of Mixed DNA Chromatograms

RipSeq (iSentio, Bergen, Norway) is a web-based application for the analysis of mixed DNA chromatograms. It opens the possibility to analyze chromatograms obtained by direct 16S rRNA gene sequencing from polybacterial human clinical samples. In this study, we used direct 16S rRNA gene sequencing to investigate 264 samples from a wide range of suspected human bacterial infections. The sequence-based identification was compared with the results from routine culture-based identification. A total of 151 samples were positive by the first PCR, producing 85 pure and 66 mixed DNA chromatograms. All mixed chromatograms were analyzed by RipSeq, although seven were so complex that only the dominant bacterial sequences could be identified. In general, sequence-based identification detected a larger number of species than did culture for samples from patients who had received antibiotics prior to sample collection and for samples containing anaerobic bacteria. RipSeq made it possible to apply this supplementary diagnostic tool to typical polybacterial specimens, such as internal abscesses, pleural fluids, and bile.Detection and identification of bacteria directly from clinical samples by broad-range PCR targeting the 16S rRNA gene and DNA sequencing (direct 16S rRNA gene sequencing) make up a well-established method in many laboratories. This method gives the possibility to identify bacteria that died during transportation or as a consequence of antibiotic treatment and to uncover bacteria with special growth requirements. The latest advances in PCR and sequencing technology also offer a more rapid identification than that obtained with standard phenotypic methods that depend on bacterial growth.Because of difficulties in the interpretation of DNA chromatograms resulting from direct sequencing of polybacterial samples, the use of this diagnostic tool has been limited to infections that are predominantly monobacterial. We earlier described RipSeq (iSentio, Bergen, Norway), a web-based application for the analysis of mixed DNA chromatograms (12). In the same article, we presented the RipSeq performance on a number of mixed DNA chromatograms obtained by direct sequencing from saline suspensions containing two and three different bacterial species and discussed the possible benefits and limitations one could experience if the method was to be applied to human clinical samples. In this study, direct 16S rRNA gene sequencing was used to investigate 264 human clinical samples from a wide range of locations, including typical polybacterial specimens such as abscesses and pleural fluids. All mixed DNA chromatograms were analyzed with the RipSeq program, and the sequence-based results were compared to routine culture-based diagnostics in our hospital laboratory.We also discuss special concerns in the selection of a lysis procedure and primers for use on polymicrobial samples as well as the establishment of a reliable negative control.     

Molecular methods to improve diagnosis and identification of mucormycosis

Mucormycosis is difficult to diagnose. Samples from suspected cases often fail to grow Mucorales in microbiologic cultures. We identified all hematologic malignancy and stem cell transplant patients diagnosed with proven mucormycosis between 2001 and 2009 at Brigham and Women's Hospital/Dana-Farber Cancer Institute. Seminested PCR targeting Mucorales 18S ribosomal DNA and sequencing were performed on formalin-fixed paraffin-embedded tissue samples. Of 29 cases of mucormycosis, 27 had tissue samples available for PCR and sequencing. Mucorales PCR was positive in 22. Among 12 culture-positive cases, 10 were PCR positive and sequencing was concordant with culture results to the genus level in 9. Among 15 culture-negative cases, PCR was positive and sequencing allowed genus identification in 12. Mucorales PCR is useful for confirmation of the diagnosis of mucormycosis and for further characterization of the infection in cases where cultures are negative.     

Enhanced fungal DNA-extraction from formalin-fixed, paraffin-embedded tissue specimens by application of thermal energy

Determining the etiology of invasive fungal infections (IFI) is critical for patient management as fungi vary in their susceptibility to antifungals. However, the etiology remains obscure in many cases due to negative culture results. The identification of fungal DNA by PCR in pathology blocks and sequencing it is an alternative approach to determine the cause of IFI. Previous studies identified fungal DNA in only 50% of samples with positive histopathology results, probably due to DNA damage by tissue fixation. We used realtime PCR to quantify human and fungal DNA from formalin-fixed, paraffin-embedded tissue specimens in order to study the effect of thermal energy during extraction on the yield of amplifiable DNA and subsequent identification of fungal DNA. Tissue sections from eight patients with proven IFI were subjected to DNA extraction with varying exposure to thermal energy. Amplifiable DNA increased up to 76-fold by increasing the incubation temperature from 65°C to 90°C and an additional increase was documented by incubating samples for up to 6 hours at this temperature. The augmented amplification of fungal DNA was associated with improved species identification by the sequencing of the PCR amplicons. This may help illuminate the etiology of IFI and thereby improve patient management by guiding antifungal therapy.     

Rapid diagnosis of bacterial meningitis by nanopore 16S amplicon sequencing: A pilot study

Early administration of antibiotics is crucial in the management of bacterial meningitis. Rapid pathogen identification helps to make a definite diagnosis of bacterial meningitis and enables tailored antibiotic treatment. We investigated if the 16S amplicon sequencing performed by MinION, a nanopore sequencer, was capable of rapid pathogen identification in bacterial meningitis. Six retrospective cases of confirmed bacterial meningitis and two prospective cases were included. The initial cerebrospinal fluid (CSF) samples of these patients were used for the experiments. DNA was extracted from the CSF, and PCR was performed on the 16S ribosomal DNA (16S rDNA). Sequencing libraries were prepared using the PCR products, and MinION sequencing was performed for up to 3 h. The reads were aligned to the bacterial database, and the results were compared to the conventional culture studies. Pathogenic bacteria were successfully detected from the CSF by 16S sequencing in all retrospective cases. 16S amplicon sequencing was more sensitive than conventional diagnostic tests and worked properly even in antibiotics-treated samples. MinION sequencing significantly reduced the turnaround time, and even 10 min of sequencing was sufficient for pathogen detection in certain cases. Protocol adjustment could further increase the sensitivity and reduce the turnaround time for MinION sequencing. Finally, the prospective application of MinION 16S sequencing was successful. Nanopore 16S amplicon sequencing is capable of rapid bacterial identification from the CSF of the bacterial meningitis patients. It may have many advantages over conventional diagnostic tests and should therefore be applied in a larger number of patients in the future.     

Increased prevalence and altered species composition of filamentous fungi in respiratory specimens from cystic fibrosis patients

Filamentous fungi cultured from respiratory tract specimens submitted to the department of clinical microbiology, Aarhus University Hospital, during 2010 were identified by morphology and by internal transcribed spacer (ITS) sequencing. Of 343 fungal isolates, discrepancies between identification methods were observed for four isolates (1.2%), while identification to species was achieved only with ITS sequencing for 16 isolates (4.7%). Filamentous fungi were isolated from 15% of cystic fibrosis (CF) respiratory samples in contrast to 2% of non‐CF samples. From CF patients, a total of nine different species were found in 188 samples from 48 patients, whereas from non‐CF patients, 24 different species were found in 155 samples from 111 patients. CF was associated with a significant overrepresentation of Aspergillus fumigatus and Scedosporium species; in contrast, the frequency of Penicillium spp. and other putative contaminants were significantly increased in non‐CF patients. The altered species variation of filamentous fungi in CF respiratory specimens is contradictory to a scenario of incidentally inhaled spores, trapped in the viscous airway mucus of these patients and subsequently expectorated; rather, our data most likely reflect both an increased prevalence and an increased proportion of truly colonizing fungi in this patient group.     

Evaluation of a PCR method to determine the clinical significance of blood cultures with Staphylococcus epidermidis in patients with hematological malignancies

The aim was to investigate whether the detection and quantification of Staphylococcus epidermidis DNA in blood could distinguish S. epidermidis blood stream infections (BSIs) from blood culture contaminations in patients with hematological malignancies. The hld gene was chosen to identify S. epidermidis DNA and DNA in blood samples was detected by real‐time PCR. Blood samples were obtained simultaneously with blood cultures positive for S. epidermidis (n = 30), during blood culture‐negative episodes (n = 10) and episodes of bacteremia with other bacteria than S. epidermidis (n = 4) and from healthy blood donors (n = 10). In addition, DNA from S. epidermidis and a selection of other bacterial species were analyzed. Three different sets of criteria were used to classify episodes with positive blood cultures with S. epidermidis as BSIs or contaminations. All DNA preparations from S. epidermidis (n = 48) were hld‐positive, but other bacterial species (n = 13) were negative. Sixteen (53%) of 30 blood samples from patients with blood cultures positive for S. epidermidis were hld‐positive, but none of the controls. There was no clear association between a positive hld PCR and episodes interpreted as BSIs. In conclusion, hld PCR failed to distinguish S. epidermidis BSIs from blood culture contaminations in patients with hematological malignancies.     

Bacterial diversity in cases of lung infection in cystic fibrosis patients: 16S ribosomal DNA (rDNA) length heterogeneity PCR and 16S rDNA terminal restriction fragment length polymorphism profiling

The leading cause of morbidity and mortality in cystic fibrosis (CF) patients stems from repeated bacterial respiratory infections. Many bacterial species have been cultured from CF specimens and so are associated with lung disease. Despite this, much remains to be determined. In the present study, we characterized without prior cultivation the total bacterial community present in specimens taken from adult CF patients, extracting DNA directly from 14 bronchoscopy or sputum samples. Bacterial 16S ribosomal DNA (rRNA) gene PCR products were amplified from extracted nucleic acids, with analyses by terminal restriction fragment length polymorphism (T-RFLP), length heterogeneity PCR (LH-PCR), and sequencing of individual cloned PCR products to characterize these communities. Using the same loading of PCR products, 12 distinct T-RFLP profiles were identified that had between 3 and 32 T-RFLP bands. Nine distinct LH-PCR profiles were identified containing between one and four bands. T-RFLP bands were detected in certain samples at positions that corresponded to pathogens cultured from CF samples, e.g., Burkholderia cepacia and Haemophilus influenzae. In every sample studied, one T-RFLP band was identified that corresponded to that produced by Pseudomonas aeruginosa. A total of 103 16S rRNA gene clones were examined from five patients. P. aeruginosa was the most commonly identified species (59% of clones). Stenotrophomonas species were also common, with eight other (typically anaerobic) bacterial species identified within the remaining 17 clones. In conclusion, T-RFLP analysis coupled with 16S rRNA gene sequencing is a powerful means of analyzing the composition and diversity of the bacterial community in specimens sampled from CF patients.     

Clinical evaluation of rapid identification of bacteria from positive-testing blood culture bottles by internal transcribed spacer PCR

Delays in diagnosis and initiation of treatment of severe infections such as sepsis greatly influence patient prognosis. Our laboratory introduced rapid identification of bacterial species by PCR for positive blood culture samples as a routine laboratory test since April 2008. We extracted DNA directly from positive blood culture bottles and amplified the internal transcribed spacer (ITS) region of pathogenic microorganisms by PCR in order to identify bacterial species from electrophoretic patterns of PCR products. Of 167 strains from 167 samples excluding three samples with polymicrobial organisms, 144 strains (86.2%) were correctly identified at species level and 17 strains (10.2%) at genus level. The time required between DNA extraction and bacterial identification was about one and one-half hours. In patients with MRSA sepsis, the time of initiation of treatments such as administration of anti-MRSA drugs and intravascular catheter removal has clearly become earlier with the introduction of ITS-PCR, resulting decreased mortality from 35.0% to 16.0%. Rapid identification of pathogens directly from blood culture bottles by ITS-PCR seems to be useful for appropriate treatment of severe infectious diseases.     

Metagenomic analysis of viral diversity in respiratory samples from patients with respiratory tract infections in Kuwait

A metagenomic approach based on target independent next‐generation sequencing has become a known method for the detection of both known and novel viruses in clinical samples. This study aimed to use the metagenomic sequencing approach to characterize the viral diversity in respiratory samples from patients with respiratory tract infections. We have investigated 86 respiratory samples received from various hospitals in Kuwait between 2015 and 2016 for the diagnosis of respiratory tract infections. A metagenomic approach using the next‐generation sequencer to characterize viruses was used. According to the metagenomic analysis, an average of 145, 019 reads were identified, and 2% of these reads were of viral origin. Also, metagenomic analysis of the viral sequences revealed many known respiratory viruses, which were detected in 30.2% of the clinical samples. Also, sequences of non‐respiratory viruses were detected in 14% of the clinical samples, while sequences of non‐human viruses were detected in 55.8% of the clinical samples. The average genome coverage of the viruses was 12% with the highest genome coverage of 99.2% for respiratory syncytial virus, and the lowest was 1% for torque teno midi virus 2. Our results showed 47.7% agreement between multiplex Real‐Time PCR and metagenomics sequencing in the detection of respiratory viruses in the clinical samples. Though there are some difficulties in using this method to clinical samples such as specimen quality, these observations are indicative of the promising utility of the metagenomic sequencing approach for the identification of respiratory viruses in patients with respiratory tract infections.

     

Detection and strain identification of Actinobacillus actinomycetemcomitans by nested PCR.

By using PCR, Actinobacillus actinomycetemcomitans strains were identified directly from plaque samples without the need to isolate or culture bacteria. DNA fragments were generated by a nested, two-step PCR amplification of the ribosomal spacer region between the 16S and 23S rRNA genes. For the first amplification, primers homologous to sequences common to all bacterial species were used. This was followed by a second amplification with primers specific to A. actinomycetemcomitans. The ribosomal DNA spacer region was amplified from as few as 10 bacterial cells within a total population of 10(8) cells (0.00001%), and cross-reactivity between species was not observed. DNA fragments specific for Porphyromonas gingivalis were generated from the same samples by using a P. gingivalis-specific primer, and equivalent sensitivity and specificity were observed. A. actinomycetemcomitans was detected in 60% and P. gingivalis was detected in 79% of 52 subjects tested. Sequence analysis of the spacer region DNA fragment for A. actinomycetemcomitans gave precise strain identification, producing unique sequences for seven reference strains and identification of nine plaque-derived isolates. A phylogenetic tree based on quantitative sequence relationships was constructed. Two-step PCR amplification directly from plaque samples combined with sequence analysis of the ribosomal DNA spacer region provides a sensitive assay for detection and strain identification of multiple species directly from a single plaque sample. This simplified approach provides a practical method for large-scale studies on the transmission and pathogenicity of periodontitis-associated bacteria.     

Use of tuf sequences for genus-specific PCR detection and phylogenetic analysis of 28 streptococcal species

A 761-bp portion of the tuf gene (encoding the elongation factor Tu) from 28 clinically relevant streptococcal species was obtained by sequencing amplicons generated using broad-range PCR primers. These tuf sequences were used to select Streptococcus-specific PCR primers and to perform phylogenetic analysis. The specificity of the PCR assay was verified using 102 different bacterial species, including the 28 streptococcal species. Genomic DNA purified from all streptococcal species was efficiently detected, whereas there was no amplification with DNA from 72 of the 74 nonstreptococcal bacterial species tested. There was cross-amplification with DNAs from Enterococcus durans and Lactococcus lactis. However, the 15 to 31% nucleotide sequence divergence in the 761-bp tuf portion of these two species compared to any streptococcal tuf sequence provides ample sequence divergence to allow the development of internal probes specific to streptococci. The Streptococcus-specific assay was highly sensitive for all 28 streptococcal species tested (i.e., detection limit of 1 to 10 genome copies per PCR). The tuf sequence data was also used to perform extensive phylogenetic analysis, which was generally in agreement with phylogeny determined on the basis of 16S rRNA gene data. However, the tuf gene provided a better discrimination at the streptococcal species level that should be particularly useful for the identification of very closely related species. In conclusion, tuf appears more suitable than the 16S ribosomal RNA gene for the development of diagnostic assays for the detection and identification of streptococcal species because of its higher level of species-specific genetic divergence.     

Prospective value of PCR amplification and sequencing for diagnosis and typing of old world Leishmania infections in an area of nonendemicity

We assessed the prospective value of PCR amplification of a repetitive sequence from Leishmania nuclear DNA and sequencing for the diagnosis and typing of Old World Leishmania infection in an area of nonendemicity. During this 42-month study, 29 of 168 consecutive samples were examined and classified as positive for Leishmania by direct examination and/or in vitro culture. This molecular approach showed excellent sensitivity (97%) and specificity (100%) compared to direct examination (86 and 100%, respectively) and in vitro culture (72 and 100%, respectively). Isoenzymatic and molecular typing allowed similar identification for 12 samples. Besides, PCR and subsequent sequencing of DNA products permitted the species identification of 14 samples for which parasite culture remained negative or did not allow isoenzymatic characterization, indicating the complementarity of parasitological and molecular tools.     

Identification of unknown ocular pathogens in clinically suspected eye infections using ribosomal RNA gene sequence analysis

DNA sequence‐based identification of pathogens from ocular samples of patients with clinically suspected eye infections was accomplished using 16S and internal transcribed spacer (ITS) ribosomal RNA gene sequence analysis. PCR was positive for 24 of 99 samples tested. Both culture and 16S rDNA sequence analysis identified Pseudomonas aeruginosa, streptococci and Enterobacteriaceae. Isolates misidentified as Burkholderia cepacia by biochemical tests were identified as Ralstonia mannitolilytica by 16S rDNA sequence analysis. Sequence analysis identified the following microorganisms from 19 culture‐negative samples: Haemophilus influenzae, Sphingomonas sp., Klebsiella pneumoniae, Staphylococcus haemolyticus, Morganella morganii, Mycobacterium sp., Chryseobacterium sp., Pseudomonas saccharophila (Xanthomonas) and the fungus, Phaeoacremonium inflatipes.     

Cell‐free fetal DNA and fetal blood group genotyping: non‐invasive prenatal testing

In transfusion medicine and clinical immunology, cell‐free fetal DNA (cffDNA) is analysed from maternal plasma of pregnant women to predict fetal blood groups with the purpose of (1) assessing the risk of haemolytic disease of the fetus and newborn (HDFN) in immunized women and (2) guiding targeted Rh prophylaxis in non‐immunized RhD‐negative women. National programmes for guiding prophylaxis are now implemented in around 6–7 European countries; assay accuracy is very high, with sensitivities of 99·9%. Sensitivity is challenged by low quantities of cffDNA, especially in early pregnancy. Specificity is challenged by the polymorphic Rh blood group system, where careful attention is needed to navigate among the many RHD variants that may complicate cffDNA analysis and interpretation of results, especially in populations with mixed ethnicities. However, fetal RHD testing is feasible when implemented with careful attention to these issues. The success of predicting fetal RhD and its successful clinical implementation should encourage widespread implementation. For blood groups that are determined by SNPs, such as KEL or Rhc, novel techniques such as next‐generation sequencing and droplet digital PCR are now providing accurate non‐invasive prediction of these fetal blood groups. Future work on non‐invasive prenatal testing of fetal blood groups determined by SNPs may consolidate the application for cell‐free DNA testing for such targets. At ISBT, the newly formed cfDNA subgroup of the Red Cell Immunogenetics and Blood Group Terminology Working Party will work to facilitate clinical applications, implementation and evaluation of cell‐free DNA testing.     

A six-month audit of the isolation of Fusobacterium necrophorum from patients with sore throat in a district general hospital

Fusobacterium necrophorum is an obligate anaerobe believed to be a member of the normal flora of the human oropharangeal and urogenital tract. It has been associated with deep-seated infections and was first described in 1936 by Lemierre, a French microbiologist. There is now strong evidence to suggest that it is also a cause of recurrent sore throat and persistent sore throat syndrome (PSTS) without leading to full systemic infection. It is considered to be the second most common cause of sore throat after group A beta-haemolytic streptococci. This study was performed over a six-month period (October 2004 to March 2005) at the Eastbourne District General Hospital. All throat swabs received in the laboratory are cultured routinely for haemolytic group A streptococci and pathogenic Corynebacteria spp. During the study period an extra fastidious anaerobic blood agar plate with neomycin was inoculated, with a 30 microg vancomycin disc placed at the junction of the second and third streaks. This was examined after 48 h for the presence of F. necrophorum. A total of 1157 swabs were processed during the study period: 156 were positive for haemolytic group A streptococci, 57 were positive for F. necrophorum, 47 for group C haemolytic streptococci, nine for group G haemolytic streptococci, and one was positive for C. ulcerans. Patient age ranged from less than a year old to 88. The majority of F. necrophorum isolates were from patients in the 11-25 age group, with an isolation rate of 9.48% (44/464). This age group accounted for 40% (464/1157) of the swabs received during the study period and 77% (44/57) of these were positive for F. necrophorum. Group A haemolytic streptococci showed an overall isolation rate of 13.5%, with peaks of 23% in the 0-10 and 26-35 age ranges. Together, these two organisms were responsible for 18.4% (213/1157) of all throat infections in this study. The results presented here indicate that F. necrophorum is second to group A haemolytic streptococci as a cause of sore throat, especially in the young adult, and introduction of routine culture should be considered.     

Presumptive identification and antibiotic susceptibility of group B streptococci.

The comparative performance of three presumptive identification tests for group B haemolytic streptococci was investigated, using 371 different clinical isolates of group B streptococci. Hippurate was hydrolysed by 96-1%, the CAMP reaction was positive in 95-0%, and pigment was produced by 97-3%. A combination of any two tests would have detected over 99-8%. On bile esculin agar 99-0% were able to grow, but non hydrolysed esculin; 5-1% were susceptible to bacitracin. The minimum inhibitory concentrations of five antibiotics for 279 group B streptococci were determined. All were susceptible to penicillin G, ampicillin, cephalothin, and erythromycin, while 80% were resistant to tetracycline. The MIC distributions were independent of the results of any identification test.     

Prevalence of hepatitis B and hepatitis D coinfection in asymptomatic blood donors in Iran

Hepatitis delta virus (HDV) is a satellite virus that needs hepatitis B virus (HBV) surface antigen for amplification and transition. HDV appears in HBsAg carriers as acute coinfection and superinfection in patients with chronic hepatitis B. This coinfection leads to chronic hepatitis, cirrhosis, and liver carcinoma. The aim of this study was to detect the prevalence of coinfection and superinfection of HBVs and HDVs in blood donor individuals in Iran. Sera from 854 asymptomatic blood donors from the Bank of positive samples storage at the National Blood Transfusion Organization of Iran that were positive for hepatitis B surface antigen were analysed. The presence of antibody against HDV in blood donors was detected using ELISA followed by conventional PCR, seminested PCR and real‐time PCR to determine coinfection and/or superinfection. Restriction fragment length polymorphism was used for HDV genotyping. All 854 samples were HBsAg and anti‐HBc positive whereas only 18 (2%) of them were positive for anti‐HDV. Of the 854 samples, 154 (18%) were HBV‐DNA positive. HDV‐RNA was detected in 0.6% of the total samples by seminested PCR and real‐time PCR and the two PCR methods produced similar results. Moreover, 16.6% and 83.4% of anti‐HDV‐positive samples exhibited coinfection and superinfection with HBV, respectively. Genotype I of HDV was determined in positive samples.     

rpoB gene mutations among Mycobacterium tuberculosis isolates from extrapulmonary sites

The aim of this study was to analyze mutations occurring in the rpoB gene of Mycobacterium tuberculosis (MTB) isolates from clinical samples of extrapulmonary tuberculosis (EPTB). Seventy formalin‐fixed, paraffin‐embedded samples and fresh tissue samples from confirmed EPTB cases were analyzed. Nested PCR based on the rpoB gene was performed on the extracted DNAs, combined with cloning and subsequent sequencing. Sixty‐seven (95.7%) samples were positive for nester PCR. Sequence analysis of the 81 bp region of the rpoB gene demonstrated mutations in 41 (61.2%) of 67 sequenced samples. Several point mutations including deletion mutations at codons 510, 512, 513 and 515, with 45% and 51% of the mutations in codons 512 and 513 respectively were seen, along with 26% replacement mutations at codons 509, 513, 514, 518, 520, 524 and 531. The most common alteration was Gln → His, at codon 513, presented in 30 (75.6%) isolates. This study demonstrated sequence alterations in codon 513 of the 81 bp region of the rpoB gene as the most common mutation occurred in 75.6% of molecularly confirmed rifampin‐resistant strains. In addition, simultaneous mutation at codons 512 and 513 was demonstrated in 34.3% of the isolates.