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Amino acid characterizations of 26 M. Of the 26 M. A total of 10 rpoB amino acid substitutions were identified in the 26 clinical isolates compared to the H37Rv wild-type strain. The common SL mutation was the most prevalent, but mutations at positions and , also known to confer resistance to rifampin, were observed Table 2.

Additionally, mutations were observed within the rpoB open reading frame but outside the bp rifampin resistance-determining region RRDR Table 2. The VI mutation observed outside the RRDR in one strain is a unique substitution that is likely not associated with rifampin resistance. Five amino acid substitutions were noted in at least one strain beyond residue of the RpoB protein. There were seven strains with an RpoB mutation 6 at position and 1 at position where a wild-type band was absent without a corresponding mutation band according to LPA.

In six of these seven isolates, Ion Torrent sequencing revealed an uncommon amino acid substitution i. Four amino acid substitutions were observed in the katG gene product, with ST, which is known to confer isoniazid resistance, present in all resistant strains Table 3. A substitution at position RL in the katG product has been previously shown to have no effect on antibiotic resistance and can be used to categorize M.

Seven nucleotide mutations were noted in at least one strain among bp comprising the full-length coding region for the pncA gene Table 4. Nine of 26 strains In one strain, a silent synonymous nucleotide mutation was characterized at a position corresponding to amino acid CT. A novel mutation, not previously reported elsewhere, including a termination codon was found in one isolate at residue QStop in the PncA protein Table 4.

Nine unique mutations were observed in the 2,bp full-length gyrA gene encoding subunit A of the DNA gyrase enzyme. Resistance to fluoroquinolones FQs was noted only in strains harboring mutations in the quinolone resistance-determining region QRDR defined by substitutions in gyrA at positions corresponding to amino acids 88, 90, and Mutation at position 95 S95T is known to have no effect on FQ resistance but can be used to categorize strains in genetic group 2 or 3.

Four nucleotide mutations were noted among the 1, bp comprising the full-length 16S rrs gene. Two other amino acid mutations CT and AC were observed but have been previously shown to not inhibit aminoglycoside efficacy. A previously uncharacterized GA nucleotide mutation was observed, but the isolate was shown to be susceptible according to DST Table 6. The protocol described in this report enables sequencing of entire coding regions implemented in M.

Rapid analysis of genes associated with drug-resistant strains is a major challenge for successful treatment of tuberculosis. In addition, real-time geographical surveillance of emerging M. Currently, available molecular methods such as the GenoType MTBDR plus LPA offer limited detection capabilities, particularly when novel or uncommon amino acid substitutions are within known drug resistance regions or when undiscovered amino acid mutations impact drug resistance.

We have established a simplified Ion Torrent sequencing protocol for rapid characterization of five full-length genes cumulatively sequencing The M. Of the 21 rifampin-resistant strains characterized in this study, 11 The most prevalent RpoB substitution observed at position is a valine DV Interestingly, Ion Torrent sequencing revealed that 6 of 7 strains contained a rarer glycine residue DG at this position Table 2. Using LPA, these 6 strains were determined to be resistant, since both mutant and wild-type bands were absent Table 2.

The DG mutation occurs less frequently than DV, is not clustered or epidemiologically significant, and has been reported to occur in isolates characterized from China, Africa, and North America 7 , 12 , Similarly, an uncommon amino acid substitution was identified at position in the rpoB gene product. However, Ion Torrent sequencing revealed that 1 of 3 isolates contained an uncommon arginine residue HR that by Hain LPA was shown to be absent for both wild-type and mutant bands Table 2.

While the absence of wild-type and mutant bands in a sample is interpreted as resistant according to LPA testing 6 , there remains ambiguity, since the type of amino acid change is not directly characterized. The absence of wild-type and mutant bands as an indication of drug resistance using the LPA test could possibly give a false-positive result if different amino acid residues in known amino acid positions affect resistance or levels of resistance to a particular drug.

This underscores the utility of Ion Torrent sequencing for directly detecting specific amino acid substitutions in positions known to confer resistance, as well as discovery of novel amino acids outside known sites that may impact resistance. The katG gene encodes catalase peroxidase, an enzyme that converts isoniazid INH into the active form. The majority of isoniazid resistance is associated with the katG codon corresponding to amino acid ST 9 , although mutations in the promoter region of inhA and nod also contribute to resistance 9 , Of the 26 strains assessed, 16 contained the characteristic serine-to-threonine amino acid substitution at position ST conferring isoniazid resistance Table 3.

Pyrazinamide PZA is a synthetic derivative of nicotinamide that has been used as a first-line drug to fight tuberculosis since PZA resistance is attributed to mutations in the pncA gene, which encodes a pyrazinamidase. However, these resistance-conferring mutations are numerous and consist of amino acid substitutions, frameshifts, and stop codon mutations Seven mutations were characterized from the 26 South African isolates assessed, including one silent mutation, five amino acid substitutions, and one chain termination mutation.

The QStop termination mutation Table 4 observed in one isolate is novel, having never been reported elsewhere. The difficulty in PZA phenotypic assessment and the variability of mutations along the pncA gene highlight the use of Ion Torrent gene sequencing to assess mutations in this hypervariable M. The primary target of fluoroquinolones FQs in M. Amino acid substitutions located within a short region of the gyrA gene known as the quinolone resistance-determining region QRDR account for the majority of known FQ-resistant M.

Substitution mutations in the QRDR at positions 88, 90, and 94 were observed in 10 of 26 sequences from this study Table 5. Three of the 10 strains contained substitutions at position 94 in the gyrA gene product; two were noted as D94G substitutions, and one was a D94Y substitution. A previous study characterized both D94G and D94Y substitutions and demonstrated that these amino acid substitutions at position 94 give rise to similar levels of FQ resistance 3.

Among the strains assessed in this study, the gyrA gene was the most variable, yielding nine amino acid substitutions in the 26 clinical isolates assessed. The majority of resistance to second-line drugs is associated with mutations in codons corresponding to amino acids AG , CT , and GT in the 16S rRNA rrs gene 11 , Analysis of African M.

Three additional nucleotide mutations affecting positions , , and were also discovered Table 6 in strains from this analysis. The overall cumulative impact of these newly discovered mutations on drug resistance remains to be established. Previous studies have shown that mutations in the katG codon corresponding to amino acid and the gyrA codon corresponding to amino acid 95 are genetic markers for categorizing strains into epidemiological genetic groups 1, 2, and 3 and that these codons have no effect on antibiotic resistance 16 , Group 1 strains are genetic ancestors of group 2 and group 3 strains that link the predominately nonhuman Mycobacterium species M.

Tracking group 1 organisms is important in terms of M. The purpose of this article was to describe the development of an Ion Torrent protocol for evaluating genotypic drug resistance using clinical isolates. We intend to apply our sequencing methodology to a much larger prospective study using samples collected from Kwa-Zulu Natal Province in South Africa. Evaluation of a larger sample size will further validate the developed methodology and make possible detection of uncommon or novel mutations in the full coding regions of these five genes.

One inherent concern with performing full-gene sequencing, especially from geographically diversified samples, is the discovery of novel mutations that may have no impact on drug susceptibility. Conversely, detected mutations outside known resistance sites may cause complete resistance or an additive resistance when combined with other mutations. Thus, genotypic identification of mutations through full-gene sequencing is the first step in understanding the complete resistance picture.

However, genotypic discovery might be complemented and validated by subsequent phenotypic MIC studies. There is potential for the Ion Torrent instrument and described methodology to be integrated into selected regional and reference settings throughout Africa, India, and China.

In contrast to most Sanger sequencers, the Ion Torrent does not utilize expensive and maintenance-requiring lasers that typically require modified fluorescence-based, light-sensitive chemistries, and it has a much smaller overall footprint. Furthermore, our described methodology does not require expensive ancillary equipment such as the Agilent BioAnalyzer, the Ion OneTouch system, ultracentrifuges, or the Pippin Prep Workstation as current Ion Torrent protocols recommend.

This is significant, since these instruments and needed accessories and consumables can be expensive, require large laboratory footprints, and often require routine maintenance. When clinical isolates derived from traditional detection methods require further genotypic drug susceptibility characterization, they can safely be inactivated in PrimeStore MTM for extraction and PCR amplification in a standard molecular biology laboratory setting prior to Ion Torrent sequencing.

Using the protocol reported here, several uncommon amino acid changes in clinical field isolates have been found. Furthermore, the extensive depth of sequence coverage from the Ion Torrent allows for discovery of heterogeneous or mixed-strain genetic populations within an isolate.

The scalability of Ion Torrent sequencing permits expansion to include megabases of additional genes on a single chip. As a proof of principle with five full-length M. This work would not have been possible without the efforts of Chris Helm and Susan Graham, who facilitated international planning meetings, travel, and teleconferences between U. We thank Richard Tamfu for his critical review of the manuscript.

Madison, WI for his bioinformatics assistance. Published ahead of print 12 September J Clin Microbiol. Luke T. Daum , a John D. Rodriguez , a Sue A. Worthy , a Nazir A. Ismail , b, c Shaheed V. Omar , b, c Andries W. Dreyer , b P. Bernard Fourie , b Anwar A. Hoosen , b James P. Chambers , a, d and Gerald W. Fischer a. John D. Sue A. Nazir A. Shaheed V. Andries W. Bernard Fourie. Anwar A. James P.

Gerald W. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Address correspondence to Luke T. Daum, moc. All Rights Reserved. This article has been cited by other articles in PMC. Abstract A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine PGM.

DNA preparation. Primer design. Open in a separate window. PCR amplification. Ion Torrent library preparation. Amplicon shearing. Adaptor ligation. Depesh is a Mid- Caps Analyst at Equirus. He has over 9 years of experience in Equity Research and has been tracking various Mid-Cap companies across sectors.

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Man and boy tony parsons ebook torrents Williams DL, continue reading al. Dugar brings along a significantly rich expertise in the field of Corporate Banking which spans across various Verticals and Geographies. Franklin estimating torrent the tube was in motion, the entire 1-ml PCR master mix solution was dispensed into the cap port and mixed for 5 min. The common SL mutation was the most prevalent, but mutations at positions andalso known to confer resistance to rifampin, were observed Table 2. We also wish to express our appreciation to Laura Cheu, Emile Bauer, and all the staff at Addison-Wesley for their quality production of the book. World Health Organization
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Golden earring complete discography torrents Chapter 2 is a review of the prerequisite continuous control; Chapter 3 introduces the key effects of sampling in order to elucidate many of the topics that follow. I'am primarily a VLSI design engineer who is working on a product involving digital control. It is very clear. Table P. The clinical specimens were initially confirmed by conventional culture methodology. Furthermore, our described methodology does not require expensive ancillary equipment such as the Agilent BioAnalyzer, the Ion OneTouch system, ultracentrifuges, or the Pippin Prep Workstation as current Ion Franklin estimating torrent protocols recommend.

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