scholarly journals Rapid Tuberculosis Diagnosis Using Reporter Enzyme Fluorescence

2019 ◽  
Vol 57 (12) ◽  
Author(s):  
Preeti Sule ◽  
Ronak Tilvawala ◽  
Toriq Mustapha ◽  
Hany Hassounah ◽  
Aneesa Noormohamed ◽  
...  
Keyword(s):  
Predictive Value ◽  
Positive Culture ◽  
Infectious Agent ◽  
Clinical Samples ◽  
Smear Microscopy ◽  
Content Type ◽  
Diagnostic Strategies ◽  
Reporter Enzyme ◽  
Tuberculosis Diagnosis ◽  
Culture Positive

ABSTRACT Tuberculosis is the most frequent cause of death in humans from a single infectious agent. Due to low numbers of bacteria present in sputum during early infection, diagnosis does not usually occur until >3 to 4 months after symptoms develop. We created a new more sensitive diagnostic that can be carried out in 10 min with no processing or technical expertise. This assay utilizes the Mycobacterium tuberculosis-specific biomarker BlaC in reporter enzyme fluorescence (REF) that has been optimized for clinical samples, designated REFtb, along with a more specific fluorogenic substrate, CDG-3. We report the first evaluation of clinical specimens with REFtb assays in comparison to the gold standards for tuberculosis diagnosis, culture and smear microscopy. REFtb assays allowed diagnosis of 160 patients from 16 different countries with a sensitivity of 89% for smear-positive, culture-positive samples and 88% for smear-negative, culture-positive samples with a specificity of 82%. The negative predictive value of REFtb for tuberculosis infection is 93%, and the positive predictive value is 79%. Overall, these data point toward the need for larger accuracy studies by third parties using a commercially available REFtb kit to determine whether incorporation of REFtb into the clinical toolbox for suspected tuberculosis patients would improve case identification. If results similar to our own can be obtained by all diagnostic laboratories, REFtb would allow proper treatment of more than 85% of patients that would be missed during their initial visit to a clinic using current diagnostic strategies, reducing the potential for further spread of disease.

scholarly journals Comparative Evaluation of Enteric Bacterial Culture and a Molecular Multiplex Syndromic Panel in Children with Acute Gastroenteritis

2019 ◽  
Vol 57 (6) ◽  
Author(s):  
Thomas Kellner ◽  
Brendon Parsons ◽  
Linda Chui ◽  
Byron M. Berenger ◽  
Jianling Xie ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Rectal Swab ◽  
Bacterial Pathogen ◽  
Positive Culture ◽  
Content Type ◽  
Negative Culture ◽  
Percent Agreement ◽  
Stool Samples ◽  
Culture Positive ◽  
Culture Negative

ABSTRACTAlthough enteric multianalyte syndromic panels are increasingly employed, direct comparisons with traditional methods and the inclusion of host phenotype correlations are limited. Luminex xTAG gastrointestinal pathogen panel (GPP) and culture results are highly concordant. However, phenotypic and microbiological confirmatory testing raises concerns regarding the accuracy of the GPP, especially forSalmonellaspp. A total of 3,089 children with gastroenteritis submitted stool specimens, rectal swab specimens, and clinical data. The primary outcome was bacterial pathogen detection agreement for shared targets between culture and the Luminex xTAG GPP. Secondary analyses included phenotype assessment, additional testing of GPP-negative/culture-positive isolate suspensions with the GPP, and in-house and commercial confirmatory nucleic acid testing of GPP-positive/culture-negative extracts. The overall percent agreement between technologies was >99% for each pathogen.Salmonellaspp. were detected in specimens from 64 participants: 12 (19%) by culture only, 9 (14%) by GPP only, and 43 (67%) by both techniques. Positive percent agreement forSalmonellaspp. was 78.2% (95% confidence interval [CI], 64.6%, 87.8%). Isolate suspensions from the 12 participants with specimens GPP negative/culture positive forSalmonellatested positive by GPP. Specimens GPP positive/culture negative forSalmonellaoriginated in younger children with less diarrhea and more vomiting. GPP-positive/culture-negative specimen extracts tested positive using additional assays for 0/2Campylobacter-positive specimens, 0/4Escherichia coliO157-positive specimens, 0/9Salmonella-positive specimens, and 2/3Shigella-positive specimens. For both rectal swab and stool samples, the median cycle threshold (CT) values, determined using quantitative PCR, were higher for GPP-negative/culture-positive samples than for GPP-positive/culture-positive samples (for rectal swabs, 36.9 [interquartile range {IQR}, 33.7, 37.1] versus 30.0 [IQR, 26.2, 33.2], respectively [P = 0.002]; for stool samples, 36.9 [IQR, 33.7, 37.1] versus 29.0 [IQR, 24.8, 30.8], respectively [P = 0.001]). GPP and culture have excellent overall agreement; however, for specific pathogens, GPP is less sensitive than culture and, notably, identifies samples false positive forSalmonellaspp.

scholarly journals Nested-PCR using MPB64 fragment improves the diagnosis of pleural and meningeal tuberculosis

2000 ◽  
Vol 33 (3) ◽  
pp. 253-257 ◽  
Author(s):  
Luiz C. Martins ◽  
Ilma A. Paschoal ◽  
Angela Von Nowakonski ◽  
Silvana A.B. Silva ◽  
Fernando F. Costa ◽  
...  
Keyword(s):  
Predictive Value ◽  
Gold Standard ◽  
Positive Culture ◽  
High Specificity ◽  
Pcr Assay ◽  
Tuberculous Pleural Effusion ◽  
Tuberculosis Diagnosis ◽  
Pcr Techniques ◽  
Meningeal Tuberculosis ◽  
Gold Standards

Fluids in which Mycobacterium tuberculosis are seldom found, such as pleural and cerebrospinal liquids, are good candidates to be studied using PCR techniques. We detail our experience with a PCR assay applied to pleural and cerebrospinal fluids using the primer MPB64. Seventy three specimens were analyzed: 30 pleural fluids (PF), 26 pleural biopsies (PB) and 17 cerebrospinal fluids (CSF). The gold standard for the diagnosis of tuberculous meningitis was the positive culture for M. tuberculosis in CSF. Tuberculous pleural effusion was diagnosed when cultures of PF and/or PB were positive for M. tuberculosis, or the PB histology showed granulomas. Our results, compared to the gold standards employed, showed a sensitivity of 70%, specificity of 88%, positive predictive value of 82% and negative predictive value of 80%. The high specificity of the MPB64 fragment while still retaining a good sensitivity makes it very well suited for pleural and cerebrospinal tuberculosis diagnosis.

scholarly journals Direct Detection of Shigella in Stool Specimens by Use of a Metagenomic Approach

2017 ◽  
Vol 56 (2) ◽  
Author(s):  
Jie Liu ◽  
Mathieu Almeida ◽  
Furqan Kabir ◽  
Sadia Shakoor ◽  
Shahida Qureshi ◽  
...  
Keyword(s):  
Direct Detection ◽  
Positive Culture ◽  
Accurate Method ◽  
Diagnostic Methods ◽  
Metagenomic Sequencing ◽  
Illumina Hiseq ◽  
Content Type ◽  
Sequence Composition ◽  
Culture Positive ◽  
Culture Negative

ABSTRACTThe underestimation ofShigellaspecies as a cause of childhood diarrhea disease has become increasingly apparent with quantitative PCR (qPCR)-based diagnostic methods versus culture. We sought to confirm qPCR-based detection ofShigellavia a metagenomics approach. Three groups of samples were selected from diarrheal cases from the Global Enteric Multicenter Study: nineShigellaculture-positive and qPCR-positive (culture+qPCR+) samples, nine culture-negative but qPCR-positive (culture−qPCR+) samples, and nine culture-negative and qPCR-negative (culture−qPCR−) samples. Fecal DNA was sequenced using paired-end Illumina HiSeq, whereby 3.26 × 108± 5.6 × 107high-quality reads were generated for each sample. We used Kraken software to compare the read counts specific to “Shigella” among the three groups. The proportions ofShigella-specific nonhuman sequence reads between culture+qPCR+(0.65 ± 0.42%) and culture−qPCR+(0.55 ± 0.31%) samples were similar (Mann-Whitney U test,P= 0.627) and distinct from the culture−qPCR−group (0.17 ± 0.15%,P< 0.05). The read counts of sequences previously targeted byShigella/enteroinvasiveEscherichia coli(EIEC) qPCR assays, namely,ipaH,virA,virG,ial,ShET2, andipaH3, were also similar between the culture+qPCR+and culture−qPCR+groups and distinct from the culture−qPCR−groups (P< 0.001). Kraken performed well versus other methods: its precision and recall ofShigellawere excellent at the genus level but variable at the species level. In summary, metagenomic sequencing indicates thatShigella/EIEC qPCR-positive samples are similar to those ofShigellaculture-positive samples inShigellasequence composition, thus supporting qPCR as an accurate method for detectingShigella.

scholarly journals Improving the Sensitivity of the Xpert MTB/RIF Assay on Sputum Pellets by Decreasing the Amount of Added Sample Reagent: a Laboratory and Clinical Evaluation

2015 ◽  
Vol 53 (4) ◽  
pp. 1258-1263 ◽  
Author(s):  
Nila J. Dharan ◽  
Danielle Amisano ◽  
Gerald Mboowa ◽  
Willy Ssengooba ◽  
Robert Blakemore ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Limit Of Detection ◽  
Smear Microscopy ◽  
Content Type ◽  
Clinical Specimens ◽  
Test Sensitivity ◽  
Smear Negative ◽  
Almost All ◽  
Culture Positive ◽  
Xpert Assay

The Xpert MTB/RIF (Xpert) assay permits rapid near-patient detection ofMycobacterium tuberculosisin sputum; however, the test sensitivity remains suboptimal in paucibacillary specimens that are negative for acid-fast bacilli using smear microscopy. Xpert testing includes dilution with sample reagent, and when processed sputum pellets are tested, the recommended sample reagent/pellet ratio is 3:1. We evaluated whether a decreased sample reagent/pellet ratio of 2:1 increased Xpert sensitivity compared to the recommended 3:1. The limit of detection was determined by inoculating serial dilutions ofM. tuberculosisinto sputum samples, preparing sputum pellets, and testing each pellet by Xpert at both sample reagent ratios. Processed sputum pellets obtained fromM. tuberculosisculture-positive clinical specimens were also tested by Xpert at both ratios. Among spiked sputum pellets, the limit of detection was 1,478 CFU/ml (95% confidence interval [CI], 1,211 to 1,943) at a 3:1 ratio and decreased to 832 CFU/ml (95% CI, 671 to 1,134) at 2:1. The proportion of specimens in whichM. tuberculosiswas detected was greater at 2:1 than at 3:1 for almost all numbers of CFU/ml; this difference was most prominent at lower numbers of CFU/ml. Among 134 concentrated sputum pellets from the clinical study, the sensitivity of Xpert at 2:1 was greater than at 3:1 overall (80% versus 72%;P= 0.03) and for smear-negative specimens (67% versus 58%;P= 0.12). For Xpert testing of sputum pellets, using a lower sample reagent/pellet ratio increasedM. tuberculosisdetection, especially for paucibacillary specimens. Our study supports use of a 2:1 sample reagent/pellet dilution for Xpert testing of sputum pellets.

scholarly journals Evaluation of a Multiplex Real-Time PCR Assay for Detecting Major Bacterial Enteric Pathogens in Fecal Specimens: Intestinal Inflammation and Bacterial Load Are Correlated in Campylobacter Infections

2016 ◽  
Vol 54 (9) ◽  
pp. 2262-2266 ◽  
Author(s):  
Nadia Wohlwend ◽  
Sacha Tiermann ◽  
Lorenz Risch ◽  
Martin Risch ◽  
Thomas Bodmer
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Bacterial Load ◽  
Positive Culture ◽  
Fecal Calprotectin ◽  
Enteric Pathogens ◽  
Significant Linear Trend ◽  
Content Type ◽  
Culture Positive ◽  
Culture Negative

A total of 1,056 native or Cary-Blair-preserved stool specimens were simultaneously tested by conventional stool culturing and by enteric bacterial panel (EBP) multiplex real-time PCR forCampylobacter jejuni,Campylobacter coli,Salmonellaspp., and shigellosis disease-causing agents (Shigellaspp. and enteroinvasiveEscherichia coli[EIEC]). Overall, 143 (13.5%) specimens tested positive by PCR for the targets named above; 3 coinfections and 109 (10.4%)Campylobacterspp., 17 (1.6%)Salmonellaspp., and 20 (1.9%)Shigellaspp./EIEC infections were detected. The respective positive stool culture rates were 75 (7.1%), 14 (1.3%), and 7 (0.7%). The median threshold cycle (CT) values of culture-positive specimens were significantly lower than those of culture-negative ones (CTvalues, 24.3 versus 28.7;P< 0.001), indicating that the relative bacterial load per fecal specimen was significantly associated with the culture results. InCampylobacterinfections, the respective median fecal calprotectin concentrations in PCR-negative/culture-negative (n =40), PCR-positive/culture-negative (n =14), and PCR-positive/culture-positive (n =15) specimens were 134 mg/kg (interquartile range [IQR], 30 to 1,374 mg/kg), 1,913 mg/kg (IQR, 165 to 3,813 mg/kg), and 5,327 mg/kg (IQR, 1,836 to 18,213 mg/kg). Significant differences were observed among the three groups (P< 0.001), and a significant linear trend was identified (P< 0.001). Furthermore, the fecal calprotectin concentrations andCTvalues were found to be correlated (r= −0.658). Our results demonstrate that molecular screening ofCampylobacterspp.,Salmonellaspp., andShigellaspp./EIEC using the BD Max EBP assay will result in timely diagnosis and improved sensitivity. The determination of inflammatory markers, such as calprotectin, in fecal specimens may aid in the interpretation of PCR results, particularly for enteric pathogens associated with mucosal damage and colonic inflammation.

scholarly journals DETECTION FAILURE OF SMEAR MICROSCOPY TO CONFIRM PULMONARY TUBERCULOSIS

2018 ◽  
Vol 6 (4) ◽  
Author(s):  
Yunusa EU ◽  
Bakare AT ◽  
Shagari GB ◽  
Abubakar AM ◽  
Sharhabila Y ◽  
...  
Keyword(s):  
Pulmonary Tuberculosis ◽  
Positive Culture ◽  
Culture Method ◽  
Molecular Diagnostic ◽  
Smear Microscopy ◽  
Reference Laboratory ◽  
Sample Collection ◽  
Cross Sectional ◽  
Detection Failure ◽  
Culture Positive

Background: The laboratories in poor resource settings commonly use smear microscopy for the diagnosis of pulmonary tuberculosis, because it is rapid, inexpensive and easy to perform. The commonly use Lowensten Jensen culture method for the diagnosis of tuberculosis is slow and usually not very sensitive. Polymerase Chain Reaction (PCR) in the diagnosis of tuberculosis (TB) has come to stay as an effective diagnostic tool for effective pulmonary TB case detections. Objectives: This is to promote and encourage utilization of molecular diagnostic method (PCR) for the diagnosis of tuberculosis especially in smear negative microscopy suspected cases to reduce the rate of missed diagnosis. Material and Methods: This was an observational cross sectional prospective study among patients who presented at tuberculosis reference laboratory with request form for the diagnosis of pulmonary tuberculosis. Consent forms were administered and only consented clients were enrolled in the study. Standard sample collection criteria were used for all collected samples subjected to standard smear microscopy, Lowenstein Jensen culture and PCR studies. Results: Direct smear microscopy (AFB) was positive in 13 (13.5%), 24 (23.3%) were detected by PCR and 20 (19.4%) were culture positive. All positive smears were found to be positive for both culture and PCR. All culture positive samples were positive by PCR, culture growth were also positive by PCR. Most of the PCR results were ready within the first day of the analysis with average of 1.2 days (P = 0.014) of the 103 samples processed. The result of smear microscopy is only ready on the second day after analysis of the second and third samples submitted with reported average of 1.7 days (P = 0.035). The fastest observable growth of positive culture was seen only within ten days and only three cases showed such. It took eight weeks for a negative growth to be regarded as negative. The culture has an average tun around ti

scholarly journals Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples

2015 ◽  
Vol 53 (7) ◽  
pp. 2230-2237 ◽  
Author(s):  
Amanda C. Brown ◽  
Josephine M. Bryant ◽  
Katja Einer-Jensen ◽  
Jolyon Holdstock ◽  
Darren T. Houniet ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Positive Culture ◽  
Clinical Samples ◽  
Whole Genome ◽  
Resistance Mutations ◽  
High Quality ◽  
Content Type ◽  
Negative Case

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistantMycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures ofM. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically forM. tuberculosisDNA to capture fullM. tuberculosisgenomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture.M. tuberculosissequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing ofM. tuberculosisgenomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.

scholarly journals Evaluation of the Xpert MTB/RIF Ultra Assay for Direct Detection ofMycobacterium tuberculosisComplex in Smear-Negative Extrapulmonary Samples

2018 ◽  
Vol 56 (9) ◽  
Author(s):  
Daniel Perez-Risco ◽  
David Rodriguez-Temporal ◽  
Ivan Valledor-Sanchez ◽  
Fernando Alcaide
Keyword(s):  
Nontuberculous Mycobacteria ◽  
Direct Detection ◽  
Extrapulmonary Tuberculosis ◽  
High Sensitivity ◽  
Rapid Diagnosis ◽  
Clinical Samples ◽  
Content Type ◽  
Smear Negative ◽  
Culture Positive ◽  
Culture Negative

ABSTRACTThe rapid detection ofMycobacterium tuberculosiscomplex (MTUBC) in clinical samples is essential for successful treatment. New techniques such as real-time PCR have been developed in order to facilitate rapid diagnosis, but their sensitivity is low in extrapulmonary specimens, due to the low bacillary load in such samples. A next-generation assay has recently been developed to try to overcome this limitation. The aim of this study was to analyze the effectiveness of the Xpert MTB/RIF Ultra (GX-Ultra) for the detection of MTUBC DNA in 108 smear-negative extrapulmonary specimens that were MTUBC culture positive. In addition, 40 extrapulmonary culture-negative samples and 20 samples with nontuberculous mycobacteria were tested to evaluate the specificity of the assay. All samples were collected between May 1999 and May 2017. The GX-Ultra detected DNA of MTUBC in 82 extrapulmonary specimens that were MTUBC culture positive (75.9% sensitivity; 95% confidence interval [CI], 66.6 to 83.4%). The assay was negative for all clinical specimens that were MTUBC culture negative and the samples with nontuberculous mycobacteria (100% specificity). Furthermore, two (1.8%) samples presented mutations related to rifampin resistance. The highest sensitivity was obtained in samples of lymph nodes (94.1%) and nonsterile fluids (93.7%), followed by tissue specimens (86.6%), stool material (80%), abscess aspirates (64.7%), and sterile fluids (60.5%). Pleural fluids, one of the least optimal samples for detecting DNA of MTUBC, were GX-Ultra positive in 10/21 (47.6%) of cases. In summary, GX-Ultra showed excellent specificity and high sensitivity in paubacillary specimens, making it a useful tool for rapid diagnosis of extrapulmonary tuberculosis.

scholarly journals Development of Novel PCR Assays To Detect Azole Resistance-Mediating Mutations of theAspergillus fumigatus cyp51AGene in Primary Clinical Samples from Neutropenic Patients

2012 ◽  
Vol 56 (7) ◽  
pp. 3905-3910 ◽  
Author(s):  
Birgit Spiess ◽  
Wolfgang Seifarth ◽  
Natalia Merker ◽  
Susan J. Howard ◽  
Mark Reinwald ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Positive Culture ◽  
Cross Reactivity ◽  
Resistant Isolate ◽  
Clinical Samples ◽  
Azole Resistance ◽  
Wild Type ◽  
Content Type ◽  
Pcr Assays

ABSTRACTThe increasing incidence of azole resistance inAspergillus fumigatuscausing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediatingcyp51Agene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of theAspergillus fumigatus cyp51Agene to establish PCR assays with consecutive DNA sequence analysis to detect and identify theA. fumigatus cyp51Atandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially dilutedA. fumigatusand human DNA,A. fumigatus cyp51Agene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg ofA. fumigatusDNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons forA. fumigatuswild-type DNA confirmed thecyp51Awild-type sequence, and PCR products from one azole-resistantA. fumigatusisolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.

scholarly journals Detection of parC gene mutations associated with quinolone resistance in Mycoplasma genitalium: evaluation of a multiplex real-time PCR assay

2021 ◽  
Vol 70 (3) ◽  
Author(s):  
Kaveesha Bodiyabadu ◽  
Jennifer Danielewski ◽  
Suzanne M. Garland ◽  
Dorothy A. Machalek ◽  
Catriona S. Bradshaw ◽  
...  
Keyword(s):  
Treatment Failure ◽  
Predictive Value ◽  
Sanger Sequencing ◽  
Mycoplasma Genitalium ◽  
Amino Acid Position ◽  
Fluoroquinolone Resistance ◽  
Clinical Samples ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Content Type

Introduction. Increasing levels of antibiotic resistance are complicating treatment for the sexually transmitted pathogen Mycoplasma genitalium . Resistance to fluoroquinolones is associated with mutations in the parC gene. Although the precise mutations conferring resistance are not fully understood, the single nucleotide polymorphism (SNP) G248T/S83I is most implicated. Aim. To evaluate the performance of the MG+parC(beta2) assay (SpeeDx, Australia), which detects single nucleotide polymorphisms (SNPs) in the parC gene at amino acid position S83 (A247C/S83R, G248T/S83I, G248A/S83N) and D87 (G259A/D87N, G259T/D87Y, G259C/D87H). Methods. Clinical samples were analysed by MG+parC(beta2) assay and results compared to Sanger sequencing. Sensitivity, specificity, and predictive value for treatment failure were calculated. Results. From analysis of 205 samples, the MG+parC(beta2) assay performed with a high sensitivity 98.2% (95% CI:90.3–100) and specificity 99.3% (95% CI:96.3–100) for parC SNP detection with a kappa of 0.97 (95% CI:0.94–1.00). The predictive value of G248T/S83I detection (the most common SNP, prevalence of 13% in the study population) was analysed with respect to treatment failure (patients received sequential doxycycline-moxifloxacin). The positive-predictive-value for moxifloxacin failure after detection of S83I was only 44% (95% CI:24.4–65.1), while negative-predictive-value was high at 96.9% (95% CI:92.7–99.0), suggesting that other SNPs are contributing to resistance. Conclusion. MG+parC(beta2) performed with high concordance compared to Sanger sequencing. Such qPCR assays can assist in understanding causes of treatment failure, inform the development of diagnostic assays, and can be applied to surveillance of mutations in populations. Due to an incomplete understanding of the basis for fluoroquinolone resistance, such tests do not appear to be ready for clinical application.

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