Detection Of Drug Resistance Research Articles

Value of routine whole genome sequencing for Mycobacterium tuberculosis drug resistance detection

Routine whole genome sequencing (WGS) of pathogens is becoming more feasible as sequencing costs decrease and access to benchtop sequencing equipment and bioinformatics pipelines increases. This study examined the added value gained from implementing routine WGS of all Mycobacterium tuberculosis isolates in New South Wales, Australia.Drug resistance markers inferred from WGS data were compared to commercial genotypic drug susceptibility testing (DST) assays and conventional phenotypic DST in all isolates sequenced between 2016 and 2019. Of the 1107 clinical M. tuberculosis isolates sequenced, 29 (2.6%) were multi-drug resistant (MDR); most belonged to Beijing (336; 30.4%) or East-African Indian (332; 30%) lineages. Compared with conventional phenotypic DST, WGS identified an additional 1% of isolates which were likely drug resistant, explained by mutations previously associated with treatment failure and mixed bacterial populations. However, WGS provided a 20% increase in drug resistance detection in comparison with commercial genotypic assays by identifying mutations outside of the classic resistance determining regions in rpoB, inhA, katG, pncA and embB genes. Gains in drug resistance detection were significant (p = 0.0137, paired t-test), but varied substantially for different phylogenetic lineages.In low incidence settings, routine WGS of M. tuberculosis provides better guidance for person-centered management of drug resistant tuberculosis than commercial genotypic assays.

Increased Isolation of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli From Community-Onset Urinary Tract Infection Cases in Uttarakhand, India.

Background: Management of community-acquired urinary tract infection (CA-UTI) relies heavily on empirical antibiotic therapy. Knowledge of the proportion of drug-resistant isolates especially extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli), and various risk factors for acquisition are essential.Method: Outpatient-treated CA-UTI cases were enrolled (continuously for three months), and microbiological analysis of urine sample was performed for significant bacterial growth followed by identification of conventional and matrix-assisted laser desorption/ionization-time of flight (MALDI-ToF) spectrometry method. Subsequent drug resistance and phenotypic ESBL detection were as per guidelines of the Clinical Laboratory Standard Institute (CLSI, USA). Univariate and multivariate analyses (logistic regression) of known and relevant risk factors of ESBL E. coli were performed as per standard statistical technique, using the SPSS computer package (IBM Corp., Armonk, NY). Results: Two hundred and forty-one samples (of 694 samples) yielded significant growth. Sixty-one of 131 (46.6%) E. coli isolates were found to be ESBL producers. Non-beta-lactam antibiotic resistance in ESBL producers was high compared to non-ESBL producers (e.g., 88.5% vs 42.3% for quinolone resistance, 80.3% vs 34.3% for gentamicin resistance, etc.). Multivariate analysis (after univariate analysis detected probable factors of a likely ESBL model) indicated significant associations of ESBL-producing E. coli with advancing age (>55 years), prior hospitalization in last one year, use of antibiotics in previous six months, and presence of comorbid illness such as diabetes mellitus and chronic lung disease.Conclusion: High proportion of our community-acquired uropathogens are ESBL-producing E. coli and likely resistant to important antimicrobial agents such as quinolones, gentamicin, etc. Factors like advancing age, prior hospitalization, and antibiotic use, as well as comorbidities such as diabetes and chronic lung disease, may be strongly associated with ESBL E. coli and should be remembered while administering or preparing guidelines for empiric management of CA-UTI subjects.

Drug resistance pattern and mutation pattern in pediatric tuberculosis: Study from north India

BackgroundThe emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB are serious threats to global TB control. Molecular tests like GenoType MTBDRplus has revolutionized MDR-TB diagnosis by rapid detection of resistance, leading to early and appropriate management of DR-TB. Information about common mutations imparting resistance to RIF and INH, helps in understanding the disease epidemiology in various regions. The study was conducted to determine the genetic mutation in drug resistant tuberculosis in children less than 12 years with pulmonary or extrapulmonary tuberculosis. Materials/methodsRetrospective analysis was done over a period of 54 months from January 2015 to June 2019 to study the resistance pattern and mutations present in DR-TB in children less than 12 years with suspected pulmonary or extrapulmonary tuberculosis using Hain's GenoType MTBDRplus VER 2.0. ResultsOver a period of 54 months, samples from 3461 patients with suspected TB were received for MGIT culture, out of which, 347 were positive for Mycobacterium tuberculosis. 250 of these 347 isolated were tested for drug resistance by Hain's GenoType MTBDRplus VER 2.0.61.1% were sensitive to isoniazid and rifampicin while 15.2% were DR-TB (38 out of 250). Out of these 38, 22 were MDR TB, 13 were isoniazid monoresistant (34.2%) and 3 were rifampicin monoresistant. The most common genotypic resistance for rifampicin was absence of rpoB WT8 band and presence of rpoB MUT 3 band (88%). 84.6% of the INH monoresistant isolates showed high level isoniazid resistant. All these isolates showed presence of katG MUT 1 band. On comparing Hain's GenoType MTBDRplus VER 2.0 with Xpert MTB/Rif Assay, most common mutation for rifampicin resistance at S531L which can be detected by Xpert MTB/Rif Assay (probe E). However, two cases with rifampicin resistance had mutation in codon region 509–513 and 513–519 which could be missed by Xpert MTB/Rif Assay. ConclusionsWe cannot solely rely on Xpert MTB/Rif Assay for detection of drug resistance due to the risk of missing the isoniazid monoresistance. GenoType MTBDRplus has revolutionized MDR-TB diagnosis by substantially reducing turn around time and leading to early management of DR-TB cases.

Detection of drug resistance mutations of hepatitis C virus in patients with failure of the treatment with direct acting antivirals

Background. The development of direct acting antivirals (DAAs) has spurred a revolution in treatment of patients with chronic hepatitis C. However, there are cases showing no response to treatment. In 5% of cases, the viral breakthrough is most likely caused by DAA resistance mutations in the hepatitis C virus genome.The purpose of the study is to detect drug resistance mutations of hepatitis C virus in patients with DAA treatment failure.Materials and methods. The study was performed on plasma samples from 3 patients diagnosed with chronic hepatitis C virus infection and demonstrating DAA virological treatment failure. All isolates had genotype 1b. Drug resistance mutations were detected by using direct sequencing of NS3, NS5A, and NS5B genome regions. The detection technique was developed at the Pasteur Research Institute of Epidemiology and Microbiology.Results. Drug resistance mutations were detected in all cases. By using the Geno2pheno [hcv] 0.92 tool, nucleotide substitutions were detected in different viral genome regions and presumably caused resistance or decreased sensitivity to antivirals both present and absent in the sofosbuvir + daclatasvir combination therapy. Antiviral treatment failure in patients with chronic hepatitis C is caused by drug resistance mutations.Conclusions. The developed technique is efficient for detection of drug resistance mutations in NS3, NS5A, and NS5B regions in cases of virological failure of DAA treatment.

A Comparative Study of Chest Computed Tomography Findings: 1030 Cases of Drug-Sensitive Tuberculosis versus 516 Cases of Drug-Resistant Tuberculosis

PurposeTo investigate the CT features of drug-resistant pulmonary tuberculosis (DR-PTB) and the diagnostic value of CT in DR-PTB diagnosis to provide imaging evidence for the timely detection of drug-resistant Mycobacterium tuberculosis.Materials and MethodsA total of 1546 cases of pulmonary tuberculosis (PTB) with complete clinical data, chest CT images and defined drug sensitivity testing results were consecutively enrolled; 516 cases of DR-PTB were included in the drug-resistant group, and 1030 cases of drug-sensitive pulmonary tuberculosis (DS-PTB) were included in the drug-sensitivity group. Comparative analyses of clinical symptoms and imaging findings were conducted. Univariate and logistic regression analyses were performed, a regression equation model was developed, and the receiver operating characteristic (ROC) curve was constructed.ResultsIn the univariate analysis, some features, including whole-lung involvement, multiple cavities, thick-walled cavities, collapsed lung, disseminated lesions along the bronchi, bronchiectasis, emphysema, atelectasis, calcification, proliferative lesions, encapsulated effusion, etc., were observed more frequently in the DR-PTB group than in the DS-PTB group, and the differences were statistically significant (p<0.05). Exudative lesions and pneumoconiosis were observed more frequently in the drug-sensitivity group than in the drug-resistant group (p<0.05). Logistic regression analysis indicated that whole-lung involvement, multiple cavities, thick-walled cavities, disseminated lesions along the bronchi, bronchiectasis, and emphysema were independent risk factors for DR-PTB, and exudative diseases were protective factors. The total prediction accuracy of the regression model was 80.6%, and the area under the ROC curve (AUC) was 82.6%.ConclusionChest CT manifestations of DR-PTB had certain characteristics that significantly indicated the possibility of drug resistance in tuberculosis patients, specifically when multifarious imaging findings, including multiple cavities, thick-walled cavities, disseminated lesions along the bronchi, whole-lung involvement, etc., coexisted simultaneously. These results may provide imaging evidence for timely drug resistance detection in suspected drug-resistant cases and contribute to the early diagnosis of DR-PTB.

Antibacterial activity of positively charged carbon quantum dots without detectable resistance for wound healing with mixed bacteria infection

Widespread bacterial infection and the spread of antibiotic resistance exhibit increasing threat to the public and thus require new antibacterial strategies. Carbon quantum dots (CQDs) have been extensively investigated to play fluorescent, catalytic roles and even potential biomedical functions containing sterilization. However, synthetic understanding of the interaction of CQDs and bacteria, the exhibition of antibacterial ability, and the risk of resistance evolution remain lacking. Herein, a simple one-pot method was fabricated to prepare positively charged CQDs (PC-CQDs) as a broad-spectrum antibacterial agent. PC-CQDs possessed effective antibacterial activity against all tested Gram-positive, Gram-negative, and drug-resistant bacteria. Investigation of the antibacterial mechanism of PC-CQDs indicated that small-sized PC-CQDs functionalized with –NH2 and –NH induced strong adherence behavior on the bacterial cell membrane. Moreover, the entry of PC-CQDs caused conformational changes in the genes and generation of reactive oxygen species in the bacteria. Safety evaluation illustrated that PC-CQDs did not trigger detectable drug resistance or hemolysis. Furthermore, PC-CQDs effectively promoted the antibacterial treatment of mixed Staphylococcus aureus and Escherichia coli infected wound in rats with low in vivo toxicity. These results suggested that PC-CQDs are a potential antibacterial candidate for real wound healing applications in complex bacterial infections and even resistant bacteria-caused infections.

Burden of Drug resistant Tuberculosis in newly diagnosed Tuber-culosis patients of Khyber Pakhtunkhwa, Pakistan

Abstract Objective: To explore the burden of drug resistant tuberculosis in Khyber Pakhtunkhwa, Pakistan. Methods: A total of 8220 referred suspected TB patient samples were collected from 1st March 2016 to 28th February 2017 from TB laboratories across all districts of Khyber Pakhtunkhwa province and processed for the detection of drug resistant tuberculosis (DR-TB). Drug Susceptibility testing (DST) on all Mycobacterium tuberculosis complex (MTC) strains was performed at Provincial TB Reference Laboratory, Peshawar. Results: Among the 8220 suspected TB samples, 1351 samples were declared positive on culture. The DST results showed that 525 samples were resistant to at least one of the first and second line anti-TB drugs. Importantly, among these 525 TB resistance cases, XDR-TB were 0.4% (n=5), MDR-TB were 4.6% (n=62), polyresistant were 18% (n=243), monoresistant was 16% (n=215) and 61% (n=826) were reported as pan-sensitive. Gender wise ratio are slightly increased in female (n= 4230; 51.5%) as compared to male (n=3990; 48.5%), while the age group 15-24 were the main target of TB disease (n=1978; 24%). Conclusion: Our data suggests that DR-TB cases especially MDR-TB and XDR-TB in newly diagnosed TB patients are alarmingly increased in Khyber Pakhtunkhwa region. Furthermore, the timely detection of drug resistance is important to optimize treatment and management of MDR-TB as well as XDR-TB. Key words: Tuberculosis; MDR-TB; XDR-TB; Continuous....

Early detection of drug-resistant Streptococcus pneumoniae and Haemophilus influenzae by quantitative flow cytometry

Early detection of drug resistance contributes to combating drug-resistant bacteria and improving patient outcomes. Microbial testing in the laboratory is essential for treating infectious diseases because it can provide critical information related to identifying pathogenic bacteria and their resistance profiles. Despite these clinical requirements, conventional phenotypic testing is time-consuming. Additionally, recent rapid drug resistance tests are not compatible with fastidious bacteria such as Streptococcus and Haemophilus species. In this study, we validated the feasibility of direct bacteria counting using highly sensitive quantitative flow cytometry. Furthermore, by combining flow cytometry and a nucleic acid intercalator, we constructed a highly sensitive method for counting viable fastidious bacteria. These are inherently difficult to measure due to interfering substances from nutrients contained in the medium. Based on the conventional broth microdilution method, our method acquired a few microliter samples in a time series from the same microplate well to exclude the growth curve inconsistency between the samples. Fluorescent staining and flow cytometry measurements were completed within 10 min. Therefore, this approach enabled us to determine antimicrobial resistance for these bacteria within a few hours. Highly sensitive quantitative flow cytometry presents a novel avenue for conducting rapid antimicrobial susceptibility tests.

The Role of Cartridge-Based Nucleic Acid Amplification Test (CBNAAT), Line Probe Assay (LPA), Liquid Culture, Acid-Fast Bacilli (AFB) Smear and Histopathology in the Diagnosis of Osteoarticular Tuberculosis.

There is a paucity of data on the role of molecular methods in the diagnosis of osteoarticular tuberculosis. The present study was conducted to define the role of molecular (CBNAAT, LPA), phenotypic (AFB smear and culture) and histopathological evaluation in the diagnosis of osteoarticular TB. Seventy-seven consecutive cases of osteoarticular tuberculosis were grouped into presumptive TB cases (group A) and presumptive drug-resistant cases (group B). Tissue samples obtained were submitted for CBNAAT, LPA, AFB smear, liquid culture and histological examinations. The diagnostic accuracy of each test was reported against histologically diagnosed cases and in all tests in tandem. Group A and group B had 65 and 12 cases, respectively. The diagnostic accuracy for tuberculosis was 84.62% by CBNAAT, 70.77% by LPA, 86.15% by molecular tests (combined), 47.69% by AFB smear, 50.77% by liquid culture and 87.69% by histology in group A, and 91.67% for CBNAAT, 83.33% for LPA, 91.67% for molecular tests (combined), 25% for AFB smear, 16.67% for liquid culture and 83.33% for histology in group B. The drug resistance detection rate was 4.62% on CBNAAT, 3.08% on LPA, 6.15% on molecular tests (combined) and 1.54% on DST in group A, while it was 33.33% on CBNAAT, 58.33% on LPA, 58.33% on molecular tests (combined) and 16.67% on DST among group B cases. Similar sensitivity rates for the various tests were obtained among both the groups on comparison with histology (taken as denominator). The addition of molecular methods increased the overall diagnostic accuracy (all tests in tandem) from 93.8 to 100% in group A and from 83.3 to 100% in group B cases. No single tests could diagnose tuberculosis in all cases; hence, samples should be evaluated by molecular tests (CBNAAT and LPA), AFB smear, culture and histological examinations simultaneously. The molecular tests have better demonstration of drug resistance from mycobacterial culture. The online version contains supplementary material available at 10.1007/s43465-020-00326-w.

Rifampin resistance among individuals with extrapulmonary tuberculosis: 4 years of experience from a reference laboratory

Information is limited about the drug resistance patterns in extrapulmonary tuberculosis (EPTB) in Iran. This study aimed to determine the prevalence of EPTB and to investigate the drug-resistance pattern in Mycobacterium tuberculosis strains collected from extrapulmonary samples at the Tehran regional TB reference laboratory. Extrapulmonary specimens from individuals with suspected TB referred to the TB reference laboratories in five cities of Iran were collected. Both standard conventional methods (culture and direct smear microscopy) and Xpert MTB/RIF assay were used for the identification of mycobacteria. Drug susceptibility testing was done using Xpert MTB/RIF. The proportion method on Lowenstein–Jensen medium was performed for confirmation. Between 2016 and 2020, a total of 12 050 clinical specimens from individuals with suspected TB were collected, of which 10 380 (86%) were pulmonary specimens and 1670 (14%) were extrapulmonary. Of the extrapulmonary specimens, 85 (5.0%) were positive for M. tuberculosis, and the remaining 1585 (95.0%) samples were negative by standard methods. Of 85 M. tuberculosis isolates, drug susceptibility testing was performed for 32 isolates, of which 1 (3.1%, 95% CI 0.0%–9.4%) was rifampin resistant and 31 (96.9%, 95% CI 90.1%–100%) were pan-susceptible. The rifampin-resistant isolate was also resistant to isoniazid, so was assigned as a multidrug-resistant TB. Our study indicated the frequency of drug-resistance among EPTB in Iran. Establishing rapid diagnostic methods for detection of drug-resistance in EPTB, performing drug susceptibility testing for all EPTB cases to provide effective treatment, and continuous monitoring of drug resistance, are suggested for prevention and control of drug resistance in EPTB in Iran.

Targeted next generation sequencing directly from sputum for comprehensive genetic information on drug resistant Mycobacterium tuberculosis

BackgroundTimely drug resistance detection is essential to global tuberculosis management. Unfortunately, rapid molecular tests assess resistance to only a few drugs, with culture required for comprehensive susceptibility test results. MethodsWe evaluated targeted next generation sequencing (tNGS) for tuberculosis on 40 uncultured sputum samples. Resistance profiles from tNGS were compared with profiles from Xpert MTB/RIF, line probe assay (LPA), pyrosequencing (PSQ), and phenotypic testing. Concordance, sensitivity, specificity, and overall test agreement were compared across assays. ResultstNGS provided results for 39 of 40 samples (97.5%) with faster turnaround than phenotypic testing (median 3 vs. 21 days, p = 0.0068). Most samples were isoniazid and rifampin resistant (N = 31, 79.5%), 21 (53.8%) were fluoroquinolone resistant, and 3 (7.7%) were also resistant to Kanamycin. Half were of the Beijing lineage (N = 20, 51.3%). tNGS from uncultured sputum identified all resistance to isoniazid, rifampin, fluoroquinolones, and second-line injectable drugs that was identified by other methods. Agreement between tNGS and existing assays was excellent for isoniazid, rifampin, and SLDs, very good for levofloxacin, and good for moxifloxacin. ConclusiontNGS can rapidly identify tuberculosis, lineage, and drug resistance with faster turnaround than phenotypic testing. tNGS is a potential alternative to phenotypic testing in high-burden settings.

KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum

BackgroundThe emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum.MethodsThree SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method.ResultsThe KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed.ConclusionsThe KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

National Proficiency Testing of Molecular Diagnostics for Tuberculosis and Drug Resistance Detection - China, 2014-2019.

What is already known about this topic? Proficiency testing (PT) is a key component of quality assurance and is essential in ensuring accurate laboratory diagnosis of tuberculosis (TB) and drug-resistant TB. The National Tuberculosis Reference Laboratory (NTRL) developed a novel PT panel to test laboratories’ proficiency for molecular TB diagnostic assays throughout the TB laboratory network. A total of 6 PT rounds for molecular diagnostics were conducted by NTRL from 2014 to 2019. What is added by this report? PT conducted using artificial sputum specimens increased from 120 in the first round to 1,835 in the sixth round. Overall, laboratories demonstrated good proficiency for MTB and drug-resistance detection by molecular diagnostics, which is evident from the qualification rates over the six rounds: 95%, 97%, 96%, 93%, 93%, and 97%, respectively. What are the implications for public health practice? The use of artificial sputum specimens for PT panel production to test TB molecular diagnostics in China is feasible. Most of the participating laboratories provided reliable molecular diagnostic results for MTB and drug-resistance detection. The TB laboratory network can be instrumental in implementing PT expansion and improving the quality of TB molecular diagnosis in China.

Extra-pulmonary tuberculosis (EPTB): Study of clinico-demographic profile, and comparison of microbiological diagnostic modalities with special emphasis on role of CBNAAT in detecting rifampicin resistance in fluid specimens

The purpose of our study was to evaluate the place of CBNAAT in the diagnosis of extra-pulmonary TB in fluid specimens, and also detecting drug resistance in comparison to the Gold Standard - MGIT Bactec Culture. Material and Methods: In prospective observational study of 2 yrs. among 165 EPTB cases, we studied the clinical presentation, compared the results of microbiological diagnostic modalities (ZN smear, CBNAAT, MGIT Culture) and evaluated the drug resistance. Statistics: Used Chi square test via software STATA 15 and calculated p value.Results: The positivity rate of CBNAAT (31.30%) was higher than ZN smear (23.92%), which was in turn higher than MGIT culture (9.9%) and CBNAAT was able to detect Rifampicin resistance in 16.66% EPTB cases. Conclusion: We concluded that, the role of CBNAAT in fluid specimens should not be undermined and should be preferred over the “Gold standard” MGIT culture.

Genexpert rapid detection of rifampicin resistance among patients with pulmonary tuberculosis

The routine conventional phenotypic bacteriological tests available for pulmonary tuberculosis (PTB) diagnosis, such as microscopy by Ziehl Neelsen staining, and Lowenstein Jensen method of solid culture, requires a long time for revealing positivity, resulting in a delayed diagnosis. After Mycobacterium tuberculosis (MTB) genome was discovered, nucleic acid amplification techniques were developed with more rapid detection and identification of rifampicin resistance in respiratory and extra-pulmonary specimens. GeneXpert is a DNA-polymerase chain reaction technique based on NAA, which allows an accurate genotypic method for a quick diagnosis of PTB diagnosis. The aim of the study was to assess retrospectively the yield of genotypic assay GeneXpert in revealing multidrug resistant TB compare to phenotypic confirmation by solid culture. 512 patients with positive and negative smears of suspected PTB were investigated by conventional phenotypic assays. PTB diagnosis assessed by positive genotypic assay and confirmed by phenotypic conventional solid culture method was 78.12%. The yield of GeneXpert in revealing rifampicin resistance was high (n=79/512; 15.43%) with 12.5% additional resistance against isoniazid revealed by positive solid cultures and drug susceptibility test. GeneXpert assay is a very useful method for rapid detection of MTB and drug resistance, which facilitates timely diagnosis and appropriate management of pulmonary tuberculosis.

Utility of GeneXpert Mycobacterium tuberculosis/rifampicin assay in smear-negative pulmonary tuberculosis

Background: Tuberculosis (TB) remains one of the world's deadliest communicable diseases. An alarming rise in the incidence of Mycobacterium tuberculosis (MTB) has prompted the need for rapid diagnostic techniques. Despite low sensitivity in MTB detection, sputum microscopy remains the main diagnostic method, especially in resource-limited settings. Mycobacterial culture is the gold standard method for TB diagnosis; however, the use in clinical practice is limited due to longer time, biosafety requirements, and high cost. In December 2010, the World Health Organization (WHO) 1st endorsed the usage of Xpert MTB/rifampicin (RIF) assay. The WHO recommends that GeneXpert test could be used as a follow on test to microscopy, especially in smear-negative specimens. Objectives: To assess percentage of Tuberculosis cases and Multi Drug Resistant (MDR) case detection at diagnosis using Xpert MTB/RIF assay in smear-negative pulmonary cases. Materials and Methodology: Detailed relevant clinical history and examination were carried out for 105 pulmonary TB suspect patients. Sputum acid-fast bacilli smear two samples and human immunodeficiency virus status were checked under RNTCP; chest X-ray PA view was done. Smear-negative patients were subjected to bronchoscopy and bronchoalveolar lavage (BAL)/bronchial washing. BAL/Bronchial washing was tested for GeneXpert. Interpretation and Conclusion: MTB was detected in 21 (20%) samples out of 105 samples and RIF resistance was detected in 1 (1%) sample. Most common chest x-ray abnormalities were cavity (33.3%) and consolidation (33.3%). 8 cases (38.1%) among Gene xpert positives had past history of Tuberculosis. Comorbidities were found in 5 cases (23.8%) among Gene Xpert positives. The present study concludes that using standard clinical assessment for selection, five patients need to be tested to detect one case of smear-negative TB. Smear-negative patients could benefit from Xpert, particularly in areas where no culture is available.

Study on Virulence Genes of Type III Secretion System of Pseudomonas aeruginosa in Xinjiang Province.

To investigate the distribution of virulence genes exoS and exoU of Pseudomonas aeruginosa type III secretion system and their antimicrobial resistance characteristics in Xinjiang Province. A total of 228 isolates of Pseudomonas aeruginosa were collected from January 2017 to April 2017 in our hospital. The VITEK2-compac system was used for strain identification and antimicrobial susceptibility test. The disk diffusion method was used for antimicrobial susceptibility supplementation. PCR method was used for detection of exoS and exoU virulence gene. Among 228 isolates of Pseudomonas aeruginosa, 178 (78.07%) were positive for exoS gene, 91 (39.91%) were positive for exoU gene, and 21.49% of the isolates carried both genes (exoU+/exoS+). A total of 30 MDR strains were detected, accounting for 13.16%. The antimicrobial resistance of the exoU+ group was 76.67%, which was significantly higher than that of the exoU-group (23.33%). The difference was statistically significant (p < 0.001). The detection rate of fluoroquinolone-insensitive strains in exoU+ group was as high as 57.45%, which was significantly higher than 42.55% in exoU-group and the difference was statistically significant (p < 0.05). The 30-day mortality rate of the exoU+ group was 8.79%, which was higher than that of the exoU-group (4.38%), and the difference was statistically significant (p < 0.05). The expression of exoU gene is associated with multidrug resistance, fluoroquinolone resistance, and prognosis. We should enhance the detection of drug resistance and study the pathogenesis and regulation mecha-nism of T3SS, in order to provide new ideas for the design of reasonable treatment strategies and the development of new therapeutic drugs.

ODELAM: Rapid Sequence-independent Detection of Drug Resistance in Mycobacterium tuberculosis Isolates.

Antimicrobial-resistant Mycobacterium tuberculosis (Mtb) causes over 200,000 deaths globally each year. Current assays of antimicrobial resistance require knowledge of the mutations that confer drug resistance or long periods of culture time to test growth under drug pressure. We present ODELAM (One-cell Doubling Evaluation of Living Arrays of Mycobacterium), a time-lapse microscopy-based method that observes individual cells growing into microcolonies. This protocol describes sample and media preparation and contains instructions for assembling the ODELAM sample chamber. The ODELAM sample chamber is designed to provide a controlled environment to safely observe the growth of Mtb by time-lapse microscopy on an inverted wide-field microscope. A brief description of the ODELAM software is also provided here. ODELAM tracks up to 1500 colony forming units per region of interest and can observe up to 96 regions for up to seven days in a single experiment. This technique allows the quantification of population heterogeneity. ODELAM enables rapid quantitative measurements of growth kinetics in as few as 30 h under a wide variety of environmental conditions. Graphic abstract: Schematic representation of the ODELAM platform.

New drugs and treatment recommendations - what are the consequences at the TB-laboratory?

Aims &amp; objectives: Resistance to TB drugs is a severe obstacle to effective TB control globally. Timely detection of multidrug-resistant TB (MDR-TB) is crucial to avoid poor cure rates and further development and transmission of increasingly drug-resistant strains of M. tuberculosis. Molecular rapid tests are recommended by WHO for the early detection of mutations associated with drug-resistance. When resistance to rifampicin and/or isoniazid is detected, additional DST should be performed for additional drugs. Optimally all drugs used in the therapy of MDR-TB should be tested, and regimens include only drugs to which the patient's isolate has documented susceptibility. The recent introduction of new anti-TB drugs made more effective and shorter treatment of MDR-TB possible. An example is the WHO recommended all-oral bedaquiline-containing MDR-TB regimen (WHO TB treatment handbook 2020) which contains bedaquiline, levofloxacin/moxifloxacin, clofazimine, ethionamide, ethambutol, isoniazid (high dose) and pyrazinamide for 4 months followed by 5 months of treatment with levofloxacin/moxifloxacin, clofazimine, ethambutol and pyrazinamide. The use of rapid diagnostic tests and more effective MDR-TB treatment regimens has the potential to improve treatment outcomes and reduce transmission of MDR-TB. Results: The introduction of new drugs and WHO recommendations for the therapy of MDR-TB put increased demands on TB-laboratories around the world to update their diagnostics algorithms to make sure they reflect new treatment regimens. If possible, DST should be performed for all drugs used in modern MDR-TB therapy. It is a challenge for TB laboratories to introduce DST for several compounds not earlier tested. On the other hand, DST of obsolete drugs such as the second line injectables, and especially kanamycin and capreomycin which are no longer recommended for use should be discontinued. Standardized protocols with critical concentrations for solid or liquid culture-based assays are needed to enable a successful re-focusing of DST at clinical TB laboratories. The possible added value of MIC determinations, as an alternative to just testing a single critical drug concentration, should be considered especially for the new drugs where we have more limited experience. Irrespectively of the method, there is a strong need for relevant drug-susceptible and drug-resistant control strains to ensure high quality when a new DST method is introduced. It is also of importance to develop and implement QC/EQA methods for all drugs tested. The need for knowledge transfer and training should not be neglected. Conclusions: Extended DST is initially a task of the NRL. In high drug-resistant TB burden areas, there might be a subsequent need for decentralization. To meet the demands of rapid detection of drug resistance there is a need for increased use of molecular tests. Such tests promise to be increasingly useful, but they can not be initiated for new drugs until we have a sufficient understanding of which genetic markers need to be examined to predict clinically relevant drug resistance in a reliable way. Research is ongoing to determine this for the new drugs and I expect to see such tests developed in the not so far future.

MfloDx® - an innovative technology for MDR-TB antibiotic resistance profiling

Aims &amp; objectives: The aim of this work was to evaluate the performance of mfloDx® MDR-TB assay kit on DNA extracted from 100 pulmonary sputum samples. mfloDx® MDR-TB is a qualitative test that can identify bacteria belonging to the Mycobacterium tuberculosis complex (MTC) as well as robustly identify single-nucleotide variants of the most common mutations in multidrug resistant M. tuberculosis DNA. The current assay can detect the most prevalent mutations causing resistance to Rifampicin (RIF) and Isoniazid (INH). Methods: Genes of interest were selectively enriched and targeted using amplifiable padlock probes (PLP). PLPs that recognize mutation or wild type were ligated upon specific recognition of the correct target sequence. Once PLPs were circularized, they underwent rolling circle amplification. Polymers resulting from this amplification were monomerized and subsequently visualized on disposable cassettes. The developed bands allowed for accurate genotyping of eleven resistance-related mutations, as well as respective wild type alleles and MTC identification. Results: From 100 samples; 81 were successfully genotyped, 16 were negative for MTC, two were inconclusive and one ran out during analysis. Among the genotyped samples; 46 were resistant to both RIF and INH, eight samples were resistant only to INH, three samples were resistant only to RIF. The remaining samples were susceptible to both drugs. Clinical sensitivity and specificity were calculated based on phenotypic drug susceptibility test results (MGIT culture) as reference. Sensitivity was 89.6% for RIF and 92% for INH resistance. Specificity was 95.5% for RIF and 89.5% for INH susceptibility. Conclusions: In conclusion, mfloDx® MDR-TB offers a reliable and affordable rapid drug-susceptibility testing for MDR-TB with potential implications in early detection of drug-resistance in resource-limited, high burden TB and MDR-TB settings. It enables clinicians to ensure the timely administration of effective drug therapy which will not only reduce the risk of treatment failure but also limit the further development and spread of dug resistant TB.