Drug susceptibility testing methods

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Discordance across Several Methods for Drug Susceptibility Testing of Drug Resistant Mycobacterium tuberculosis Isolates in a Single Laboratory

Discordance across Several Methods for Drug Susceptibility Testing of Drug Resistant Mycobacterium tuberculosis Isolates in a Single Laboratory

We think it is both intuitive and logical to use a quantitative susceptibility method for MDR-TB detection, such as the Trek MycoTB plate or the phage qPCR method. The clinician may seek to maximize dosing for an isolate with a MIC or quantitative result indicating a borderline susceptible range, as discussed for the fluo- roquinolone, or even continue a medication if the MIC or quan- titative result is at the lower end of the resistance range in the setting of extensively drug-resistant TB or within a limited for- mulary. Both of these methods were readily established in the laboratory, provided information for a broad range of drugs, were relatively rapid, and could be useful for DST surveillance or individualization of multidrug regimens. Of course, interpre- tation of a quantitative range is new territory for the TB field, but so are the issues of complex drug resistance that we are now facing. Some operational aspects of the methods should be men- tioned. MTBDRplus and the Xpert MTB/RIF can of course be performed on sputum samples (preferably smear positive) as a direct DST, which saves time over methods requiring culture. The Xpert MTB/RIF method was the fastest (2 h 40 min) and required the fewest repeats, but it is limited to RIF susceptibility testing. The MTBDRplus line probe assay additionally yields INH suscep- tibility information, but it required more repeats due to missing control bands. All other methods (L-J proportion, MGIT 960, MycoTB, and phage qPCR), being culture based, required time to obtain an adequate isolate. With the MGIT 960 SIRE AST method, when a valid result could be obtained using a seed tube from the primary MGIT culture, the turnaround time was good; however this method required the most repeat testing due to contamina- tion. The MycoTB MIC plate method required the least special- ized equipment of all the methods: an incubator and a multichan- nel pipette. When this test was valid on the first try, the turnaround time was 21 days when we used growth from solid medium. However, contamination or no growth may not be ap- parent for 21 days with this method; thus, when repeat testing is required, the turnaround time doubles to 42 days. For the D29 phage method, contamination was less problematic, due to the short incubation times and specificity of the D29 phage, and this is the easiest method to customize for laboratory-specific drug pan- els. We found that this and other culture-based methods per- formed best when our slow-growing MDR and extremely drug- resistant isolates were subcultured on 7H11 agar.
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Comparative Evaluation of FUNGITEST and Broth Microdilution Methods for Antifungal Drug Susceptibility Testing of Candida Species and Cryptococcus neoformans

Comparative Evaluation of FUNGITEST and Broth Microdilution Methods for Antifungal Drug Susceptibility Testing of Candida Species and Cryptococcus neoformans

Analysis of results. Tentative breakpoints have been established for flucon- azole and itraconazole, allowing isolates of Candida spp. tested according to NCCLS guidelines to be classified as susceptible, susceptible dependent upon dose, or resistant (15). However, these breakpoints do not correspond to the breakpoint concentrations selected for the FUNGITEST method. Moreover, NCCLS breakpoints have not been established for the other four drugs included in FUNGITEST or for C. neoformans. For these reasons we classified isolates that were inhibited from growth at the lower of the two drug concentrations used in FUNGITEST as susceptible, those inhibited from growth at the higher of the two concentrations as intermediate, and those inhibited from growth at neither of the two concentrations as resistant. To permit comparisons with the broth microdilution reference method, the same breakpoints were applied to both tests. Major discrepancies were defined as results that classified the isolate as susceptible by one method and resistant by the other, and minor discrepancies were defined as variations from resistant to intermediate or intermediate to susceptible between the two methods.
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Theoretical Investigation on Antimicrobial Susceptibility Testing Methods

Theoretical Investigation on Antimicrobial Susceptibility Testing Methods

AMR [Resistance to antimicrobial agents] has resulted in morbidity and transience from treatment failures and improved health awareness expenses. Suitable antimicrobial drug use has unquestionable advantage, but physicians and the public frequently use these agents inappropriately. Inappropriate use results from physicians providing antimicrobial drugs to indulge viral infections, using insufficient criteria for diagnosis of infections that potentially have a bacterial aetiology, gratuitously prescribing expensive, broad-spectrum agents, and not following recognized recommendations for using chemo prophylaxis. The simple accessibility of antimicrobial drugs leads to their incorporation into herbal or "folk" remedies that also increase unsuitable use of these agents. Antibiotic usage exerts a selective pressure that acts as a driving force in the progress of antibiotic resistance. The connection between increased rates of antimicrobial use and resistance has been predictable for nosocomial infections as well as for resistant area acquired infections. Resistance factors carried on mobile elements, can spread rapidly within human and animal
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Interlaboratory drug susceptibility testing of Mycobacterium tuberculosis by a radiometric procedure and two conventional methods

Interlaboratory drug susceptibility testing of Mycobacterium tuberculosis by a radiometric procedure and two conventional methods

A total of 224 recent isolates of Mycobacterium tuberculosis from 163 patients selected to have multidrug resistance were tested against streptomycin SM, isoiiazid, rifampin, and ethambu[r]

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Direct susceptibility testing for multi drug resistant tuberculosis: A meta analysis

Direct susceptibility testing for multi drug resistant tuberculosis: A meta analysis

We presented data for TTR for 100% of the DST results to permit comparison of rapidity between the different tests. For the MODS and NRA tests, the average TTR was within 23 days compared with the 2 months required for conven- tional indirect testing. Moreover, most results were ready in 7–14 days for MODS (data not shown). Contamina- tion and indeterminate results in phenotypic methods may prolong the time to the final result but this was diffi- cult to quantify in this study. For the genotypic assays, the only study that indicated TTR reported 2 days, but the pro- tocol of these genotypic assays allows DST results within 1–2 days [36]. The risk of amplicon contamination is a problem in PCR-based tests. This could prolong the time to results as repeat testing or new samples have to be ana- lysed. From this study however, it was evident that direct testing with any of the studied tests significantly shortens the time to detection of MDR TB, and would permit timely decision on patient management. This is supported by a retrospective study of the impact of direct MODS assay in a clinical setting where DST results in 82.8% of the cases were available before those of any standard method. In 41% of these cases, the rapid results should have prompted a timely modification in patient manage- ment [37].
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Rapid identification of extensively and extremely drug resistant tuberculosis from multidrug resistant strains; using PCR-RFLP and PCR-SSCP.

Rapid identification of extensively and extremely drug resistant tuberculosis from multidrug resistant strains; using PCR-RFLP and PCR-SSCP.

respectively) and inactivate it (7). The quinolone- resistance determining region (QRDR) of the gyrA and gyrB genes is the conserved region and mutation in this region is responsible for resistance to FQs. Mutation were frequently reported at codon 88 to 94 of the gyrA gene, however, a less frequently mutation was also seen at codon 495, 516, 533 of gyrB gene (8). Resistance to amikacin (AMK) is associated with nucleotide change at positions 1400 (substitution A to G) 1401 (substitution C to A) and 1483 (substitution G to T) in the rrs gene (that encoding 16s rRNA) (9). At present, detection of drug resistance is performed by proportional methods. It takes at least 6 to 12 weeks to determine the susceptibility patterns. In the present study, we tried to identify the MDR and XDR-TB isolates using molecular techniques, i.e., PCR-RFLP (PCR-Restriction fragment length polymorphism) and PCR-SSCP (PCR-Single-strand conformation polymorphism). Thereafter, the results were compar- ed with sequencing and classical susceptibility testing. MATeRIALS AND MeThODS
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Evaluation of microscopic observation drug susceptibility assay for diagnosis of multidrug resistant Tuberculosis in Viet Nam

Evaluation of microscopic observation drug susceptibility assay for diagnosis of multidrug resistant Tuberculosis in Viet Nam

Detection rates for MODS, MGIT and LJ were summar- ized and compared between methods using McNemar ’ s test. The accuracy of MODS for diagnosis of TB was then assessed using MGIT and LJ as the gold-standard refer- ence test, i.e. a sample was defined as positive by the refer- ence test if either MGIT or LJ (or both) were positive. The accuracy of MODS for drug-susceptibility testing was assessed in samples with a valid drug susceptibility test result by both MODS and DST-LJ. The gold-standard reference test was the DST-LJ result. We also summarized agreement between DST-MODS and DST-LJ as raw agreement and by Cohen’s kappa. Confidence intervals for accuracy measures (sensitivities, specificities, positive and negative predictive values) were calculated according to the method of Pearson and Clopper. Finally, we compared the time to a positive test for MGIT and MODS, respec- tively, in samples positive by both methods using the Wilcoxon signed rank test and visualized it using the empirical cumulative distribution.
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Testing of Inoculation Methods and Susceptibility Testing of Perspective Cabbage Breeding Lines (Brassica Oleracea convar. Capitata) to the Black Rot Disease Caused by Xanthomonas Campestris pv. Campestris

Testing of Inoculation Methods and Susceptibility Testing of Perspective Cabbage Breeding Lines (Brassica Oleracea convar. Capitata) to the Black Rot Disease Caused by Xanthomonas Campestris pv. Campestris

All inoculation methods proved exploitability of artificial infection with Xcc and led to the expression of symptoms. Presented symptoms ranged within the entire scale. Methods were compared by the total reaction of all cultivars to the infection (Fig. 1). The carborundum abrasion method (no. 4) could not be evaluated because of significant damage of leaves by this method. The pressurized spraying (no. 1) showed the statistical difference from other inoculation methods and led to the lowest symptoms. Otherwise it is a useful tool for non‑invasive inoculation by hydathodes because the symptom expression was obtained for all tested cultivars. In case of methods using inoculation by wounds, statistical differences were not found. The inoculation by injection (no. 2) recorded commonly higher disease severity than multiple pricking (no. 3) and scissor clipping (no. 5). Regarding the aspects of inoculating time, equipment and clarity between symptoms caused by pathogen and by plant reaction to wound, the multiple pricking method proved the highest exploitability. Based on these results, the spraying (non‑invasive method) and multiple pricking (invasive method) were considered to be suitable for resistance testing.
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Extensively drug resistant tuberculosis in Mali: a case report

Extensively drug resistant tuberculosis in Mali: a case report

Data on XDR-TB are scarce in Africa, especially in West African countries [2, 3]. We describe here the first docu- mented XDR patients in Mali. Extensively drug resistant TB patients were identified in neighbouring countries Burkina Faso in 2010 and Cote d’Ivoire in 2015 [5, 6]. As culture and DST were not performed during the first episodes of TB infection in the patients presented here, we cannot exclude primary pre-XDR resistance. Also in the XDR patients in Burkina Faso and Cote d’Ivoire [5, 6] baseline resistance tests were missing, whereas in South Africa primary resistance was well documented in XDR patients [7]. The NTM isolated from the first culture of patient 1 may have been a ‘colonizer’ that obscured ongo- ing TB disease, or may have contributed to chronic pul- monary infection in this HIV co-infected patient [8]. These patients also highlight the need for new diagnos- tic tools that could simultaneously detect the MTBc and NTM. In addition, both patients experienced treatment interruptions, and patient 1 was inappropriately treated with a weak regimen based on kanamycin for more than 6  months after diagnosis of high level fluoroquinolone resistance [9], which likely caused the additional resist- ance to injectables, resulting in XDR-TB. Lastly, the inef- fective treatment and poor hospital infection control likely permitted the possible nosocomial superinfection from patient 1 to patient 2 and 3. Urgent treatment ini- tiation limits morbidity, mortality, and ongoing trans- mission [10, 11]. Here, early initiation of appropriate treatment could have stopped the possible nosocomial transmission and may have prevented the third XDR-TB patient. Despite the resource limited condition with the Mali NTP, it is high time that all recommended steps for programmatic management of drug-resistant tuberculo- sis (PMDT) implementation for strengthening the MDR TB program are taken to serve patients with rifampicin resistance in Mali.
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Clonal expansion of Mycobacterium tuberculosis isolates and coexisting drug resistance in patients newly diagnosed with pulmonary tuberculosis in Hanoi, Vietnam

Clonal expansion of Mycobacterium tuberculosis isolates and coexisting drug resistance in patients newly diagnosed with pulmonary tuberculosis in Hanoi, Vietnam

Methods: We collected 346 clinical isolates from previously untreated patients with smear-positive active TB in Hanoi, the capital of Vietnam. Of these, 339 were tested for susceptibility to four first-line anti-TB drugs, including isoniazid (INH), rifampicin (RMP), streptomycin (SM), and ethambutol (EMB), using the proportion method. A pyrazi- namidase (PZase) test was used to assess pyrazinamide (PZA) resistance. Results of the culture-based drug suscepti- bility tests were confirmed by those from reverse hybridization-based line probe assays (LiPAs) that detected mutations associated with RMP, INH, PZA, and fluoroquinolone (FQ) resistance. To investigate a diversity of these strains, IS6110-probed restriction fragment length polymorphisms (RFLPs) were analyzed. Nucleotide sequences for furA-katG and fabG1-inhA operons, transcription units responsible for INH resistance, were also determined. Results: Of the isolates tested, 127 (37.5%) were resistant to at least one of the four drugs, which included 93 (27.4%) isolates that were resistant to INH. RFLP analysis identified four clusters defined by similarity of the band patterns, which accounted for 46.1% of the tested isolates. Among the clustered isolates, 37.7% were resistant to INH, most of which (85.4%) carried a g944c mutation, which causes an S315T amino acid substitution, in the katG gene.
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Conventional and radiometric drug susceptibility testing of Mycobacterium tuberculosis complex

Conventional and radiometric drug susceptibility testing of Mycobacterium tuberculosis complex

The following characteristics were determined for each test: sensitivity, the capacity of the test method to distinguish correctly resistant strains = D/C + D; specificity, the capacity [r]

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Screening for Plasmid-Mediated Multidrug Resistant Bacteria in Ikpoba River Water Samples

Screening for Plasmid-Mediated Multidrug Resistant Bacteria in Ikpoba River Water Samples

Plasmid curing was carried out on the multi-drug resistant isolates using the methods earlier described by [17] and used by [18]. The curing agent used was sodium dodecyl sulphate (SDS). Physical evidence for the presence or loss of plasmid(s) in cured and non-cured isolates was obtained by alkaline phosphate method of rapid DNA isolation technique [19,20]. This involved four basic steps – cell harvest, lysis, deproteination and decontamination. The plasmids were characterized using agarose gel electrophoresis [21]. The molecular weights of plasmids were visualized using UV transilluminator (AlphaImager TM 2200) at 302-365nm. Thereafter the susceptibility patterns of the plasmid cured isolates was performed and compared with the original isolates.
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Evaluation of Molecular Tools for Detection and Drug Susceptibility Testing of Mycobacterium tuberculosis in Stool Specimens from Patients with Pulmonary Tuberculosis

Evaluation of Molecular Tools for Detection and Drug Susceptibility Testing of Mycobacterium tuberculosis in Stool Specimens from Patients with Pulmonary Tuberculosis

In resource-poor settings, patients are often tested only for MDRTB if they have known risk factors for MDRTB, such as past tuberculosis treatment, or if their disease does not im- prove during the first months of therapy. The latter strategy for selective MDRTB testing of follow-up samples collected dur- ing therapy is microbiologically challenging because first-line tuberculosis therapy administered empirically for unrecog- nized MDRTB often causes sputum cultures to become neg- ative despite not achieving a long-term cure (16). Stool PCR tests for the presence of M. tuberculosis DNA derived from living or dead mycobacteria in swallowed sputum and may be particularly well suited to MDRTB testing of follow-up sam- ples collected during therapy. We therefore tested approxi- mately equal numbers of “diagnostic samples” obtained from newly diagnosed patients and “follow-up samples” obtained from patients who were already established on tuberculosis treatment.
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Epidemiological trends and outcomes of extensively drug resistant tuberculosis in Shandong, China

Epidemiological trends and outcomes of extensively drug resistant tuberculosis in Shandong, China

Shandong province lies in the east coast of China and is the second largest province with a population of 95 million. In 2013, 35,971 people in Shandong were infected with latent TB. Shandong Provincial Chest Hospital (SPCH) is the only provincial-level hospital specializing in TB clinical service and control. This retrospective study enrolled con- secutive human immunodeficiency virus (HIV)-uninfected culture-positive Mycobacterium tuberculosis cases con- firmed and treated in SPCH between January 2008 and December 2015. Trained research clinicians reviewed the medical records of these TB patients and abstracted their drug susceptibility test (DST) results along with the corre- sponding sociodemographic and clinical information using a standard case report form.
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Antituberculosis Drugs Resistance and Treatment Outcomes Among Retreatment Patients in Guinea: A Five-Year Retrospective Cohort Study

Antituberculosis Drugs Resistance and Treatment Outcomes Among Retreatment Patients in Guinea: A Five-Year Retrospective Cohort Study

Globally in 2017, there were an estimated 558 000 new cases of rifampicin resistant TB (RR-TB), of which almost 82% were multidrug-resistant TB (MDR-TB) [2]. Inadequate treatment and non-adherence to treatment result in drug resistance in patients undergoing retreatment, especially those admitted for retreatment failure [8-10]. Retreatment TB patients represent those who have been treated previously for one month or more with anti-TB drugs and who have been diagnosed once again with the disease. These patients mainly include relapses, treatment after failure, or loss to follow-up on a first-line treatment regimen [1]. The number of these patients is not negligible. Worldwide in 2017, of the 6.7 million TB cases that were officially notified by national TB programmes to the WHO, 260,000 patients were already previously treated [2]. It has been globally estimated that 20% of previously treated tuberculosis cases would have developped MDR-TB [6].
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Validation of the GenoType®MTBDRplus assay for diagnosis of multidrug resistant tuberculosis in South Vietnam

Validation of the GenoType®MTBDRplus assay for diagnosis of multidrug resistant tuberculosis in South Vietnam

mated prevalence of 92.8 per 100,000 (NTP, unpublished data, 2006). M. tuberculosis resistance to INH is common (16-25% among new patients) [4,5]. Among patients experiencing a first episode of TB these two studies reported MDR-TB rates of 2 and 4%, and of 23% and 27% among previously treated patients, respectively [4,5], whereas 80% chronic patients had MDR-TB [6]. Geneti- cally, approximately half of the strains belongs to the East-African Indian clade whereas the other half are of the Beijing genotype, which was found to be strongly associated with (multi-)drug resistance [7,8].

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An investigation of Stenotrophomonas maltophilia positive culture caused by fiberoptic bronchoscope contamination

An investigation of Stenotrophomonas maltophilia positive culture caused by fiberoptic bronchoscope contamination

SMA, the sole species in the genus Stenotrophomonas, is a major conditional pathogen and agent of nosocomial infection. SMA infection rates are higher for elderly patients undergoing invasive procedures such as trache- otomy, endotracheal intubation and ventilator assisted ventilation. China’s CHINET annual drug resistance monitoring data for 2011 showed SMA accounting for 4.45% of all gram-negative bacteria and 11.61% of non- fermentative bacteria [5]. SMA is highly resistant to most antibacterial drugs used in clinical practice. Re- cently, with the widespread use of broad-spectrum anti- bacterial drugs and the implementation of invasive diagnostic techniques, both the incidence of SMA infec- tions and drug resistance has increased every year [6].
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Comparison of Nine Phenotypic Methods for Detection of Extended Spectrum β Lactamase Production by Enterobacteriaceae

Comparison of Nine Phenotypic Methods for Detection of Extended Spectrum β Lactamase Production by Enterobacteriaceae

VITEK2 system (bioMe ´rieux). We tested four Vitek2 antimicrobial suscepti- bility test cards, including two standard cards, AST-N017 and AST-N052, as well as their corresponding extended cards, AST-EXN3 and EXN5. N017 and N052 include ␤ -lactam antibiotics, such as CTX, CAZ, and cefoxitin, and differ only by the replacement of ticarcillin-clavulanic acid (TIM) by ertapenem in N052. EXN3 and EXN5 cards test for susceptibility to ceftriaxone, FEP, and ATM, and, in addition, EXN5 tests for susceptibility to three cephalosporins with or without clavulanic acid (CLA) to detect the production of ESBLs. The EXN5 extended card is currently only validated for detection of ESBLs in E. coli, K. pneumoniae, and Klebsiella oxytoca. An expert system (AES) interprets the re- sults obtained with Vitek2 by using nine different phenotypes relevant to ␤-lac- tam antibiotics, including the wild type, and ESBL production. For each card, we considered a strain ESBL positive if the phenotypic interpretation by the AES included ESBL with or without decreased outer membrane permeability (i.e., porin loss) and negative if only the wild type or ␤-lactamases other than ESBLs were proposed by AES. All other interpretation results were considered inde- terminate (ND).
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Tackling tuberculosis: insights from an international TB Summit in London

Tackling tuberculosis: insights from an international TB Summit in London

Anti-TNF therapies for rheumatism have been very effective and have led to the development of several artificial monoclonal antibodies such as infliximab and rituximab that act as TNF ago- nists. However, TNF is pivotal in the immune response to M. tuberculosis and helps contain the infection. 72 Therefore, the use of these agonists may increase the risk of patients developing TB or other complications from infections by non-tuberculous mycobacteria. Keane et al. showed that there were 70 reported cases of TB, usually extra-pulmonary, from the 147,000 patients administered with infliximab worldwide between 1998–2001. 73 Almost all of the TB cases were reported in countries with low incidence of TB making this association a highly alarming one. Etarnecept, another TNF antagonist was found to fare marginally better than infliximab and adalimumab, but the TB incidences still remain high. 74,75 Professor O.M. Kon, Imperial College Healthcare NHS Trust (UK), highlighted the probability of most of these cases being LTBI that are activated as the host immune surveillance drops and recommend testing for it before initiating treatment for rheumatism. Therefore diagnosing LTBI in these patients is a high priority. Patients are usually risk- assessed, taking into account the risk of prophylaxis versus devel- oping TB and then put forward for diagnosis. However, risk stratification misses 2-thirds of the cases thereby lowering the sensitivity manifold. For diagnosis, chest X-rays, TST and IGRA are the methods of choice. The TST can be used for diagnosis but patients with rheumatism usually have an attenuated response to the test. 76 IGRAs in this case are highly specific for the detection of LTBI unless steroids are administered to the patient. In the absence of guidelines for practitioners, Professor Kon suggested a thorough follow-up and surveillance of patients by taking into account their contact and travel histories as treat- ment with biologics has been noted to be associated with reacti- vation of TB many years post cessation of treatment.
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Inaccuracy of the Disk Diffusion Method Compared with the Agar Dilution Method for Susceptibility Testing of Campylobacter spp

Inaccuracy of the Disk Diffusion Method Compared with the Agar Dilution Method for Susceptibility Testing of Campylobacter spp

In conclusion, our results show that the disk diffusion method may not be a reliable tool for susceptibility testing of Campylobac- ter spp. This is a major concern due to the wide use of the disk diffusion method in routine clinical laboratories as well as in some research laboratories. Accurate determination of Campylobacter susceptibility and resistance is of vital importance to ensure an adequate antimicrobial therapy for patients with severe forms of the disease and, also, to efficiently monitor the antimicrobial re- sistance situation of Campylobacter spp. worldwide. Further stud- ies are needed to assess whether the disk diffusion test method could be improved or whether all susceptibility testing of campy- lobacters should be done using an MIC-based method.
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