Xpert Assay Research Articles

Diagnostic Performance of Xpert MTB/RIF Ultra Compared with Predecessor Test, Xpert MTB/RIF, in a Low TB Incidence Setting: a Retrospective Service Evaluation.

ABSTRACTThe aim of this study was to evaluate the performance of Xpert MTB/RIF Ultra (Ultra) compared with its predecessor, Xpert MTB/RIF (Xpert), in the diagnosis of tuberculosis (TB) in a low TB incidence country. Retrospective analysis was performed on 689 clinical samples received between 2015 and 2018, on which Xpert was performed, and on 715 samples, received between 2018 and 2020, on which Ultra was performed. Samples were pulmonary (n = 830) and extrapulmonary (n = 574) in nature, and a total of 264 were culture positive for Mycobacterium tuberculosis complex (MTBC). The diagnostic performance of both assays was analyzed using culture as the reference standard. The sensitivity of Ultra for culture positive (smear positive and smear negative) MTBC samples, was 93.2% (110/118) compared with 82.2% (120/146) for Xpert (P = 0.0078). In smear negative-culture positive samples, Ultra had a sensitivity of 74.2% (23/31) versus 36.11% (13/36) for Xpert (P = 0.0018). Specificity of both assays was comparable at 94.8% (566/597) for Ultra and 95.8% (520/543) for Xpert (P = 0.4475). The sensitivity of Ultra and Xpert assays among exclusively pulmonary samples was 95.3% (82/86) and 90.3% (84/93), respectively (P = 0.1955), and 87.5% (28/32) and 67.9% (36/53), respectively, among extrapulmonary samples (P = 0.0426). Ultra showed improved performance compared with Xpert in a low TB incidence setting, particularly in smear negative and extrapulmonary MTBC disease. The specificity of Ultra was lower than Xpert, however, this was not statistically significant.IMPORTANCE The study demonstrates the improved sensitivity of the Ultra compared with the Xpert, particularly in smear negative TB disease, for both pulmonary and extrapulmonary samples in a low TB incidence setting. Cycle threshold (Ct) value for both assays was found to positively correlate with time to TB culture positivity, suggesting that Ct and semiquantitative results could be used as indicators of sample MTBC bacillary burden, and thus, perhaps, of transmission potential. This may have implications for the designation of patient isolation precautions.

Comparison of BD MAX Check-Points CPO assay with Cepheid Xpert Carba-R assay for the detection of carbapenemase-producing Enterobacteriaceae directly from rectal swabs

We compared the performance of 2 automated systems for detection of carbapenemase-producing Enterobacteriaceae, BD MAX Check-Points CPO (CPO assay) and Xpert Carba-R assay, with culture confirmed by polymerase chain reaction as the reference method. Using 867 samples from 627 patients, the overall sensitivity, specificity, total positive predictive value, and negative predictive value of the CPO assay were 95.7%, 96.5%, 60.8% and 99.8% and for the Xpert assay were 97.9%, 99.8%, 95.8%, and 99.9%, respectively. The cycle threshold values (Ct value) of the false-positive CPO assay results were significantly higher than those of true-positive cases (P < 0.001). By applying a modified cut-off Ct value of 37.3 for klebsiella pneumoniae carbapenemases (KPC), the positive predictive value for KPC was improved from 52.9% to 89.5%. The CPO assay will be useful when handling many specimens, as tests are conducted in batches. However, positive cases showing high Ct values should be confirmed by another assay to rule out false positivity.

Usability of HIV-1 Assay Using DBS for Early Infant Diagnosis in Field Settings in Kenya.

The Xpert HIV-1 Qualitative assay has been in use in Kenya since 2016 for infant diagnosis of HIV. Recently, the assay has been improved and its impact of this on ease of use is yet to be determined. We sought to determine the usability of Xpert® HIV-1 Qual XC assay using dried blood spots (DBS) for early infant diagnosis following this improvement. This was a cross-sectional usability study undertaken in 2 selected health facilities in Kenya from October 2020 to February 2021. The laboratory technicians were retrained for this study. HIV-exposed infants were recruited with the consent of their parents. Patient data were recorded, and DBS samples were collected from the infants and tested for HIV on the improved assay. Each laboratory technician performing the assay documented usability characteristics on the provided questionnaire. Data on test errors were collected from the machine logs and analyzed using STATA for Windows. Of 313 test cartridges, 265 (84.66%) were successfully tested on the GeneXpert platform, and 263 valid outcomes were used for comparison with the Roche CAP/CTM HIV-1 Qualitative assay. The sensitivity, specificity, and accuracy of the Xpert HIV-1 Qualitative assay on DBS was 100%. Overall, 48 (15.34%) errors were recorded; 40 (83.33%) were user related and 8 (16.67%) were hardware related. All 4 (4/4, 100%) participating laboratory technicians said the assay had a simple workflow, was easy to use, the tests results were easy to interpret, and the assay throughput was sufficient for their workload. The improved Xpert HIV-1 Qual XC assay is highly accurate, has a simple workflow, and is easy to use and easy to interpret. Both hardware- and user- related errors are common.

Diagnostic role of Xpert-MTB RIF assay in osteoarticular tuberculosis: A retrospective study

BACKGROUNDOsteoarticular tuberculosis (OATB) is a severe form of extrapulmonary tuberculosis (TB), which causes notable morbidity and warrants a high index of suspicion for prompt management. The diagnosis of OATB poses a challenge, because of the difficulty of collecting the samples and, secondarily, the paucibacillary nature of lesion, which gives poor sensitivity and reproducibility, with long turnaround time of routine/conventional laboratory tests and the requirement for invasive procedures and expertise. The Xpert MTB/RIF assay has been approved by the World Health Organization as a rapid diagnostic tool for diagnosing pulmonary and extrapulmonary TB.AIMTo emphasize the diagnostic efficiency of gene Xpert for OATB in suspected patients in a tertiary care hospital of Eastern India.METHODSThis retrospective study was conducted in the Department of Microbiology and Orthopaedics by analyzing the data of the gene Xpert assay over a 3-year duration from January 2018 to February 2021. Demographic and clinical data were recorded. The diagnostic efficiency of gene Xpert was evaluated against the composite reference standard (CRS).RESULTSA total of 37 cases fell into positive, probable, and possible categories of osteoarticular TB out of 112 patients included in the study by CRS; gene Xpert result was positive in 35 out of the 37 different CRS categorized cases. Of the 112 cases, culture was put in 40 cases, and, of these cultures, 5 cases showed the growth of MTB. Of these, 4 cases were included in the 35 cases diagnosed by gene Xpert. Smear microscopy was positive in 6 out of 37 CRS categorized cases. When compared with CRS, the sensitivity of gene Xpert assay, culture, and smear was found to be 94.6%, 13.5%, and 16.2%, respectively, while specificity in all the three types of tests was 100%. When kappa statistics were applied, the percentage of agreement gene Xpert, culture, and microscopy with CRS was found to be 95%, 20%, and 22.6%, respectively. Follow-up of the gene Xpert positive patients after getting anti-tubercular treatment revealed improved conditions.CONCLUSIONGene Xpert could detect 31 extra cases with a low and very low mycobacterial load that were missed by the routine culture methods. Hence, more samples should be processed for molecular diagnostic methods like gene Xpert along with other conventional methods for the validation of the molecular test prospectively for the timely diagnosis of osteoarticular TB.

Performance assessment of the new Xpert® HIV-1 viral load XC assay for quantification of HIV-1 viral loads

BackgroundHIV-1 RNA quantification is a key component of treatment monitoring. ObjectivesTo assess the performance of a redesigned HIV-1 RNA quantitative assay that uses a dual-target approach: Xpert® HIV-1 Viral Load (VL) XC. Study designFresh and frozen samples (N = 533) from HIV-1 positive patients tested with Abbott HIV-1 assays (Alinity m and RealTime [m2000]) were retested using the new Xpert XC assay. Three samples with known underquantification using the previous single-target Xpert assay were retested. ResultsThe Xpert XC assay yielded valid results in 98.5% (N = 528/536) of cases and showed high sensitivity in 80 fresh samples that had undetectable VLs or ≤1.7 log copies/mL with Alinity m. Linear regression and Bland-Altman analyses showed high concordance with the Abbott tests for quantified samples over a wide VL range (1.6–6.9 copies/mL), including non-B subtypes (mean difference=-0.1±0.23 log copies/mL). Mutations associated with integrase resistance did not impact the results. Very good linearity and reproducibility was shown for the tested subtypes B, CRF06_cpx, and CRF02_AG. Xpert XC VLs in samples that were previously underquantified using the original single-target Xpert assay were similar to those detected by the Abbott assays (±0.11 log copies/mL). ConclusionsThe Xpert XC assay showed excellent correlation with the Abbott assays for all tested HIV-1 subtypes. Sensitivity, linearity and accuracy were high in the therapeutically relevant VL range. With a time to result of only 90 min, this on-demand decentralized assay is a safe, reliable and fast option for VL monitoring in HIV-1-infected patients.

Performance of WHO-Endorsed Rapid Tests for Detection of Susceptibility to First-Line Drugs in Patients with Pulmonary Tuberculosis in Bangladesh.

The fast and accurate detection of susceptibility in drugs is a major challenge for a successful tuberculosis (TB) control programme. This study evaluated the performance of WHO-endorsed rapid diagnostic tools, such as BACTEC MGIT 960 SIRE (MGIT SIRE), GenoType MTBDRplus (MTBDRplus) and Xpert MTB/RIF (Xpert), for detecting susceptibility to first-line anti-TB drugs among pulmonary TB patients in Bangladesh. A total of 825 sputum samples with results from drug susceptibility testing (DST) against first-line anti-TB drugs in the MGIT SIRE, MTBDRplus and Xpert assays were evaluated and compared with the gold standard proportion susceptibility method of the Lowenstein–Jensen (LJ) medium. The overall sensitivities of MGIT SIRE were 97.6%, 90.0%, 61.3% and 44.9%, while specificities were 89.9%, 94.5%, 91.3% and 92.2% for detection of susceptibility to isoniazid (INH), rifampicin (RIF), streptomycin (STR) and ethambutol (EMB), respectively. For MTBDRplus, the sensitivities were 88.0% and 88.7%, and the specificities were 97.4% and 97.8% for the detection of susceptibility to INH and RIF, respectively. Xpert demonstrated a sensitivity and specificity of 94.8% and 99.5%, respectively, for the detection of RIF susceptibility. All tests performed significantly better in retreated TB patients compared with primary TB cases. For detection of RIF and INH susceptibility, all three assays showed almost perfect agreement with the LJ method, although MGIT SIRE exhibited low agreement for STR and EMB. Considering the high performance, shorter turnaround time and ease of use, molecular-based approaches Xpert and MTBDRplus can be widely implemented throughout the country for the rapid detection of drug-resistant TB.

Evaluating NG-Test CARBA 5 Multiplex Immunochromatographic and Cepheid Xpert CARBA-R Assays among Carbapenem-Resistant Enterobacterales Isolates Associated with Bloodstream Infection.

ABSTRACTDecreased susceptibility to carbapenems in Enterobacterales is an emerging concern. Conventional methods with short turnaround times are crucial for therapeutic decisions and infection control. In the current study, we used the Xpert CARBA-R (Cepheid, Sunnyvale, CA, USA) and the NG-Test CARBA 5 (NG Biotech, Guipry, France) assays for carbapenemase detection in 214 carbapenem-resistant Enterobacterales (CRE) blood isolates. We used the modified carbapenem inactivation method, conventional PCR, and sequencing to determine the production of five common carbapenemase families and their subtypes. We performed wzc-genotyping for all CR-Klebsiella pneumoniae (CRKP) and multilocus sequence typing for all carbapenemase-producing CRE isolates to reveal their genetic relatedness. The results showed a sensitivity of 99.8% and a specificity of 100% by the Xpert assay, and a sensitivity of 100% and a specificity of 99% by the NG-Test in detecting carbapenemases of 84 CRKP isolates with only one (VIM-1+IMP-8) failure in both tests. For CR-Escherichia coli, four carbapenemase-producing isolates were detected accurately for their subtypes. The two major clones of carbapenemase-producing CRKP isolates in Taiwan were ST11-K47 producing KPC-2 (n = 47) and ST11-K64 producing OXA-48-like (n = 9). Our results support the use of either test in routine laboratories for the rapid detection of common carbapenemases. Caution should be taken using the Xpert assay in areas with a high prevalence of CRE carrying blaIMP-8.IMPORTANCE Carbapenemase-producing Enterobacterales (CPE) are emerging worldwide, causing nosocomial outbreaks and even community-acquired infections since their appearance 2 decades ago. Our previous national surveillance of CPE isolates in Taiwan identified five carbapenemase families (KPC, OXA, NDM, VIM, and IMP) with the KPC-2 and OXA-48-like types predominant. Timely detection and classification of carbapenemases in CPE may be a useful test to guide optimal therapy and infection control. Genetic detection methods using the Xpert CARBA-R assay and the immunochromatographic assay using the NG-Test CARBA 5 have been validated with the advantage of short turnaround time. Our study demonstrated that the NG and Xpert assays are convenient methods to accurately identify carbapenemases in carbapenem-resistant Klebsiella pneumoniae and carbapenem-resistant Escherichia coli blood isolates. Detecting IMP variants remains challenging, and the results of Xpert CARBA-R assay should be carefully interpreted.

Rifampicin Resistance by Xpert MTB/RIF Assay in Pulmonary Tuberculosis- Is there a Need for Confirmation by Retesting?

Introduction: Xpert Mycobacterium tuberculosis/ resistance to Rifampicin (MTB/RIF) assay detects MTB complex (MTBC) and rifampicin resistance simultaneously. In high prevalence countries like India, detection of rifampicin resistance in sputum specimen of a newly diagnosed case of pulmonary TB with a low pretest probability needs to be confirmed by retesting. Aim: To evaluate the results of retesting of rifampicin resistant specimens in newly diagnosed pulmonary TB cases. Materials and Methods: A retrospective analysis of the data of Xpert assay was performed on specimens received in Department of Microbiology, Seth G.S. Medical College and KEM Hospital, Mumbai, Maharashtra, India. If rifampicin resistance was detected in a newly diagnosed case of Tuberculosis (TB), a second specimen was retested by Xpert assay for confirmation. Concordance of retesting was seen with results of Line Probe Assay (LPA). Results: Total 27,429 specimens were processed by Xpert assay of which 803 specimens showed rifampicin resistance, 157 sputum specimens fulfilling criteria of Programmatic Management of Drug resistant Tuberculosis (PMDT) guidelines were retested. High, medium, low and very low bacterial load was observed in 30, 51, 34 and 42 specimens’ respectively. All specimens having high or medium bacillary load showed rifampicin resistant result on retesting. On retesting 34 sputum specimens with low bacterial load, rifampicin resistance was confirmed in 30 specimens. LPA done after growing them by liquid culture confirmed rifampicin resistance in remaining four specimens. Conclusion: Xpert assay is recommended when the bacterial load identified by Xpert assay is very low and when there is discordance between Xpert results of rifampicin resistance and the reflex LPA testing.

Clinical Impact of the Line Probe Assay and Xpert® MTB/RIF Assay in the Presumptive Diagnosis of Drug-Resistant Tuberculosis in Brazil: A Pragmatic Clinical Trial.

ABSTRACTBackground: Rapid molecular methods such as the line probe assay (LPA) and Xpert® MTB/RIF assay (Xpert) have been recommended by the World Health Organization for drug-resistant tuberculosis (DR-TB) diagnosis. We conducted an interventional trial in DR-TB reference centers in Brazil to evaluate the impact of the use of LPA and Xpert. Methods: Patients with DR-TB were eligible if their drug susceptibility testing results were available to the treating physician at the time of consultation. The standard reference MGITTM 960 was compared with Xpert (arm 1) and LPA (arm 2). Effectiveness was considered as the start of the appropriate TB regimen that matched drug susceptibility testing (DST) and the proportions of culture conversion and favorable treatment outcomes after 6 months. Results: A higher rate of empirical treatment was observed with MGIT alone than with the Xpert assay (97.0% vs. 45.0%) and LPA (98.2% vs. 67.5%). Patients started appropriate TB treatment more quickly than those in the MGIT group (median 15.0 vs. 40.5 days; p<0.01) in arm 1. Compared to the MGIT group, culture conversion after 6 months was higher for Xpert in arm 1 (90.9% vs. 79.3%, p=0.39) and LPA in arm 2 (80.0% vs. 83.0%, p=0.81). Conclusions: In the Xpert arm, there was a significant reduction in days to the start of appropriate anti-TB treatment and a trend towards greater culture conversion in the sixth month.

Multiple Cross Displacement Amplification Combined With Real-Time Polymerase Chain Reaction Platform: A Rapid, Sensitive Method to Detect Mycobacterium tuberculosis.

In this study, we evaluated the diagnostic accuracy of multiple cross displacement amplification (MCDA) combined with real-time PCR platform in pulmonary tuberculosis (PTB) patients. Total 228 PTB patients and 141 non-TB cases were enrolled. Based on the analysis of the first available sample of all participants, MCDA assay showed a higher overall sensitivity (64.0%), with a difference of more than 10% compared with Xpert MTB/RIF (Xpert) assay (51.8%, P < 0.05) and combined liquid and solid culture (47.8%, P < 0.001) for PTB diagnosis. In particular, MCDA assay detected 31 probable TB patients, which notably increased the percentage of confirmed TB from 57.9% (132/228) to 71.5% (163/228). The specificities of microscopy, culture, Xpert and MCDA assay were 100% (141/141), 100% (141/141), 100% (141/141), and 98.6% (139/141), respectively. Among the patients with multiple samples, per patient sensitivity of MCDA assay was 60.5% (52/86) when only the first available sputum sample was taken into account, and the sensitivity increased to 75.6% (65/86) when all samples tested by MCDA assay were included into the analysis. Therefore, MCDA assay established in this study is rapid, accurate and affordable, which has the potential in assisting the accurate and rapid diagnosis of PTB and speed up initiation of TB treatment in settings equipped with real-time PCR platform.

The impact of changing the diagnostic algorithm for TB in Manicaland, Zimbabwe.

Governmental health facilities performing TB diagnostics in Manicaland, Zimbabwe. To investigate the effect of making Xpert® MTB/RIF the primary TB diagnostic for all patients presenting with presumptive TB on 1) the number of samples investigated for TB, 2) the proportion testing TB-positive, and 3) the proportion of unsuccessful results over time. This retrospective study used data from GeneX-pert downloads, laboratory registers and quality assurance reports between 1 January 2017 and 31 December 2018. The total number of Xpert tests performed in Manicaland increased from 3,967 in the first quarter of 2017 to 7,011 in the last quarter of 2018. Mycobacterium tuberculosis DNA was detected in 4.9-8.6% of the samples investigated using Xpert, with a higher yield in 2017 than in 2018. The overall proportion of unsuccessful Xpert assays due to "no results", errors and invalid results was 6.3%, and highly variable across sites. Roll out of more sensitive TB diagnostics does not necessarily result in an increase of microbiologically confirmed TB diagnosis. While the number of samples tested using Xpert increased, the proportion of TB-positive tests decreased. GeneXpert soft- and hardware infrastructure needs to be strengthened to reduce the rate of unsuccessful assays and therefore, costs and staff time.

COMPARATIVE ASSESSMENT OF DIFFERENT DIAGNOSTIC TECHNIQUES IN THE IDENTIFICATION OF MYCOBACTERIUM TUBERCULOSIS IN SUSPECTED CASES OF TUBERCULOSIS

Statement of the Problem: Tuberculosis remains a serious public-health threat in developing countries though it has been eradicated in some advanced countries. This disease constitutes a significant threat to global health, being the second highest cause of morbidity and mortality resulting from infectious agents. Prompt diagnosis of active TB facilitates timely therapeutic intervention and minimizes community transmission. Aim: This study aimed at determining a ‘Point of Care’ diagnostic tool for pulmonary tuberculosis (PTB) by comparing the efficiency of four different PTB diagnostic tools for different age groups. Methodology: Zeihl Nelson (ZN) staining, culture, Gene xpert (GX) and Lipoarabinomanan (LAM) assay were employed in this study The culture method was used for confirmation. Sputum and urine samples were collected from each of 100 patients symptomatically diagnosed of PTB. Findings: Fifty-seven percent of the population was male while 43% were female. Mycobacterium tuberculosis was isolated from 9 (9%) of 100 patients. Similarly, GX detected Mycobacterium tuberculosis in 9 (9%) of the patients while the rate of detection using LAM was 10% and with ZN it was 7%. Gene xpert produced no true or false positive and negative result, LAM had one false positive result and ZN had two false negative results. The maximum time frame to generate result was 25 minutes for LAM, two hours for Gene xpert, eight weeks for culture and two days for ZN. Two positive isolates were observed at the same frequency for age group 21-30 and 31- 40 while age groups 1-10, 10-20, 41-50, 50-60 and above has 1 positive result each. Gene xpert had 98.11% sensitivity while LAM had 96.23% and ZN had 86.79%. The choice of ‘Point of Care’ diagnostic tool is of great concern to clinicians and the general public. Conclusion &amp; Significance: This study identified LAM assay as suitable ‘Point of Care’ diagnostic and an add-on tool for PTB diagnosis because of its relatively high sensitivity and short maximum time frame to generate result compare to other three diagnostic techniques.&#x0D; &#x0D; &#x0D;

Visual Diagnosis of Pleural Tuberculosis and its Association with Tissue Biopsy, Culture and Xpert Assay.

The diagnosis of pleural tuberculosis remains a clinical challenge due to the paucibacillary nature of disease. Medical thoracoscopy remains the gold standard in diagnosing tuberculous pleuritis. To establish the diagnostic yield of sago-seed thoracoscopic appearance of pleura in tuberculosis and its correlation with histopathology, tissue AFB culture and tissue Xpert MTB/Rif assay. All consecutive patients with lymphocytic exudative pleural effusion, who fulfilled inclusion criteria of the study underwent medical thoracoscopy under local anesthesia and pleural tissue was sent for histopathology, AFB culture and Xpert MTB/Rif assay. Chronic granulomatous inflammation on histopathology and response to anti-tuberculous treatment was taken as reference standard for diagnosis of tuberculous pleurisy. A total of 249 patients were included in the study, out of which 168 had effusion secondary to tuberculosis. Sago-like nodules visualized on thoracoscopy had a sensitivity of 58.9 %, specificity of 92.6 % and diagnostic accuracy of 69.88 % for pleural tuberculosis. There is a strong association between the presence of sago-like nodules and detection of mycobacterium tuberculosis on Xpert MTB/Rif assay and AFB culture of pleura (p-value 0.007). Sago seed nodules on pleura have a high positive predictive value for tuberculous pleurisy. In high endemic countries patients with this finding on thoracoscopy can be commenced on anti-tuberculous treatment before histopathology or culture results are available.

The Practicability of the Xpert HCV Viral Load Fingerstick Point-of-Care Assay in Primary Care Settings.

Linkage to care presents one obstacle toward eliminating HCV, and the current two-step pathway (anti-HCV, followed by HCV-RNA testing) results in the loss of patients. HCV screening was tested in the primary care setting with the fingerstick Xpert HCV viral load point-of-care assay to analyze the practicability of immediate diagnosis. Anti-HCV (Cobas) and HCV-RNA (Cobas Amplicor version 2.0, only performed if anti-HCV was positive) were analyzed centrally as the gold standard. The Xpert assay was performed by 10 primary care private practices. In total, 622 patients were recruited. Five individuals (0.8%) were anti-HCV positive, and one was HCV-RNA positive. The Xpert test was valid in 546/622 (87.8%) patients. It was negative in 544 and positive in 2 cases, both of whom were anti-HCV negative. The HCV-RNA PCR and the Xpert test were both negative in 4/5 anti-HCV-positive cases, and the individual with HCV-RNA 4.5 × 106 IU/mL was not detected by the Xpert test. Primary care physicians rated the Xpert test practicability as bad, satisfactory, or good in 6%, 13%, and 81%, respectively, though 14/29 (48%) bad test ratings were assigned by a single practice. Despite adequate acceptance, interpretability and diagnostic performance in primary care settings should be further evaluated before its use in HCV screening can be recommended.

Head-to-Head Comparison between Xpert MTB/RIF Assay and Real-Time Polymerase Chain Reaction Assay Using Bronchial Washing Specimens for Tuberculosis Diagnosis.

Background With the introduction of Xpert MTB/RIF assay (Xpert), its incorporation into tuberculosis (TB) diagnostic algorithm has become an important issue. The aim of this study was to evaluate the performance of the Xpert assay in comparison with a commercial polymerase chain reaction (PCR) assay.Methods Medical records of patients having results of both Xpert and AdvanSure TB/NTM real-time PCR (AdvanSure) assays using the same bronchial washing specimens were retrospectively reviewed.Results Of the 1,297 patients included in this study, 205 (15.8%) were diagnosed with pulmonary TB. Using mycobacterial culture as the reference method, sensitivity of the Xpert assay using smear-positive specimens was 97.5%, which was comparable to that of the AdvanSure assay (96.3%, p=0.193). However, the sensitivity of the Xpert assay using smear-negative specimens was 70.6%, which was significantly higher than that of the AdvanSure assay (52.9%, p=0.018). Usng phenotypic drug susceptibility testing as the reference method, sensitivity and specificity for detecting rifampicin resistance were 100% and 99.1%, respectively. Moreover, a median turnaround time of the Xpert assay was 1 day, which was significantly shorter than 3 days of the AdvanSure assay (p<0.001).Conclusion In comparison with the AdvanSure assay, the Xpert assay had a higher sensitivity using smear-negative specimens, a shorter turnaround time, and could reliably predict rifampin resistance. Therefore, the Xpert assay might be preferentially recommended over TB-PCR in Korean TB diagnostic algorithm.

Performance of loop-mediated isothermal amplification assay for diagnosis of extrapulmonary tuberculosis and antituberculosis treatment initiation.

Extra-pulmonary TB(EPTB) accounts for 15-20% of total TB cases in India. Many cases remain undiagnosed due to poor sensitivity/long turn-around-time of conventional diagnostic tests. Molecular tests offer rapidity, improved sensitivity and exquisite specificity, but are expensive, require skilled manpower and enhanced laboratory infrastructure. Loop-mediated isothermal amplification (LAMP) assay is a unique, temperature-independent DNA amplification test facilitated by visual optic-readout. WHO has recommended use of LAMP for pulmonary TB diagnosis in 2016. For END-TB strategy to succeed, its necessary to capture all forms of TB. The aim of the study was to determine the sensitivity and specificity of LAMP assay against culture, Xpert MTB/RIF assay and Composite Reference Standard(CRS) for diagnosis of EPTB. In a cross-sectional study hundred consecutive EPTB specimens were processed for microscopy, culture, Xpert and LAMP assay. Standard formulae of sensitivity and specificity and McNemar chi square test of significance was applied. Hundred specimens included in the study were fluids(65), pus(19) and tissue(16). TB was detected in 38 specimens by any of the four methods. Positivity of microscopy-5%, culture-28%, Xpert-25% and LAMP-32%. Sensitivity and specificity of LAMP against culture was 85.71% and 88.89%; against Xpert was 88% and 86.67%; against CRS was 80% and 88.6% respectively. LAMP detected TB in 32 patients of which 28 were put on anti-TB treatment (ATT). Of the 62 patients with negative results in all the tests, 22 were put on ATT. LAMP has good sensitivity for EPTB diagnosis. Further studies are required to establish utility of LAMP as EPTB diagnostic tool.

Evaluation of Xpert GBS assay and Xpert GBS LB assay for detection of Streptococcus agalactiae

BackgroundGroup B Streptococcal (GBS) infection is the primary agent of neonatal morbidity and mortality. Rapid and simple methods to detect GBS are Xpert GBS and GBS LB assays based on real-time polymerase chain reaction (PCR). However, since the diagnostic accuracy of the two techniques in diagnosing GBS remains unclear, we designed this study to appraise the diagnostic accuracy of the aforementioned.MethodsA systematic search of all literature published before July 16, 2020 was conducted using Embase, PubMed, Web of Science, and Cochrane Library. The study quality was evaluated through Review Manager 5.3. Accordingly, data extracted in the included studies were analyzed using Meta-DiSc 1.4 and Stata 12.0 software. The diagnosis odds ratio (DOR) and bivariate boxplot were utilized to evaluate the heterogeneity. Publication bias was appraised by using Deeks’ funnel plot.ResultsA total of 13 studies were adopted and only 19 sets of data met the criteria. The sensitivity and specificity of Xpert GBS were 0.91 (95% CI 0.89–0.92) and 0.93 (95% CI 0.92–0.94). The area under the curve (AUC) was 0.9806. The sensitivity and specificity results of Xpert GBS LB were 0.96 (95% CI 0.95–0.98) and 0.94 (95% CI 0.92–0.95), respectively. The AUC was 0.9950. No publication bias was found.ConclusionsThe Xpert GBS and GBS LB assays are valuable alternative methods with high sensitivity and specificity. However, determining whether they can be used as clinical diagnostic standards for GBS is essential for the future.

TB or NTM: Can a new multiplex PCR assay be the answer?

TB or NTM: Can a new multiplex PCR assay be the answer?

Viability assessment of Mycobacterium tuberculosis complex in OMNIgene • SPUTUM reagent using the BACTEC MGIT 960 System and Xpert MTB/RIF assay.

The World Health Organization advocates that sputum specimens submitted to tuberculosis (TB) diagnostic should be processed within 48 h after collection and be stored under cooling. We aimed to assess the performance of OMNIgene • SPUTUM reagent in maintaining viable specimens of Mycobacterium tuberculosis complex (MTBC) during transportation of sputum samples without refrigeration, in comparison to the standard protocol of the National TB Control Program. Sputum samples obtained in southeastern Brazil (June 2017 to July 2018) from 100 sequential patients with positive acid-fast bacillus smear microscopy were divided into two portions. Portion 1 continued to be cooled (standard protocol, STA), but portion 2 was added to OMNIgene • SPUTUM reagent (alternative protocol, OMS) until concomitant further processing. Both portions of all samples were cultured using MGIT and tested by Xpert MTB/RIF assay. Growth of MTBC in the first 42 days was detected in 96% of the cultures under the STA and 88% under the OMS. Intervals between processing and detecting MTBC growth in the two portions significantly differed (p = 0.0001). Portions under the two protocols showed similar results in the MTBC detection by Xpert assay and culture contamination by non-MTBC. The OMNIgene reagent liquefies and decontaminates sputum leading to a decrease in processing time. Although there was a small delay in mycobacterial growth, the OMNIgene reagent can be useful in specimens transported from collection sites over a long distance to centralized testing centers, maintaining viable MTBC for at least 8 days at room temperature.

Time to Detection of Growth for Mycobacterium tuberculosis in a Low Incidence Area.

BackgroundDiagnosis of Mycobacterium tuberculosis (MTB) infection can be confirmed by Xpert assays within hours. However, when sample size does not allow performing both culture and Xpert, or if Xpert is negative, then formal diagnosis of MTB relies on culture and time to detection of growth (TDG) becomes critical for clinical management.ObjectivesTo determine TDG in Xpert negative samples, or in samples in which Xpert could not be performed, in a low-incidence area for MTB.MethodsRetrospective analysis (2015-2020) of a database including all cultures for mycobacteria in a University Hospital covering approximately 500’000 inhabitants. Analysis was restricted to culture positive (C+) samples for MTB for which 1/Xpert was negative or could not be performed because of limited sample volume, and 2/collected from subjects treated less than 24 hours. TDG was analyzed according to microscopy, origin of sample (pulmonary or not) and presence of cavitation.ResultsAmong 837 C+ samples for MTB, 236 samples (80% of respiratory origin) from 147 patients fulfilled study criteria; 78 samples (49 patients, 33%) were acid-fast bacilli (AFB) positive. Median (IQR) TDG was 25 (17; 40) days for all samples. TDG exceeded 28 days in 43% of samples and was significantly shorter in AFB+ vs AFB- samples, and samples from cavitary vs non cavitary or extra-thoracic disease.ConclusionsIn Xpert negative samples, or samples for which Xpert could not be performed, TDG exceeded 4 weeks in 43% of samples. AFB+ and samples from cavitary lung disease had a significantly shorter TDG.