Impact of line probe assay-based molecular testing on individualized treatment in patients with rifampicin-resistant tuberculosis: data from the prospective INNOVA4TB cohort study in Ukraine.

BACKGROUND: Ukraine remains a high World Health Organization priority country for drug-resistant tuberculosis (TB). Rifampicin-resistant TB (RR-TB) has a more protracted, more complicated, and more expensive treatment. In 2021, Ukraine reported 4025 RR-TB cases - 5.4 times more (751) than all 30 European Union/ European Economic Area countries together. OBJECTIVES: The objective of the study was to determine the diagnostic accuracy of line probe assay (LPA), AID Autoimmun Diagnostika GmbH, for detecting resistance to anti-TB drugs and its clinical application for selecting treatment regimens. DESIGN: A prospective observational cohort study. METHODS: From May 2019 to June 2020, we consecutively enrolled patients with active TB hospitalized at the Regional Phthisiopulmonology Center (Vinnytsia, Ukraine), aged between 18 and 82 years. The LPA was performed in the Genetic Research Laboratory at National Pirogov Memorial Medical University, Vinnytsia, Ukraine. RESULTS: A total of 84 clinical specimens and 97 culture isolates from 126 TB patients were tested during the study. Accuracy (95% confidence interval) of LPA for clinical samples in comparison with phenotypic drug susceptibility test (DST) was 80.1 (68.5-89.0) for isoniazid (H), 74.7 (62.4-84.6) for rifampicin (R), 74.4 (62.5-84.1) for ethambutol, 71.4 (41.9-91.6) for streptomycin, 84.6 (62.4-96.5) for prothionamide/ethionamide, and 84.6 (73.6-92.3) for levofloxacin (Lfx), respectively. We found a significantly higher sensitivity of LPA for H, R, and Lfx for the culture isolates compared to clinical specimens (p < 0.05). LPA detected different mutations in 6 out of 17 (35.5%) patients susceptible to R by Xpert. A shorter treatment regimen with an injectable agent demonstrated a low suitability rate of 5% (8/156) in a cohort of RR-TB patients from Ukraine. CONCLUSION: Initial LPA testing accurately identifies resistance to anti-TB drugs and facilitates the selection of an appropriate treatment regimen, minimizing exposure to empirical therapy.

Study about the impact of rapid resistance detection on the treatment of patients with tuberculosis in Ukraine written by healthcare and biomedical professionals to better understand how we can improve the results of treatment and to prevent spreading of resistant bacteriaWhy was the study done? Ukraine has over 4000 patients with tuberculosis (TB) resistant to at least one drug (rifampicin) - five times that of all 30 European Union/European Economic Area countries combined. Unfortunately, only about 60% of such patients have been successfully treated in 2019. At that time, the majority of people suffering from tuberculosis in Ukraine, after checking resistance to rifampicin, initially received standard combinations of the first-line or second-line anti-TB medicines before the result of traditionally used tests (usually few weeks later) became available to individualize the treatment. Alternatively, the sputum could be transported to some overloaded reference laboratories located hundreds of km away from the treatment places.What did the researchers do? The INNOVA4TB team implemented rapid diagnostics of drug resistance in routine practice, guiding key antibiotics use in TB patients. A total of 181 samples from 126 individuals were tested during 2019-2020.What did the researchers find? This new diagnostic technology accurately detected resistance to 9 anti-TB drugs in sputum samples. It could be helpful to select appropriate TB treatment regimens, reducing time for decision from 1 month up to 2 days. Recommended at the study time 9-month shorter standardized treatment regimen with injectable agent was suitable only for 5% of patients for whom it was indicated in Vinnytsia region of Ukraine.What do the findings mean? The study has demonstrated successful implementation of the new molecular diagnostic technology from scratch in a country with restricted resources and limited TB laboratory capacity. This test can facilitate optimal distribution of available wards among patients with different profiles of resistance and correct choice between treatment options.

Clinical performance of the HPV DNA Array genotyping assay in detection of CIN2+ lesions with BS GP5+/6+ MPG Luminex tested cervical samples.

Human papillomavirus (HPV) detection is used for screening of cervical cancer and genotype-specific persistence has shown to be mandatory for dysplasia development. Aim of this study was to evaluate the clinical performance of HPV DNA Array for cervical intraepithelial neoplasia 2+ (CIN2+) lesion detection. HPV DNA Array is a polymerase chain reaction-based assay that targets E1 sequences of 29 HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, 85, and 97). The clinical evaluation was performed against the reference assay, BS-GP5+/6+ multiplex genotyping (MPG)-Luminex, with 600 cervical smear samples of a referral population. HPV DNA Array detected CIN2+ lesions with a sensitivity of 90.2%, identical to that of MPG-Luminex. Detection of CIN3+ lesions was with a sensitivity of 90.3%, as compared with 88.7% of MPG-Luminex. It demonstrated very good agreement for HPV detection, irrespective of type, of 91.5% (κ = 0.832). HPV DNA Array is a simple and robust assay, with a short protocol of 4 hours hands-on time and automated readout by ELISpot AiDot software. It permits testing of up to 96 samples in one run and may be considered for use in organized screening programs and low resource settings.

Analytical Evaluation of the Human Papillomavirus HPV DNA Array E1-Based Genotyping Assay.

BACKGROUND: Cervical cancer is caused by a persistent infection of human papillomavirus (HPV). Therefore, tests which detect the carcinogenic virus can be used for cervical cancer screening. OBJECTIVE: This is the first evaluation of the HPV DNA Array (AID Diagnostika, Strassberg, Germany), an E1-based genotyping polymerase chain reaction (PCR) test for identification of 29 HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 67, 68, 69, 70, 73, 82, 85, and 97). METHODS: Analytical performance of the assay was assessed with cervical cancer cell lines with known HPV status, and preselected clinical cervical scrapings genotyped by multiplexed genotyping (MPG) with a Luminex readout (validated in-house assay). Intra- and inter-laboratory reproducibility experiments were performed to ensure the reliability of the assay. RESULTS: HPV DNA Array identified the intrinsic HPV genotype in all cervical cancer cell lines and demonstrated a high sensitivity for HPV16 probe (1 cell per PCR reaction), as well as HPV18 and 45 probes (100 cells per PCR reaction). When compared with MPG, HPV DNA Array showed a good agreement of 92.2% for HPV detection irrespective of type (κ = 0.601), and demonstrated high agreement for HPV16 (80.7%, κ = 0.836) and HPV18 (86.7%, κ = 0.925). Furthermore, high intra-/inter-laboratory reproducibility was observed (90.9-100%). CONCLUSION: HPV DNA Array showed high sensitivity for correct HPV genotype detection in experimental and clinical samples with a good correlation to the reference test. Since HPV DNA Array is based on a simple multiplexed PCR followed by reverse hybridization in a 96-well format and automated visual readout by AID ELISpot reader, it is capable of high throughput in a time-effective manner. HPV DNA Array could be considered for extended HPV genotyping of cervical smears.

CIN2+ detection of the HPV DNA Array genotyping assay in comparison with the Cobas 4800 HPV test and cytology.

BACKGROUND: HPV DNA Array is an E1-targeting PCR genotyping test, with capability of distinguishing 18 high-risk (16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82) and 11 low-risk HPV types (6, 11, 40, 42, 44, 54, 67, 69, 70, 85, 97). HPV DNA Array uses multiplex PCR for E1-gene sequence amplification. The amplicons are detected and genotyped by reverse hybridization to immobilized DNA probes spotted as triplets in single 96 well-plate wells and read by AID ELISPOT reader. METHODS: Aim of the study was to evaluate the clinical performance of the assay against internationally accepted and FDA approved Cobas 4800 HPV test (Roche Diagnostics). Study population comprised of 500 cervical samples. RESULTS: HPV DNA Array demonstrated a very high sensitivity of 100% for CIN2+ and 100% for CIN3+ detection, same as Cobas 4800. HPV DNA Array showed greater sensitivity for CIN2+ detection than cytology (100% vs. 13.6%). The agreement to Cobas 4800 for HPV detection, irrespective of type, was 81.4% with κ = 0.613. The agreement for HPV 16 was 92.8% (κ = 0.929), and for HPV 18 54.2% (κ = 0.681). CONCLUSION: HPV DNA Array demonstrated good clinical performance for detection of high-grade lesions, and may be considered for usage in a screening setting.

Specific magnetic bead-based capture of free fetal DNA from maternal plasma.

An automated magnetic capture hybridization (MCH) method for the extraction and enrichment of fetal RHD specific DNA fragments from maternal plasma was developed using plasma from 1000 D-negative pregnant women. A real time PCR protocol for RHD exon 7 was applied. MCH was compared with the QIAamp DSP Virus Kit (QIAamp) as a reference. Compared with the QIAamp method, the percentage of fetal DNA increased from 2.86% to 4.83% (p<0.05, n=8). The 95% detection limit of MCH was determined at 286 pg/ml (43 geg/ml) compared with 138 pg/ml (21 geq/ml) for the QIAamp DSP Virus Kit.