Real-time PCR of whole blood specimens transported in PrimeStore MTM® to detect and monitor MTB bacteremia

Abstract

studied. Aerosol therapy in mice is problematic due to the high cost of the required apparatus. The purpose of this study was to explore the efficacy of intranasal (IN) administration (as a surrogate for aerosol delivery) of isoniazid (INH) and rifalazil (RZL) in a murine tuberculosis model compared to oral delivery.Methods & Materials: Six week old female Balb/c mice (purchased from Charles River Laboratories, Wilmington, MA) were infected intranasally with about 10 3 CFU of Mycobacterium tuberculosis (MTB) ATCC 27294 (H37Rv) for experiment 1 or about 10 6 CFU of MTB ATCC 35801 (Erdman) for experiment 2. One week post infection mice in experiment 1 were treated with INH 5mg/kg orally by gavage or 5mg/kg IN for 3 days and mice in experiment 2 were treated with RZL 5 mg/kg orally by gavage 5 days/week or 5 mg/kg IN Mon, Wed, and Fri for 2 weeks. At the initiation of therapy in each experiment a group of mice (early controls) were euthanized by CO 2 inhalation and their lungs were collected. At the completion of therapy an untreated group of mice, late controls (LC), and treated mice were euthanized. Mycobacterial loads in right lungs were measured by serial dilution and plating on Middlebrook 7H10 agar plates.Results: The mycobacterial loads (log CFU) for the EC, LC, INH oral and INH IN were 3.34, 4.49, 2.94, and 2.82 respectively (exp. 1). The mycobacterial loads for the EC, LC, RZL oral, and RZL IN were 6.26, 8.93, 4.30, and 4.58 respectively (exp. 2). The LC group was euthanized 3 days early due to their advanced illness. IN delivery of INH and RIF was significantly better than the untreated late controls. Conclusion: The activities of INH and RZL by IN delivery in these experiments suggest that other agents that cannot be given orally could be evaluated for their potential therapeutic activities by IN administration. http://dx.Background: Mycobacterium tuberculosis (MTB) is an important cause of bacteremia and sepsis in HIV patients residing in sub-Saharan Africa. Many patients with MTB sepsis go undiagnosed and die within 18 days of presentation, making culture inadequate for detecting MTB in the blood. Objective:To determine the feasibility of ambient temperature transport of blood in PrimeStore MTM ® to a distant lab for real-time PCR detection of MTB bacteremia and to monitor clearance of MTB from the blood after therapy.Methods & Materials: BALB/c female mice were injected intravenously with 0.2 mls of ethanol killed MTB (approximately 10 5

Evaluation of Commercially Available Viral Transport Medium (VTM) for SARS-CoV-2 Inactivation and Use in Point-of-Care (POC) Testing

“…Several commercially available Viral Transport Mediums (VTMs) contain a chemical inactivation ingredient. These include the Primestore ® Molecular Transport Medium (MTM) [ 6 ], which is a United States Food and Drug Administration (FDA)-registered VTM (USA) that has been demonstrated to effectively inactivate and preserve multiple pathogens, including mycobacterium tuberculosis (MTB) [ 7 ] and influenza virus [ 8 ], and does not compromise the sensitivity of the GeneXpert platform in the detection of MTB or first-line MTB rifampicin (RIF) resistance (MTB/RIF assay) [ 9 , 10 ]. COPAN™, another VTM, is registered as a non-propagating transport and culture medium with the FDA but does not contain any inactivating agents [ 11 ].…”

Diagnostic Evaluation of the IS1081-Targeted Real-Time PCR for Detection of Mycobacterium bovis DNA in Bovine Milk Samples

“…The secondary objective of this study was to evaluate the ability of MTM to inactivate BCG in spiked milk samples as well as its ability to preserve BCG DNA for PCR-based diagnostics. According to previous reports, PrimeStore ® MTM was an efficient storage and transport medium for the inactivation of bacteria, fungi, and viruses as well as stabilizing and preserving the nucleic acids for molecular testing [ 26 , 27 , 28 ]. In line with other studies, samples preserved in MTM showed no mycobacterial growth after 8-week incubation on solid media compared to the positive control where mycobacterial colonies were observed after 2–3 weeks incubation [ 44 , 45 ].…”

“…Prime Store MTM is composed of an optimized blend of chelating, chaotrophic detergents, and electrolyte reagents specifically formulated for sample inactivation, lysis of biological pathogens, and stabilizing and preserving released nucleic acids for a prolonged time. Previous studies have shown that MTM effectively inactivated several viruses, fungi, and bacteria, including M. tuberculosis , and preserved nucleic acids at ambient temperature for downstream molecular testing [ 26 , 27 , 28 ]. Based on the potential utility of MTM for collection, transportation, and inactivation of potentially infectious pathogens, the secondary objective of this study was to evaluate the ability of MTM to inactivate M. bovis BCG, as well as to preserve BCG DNA for PCR-based diagnostics.…”

Rotavirus Samples processing using Indigenously Developed Molecular Transport Medium (IMTM)

“…We propose the use of IMTM for other gastrointestinal pathogen detection like norovirus, astrovirus, sapovirus, adenovirus and other enteroviruses. A similar product Longhorn PrimeStore® has been used for Mycobacterium tuberculosis, vibrio, rotavirus and influenza virus detection through Genexpert and next generation sequencing 4,5,6,7,8,9 (NGS).…”

“…Longhorn PrimeStore® is an approved molecular transport medium from Center for Disease Control CDC, USA having guanidine thiocyanide as basic ingredient which is utilized to collect and transport good quality nucleic acid for PCR based molecular diagnostics. It is also proven to be used for genomic applications other 4,5,6,7,8,9 than PCRs.…”