A Cold Chain-Independent Specimen Collection and Transport Medium Improves Diagnostic Sensitivity and Minimizes Biosafety Challenges of COVID-19 Molecular Diagnosis

Approximately forty-four percent of the global population lives in villages, including 59% in Africa ( https://unhabitat.org/World%20Cities%20Report%202020 ). The fast-evolving nature of SARS-CoV-2 and its extremely contagious nature warrant early and accurate COVID-19 diagnostics across rural and urban population as a key to prevent viral transmission. Unfortunately, lack of adequate infrastructure, including the availability of biosafety-compliant facilities and an end-to-end cold chain availability for COVID-19 molecular diagnosis, limits the accessibility of testing in these countries.

Newborn calf serum supplemented by tellurite as alternative transport medium for Corynebacterium diphtheriae

Indonesia is one of the five countries with highest diphtheria cases in the world. Laboratory confirmation by culture method as a gold standard requires bacterial survival. Indonesia’s geographical condition as an archipelagic country and difficulties in transporting clinical samples are often obstacles in maintaining bacterial survival. This study aims to evaluate the ability of several transport mediums to maintain the survival of Corynebacterium diphtheriae. A total of 90 isolates were divided into nine groups of transport mediums. Samples were divided into 2 treatment groups, namely room temperature and temperature 2-8 ºC. On day 2, 4, 8, 16, and 32, 1 isolate from each group with 2 different incubation temperatures was cultured on blood agar medium and incubated for 24 hours at 37 ºC. Bacterial survival was indicated by the growth of suspect colonies which were identified by microscopic and biochemical tests. Results show serum with tellurite can be used with viability lower than silica gel, but higher than other media. Meanwhile at a temperature of 2-8 ºC, there are 2 types of the best transport medium, namely serum with tellurite and open silica gel in aluminum foil. Newborn Calf Serum supplemented with Tellurite can be used as an alternative transport medium for Corynebacterium diphtheriae, both at room temperature and at 2-8 ºC.

Development and validation of the method for the quantitative determination of methotrexate in a transport medium by HPLC-MS/MS

Relevance. BCRP is an efflux transporter protein that plays an important role in the pharmacokinetics of a wide range of drugs. The BCRP activity in vitro experiments is assessed by the transport of transporter protein substrates (methotrexate, etc.) across the bilipid membrane of cells overexpressingBCRP, for example, Caco-2 cells. The aim is to develop and validate a method for the quantitative determination of the BCRP substrate, methotrexate, in the transport medium of Caco-2 cells by HPLC-MS/MS. Methods. The work was performed on an Ultimate 3000 HPLC chromatograph (ThermoFisher, USA) with a TSQ Fortis tandem mass-selective detector (ThermoFisher, USA). The conditions of chromatographic analysis were as follows: column UCT Selectra C18 4.6 mm * 100 mm 5um, 100A, Selectra C18 Guard Cartridges SLC-18GDC46-5UM, separation temperature 35 °С, flow rate 0.3 ml/min, injected sample volume - 2 μl, analysis time - 10 min. Used a gradient elution: the ratio of the solution of 0.1 % formic acid and acetonitrile was at 0 min 75 and 25 %; 0.4 min 60 and 40 %; 6 minutes 20 and 80 %; 8 minutes 75 and 25 %. Under these conditions, the retention time of methotrexate is 3.11 minutes. Detection conditions: methotrexate - positive ionization mode, 455.15 m / z → 308.125 m / z, collision energy 22.99 V, source fragmentation 5, CID gas pressure 2 mTorr. The extraction of methotrexate from the transport medium (Hanks solution with 25 mM Hepes and 1% dimethyl sulfoxide) after incubation with Caco-2 cells for 3 h was carried out with a mixture of methanol + water in a ratio of 1: 1. Results. The developed method was validated according to the following parameters: selectivity, linearity, accuracy, precision, limit of quantitative determination, sample transfer, sample stability. The confirmed analytical range of the method was 60 -10,000 nmol / L in the transport medium. Conclusions: a method for the quantitative determination of methotrexate in the transport medium of Caco-2 cells by HPLC-MS / MS was developed and validated.

Evaluation of an Environmental Transport Medium for Legionella pneumophila Recovery

The collection and storage of water-related matrices such as biofilm from collection to processing are critical for the detection of Legionella pneumophila by cultural and molecular tests. SRK™ is a liquid medium that acts both as an antimicrobial neutralizing agent and a transport medium for bacterial culture enumeration and is useful to maintain the stability of the sample from collection to analysis. The aims of this study were to evaluate Legionella pneumophila viability and bacterial nucleic acids’ stability in SRK™ medium over time at different storage conditions. Artificial bacterial inoculates with an approximate concentration of 104, 103 and 102 CFU/mL were made using Legionella pneumophila certified reference material suspended in SRK™ medium. Bacteria recovery was analyzed by cultural and molecular methods at time 0, 24 and 48 h at room temperature and at 0, 24, 48 and 72 h at 2–8 °C, respectively. SRK™ medium supported Legionella pneumophila culture viability with CFU counts within the expected range. The recovery after 72 h at 2–8 °C was 83–100% and 75–95% after 48 h at room temperature. Real-time PCR appropriately detected Legionella pneumophila DNA at each temperature condition, dilution and time point. Results demonstrated a good performance of SRK™ medium for the reliable recovery of environmental Legionella.

ANALYSIS OF FOUR DIFFERENT TRANSPORT AND PRESERVATION MEDIUM KITS FOR SARS-COV-2 DIAGNOSIS FROM NASOPHARYNGEAL SWAB BY REAL-TIME PCR: ADAPTING TO THE CONSTANTLY INCREASING DEMAND OF SAMPLING PROCESSING AND STOCK-OUTS DURING THE PANDEMIC

The high demand for supplies during the COVID19-pandemic has generated several stock-out of material and essential reagents needed to meet the current high demand for diagnosis in the worldwide population. In this way, there is limited information regarding the performance of different virus transport medium (VTM) for nasopharyngeal swab sampling (NPS) aimed for SARS-CoV-2 detection. We compared the RT-qPCR amplification profile of four different commercial transport medium kits, including DNA/RNA Shield, NAT, VTM, and Phosphate-buffered saline (PBS) transport medium, for NPSs samples from Central Metropolitan Health Service, Santiago, Chile. The RT-qPCR showed a slight lower RNase P Cq value of the samples preserved and transported in DNA/RNA Shield compared to NAT medium. By contrast, a marked increase in the RNase P Cq value was registered in the samples transported with VTM compared to DNA/RNA Shield medium. For PBS-preserved NPS, the performance of two strategies were assessed due to the potential presence of any remaining active virus in the sample: (1) thermal inactivation; and (2) thermal inactivation treatment followed by RNA extraction. The heat inactivation showed a significantly lower Cq value for RNase P and viral ORF1ab Cq compared to the followed by RNA extraction. This study indicates that new medium alternatives could be used if supplies run out to diagnose COVID19

Optimization of SARS-CoV-2 laboratory testing in a rural healthcare facility in the United States

Background: The diagnostic testing for SARS-COV-2 (COVID-19) presented a profound challenge to the entire world, dominating the concern of most governments and public health systems, particularly rural community hospitals in the United States. Indiana University of Pennsylvania (IUP) in partnership with Indiana Regional Medical Center (IRMC) began on site, same-day COVID-19 testing in efforts to not only combat the challenges that health providers faced in rural Indiana community but also help to strengthen global diagnostic capacity.Methods: Clinical samples were collected as dry swabs from the nasopharyngeal (NP) regions and processed in phosphate buffer saline (PBS). The crude RNA was directly tested using real-time (RT) reverse transcription quantitative polymerase chain reaction (RT-qPCR) with PrimeDirect probe RT-qPCR Mix (Takara Bio USA) and optimized with probe-primer sets [Integrated DNA Technologies (IDT)].Results: Validation experiments with dry swabs from NP clinical samples showed no difference in the testing accuracy to those collected in viral transport medium or universal transport medium. Extraction of COVID-19 RNA in PBS reduced processing time of a batch of 50 NP clinical samples from 6 hours to an hour. This allowed for rapid diagnostic testing of nearly 200 clinical samples per day. Optimization of analytical variables helped to detect virus loads up to 2.0 copies/μl during routine diagnostic testing.Conclusions: During an infectious outbreak, the ideal response by public health authorities is rapid testing. The collaboration between IUP and IRMC attests to the importance of teamwork between local initiatives to detect and prevent further spread within a rural community.