Standard PCR Research Articles

DNA Extraction from Clinical Specimens for the Direct Detection of Mycobacterium ulcerans by Real-Time PCR.

Mycobacterium ulcerans is a slow-growing environmental bacterium that causes a severe skin disease known as Buruli ulcer (BU). Rapid detection of M. ulcerans in clinical specimens is essential for early diagnosis so that patients can be treated appropriately as soon as possible. This chapter describes suitable methods for the extraction of M. ulcerans DNA from the most common specimens submitted to the laboratory for confirmation of BU: swabs, fresh tissue biopsies, and fixed tissue sections. The resulting DNA extracts may be used for downstream procedures including standard gel-based PCR and real-time PCR assays. Protocols for direct detection of M. ulcerans DNA by real-time PCR are described in Chapter 8 .

Bio-O1-01 - Diagnostic performance of high throughput sequencing of the T-cell receptor beta gene for the diagnosis of cutaneous T-cell lymphoma

Bio-O1-01 - Diagnostic performance of high throughput sequencing of the T-cell receptor beta gene for the diagnosis of cutaneous T-cell lymphoma

Diarrhoea and Associated Clinical Features in Different Pathotypes of Diarrheagenic E. coliIsolated in Children: A Case-Control Study in a Tertiary Care Hospital

Background: Acute gastroenteritis remains to be a major health problem in children causing high morbidity and mortalityIn India, diarrhoea is the third most common cause of death in children younger than 5 years of age, with an estimation of 300,000 deaths each year. Diarrheagenic Escherichia coli (DEC) being one of the important bacterial agents, the present hospital-based study was done to enlighten on the occurrence of different pathotypes and clinical features associated with DEC related diarrhoea. Methods:The case-control study was carried out in SCB Medical College, Odisha from October 2014 to September 2016 on the childhood diarrhoea cases (≤ 14 years of age). Stool samples were collected and processed to isolate the causative bacterial agent by standard biochemical test, serotyping and multiplex PCR. Results: 350 cases and 100 controls were included in the study. The different pathotypes of DEC were isolated significantly more in cases than control group (P value = 0.0205) with an isolation rate of 7.4% in cases. 12 (46.2%) of DEC were in 0-1 year age group followed by 1-5 year of age group i.e. 11 (42.3%) and least in 5-14 years of age group i.e. 3 (11.5%). The most common pathotype was Enterotoxigenic E. coli (ETEC) followed by Enteropathogenic E.coli (EPEC). Fever is the most common symptom associated with DEC diarrhoea; the other two common symptoms were watery diarrhoea and vomiting. Exclusive breastfeeding was the protective factor. Seasonal variation of DEC was found more among the cases in the rainy season. Conclusion: Different pathotypes of DEC being associated commonly with childhood diarrhoea, the correct identification of various types of DEC along with the clinical knowledge is important to reduce the morbidity and mortality caused by it.

Multiple vector-borne pathogens of domestic animals in Egypt.

Vector Borne Diseases (VBDs) are considered emerging and re-emerging diseases that represent a global burden. The aim of this study was to explore and characterize vector-borne pathogens in different domestic animal hosts in Egypt. A total of 557 blood samples were collected from different animals using a convenience sampling strategy (203 dogs, 149 camels, 88 cattle, 26 buffaloes, 58 sheep and 33 goats). All samples were tested for multiple pathogens using quantitative PCR and standard PCR coupled with sequencing. We identified Theileria annulata and Babesia bigemina in cattle (15.9 and 1.1%, respectively), T. ovis in sheep and buffaloes (8.6 and 7.7%, respectively) and Ba. canis in dogs (0.5%) as well as Anaplasma marginale in cattle, sheep and camels (20.4, 3.4 and 0.7%, respectively) and Coxiella burnetii in sheep and goats (1.7 and 3%; respectively). New genotypes of An. centrale, An. ovis, An. platys-like and Borrelia theileri were found in cattle (1.1,3.4, 3.4 and 3.4%, respectively), An. platys-like in buffaloes (7.7%), An. marginale, An. ovis, An. platys-like and Bo. theileri in sheep (3.4, 1.7, 1.7 and 3.4%, respectively), An. platys, An. platys-like and Setaria digitata in camels (0.7, 5.4 and 0.7%, respectively) and Rickettsia africae-like, An. platys, Dirofilaria repens and Acanthocheilonema reconditum in dogs (1.5, 3.4, 1 and 0.5%, respectively). Co-infections were found in cattle, sheep and dogs (5.7, 1.7, 0.5%, respectively). For the first time, we have demonstrated the presence of several vector-borne zoonoses in the blood of domestic animals in Egypt. Dogs and ruminants seem to play a significant role in the epidemiological cycle of VBDs.

Evaluating the Sensitivity and Specificity of Siemens Clinitest Lateral Flow Test and the Simple AMplification-Based Assay (SAMBA)-2 PCR Test for SARS-CoV-2 Infection.

IntroductionAccurate point-of-care testing for SARS-CoV-2 could quickly identify which patients need to be isolated and improve flow for patients being admitted as an emergency to the hospital. We evaluated two diagnostic tests with shorter turnaround times, the Siemens Clinitest Lateral Flow (Siemens Healthineers AG, Erlangen, Germany) and the Simple AMplification-Based Assay (SAMBA)-2 PCR test against a standard laboratory PCR test.MethodsWe conducted a prospective diagnostic cohort study in a single English emergency department. Adult participants underwent three swabs: the Siemens Clinitest Lateral Flow Test, the SAMBA-2 and a standard laboratory PCR test.ResultsA total of 212 participants were recruited. The sensitivity and specificity of the Siemens Clinitest Lateral Flow Test against the laboratory PCR test was 55.6% (95% CI 30.8-78.5) and 100% (95% CI 98.1-100) respectively. The sensitivity and specificity of the SAMBA-2 PCR test against the laboratory PCR test was 60.0% (95% CI 32.3-83.7) and 100% (95% CI 97.9-100) respectively.ConclusionNeither the Siemens Clinitest Lateral Flow Test nor the SAMBA-2 PCR test demonstrated sufficient sensitivity to rule out active SARS-CoV-2 infection. Both tests demonstrated high specificity.

A Numerical Procedure for Multivariate Calibration Using Heteroscedastic Principal Components Regression

Many methods have been developed to allow for consideration of measurement errors during multivariate data analyses. The incorporation of the error structure into the analytical framework, usually described in terms of the covariance matrix of measurement errors, can provide better model estimation and prediction. However, little effort has been made to evaluate the effects of heteroscedastic measurement uncertainties on multivariate analyses when the covariance matrix of measurement errors changes with the measurement conditions. For this reason, the present work describes a new numerical procedure for analyses of heteroscedastic systems (heteroscedastic principal component regression or H-PCR) that takes into consideration the variations of the covariance matrix of measurement fluctuations. In order to illustrate the proposed approach, near infrared (NIR) spectra of xylene and toluene mixtures were measured at different temperatures and stirring velocities and the obtained data were used to build calibration models with different multivariate techniques, including H-PCR. Modeling of available xylene–toluene NIR data revealed that H-PCR can be used successfully for calibration purposes and that the principal directions obtained with the proposed approach can be quite different from the ones calculated through standard PCR, when heteroscedasticity is disregarded explicitly.

Parasites Circulating in Wild Synanthropic Capybaras (Hydrochoerus hydrochaeris): A One Health Approach.

Capybaras (Hydrochoerus hydrochaeris) are affected by a wide range of protozoan and metazoan-derived parasitic diseases. Among parasites of free-ranging capybaras are soil-, water-, food- and gastropod-borne parasitosis, today considered as opportunistic infections in semiaquatic ecosystems. The overlapping of the capybara’s natural ecological habitats with human and domestic animal activities has unfortunately increased in recent decades, thereby enhancing possible cross- or spillover events of zoonotic parasites. Due to this, three synanthropic wild capybara populations in the Orinoco Basin were studied for the occurrence of gastrointestinal parasite infections. A total of forty-six fecal samples were collected from free-ranging capybaras in close proximity to livestock farms. Macroscopical analyses, standard copromicroscopical techniques, coproELISA, PCR, and phylogenetic analysis revealed thirteen parasite taxa. In detail, the study indicates stages of five protozoans, four nematodes, one cestode, and three trematodes. Two zoonotic parasites were identified (i.e., Plagorchis muris, and Neobalantidium coli). The trematode P. muris represents the first report within South America. In addition, this report expands the geographical distribution range of echinocoelosis (Echinocoleus hydrochoeri). Overall, parasitological findings include two new host records (i.e., P. muris, and Entamoeba). The present findings collectively constitute baseline data for future monitoring of wildlife-derived anthropozoonotic parasites and call for future research on the health and the ecological impact of this largest semiaquatic rodent closely linked to humans, domestic and wild animals.

Species identification of blackcurrant anthracnose agent <i>Colletotrichum floriniae</i> Marcelino & Gouli

A first Russian isolate of the anthracnose fruit rot-causing Colletotrichum fungus was obtained from blackcur rant of Moscow and Bryansk regions. In the Bryansk Region, Colletotrichum sp. was isolated from berries of ten blackcur­ rant varieties, including Bagira, Mriya, Mriya-5, Selechenskaya, Litvinovskaya, Nadina, Nara, Yadrenaya, Sofievskaya and Ben Hopen. The Colletotrichum genus fungi were observed on Sadko and Bryanskiy Agat blackcurrants in the Moscow Region and on Dobrynya and Yadrenaya — at the Timiryazev Academy station in Moscow. Colletotrichum species endure mainly on mummified persistent blackcurrant berries in a viable form for over two years. Saprotrophic yeast-like fungiof the genera Cryptococcus and Rhodotorula largely compound the blackcurrant-infesting micromycete flora, aside to the hyphomycete Auerobasidium pullans and related genera Penicillium, Fusarium, Alternaria and Cladosporium. Some of these fungi are antagonistic to suppress the Colletotrichum genus fungi. A standard PCR sequencing was employed for the species identification of two Colletotrichum isolates from Bryansk and Moscow regions. Both isolates were found to represent the same species Colletotrichum fioriniae Marcelino & Gouli.

Children with Plasmodium vivax infection previously observed in Namibia, were Duffy negative and carried a c.136G\u2009>\u2009A mutation

BackgroundIn a previous study, using a molecular approach, we reported the presence of P. vivax in Namibia. Here, we have extended our investigation to the Duffy antigen genetic profile of individuals of the same cohort with and without Plasmodium infections.MethodsParticipants with P. vivax (n = 3), P. falciparum (n = 23) mono-infections and co-infections of P. vivax/P. falciparum (n = 4), and P. falciparum/P. ovale (n = 3) were selected from seven regions. Participants with similar age but without any Plasmodium infections (n = 47) were also selected from all the regions. Duffy allelic profile was examined using standard PCR followed by sequencing of amplified products. Sequenced samples were also examined for the presence or absence of G125A mutation in codon 42, exon 2.ResultsAll individuals tested carried the − 67 T > C mutation. However, while all P. vivax infected participants carried the c.G125A mutation, 7/28 P. falciparum infected participants and 9/41 of uninfected participants did not have the c.G125A mutation. The exon 2 region surrounding codon 42, had a c.136G > A mutation that was present in all P. vivax infections. The odds ratio for lack of this mutation with P. vivax infections was (OR 0.015, 95% CI 0.001–0.176; p = 0.001).ConclusionWe conclude that P. vivax infections previously reported in Namibia, occurred in Duffy negative participants, carrying the G125A mutation in codon 42. The role of the additional mutation c.136 G > A in exon 2 in P. vivax infections, will require further investigations.

Influence of HIV infection on the distribution of high-risk HPV types among women with cervical precancerous lesions in Yaounde, Cameroon

ObjectivesTo characterize high-risk HPV types associated with cervical precancerous lesions in women living in Yaounde, Cameroon, and to determine their distribution with HIV status. MethodsWomen with abnormal pap smears recorded from February 2015 to May 2019 at Saint Martin de Porres' Health Centre, Yaounde, Cameroon, were recruited in this study after obtaining informed consent. Pap smears were collected and re-examined. Human immunodeficiency virus (HIV) serology was determined. HPV16, 18, 33, and 45 were assessed using standard PCR. ResultsAll included participants (370) were HPV-positive and had either low grade squamous intraepithelial lesions (67.03%) or high grade squamous intraepithelial lesions (31.35%). They were subdivided into HIV-positive (N =102) and HIV-negative (N =268). In the HIV-negative subgroup, we observed 66.04% HPV16-positve, 41.79% HPV18-positve, 21.27% HPV33-positve and 8.21% HPV45-positve. In the HIV-positive subgroup, we observed 22.55% HPV16-positve, 5.88% HPV18-positve, 75.49% HPV33-positve, and 49.02% HPV45-positve. Married HIV-positive participants (47.14 ± 1.19) were older than both their single counterparts (34.94±1.22, P = 0.0008) and HIV-negative participants (41.43 ± 0.79, P = 0.0001). Single HIV-positive women reported higher numbers of miscarriages (P = 0.0023), and had later first sexual intercourse than HIV-negative (P = 0.0079) women. ConclusionOur study suggested differential expressions in high-risk HPV types with HIV status and cervical precancerous lesions and warrants more extensive studies.

Positive rate of COVID 19 in the setting of GGH Ghulam Muhammadabad

Background: The corona virus disease also known as COVID-19 has opened gates to a lot of research about detection, treatment and prevention in the last past year due to lack of information regarding the SARS-CoV-2 virus. PCR corona via nasopharyngeal swab is the standard method of detection in our set-up. Materials and methods: Nasopharyngeal swabs were taken by ENT department using precautions and following proper SOPs. Swabs were sent for rRT-PCR tests. Data was collected and analyzed. All the subjects meeting the inclusion criteria were included in study. Data was analyzed using SPSS version 20. Frequency and percentage was calculated for gender. Positivity rate was calculated using CDC formula. Study design: - Descriptive studies Place and Duration of Study: - Government General Hospital, Ghulam Muhammadabad, Faisalabad. 889 samples were taken from 7.1.2020 to 6.12.2020. Results: Standard PCR for corona test by nasopharyngeal route was taken of suspected corona patients and of patients with contact with corona positive patients. 894 samples were taken from 7.1.2020 to 6.12.2020. 263 were positive for corona. The calculated positive rate is 29.41%. Conclusions: Calculation of positive rates is a very easy metric to give us a quick overview of the spread of the virus. Keywords: COVID-19, Positive rates, PCR-Corona

Rapid Detection of New Delhi Metallo-β-Lactamase Gene Using Recombinase-Aided Amplification Directly on Clinical Samples From Children.

New Delhi metallo-β-lactamase, a metallo-β-lactamase carbapenemase type, mediates resistance to most β-lactam antibiotics including penicillins, cephalosporins, and carbapenems. Therefore, it is important to detect blaNDM genes in children’s clinical samples as quickly as possible and analyze their characteristics. Here, a recombinase-aided amplification (RAA) assay, which operates in a single one-step reaction tube at 39°C in 5−15 min, was established to target blaNDM genes in children’s clinical samples. The analytical sensitivity of the RAA assay was 20 copies, and the various bacterial types without blaNDM genes did not amplify. This method was used to detect blaNDM genes in 112 children’s stool samples, 10 of which were tested positive by both RAA and standard PCR. To further investigate the characteristics of carbapenem-resistant bacteria carrying blaNDM in children, 15 carbapenem-resistant bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Citrobacter freundii, Klebsiella oxytoca, Acinetobacter junii, and Proteus mirabilis) were isolated from the 10 samples. Notably, more than one bacterial type was isolated from three samples. Most of these isolates were resistant to cephalosporins, cefoperazone-sulbactam, piperacillin-tazobactam, ticarcillin-clavulanic acid, aztreonam, co-trimoxazole, and carbapenems. blaNDM–1 and blaNDM–5 were the two main types in these samples. These data show that the RAA assay has potential to be a sensitive and rapid blaNDM gene screening test for clinical samples. The common existence of blaNDM and multi-drug resistance genes presents major challenges for pediatric treatment.

A More Universal Approach to Comprehensive Analysis of Thalassemia Alleles (CATSA)

The aim of the study was to assess the clinical utility of a third-generation sequencing (TGS) approach termed comprehensive analysis of thalassemia alleles (CATSA) for identifying both α and β thalassemia genetic carrier status. Prospective blood samples (n=1759) with abnormal hemoglobin parameters were screened for pathogenic thalassemia variants by CATSA on the PacBio TGS platform. In 1159 individuals, a total of 1317 pathogenic thalassemia variants were identified and confirmed by independent PCR-based tests. Of the total thalassemia variants detected, the α-variant --SEA (35.4%) and β-variant c.126_129delCTTT (15%) were the most common. CATSA was also able to detect three types of rare HBA structural variants as well as five rare HBA2, three HBA1, and 10 HBB single-nucleotide variations/insertions and deletions. Compared with standard thalassemia variant PCR panel testing, CATSA identified all panel variants present, with no false-negative results. Carrier assignment was improved through identification of rare variants missed by the panel test. On the basis of allelic coverage, reliability, and accuracy, TGS with long-range PCR presents a comprehensive approach with the potential to provide a universal solution for thalassemia genetic carrier screening. It is proposed that CATSA has immediate clinical utility as an effective carrier screening approach for at-risk couples.

Stimuli-Responsive Autonomous-Motion Molecular Machine for Sensitive Simultaneous Fluorescence Imaging of Intracellular MicroRNAs.

DNAzymes with enzymatic activity identified from random DNA pools by in vitro selection have recently attracted considerable attention. In this work, a DNAzyme-based autonomous-motion (AM) molecular machine is demonstrated for sensitive simultaneous imaging of different intracellular microRNAs (miRNAs). The AM molecular machine consists of two basic elements, one of which is a target-analogue-embedded double-stem hairpin substrate (TDHS) and the other is a locking-strand-silenced DNAzyme (LSDz). LSDz can be activated by target miRNA and catalytically cleave TDHS, generating Clv-TDHS and releasing free target analogue capable of triggering the next round of cleavage reaction. As such, the molecular machine can exert sustainable autonomous operation, producing an enhanced signal. Because the active target analogue comes from the machine itself and offers cyclical stimulation in a feedback manner, this target-induced autonomous cleavage circuit is termed a self-feedback circuit (SFC). The SFC-based molecular machine can be used to quantify miRNA-21 down to 10 pM without interference from nontarget miRNAs, indicating a substantial improvement in assay performance compared with its counterpart system without an SFC effect. Moreover, due to the enzyme-free process, the AM molecular machine is suitable for miRNA imaging in living cells, and the quantitative results are consistent with the gold standard PCR assay. More interestingly, the AM molecular machine can be used for the simultaneous fluorescence imaging of several intracellular miRNAs, enabling the accurate discrimination of cancerous cells (e.g., HeLa and MCF-7) from healthy cells. The SFC-based autonomous-motion machine is expected to be a promising tool for the research of molecular biology and early diagnosis of human diseases.

Verification of CRISPR editing and finding transgenic inserts by Xdrop indirect sequence capture followed by short- and long-read sequencing.

Validation of CRISPR-Cas9 editing typically explores the immediate vicinity of the gene editing site and distal off-target sequences, which has led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kilobases-long and only affect one allele. The gold standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach allows the detection of small editing events but fails in detecting large rearrangements, in particular when only one allele is affected. Detection of large rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we implemented Xdrop™, a new microfluidic technology that allows targeted enrichment of long regions (~100kb) using just a single standard PCR primer set. Sequencing of the enriched CRISPR-Cas9 gene-edited region in four cell lines on long- and short-read sequencing platforms unravelled unknown and unintended genome editing events. The analysis revealed accidental kilobases-large insertions in three of the cell lines, which remained undetected using standard procedures. We also applied the targeted enrichment approach to identify the integration site of a transgene in a mouse line. The results demonstrate the potential of this technology in gene editing validation as well as in more classic transgenics.

DNA-only bioassay for simultaneous detection of proteins and nucleic acids.

Testing multiple biomarkers, as opposed to one, has become a preferred approach for diagnosing many heterogeneous diseases, such as cancer and infectious diseases. However, numerous technologies, including gold standard ELISA and PCR, can detect only one type of biomarker, either protein or nucleic acid (NA), respectively. In this work, we report for the first time simultaneous detection of proteins and NAs in the same solution, using solely functional NA (FNA) molecules. In particular, we combined the thrombin binding aptamer (TBA) and the 10-23 RNA-cleaving DNA enzyme (DNAzyme) in a single aptazyme molecule (Aptazyme1.15-3′), followed by extensive optimization of buffer composition, sequences and component ratios, to establish a competitive bioassay. Subsequently, to establish a multiplex bioassay, we designed a new aptazyme (Aptazyme2.20-5′) by replacing the target recognition and substrate sequences within Aptazyme1.15-3′. This designing process included an in silico study, revealing the impact of the target recognition sequence on the aptazyme secondary structure and its catalytic activity. After proving the functionality of the new aptazyme in a singleplex bioassay, we demonstrated the capability of the two aptazymes to simultaneously detect thrombin and NA target, or two NA targets in a multiplex bioassay. High specificity in target detection was achieved with the limits of detection in the low nanomolar range, comparable to the singleplex bioassays. The presented results deepen the barely explored features of FNA for diagnosing multiple targets of different origins, adding an extra functionality to their catalogue.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00216-021-03458-6.

Molecular Characterization of Post-Transplant Plasmablastic Lymphomas Implicates RAS, TP53, and NOTCH Mutations and MYC Deregulation in Disease Pathogenesis

Background: Plasmablastic lymphoma (PBL) is a rare subtype of aggressive B-cell non-Hodgkin lymphoma (B-NHL) that typically occurs in immunocompromised individuals, especially those with HIV infection or patients receiving immunosuppressive therapy for autoimmune diseases or organ allografts. Cytogenetic and gene expression profile analyses of PBL have provided some biological insights. However, the molecular and genetic alterations underlying PBL are not known. In this study, we assessed the pathologic features and spectrum of genetic mutations in a series of post-transplant PBL (PT-PBL) in order to identify molecular pathways potentially contributing to disease pathogenesis.Methods: We searched our departmental archives for cases of PT-PBL diagnosed over the last 17 years. Morphologic and immunophenotypic features and results of in situ hybridization for EBV were evaluated. C-MYC translocations and copy number abnormalities were assessed by fluorescence in situ hybridization (FISH). Targeted next-generation sequencing of a large panel of cancer-associated genes was performed on DNA extracted from formalin-fixed paraffin-embedded tumor samples as well as corresponding non-tumor tissue in 8 of 9 cases. Microsatellite instability and immunoglobulin heavy chain (IGH ) gene rearrangements were assessed using standard PCR based methodologies.Results: Fourteen PT-PBL samples from 9 recipients of solid organ allografts (2 lung, 3 cardiac, 3 renal, and 1 combined liver and renal) were analyzed. The median time to development of PBL after transplant was 8 years (range 0.5 - 11.9 years). All occurred at extranodal sites. Eight of 9 patients developed de novo PT-PBL and one case represented transformation of a clonally-related plasmacytoma-like lesion. All cases showed plasmablastic morphology and 5 of 9 cases had a variable but small component of mature plasma cells (PBL with plasmacytic differentiation). All cases demonstrated a plasmablastic immunophenotype; 6/9 cases expressed CD138 and 6/9 expressed IgG. A minority of cases (2/9) were CD56 positive. Five of 9 cases were EBV positive. Four (44%) cases harbored MYC rearrangements, which were detected at diagnosis in 3 cases and acquired at disease recurrence in 1 case; 3 of these 4 cases were EBV negative. Gains of MYC were observed in an EBV positive case. Non-synonymous pathogenic somatic mutations were identified in 7/9 PT-PBL. Recurrent mutations of the RAS/MAP kinase pathway genes were the most frequent, being detected in 4/9 patients (3 KRAS , 1 with both NRAS and HRAS ). TP53 mutations occurred in 3/9 cases and NOTCH1 was mutated in 2/9 cases. Two of 9 cases had mutations in DNA mismatch repair genes and showed high level microsatellite instability, which was associated with a high burden of somatic mutations. No PRDM1 (BLIMP1 ) mutations were observed.Conclusions: The mutational profiles of PT-PBL differ from those reported for other types of aggressive B-NHL and implicate deregulation of MYC as well as genetic aberrations of the RAS/MAPK, TP53, and NOTCH signaling pathways in disease pathogenesis. Further studies are warranted to determine if the mutational spectrum of PT-PBL is similar to or distinct from that of PBL arising in the context of other diseases. DisclosuresNo relevant conflicts of interest to declare.

Rapid and Safe Detection of SARS-CoV-2 and Influenza Virus RNA Using Onsite Quantitative PCR Diagnostic Testing From Clinical Specimens Collected in Molecular Transport Medium.

BACKGROUNDThe ability to rapidly detect SARS-CoV-2 and influenza virus infection is vital for patient care due to overlap in clinical symptoms. Roche’s cobas® Liat® SARS-CoV-2 & Influenza A/B Nucleic Acid Test used on the cobas® Liat® was granted approval under FDA’s Emergency Use Authorization (EUA) for nasopharyngeal (NP) and nasal swabs collected in viral/universal transport medium (VTM/UTM). However, there is a critical need for media that inactivates the virus, especially when specimens are collected in decentralized settings. This study aimed to investigate the use of PrimeStore Molecular Transport Medium® (PS-MTM®), designed to inactivate/kill and stabilize RNA/DNA for ambient transport and pre-processing of collected samples.METHODSA limit of detection (LOD) using serially diluted SARS-CoV-2 RNA in PS-MTM® and routine UTM was established using standard qPCR. Additionally, a clinical panel of NP and oral swabs collected in PS-MTM® collected during the 2020 coronavirus disease 2019 (COVID-19) pandemic were evaluated on the cobas® Liat® and compared to ‘gold standard’ qPCR on an ABI-7500 instrument.RESULTSSARS-CoV-2 RNA LOD using standard qPCR was equivalent on the cobas® Liat® instrument. cobas® Liat® detection from oral/NP swabs in PS-MTM® media exhibited equivalent positive percent agreement (100%) and negative percent agreement (96.4%).CONCLUSIONPS-MTM® and the Roche cobas® Liat® are compatible and complimentary devices for respiratory specimen collection and rapid disease detection, respectively. PS-MTM® is equivalent to standard VTM/UTM with the added benefit of safe, non-infectious sample processing for near-patient testing.

FnCas9-based CRISPR diagnostic for rapid and accurate detection of major SARS-CoV-2 variants on a paper strip.

The COVID-19 pandemic originating in the Wuhan province of China in late 2019 has impacted global health, causing increased mortality among elderly patients and individuals with comorbid conditions. During the passage of the virus through affected populations, it has undergone mutations, some of which have recently been linked with increased viral load and prognostic complexities. Several of these variants are point mutations that are difficult to diagnose using the gold standard quantitative real-time PCR (qRT-PCR) method and necessitates widespread sequencing which is expensive, has long turn-around times, and requires high viral load for calling mutations accurately. Here, we repurpose the high specificity of Francisella novicida Cas9 (FnCas9) to identify mismatches in the target for developing a lateral flow assay that can be successfully adapted for the simultaneous detection of SARS-CoV-2 infection as well as for detecting point mutations in the sequence of the virus obtained from patient samples. We report the detection of the S gene mutation N501Y (present across multiple variant lineages of SARS-CoV-2) within an hour using lateral flow paper strip chemistry. The results were corroborated using deep sequencing on multiple wild-type (n = 37) and mutant (n = 22) virus infected patient samples with a sensitivity of 87% and specificity of 97%. The design principle can be rapidly adapted for other mutations (as shown also for E484K and T716I) highlighting the advantages of quick optimization and roll-out of CRISPR diagnostics (CRISPRDx) for disease surveillance even beyond COVID-19. This study was funded by Council for Scientific and Industrial Research, India.

Molecular Identification of Virulence and Antibiotic-Resistant Gene in Escherichia coli O157 and Non-O157 Recovered from Water Samples

Background: This research work focused on ascertaining the presence of virulence and antibiotic-resistant genes in Escherichia coli (E. coli) O157 and non-O157 recovered from drinking water sources. Methods: Identification of E. coli O157 and non-O157 was carried out using standard serological and PCR techniques. Virulence genes (rfb O157, fliC H7, stx1, stx2, eae and hly genes) and antibiotic-resistant gene (BlaTEM) were detected using PCR method on selected isolates (n= 15) from different water sources which demonstrated multiple antibiotic-resistance in a previous study. Results: The serological identification result revealed that a total of 68 out of 382 E. coli isolates, recovered in a previous work, were identified as a presumptive E. coli O157. These included 19.1 %, 21.7 %, 33.3 %, 14.3 % and 9.1 % of E. coli isolates from wells, boreholes, sachets, streams and pipe-borne respectively. Statistical analysis revealed that there was no significant difference in the frequency of E. coli O157 from the different water sources (p > 0.05). Also, there was a statistically significant positive correlation between the E. coli isolates and E. coli O157 (Pearson’s r = 0.996). Detection of virulence and antibiotic-resistant genes showed that only 46.7 %, 33.3 %, 33.3 %, 93.3 %, and 66.7 % carried rfb O157, fliC H7, stx1, stx2 and rpoS gene respectively. In contrast, all the isolates possessed hly and BlaTEM genes but none had eae gene. Conclusion: The presence of one or combination of these genes in these isolates depicts their virulence and resistance nature.