Nucleic Acid Amplification Assays Research Articles

Large-volume RT-LAMP enables extraction-free amplification of HIV RNA from fingerstick plasma

BackgroundPoint-of-care (POC) nucleic acid amplification tests (NAAT) can significantly expand testing coverage, which is critical for infectious disease diagnostics and monitoring. The development of various isothermal amplification techniques greatly simplifies NAATs, but the cumbersome nucleic acid extraction step remains a bottleneck for the POC. Alternatively, extraction-free amplification, where crude samples are directly added into the assay, substantially simplifies the workflow. However, sample dilution is often needed in extraction-free amplification to reduce assay inhibition from sample matrices. Since NAATs are typically run at small volumes around 20 μL, the input sample quantity is therefore limited, resulting in an inevitable sensitivity loss. ResultsHere we explore the potential to perform isothermal amplification in larger reaction volumes to accommodate larger sample quantities, thereby improving sensitivity in extraction-free amplification. We demonstrated the approach by developing large-volume reverse transcription loop-mediated isothermal amplification (RT-LAMP) for HIV RNA detection from fingerstick plasma. We found that LAMP at reaction volumes up to 1 mL maintained the same performance. We then identified plasma dilution conditions needed to maintain the limit of detection in RT-LAMP. Subsequently, using inactivated HIV virus, we showed the successful detection of 24 HIV RNA copies in a 500 μL RT-LAMP reaction in the presence of 20 μL plasma (fingerstick volumes), translating to a viral load of 1200 copies per mL. To reduce the increased reagent cost with expanded reaction volumes, we further identified lower-cost reagents with maintained assay performance. Moreover, we showed that large-volume LAMP, compared to 20 μL reactions, could tolerate higher concentrations of various inhibitors in the sample, such as albumin and GuSCN. Significance and noveltyNAATs are conventionally conducted at small reaction volumes. Here we demonstrated that LAMP can be run at large reaction volumes (over 100 μL) with maintained assay performance, allowing sample inhibition to be mitigated while accommodating larger sample quantities. The same strategy of expanding reaction volumes could be applied to other isothermal amplification methods and various POC applications, to streamline test workflows and/or improve assay sensitivity.

Ligation-Based High-Performance Mimetic Enzyme Sensing Platform for Nucleic Acid Detection.

G-quadruplex (G4)/hemin DNAzyme is a promising candidate to substitute horseradish peroxidase in biosensing systems, especially for the detection of nucleic acids. However, the relatively suboptimal catalytic capacity limits its potential applications. This makes it imperative to develop an ideal signal for the construction of highly sensitive biosensing platforms. Herein, we integrated a novel chimeric peptide-DNAzyme (CPDzyme) with the ligase chain reaction (LCR) for the cost-efficient and highly sensitive detection of nucleic acids. By employing microRNA (miRNA) and single-nucleotide polymorphism detection as the model, we designed a G4-forming sequence on the LCR probe with a terminally labeled amino group. Subsequently, asymmetric hemin with carboxylic arms allowed assembly with the LCR products and peptide to form CPDzyme, followed by the magnetic separation of the extraneous components and chemiluminescence detection. Compared with the conventional G4/hemin signaling-based method, the LCR-CPDzyme system demonstrated 3 orders of magnitude improved sensitivity, with accurate quantification of as low as 25 aM miRNA and differentiation of 0.1% of mutant DNA from the pool containing a large amount of wild-type DNA. The proposed LCR-CPDzyme strategy is a potentially powerful method for in vitro diagnostics and serves as a reference for the development of other ligation- or hybridization-based nucleic acid amplification assays.

Abstract C136: Magnetic nanoparticles and quantum dots coupled visual confirmation of cervical cancer cells from cytology samples: A cost-effective, quick and suitable test for rural Indian set-up

Abstract Introduction Screening and management of cervical cancer is a National health priority of developed and developing countries like India. Cervical cancer screening and diagnosis are currently done by cervical pap screening, colposcopy, and nucleic acid hybridization and amplification assays, which are time-consuming and require specialized facilities. These shortcomings pose severe restrictions on the feasibility of the techniques for a country like India.Aim and objectives Bearing this in mind, we aimed to develop a simple, cost-effective, and visual detection of cervical cancer, based on magnetic nanoparticles coupled quantum dots immuno-nano-fluorescence assay (MNCQDINFA).Materials and methods The detection system comprised of magnetic nanoparticles(MNP) and quantum dots (QD) conjugated specific polyclonal antibodies complex generated against recombinantly purified individual domains (domainN and domainC) of HPV-16 oncoprotein E7, which is deregulated and overexpressed to detectable limits by transformed cervical cancer cells only [1-3]. The assay procedure involves incubating processed cervical cytology samples with MNP-Ab (domainN) complex, magnet-induced pelleting, incubation with QD-Ab (domainC) complex, magnet-assisted washing, and final reconstitution in sample buffer and visualization under UV (Figure). Reconstituted sample in the presence of UV gives fluorescence as an indication of the presence of E7 protein thus cervical cancer cells. Results This method produces a visual fluorescence exclusively in the presence of transformed cervical cancer cells under UV light. Analytical assays showed that a minimum of 0.1µg of E7 protein could be detected specifically from a pool of cellular proteins. HPV 16-positive cervical cancer cell lysates showed concordant results. Both the diagnostic sensitivity and specificity of the assay were determined to be 100%. Conclusion The proposed invention is a simple, visual (naked eye), quick (4 hours), and cost-effective (INR 200-300) cervical cancer cells detection method necessary to identify the candidates at risk so that suitable and timely intervention can be provided.

647. Comparative Analysis of Two Sample -to - Answer Nucleic Acid Amplification Assays for the Detection of Respiratory Viral Pathogens from Pediatric Respiratory Specimens

Abstract Background Rapid and accurate diagnosis of acute respiratory infection (ARI) in children is crucial for proper patient management, and nucleic acid amplification tests are the gold standard. Nasal swabs are preferred for surveillance studies due to their ease of use, mainly in children. Our study compares the performance of two sample-to-answer (STA) platforms for detecting multiple viruses in self-collected nasal swabs from symptomatic children. Methods Between January 2023 and March 2023, self-collected nasal swabs in UTM from children and staff with ARI symptoms were collected as part of an ongoing school-based surveillance study in a large Missouri school district. Samples were tested on the QIAstat-Dx Respiratory SARS-CoV-2 Panel and the fully automated Hologic Panther Fusion system, which can detect a total of 21 respiratory viruses. Percent Agreement analysis was performed to compare the performance of each platform. Results Of 200 samples collected, QIAstat detected viruses in 111 samples (55.5%) and Hologic in 115 samples (57.5%). Both assays showed high overall agreement (90%), with a PPA (Positive percentage agreement) of 89%, an NPA (Negative percentage agreement) of 94%. Parainfluenza (PIV) showed 100% PPA and NPA followed by adenovirus (Adv) (PPA: 100%; NPA: 97.9%) and rhinovirus (RV) (PPA: 92% and NPA: 94%). Human metapneumovirus (hMPV) had the lowest PPA (47%) but a high NPA (99%) (Table 1). The median CT values for QIAstat and Hologic were 28.8 (IQR 23.65 - 32.5) and 29.3 (IQR 24.2 – 34.5), respectively. QIAstat missed 19 detections, including hMPV (9, 53%), coronavirus NL63 (4, 16%), and RV (4, 8%), with a corresponding median Ct of 39.1 (IQR 34.5 – 41.2) on Hologic. Hologic missed 12 detections, with RV being the most common target (9, 17%), and a corresponding median Ct of 32.9 (IQR: 30.3-35.5) on QIAstat. Conclusion QIAstat and Hologic exhibit comparable performance (overall agreement 90%) for detecting viral respiratory pathogens from self-collected nasal swabs. Viral detections missed by QIAstat assay had low viral loads, while targets missed by Hologic assay had moderate viral load; additional investigation is needed. Our findings suggest that both STA platforms are suitable for diagnosing respiratory viral infections in self-collected nasal swabs in school-setting. Disclosures Rangaraj Selvarangan, BVSc, PhD, D(ABMM), FIDSA, FAAM, Abbott: Honoraria|Altona Diagnostics: Grant/Research Support|Baebies Inc: Advisor/Consultant|BioMerieux: Advisor/Consultant|BioMerieux: Grant/Research Support|Bio-Rad: Grant/Research Support|Cepheid: Grant/Research Support|GSK: Advisor/Consultant|Hologic: Grant/Research Support|Lab Simply: Advisor/Consultant|Luminex: Grant/Research Support

Considerations for colorblind individuals on selecting colorimetric or fluorescent dye assay outcomes.

A disadvantage of colorimetric detection in nucleic acid amplification assays is the possibility thata colorblind individual may interpret colors differently than observers with full-color vision. Using an isothermal amplification assay, the ability of colorblind individuals to distinguish between positive and negative results for four dyeswas tested. Five individuals with self-reported colorblindness and four with full-color vision reported their observations of the color of the solution. Although colorblind individuals may accurately interpret assay results, they were often not accurate in reporting the color. Hydroxynaphthol blue was the most problematic dye, and both phenol red and SYBR™ green were less troublesome. Consideration for colorblind individuals is warranted when developing an assay and training staff in its performance.

Self- and physician-collected high-risk human papillomavirus (HPV) testing to detect high-grade cervical lesions among Thai women

ObjectiveWe compared the performance of high-risk human papillomavirus (HPV) messenger RNA testing of physician- and self-collected specimens for detecting histological grade 2 or higher cervical intraepithelial neoplasia (CIN) among women...

Laboratory diagnostics for primary spinal infections in pediatric and adult populations: a narrative review

Primary spinal infection (PSI) is a generic term covering a heterogeneous group of infections that can affect the vertebral body, intervertebral disks, the content of the medullary cavity, and adjacent paraspinal tissues. Patients’ characteristics can vary significantly, notably according to their age, and some of these characteristics undoubtedly play a primordial role in the occurrence of a PSI and in the type of offending pathogen. Before approaching the subject of laboratory diagnostics, it is essential to define the characteristics of the patient and their infection, which can then guide the physician toward specific diagnostic approaches. This review critically examined the roles and usefulness of traditional and modern laboratory diagnostics in supporting clinicians’ decision-making in cases of pediatric and adult primary spinal infection (PSI). It appears impossible to compare PSIs in children and adults, whether from an epidemiological, clinical, bacteriological, or biological perspective. The recipients are really too different, and the responsible germs are closely correlated to their age. Secondly, the interpretation of traditional laboratory blood tests appears to contribute little guidance for clinicians attempting to diagnose a PSI. Biopsy or needle aspiration for bacterial identification remains a controversial subject, as the success rates of these procedures for identifying causative organisms are relatively uncertain in pediatric populations.Using nucleic acid amplification assays (NAAAs) on biopsy samples has been demonstrated to be more sensitive than conventional cultures for diagnosing PSI. Recent advances in next-generation sequencing (NGS) are particularly interesting for establishing a microbiological diagnosis of a PSI when standard cultures and NAAAs have failed to detect the culprit. We can even imagine that plasma metagenomic NGS using plasma (known as “liquid biopsy”) is a diagnostic approach that can detect not only pathogens circulating in the bloodstream but also those causing focal infections, and thus eliminate the need for source sample collection using costly invasive surgical procedures.

Nanoplasmonic amplification in microfluidics enables accelerated colorimetric quantification of nucleic acid biomarkers from pathogens.

Deployment of nucleic acid amplification assays for diagnosing pathogens in point-of-care settings is a challenge due to lengthy preparatory steps. We present a molecular diagnostic platform that integrates a fabless plasmonic nano-surface into an autonomous microfluidic cartridge. The plasmonic 'hot' electron injection in confined space yields a ninefold kinetic acceleration of RNA/DNA amplification at single nucleotide resolution by one-step isothermal loop-mediated and rolling circle amplification reactions. Sequential flow actuation with nanoplasmonic accelerated microfluidic colorimetry and in conjugation with machine learning-assisted analysis (using our 'QolorEX' device) offers an automated diagnostic platform for multiplexed amplification. The versatility of QolorEX is demonstrated by detecting respiratory viruses: SARS-CoV-2 and its variants at the single nucleotide polymorphism level, H1N1 influenza A, and bacteria. For COVID-19 saliva samples, with an accuracy of 95% on par with quantitative polymerase chain reaction and a sample-to-answer time of 13 minutes, QolorEX is expected to advance the monitoring and rapid diagnosis of pathogens.

DNA-templated silver nanocluster-based cleavable probes for nucleic acid detection.

DNA or RNA probes that fluoresce upon enzymatic digestion, such as TaqMan probes and DNaseAlert, allow for not only nuclease activity detection but also nucleic acid amplification assays. However, relying on FRET as a signal transduction mechanism, these short oligonucleotide probes need to be dually labeled with a donor and a quencher, thus suffering from the issues of high cost and singly labeled impurities. Here we demonstrate a new class of probe, based on DNA-templated silver nanoclusters (DNA/AgNCs). These nanoclusters exhibit fascinating photophysical properties that depend on the arrangement of the nucleobases in the DNA ligand. Not using FRET, our probes not only turn on upon nuclease cleavage but display a significantly altered fluorescence color. Although a variety of DNA/AgNC probes exist to detect proteins and nucleic acids, no AgNC probes have been designed to detect nuclease activity (enzymes that cleave the bonds between the nucleotides of nucleic acids such as DNA). Due to their multicolor nature, our probes also enable ratiometric sensing for the highly quantitative detection of nuclease activities. Here we show that our silver cluster probes can replace DNaseAlert for the enzymatic activity assay of DNase I. The electrophoresis gel shows clear digestion of our silver cluster probes by DNase I and the digested products have a significantly enhanced fluorescence with 700% increase in intensity and different emission color (90 nm red-shift). In addition, our silver cluster probes can substitute the FRET probes in novel CRISPR-Cas assays (such as DETECTR and SHERLOCK) for nucleic acid detection. We demonstrate that we can selectively detect SARS-CoV-2 cDNA in combination with CRISPR-Cas12a. Our results not only demonstrate that DNA-templated silver nanocluster probes can be a versatile nuclease activity probe but also provide insights into the interactions between nucleases and metallo-DNA nanomaterials.

Cerebrospinal fluid lactate as a predictive biomarker for tuberculous meningitis diagnosis.

The definitive diagnosis of tuberculous meningitis (TBM) is achieved by identifying Mycobacterium tuberculosis (MTb) in cerebrospinal fluid (CSF); however, diagnostic confirmation is difficult due to the inability ofcurrent tests for an effective diagnosis. Our objective was to retrospectively assess the characteristics of CSF lactate (CSF-LA) as an adjunct biomarker in the diagnosis of TBM. 608 CSF laboratory reports were assessed. Of these, 560 had clinically suspected TBM. These were classified as definite (n=36), probable (23), possible (278),or non-TBM (223) according to the international consensus TBM case definitions. An additional 48 CSF samples were negative controls with normal CSF. Against a reference standard of definite TBM, the cut-off value for CSF-LA was 4.0mmol/L, the area under the ROC curve was 0.88 (95% CI, 0.82-0.94; p=0.0001), sensitivity was 69%, specificity 90%, negative predictive value 98%. These diagnostic parameters decreased when calculated against those of the other categories of TBM. CSF-LA exhibited high specificity, efficiency, negative predictive value, and clinical utility index in all the groups studied. CSF-LA is a useful diagnostic marker to rule out TBM when associated with conventional microbiology tests, nucleic acid amplification assays, and clinical algorithms, particularly in endemic areas.

Clinical and Biologic Characteristics of Kingella kingae -Induced Septic Arthritis of the Knee in Young Children.

Septic arthritis of the knee is presumed to be the most frequent form of Kingella kingae -induced osteoarticular infection. This study aimed to report on the clinical course, biological parameters, and results of microbiological investigations among children with K. kingae -induced septic arthritis of the knee. It also assessed the modified Kocher-Caird criteria's ability to predict K. kingae -induced septic arthritis of the knee. The medical charts of 51 children below 4 years old with confirmed or highly probable K. kingae -induced arthritis of the knee were reviewed. Data were gathered on the five variables in the commonly-used Kocher-Caird prediction algorithm (body temperature, refusal to bear weight, leukocytosis, erythrocyte sedimentation rate, and C-reactive protein level). Patients with K. kingae -induced arthritis of the knee usually presented with a mildly abnormal clinical picture and normal or near-normal serum levels of acute-phase reactants. Data on all five variables were available for all the children: 7 children had zero predictors; 8, 20, 12, and 4 children had 1, 2, 3, and 4 predictors, respectively; no children had 5 predictors. This gave an average of 1.96 predictive factors and a subsequent probability of ≤ 62.4% of infectious arthritis in this pediatric cohort. Because the clinical features of K. kingae -induced arthritis of the knee overlap with many other conditions affecting this joint, the Kocher-Caird prediction algorithm is not sensitive enough to effectively detect K. kingae -induced septic arthritis of the knee. Excluding K. kingae -induced arthritis of the knee requires performing nucleic acid amplification assays on oropharyngeal swabs and joint fluid from those young children presenting with effusion of the knee, even in the absence of fever, leukocytosis, or a high Kocher-Caird score.

A robust, low-cost instrument for real-time colorimetric isothermal nucleic acid amplification.

The COVID-19 pandemic has highlighted the need for broader access to molecular diagnostics. Colorimetric isothermal nucleic acid amplification assays enable simplified instrumentation over more conventional PCR diagnostic assays and, as such, represent a promising approach for addressing this need. In particular, colorimetric LAMP (loop-mediated isothermal amplification) has received a great deal of interest recently. However, there do not currently exist robust instruments for performing these kinds of assays in high throughput with real-time readout of amplification signals. To address this need, we developed LARI, the LAMP Assay Reader Instrument. We have deployed over 50 LARIs for routine use in R&D and production environments, with over 12,000 assays run to date. In this paper, we present the design and construction of LARI along with thermal, optical, and assay performance characteristics. LARI can be produced for under $1500 and has broad applications in R&D, point-of-care diagnostics, and global health.

Mobilefuge: A low-cost, portable, open source, 3D-printed centrifuge that can be powered by a mobile phone

Background: A centrifuge is an essential piece of equipment for several biology and diagnostic applications such as pelleting cells and purifying nucleic acids, etc. In recent years, there has been an increasing interest for low-cost centrifuges that are alternatives to the commercially available centrifuges. In this article, we present a low-cost, open-source, 3D printed centrifuge (mobilefuge) that is portable, robust, stable, safe, easy to build and operate. Methods: The mobilefuge was designed such that it doesn't require soldering or programming skills and can be built without any specialised equipment yet practical enough for high throughput use. More importantly, the mobilefuge can be powered from widely available 5V USB ports, including mobile phones and associated power supplies. This allows the mobilefuge to be used even in off-grid and resource limited settings. Results: Performance of the mobilefuge is demonstrated by separating silica particles are from a suspension consisting of water and “glassmilk”. This separation process is one of the crucial steps in nucleic-acid amplification assays for reliable, low-cost diagnostic applications such as SARS-CoV2. Conclusions: We believe that beyond the nucleic-acid assays, the mobilefuge can have several applications as a microcentrifuge in the field of biomedical research and diagnostics.

Amine-Functionalized Quantum Dots as a Universal Fluorescent Nanoprobe for a One-Step Loop-Mediated Isothermal Amplification Assay with Single-Copy Sensitivity.

Loop-mediated isothermal amplification (LAMP) has received considerable attention for decentralized (point-of-care and on-site) nucleic acid testing in view of its simple temperature control (60-65 °C) and short assay time (15-60 min). There remains a challenge in its wide adoption and acceptance due to the limitations of the existing amplification result reporter probes, e.g., photobleaching of organic fluorophore and reduced sensitivity of the pH-sensitive colorimetric dye. Herein, we demonstrate CdSeS/ZnS quantum dots (semiconductor fluorescent nanocrystals with superior photostability than organic fluorophore) with surface modification of cysteamine (amine-QDs) as a new reporter probe for LAMP that enabled single-copy sensitivity (limit of detection of 83 zM; 20 μL reaction volume). For a negative LAMP sample (absence of target sequence), positively charged amine-QDs remained dispersed due to interparticle electrostatic repulsion. While for a positive LAMP sample (presence of target sequence), amine-QDs became precipitated. The characterization data showed that amine-QDs were embedded in magnesium pyrophosphate crystals (generated during positive LAMP), thus leading to their coprecipitation. This amine-QD-based one-step LAMP assay advances the field of QD-based nucleic acid amplification assays in two aspects: (1) compatibility─one-step amplification and detection (versus separation of amplification and detection steps); and (2) universality─the same amine-QDs for different target sequences (versus different oligonucleotide-modified QDs for different target sequences).

Tuberculosis diagnostics to reduce HIV-associated mortality

Tuberculosis (TB) was declared a global emergency in 1993 by the World health Organization (WHO), with global and African regional TB incidence rates driven by the HIV epidemic. Much of this burden lay in health facilities in sub-Saharan Africa, with post-mortem studies showing almost half of fatal TB goes undiagnosed, reflecting a failure in the approach to TB diagnostics. HIV-associated TB is often under the clinical radar, and this paper describes strategies to improve TB diagnostics to expedite treatment, reduce the amount of undiagnosed disease and ultimately reduce mortality. Through studies assessing the diagnostic accuracy and yield of new, rapid TB diagnostics, including the Xpert MTB/RIF nucleic acid amplification and urinary lipoarabinomannan (LAM) lateral flow assays, and clinical trials to measure impact on mortality and clinically relevant outcomes, this paper describes how TB diagnostics can reduce HIV-associated morbidity and mortality. With improved TB diagnostics in the pipeline, the future of urine-LAM assays for TB are also discussed.

Label-free electrical monitoring of nucleic acid amplification with integrated hydrogel ionic diodes.

We demonstrate here for the first time the utility of a monolithically integrated hydrogel ionic diode for label-free quantitative DNA detection and real-time monitoring of nucleic acid amplification. The hydrogel ionic diode presented herein, unlike nanomaterial-based field-effect biosensors, features high cost-effectiveness and convenient fabrication. This is realized by patterning a micrometer-sized heterojunction consisting of adjacent segments of polycationic and polyanionic hydrogels on a microfluidic chip through simple photocuring steps. The integrated diode rectifies ionic currents being sensitive to the charge of DNA adsorbed onto the polycationic chains through electrostatic associations. Based on the mechanism, we show that the ionic biosensor can electrically quantify DNA in a dynamic range relevant to typical nucleic acid amplification assays. Utilizing the device, we demonstrate the evaluation of a PCR assay amplifying a 500-bp DNA fragment of E. coli, an infection-causing pathogen, and real-time in situ monitoring of an isothermal assay amplifying E. coli whole genome. We anticipate that the device could potentially pave the way for miniaturized optics-free platforms for quantifying nucleic acid amplification at point-of-care.

Use of a FluoroType® System for the Rapid Detection of Patients with Multidrug-Resistant Tuberculosis—State of the Art Case Presentations

According to the World Health Organization (WHO), there were 465,000 cases of tuberculosis caused by strains resistant to at least two first-line anti-tuberculosis drugs: rifampicin and isoniazid (MDR-TB). In light of the growing problem of drug resistance in Mycobacterium tuberculosis across laboratories worldwide, the rapid identification of drug-resistant strains of the Mycobacterium tuberculosis complex poses the greatest challenge. Progress in molecular biology and the development of nucleic acid amplification assays have paved the way for improvements to methods for the direct detection of Mycobacterium tuberculosis in specimens from patients. This paper presents two cases that illustrate the implementation of molecular tools in the recognition of drug-resistant tuberculosis.

Effect of not vortexing nasopharyngeal and throat swabs on SARS-CoV-2 nucleic acid detection: A pilot study

The processing of swabs for respiratory virus detection involves vortexing while still in the viral transport medium (VTM). The effect of not vortexing swabs prior to analysis has not been studied extensively for SARS-CoV-2 detection, and presents an opportunity to improve pre-analytic laboratory workflow. We aimed to assess the impact of not vortexing nasopharyngeal/throat swabs submitted in VTM for SARS-CoV-2 testing. To assess the impact of not vortexing swabs, 277 swab samples were tested for SARS-CoV-2 RNA in paired vortexed and non-vortexed aliquots using eight routine nucleic acid amplification assays. We compared the qualitative (positive/negative) and semi-quantitative (cycle threshold, Ct) results. Following discordant analysis, all but one non-vortexed sample had the same qualitative result as the vortexed sample. 27.4 % of samples were SARS-CoV-2 positive. Comparison of Ct values revealed an apparent reduction in human cellular nucleic acid in the non-vortexed samples (mean Ct values of 24.0 and 26.5 for vortexed and non-vortexed samples, respectively, p < 0.0001) and increased Ct values for non-vortexed samples using a laboratory-developed SARS-CoV-2 assay (mean Ct values of 4.1 and 4.2 for vortexed and non-vortexed samples, respectively; p < 0.0001), but this was not observed for a more automated commercial SARS-CoV-2 assay (mean Ct values of 15.2 for both vortexed and non-vortexed samples, respectively; p = 0.68). While vortexing swabs appears to improve the recovery of cellular material, it does not have an appreciable impact on the qualitative sensitivity of SARS-CoV-2 nucleic acid tests, which may support omission of this step and simplification of front-end sample processing.

First report of Kingella kingae infection in a paediatric population in Accra, Ghana.

IntroductionKingella kingae is recognized as a frequent source of childhood bacteremia and the commonest agent of skeletal system infections in children 6 months - 4 years old. Several factors, including difficulty in detecting this fastidious organism in routine laboratory assays, result in underdiagnosis of the infections. Species-specific nucleic acid amplification assays, however, significantly improve the detection of K. kingae in blood samples. The aim of this study was to detect K. kingae infection in young children in Accra, Ghana.Methodsa cross-sectional based study was carried out in three hospitals in Accra. Children with febrile illness and directed by a clinician for blood culture were recruited. Blood samples collected were analysed by culture and polymerase chain reaction (PCR), using universal prokaryotic and K. kingae rtxA primers.Resultsblood samples from 232 children (mean age 20.10 ± 12.57 months) were analysed. Bacteremia (72.4%) was the highest clinical diagnosis particularly in the 12-24 months age group. Only 7 (3.1%) samples showed bacterial growth and were negative for Kingella. PCR with universal prokaryotic primers succeeded in 223 (96.1%) out of 232 samples. PCR with K. kingae rtxA toxin primers was positive for 12 (5.4%) samples, all diagnosed as bacteremia, out of the 223 samples. Eleven (91.7%) out of the 12 K. kingae PCR positives were culture-negative.ConclusionKingella kingae was detected only by PCR specific for the K. kingae rtxA toxin. Kingella kingae may be a potential cause of bacteremia and hence febrile illness in young children living in Accra, Ghana.

Comparative Evaluation of Allplex Respiratory Panels 1, 2, 3, and BioFire FilmArray Respiratory Panel for the Detection of Respiratory Infections.

Multiplex nucleic acid amplification assays that simultaneously detect multiple respiratory pathogens in a single nasopharyngeal swab (NPS) specimen are widely used for rapid clinical diagnostics. We evaluated Allplex Respiratory Panel (RP) 1, 2, 3, and the BioFire FilmArray RP assay for detecting respiratory pathogens from NPS specimens. In all, 181 NPS specimens obtained from patients suspected of having respiratory infections during the non-influenza season (August–December 2019) were included. The Allplex RP 1, 2, and 3 detected 154 samples positive for respiratory viruses, whereas the BioFire FilmArray detected viruses in 98 samples. Co-infection with two or more viruses was detected in 41 and 17 NPS specimens by Allplex RP and the BioFire FilmArray RP, respectively. For adenoviruses, Allplex RP 1 detected 31 specimens, compared to 34 by the BioFire FilmArray. In all, 64 NPS specimens were positive for human enterovirus (HEV) and human rhinovirus (HRV) on the Allplex RP, in contrast to 39 HEV/HRV on the BioFire FilmArray. The parainfluenza virus (PIV-1–4) detection rate differed between the two systems. Most discrepant results were observed for NPS specimens with high cycle threshold values obtained by Allplex RP. This study showed concordant performance of the Allplex RP 1, 2, 3, and the BioFire FilmArray RP for the simultaneous detection of multiple respiratory viruses.