Rapid Molecular Diagnostic Tests Research Articles

Tackling Infectious Diseases with Rapid Molecular Diagnosis and Innovative Prevention.

Infectious diseases (IDs) are a leading cause of death. The diversity and adaptability of microbes represent a continuing risk to health. Combining vision with passion, our transdisciplinary medical research team has been focussing its work on the better management of infectious diseases for saving human lives over the past five decades through medical discoveries and innovations that helped change the practice of medicine. The team used a multiple-faceted and integrated approach to control infectious diseases through fundamental discoveries and by developing innovative prevention tools and rapid molecular diagnostic tests to fulfill the various unmet needs of patients and health professionals in the field of ID. In this article, as objectives, we put in context two main research areas of ID management: innovative infection prevention that is woman-controlled, and the rapid molecular diagnosis of infection and resistance. We also explain how our transdisciplinary approach encompassing specialists from diverse fields ranging from biology to engineering was instrumental in achieving success. Furthermore, we discuss our vision of the future for translational research to better tackle IDs.

Effect of mixed Mycobacterium tuberculosis infection on rapid molecular diagnostics among patients starting MDR-TB treatment in Uganda

BackgroundMixed M. tuberculosis (MTB) infection occurs when one is infected with more than one clonally distinct MTB strain. This form of infection can assist MTB strains to acquire additional mutations, facilitate the spread of drug-resistant strains, and boost the rate of treatment failure. Hence, the presence of mixed MTB infection could affect the performance of some rapid molecular diagnostic tests such as Line Probe Assay (LPA) and GeneXpert MTB/RIF (Xpert) assays.MethodsThis was a cross-sectional study that used sputum specimens collected from participants screened for STREAM 2 clinical trial between October 2017 and October 2019. Samples from 62 MTB smear-positive patients and rifampicin-resistant patients from peripheral health facilities were processed for Xpert and LPA as screening tests for eligibility in the trial. From November 2020, processed stored sputum samples were retrieved and genotyped to determine the presence of mixed-MTB strain infection using a standard 24-locus Mycobacterial Interspersed Repetitive Unit–Variable Number Tandem-Repeat (MIRU-VNTR). Samples with at least 20/24 MIRU-VNTR loci amplified were considered for analysis. Agar proportional Drug Susceptibility Test (DST) was performed on culture isolates of samples that had discordant results between LPA and Xpert. The impact of the presence of mixed-MTB strain on Xpert and LPA test interpretation was analyzed.ResultsA total of 53/62 (85%) samples had analyzable results from MIRU-VNTR. The overall prevalence of mixed-MTB infection was 5/53 (9.4%). The prevalence was highest among male’s 3/31 (9.7%) and among middle-aged adults, 4/30 (33.3%). Lineage 4 of MTB contributed 3/5 (60.0%) of the mixed-MTB infection prevalence. Having mixed MTB strain infection increased the odds of false susceptible Xpert test results (OR 7.556, 95% CI 0.88–64.44) but not for LPA. Being HIV-positive (P = 0.04) independently predicted the presence of mixed MTB infection.ConclusionsThe presence of mixed-MTB strain infection may affect the performance of the GeneXpert test but not for LPA. For patients with high pre-test probability of rifampicin resistance, an alternative rapid method such as LPA should be considered.

Oral swabs with a rapid molecular diagnostic test for pulmonary tuberculosis in adults and children: a systematic review

Tuberculosis is a leading cause of infectious disease mortality worldwide, but diagnosis of pulmonary tuberculosis remains challenging. Oral swabs are a promising non-sputum alternative sample type for the diagnosis of pulmonary tuberculosis. We aimed to assess the diagnostic accuracy of oral swabs to detect pulmonary tuberculosis in adults and children and suggest research implications. In this systematic review, we searched published and preprint studies from Jan 1, 2000, to July 5, 2022, from eight databases (MEDLINE, Embase, Scopus, Science Citation Index, medRxiv, bioRxiv, Global Index Medicus, and Google Scholar). We included diagnostic accuracy studies including cross-sectional, cohort, and case-control studies in adults and children from which we could extract or derive sensitivity and specificity of oral swabs as a sample type for the diagnosis of pulmonary tuberculosis against a sputum microbiological (nucleic acid amplification test [NAAT] on sputum or culture) or composite reference standard. Of 550 reports identified by the search, we included 16 eligible reports (including 20 studies and 3083 participants) that reported diagnostic accuracy estimates on oral swabs for pulmonary tuberculosis. Sensitivity on oral swabs ranged from 36% (95% CI 26-48) to 91% (80-98) in adults and 5% (1-14) to 42% (23-63) in children. Across all studies, specificity ranged from 66% (95% CI 52-78) to 100% (97-100), with most studies reporting specificity of more than 90%. Meta-analysis was not performed because of sampling and testing heterogeneity. Sensitivity varies in both adults and children when diverse methods are used. Variability in sampling location, swab type, and type of NAAT used in accuracy studies limits comparison. Although data are suggestive that high accuracy is achievable using oral swabs with molecular testing, more research is needed to define optimal methods for using oral swabs as a specimen for tuberculosis detection. The current data suggest that tongue swabs and swab types that collect increased biomass might have increased sensitivity. We would recommend that future studies use these established methods to continue to refine sample processing to maximise sensitivity. Bill and Melinda Gates foundation (INV-045721) and FIND (Netherlands Enterprise Agency on behalf of the Minister for Foreign Trade and Development Cooperation [NL-GRNT05] and KfW Development Bank, German Federal Ministry of Education and Research [KFW-TBBU01/02]).

Clinical Differences between SARS-CoV-2 and RSV Infections in Infants: Findings from a Case-Control Study.

Infants are a unique pediatric group due to their high hospitalization rates and unfavorable outcomes from acute infectious diseases. Understanding the clinical differences and aftereffects of SARS-CoV-2 in comparison to other prevalent viruses in this age group, like RSV, is crucial for effective management. We conducted a retrospective case-control study of infants hospitalized with SARS-CoV-2 or respiratory syncytial virus (RSV) infection in one year, in a tertiary pediatric hospital in Bucharest, Romania. A total of 188 infants were included in the analysis in a 1:1 ratio (94 with SARS-CoV-2 infection and 94 with RSV infection). Infants with COVID-19 were 10.2 times more likely to have fever (p < 0.001) and 2.4 times more likely to have diarrhea (p = 0.016). Conversely, infants with RSV were 2.5 times more likely to have a cough (p < 0.001), 3.0 times more likely to have nasal congestion (p < 0.001), and 14.7 times more likely to present with dyspnea (p < 0.001). Increased lymphocyte count was more common in infants with RSV (p = 0.008), while lymphopenia was more frequent in infants with SARS-CoV-2 (p = 0.011). The median length of hospital stay was one day longer in infants with RSV infection (5 days vs. 4 days). Overall, infants with RSV infection had a 27.3-fold increased risk of developing respiratory failure (p < 0.001), while infants with COVID-19 had a 5.8-fold increased risk of laryngitis (p = 0.003). Our findings suggest that infants with SARS-CoV-2 infection may present with polymorphic symptoms, mostly dominated by fever, whereas infants with RSV often present with respiratory symptoms. Laboratory differentiation between the two infections is challenging; therefore, the use of rapid antigen or molecular diagnostic tests is crucial for accurate diagnosis, epidemiologically appropriate measures, and effective management. Continued surveillance of both viruses in infants, and beyond, and the implementation of specific control measures are needed to mitigate their impact on this vulnerable pediatric group.

Handheld Ultra-Fast Duplex Polymerase Chain Reaction Assays and Lateral Flow Detection and Identification of Leishmania Parasites for Cutaneous Leishmaniases Diagnosis.

Early and accurate detection of infectious diseases is a key step for surveillance, epidemiology and control, notably timely disease diagnosis, patient management and follow-up. In this study, we aimed to develop handheld ultra-fast duplex PCR assays coupled to amplicon detection by lateral flow (LF) immunoassay to deliver a rapid and simple molecular diagnostic test for concomitant detection and identification of the main Leishmania parasites encountered in Tunisia. We selected two DNA targets to amplify L. major/L. tropica and L. infantum/L. tropica groups of species DNAs, respectively. We optimized the experimental conditions of a duplex ultra-fast PCR. The amplification is performed using a portable Palm convection PCR machine within 18 min, and the products are detected using an LF cassette within 10 min. The test allows the identification of the infecting species according to the position and number of test lines revealed. Tested on a selection of DNAs of representative Leishmania strains of the three studied species (N = 37), the ultra-fast duplex PCR-LF showed consistent, stable and reproducible results. The analytical limit of detection of the test was 0.4 pg for L. major, 4 pg for L. infantum and 40 pg for L. tropica.

Simple Point-of-Care Nucleic Acid Amplification Test for Rapid SARS-CoV-2 Infection Diagnosis.

After three years of the SARS-CoV-2 pandemic, the demand for developing field-deployable point-of-care (PoC) molecular diagnostic tests has increased. Although RT-qPCR is the molecular diagnostic gold standard and is accurate, it is not readily applied to point-of-care testing (POCT). Meanwhile, rapid diagnostic kits have the disadvantage of low sensitivity. Recently, rapid isothermal nucleic acid amplification technology has emerged as an alternative for rapid diagnosis. Here, we developed a rapid SARS-CoV-2 reverse transcription loop-mediated isothermal amplification (RT-LAMP)-lateral flow assay (LFA) kit. This kit includes a Chelex-100/boiling nucleic acid extraction device and a one-step amplification detection apparatus capable of performing the entire process, from RNA extraction to detection, and diagnosing SARS-CoV-2 infection within 40 min without contamination. The detection limits of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 100 plaque-forming units (PFUs) mL-1 and 10-1 PFU mL-1 for RNA samples extracted using the Chelex-100/boiling nucleic acid extraction device and commercial AdvansureTM E3 system, respectively. The sensitivity and specificity of the rapid SARS-CoV-2 RT-LAMP-LFA kit were 97.8% and 100%, respectively. Our SARS-CoV-2 RT-LAMP-LFA kit exhibited high sensitivity and specificity within 40 min without requiring laboratory instruments, suggesting that the kit could be used as a rapid POC molecular diagnostic test for SARS-CoV-2.

Performance of the Cue COVID-19 point-of-care molecular test: insights from a multi-site clinic service model.

The COVID-19 pandemic highlighted the critical need for rapid and accurate molecular diagnostic testing. The Cue COVID-19 Point-of-Care Test (Cue POCT) is a nucleic acid amplification test (NAAT), authorized by Health Canada and FDA as a POCT for SARS-CoV-2 detection. Cue POCT was deployed at a network of clinics in Ontario, Canada with n = 13,848 patrons tested between 17 July 2021 and 31 January 2022. The clinical performance and operational experience with Cue POCT were examined for this testing population composed mostly of asymptomatic individuals (93.7%). A head-to-head prospective clinical verification was performed between 17 July and 4 October for all POCT service clients (n = 3,037) with paired COVID-19 testing by Cue and RT-PCR. Prospective verification demonstrated a clinical sensitivity of 100% and clinical specificity of 99.4% for Cue COVID-19 POCT. The lack of false negatives and low false positive rate (0.64%), underscores the high accuracy (99.4%) of Cue POCT to provide rapid PCR quality results. Low error rates (cancellation rate of 0% and invalid rate of 0.63%) with the current software version were additionally noted. Taken together, these findings highlight the value of accurate molecular COVID-19 POCT in a distributed service delivery model to rapidly detect cases in the community with the potential to curb transmission in high-exposure settings (i.e., in-flight, congregate workplace, and social events). The insights gleaned from this operational implementation are readily transferable to future POCT diagnostic services. IMPORTANCE This manuscript reports on the findings of a large asymptomatic population who underwent surveillance COVID testing on the Cue COVID-19 Point-of-Care Test (POCT). Review of test performance of this rapid molecular POCT, as compared to gold standard RT-PCR, is valuable to many audiences, including public health, emergency testing services, employers, and the general population of consumers who are seeking a user-friendly, accurate, cost-effective, and sustainable testing model for COVID screening. The findings from this operational experience also inform future models of POCT services beyond COVID.

Implementation of large-scale pooled testing to increase rapid molecular diagnostic test coverage for tuberculosis: a retrospective evaluation

In 2021, only 6.4 million of the 10.6 million people with tuberculosis (TB) were diagnosed and treated for the disease. Although the World Health Organization recommends initial diagnostic testing using a rapid sensitive molecular assay, only 38% of people diagnosed with TB benefited from these, due to barriers including the high cost of available assays. Pooled testing has been used as an approach to increase testing efficiency in many resource-constrained situations, such as the COVID-19 pandemic, but it has not yet been widely adopted for TB diagnostic testing. Here we report a retrospective analysis of routine pooled testing of 10,117 sputum specimens using the Xpert MTB/RIF and Xpert MTB/RIF Ultra assays that was performed from July 2020 to February 2022. Pooled testing saved 48% of assays and enabled rapid molecular testing for 4156 additional people as compared to individual testing, with 6.6% of specimens positive for TB. From an in silico analysis, the positive percent agreement of pooled testing in pools of 3 as compared with individual testing for the Xpert MTB/RIF Ultra assay was estimated as 99.4% (95% CI, 96.6% to 100%). These results support the scale-up of pooled testing for efficient TB diagnosis.

Rapid molecular diagnostic tests fail to PERFORM in febrile children.

Rapid molecular diagnostic tests fail to PERFORM in febrile children.

A novel multiplex qPCR‑HRM assay for the simultaneous detection of four abortive zoonotic agents in cattle, sheep, and goats

Abortifacient pathogens induce substantial economic losses in the livestock industry worldwide, and many of these pathogens are zoonotic, impacting human health. As Brucella spp., Coxiella burnetii, Leptospira spp., and Listeria monocytogenes cause abortion, rapid differential molecular diagnostic tests are needed to facilitate early and accurate detection of abortion to establish effective control measures. However, the available molecular methods are laborious, time-consuming, or costly. Therefore, we developed and validated a novel multiplex real-time polymerase chain reaction (qPCR) method based on high-resolution melting (HRM) curve analysis to simultaneously detect and differentiate four zoonotic abortifacient agents in cattle, goats, and sheep. Our HRM assay generated four well-separated melting peaks allowing the differentiation between the four zoonotic abortifacients. Out of 216 DNA samples tested, Brucella spp. was detected in 45 samples, Coxiella burnetii in 57 samples, Leptospira spp. in 12 samples, and Listeria monocytogenes in 19 samples, co-infection with Brucella spp. and Coxiella burnetii in 41 samples, and 42 samples were negative. This assay demonstrated good analytical sensitivity, specificity, and reproducibility. This is a valuable rapid, cost-saving, and reliable diagnostic tool for detecting individual and co-infections for zoonotic abortifacient agents in ruminants.

Mortality due to Multidrug-Resistant Gram-Negative Bacteremia in an Endemic Region: No Better than a Toss of a Coin.

The incidence of multidrug-resistant (MDR) bloodstream infections (BSIs) is associated with high morbidity and mortality. Little evidence exists regarding the epidemiology of BSIs and the use of appropriate empirical antimicrobial therapy in endemic regions. Novel diagnostic tests (RDTs) may facilitate and improve patient management. Data were assessed from patients with MDR Gram-negative bacteremia at a university tertiary hospital over a 12-month period. In total, 157 episodes of MDR Gram-negative BSI were included in the study. The overall mortality rate was 50.3%. Rapid molecular diagnostic tests were used in 94% of BSI episodes. In univariate analysis, age (OR 1.05 (95% CI 1.03, 1.08) p < 0.001), Charlson Comorbidity Index (OR 1.51 (95% CI 1.25, 1.83) p < 0.001), procalcitonin ≥ 1(OR 3.67 (CI 95% 1.73, 7.79) p < 0.001), and monotherapy with tigecycline (OR 3.64 (95% CI 1.13, 11.73) p = 0.030) were the only factors associated with increased overall mortality. Surprisingly, time to appropriate antimicrobial treatment had no impact on mortality. MDR pathogen isolation, other than Klebsiella pneumoniae and Acinetobacter baumanii, was associated with decreased mortality (OR 0.35 (95% CI 0.16, 0.79) p = 0.011). In multivariate analysis, the only significant factor for mortality was procalcitonin ≥ 1 (OR 2.84 (95% CI 1.13, 7.11) p = 0.025). In conclusion, in an endemic area, mortality rates in MDR BSI remain notable. High procalcitonin was the only variable that predicted death. The use of rapid diagnostics did not improve mortality rate.

Diagnostic Tests for SARS COV-2: Application, Challenges and Recommendations

Background: The recent pandemic of human coronavirus infections, at the beginning of the 21st century, have shown the prominent roles of rapid and accurate diagnostic technologies to contain the emerging and re-emerging infections. To control such a large-scale crisis, early identification and prevention of infection are essential. Prompt identification of cases relies on the accurate diagnostic technologies. Before analysis, appropriate sample collection is essential for making a precise molecular diagnosis of SARS-COV-2 (Severe Acute Respiratory Syndrome Coronavirus-2). To prevent further spread of infection and obtain consistent results, proper preventive measures must be taken into consideration to keep laboratory staff safe. Despite the development of specific and sensitive point-of-care tests and serological immunoassays which are considered supplementary methods for diagnosing SARS-COV-2, RT-PCR (Reverse-Transcriptase Polymerase-Chain-Reaction) remains the gold standard performed on respiratory specimens. Upon completion of the sample analysis, testing results must be interpreted using both molecular and serological findings. Finally, random-access, integrated devices available for the purpose of care with scalable capacities will facilitate the rapid and accurate diagnosis and monitoring of Severe Acute Respiratory Syndrome Corona Virus-2 (SARS CoV-2) infections. Objectives: This review covers the types of sample collection, current diagnostic techniques for COVID-19 and challenges in their application Methods: A literature search was performed on the lines for a narrative review, but including features of systematic review methodology (PRISMA flow diagram). Two authors searched three electronic databases for literature review, i.e., PubMed, HINARI, and Google Scholar using Mesh-terms, specimen types or molecular diagnostic tests or serological tests in link with COVID-19 or SARS CoV-2, with different combinations, between March 21, 2020 and March 8, 2023. Conclusion: To control the current pandemic of COVID-19 and reduce its burden on healthcare systems around the world, the capacity of diagnostic tests should be improved. Rapid molecular diagnostic tests that can be fast, reliable, cost-effective, available, sensitive, and specific and point of care, must be urgently developed, produced, and widely distributed all over the globe.

Optimizing Treatment of Staphylococcus aureus Bloodstream Infections Following Rapid Molecular Diagnostic Testing and an Antimicrobial Stewardship Program Intervention.

Pending antibiotic susceptibility results, vancomycin is often used for bloodstream infections (BSIs) to ensure treatment of methicillin-resistant Staphylococcus aureus (MRSA). As rapid discrimination of methicillin-susceptible S. aureus (MSSA) from MRSA in BSIs could decrease vancomycin use and allow early optimization of beta-lactam therapy, this study evaluated the impact of the use of rapid molecular testing for MSSA and MRSA coupled with an antimicrobial stewardship program (ASP) intervention. Between January and July 2020, the Cepheid Xpert MRSA/SA blood culture assay was performed on blood cultures with Gram-positive cocci in clusters that were identified as S. aureus using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The ASP team member then consulted with the treating physician. The time to optimal therapy (TTOT) and clinical outcomes, including length of hospital stay (LOS), were compared between the intervention (n = 29) and historical (n = 27) cohorts. TTOT was defined as the time from the first blood culture draw to the use of appropriately dosed antistaphylococcal beta-lactam monotherapy without vancomycin. Molecular testing significantly reduced the median time to MSSA and MRSA discrimination to 7.8 h, compared to 24.3 h with culture-based methods (P < 0.001). Compared to the control group, the median TTOT in the ASP intervention group was significantly shorter (P = 0.041) at 38.0 h (versus 50.1 h). Rapid discrimination between MRSA and MSSA using molecular testing, paired with an ASP intervention, significantly reduced the TTOT in patients with MSSA BSIs. IMPORTANCE Our research shows that time to optimal antibiotic treatment for serious bloodstream infections can be improved with rapid molecular sensitivity testing and feedback to prescribers. This can be implemented in laboratories without full microbiology services or training to improve patient outcomes by improving antimicrobial use.

Evaluation of three rapid low-resource molecular tests for Nipah virus.

Accurate and timely diagnosis of Nipah virus (NiV) requires rapid, inexpensive, and robust diagnostic tests to control spread of disease. Current state of the art technologies are slow and require laboratory infrastructure that may not be available in all endemic settings. Here we report the development and comparison of three rapid NiV molecular diagnostic tests based on reverse transcription recombinase-based isothermal amplification coupled with lateral flow detection. These tests include a simple and fast one-step sample processing step that inactivates the BSL-4 pathogen, enabling safe testing without the need for multi-step RNA purification. The rapid NiV tests targeted the Nucleocapsid protein (N) gene with analytical sensitivity down to 1,000 copies/μL for synthetic NiV RNA and did not cross-react with RNA of other flaviviruses or Chikungunya virus, which can clinically present with similar febrile symptoms. Two tests detected 50,000-100,000 TCID50/mL (100-200 RNA copies/reaction) of the two distinct strains of NiV, Bangladesh (NiVB) and Malaysia (NiVM), and took 30 min from sample to result, suggesting these tests are well suited for rapid diagnosis under resource-limited conditions due to rapidity, simplicity, and low equipment requirements. These Nipah tests represent a first step toward development of near-patient NiV diagnostics that are appropriately sensitive for first-line screening, sufficiently robust for a range of peripheral settings, with potential to be safely performed outside of biohazard containment facilities.

Clinical impact of the rapid molecular detection of RSV and influenza A and B viruses in the emergency department.

IntroductionUsing respiratory virus rapid diagnostic tests in the emergency department could allow better and faster clinical management. Point-of-care PCR instruments now provide results in less than 30 minutes. The objective of this study was to assess the impact of the use of a rapid molecular diagnostic test, the cobas® Influenza A/B & RSV Assay, during the clinical management of emergency department patients.MethodsPatients (adults and children) requiring admission or suffering from an underlying condition at risk of respiratory complications were prospectively recruited in the emergency department of four hospitals in the Brussels region. Physicians’ intentions regarding admission, isolation, antibiotic, and antiviral use were collected before and after performing the rapid molecular test. Additionally, a comparison of the analytical performance of this test against antigen rapid tests and viral culture was performed as well as a time-to-result evaluation.ResultsAmong the 293 patients recruited, 90 had a positive PCR, whereas 44 had a positive antigen test. PCR yielded a sensitivity of 100% for all targets. Antigen tests yielded sensitivities ranging from 66.7% for influenza B to 83.3% for respiratory syncytial virus (RSV). The use of PCR allowed a decrease in the overall need for isolation and treatment by limiting the isolation of negative patients and antibiotic use for positive patients. Meanwhile, antiviral treatments better targeted patients with a positive influenza PCR.ConclusionThe use of a rapid influenza and RSV molecular test improves the clinical management of patients admitted to the emergency department by providing a fast and reliable result. Their additional cost compared to antigen tests should be balanced with the benefit of their analytical performance, leading to efficient reductions in the need for isolation and antibiotic use.

Rapid Molecular Assays for the Diagnosis of Drug-Resistant Tuberculosis.

The recognition that drug-resistant tuberculosis (DR-TB) poses a major threat to global tuberculosis (TB) control efforts has catalysed the development of new and urgently needed TB diagnostics. The full beneficial impact of the subsequent flood of new TB diagnostic tests into the market can only be realised if these diagnostic tests are readily accessible to TB programs and contribute to improved patient outcomes. Although phenotypic drug-susceptibility testing remains the gold standard, an improved understanding of the relationship between mutations and different levels of drug resistance coupled with the advantages of molecular diagnostics could result in rapid molecular diagnostic tests replacing phenotypic drug-susceptibility testing. Successful diagnostics need to diagnose all forms of drug-resistant TB prevalent in each geographic region. Given the finite number and often limited availability of effective drugs for DR-TB, the diagnostic test must be able to detect all clinically important types of resistance to available anti-TB drugs. However, less comprehensive resistance profiling may be sufficient in settings where extensively drug-resistant TB (XDR-TB) and pre-XDR are absent. Rapid molecular diagnostic tests for DR-TB detection suitable for DR-TB endemic settings should be accurate, inexpensive, suitable to be performed on an easily accessible sample, detect prevalent circulating drug-resistant strains, and provide results within a short turnaround time to enable timely treatment initiation. In this review, we appraise the wide range of molecular diagnostics for DR-TB endorsed by the World Health Organisation, discuss the challenges in the development and rollout of rapid molecular DR-TB tests in low- and middle-income countries, and highlight user perspectives and cost-effectiveness factors that influence their utility.

Rapid Diagnosis of XDR and Pre-XDR TB: A Systematic Review of Available Tools

IntroductionNo previous systematic reviews have comprehensively investigated the features of Xpert MTB/XDR and other rapid tests to diagnose pre-XDR/XDR-TB. The aim of this systematic review is to assess existing rapid diagnostics for pre-XDR/XDR-TB from a point-of-care perspective and describe their technical characteristics (i.e., sensitivity, specificity, positive and negative predictive values). MethodsEmbase, PubMed, Scopus, and Web of Science were searched to detect the articles focused on the accuracy of commercially available rapid molecular diagnostic tests for XDR-TB according to PRISMA guidelines. The analysis compared the diagnostic techniques and approaches in terms of sensitivity, specificity, laboratory complexity, time to confirmed diagnosis. ResultsOf 1298 records identified, after valuating article titles and abstracts, 97 (7.5%) records underwent full-text evaluation and 38 records met the inclusion criteria.Two rapid World Health Organization (WHO)-endorsed tests are available: Xpert MTB/XDR and GenoType MTBDRsl (VER1.0 and VER 2.0). Both tests had similar performance, slightly favouring Xpert, although only 2 studies were available (sensitivity 91.4–94; specificity 98.5–99; accuracy 97.2–97.7; PPV 88.9–99.1; NPV 95.8–98.9). ConclusionsXpert MTB/XDR could be suggested at near-point-of-care settings to be used primarily as a follow-on test for laboratory-confirmed TB, complementing existing rapid tests detecting at least rifampicin-resistance.Both Xpert MTB/XDR and GenoType MTBDRsl are presently diagnosing what WHO defined, in 2021, as pre-XDR-TB.

Frequency of rpoB, katG, and inhA Gene Polymorphisms Associated with Multidrug-Resistant Mycobacterium tuberculosis Complex Isolates among Ethiopian TB Patients: A Systematic Review.

Tuberculosis (TB) is one of the top 10 causes of mortality and the first killer among infectious diseases of poverty (IDoPs) worldwide. It disproportionately affects on-third of the world's low-income countries including Ethiopia. One of the factors driving the TB epidemic is the global rise of MDR/XDR-TB and their low detection affect the global TB control progress. Recently, the resistance-associated genetic mutations in MTBC known to confer drug resistance have been detected by rapid molecular diagnostic tests and sequencing methods. In this article, the published literature searched by PubMed database from 2010 to 2021 and English language were considered. The aim of this systematic review was to assess the prevalence of the most common rpoB, katG, and inhA gene mutations associated with multidrug resistance in MTBC clinical strains among TB patients in Ethiopia. Though 22 studies met our eligibility criteria, only 6 studies were included in the final analysis. Using the molecular GenoType MTBDRplus and MTBDRsl line probe assay and sequencing procedures, a total of 932 culture-positive MTBC isolates were examined to determine RIF, INH, and MDR-TB resistance patterns along with rpoB, katG, and inhA gene mutation analysis. As a result, among the genotypically tested MTBC isolates, 119 (12.77%), 83 (8.91%), and 73 (7.32%) isolates were INH, RIF, and MDR-TB resistant, respectively. In any RIF-resistant MTBC strains, the most common single point mutations were in codon 531 (S531L) followed by codon 526 (H526Y) of the rpoB gene. Besides, the most common mutations in any INH-resistant MTBC were strains observed at codon 315 (S315T) and WT probe in the katG gene and at codon C15T and WT1 probe in the inhA promoter region. Detection of resistance allele in rpoB, KatG, and inhA genes for RIF and INH could serve as a marker for MDR-TB strains. Tracking the most common S531L, S315T, and C15T mutations in rpoB, katG, and inhA genes among RIF- and INH-resistant isolates would be valuable in TB diagnostics and treatment regimens, and could reduce the development and risk of MDR/XDR-TB drug-resistance patterns.

Guidelines for COVID-19 Laboratory Testing for Emergency Departments From the New Diagnostic Technology Team of the Taiwan Society of Emergency Medicine.

COVID-19 tests have different turnaround times (TATs), accuracy levels, and limitations, which emergency physicians should be aware of. Nucleic acid amplification tests (NAATs) can be divided into standard high throughput tests and rapid molecular diagnostic tests at the point of care (POC). The standard NAAT has the advantages of high throughput and high accuracy with a TAT of 3-4 hours. The POC molecular test has the same advantages of high accuracy as standard high throughput PCR, but can be done in 13-45 minutes. Roche cobas Liat is the most commonly used machine in Taiwan, displaying 99%-100% sensitivity and 100% specificity, respectively. Abbott ID NOW is an isothermal PCR-based POC machine with a sensitivity of 79% and a specificity of 100%. A high rate of false positives and false negatives is associated with rapid antigen testing. Antibody testing is mostly used as part of public health surveys and for testing for immunity.

Rotational diffusometric sensor with isothermal amplification for ultra-sensitive and rapid detection of SARS-CoV-2 nsp2 cDNA

In the wake of a pandemic, the development of rapid, simple, and accurate molecular diagnostic tests can significantly aid in reducing the spread of infections. By combining particle imaging with molecular assays, a quick and highly sensitive biosensor can readily identify a pathogen at low concentrations. Here, we implement functionalized particle-enabled rotational diffusometry in combination with loop-mediated isothermal amplification for the rapid detection of the SARS-CoV-2 nsp2 gene in the recombinant plasmid as a proof of concept for COVID-19 diagnostics. By analyzing the images of blinking signals generated by these modified particles, the change in micro-level viscosity due to nucleic acid amplification was measured. The high sensitivity of rotational diffusometry enabled facile detection within 10 min, with a limit of detection of 70 ag/μL and a sample volume of 2 μL. Tenfold higher detection sensitivity was observed for rotational diffusometry in comparison with real-time PCR. In addition, the system stability and the effect of temperature on rotational diffusometric measurements were studied and reported. These results demonstrated the utility of a rotational diffusometric platform for the rapid and sensitive detection of SARS-CoV-2 cDNA fragments.