Performances of rapid and connected salivary RT-LAMP diagnostic test for SARS-CoV-2 infection in ambulatory screening.

In the context of social events reopening and economic relaunch, sanitary surveillance of SARS-CoV-2 infection is still required. Here, we evaluated the diagnostic performances of a rapid, extraction-free and connected reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay on saliva. Nasopharyngeal (NP) swabs and saliva from 443 outpatients were collected simultaneously and tested by reverse-transcription quantitative PCR (RT-qPCR) as reference standard test. Seventy-one individuals (16.0%) were positive by NP and/or salivary RT-qPCR. Sensitivity and specificity of salivary RT-LAMP were 85.9% (95%CI 77.8-94.0%) and 99.5% (98.7-100%), respectively. Performances were similar for symptomatic and asymptomatic participants. Moreover, SARS-CoV-2 genetic variants were analyzed and no dominant mutation in RT-LAMP primer region was observed during the period of the study. We demonstrated that this RT-LAMP test on self-collected saliva is reliable for SARS-CoV-2 detection. This simple connected test with optional automatic results transfer to health authorities is unique and opens the way to secure professional and social events in actual context of economics restart.

Automatic system for high-throughput and high-sensitivity diagnosis of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had severe consequences for health and the global economy. To control the transmission, there is an urgent demand for early diagnosis and treatment in the general population. In the present study, an automatic system for SARS-CoV-2 diagnosis is designed and built to deliver high specification, high sensitivity, and high throughput with minimal workforce involvement. The system, set up with cross-priming amplification (CPA) rather than conventional reverse transcription-polymerase chain reaction (RT-PCR), was evaluated using more than 1000 real-world samples for direct comparison. This fully automated robotic system performed SARS-CoV-2 nucleic acid-based diagnosis with 192 samples in under 180 min at 100 copies per reaction in a "specimen in data out" manner. This throughput translates to a daily screening capacity of 800-1000 in an assembly-line manner with limited workforce involvement. The sensitivity of this device could be further improved using a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based assay, which opens the door to mixed samples, potentially include SARS-CoV-2 variants screening in extensively scaled testing for fighting COVID-19.

A rapid and highly effective approach for SARS-CoV-2 nucleic acid daily testing in more than four thousand single-tube samples.

BACKGROUND: Presently, the global spread of COVID-19 is still going on, with more than 0.6 million new cases confirmed per day (as of November 20, 2021). However, since China entered a post-epidemic phase in mid-March 2020, the daily number of new domestic infections in the Chinese mainland has been maintained at almost zero or single digits, which was attributed to a series of effective measures for COVID-19 prevention and control adopted by the Chinese government. Among these measures, SARS-CoV-2 nucleic acid testing holds key role for the timely confirmation and isolation of the infections to prevent further transmission. METHODS: Referring to the national policy requirements, since April 30, 2020, The Affiliated Hospital of Qingdao University has conducted SARS-CoV-2 nucleic acid testing in its PCR laboratory for patients and social workers, as well as for environmental monitoring and employee screening. As of mid-November 2020, the daily amount of single-tube samples for nucleic acid testing rose above 4,000. RESULTS: In this article, a rapid and highly effective approach for SARS-CoV-2 nucleic acid daily testing is presented, allowing five technicians to complete nucleic acid testing in 6,500 single-tube samples in one day with a high level of quality. Using this approach, since the samples entered the PCR laboratory, all testing results were reported in 2.5-3 h with satisfactory quality control and precise reporting criterion as prerequisites. CONCLUSION: This testing approach provides a referable workflow for other testing institutions and is expected to play an important role in COVID-19 prevention and control.

Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection.

Verifying the authenticity of food products is essential due to the recent increase in counterfeit meat-containing food products. The existing methods of detection have a number of disadvantages. Therefore, simple, cheap, and sensitive methods for detecting various types of meat are required. In this study, we propose a rapid full-cycle technique to control the chicken or pig adulteration of meat products, including 3 min of crude DNA extraction, 20 min of recombinase polymerase amplification (RPA) at 39 °C, and 10 min of lateral flow assay (LFA) detection. The cytochrome B gene was used in the developed RPA-based test for chicken and pig identification. The selected primers provided specific RPA without DNA nuclease and an additional oligonucleotide probe. As a result, RPA-LFA, based on designed fluorescein- and biotin-labeled primers, detected up to 0.2 pg total DNA per µL, which provided up to 0.001% w/w identification of the target meat component in the composite meat. The RPA-LFA of the chicken and pig meat identification was successfully applied to processed meat products and to meat after heating. The results were confirmed by real-time PCR. Ultimately, the developed analysis is specific and enables the detection of pork and chicken impurities with high accuracy in raw and processed meat mixtures. The proposed rapid full-cycle technique could be adopted for the authentication of other meat products.

Performance of rapid diagnostic tests, microscopy, loop-mediated isothermal amplification (LAMP) and PCR for malaria diagnosis in Ethiopia: a systematic review and meta-analysis.

BACKGROUND: Rapid accurate diagnosis followed by effective treatment is very important for malaria control. Light microscopy remains the "golden standard" method for malaria diagnosis. Diagnostic test method must have sufficient level of accuracy for detecting malaria parasites. Therefore, this study aimed to investigate the diagnostic accuracy of rapid diagnostic tests (RDTs), microscopy, loop-mediated isothermal amplification (LAMP) and/or polymerase chain reaction (PCR) for the malaria diagnosis in Ethiopia. METHODS: Data bases such as PubMed, PubMed central, Science direct databases, Google scholar, and Scopus were searched from September to October, 2020 for studies assessing the diagnostic accuracy of RDTs, microscopy, LAMP and PCR methods for malaria diagnosis. RESULTS: A total of 29 studies published between 2001 and 2020 were analysed using review manager, Midas (Stata) and Meta-disc. The sensitivity and specificity of studies comparing RDT with microscopy varies from 79%-100% to 80%-100%, respectively. The sensitivity of LAMP (731 tests) was 100% and its specificity was varies from 85 to 99% when compared with microscopy and PCR. Considerable heterogeneity was observed between studies included in this meta-analysis. Meta-regression showed that blinding status and target antigens were the major sources of heterogeneity (P < 0.05). RDT had an excellent diagnostic accuracy (Area under the ROC Curve = 0.99) when compared with microscopy. Its specificity was quite good (93%-100%) except for one outlier (28%), but lower "sensitivity" was observed when PCR is a reference test. This indicates RDT had a good diagnostic accuracy (AUC = 0.83). Microscopy showed a very good diagnostic accuracy when compared with PCR. CONCLUSIONS: The present study showed that microscopy and RDTs had high efficiency for diagnosing febrile malaria patients. The diagnostic accuracy of RDT was excellent when compared with microscopy. This indicates RDTs have acceptable sensitivities and specificities to be used in resource poor settings as an alternative for microscopy. In this study, LAMP showed an excellent sensitivities and specificities. Furthermore, the need of minimum equipment and relatively short time for obtaining results can made LAMP one of the best alternatives especially for accurate diagnosis of asymptomatic malaria.

Effect of a confirmatory testing algorithm on early acute HIV diagnosis in Korea.

ABSTRACT: The 17 Provincial Institutes of Health and Environment (PIHEs) in Korea use HIV antibody, antigen, and Western blot assays for confirmatory testing of HIV infection. The Korea Disease Control and Prevention Agency (KDCA) has further included p24 antigen neutralization and nucleic acid tests (NATs) since 2015. Our study aimed to investigate the effect of this new testing algorithm on the confirmation rate of HIV infection.Annual changes, from 2012 through 2017, in positive or indeterminate HIV confirmatory results were compared for the two algorithms between the PIHEs and the KDCA. Fiebig stages and Western blot p31 band were used to identify the diagnostic proportions of acute or early chronic HIV for the two algorithms.The number of positive cases in the samples requested from PIHEs for reconfirmation by the KDCA has steadily increased from 10.3% in 2014 to 33.3% in 2017. However, the number of indeterminate cases dropped sharply, from 71.9% in 2014 to 14.0% in 2017. The results for the p31 reactive band were 27.4% and 88.4% for the KDCA and PIHEs, respectively. Of positive cases reported by the KDCA, 22.9% were in the early acute stage and Fiebig stages I to II.The new testing algorithm has improved the diagnosis of HIV infections in the early acute stage. Early confirmatory diagnosis can prevent secondary transmission of HIV and provide early treatment opportunities for people living with HIV infection.

Fluorescence polarization system for rapid COVID-19 diagnosis.

Prompt diagnosis, patient isolation, and contact tracing are key measures to contain the coronavirus disease 2019 (COVID-19). Molecular tests are the current gold standard for COVID-19 detection, but are carried out at central laboratories, delaying treatment and control decisions. Here we describe a portable assay system for rapid, onsite COVID-19 diagnosis. Termed CODA (CRISPR Optical Detection of Anisotropy), the method combined isothermal nucleic acid amplification, activation of CRISPR/Cas12a, and signal generation in a single assay, eliminating extra manual steps. Importantly, signal detection was based on the ratiometric measurement of fluorescent anisotropy, which allowed CODA to achieve a high signal-to-noise ratio. For point-of-care operation, we built a compact, standalone CODA device integrating optoelectronics, an embedded heater, and a microcontroller for data processing. The developed system completed SARS-CoV-2 RNA detection within 20 min of sample loading; the limit of detection reached 3 copy/µL. When applied to clinical samples (10 confirmed COVID-19 patients; 10 controls), the rapid CODA test accurately classified COVID-19 status, in concordance with gold-standard clinical diagnostics.

Sequence-specific and multiplex detection of COVID-19 virus (SARS-CoV-2) using proofreading enzyme-mediated probe cleavage coupled with isothermal amplification.

The outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been challenging human health worldwide. Loop-mediated isothermal amplification (LAMP) has been promptly applied to the detection of SARS-CoV-2 owing to its high amplification efficacy and less requirement of the thermal cycler. However, the vast majority of these LAMP-based assays depend on the non-specific detection of LAMP products, which can not discern the undesirable amplificons, likely to yield unreliable results. Herein, a sequence-specific LAMP assay was reported to detect SARS-CoV-2 using proofreading enzyme-mediated probe cleavage (named Proofman), which could realize real-time and visual detection without uncapping. This assay, introducing a proofreading enzyme and the fluorogenic probe to reverse-transcription LAMP (RT-Proofman-LAMP), can specifically detect the SARS-CoV-2 RNA with a detection limit of 100 copies. In addition to the real-time analysis, the assay is capable of endpoint visualization under a transilluminator within 50 min, providing a convenient reporting manner under the setting of point-of-care testing (POCT). In combination with different fluorophores, the one-pot multiplex assay was successfully achieved to detect multiple targets of SARS-CoV-2 and inner control simultaneously. In summary, the development of RT-Proofman-LAMP offers a versatile and highly-specific method for fast field screening and laboratory testing of SARS-CoV-2, making it a promising platform in COVID-19 diagnosis.

Xpert MTB/RIF Ultra and Xpert MTB/RIF assays for extrapulmonary tuberculosis and rifampicin resistance in adults.

BACKGROUND: Xpert MTB/RIF Ultra (Xpert Ultra) and Xpert MTB/RIF are World Health Organization (WHO)-recommended rapid nucleic acid amplification tests (NAATs) widely used for simultaneous detection of Mycobacterium tuberculosis complex and rifampicin resistance in sputum. To extend our previous review on extrapulmonary tuberculosis (Kohli 2018), we performed this update to inform updated WHO policy (WHO Consolidated Guidelines (Module 3) 2020). OBJECTIVES: To estimate diagnostic accuracy of Xpert Ultra and Xpert MTB/RIF for extrapulmonary tuberculosis and rifampicin resistance in adults with presumptive extrapulmonary tuberculosis. SEARCH METHODS: Cochrane Infectious Diseases Group Specialized Register, MEDLINE, Embase, Science Citation Index, Web of Science, Latin American Caribbean Health Sciences Literature, Scopus, ClinicalTrials.gov, the WHO International Clinical Trials Registry Platform, the International Standard Randomized Controlled Trial Number Registry, and ProQuest, 2 August 2019 and 28 January 2020 (Xpert Ultra studies), without language restriction. SELECTION CRITERIA: Cross-sectional and cohort studies using non-respiratory specimens. Forms of extrapulmonary tuberculosis: tuberculous meningitis and pleural, lymph node, bone or joint, genitourinary, peritoneal, pericardial, disseminated tuberculosis. Reference standards were culture and a study-defined composite reference standard (tuberculosis detection); phenotypic drug susceptibility testing and line probe assays (rifampicin resistance detection). DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and assessed risk of bias and applicability using QUADAS-2. For tuberculosis detection, we performed separate analyses by specimen type and reference standard using the bivariate model to estimate pooled sensitivity and specificity with 95% credible intervals (CrIs). We applied a latent class meta-analysis model to three forms of extrapulmonary tuberculosis. We assessed certainty of evidence using GRADE. MAIN RESULTS: 69 studies: 67 evaluated Xpert MTB/RIF and 11 evaluated Xpert Ultra, of which nine evaluated both tests. Most studies were conducted in China, India, South Africa, and Uganda. Overall, risk of bias was low for patient selection, index test, and flow and timing domains, and low (49%) or unclear (43%) for the reference standard domain. Applicability for the patient selection domain was unclear for most studies because we were unsure of the clinical settings. Cerebrospinal fluid Xpert Ultra (6 studies) Xpert Ultra pooled sensitivity and specificity (95% CrI) against culture were 89.4% (79.1 to 95.6) (89 participants; low-certainty evidence) and 91.2% (83.2 to 95.7) (386 participants; moderate-certainty evidence). Of 1000 people where 100 have tuberculous meningitis, 168 would be Xpert Ultra-positive: of these, 79 (47%) would not have tuberculosis (false-positives) and 832 would be Xpert Ultra-negative: of these, 11 (1%) would have tuberculosis (false-negatives). Xpert MTB/RIF (30 studies) Xpert MTB/RIF pooled sensitivity and specificity against culture were 71.1% (62.8 to 79.1) (571 participants; moderate-certainty evidence) and 96.9% (95.4 to 98.0) (2824 participants; high-certainty evidence). Of 1000 people where 100 have tuberculous meningitis, 99 would be Xpert MTB/RIF-positive: of these, 28 (28%) would not have tuberculosis; and 901 would be Xpert MTB/RIF-negative: of these, 29 (3%) would have tuberculosis. Pleural fluid Xpert Ultra (4 studies) Xpert Ultra pooled sensitivity and specificity against culture were 75.0% (58.0 to 86.4) (158 participants; very low-certainty evidence) and 87.0% (63.1 to 97.9) (240 participants; very low-certainty evidence). Of 1000 people where 100 have pleural tuberculosis, 192 would be Xpert Ultra-positive: of these, 117 (61%) would not have tuberculosis; and 808 would be Xpert Ultra-negative: of these, 25 (3%) would have tuberculosis. Xpert MTB/RIF (25 studies) Xpert MTB/RIF pooled sensitivity and specificity against culture were 49.5% (39.8 to 59.9) (644 participants; low-certainty evidence) and 98.9% (97.6 to 99.7) (2421 participants; high-certainty evidence). Of 1000 people where 100 have pleural tuberculosis, 60 would be Xpert MTB/RIF-positive: of these, 10 (17%) would not have tuberculosis; and 940 would be Xpert MTB/RIF-negative: of these, 50 (5%) would have tuberculosis. Lymph node aspirate Xpert Ultra (1 study) Xpert Ultra sensitivity and specificity (95% confidence interval) against composite reference standard were 70% (51 to 85) (30 participants; very low-certainty evidence) and 100% (92 to 100) (43 participants; low-certainty evidence). Of 1000 people where 100 have lymph node tuberculosis, 70 would be Xpert Ultra-positive and 0 (0%) would not have tuberculosis; 930 would be Xpert Ultra-negative and 30 (3%) would have tuberculosis. Xpert MTB/RIF (4 studies) Xpert MTB/RIF pooled sensitivity and specificity against composite reference standard were 81.6% (61.9 to 93.3) (377 participants; low-certainty evidence) and 96.4% (91.3 to 98.6) (302 participants; low-certainty evidence). Of 1000 people where 100 have lymph node tuberculosis, 118 would be Xpert MTB/RIF-positive and 37 (31%) would not have tuberculosis; 882 would be Xpert MTB/RIF-negative and 19 (2%) would have tuberculosis. In lymph node aspirate, Xpert MTB/RIF pooled specificity against culture was 86.2% (78.0 to 92.3), lower than that against a composite reference standard. Using the latent class model, Xpert MTB/RIF pooled specificity was 99.5% (99.1 to 99.7), similar to that observed with a composite reference standard. Rifampicin resistance Xpert Ultra (4 studies) Xpert Ultra pooled sensitivity and specificity were 100.0% (95.1 to 100.0), (24 participants; low-certainty evidence) and 100.0% (99.0 to 100.0) (105 participants; moderate-certainty evidence). Of 1000 people where 100 have rifampicin resistance, 100 would be Xpert Ultra-positive (resistant): of these, zero (0%) would not have rifampicin resistance; and 900 would be Xpert Ultra-negative (susceptible): of these, zero (0%) would have rifampicin resistance. Xpert MTB/RIF (19 studies) Xpert MTB/RIF pooled sensitivity and specificity were 96.5% (91.9 to 98.8) (148 participants; high-certainty evidence) and 99.1% (98.0 to 99.7) (822 participants; high-certainty evidence). Of 1000 people where 100 have rifampicin resistance, 105 would be Xpert MTB/RIF-positive (resistant): of these, 8 (8%) would not have rifampicin resistance; and 895 would be Xpert MTB/RIF-negative (susceptible): of these, 3 (0.3%) would have rifampicin resistance. AUTHORS' CONCLUSIONS: Xpert Ultra and Xpert MTB/RIF may be helpful in diagnosing extrapulmonary tuberculosis. Sensitivity varies across different extrapulmonary specimens: while for most specimens specificity is high, the tests rarely yield a positive result for people without tuberculosis. For tuberculous meningitis, Xpert Ultra had higher sensitivity and lower specificity than Xpert MTB/RIF against culture. Xpert Ultra and Xpert MTB/RIF had similar sensitivity and specificity for rifampicin resistance. Future research should acknowledge the concern associated with culture as a reference standard in paucibacillary specimens and consider ways to address this limitation.

Rapid Detection of mrp, epf, and sly Genes by Loop-Mediated Isothermal Amplification in Streptococcus suis.

Streptococcus suis remains a serious threat to the worldwide swine industry and human health. In this study, rapid assays for the detection of three common virulence-related factors (mrp, epf, and sly) were developed, evaluated, and applied. Loop-mediated isothermal amplification (LAMP) primers were designed using Primer Explorer V5 software. The sensitivity and specificity of the LAMP assays were determined based on sample turbidity. For all three genes, LAMP assays were performed at 62°C with a reaction time of 60 min. The detection limit of conventional polymerase chain reaction (PCR) was 1 ng/µL, 10 pg/µL, and 100 fg/µL for the epf, sly, and mrp genes, respectively. For the LAMP assays, the detection limits were 10 pg/µL, 10 fg/µL, and 100 fg/µL for epf, sly, and mrp, respectively, representing sensitivities 100-1000 times higher than those of the PCR assay. Furthermore, when the LAMP assays were applied to clinical strains, the results were consistent with those of the PCR assay, confirming the LAMP assays as rapid and reliable detection techniques. In conclusion, the LAMP assays described in this study have the potential to become standard methods to detect the virulence factors mrp, epf, and sly. To the best of our knowledge, this is the first study to report the application of LAMP to detect the mrp, epf, and sly genes.

opvCRISPR: One-pot visual RT-LAMP-CRISPR platform for SARS-cov-2 detection.

The 2019 novel coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected all aspects of human life. Rapid, accurate, sensitive and user friendly detection method is urgently needed to facilitate early intervention and control the spread of SARS-CoV-2. Here, we propose a one-pot visual SARS-CoV-2 detection system named "opvCRISPR" by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) and Cas12a cleavage in a single reaction system. We demonstrate that the collateral activity against single-stranded DNA (ssDNA) reporters of activated Cas12a triggered by RT-LAMP amplicon increases detection sensitivity and makes detection results observable with naked eye. The opvCRISPR enables detection at nearly single molecule level in 45 min. We validate this method with 50 SARS-CoV-2 potentially infected clinical samples. The opvCRISPR diagnostic results provide 100% agreement with the Centers for Disease Control and Prevention (CDC)-approved quantitative RT-PCR assay. The opvCRISPR holds great potential for SARS-CoV-2 detection in next-generation point-of-care molecular diagnostics.

Nucleic Acid Amplification Testing Compared With Cultures, Gram Stain, and Microscopy in the Diagnosis of Vaginitis.

OBJECTIVE: The aim of the study was to evaluate the performance of nucleic acid amplification testing (NAAT) for the diagnosis of vulvovaginal candidiasis (VVC), bacterial vaginosis, and Trichomonas vaginalis. METHODS: A cross-sectional analysis of women with (n = 200) and without (n = 100) vulvovaginal symptoms was enrolled from outpatient gynecology offices and a vulvovaginal referral clinic. Vaginal swabs were analyzed by wet mount microscopy, yeast culture, Gram stain, T. vaginalis culture, and NAAT. Sensitivity and specificity analyses were performed. RESULTS: Among symptomatic women, the sensitivity of microscopy was 48.5% for VVC and 75% for T. vaginalis. Sensitivities of NAAT and culture for diagnosing VVC were 92.4% and 83.3%, respectively, whereas these methods were 100% and 93.8% for T. vaginalis. The sensitivity for bacterial vaginosis diagnosis by clinical criteria ("Amsel criteria"), Gram stain, and NAAT were 98.7%, 82.7%, and 78.7%, respectively. Test concordance rates were high between culture and NAAT for Candida species (91%) and between Gram stain and NAAT for the detection of bacterial vaginosis (88%). Among asymptomatic women, 20%-21% tested positive for bacterial vaginosis by Gram stain or NAAT, and 8%-13% were colonized with Candida species based on culture or NAAT. CONCLUSIONS: Given the limitations of wet mount sensitivity for VVC and T. vaginalis, culture or NAAT testing should be considered when evaluating women with symptoms of vaginitis who test negative by microscopy. Although Amsel criteria accurately diagnosed bacterial vaginosis, NAAT is preferred for detection of T. vaginalis and performed similarly to culture for the diagnosis of VVC.

Performance of human papillomavirus DNA detection in residual specimens taken for Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid amplification testing in men who have sex with men.

OBJECTIVES: Rectal swab specimens, either alone or pooled with first-void urine (FVU) and pharyngeal swab specimens, are used to test for Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infection in men who have sex with men (MSM). Following introduction of human papillomavirus (HPV) vaccination for MSM attending UK sexual health services (SHSs), HPV testing of residual CT/NG test specimens has been proposed to monitor HPV prevalence in this population. Performance of HPV detection in such specimens has not been evaluated previously. METHODS: MSM attending a UK SHS provided three specimens: (1) rectal swab for CT/NG, (2) pooled rectal/pharyngeal/FVU specimen for CT/NG and (3) dedicated anal swab for HPV. Specimen 3 and residual material from specimens 1 and 2 were tested for type-specific HPV DNA. HPV detection was by an in-house multiplex PCR and luminex-based genotyping assay. RESULTS: A total of 129 MSM were recruited with a mean age of 38.1 years; 24% were HIV-positive. Of the 129 MSM, 92 (71%) had any type-specific HPV DNA in ≥1 specimen; 80 (62%) had high risk (HR) HPV. Of 123 participants with sufficient residual pooled and dedicated specimens, 70 (56.9%) had detectable HPV on both, and 40 (32.5%) were negative on both; overall concordance was 89% (95% CI 83% to 94%), and kappa statistic was 0.78 (95% CI 0.66 to 0.89). Pooled samples had a 4.1% (95% CI -1.9% to 10.0%) higher test positivity rate than dedicated samples.Of 125 participants with sufficient residual rectal and specimens, 74 (59.2%) had detectable HPV on both, and 36 (28.8%) were negative on both; overall concordance was 88% (95% CI 81% to 93%), and kappa statistic was 0.74 (95% CI 0.61 to 0.86). Residual rectal samples had 5.6% (95%CI -0.6% to 11.8%) higher test positivity than dedicated samples. CONCLUSIONS: We observed high concordance between the dedicated and residual STI test specimens. Our data support the strategy of testing residual specimens for HPV prevalence monitoring in MSM to evaluate the impact of the targeted vaccination programme.

Molecular analysis of sentinel lymph nodes in patients with breast cancer using one-step nucleic acid amplification (OSNA): Does not lead to overtreatment in the current era of de-escalating axillary management.

PURPOSE: OSNA is a molecular technique for sentinel lymph node (SN) analysis in breast cancer. Compared to histology, OSNA may yield more (micro)metastases and thereby result in more axillary lymph node dissections or radiotherapy. We investigated whether axillary treatments increase when using OSNA, applying current guidelines for de-escalating axillary management. METHODS: All patients treated for cT1-3N0 breast cancer in our hospital between December 2013 and February 2016 were included. In 148 prospectively included patients (January 2015-February 2016), SN's were examined with OSNA. In a retrospective cohort of 123 patients (December 2013-December 2014), SN's were examined with conventional histology. Outcomes were: number of macro and micrometastases, amount of patients receiving axillary dissection or irradiation, number of patients receiving adjuvant systemic therapy. Data were analyzed using Mann-Whitney and Pearson Chi-square test. P < 0.05 was considered statistically significant. RESULTS: 230 SN's from 123 patients were examined with conventional histology. 229 SN's from 148 patients were evaluated with OSNA. Amount of macrometastases was equal between groups (histology 17.9% versus OSNA 16.2%, p = 0.715). We found significantly more micrometastases when using OSNA (histology 11.4% versus OSNA 25.0%, p = 0.004). Total number of axillary lymph node dissections was comparable in both groups (histology 12.2%, OSNA 12.2%, p = 0.993), as well as number of axillary radiations (histology 8.9%, OSNA 11.5%, p = 0.493). Also, the number of patients receiving adjuvant systemic therapy was similar between conventional histology and OSNA (histology 53.7% versus OSNA 58.1%, p = 0.462). CONCLUSION: OSNA analysis for SN in breast cancer is a highly sensitive technique, detecting more micrometastases than standard histology. When applying current guidelines, OSNA analysis does not lead to overtreatment with more axillary dissections or irradiation.

Active case detection of malaria in pregnancy using loop-mediated amplification (LAMP): a pilot outcomes study in South West Ethiopia.

BACKGROUND: 125 million women are pregnant each year in malaria endemic areas and are, therefore, at risk of Malaria in Pregnancy (MiP). MiP is the direct consequence of Plasmodium infection during pregnancy. The sequestration of Plasmodium falciparum parasites in the placenta adversely affects fetal development and impacts newborn birth weight. Importantly, women presenting with MiP commonly develop anaemia. In Ethiopia, the Ministry of Health recommends screening symptomatic women only at antenatal care visits with no formal intermittent preventive therapy. Since MiP can display low-level parasitaemia, current tests which include microscopy and RDT are challenged to detect these cases. Loop mediated isothermal Amplification (LAMP) technology is a highly sensitive technique for DNA detection and is field compatible. This study aims to evaluate the impact of active malaria case detection during pregnancy using LAMP technology in terms of birth outcomes. METHODS: A longitudinal study was conducted in two health centres of the Kafa zone, South West Ethiopia. Both symptomatic and asymptomatic pregnant women were enrolled in the first or second trimester and allocated to either Standard of Care (SOC-microscopy and RDT) or LAMP (LAMP, microscopy and RDT). Women completed at least three visits prior to delivery, and the patient was referred for treatment if Plasmodium infection was detected by any of the testing methods. The primary outcome was to measure absolute birth weight, proportion of low birth weight, and maternal/neonatal haemoglobin in each arm. Secondary outcomes were to assess the performance of microscopy and RDT versus LAMP conducted in the field. RESULTS: One hundred and ninety-nine women were included and assigned to either LAMP or SOC. Six were lost to follow up. In this cohort, 66.8% of women did not display any clinical symptoms and 70.9% were multi-parous. A reduced proportion of low birth weight newborns was observed in the LAMP group (0%) compared to standard of care (14%) (p <0.001). Improved neonatal haemoglobin was observed in the LAMP (13.1 g/dL) versus the SOC (12.8 g/dL) (p = 0.024) arm. RDT and microscopy had an analytical sensitivity of 66.7% and 55.6% compared to LAMP as a reference standard. CONCLUSIONS: These results support the use of highly sensitive tools for rapid on-site active case detection of MiP which may improve birth outcomes in the absence of IPT. However, further large-scale studies are required to confirm this finding.

A reverse-transcription recombinase-aided amplification assay for the rapid detection of N gene of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).

The current outbreak of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China firstly. A rapid, highly sensitive, specific, and simple operational method was needed for the detection of SARS-CoV-2. Here, we established a real-time reverse-transcription recombinase-aided amplification assay (RT-RAA) to detect SARS-CoV-2 rapidly. The primers and probe were designed based on the nucleocapsid protein gene (N gene) sequence of SARS-CoV-2. The detection limit was 10 copies per reaction in this assay, which could be conducted within 15 min at a constant temperature (39 °C), without any cross-reactions with other respiratory tract pathogens, such as other coronaviruses. Furthermore, compared with commercial real-time RT-PCR assay, it showed a kappa value of 0.959 (p < 0.001) from 150 clinical specimens. These results indicated that this real-time RT-RAA assay may be a valuable tool for detecting SARS-CoV-2.

Clinical Evaluation of Self-Collected Saliva by Quantitative Reverse Transcription-PCR (RT-qPCR), Direct RT-qPCR, Reverse Transcription-Loop-Mediated Isothermal Amplification, and a Rapid Antigen Test To Diagnose COVID-19.

The clinical performances of six molecular diagnostic tests and a rapid antigen test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were clinically evaluated for the diagnosis of coronavirus disease 2019 (COVID-19) in self-collected saliva. Saliva samples from 103 patients with laboratory-confirmed COVID-19 (15 asymptomatic and 88 symptomatic) were collected on the day of hospital admission. SARS-CoV-2 RNA in saliva was detected using a quantitative reverse transcription-PCR (RT-qPCR) laboratory-developed test (LDT), a cobas SARS-CoV-2 high-throughput system, three direct RT-qPCR kits, and reverse transcription-loop-mediated isothermal amplification (RT-LAMP). The viral antigen was detected by a rapid antigen immunochromatographic assay. Of the 103 samples, viral RNA was detected in 50.5 to 81.6% of the specimens by molecular diagnostic tests, and an antigen was detected in 11.7% of the specimens by the rapid antigen test. Viral RNA was detected at significantly higher percentages (65.6 to 93.4%) in specimens collected within 9 days of symptom onset than in specimens collected after at least 10 days of symptoms (22.2 to 66.7%) and in specimens collected from asymptomatic patients (40.0 to 66.7%). Self-collected saliva is an alternative specimen option for diagnosing COVID-19. The RT-qPCR LDT, a cobas SARS-CoV-2 high-throughput system, direct RT-qPCR kits (except for one commercial kit), and RT-LAMP showed sufficient sensitivities in clinical use to be selectively used in clinical settings and facilities. The rapid antigen test alone is not recommended for an initial COVID-19 diagnosis because of its low sensitivity.

Increasing Vaginal Chlamydia Trachomatis Testing in Adolescent and Young Adults.

OBJECTIVE: The Centers for Disease Control and Prevention recommend testing for Chlamydia trachomatis in sexually active female patients <25 years old using nucleic-acid amplification tests (NAAT) from a vaginal swab. Our providers were typically testing using the less sensitive urine NAATs. We aimed to increase the percentage of urogenital C trachomatis NAATs performed by using vaginal swabs in adolescent female patients ages 10 through 20 years from 1.4% to 25%. METHODS: We implemented 3 interventions at 3 pediatric practices over 12 months including education, process standardization, and cross-training. We used statistical process control to analyze the effect of interventions on our primary outcome: the percentage of urogenital C trachomatis tests performed with a vaginal swab. Our balance measure was the total number of urogenital C trachomatis tests. RESULTS: There were 818 urogenital C trachomatis tests performed: 289 before and 529 after the first intervention. Of urogenital C trachomatis tests in the preintervention time period, 1.4% were performed by using vaginal swabs. We surpassed our aim of 25% 6 weeks after the first intervention. We noted sustained improvement after the second intervention, with an average of 68.3% of tests performed by using vaginal swabs for the remaining postintervention period. There was no difference in the overall number of urogenital C trachomatis tests pre- and postintervention. CONCLUSIONS: Using quality improvement methodology and implementing easily replicable interventions, we significantly and sustainably increased use of vaginal swabs. The interventions standardizing processes were associated with a higher impact than the educational intervention.

A colorimetric RT-LAMP assay and LAMP-sequencing for detecting SARS-CoV-2 RNA in clinical samples.

The coronavirus disease 2019 (COVID-19) pandemic caused by the SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) coronavirus is a major public health challenge. Rapid tests for detecting existing SARS-CoV-2 infections and assessing virus spread are critical. Approaches to detect viral RNA based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) have potential as simple, scalable, and broadly applicable testing methods. Compared to RT quantitative polymerase chain reaction (RT-qPCR)-based methods, RT-LAMP assays require incubation at a constant temperature, thus eliminating the need for sophisticated instrumentation. Here, we tested a two-color RT-LAMP assay protocol for detecting SARS-CoV-2 viral RNA using a primer set specific for the N gene. We tested our RT-LAMP assay on surplus RNA samples isolated from 768 pharyngeal swab specimens collected from individuals being tested for COVID-19. We determined the sensitivity and specificity of the RT-LAMP assay for detecting SARS-CoV-2 viral RNA. Compared to an RT-qPCR assay using a sensitive primer set, we found that the RT-LAMP assay reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a sensitivity of 97.5% and a specificity of 99.7%. We also developed a swab-to-RT-LAMP assay that did not require a prior RNA isolation step, which retained excellent specificity (99.5%) but showed lower sensitivity (86% for CT < 30) than the RT-LAMP assay. In addition, we developed a multiplexed sequencing protocol (LAMP-sequencing) as a diagnostic validation procedure to detect and record the outcome of RT-LAMP reactions.

Nanopore Targeted Sequencing for the Accurate and Comprehensive Detection of SARS-CoV-2 and Other Respiratory Viruses.

The ongoing global novel coronavirus pneumonia COVID-19 outbreak has engendered numerous cases of infection and death. COVID-19 diagnosis relies upon nucleic acid detection; however, currently recommended methods exhibit high false-negative rates and are unable to identify other respiratory virus infections, thereby resulting in patient misdiagnosis and impeding epidemic containment. Combining the advantages of targeted amplification and long-read, real-time nanopore sequencing, herein, nanopore targeted sequencing (NTS) is developed to detect SARS-CoV-2 and other respiratory viruses simultaneously within 6-10 h, with a limit of detection of ten standard plasmid copies per reaction. Compared with its specificity for five common respiratory viruses, the specificity of NTS for SARS-CoV-2 reaches 100%. Parallel testing with approved real-time reverse transcription-polymerase chain reaction kits for SARS-CoV-2 and NTS using 61 nucleic acid samples from suspected COVID-19 cases show that NTS identifies more infected patients (22/61) as positive, while also effectively monitoring for mutated nucleic acid sequences, categorizing types of SARS-CoV-2, and detecting other respiratory viruses in the test sample. NTS is thus suitable for COVID-19 diagnosis; moreover, this platform can be further extended for diagnosing other viruses and pathogens.