Evolution of the Probe-Based Loop-Mediated Isothermal Amplification (LAMP) Assays in Pathogen Detection.

Loop-mediated isothermal amplification (LAMP), as the rank one alternative to a polymerase chain reaction (PCR), has been widely applied in point-of-care testing (POCT) due to its rapid, simple, and cost-effective characteristics. However, it is difficult to achieve real-time monitoring and multiplex detection with the traditional LAMP method. In addition, these approaches that use turbidimetry, sequence-independent intercalating dyes, or pH-sensitive indicators to indirectly reflect amplification can result in false-positive results if non-specific amplification occurs. To fulfill the needs of specific target detection and one-pot multiplex detection, a variety of probe-based LAMP assays have been developed. This review focuses on the principles of these assays, summarizes their applications in pathogen detection, and discusses their features and advantages over the traditional LAMP methods.

COVID-19 public health measures reduce the incidence of respiratory infectious diseases.

BACKGROUND: Children and the elderly are two special subpopulations for coronavirus disease 2019 (COVID-19) and respiratory tract infections (RTIs). The study aimed to evaluate the effect of COVID-19 public health measures on the burden of RTIs in China by performing a two-center investigation. METHODS: The electronic medical records of all inpatients in departments of pediatrics and respiratory medicine of Taizhou Fourth People's Hospital (Taizhou, China) and Shaanxi Provincial People's Hospital (Xi'an, China) during January 1, 2019 to June 30, 2021 were analyzed. A total of 18,084 child inpatients and 14,802 adult inpatients were included. RESULTS: The vast majority (88.3%-90.6%) of the adult inpatients were the elderly, aged over 50 years. The numbers of child and adult (elderly) inpatients, and the proportions of RTI-associated diseases substantially decreased during COVID-19 pandemic (2020-2021) compared to that before the pandemic (2019) in Taizhou and Xi'an. A significantly higher proportion of LRTI-associated diseases was observed in elderly female inpatients (53.4-55.6%) than elderly male inpatients (34.3-41.5%) (p < 0.001) in spite of more male inpatients than female inpatients (1.94-1.95:1). CONCLUSIONS: COVID-19-related interventions provide an additional beneficial effect on reduction of RTI-associated diseases in both children and the elderly.

A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2.

COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 µL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 µL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

Progressive deterioration of the upper respiratory tract and the gut microbiomes in children during the early infection stages of COVID-19.

Children are less susceptible to coronavirus disease 2019 (COVID-19), and they have manifested lower morbidity and mortality after infection, for which a multitude of mechanisms may be considered. Whether the normal development of the gut-airway microbiome in children is affected by COVID-19 has not been evaluated. Here, we demonstrate that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection alters the upper respiratory tract and the gut microbiomes in nine children. The alteration of the microbiome is dominated by the genus Pseudomonas, and it sustains for up to 25-58 days in different individuals. Moreover, the patterns of alternation are different between the upper respiratory tract and the gut. Longitudinal investigation shows that the upper respiratory tract and the gut microbiomes are extremely variable among children during the course of COVID-19. The dysbiosis of microbiome persists in 7 of 8 children for at least 19-24 days after discharge from the hospital. Disturbed development of both the gut and the upper respiratory microbiomes and prolonged dysbiosis in these nine children imply possible long-term complications after clinical recovery from COVID-19, such as predisposition to the increased health risk in the post-COVID-19 era.

Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe.

Viral evolution impacts diagnostic test performance through the emergence of variants with sequences affecting the efficiency of primer binding. Such variants that evade detection by nucleic acid-based tests are subject to selective pressure, enabling them to spread more efficiently. Here, we report a variant-tolerant diagnostic test for SARS-CoV-2 using a loop-mediated isothermal nucleic acid-based amplification (LAMP) assay containing high-fidelity DNA polymerase and a high-fidelity DNA polymerase-medicated probe (HFman probe). In addition to demonstrating a high tolerance to variable SARS-CoV-2 viral sequences, the mechanism also overcomes frequently observed limitations of LAMP assays arising from non-specific amplification within multiplexed reactions performed in a single "pot". Results showed excellent clinical performance (sensitivity 94.5%, specificity 100%, n = 190) when compared directly to a commercial gold standard reverse transcription quantitative polymerase chain reaction assay for the extracted RNA from nasopharyngeal samples and the capability of detecting a wide range of sequences containing at least alpha and delta variants. To further validate the test with no sample processing, directly from nasopharyngeal swabs, we also detected SARS-CoV-2 in positive clinical samples (n = 49), opening up the possibility for the assay's use in decentralized testing.

Longitudinal immune profiling reveals dominant epitopes mediating long-term humoral immunity in COVID-19-convalescent individuals.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic and contagious coronavirus that caused a global pandemic with 5.2 million fatalities to date. Questions concerning serologic features of long-term immunity, especially dominant epitopes mediating durable antibody responses after SARS-CoV-2 infection, remain to be elucidated. OBJECTIVE: We aimed to dissect the kinetics and longevity of immune responses in coronavirus disease 2019 (COVID-19) patients, as well as the epitopes responsible for sustained long-term humoral immunity against SARS-CoV-2. METHODS: We assessed SARS-CoV-2 immune dynamics up to 180 to 220 days after disease onset in 31 individuals who predominantly experienced moderate symptoms of COVID-19, then performed a proteome-wide profiling of dominant epitopes responsible for persistent humoral immune responses. RESULTS: Longitudinal analysis revealed sustained SARS-CoV-2 spike protein-specific antibodies and neutralizing antibodies in COVID-19 patients, along with activation of cytokine production at early stages after SARS-CoV-2 infection. Highly reactive epitopes that were capable of mediating long-term antibody responses were shown to be located at the spike and ORF1ab proteins. Key epitopes of the SARS-CoV-2 spike protein were mapped to the N-terminal domain of the S1 subunit and the S2 subunit, with varying degrees of sequence homology among endemic human coronaviruses and high sequence identity between the early SARS-CoV-2 (Wuhan-Hu-1) and current circulating variants. CONCLUSION: SARS-CoV-2 infection induces persistent humoral immunity in COVID-19-convalescent individuals by targeting dominant epitopes located at the spike and ORF1ab proteins that mediate long-term immune responses. Our findings provide a path to aid rational vaccine design and diagnostic development.

Potential Achilles heels of SARS-CoV-2 are best displayed by the base order-dependent component of RNA folding energy.

The base order-dependent component of folding energy has revealed a highly conserved region in HIV-1 genomes that associates with RNA structure. This corresponds to a packaging signal that is recognized by the nucleocapsid domain of the Gag polyprotein. Long viewed as a potential HIV-1 "Achilles heel," the signal can be targeted by a new antiviral compound. Although SARS-CoV-2 differs in many respects from HIV-1, the same technology displays regions with a high base order-dependent folding energy component, which are also highly conserved. This indicates structural invariance (SI) sustained by natural selection. While the regions are often also protein-encoding (e. g. NSP3, ORF3a), we suggest that their nucleic acid level functions can be considered potential "Achilles heels" for SARS-CoV-2, perhaps susceptible to therapies like those envisaged for AIDS. The ribosomal frameshifting element scored well, but higher SI scores were obtained in other regions, including those encoding NSP13 and the nucleocapsid (N) protein.

Correction to: Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2.

The corrected legend is given below.

Temporal association between human upper respiratory and gut bacterial microbiomes during the course of COVID-19 in adults.

SARS-CoV-2 is the cause of COVID-19. It infects multiple organs including the respiratory tract and gut. Dynamic changes of regional microbiomes in infected adults are largely unknown. Here, we performed longitudinal analyses of throat and anal swabs from 35 COVID-19 and 19 healthy adult controls, as well as 10 non-COVID-19 patients with other diseases, by 16 S rRNA gene sequencing. The results showed a partitioning of the patients into 3-4 categories based on microbial community types (I-IV) in both sites. The bacterial diversity was lower in COVID-19 patients than healthy controls and decreased gradually from community type I to III/IV. Although the dynamic change of microbiome was complex during COVID-19, a synchronous restoration of both the upper respiratory and gut microbiomes from early dysbiosis towards late more diverse status was observed in 6/8 mild COVID-19 adult patients. These findings reveal previously unknown interactions between upper respiratory and gut microbiomes during COVID-19.

Comparative evaluation of 19 reverse transcription loop-mediated isothermal amplification assays for detection of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has caused a global pandemics. To facilitate the detection of SARS-CoV-2 infection, various RT-LAMP assays using 19 sets of primers had been developed, but never been compared. We performed comparative evaluation of the 19 sets of primers using 4 RNA standards and 29 clinical samples from COVID-19 patients. Six of 15 sets of primers were firstly identified to have faster amplification when tested with four RNA standards, and were further subjected to parallel comparison with the remaining four primer sets using 29 clinical samples. Among these 10 primer sets, Set-4 had the highest positive detection rate of SARS-CoV-2 (82.8%), followed by Set-10, Set-11, and Set-13 and Set-17 (75.9%). Set-14 showed the fastest amplification speed (Tt value < 8.5 min), followed by Set-17 (Tt value < 12.5 min). Based on the overall detection performance, Set-4, Set-10, Set-11, Set-13, Set-14 and Set-17 that target Nsp3, S, S, E, N and N gene regions of SARS-CoV-2, respectively, were determined to be better than the other primer sets. Two RT-LAMP assays with the Set-4 primers in combination with any one of four other primer sets (Set-14, Set-10, Set-11, and Set-13) were recommended to be used in the COVID-19 surveillance.

Epidemiological and clinical characteristics of COVID-19 patients in Nantong, China.

INTRODUCTION: COVID-19 is a newly emerging life-threatening respiratory disease caused by a newly identified coronavirus SARS-CoV-2. METHODOLOGY: We included 28 COVID-19 patients admitted to Nantong Third Hospital from January 23 to February 26, 2020. SARS-CoV-2 infection was confirmed using real-time RT-PCR. The demographic, epidemiological, clinical, laboratory parameters were obtained from each patient. RESULTS: The vast majority (71.4%) of confirmed COVID-19 patients were brought in from outside of the city, and all others had contact history with these confirmed cases. The median age of patients was 50 years old and half had underlying diseases. The most common symptoms at the onset of illness were fever (96.4%), cough (67.9%), and chilly (28.6%), and 75.0% patients had two or more symptoms. Increased erythrocyte sedimentation rate, serum ferritin and C-reactive protein levels, and reduced absolute counts of total lymphocytes and T lymphocyte subsets were observed among the patients. The vast majority (85.7%) of patients showed bilateral or unilateral pneumonia, and three symptomatic patients and one asymptomatic case did not show abnormalities in their CT image. Among the 28 admitted patients, 24 were discharged as of February 26, 2020, with an average hospital stay of 14.96 (±4.27) days, which was not significantly associated with the interval between the onset of symptoms and admission. CONCLUSIONS: In the absence of specific antiviral drugs or a vaccine, quarantine or isolation is the most effective intervention strategy for preventing the spread of the virus. Adequate supportive medical care is crucial for good prognosis of COVID-19 patients.