Lowering the Acquisition of Multi-drug Resistant Organism (MDROs) with Pulsed-xenon (LAMP) Study: a cluster randomized controlled, double-blinded, interventional crossover trial.

BACKGROUND: Environmental disinfection is essential for reducing spread of healthcare associated infections (HAIs). Previous studies report conflicting results regarding the effects of ultraviolet light (UV) in reducing infections. This trial evaluated the impact of adding pulsed xenon UV (PX-UV) to standard terminal cleaning in reducing environmentally-implicated HAIs (eiHAIs). METHODS: The LAMP trial was conducted in 2 hospitals (15 inpatient wards) utilizing a cluster randomized controlled, double-blinded, interventional crossover trial comparing standard terminal cleaning followed by either pulsed xenon ultraviolet (PX-UV) disinfection (intervention arm) or sham disinfection (control arm). The primary outcome was incidence of eiHAIs from clinical microbiology tests on the 4th day of stay or later or within 3 days after discharge from the study unit. EiHAIs included clinical cultures positive for vancomycin-resistant enterococci (VRE), extended spectrum beta-lactamase-producing Escherichia coli or Klebsiella pneumonia, methicillin-resistant Staphylococcus aureus (MRSA), and Acinetobacter baumannii, and stool PCR positive for Clostridiodes difficile. FINDINGS: Between May 18, 2017 to Jan 7, 2020, 25,732 patients were included, with an incidence of 601 eiHAI and 180,954 patient days. There was no difference in the rate of eiHAIs in the intervention and sham arms (3.49 vs 3.17 infections/1000 patient days respectively, RR 1.10 CI (0.94, 1.29, p= 0.23)). Study results were similar when stratified by eiHAI type, hospital, and unit type. CONCLUSION: The LAMP study failed to demonstrate an effect of the addition of UV light disinfection following terminal cleaning on reductions in rates of eiHAIs. Further investigations targeting hospital environmental surfaces and the role of no touch technology to reduce HAIs are needed.

A New Method to Detect Variants of SARS-CoV-2 Using Reverse Transcription Loop-Mediated Isothermal Amplification Combined with a Bioluminescent Assay in Real Time (RT-LAMP-BART).

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), of which there are several variants. The three major variants (Alpha, Delta, and Omicron) carry the N501Y, L452R, and Q493R/Q498R mutations, respectively, in the S gene. Control of COVID-19 requires rapid and reliable detection of not only SARS-CoV-2 but also its variants. We previously developed a reverse transcription loop-mediated isothermal amplification assay combined with a bioluminescent assay in real time (RT-LAMP-BART) to detect the L452R mutation in the SARS-CoV-2 spike protein. In this study, we established LAMP primers and peptide nucleic acid probes to detect N501Y and Q493R/Q498R. The LAMP primer sets and PNA probes were designed for the N501Y and Q493R/Q498R mutations on the S gene of SARS-CoV-2. The specificities of RT-LAMP-BART assays were evaluated using five viral and four bacterial reference strains. The sensitivities of RT-LAMP-BART assays were evaluated using synthetic RNAs that included the target sequences, together with RNA-spiked clinical nasopharyngeal and salivary specimens. The results were compared with those of conventional real-time reverse transcription-polymerase chain reaction (RT-PCR) methods. The method correctly identified N501Y and Q493R/Q498R. Within 30 min, the RT-LAMP-BART assays detected up to 100-200 copies of the target genes; conventional real-time RT-PCR required 130 min and detected up to 500-3000 copies. Surprisingly, the real-time RT-PCR for N501Y did not detect the BA.1 and BA.2 variants (Omicron) that exhibited the N501Y mutation. The novel RT-LAMP-BART assay is highly specific and more sensitive than conventional real-time RT-PCR. The new assay is simple, inexpensive, and rapid; thus, it can be useful in efforts to identify SARS-CoV-2 variants of concern.

Assessment of the Legionnaires' disease outbreak in Flint, Michigan.

The 2014-2015 Legionnaires' disease (LD) outbreak in Genesee County, MI, and the outbreak resolution in 2016 coincided with changes in the source of drinking water to Flint's municipal water system. Following the switch in water supply from Detroit to Flint River water, the odds of a Flint resident presenting with LD increased 6.3-fold (95% CI: 2.5, 14.0). This risk subsided following boil water advisories, likely due to residents avoiding water, and returned to historically normal levels with the switch back in water supply. During the crisis, as the concentration of free chlorine in water delivered to Flint residents decreased, their risk of acquiring LD increased. When the average weekly chlorine level in a census tract was <0.5 mg/L or <0.2 mg/L, the odds of an LD case presenting from a Flint neighborhood increased by a factor of 2.9 (95% CI: 1.4, 6.3) or 3.9 (95% CI: 1.8, 8.7), respectively. During the switch, the risk of a Flint neighborhood having a case of LD increased by 80% per 1 mg/L decrease in free chlorine, as calculated from the extensive variation in chlorine observed. In communities adjacent to Flint, the probability of LD occurring increased with the flow of commuters into Flint. Together, the results support the hypothesis that a system-wide proliferation of legionellae was responsible for the LD outbreak in Genesee County, MI.

Initial and Repeated Point Prevalence Surveys to Inform SARS-CoV-2 Infection Prevention in 26 Skilled Nursing Facilities - Detroit, Michigan, March-May 2020.

Skilled nursing facilities (SNFs) are focal points of the coronavirus disease 2019 (COVID-19) pandemic, and asymptomatic infections with SARS-CoV-2, the virus that causes COVID-19, among SNF residents and health care personnel have been described (1-3). Repeated point prevalence surveys (serial testing of all residents and health care personnel at a health care facility irrespective of symptoms) have been used to identify asymptomatic infections and have reduced SARS-CoV-2 transmission during SNF outbreaks (1,3). During March 2020, the Detroit Health Department and area hospitals detected a sharp increase in COVID-19 diagnoses, hospitalizations, and associated deaths among SNF residents. The Detroit Health Department collaborated with local government, academic, and health care system partners and a CDC field team to rapidly expand SARS-CoV-2 testing and implement infection prevention and control (IPC) activities in all Detroit-area SNFs. During March 7-May 8, among 2,773 residents of 26 Detroit SNFs, 1,207 laboratory-confirmed cases of COVID-19 were identified during three periods: before (March 7-April 7) and after two point prevalence surveys (April 8-25 and April 30-May 8): the overall attack rate was 44%. Within 21 days of receiving their first positive test results, 446 (37%) of 1,207 COVID-19 patients were hospitalized, and 287 (24%) died. Among facilities participating in both surveys (n = 12), the percentage of positive test results declined from 35% to 18%. Repeated point prevalence surveys in SNFs identified asymptomatic COVID-19 cases, informed cohorting and IPC practices aimed at reducing transmission, and guided prioritization of health department resources for facilities experiencing high levels of SARS-CoV-2 transmission. With the increased availability of SARS-CoV-2 testing, repeated point prevalence surveys and enhanced and expanded IPC support should be standard tools for interrupting and preventing COVID-19 outbreaks in SNFs.

Stress, Coping, Resilience and Trust during the Flint Water Crisis.

In 2014, government officials in the City of Flint, Michigan switched the municipal water source from the Detroit Water System (water source: Lake Huron) to the Flint River. During this time, an estimated 102,000 Flint residents were potentially exposed to multiple chemical (e.g., lead) and biological threats (e.g., Legionella). After the switch to water sourced from the Flint River, Flint residents consistently reported concerns over water quality while also experiencing rashes, hair loss, and other health problems, including anxiety and depression. This study 1) reports on the Flint Water Crisis and its subsequent impact on residents' stress, coping, resilience and trust and 2) describes a process methodology that trained, hired and deployed Flint residents as members of a multidisciplinary research team. A random sample of 320 Flint residents underwent household-based interviews to assess their health and mental health needs. Concomitantly, household water samples were obtained and residents were connected to known resources based on interview responses relative to need. This study found that declines in health and mental health status were correlated with increased stressors (i.e., fatigue, financial concern, anxiety), coping and less resilience or the capacity to recover. Perceived trust in government officials was significantly lower after the water crisis. While the water crisis generated numerous stressors, the event also galvanized community competence to engage in solution-focused coping and other adaptive capacities. By assessing and building upon Flint residents' resilience, community resource specialists, identified and subsequently strengthened city residents' ability to survive devastating challenges.

Pertussis: Microbiology, Disease, Treatment, and Prevention.

Pertussis is a severe respiratory infection caused by Bordetella pertussis, and in 2008, pertussis was associated with an estimated 16 million cases and 195,000 deaths globally. Sizeable outbreaks of pertussis have been reported over the past 5 years, and disease reemergence has been the focus of international attention to develop a deeper understanding of pathogen virulence and genetic evolution of B. pertussis strains. During the past 20 years, the scientific community has recognized pertussis among adults as well as infants and children. Increased recognition that older children and adolescents are at risk for disease and may transmit B. pertussis to younger siblings has underscored the need to better understand the role of innate, humoral, and cell-mediated immunity, including the role of waning immunity. Although recognition of adult pertussis has increased in tandem with a better understanding of B. pertussis pathogenesis, pertussis in neonates and adults can manifest with atypical clinical presentations. Such disease patterns make pertussis recognition difficult and lead to delays in treatment. Ongoing research using newer tools for molecular analysis holds promise for improved understanding of pertussis epidemiology, bacterial pathogenesis, bioinformatics, and immunology. Together, these advances provide a foundation for the development of new-generation diagnostics, therapeutics, and vaccines.

Recurrent pneumonia among Japanese adults: disease burden and risk factors.

BACKGROUND: In Japan and other societies with rapidly aging populations, recurrent pneumonia (RP) is a major clinical problem yet only limited information exists regarding the burden of this disease. METHODS: A prospective study of adult pneumonia was conducted to investigate the incidence of RP and potential risk factors. From February 1, 2012 to January 31, 2013, patients aged ≥ 15 years who were diagnosed with pneumonia were prospectively enrolled in a representative community hospital located in central Japan. Patients were followed for one-year to evaluate the recurrence of pneumonia and characteristics associated with RP. Cox proportional hazards models were constructed to compute adjusted hazard ratios (aHR) and ascertain risk factors significantly associated with RP. RESULTS: In total, 841 patients with a median age of 73 years (range 15-101 years) were enrolled totaling 1,048 person-years of observation with a median follow-up time of 475 days. A total of 137 patients had at least one recurrent episode with an incidence rate of 13.1 per 100 person-years (95% confidence interval: 11.1-15.5). In multivariate analysis, a past history of pneumonia (aHR 1.95, 95% CI: 1.35-2.8), chronic pulmonary disease (aHR 1.86, 1.24-2.78) and inhaled corticosteroid usage (aHR 1.78, 1.12-2.84) and hypnotic/sedative medication usage (aHR 2.06, 1.28-3.31) were identified as independent risk factors for recurrent pneumonia, whereas angiotensin converting enzyme-inhibitors usage was associated with a reduction of the risk of RP (aHR 0.22, 0.05-0.91). The detection of P. aeruginosa was significantly associated with RP even after adjusting for chronic pulmonary diseases (aHR = 2.37). CONCLUSIONS: Recurrent pneumonia constitutes a considerable proportion of the pneumonia burden in Japan. A past history of pneumonia, chronic pulmonary disease, inhaled corticosteroid and hypnotic/sedative medication usage and detection of P. aeruginosa were identified as independent risk factors for recurrent pneumonia and special attention regarding the use of medications in this vulnerable population is needed to reduce the impact of this disease in aging populations.

Impact of time to treatment of oseltamivir on influenza hospitalization cost among Korean children.

BACKGROUND: Although oseltamivir is a common influenza treatment, there is a lack of data on the economic benefits of timely oseltamivir treatment. METHODS: From February 2004 through June 2007, 116 hospitalized children ≤ 15 years of age with laboratory-confirmed influenza who received oseltamivir were identified via retrospective medical chart review. Demographic, clinical, and cost data were abstracted and multivariate linear regression was used to assess the association between oseltamivir time to treatment and treatment-related costs among hospitalized children with laboratory-confirmed influenza. RESULTS: Overall, 28% (n = 33) of patients were treated with oseltamivir ≥ day 3 of admission. Rapid influenza diagnostic test was used in a significantly lower proportion of patients treated with oseltamivir ≥ day 3 of admission compared with those who received oseltamivir earlier. On multivariate linear regression, initiation of oseltamivir ≥ day 3 of admission was associated with a 60.84% increase (95%CI: 32.59-95.11) in treatment-related hospital costs, compared with initiation on admission. CONCLUSION: Delayed initiation of oseltamivir was found to be associated with increased treatment-related hospital costs among children hospitalized with laboratory-confirmed influenza.

The association between influenza treatment and hospitalization-associated outcomes among Korean children with laboratory-confirmed influenza.

There are limited data evaluating the relationship between influenza treatment and hospitalization duration. Our purpose assessed the association between different treatments and hospital stay among Korean pediatric influenza patients. Total 770 children ≤ 15 yr-of-age hospitalized with community-acquired laboratory-confirmed influenza at three large urban tertiary care hospitals were identified through a retrospective medical chart review. Demographic, clinical, and cost data were extracted and a multivariable linear regression model was used to assess the associations between influenza treatment types and hospital stay. Overall, there were 81% of the patients hospitalized with laboratory-confirmed influenza who received antibiotic monotherapy whereas only 4% of the patients received oseltamivir monotherapy. The mean treatment-related charges for hospitalizations treated with antibiotics, alone or with oseltamivir, were significantly higher than those treated with oseltamivir-only (P < 0.001). Influenza patients treated with antibiotics-only and antibiotics/oseltamivir combination therapy showed 44.9% and 28.2%, respectively, longer duration of hospitalization compared to those treated with oseltamivir-only. Patients treated with antibiotics, alone or combined with oseltamivir, were associated with longer hospitalization and significantly higher medical charges, compared to patients treated with oseltamivir alone. In Korea, there is a need for more judicious use of antibiotics, appropriate use of influenza rapid testing.

Factors influencing healthcare utilization among children with pneumonia in Muntinlupa City, the Philippines.

We evaluated the factors influencing healthcare utilization among children aged < 5 years with pneumonia symptoms in Muntinlupa City, the Philippines. We interviewed 1,330 caregivers from 190 households per district in seven districts from March to April, 2009 using a standardized questionnaire to obtain information about demographic characteristics, access to healthcare facilities, and potential barriers to healthcare utilization. The mean age of the children was 32 months; 54.7% were boys. Seventy-four point three percents (n = 972) of caregivers stated when their child had pneumonia symptoms they brought them to a government health center, 14.1% (n = 184) went to a private clinic, 8.0% (n = 104) went to a hospital, 3% (n = 40) were treated at home and 0.5% (n = 7) received traditional medicine from a local healer. Caregivers required an average of 12 minutes to reach a healthcare facility. Reasons given by caregivers for their choice of healthcare facility were low cost (47.5%), availability and accessibility to transportation (29.6%) and perceived good quality of care (16.5%). In conclusion, nearly three quarters of caregivers interviewed brought their child with pneumonia symptoms to a government health center for treatment. Cost was the main factor influencing choice, followed by transportation availability and quality of care.

Control of SARS-CoV-2 infection in skilled nursing facilities in Detroit, Michigan: a model for emerging infectious diseases.

An infection prevention bundle that consisted of the development of a response team, public-academic partnership, daily assessment, regular testing, isolation, and environmental controls was implemented in 26 skilled nursing facilities in Detroit, Michigan (March 2020-April 2021). This intervention was associated with sustained control of severe acute respiratory coronavirus virus 2 infection among residents and staff.

Impact of the Tohoku earthquake and tsunami on pneumonia hospitalisations and mortality among adults in northern Miyagi, Japan: a multicentre observational study.

BACKGROUND: On 11 March 2011, the Tohoku earthquake and tsunami struck off the coast of northeastern Japan. Within 3 weeks, an increased number of pneumonia admissions and deaths occurred in local hospitals. METHODS: A multicentre survey was conducted at three hospitals in Kesennuma City (population 74 000), northern Miyagi Prefecture. All adults aged ≥18 years hospitalised between March 2010 and June 2011 with community-acquired pneumonia were identified using hospital databases and medical records. Segmented regression analyses were used to quantify changes in the incidence of pneumonia. RESULTS: A total of 550 pneumonia hospitalisations were identified, including 325 during the pre-disaster period and 225 cases during the post-disaster period. The majority (90%) of the post-disaster pneumonia patients were aged ≥65 years, and only eight cases (3.6%) were associated with near-drowning in the tsunami waters. The clinical pattern and causative pathogens were almost identical among the pre-disaster and post-disaster pneumonia patients. A marked increase in the incidence of pneumonia was observed during the 3-month period following the disaster; the weekly incidence rates of pneumonia hospitalisations and pneumonia-associated deaths increased by 5.7 times (95% CI 3.9 to 8.4) and 8.9 times (95% CI 4.4 to 17.8), respectively. The increases were largest among residents in nursing homes followed by those in evacuation shelters. CONCLUSIONS: A substantial increase in the pneumonia burden was observed among adults after the Tohoku earthquake and tsunami. Although the exact cause remains unresolved, multiple factors including population aging and stressful living conditions likely contributed to this pneumonia outbreak.

Incidence of radiologically-confirmed pneumonia and Haemophilus influenzae type b carriage before Haemophilus influenzae type b conjugate vaccine introduction in Central Vietnam.

OBJECTIVES: To determine the incidence of radiologically-confirmed pneumonia (RCP) and Haemophilus influenzae type b (Hib) carriage in central Vietnam as a baseline data before Hib conjugate vaccine introduction. STUDY DESIGN: In the context of ongoing population-based prospective, hospitalized acute respiratory infection surveillance study, a cross-sectional Hib carriage study was conducted among 1000 children < 5 years of age living in NhaTrang, Vietnam in June 2010, 1 month before the nationwide introduction of Hib conjugate vaccine in Vietnam. RESULTS: The incidence of RCP hospitalizations among children < 5 years of age was 3.3 per 1000 children. The highest incidence was observed among children 12-23 month age group (8.3 per 1000). Haemophilus influenzae carriage was detected in 37% of the children and Hib carriage rate was 3%. Eighty-two percent of the Haemophilus influenzae had TEM ß-lactamase resistance gene. The presence of 6 or more family members was associated with an increased rate of Hib carriage (P = .04). CONCLUSIONS: Incidence of RCP and Hib carriage in this cross-sectional survey are lower compared with other studies. Continued surveillance for invasive Hib disease and sequential Hib carriage surveys are needed to support future assessments of the impact of Hib conjugate vaccine in Vietnam.

The incidence and aetiology of hospitalised community-acquired pneumonia among Vietnamese adults: a prospective surveillance in Central Vietnam.

BACKGROUND: Lower respiratory tract infection (LRTI) including Community-acquired pneumonia (CAP) is a common infectious disease that is associated with significant morbidity and mortality. The patterns of aetiological pathogens differ by region and country. Special attention must be paid to CAP in Southeast Asia (SEA), a region facing rapid demographic transition. Estimates burden and aetiological patterns of CAP are essential for the clinical and public health management. The purposes of the study are to determine the incidence, aetiological pathogens, clinical pictures and risk factors of community-acquired pneumonia (CAP) in the Vietnamese adult population. METHODS: A prospective surveillance for hospitalised adult CAP was conducted in Khanh Hoa Province, Central Vietnam. All adults aged ≥15 years with lower respiratory tract infections (LRTI) admitted to a provincial hospital from September 2009 to August 2010 were enrolled in the study. Patients were classified into CAP and non-pneumonic LRTI (NPLRTI) according to the radiological findings. Bacterial pathogens were identified from sputum samples by the conventional culture and polymerase chain reaction (PCR) for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis; 13 respiratory viruses were identified from nasopharyngeal specimens by PCR. RESULTS: Of all 367 LRTI episodes examined, 174 (47%) were CAP. Older age, the presence of underlying respiratory conditions, and higher index score of smoking were associated with CAP. The one-year estimated incidence of hospitalised adult CAP in our study population was 0.81 per 1,000 person years. The incidence increased considerably with age and was highest among the elderly. The case fatality proportion of hospitalised CAP patients was 9.8%. Among 286 sputum samples tested for bacterial PCR, 79 (28%) were positive for H. influenzae, and 65 (23%) were positive for S. pneumoniae. Among 357 samples tested for viral PCR, 73 (21%) were positive for respiratory viruses; influenza A (n = 32, 9%) was the most common. CONCLUSIONS: The current adult CAP incidence in Vietnam was relatively low; this result was mainly attributed to the young age of our study population.

Pandemic response gaps: Infection prevention and control lessons learned during coronavirus disease 2019 (COVID-19) outbreaks in skilled nursing facilities in Detroit, Michigan.

BACKGROUND: Hospitalizations among skilled nursing facility (SNF) residents in Detroit increased in mid-March 2020 due to the coronavirus disease 2019 (COVID-19) pandemic. Outbreak response teams were deployed from local healthcare systems, the Centers for Disease Control and Prevention (CDC), and the Detroit Health Department (DHD) to understand the infection prevention and control (IPC) gaps in SNFs that may have accelerated the outbreak. METHODS: We conducted 2 point-prevalence surveys (PPS-1 and PPS-2) at 13 Detroit SNFs from April 8 to May 8, 2020. The DHD and partners conducted facility-wide severe acute respiratory coronavirus virus 2 (SARS-CoV-2) testing of all residents and staff and collected information regarding resident cohorting, staff cohorting, and personnel protective equipment (PPE) utilized during that time. RESULTS: Resident cohorting had been implemented in 7 of 13 (58.3%) SNFs prior to point-prevalence survey 1 (PPS-1), and other facilities initiated cohorting after obtaining PPS-1 results. Cohorting protocols of healthcare practitioners and environmental service staff were not established in 4 (31%) of 13 facilities, and in 3 facilities (23.1%) the ancillary staff were not assigned to cohorts. Also, 2 SNFs (15%) had an observation unit prior to PPS-1, 2 (15%) had an observation unit after PPS-1, 4 (31%) could not establish an observation unit due to inadequate space, and 5 (38.4%) created an observation unit after PPS-2. CONCLUSION: On-site consultations identified gaps in IPC knowledge and cohorting that may have contributed to ongoing transmission of SARS-CoV-2 among SNF residents despite aggressive testing measures. Infection preventionists (IPs) are critical in guiding ongoing IPC practices in SNFs to reduce spread of COVID-19 through response and prevention.

An expanded age range for meningococcal meningitis: molecular diagnostic evidence from population-based surveillance in Asia.

BACKGROUND: To understand epidemiologic patterns of meningococcal disease in Asia, we performed a retrospective molecular analysis of cerebrospinal fluid (CSF) specimens collected in prospective surveillance among children aged < 5 years of age in China, South Korea, and Vietnam. METHODS: A total of 295 isolates and 2,302 CSFs were tested by a meningococcal species- and serogroup-specific polymerase chain reaction (PCR) assay targeting the Neisseria meningitidis (Nm) ctrA gene. Multi-locus sequence typing (MLST) was performed in Nm gene amplification analysis and incidence rates for meningococcal meningitis were estimated. RESULTS: Among 295 isolates tested, 10 specimens from Vietnam were confirmed as serogroup B and all were Sequence Type (ST) 1576 by MLST. Among the 2,032 CSF specimen tested, 284 (14%) were confirmed by PCR (ctrA gene), including 67 (23.6%) from China, 92 (32.4%) from Korea, and 125 (44.0%) from Vietnam. Neonates and infants aged < 6 months of age accounted for more than 50% of Nm-PCR positive CSF. Two CSF specimens from Vietnam were identified as serogroup B using MLST. In addition, 44 specimens underwent sequencing to confirm meningococcal serogroup; of these, 21 (48%) were serogroup C, 12 (27%) were serogroup X, 9 (20%) were serogroup Y and 2 (5%) were serogroup B. The incidence rates of meningococcal meningitis among children < 5 years of age was highest in Vietnam (7.4/100,000 [95% CI, 3.6-15.3] followed by Korea (6.8/100,000 [95% CI, 3.5-13.5] and China (2.1/100,000) [95% CI, 0.7-6.2]). CONCLUSIONS: These results suggest that there is a previously undetected, yet substantial burden of meningococcal meningitis among infants and young children. Standardized, sensitive and specific molecular diagnostic assays with Nm serogrouping capacity are needed throughout Asia to understand the true burden of N. meningitidis disease.

Corrigendum: Molecular Serotype-Specific Identification of Non-type b Haemophilus influenzae by Loop-Mediated Isothermal Amplification.

[This corrects the article DOI: 10.3389/fmicb.2017.01877.].

Detection of SARS-CoV-2 and the L452R spike mutation using reverse transcription loop-mediated isothermal amplification plus bioluminescent assay in real-time (RT-LAMP-BART).

The new coronavirus infection (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be fatal, and several variants of SARS-CoV-2 with mutations of the receptor-binding domain (RBD) have increased avidity for human cell receptors. A single missense mutation of U to G at nucleotide position 1355 (U1355G) in the spike (S) gene changes leucine to arginine (L452R) in the spike protein. This mutation has been observed in the India and California strains (B.1.617 and B.1.427/B.1.429, respectively). Control of COVID-19 requires rapid and reliable detection of SARS-CoV-2. Therefore, we established a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay plus a bioluminescent assay in real-time (BART) to detect SARS-CoV-2 and the L452R spike mutation. The specificity and sensitivity of the RT-LAMP-BART assay was evaluated using synthetic RNAs including target sequences and RNA-spiked clinical nasopharyngeal and saliva specimens as well as reference strains representing five viral and four bacterial pathogens. The novel RT-LAMP-BART assay to detect SARS-CoV-2 was highly specific compared to the conventional real-time RT-PCR. Within 25 min, the RT-LAMP-BART assay detected 80 copies of the target gene in a sample, whereas the conventional real-time RT-PCR method detected 5 copies per reaction within 130 min. Using RNA-spiked specimens, the sensitivity of the RT-LAMP-BART assay was slightly attenuated compared to purified RNA as a template. The results were identical to those of the conventional real-time RT-PCR method. Furthermore, using a peptide nucleic acid (PNA) probe, the RT-LAMP-BART method correctly identified the L452R spike mutation. This is the first report describes RT-LAMP-BART as a simple, inexpensive, rapid, and useful assay for detection of SARS-CoV-2, its variants of concern, and for screening of COVID-19.

Development of a novel loop-mediated isothermal amplification assay for ß-lactamase gene identification using clinical isolates of Gram-negative bacteria.

Rapid evaluation of antimicrobial susceptibility is important in the treatment of nosocomial infections by Gram-negative bacteria, which increasingly carry carbapenemases and metallo-ß-lactamases. We developed loop-mediated isothermal amplification (LAMP)-based assays for four ß-lactamase genes (bla KPC, bla NDM-1, bla IMP-1 group, and bla VIM). The assays were evaluated using eight reference bacterial strains (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter bereziniae) harboring six ß-lactamase genes. A total of 55 Gram-negative bacterial strains, including 47 clinical P. aeruginosa isolates, fully characterized by next-generation sequencing (NGS), were used to evaluate the LAMP assays. The results were compared to those of conventional PCR. The LAMP assays were able to detect as few as 10 to 100 copies of a gene, compared to 10 to 104 copies for conventional PCR. The LAMP assay detected four ß-lactamase genes with a sensitivity similar to that using purified DNA as the template in DNA-spiked urine, sputum, and blood specimens. By contrast, the sensitivity of PCR was 1- to 100-fold lower with DNA-spiked clinical specimens. Therefore, the LAMP assays were proved to be an appropriate tool for the detection of four ß-lactamases.