Strategic Retesting to Reduce False Positive SARS-CoV-2 PCR Test Results.

BACKGROUND: Nucleic acid amplification testing is the gold standard for SARS-CoV-2 diagnostics, although it may produce a certain number of false positive results. There has not been much published about the characteristics of false positive results. In this study, based on retesting, specimens that initially tested positive for SARS-CoV-2 were classified as true or false positive groups to characterize the distribution of cycle threshold (CT) values for N1 and N2 targets and number of targets detected for each group. METHODS: Specimens that were positive for N-gene on retesting and accompanied with S-gene were identified as true positives (true positive based on retesting, rTP), while specimens that retested negative were classified as false positives (false positive based on retesting, rFP). RESULTS: Of the specimens retested, 85/127 (66.9%) were rFP, 16/47 (34.0%) specimens with both N1 and N2 targets initially detected were rFP, and the CT values for each target was higher in rFP than in rTP. ROC curve analysis showed that optimal cutoff values of CT to differentiate between rTP and rFP were 34.8 for N1 and 33.0 for N2. With the optimal cutoff values of CT for each target, out of the 24 specimens that were positive for both N1 and N2 targets and classified as rTP, 23 (95.8%) were correctly identified as true positives. rFP specimens had a single N1 target in 52/61 (85.2%) and a single N2 target in 17/19 (89.5%). Notably, no true positive results were obtained from any specimens with only N2 target detected. CONCLUSIONS: These results suggest that retesting should be performed for positive results with a CT value greater than optimal cutoff value for each target or with a single N1 target amplified, considering the possibility of a false positive. This may provide guidance on indications to perform retesting to minimize the number of false positives.

High-Performance Genetically Encoded Green Fluorescent Biosensors for Intracellular l-Lactate.

l-Lactate is a monocarboxylate produced during the process of cellular glycolysis and has long generally been considered a waste product. However, studies in recent decades have provided new perspectives on the physiological roles of l-lactate as a major energy substrate and a signaling molecule. To enable further investigations of the physiological roles of l-lactate, we have developed a series of high-performance (ΔF/F = 15 to 30 in vitro), intensiometric, genetically encoded green fluorescent protein (GFP)-based intracellular l-lactate biosensors with a range of affinities. We evaluated these biosensors in cultured cells and demonstrated their application in an ex vivo preparation of Drosophila brain tissue. Using these biosensors, we were able to detect glycolytic oscillations, which we analyzed and mathematically modeled.

Lactate biosensors for spectrally and spatially multiplexed fluorescence imaging.

L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.

Evaluation of the Analytical Performance of the Fully Automated Gene Analyzer µTASWako g1 for the Detection of SARS-CoV-2.

BACKGROUND: The worldwide spread of coronavirus disease 2019 (COVID-19) has led to an urgent need for nucleic acid amplification test (NAAT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because NAAT has many manual processes, results may vary depending on the operator. Therefore, it has been required to develop a fully automated testing device and reagent that detects genetic material from SARS-CoV-2. The µTASWako g1 system (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), a genetic analyzer, provides results in 75 minutes by performing a fully automated PCR process. METHODS: We evaluated the analytical and clinical performance of the µTASWako g1 system for the detection of SARS-CoV-2 RNA. RESULTS: The µTASWako g1 system had the limit of detection at 2,000 copies/mL using a known concentration of RNA. In clinical samples, the µTASWako g1 system had a sensitivity of 88.0% and 100% specificity compared to conventional RT-PCR. The µTAS Wako g1 system could detect three variants of concern carrying spike mutations including N501Y, E484K, and L452R. CONCLUSIONS: As the assay on the µTASWako g1 system is highly accurate for the detection of SARS-CoV-2 regardless of the experience of operator, it can be widely applicable in clinical laboratories.

A Polyimide Film-Based Simple Force Plate for Measuring the Body Mass of Tiny Insects.

Insects exhibit excellent maneuvers such as running and flying despite their small bodies; therefore, their locomotion mechanism is expected to provide a design guideline for micromachines. Numerical simulations have been performed to elucidate this mechanism, whereby it is important to develop a model that is physically identical to the target insect's parts to reproduce kinematic dynamics. In particular, in flight, the shape and mass of wings, which flap at high frequencies, are significant parameters. However, small insects such as fruit flies have small, thin, and light wings; thus, their mass cannot be easily measured. In this study, we proposed a high-resolution and simple force plate to measure the mass of each part of a tiny insect. The device consists of a circular plate supported by flat spiral springs made of polyimide film, and a laser displacement meter that detects the displacement of the center of the plate. The simple plate fabrication process requires only a couple of minutes. A fabricated force plate with a sub-N/m spring constant achieved a resolution of less than 2 µg. As a demonstration, the wing mass of the fruit flies was measured. The experimental results suggest that the wings accounted for approximately 0.4% of the body mass.

Short and long sleeping mutants reveal links between sleep and macroautophagy.

Sleep is a conserved and essential behavior, but its mechanistic and functional underpinnings remain poorly defined. Through unbiased genetic screening in Drosophila, we discovered a novel short-sleep mutant we named argus. Positional cloning and subsequent complementation, CRISPR/Cas9 knock-out, and RNAi studies identified Argus as a transmembrane protein that acts in adult peptidergic neurons to regulate sleep. argus mutants accumulate undigested Atg8a(+) autophagosomes, and genetic manipulations impeding autophagosome formation suppress argus sleep phenotypes, indicating that autophagosome accumulation drives argus short-sleep. Conversely, a blue cheese neurodegenerative mutant that impairs autophagosome formation was identified independently as a gain-of-sleep mutant, and targeted RNAi screens identified additional genes involved in autophagosome formation whose knockdown increases sleep. Finally, autophagosomes normally accumulate during the daytime and nighttime sleep deprivation extends this accumulation into the following morning, while daytime gaboxadol feeding promotes sleep and reduces autophagosome accumulation at nightfall. In sum, our results paradoxically demonstrate that wakefulness increases and sleep decreases autophagosome levels under unperturbed conditions, yet strong and sustained upregulation of autophagosomes decreases sleep, whereas strong and sustained downregulation of autophagosomes increases sleep. The complex relationship between sleep and autophagy suggested by our findings may have implications for pathological states including chronic sleep disorders and neurodegeneration, as well as for integration of sleep need with other homeostats, such as under conditions of starvation.

Evolutionary Origin of Distinct NREM and REM Sleep.

Sleep is mandatory in most animals that have the nervous system and is universally observed in model organisms ranging from the nematodes, zebrafish, to mammals. However, it is unclear whether different sleep states fulfill common functions and are driven by shared mechanisms in these different animal species. Mammals and birds exhibit two obviously distinct states of sleep, i.e., non-rapid eye movement (NREM) sleep and rapid eye movement (REM) sleep, but it is unknown why sleep should be so segregated. Studying sleep in other animal models might give us clues that help solve this puzzle. Recent studies suggest that REM sleep, or ancestral forms of REM sleep might be found in non-mammalian or -avian species such as reptiles. These observations suggest that REM sleep and NREM sleep evolved earlier than previously thought. In this review, we discuss the evolutionary origin of the distinct REM/NREM sleep states to gain insight into the mechanistic and functional reason for these two different types of sleep.

Epidemiological and molecular characterization of invasive Streptococcus pneumoniae isolated following introduction of 7-valent conjugate vaccine in Kinki region, Japan, 2008-2013.

Streptococcus pneumoniae is one of the most common bacteria causing community-acquired pneumonia and meningitis. The use of 7-valent pneumococcal conjugate vaccine (PCV7) has reduced the incidence of pneumococcal disease while changing pneumococcal population through herd immunity and non-vaccine pneumococci replacement. This study investigated molecular epidemiologic characteristics of pneumococcal strains in the Kinki region of Japan from 2008 to 2013. A total of 159 invasive pneumococcal isolates were characterized by serotyping, antibiotic susceptibility testing, PCR analysis of penicillin-binding protein genes, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). In adult populations, pediatric PCV7 introduction decreased isolates expressing PCV7 serotypes via herd immunity and increased isolates expressing non-PCV7 serotypes. The rate of penicillin resistance and isolates with alterations in all three pbp genes was higher in PCV7 type isolates than in non-PCV7 type isolates. In MLST analysis, all of serotype 19F isolates were of the same sequence type, ST236, which is the antimicrobial-resistant clone Taiwan19F-14, and the majority of serotypes 23F and 19A isolates were of ST1437 and ST3111 respectively, which are the predominant clones of antimicrobial-resistant pneumococci in Japan. In PFGE profiles, serotype 6B-ST2224, serotype 19F-ST236, serotype 19A-ST3111, and serotype 23F-ST1437 formed six separate clusters composed of genetically identical strains, and genetically identical serotype 22F-ST433 formed two different clusters between the pre- and post-PCV7 period. The results of molecular analysis suggest the spread and persistence of these identical antimicrobial resistant clones in the Kinki region and genetic changes of epidemic clone serotype 22F-ST433 before and after pediatric PCV7 introduction.

Antimicrobial susceptibility surveillance of obligate anaerobic bacteria in the Kinki area.

Obligate anaerobes exist as resident flora in various sites in humans, but they are also emphasized as endogenous causative microorganism of infections. We performed surveillance to understand the trend of drug susceptibility in obligate anaerobic bacteria in the Kinki area of Japan. In the experiment, we used 156 obligate anaerobe isolates collected from 13 institutions that participated in the Study of Bacterial Resistance Kinki Region of Japan. MALDI Biotyper was used to identify the collected strains, and among the 156 test strains, those that could be identified with an accuracy of Score Value 2.0 or more included 6 genera, 30 species, and 144 strains (Bacteroides spp. 77 strains, Parabacteroides sp. 2 strains, Prevotella spp. 29 strains, Fusobacterium spp. 14 strains, Porphyromonas spp. 2 strains, and Clostridioides difficile 20 strains), and they were assigned as subject strains for drug susceptibility testing. The drug susceptibility test was carried out by broth microdilution method using Kyokuto Opt Panel MP ANA (Kyokuto Pharmaceutical Industrial Co., Ltd., Tokyo, Japan) and judged according to CLSI criteria. As a result, Bacteroides and Parabacteroides species showed good sensitivities to tazobactam-piperacillin, imipenem, metronidazole and chloramphenicol, and low sensitivities to ampicillin, cefoperazone and vancomycin. Prevotella species showed good sensitivities to sulbactam-ampicillin, tazobactam-piperacillin, cefmetazole, imipenem, doripenem and metronidazole. Susceptibility rates to other drugs were slightly different depending on the bacterial species. Both Fusobacterium spp. and Porphyromonas spp. showed high sensitivities to many drugs. C. difficile was highly sensitive to vancomycin and metronidazole, having MIC90s of 0.5 µg/mL and ≤2 µg/mL, respectively.

A sleep-inducing gene, nemuri, links sleep and immune function in Drosophila.

Sleep remains a major mystery of biology. In particular, little is known about the mechanisms that account for the drive to sleep. In an unbiased screen of more than 12,000 Drosophila lines, we identified a single gene, nemuri, that induces sleep. The NEMURI protein is an antimicrobial peptide that can be secreted ectopically to drive prolonged sleep (with resistance to arousal) and to promote survival after infection. Loss of nemuri increased arousability during daily sleep and attenuated the acute increase in sleep induced by sleep deprivation or bacterial infection. Conditions that increase sleep drive induced expression of nemuri in a small number of fly brain neurons and targeted it to the sleep-promoting, dorsal fan-shaped body. We propose that NEMURI is a bona fide sleep homeostasis factor that is particularly important under conditions of high sleep need; because these conditions include sickness, our findings provide a link between sleep and immune function.

Laboratory surveillance of antimicrobial resistance and multidrug resistance among Streptococcus pneumoniae isolated in the Kinki region of Japan, 2001-2015.

The 7-valent pneumococcal conjugate vaccine (PCV7) was introduced among children in Japan in 2010. There are no long-term multicenter surveillance studies of antimicrobial resistance in S. pneumoniae before and after the introduction of PCV7. Therefore, we examined chronological trends in antimicrobial resistance among 4534 strains of S. pneumoniae isolated from both children and adults in the Kinki region of Japan during 2001-2015. High-level penicillin and third-generation cephalosporin resistance in S. pneumoniae increased among both children and adults during the period before the introduction of PCV7 (2001-2010). Besides penicillin and cephalosporin, pneumococcal carbapenem and macrolide resistance increased among children. The rate of resistance to these antibiotics was higher among children than among adults. The introduction of PCV7 decreased the rate of non-susceptibility to ß-lactam antibiotics and the rate of multidrug resistant S. pneumoniae among children, but not among adults.

Genetic Mechanisms Underlying Sleep.

Sleep is important for cognitive ability, and perturbations of sleep are associated with a myriad of brain disorders. However, how sleep promotes health and function during wake is poorly understood. To address the cellular and molecular mechanisms underlying sleep, we use the fruit fly Drosophila melanogaster as a genetic model. Forward genetic approaches in flies were critical for deciphering molecular mechanisms of the circadian clock. Using similar approaches, we and others are gaining insights into the pathways that control sleep amount.

Evaluation of the modified carbapenem inactivation method for the detection of carbapenemase-producing Enterobacteriaceae.

Carbapenemase-producing Enterobacteriaceae (CPE) are increasing worldwide. Rapid and accurate detection of CPE is necessary for appropriate antimicrobial treatment and hospital infection control. However, CPE contains some strains that are difficult to detect depending on genotype and MIC value of carbapenem, and a detection method has not been established. The recently reported modified carbapenem inactivation method (mCIM) has been developed in CLSI M100-S27 as a phenotypic technique for detecting carbapenemase activity. In the present study, we examined mCIM as a new CPE detection method using 207 Enterobacteriaceae isolates in comparison with the three existing screening methods of modified Hodge test, Carba NP test and carbapenem inactivation method and evaluated its performance. Consequently, both the sensitivity and specificity of mCIM were 100%, indicating better results than the conventional screening methods. The mCIM is a useful tool for microbiology laboratories due to its simplicity, clear criteria, cost-effectiveness and availability at any laboratory.

Nosocomial spread of Klebsiella pneumoniae isolates producing blaGES-4 carbapenemase at a Japanese hospital.

Six Klebsiella pneumoniae clinical isolates resistant to various cephalosporins and cephamycins were identified in a Japanese general hospital, a tertiary care hospital, between November 2009 and April 2010. All K. pneumoniae isolates carried blaGES-4 and blaSHV-1, while 2 K. pneumoniae isolates also harbored blaCTX-M-15. The pulsed-field gel electrophoresis patterns revealed that these 6 K. pneumoniae isolates were almost identical, suggesting their clonal relatedness. Plasmid profiles and conjugation assays revealed that these blaGES-4 genes were located on similar conjugative plasmids. These data indicate that nosocomial spread caused by K. pneumoniae isolates producing blaGES-4 carbapenemase occurred at a Japanese general hospital. K. pneumoniae isolate harboring blaGES-4 is rarely reported in Japan, and, to the best of our knowledge, this is the second report of K. pneumoniae isolates harboring blaGES-4 that occurred nosocomial spread in Japan.

[The annual changes in antimicrobial susceptibility test results of Pseudomonas aeruginosa isolates from the Kinki district].

A study was conducted of the 1,225 Pseudomonas aeruginosa strains that were isolated at 20 medical institutions in the Kinki district between 2011 and 2013 to determine their antimicrobial susceptibility and to characterize the strains of multidrug-resistant Pseudomonas aeruginosa (MDRP) and the metallo-ß-lactamase (MBL) -producing strains. The MIC50/MIC90 values (µg/mL) of the various antimicrobial agents were as follows: imipenem, 2/>8; meropenem, 1/>8; doripenem, 0.5/8; biapenem, 1/>8; tazobactam/piperacillin, 8/>64; piperacillin, 8/>64; sulbactam/cefoperazone, 8/64; cefepime, 4/16; cefozopran, 2/>16; aztreonam, 8/>16; amikacin, 4/16; levofloxacin, 1/>4; and ciprofloxacin, 0.25/>2. From the viewpoint of the annual changes in the susceptibility rates (according to the CLSI guidelines [M100-S22]), the susceptibility to tazobactam/piperacillin, piperacillin, cefepime, cefozopran and aztreonam decreased in 2013. On the other hand, two antimicrobial agents showed high susceptibility rates each year; amikacin (94.0-95.6%) showed the highest rate, followed by doripenem (80.3-82.6%). With the exception of amikacin, there were substantial inter-institutional differences in antimicrobial susceptibility. In comparison to the previous CLSI guidelines (M100-S21), the new CLSI guidelines (M100-S22) on the use of carbapenems and penicillins show that the MIC80 has been affected. The MDRP detection rates in 2011, 2012 and 2013 were 1.8% (8 strains), 1.8% (8 strains), and 2.8% (10 strains), respectively. The MBL detection rates were as follows: bla(VIM-2), 0.2% (1 strain) in 2011; bla(IMP-1), 0.9% (4 strains) in 2012, and 1.7% (6 strains, including bla(IMP-1) [3 strains], bla(IMP-2) [2 strains] and bla(VIM-2) [1 strain]) in 2013.

Susceptibility of various oral antibacterial agents against extended spectrum ß-lactamase producing Escherichia coli and Klebsiella pneumoniae.

With the increase in extended spectrum ß-lactamase (ESBL)-producing bacteria in the community, cases are often seen in which treatment of infectious diseases with oral antimicrobial agents is difficult. Therefore, we measured the antimicrobial activities of 14 currently available oral antimicrobial agents against ESBL-producing Escherichia coli and Klebsiella pneumoniae. Based on the standard of the Clinical and Laboratory Standards Institute (CLSI), E. coli showed high susceptibility rates of 99.4% to faropenem (FRPM). In terms of fluoroquinolones, the susceptibility rate of E. coli to levofloxacin (LVFX) was low at 32.2%, whereas it showed a good susceptibility rate of 93.1% to sitafloxacin (STFX). With respect to other antimicrobial agents, susceptibility rates to fosfomycin (FOM) and colistin (CL) were more than 90% each, whereas rates of the two antimicrobial agents expected as therapeutic agents, minocycline (MINO) and sulfamethoxazole-trimethoprim (ST), were low at 62.4% and 44.3%, respectively. Based on the CLSI standard, K. pneumoniae showed high susceptibility rates to ceftibuten (CETB) (91.89%), LVFX (86.49%), and STFX (94.6%), indicating that K. pneumoniae showed higher rates than those of E. coli, particularly to fluoroquinolones. Comparison of susceptibility rates according to E. coli genotype showed that many antimicrobial agents existed to which the CTX-M-9 group showed high susceptibility rates. However, there were many agents to which the CTX-M-1 group showed low susceptibility rates, particularly to CETB (51.1%) and LVFX (17.0%). Although there was no significant difference by genotype between FRPM, STFX, and FOM, a significant difference was observed between LVFX, MINO, and ST. Antibiotic-resistant bacteria with highly pathogenic strains have spread in the community, appropriate use of oral antimicrobial agents is required.

The Drosophila female aphrodisiac pheromone activates ppk23(+) sensory neurons to elicit male courtship behavior.

Females of many animal species emit chemical signals that attract and arouse males for mating. For example, the major aphrodisiac pheromone of Drosophila melanogaster females, 7,11-heptacosadiene (7,11-HD), is a potent inducer of male-specific courtship and copulatory behaviors. Here, we demonstrate that a set of gustatory sensory neurons on the male foreleg, defined by expression of the ppk23 marker, respond to 7,11-HD. Activity of these neurons is required for males to robustly court females or to court males perfumed with 7,11-HD. Artificial activation of these ppk23(+) neurons stimulates male-male courtship even without 7,11-HD perfuming. These data identify the ppk23(+) sensory neurons as the primary targets for female sex pheromones in Drosophila.

Epidemiology of Escherichia coli, Klebsiella species, and Proteus mirabilis strains producing extended-spectrum ß-lactamases from clinical samples in the Kinki Region of Japan.

In the present study, nonduplicate, clinical isolates of extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli, Klebsiella spp, and Proteus mirabilis were collected during a 10-year period from 2000 to 2009 at several hospitals in the Kinki region, Japan. The detection rate of E coli markedly increased from 0.24% to 7.25%. The detection rate of Klebsiella pneumoniae increased from 0% to 2.44% and that of P mirabilis from 6.97% to 12.85%. The most frequently detected genotypes were the CTX-M9 group for E coli, the CTX-M2 group for K pneumoniae, and the CTX-M2 group for P mirabilis. E coli clone O25:H4-ST131 producing CTX-M-15, which is spreading worldwide, was first detected in 2007. The most common replicon type of E coli was the IncF type, particularly FIB, detected in 466 strains (69.7%). Of the K pneumoniae strains, 47 (55.3%) were of the IncN type; 77 P mirabilis strains (96.3%) were of the IncT type. In the future, the surveillance of various resistant bacteria, mainly ESBL-producing Enterobacteriaceae, should be expanded to prevent their spread.

[Clinical microbiological investigation of vancomycin intermediate Staphylococcus aureus during glycopeptide therapy].

We isolated three strains of vancomycin intermediate Staphylococcus aureus (VISA) from a blood sample of a patient with infective endocarditis (VISA-1), postoperative pneumonia sputum (VISA-2), and pyogenic spondylitis blood sample (VISA-3). These VISA strains did not carry vanA, vanB, vanC1, or vanC2/C3 genes. Cell wall thickening was observed. VISA-1 and VISA-3 PFGE patterns showed the completely same pattern compared to the PFGE pattern of methicillin-resistant Staphylococcus aureus first isolated from patients 1 and 3. After 10 days on brain heart infusion agar, wall thickening in all three type of VISA was unchanged, but VISA-2 and VISA-3 reversed vancomycin susceptibility. The most suitable use of vancomycin in patients with MRSA infection thus appears to be in reducing the opportunity for cell wall thickening.