Amino acid metabolism and MAP kinase signaling pathway play opposite roles in the regulation of ethanol production during fermentation of sugarcane molasses in budding yeast.

Sugarcane molasses is one of the main raw materials for bioethanol production, and Saccharomyces cerevisiae is the major biofuel-producing organism. In this study, a batch fermentation model has been used to examine ethanol titers of deletion mutants for all yeast nonessential genes in this yeast genome. A total of 42 genes are identified to be involved in ethanol production during fermentation of sugarcane molasses. Deletion mutants of seventeen genes show increased ethanol titers, while deletion mutants for twenty-five genes exhibit reduced ethanol titers. Two MAP kinases Hog1 and Kss1 controlling the high osmolarity and glycerol (HOG) signaling and the filamentous growth, respectively, are negatively involved in the regulation of ethanol production. In addition, twelve genes involved in amino acid metabolism are crucial for ethanol production during fermentation. Our findings provide novel targets and strategies for genetically engineering industrial yeast strains to improve ethanol titer during fermentation of sugarcane molasses.

Polymer Microspheres Copolymerized with Deep Red Fluorescent Molecules as a Label for Lateral Flow Immunochromatography.

The development of fluorescently labeled microspheres is a critical aspect of advancing the technology of lateral flow immunochromatography (LFIA) for biological detection. Nevertheless, potential interference posed by the background fluorescence originating from the nitrocellulose (NC) membrane would significantly impact the sensitivity and accuracy of microsphere-based detection in LFIA. In this work, an attempt was made to extend the π-conjugated system and asymmetric structure of rhodamine fluorophore, resulting in the synthesis of dye molecules (RB2) incorporating double bonds, which can reach an absolute photoluminescence quantum yield (PLQY) of 30.01% in EtOH. Subsequently, carboxyl group functionalized fluorescent microspheres were prepared in a two-step copolymerization via soap-free emulsion polymerization. The obtained microspheres were characterized by scanning electron microscopy, transmission electron microscopy, DLS, Fourier transform infrared spectroscopy, ultraviolet spectrophotometry, and fluorescence spectrophotometry. The results showed that RB2 was successfully copolymerized into the microspheres, and the resulting microspheres had good dispersion and stability with high red fluorescence intensity (λabs ∼ 610 nm, λem ∼ 660 nm). Utilizing these microspheres, the resulting lateral flow immunoassay was successfully found to detect SARS-CoV-2 N protein with a detection limit of 2.5 pg/mL and the linear concentration spanning from 2.5 pg/mL to 10 ng/mL. The results confirm the effectiveness of the synthetic fluorescent microspheres as the label for LFIA.

Clinical evaluation of metagenomic next-generation sequencing in unbiased pathogen diagnosis of urinary tract infection.

BACKGROUND: Early availability of pathogen identification in urinary tract infections (UTIs) has critical importance in disease management. Metagenomic next-generation sequencing (mNGS) has the potential to transform how acute and serious infections are diagnosed by offering unbiased and culture-free pathogen detection. However, clinical experience with application of the mNGS test is relatively limited. METHODS: We therefore established a MinION-based mNGS pathogens diagnostic platform and evaluated its potential for clinical implementation in UTIs with clinical samples. 213 urine samples from patients with suspected UTIs were included and subjected to mNGS testing using the MinION platform. mNGS results were compared to the gold standard of clinical culture and composite standard of combining clinical testing, confirmatory qPCR testing, and clinical adjudication by doctors. RESULTS: The mNGS exhibited a sensitivity of 81.4% and a specificity of 92.3%, along with a positive predictive value of 96.6%, a negative predictive value of 64.9%, and an overall accuracy of 84.4%, all of which were determined based on the gold standard of routine culture results. When assessed against the composite standard, the sensitivity and specificity both increased to 89.9% and 100%, respectively, while the accuracy rose to 92.4%. Notably, the positive predictive value and negative predictive value also saw improvements, reaching 100% and 76.8%, respectively. Moreover, this diagnostic platform successfully identified dsDNA viruses. Among the 65 culture-negative samples, the viral detection rate reached 33.8% (22/65) and was subsequently validated through qPCR. Furthermore, the automatic bioinformatics pipeline we developed enabled one-click analysis from data to results, leading to a significant reduction in diagnosis time. CONCLUSION: These results demonstrate that the pathogen detection performance of mNGS is sufficient for diagnostic testing in clinical settings. As the method is generally unbiased, it can improve diagnostic testing of UTIs and other microbial infections.

Targeting hnRNPC suppresses thyroid follicular epithelial cell apoptosis and necroptosis through m6A-modified ATF4 in autoimmune thyroid disease.

Both environmental and genetic factors contribute to the etiology of autoimmune thyroid disease (AITD) including Graves' disease (GD) and Hashimoto's thyroiditis (HT). However, the exact pathogenesis and interactions that occur between environmental factors and genes remain unclear, and therapeutic targets require further investigation due to limited therapeutic options. To solve such problems, this study utilized single-cell transcriptome, whole transcriptome, full-length transcriptome (Oxford nanopore technology), and metabolome sequencing to examine thyroid lesion tissues from 2 HT patients and 2 GD patients as well as healthy thyroid tissue from 1 control subject. HT patients had increased ATF4-positive thyroid follicular epithelial (ThyFoEp) cells, which significantly increased endoplasmic reticulum stress. The enhanced sustained stress resulted in cell death mainly including apoptosis and necroptosis. The ATF4-based global gene regulatory network and experimental validation revealed that N6-methyladenosine (m6A) reader hnRNPC promoted the transcriptional activity, synthesis, and translation of ATF4 through mediating m6A modification of ATF4. Increased ATF4 expression initiated endoplasmic reticulum stress signaling, which when sustained, caused apoptosis and necroptosis in ThyFoEp cells, and mediated HT development. Targeting hnRNPC and ATF4 notably decreased ThyFoEp cell death, thus ameliorating disease progression. Collectively, this study reveals the mechanisms by which microenvironmental cells in HT and GD patients trigger and amplify the thyroid autoimmune cascade response. Furthermore, we identify new therapeutic targets for the treatment of autoimmune thyroid disease, hoping to provide a potential way for targeted therapy.

The Double-Edged Impact of the COVID-19 Pandemic on Chinese Family Relationships.

To comprehensively understand the Chinese family relationships (i.e., marital relationship, parent-child relationship, sibling relationship, and grandparent-grandchild relationship) during the COVID-19 pandemic, this study investigated the changes of family relationships and the individual differences related to knowledge of the COVID-19, personality traits (i.e., neuroticism and optimism), and emotional characteristics (i.e., emotion regulation and negative emotional reactions). From March 1 to April 5, 2020, 8821 participants were involved, including 3995 teenagers, 1146 unmarried young adults, 3571 married adults, and 109 grandparents. Results revealed a double-edged pattern that people experienced both positive changes and negative changes during the pandemic. Teenagers reported significant negative changes in the relationships with their parents. Peoples' knowledge of the COVID-19, neuroticism, optimism, emotion regulation, and negative emotional reactions were in varying extents to which accounted for the individual differences in the changes of family relationships. These findings help recognize the overall Chinese family relationships during the hard period.

Caregivers' joint depressive symptoms and preschoolers' daily routines in Chinese three-generation families: Does household chaos matter?

The benefits of routines for children have been consistently demonstrated in previous literature. However, factors that may confer risks for child routines have seldom been examined, particularly in families where parents and grandparents co-care the children. This study aimed to investigate the associations of parents' and grandparents' depressive symptoms with preschoolers' daily routines in Chinese three-generation families and to determine whether household chaos mediated or moderated the associations. The participants were from 171 urban three-generation families where mothers, fathers, and grandmothers (97 paternal and 74 maternal) were primary caregivers. Mothers, fathers, and grandmothers reported their depressive symptoms at Wave 1; at Wave 2 (during the COVID-19 pandemic), caregivers reported household chaos and child routines. The results revealed that child routines were negatively predicted by parents' joint depressive symptoms rather than grandmothers' depressive symptoms. In the associations, household chaos acted as a mediator rather than a moderator. Specifically, household chaos marginally mediated the associations between parents' and grandmothers' depressive symptoms and child routines only in maternal three-generation families. These findings suggest that in three-generation families, caregivers with more depressive symptoms may elicit more chaotic family environments, which may in turn compromise their children's daily routines.

Dictamnine Inhibits the Adhesion to and Invasion of Uropathogenic Escherichia Coli (UPEC) to Urothelial Cells.

Uropathogenic Escherichia coli (UPEC) is the most common pathogenic bacteria associated with urinary tract infection (UTI). UPEC can cause UTI by adhering to and invading uroepithelial cells. Fimbriae is the most important virulence factor of UPEC, and a potentially promising target in developing novel antibacterial treatments. In this study, the antibacterial properties and effects of the compound dictamnine, extracted from the traditional Chinese medicine Cortex Dictamni, on the bacterial morphology, cell adhesion, and invasion of UPEC were studied. Dictamnine exhibited no obvious antibacterial activity against UPEC, but significantly impeded the ability of UPEC to adhere to and invade uroepithelial cells. RT-qPCR analysis showed that treatment downregulated the expression of type 1 fimbriae, P fimbriae, and curli fimbriae adhesion genes, and also downregulated adhesion-related receptor genes of uroepithelial cells. Transmission electron microscopy showed that dictamnine destroyed the structure of the fimbriae and the surface of the bacteria became smooth. These results suggest that dictamnine may help to prevent UTI by simultaneously targeting UPEC fimbriae and urothelial adhesin receptors, and may have a potential use as a new anti-UPEC drug.

Annexin A1 Attenuates Neutrophil Migration and IL-6 Expression through Fpr2 in a Mouse Model of Streptococcus suis-Induced Meningitis.

Streptococcus suis serotype 2 is a crucial pathogenic cause of bacterial meningitis, a life-threatening disease with neurological sequelae and high rates of mortality. Inflammation triggered by S. suis infection must be precisely regulated to prevent further tissue damage. As a glucocorticoid anti-inflammatory mediator, annexin A1 (AnxA1) mainly acts through formyl peptide receptor 2 (Fpr2) to alleviate inflammation in the peripheral system. In this study, we evaluated the roles of AnxA1 and Fpr2 in a mouse model of S. suis meningitis created via intracisternal infection in Fpr2-deficient (Fpr2-/-) and wild-type (WT) mice. We revealed that Fpr2-/- mice were highly susceptible to S. suis meningitis, displaying increased inflammatory cytokine levels, bacterial dissemination, and neutrophil migration compared with WT mice. Additionally, AnxA1 exerted anti-inflammatory effects through Fpr2, such as attenuation of leukocyte infiltration, inflammatory mediator production, and astrocyte or microglial activation in the brain. Importantly, we found that the antimigratory function of AnxA1 decreases neutrophil adherence to the endothelium through Fpr2. Finally, an in vitro study revealed that AnxA1 potentially suppresses interleukin-6 (IL-6) expression through the Fpr2/p38/COX-2 pathway. These data demonstrated that Fpr2 is an anti-inflammatory receptor that regulates neutrophil migration in mice with S. suis meningitis and identified AnxA1 as a potential therapeutic option.

Metagenomic diagnosis of severe psittacosis using multiple sequencing platforms.

BACKGROUND: Chlamydia psittaci is an avian pathogen that can cause lethal human infections. Diagnosis of C. psittaci pneumonia is often delayed due to nonspecific clinical presentations and limited laboratory diagnostic techniques. RESULTS: The MinION platform established the diagnosis in the shortest time, while BGISEQ-500 generated additional in-depth sequence data that included the rapid characterization of antibiotic susceptibility. Cytopathy appeared only in cell cultures of BALF. BALF yielded a higher bacterial load than sputum or blood, and may be the most suitable clinical specimen for the genomic diagnosis of severe pneumonia. CONCLUSIONS: This study indicated that the benefits of metagenomic sequencing include rapid etiologic diagnosis of unknown infections and the provision of additional relevant information regarding antibiotic susceptibility. The continued optimization and standardization of sampling and metagenomic analysis promise to enhance the clinical utility of genomic diagnosis.

Selection of an artificial paraquat-specific binding protein from a ribosome display library based on a lipocalin scaffold.

In this study, an anticalin that could specifically bind paraquat (PQ), a quaternary nitrogen herbicide, as a new set of engineered receptor protein with antibody-like properties was generated to detect PQ concentration. To this end, a native and random library was constructed and engineered to allow in vitro transcription and translation using an Escherichia coli lysate system. Meanwhile, a PQ derivative that carries an active aliphatic carboxylate group at the end of an aliphatic spacer arm was synthesized. Then, this compound was coupled covalently to the carrier protein bovine serum albumin/ovalbumin and amino-functionalized paramagnetic beads. Alternating selection in solution and immobilization in microtiter wells were used to pan mRNA-ribosome-antibody complexes. After several rounds of ribosome display, three variants were selected from a random library of the bilin-binding protein. The variants that could bind complex PQ with high affinity and exhibit IC50 values as low as 14.039 ± 0.970 ng/mL were identified. Moreover, the limits of detection reached 0.083 ± 0.011 ng/mL. Our data suggest that the generation of anticalins may provide a promising alternative to recombinant antibody fragments to create a stable receptor protein against hapten with bioanalytical relevance.

TRIM32 Drives Pathogenesis in Streptococcal Toxic Shock-Like Syndrome and Streptococcus suis Meningitis by Regulating Innate Immune Responses.

Streptococcus suis is an emerging zoonotic agent that causes streptococcal toxic shock-like syndrome (STSLS) and meningitis in humans, with high mortality and morbidity. The pathogenesis of both STSLS and central nervous system (CNS) infections caused by S. suis is not well understood. TRIM32, a member of the tripartite motif (TRIM) protein family, has been reported to regulate host inflammatory responses. In this study, we showed that TRIM32 deficiency significantly reduced the level of bacteremia and the production of proinflammatory cytokines following severe S. suis infection, protecting infected mice from STSLS. The influence of TRIM32 gene deletion on a range of processes known to be involved in S. suis meningitis was also examined. Both levels of bacterial loads and indications of brain hemorrhage were reduced in infected Trim32-/- mice compared with infected wild-type (WT) controls. We also found that TRIM32 deficiency increased the permeability of the blood-brain barrier (BBB) and the recruitment of inflammatory monocytes during the early course of S. suis infection, potentially limiting the development of S. suis meningitis. Our results suggest that TRIM32 sensitizes S. suis-induced infection via innate immune response regulation.

Next-generation sequencing technology as a powerful detection and semi-quantitative method for herpes simplex virus type 1 in pediatric encephalitis.

This case report presents a 1-year-old boy from China, with sudden onset of fever, convulsion, and sleepiness, screened for viral DNA in blood and cerebrospinal fluid (CSF) sample using next-generation sequencing (NGS) to diagnose herpes simplex virus type 1 (HSV-1) encephalitis, further validated by PCR. After acyclovir treatment, the patient's symptom disappeared and HSV-1 DNA unique reads decreased from 4290 to zero in CSF, and from 23 to zero in blood detected by NGS. The clinical presentation and outcome were consistent with the pathogenic diagnostic results of NGS. NGS of CSF samples can be used as a diagnostic assay for HSV-1 encephalitis and also might be a semi-quantitative method for evaluation of treatment effect.

Observation of spatial self-phase modulation induced via two competing mechanisms.

Two spatial self-phase modulation (SSPM) patterns were observed in suspensions of Bi2TeSe2 nanosheets. Two mechanisms were found to produce SSPM with different occurrence times and power dependence. The Type I (narrow) rings are attributed to the coherent third-order nonlinear optical Kerr effect, which induces self-diffraction in 2D materials, and the Type II (wide) rings are assigned to the contribution of a thermal effect. The nonlinear refractive index of Bi2TeSe2 is found to be 2.30×10-5cm2W-1 at 700 nm. The findings described here provide an explanation for the formation of rings in 2D systems due to SSPM.

Evaluation of TaqMan Array card (TAC) for the detection of 28 respiratory pathogens.

BACKGROUND: Respiratory infections are a serious threat to human health. So, rapid detection of all respiratory pathogens can facilitate prompt treatment and prevent the deterioration of respiratory disease. Previously published primers and probes of the TaqMan array card (TAC) for respiratory pathogens are not sensitive to Chinese clinical specimens. This study aimed to develop and improve the TAC assay to detect 28 respiratory viral and bacterial pathogens in a Chinese population. METHODS: To improve the sensitivity, we redesigned the primers and probes, and labeled the probes with minor groove binders. The amplification efficiency, sensitivity, and specificity of the primers and probes were determined using target-gene containing standard plasmids. The detection performance of the TAC was evaluated on 754 clinical specimens and the results were compared with those from conventional methods. RESULTS: The performance of the TAC assay was evaluated using 754 clinical throat swab samples and the results were compared with those from gold-standard methods. The sensitivity and specificity were 95.4 and 96.6%, respectively. The lowest detection limit of the TAC was 10 to 100 copies/µL. CONCLUSIONS: TAC is an efficient, accurate, and high-throughput approach to detecting multiple respiratory pathogens simultaneously and is a promising tool for the identification of pathogen outbreaks.

Self-compassion and family cohesion moderate the association between suicide ideation and suicide attempts in Chinese adolescents.

BACKGROUND: In recent years, many studies have examined risks factors that facilitated the transition from suicide ideation to suicide attempts. Few studies, however, have examined protective factors against this transition. The current study thus assessed two protective factors, self-compassion and family cohesion, in buffering the transition from suicide ideation to suicide attempts. METHOD: A number of 520 Chinese adolescents (43.46% females, mean age = 12.96 years) completed questionnaires assessing self-compassion, family cohesion, suicide ideation, and suicide attempts two times with a 12-month interval. RESULTS: Self-compassion significantly moderated the association between Wave 1 SI and later SA. The positive dimension of self-compassion thwarted the transition from SI and SA, and the negative dimension of self-compassion strengthened the associations. In addition, family cohesion also significantly moderated the transition from SI to SA. CONCLUSION: Increasing the levels of self-compassion and family cohesion may be the targets for treating adolescents with suicide ideation to prevent them from attempting suicide.

Parenting Stress and Depressive Symptoms Among Chinese Parents of Children With and Without Oppositional Defiant Disorder: A Three-Wave Longitudinal Study.

Parents of children with oppositional defiant disorder (ODD) experience greater stress in parenting and more parental depressive symptoms. The study examined the longitudinal and bidirectional associations between three dimensions of parenting stress (i.e., parental distress, parent-child dysfunctional interaction, and difficult child) and parental depressive symptoms from a sample of Chinese parents of children with or without ODD. The sample included 256 parents of children with ODD and 265 parents of children without ODD, along with children's teachers. Using a three wave, cross-lagged design, results showed that parents of children with ODD suffered higher levels of parenting stress across three dimensions. For both groups, the links between parental depressive symptoms and subsequent parental distress and difficult child were unidirectional, whereas the relation between parental depressive symptoms and parent-child dysfunctional interaction was bidirectional. Multi-group analysis found that there was no significant difference in the relations between parenting stress and depressive symptoms between the ODD and non-ODD groups. The findings indicated that children with ODD require comprehensive services to address the stress of their parents. The study also provided support for the dynamic and longitudinal relations between specific dimensions of parenting stress and depressive symptoms among parents of children with or without ODD.

Actor-Partner Interdependence of Compassion toward Others with Qualities of Marital Relationship and Parent-Child Relationships in Chinese Families.

Considering the nature of compassion and cultural and times characteristics of Chinese families, individuals' general dispositional compassion toward others may have potential benefits for relationship qualities in Chinese families. In this study, we explored how men's and women's compassion related to their own (actor effects) and the partners' (partner effects) marital relationship and parent-child relationships, respectively, using the Actor-Partner Interdependent Model. Participants were from a cross-sectional sample of 534 Chinese heterosexual married couples (females' mean age = 37.20, SD = 4.28; males' mean age = 40.29, SD = 5.39) whose biological children were pupils (mean age = 9.28, SD = 1.11). Results revealed that: (a) within marital relationships, couple members' actor effects and partner effects were found both significant; but (b) within parent-child relationships, couple members' actor effects were significant, whereas only mothers' partner effect of compassion on father-child relationship was significant. These findings support the benefits of couple members' individual compassion with respect to family relationship quality in contemporary China. Limitations, implications, and future directions are discussed.

Teniendo en cuenta la índole de la compasión y de las características culturales y temporales de las familias chinas, la compasión disposicional general de una persona hacia las demás puede tener posibles beneficios para la calidad de la relación en las familias chinas. En este estudio, analizamos cómo la compasión de los hombres y de las mujeres está relacionada con la relación conyugal y las relaciones entre los padres y los hijos, tanto propias (efectos del actor) como de la pareja (efectos de la pareja), respectivamente, utilizando el modelo de interdependencia actor-pareja. Los participantes fueron de una muestra transversal de 534 parejas chinas heterosexuales casadas (edad promedio de las mujeres = 37.20, Desviación Típica = 4.28; edad promedio de los hombres = 40.29, Desviación Típica = 5.39) cuyos hijos biológicos eran pupilos (edad promedio = 9.28, Desviación Típica = 1.11). Los resultados revelaron que: (a) dentro de las relaciones conyugales, se descubrió que los efectos del actor y los efectos de la pareja de los integrantes de la pareja fueron ambos significativos; pero (b) dentro de las relaciones entre padres e hijos, los efectos del actor de los integrantes de la pareja fueron significativos, mientras que solo el efecto de la pareja relativo a la compasión de las madres por la relación padre e hijo fue significativo. Estos resultados respaldan los beneficios de la compasión individual de los integrantes de la pareja con respecto a la calidad de la relación familiar en la China contemporánea. Se explican las limitaciones, las implicancias y las futuras direcciones.

The Associations between Mothers' and Grandmothers' Depressive Symptoms, Parenting Stress, and Relationship with Children: An Actor-Partner Interdependence Mediation Model.

Three-generation households that include parents and grandparents raising children together have become increasingly common in China. This study examined the relations among depressive symptoms, parenting stress, and caregiver-child relationships in the mother-grandmother dyadic context. Participants were mothers and grandmothers from 136 three-generation households. Results from Actor-Partner Interdependence Mediation Modeling indicated that mothers' depressive symptoms were indirectly related to mother-child conflict/closeness through own parenting stress; grandmothers' depressive symptoms were indirectly related to grandmother-child conflict through own parenting stress. Mothers' depressive symptoms were indirectly related to grandmothers' conflict with children through grandmothers' parenting stress, and grandmothers' depressive symptoms were indirectly related to mothers' conflict/closeness with children through mothers' parenting stress. The relation between mothers' parenting stress and mother-child closeness was stronger than the relation between grandmothers' parenting stress and grandmother-child closeness. Findings highlight the implications of using a family system perspective and the dyadic approach in understanding and improving family functioning in Chinese three-generation households.

Los hogares de tres generaciones integrados por padres y abuelos que crían a niños juntos son cada vez más comunes en China. Este estudio examinó las relaciones entre los síntomas depresivos, el estrés por la crianza y las relaciones entre cuidadores y niños en el contexto diádico madre-abuela. Las participantes fueron madres y abuelas de 136 hogares de tres generaciones. Los resultados del modelo de mediación de la interdependencia actor-pareja indicaron que los síntomas depresivos de las madres estuvieron relacionados indirectamente con el conflicto/la cercanía entre madre e hijo mediante el propio estrés por la crianza; los síntomas depresivos de las abuelas estuvieron indirectamente relacionados con el conflicto entre la abuela y el niño mediante el propio estrés por la crianza. Los síntomas depresivos de las madres estuvieron indirectamente relacionados con el conflicto de las abuelas con los niños mediante el estrés por la crianza de las abuelas, y los síntomas depresivos de las abuelas estuvieron indirectamente relacionados con el conflicto/la cercanía de las madres con los niños mediante el estrés por la crianza de las madres. La relación entre el estrés por la crianza de las madres y la cercanía entre madre e hijo fue más fuerte que la relación entre el estrés por la crianza de las abuelas y la cercanía entre abuela y niño. Los resultados destacan las implicancias de usar una perspectiva de sistema familiar y el enfoque diádico a la hora de comprender y mejorar el funcionamiento familiar en los hogares chinos de tres generaciones.

Development of a TaqMan Array card to target 21 purulent meningitis-related pathogens.

BACKGROUND: Purulent meningitis (PM) is a serious life-threatening infection of the central nervous system (CNS) by bacteria or fungi and associated with high mortality and high incidence of CNS sequelae in children. However, the conventional cerebrospinal fluid (CSF) culture method is time-consuming and has a low sensitivity. METHODS: Our study developed a real-time PCR-based purulent meningitis-TaqMan array card (PM-TAC) that targeted 21 PM-related pathogens and could produce results within 3 h. Primers and probes were adapted from published sources possibly. The performance of them were evaluated and optimized and then they were spotted on TAC. RESULTS: The PM-TAC showed a sensitivity and specificity of 95 and 96%, respectively. For all of the 21 targeted pathogens, the PM-TAC assay had a LOD ranging from 5 copies/reaction to 100 copies/reaction, an intra-assay variation of 0.07-4.45%, and an inter-assay variation of 0.11-6.81%. Of the 15 CSF samples collected from patients with PM after empiric antibiotic therapies, the positive rate was 53.3% (8/15) for our PM-TAC assay but was only 13.3% (2/15) for the CSF culture method. Of the 17 CSF samples showing negative CSF culture, the PM-TAC assay identified a case of Neisseria meningitidis infection. Furthermore, all of the 10 CSF samples from patients without CNS infection showed negative for the PM-TAC assay. CONCLUSIONS: Our PM-TAC assay also demonstrated that the pathogen loads in the CSF samples correlated with the severity of PM. Thus, the PM-TAC may be helpful to improve the prognosis of PM and clinical outcomes from antibiotic therapies.

The diagnostic value of metagenomic next-generation sequencing for identifying Streptococcus pneumoniae in paediatric bacterial meningitis.

BACKGROUND: There is currently no research on the diagnostic value of metagenomic next-generation sequencing (mNGS) for a single pathogens in CSF. The aim of this study was to analyse the value of mNGS for identifying Streptococcus pneumoniae (S. pneumoniae) in paediatric bacterial meningitis. METHODS: Bacterial meningitis (BM) cases from October 23, 2014, to December 31, 2016, and December 1, 2017, to July 31, 2018 at Beijing Children's Hospital were reviewed. Clinical features and pathogens were analysed. RESULTS: We diagnosed 135 patients with BM in this study. A total of 43 S. pneumoniae were identified by combination methods. 26/135 (19.3%) patients had positive results in S. pneumoniae by blood and/or cerebrospinal fluid (CSF) culture. Alere BinaxNow®Streptococcus pneumoniae Antigen test was positive in 35/135(25.9%) cases. 32/135 (23.7%) S. pneumoniae were identified by mNGS. Six CSF samples were identified as S. pneumoniae only by mNGS technology. Taking culture as the gold standard, the sensitivity and specificity of mNGS for diagnosing S. pneumoniae meningitis were 73.1 and 88.1%, respectively. The positive predictive value (PPV) and negative predictive value (NPV) of diagnosing S. pneumoniae meningitis by mNGS were 59.4 and 93.2%, respectively. When comparison between mNGS and combined tests (culture and Alere BinaxNow®Streptococcus pneumoniae Antigen test), the sensitivity and specificity of mNGS for S. pneumoniae identification were 70.3 and 93.9%, the PPV and NPV in the identification of S. pneumoniae by mNGS were 81.4 and 89.3%, respectively. The difference in number of unique reads of S. pneumoniaein from CSF sample (< 14 days onset) and CSF sample (> 14 days from onset) was statistically significant (170.5 VS. 13, P = 0.019). The difference in the collected time of CSF for culture and mNGS was statistically significant (4 days VS. 14 days, P < 0.001). CONCLUSIONS: mNGS has high sensitivity and specificity for S. pneumoniae identification. The pathogen load (number of unique reads) of S. pneumonia is related to the CSF collection time. mNGS was less affected than culture by the use of antibiotics before CSF collection.