Causative Pathogen Research Articles

Comparative Evaluation of PCR-Based, LAMP and RPA-CRISPR/Cas12a Assays for the Rapid Detection of Diaporthe aspalathi.

Southern stem canker (SSC) of soybean, attributable to the fungal pathogen Diaporthe aspalathi, results in considerable losses of soybean in the field and has damaged production in several of the main soybean-producing countries worldwide. Early and precise identification of the causal pathogen is imperative for effective disease management. In this study, we performed an RPA-CRISPR/Cas12a, as well as LAMP, PCR and real-time PCR assays to verify and compare their sensitivity, specificity and simplicity and the practicality of the reactions. We screened crRNAs targeting a specific single-copy gene, and optimized the reagent concentrations, incubation temperatures and times for the conventional PCR, real-time PCR, LAMP, RPA and Cas12a cleavage stages for the detection of D. aspalathi. In comparison with the PCR-based assays, two thermostatic detection technologies, LAMP and RPA-CRISPR/Cas12a, led to higher specificity and sensitivity. The sensitivity of the LAMP assay could reach 0.01 ng μL-1 genomic DNA, and was 10 times more sensitive than real-time PCR (0.1 ng μL-1) and 100 times more sensitive than conventional PCR assay (1.0 ng μL-1); the reaction was completed within 1 h. The sensitivity of the RPA-CRISPR/Cas12a assay reached 0.1 ng μL-1 genomic DNA, and was 10 times more sensitive than conventional PCR (1.0 ng μL-1), with a 30 min reaction time. Furthermore, the feasibility of the two thermostatic methods was validated using infected soybean leaf and seeding samples. The rapid, visual one-pot detection assay developed could be operated by non-expert personnel without specialized equipment. This study provides a valuable diagnostic platform for the on-site detection of SSC or for use in resource-limited areas.

Insect Vector and Reservoir Plant of 'Fragaria × ananassa' Phyllody Phytoplasma (16SrXIII-F) in Central Region of Chile.

Strawberry phyllody has emerged as a prevalent disease affecting Chilean strawberry in recent years. The causal pathogen, 'Fragaria × ananassa' phyllody phytoplasma (StrPh), is categorized within the 16S ribosomal group XIII that is exclusively found in the Americas. In the context of economically significant crops, hemipteran insect vectors and alternative host plants play a pivotal role in their natural dissemination. This study comprehensively examined the key epidemiological facets of StrPh in the central region of Chile: the insect vector and alternative hosts. Through field surveys, we identified an abundance of an insect species, Cixiosoma sp., in an StrPh-infected strawberry field and confirmed its role as a vector of this phytoplasma through subsequent transmission assays. Moreover, we found a spontaneous weed species, Galega officinalis, to be infected with StrPh, raising the possibility of it being a potential alternative host plant for this phytoplasma. StrPh was also detected in cold-stored strawberry runners purchased from a nursery that supplies the local strawberry cultivation, suggesting a potential source of this phytoplasma in Chile. Collectively, these findings provide a significant epidemiological source of StrPh dissemination in central Chile.

Identification and fungicides sensitivity evaluation of the causal agent of cobweb disease on Lyophyllum decastes in China

BackgroundCobweb disease is a fungal disease that commonly affects the cultivation and production of edible mushrooms, leading to serious yield and economic losses. It is considered a major fungal disease in the realm of edible mushrooms. The symptoms of cobweb disease were found during the cultivation of Lyophyllum decastes. This study aimed to identify the causative pathogen of cobweb disease and evaluate effective fungicides, providing valuable insights for field control and management of L. decastes cobweb disease.ResultsThe causal agent of cobweb disease was isolated from samples infected and identified as Cladobotryum mycophilum based on morphological and cultural characteristics, as well as multi-locus phylogeny analysis (ITS, RPB1, RPB2, and TEF1-α). Pathogenicity tests further confirmed C. mycophilum as the responsible pathogen for this condition. Among the selected fungicides, Prochloraz-manganese chloride complex, Trifloxystrobin, tebuconazole, and Difenoconazole exhibited significant inhibitory effects on the pathogen’s mycelium, with EC50 values of 0.076 µg/mL, 0.173 µg/mL, and 0.364 µg/mL, respectively. These fungicides can serve as references for future field control of cobweb disease in L. decastes.ConclusionThis study is the first report of C. mycophilum as the causing agent of cobweb disease in L. decastes in China. Notably, Prochloraz-manganese chloride complex demonstrated the strongest inhibitory efficacy against C. mycophilum.

Antifungal drug penetration in soft tissue abscesses: a comparative analysis.

Invasive fungal infections (IFIs) are severe and difficult-to-treat infections affecting immunocompromised patients. Antifungal drug penetration at the site of infection is critical for outcome and may be difficult to achieve. Data about antifungal drug distribution in infected human tissues under real circumstances of IFI are scarce. Multiple samples were obtained from soft tissue abscesses of a lung transplant patient with Candida albicans invasive candidiasis who underwent recurrent procedures of drainage, while receiving different consecutive courses of antifungal therapy [itraconazole (ITC), fluconazole, caspofungin]. Antifungal drug concentrations were measured simultaneously at the site of infection (surrounding inflammatory tissue and fluid content of the abscess) and in plasma for calculation of the tissue/plasma ratio (R). The concentration within the infected tissue was interpreted as appropriate if it was equal or superior to the MIC of the causal pathogen. A total of 30 tissue samples were collected for measurements of ITC (n = 12), fluconazole (n = 17) and caspofungin (n = 1). Variable concentrations were observed in the surrounding tissue of the lesions with median R of 2.79 (range 0.51-15.9) for ITC and 0.94 (0.21-1.37) for fluconazole. Concentrations ranges within the fluid content of the abscesses were 0.39-1.83 for ITC, 0.66-1.02 for fluconazole and 0.23 (single value) for caspofungin. The pharmacodynamic target (tissue concentration ≥ MIC) was achieved in all samples for all three antifungal drugs. This unique dataset of antifungal drug penetration in infected human soft tissue abscesses suggests that ITC, fluconazole and caspofungin could achieve appropriate concentrations in soft tissue abscesses.

End-organ damage from neonatal invasive fungal infection: a 14-year retrospective study from a tertiary center in China

BackgroundInvasive fungal infection (IFI) has become an increasing problem in NICU neonates, and end-organ damage (EOD) from IFI is one of the leading causes of morbidity and mortality in neonates. This study was conducted to summarize clinical data on epidemiology, risk factors, causative pathogens, and clinical outcomes of IFI-associated EOD among neonates in a center in China for the sake of providing references for prevention and treatment of fungal infections in neonates in future.MethodsThe clinical data of IFI neonates who received treatment in a tertiary NICU of China from January 2009 to December 2022 were retrospectively analyzed, including causative pathogens and the incidence of EOD. The neonates were divided into EOD group and non-EOD (NEOD) group. The general characteristics, risk factors and clinical outcomes of the two groups were compared.ResultsIncluded in this study were 223 IFI neonates (137 male and 86 female) with a median gestational age (GA) of 30.71 (29,35) weeks and a median birth weight (BW) of 1470 (1120,2150) g. Of them, 79.4% were preterm infants and 50.2% were born at a GA of ≥ 28, <32 weeks, and 37.7% with BW of 1000–1499 g. Candida albicans (C. albicans) was the most common Candida spp. in these neonates, accounting for 41.3% of all cases, followed by C. parapsilosis (30.5%) and C. glabrata (7.2%). EOD occurred in 40 (17.9%) of the 223 cases. Fungal meningitis was the most common EOD, accounting for 13.5% of the 40 EOD cases. There was no significant difference in the premature birth rate, delivery mode, GA and BW between EOD and NEOD groups, but the proportion of male infants with EOD was higher than that without. There was no significant difference in antenatal corticosteroid use, endotracheal intubation, invasive procedures, use of antibiotics, total parenteral nutrition, blood transfusion, postnatal corticosteroid use, fungal prophylaxis and the incidence of necrotizing enterocolitis between the two groups, but the proportion of C. albicans infection cases in EOD group was higher than that in NEOD group (57.5% vs. 37.7%). Compared with NEOD group, the proportion of cured or improved infants in EOD group was significantly lower (P < 0.05), and the number of infants who died or withdrew from treatment was larger (P < 0.05).ConclusionsOur retrospective study showed that preterm infants were prone to fungal infection, especially very preterm infants. C. albicans was the most common Candida spp. for IFI, and was a high-risk factor for EOD. EOD can occur in both full-term and premature infants, so the possibility of EOD should be considered in all infants with IFI.

Morphological and pathogenic characterization of Fusarium species causing common bean root rot in Uganda

Fusarium root rot (FRR) of common bean occurs in Africa, Central and South America, and causes yield losses of up to 86%. Recently, FRR-like wilt symptoms were observed in Uganda’s agroecology zones. To identify the causal pathogen, we conducted surveys in seven agroecology zones to determine the prevalence and incidence of the reported disease. During the surveys, diseased roots were collected for pathogen isolation. Fungal strains were characterized using colony color, mycelial growth rate and microscopic structures such as conidia and microconidia. The pathogenicity of 99 strains on five bean varieties was determined in artificially inoculated soils in the screenhouse. Based on field symptoms, the observed wilting was identified to be Fusarium root rot, the prevalence of which varied across agroecologies, with the highest (95%) in the Karamoja pastoral zone (KP) and the lowest (40%) in West Nile farming system (WN). Similarly, the incidence of FRR was highest (87%) in KP, and lowest (20%) in WN. Fusarium strains differed in growth rate, colony color, shape and size of microscopic structures. All evaluated strains were pathogenic on common bean and caused severities of 0.9 to 98.3%. Further studies are required to identify the isolated strains at the species level using molecular tools.

PSMC5 insufficiency and P320R mutation impair proteasome function.

The ubiquitin-proteasome system mediates the degradation of a wide variety of proteins. Proteasome dysfunction is associated with neurodegenerative diseases and neurodevelopmental disorders in humans. Here we identified mutations in PSMC5, an AAA ATPase subunit of the proteasome 19S regulatory particle, in individuals with neurodevelopmental disorders, which were initially considered as variants of unknown significance. We have now found heterozygotes with the following mutations: P320R (6 individuals), R325W, Q160A, and one nonsense mutation at Q69. We focused on understanding the functional consequence of PSMC5 insufficiency and the P320R mutation in cells and found that both impair proteasome function and activate apoptosis. Interestingly, the P320R mutation impairs proteasome function by weakening the association between the 19S regulatory particle and the 20S core particle. Our study supports that proteasome dysfunction is the pathogenic cause of neurodevelopmental disorders in individuals carrying PSMC5 variants.

Comparison of plasma and blood cell samples in metagenomic next-generation sequencing for identification of the causative pathogens of fever

BackgroundMetagenomic next-generation sequencing (mNGS) of plasma DNA has become an attractive diagnostic method for infectious diseases; however, the rate of false-positive results is high. This study aims to evaluate the diagnostic accuracy of mNGS in plasma versus blood cell samples for immunocompromised children with febrile diseases. MethodsThe results of conventional microbiological test (CMT) and mNGS using plasma and blood cells in 106 patients with 128 episodes of febrile diseases from the Department of Hematology/Oncology were analyzed and described. ResultsThe positivity rates for CMT and mNGS of plasma and blood cells were 35.9 %, 84.4 % and 46.9 %, respectively (P < 0.001). Notably, mNGS identified multiple pathogens in a single specimen in 68.5 % of plasma samples and 38.3 % of blood cell samples (P < 0.001). Furthermore, plasma and blood cell mNGS identified causative pathogens in 58 and 46 cases, accounting for 53.7 % and 76.7 % of the mNGS-positive cases for each sample type, respectively (P = 0.002). By integrating results from both plasma and blood cell samples, causative pathogens were identified in 77 cases (60.2 %), enhancing sensitivity to 87.5 % but reducing specificity to 15.0 %, compared to plasma (65.9 % sensitivity and 20.0 % specificity) and blood cell samples (52.3 % sensitivity and 80.0 % specificity). ConclusionsmNGS of plasma is sensitive but has a high false-positive rate, while mNGS of blood cells has low sensitivity but higher specificity.

A rapid simultaneous antigen detection of Haemophilus influenzae and Streptococcus pneumoniae for predicting the prognosis of acute otitis media

BackgroundRapid identification of causative bacteria in treatment of acute otitis media (AOM) is of paramount importance for appropriate antibiotic use. Materials and methodsThis prospective observational study was conducted in 15 hospitals and clinics in Japan between 2018 and 2020. A new rapid antigen test kit (AOS-116), which simultaneously detects antigens for Streptococcus pneumoniae (Sp) and Haemophilus influenzae (Hi), was applied for middle ear fluids (MEFs) and nasopharyngeal secretions (NPSs) in patients with moderate to severe AOM. We investigated relationship between the results of rapid test, severity at initial visit, and clinical course. ResultsRegarding performance accuracy based on culture results, AOS-116 showed 1) high (>80%) sensitivity, specificity, and negative predictive value (NPV) in MEFs for both antigens, 2) high sensitivity, specificity, and positive predictive value (PPV) in NPSs for Hi antigen, and 3) high specificity, and PPV in NPSs for Sp antigen. Regarding predictive value of nasopharyngeal culture and antigen detection for causative middle ear pathogens, similar results were observed between AOS-116 and culture, which was characterized with high sensitivity and NPV for both pathogens. MEFs/NPSs positive for Hi antigen were significantly associated with eardrum findings, and severity. MEFs/NPSs positive for pneumococcal antigen were significantly associated with severity of otalgia, fever, and otorrhea. Among patients with prior antimicrobial treatment, improvement tended to be slower in cases positive for Hi than in cases negative. ConclusionThe rapid antigen detection test is useful as a decision-making tool for prescribing antimicrobial agents and may play an important role in promoting appropriate antimicrobial use.

Identification of circRNA Expression Profile and Potential Systemic Immune Imbalance Modulation in Premature Rupture of Membranes.

Premature rupture of membrane (PROM) refers to the rupture of membranes before the onset of labor which increases the risk of perinatal morbidity and mortality. Recently, circular RNAs (circRNAs) have emerged as promising regulators of diverse diseases. However, the circRNA expression profiles and potential circRNA-miRNA-mRNA regulatory mechanisms in PROM remain enigmatic. In this study, we displayed the expression profiles of circRNAs and mRNAs in plasma and fetal membranes of PROM and normal control (NC) groups based on circRNA microarray, the Gene Expression Omnibus database, and NCBI's Sequence Read Archive. A total of 1,459 differentially expressed circRNAs (DECs) in PROM were identified, with 406 upregulated and 1,053 downregulated. Then, we constructed the circRNA-miRNA-mRNA network in PROM, encompassing 22 circRNA-miRNA pairs and 128 miRNA-mRNA pairs. Based on the analysis of gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene set enrichment analysis (GSEA), DECs were implicated in immune-related pathways, with certain alterations persisting even postpartum. Notably, 11 host genes shared by DECs of fetal membrane tissue and prenatal plasma in PROM were significantly implicated in inflammatory processes and extracellular matrix regulation. Our results suggest that structurally stable circRNAs may predispose to PROM by mediating systemic immune imbalances, including peripheral leukocyte disorganization, local immune imbalance at the maternal-fetal interface, and local collagen disruption. This is the first time to decipher a landscape on circRNAs of PROM, reveals the pathogenic cause of PROM from the perspective of circRNA, and opens up a new direction for the diagnosis and treatment of PROM.

“Why Should I Be Scared?” Epidemic Uncertainties and Risk Construction in Emerging Infectious Disease Epidemics

ABSTRACT The onset of emerging infectious disease epidemics is marked by uncertainties regarding the nature of the causative pathogen, mode of transmission, and preventive measures among other factors. While scientists are developing knowledge on these subjects, people must adapt to these uncertainties. We explore risk construction in the epidemic context by conceptualizing risk as a knowledge construct and identifying the constitutive elements that influence its construction. Using qualitative interviews and comparing three locations in Ghana during the 2014–2016 Ebola epidemic in West Africa, we argue that the construction of risk is influenced by a combination of disease, networked information, and information on enabled factors—factors triggered by the epidemic context (geographic location, government policies, absence of confirmed cases). These enabled factors, we suggest, shape the disease risk characteristics that amplify or attenuate risk perception and influence health behavior.

Maize Kernel-Associated Metagenomes Reveal Potential Microbe–Microbe Interactions that Underlie Fusarium Ear Rot Disease

As an alternative to host resistance, transgenic expression of entomocidal and antimicrobial proteins from Bacillus thuringiensis ( Bt) in maize can mitigate Fusarium ear rot (FER). This study evaluated FER in Bt and conventional maize and the role of microbes in the kernel-associated metagenome using quantitative reduced representation sequencing. Our results revealed significant differences in FER severity across environments and varieties and between inoculation treatments. The lower FER scores of conventional maize Spectrum 6416 relative to other varieties highlighted resistant alleles in maize germplasm, whereas strong correlations indicated FER-induced yield loss. Mitigation of FER by transgenes was validated by about threefold enrichment of Fusarium verticillioides ( Fv) postinoculation compared with nonsignificant Fv enrichment in conventional maize. Although the major causal pathogen of FER, Fv, was the most abundant species in the metagenomes ( r = 0.41 to 0.49), a similar degree of correlation was observed between FER and several Fusarium spp. ( r = 0.2 to 0.56). The potential FER-suppressing properties of Talaromyces stipitatus and Ustilago maydis were indicated by negative correlations with FER ( r = –0.22 to –0.42), Fv, and some Fusarium spp. The more FER-resistant varieties consistently enriched for potential FER-suppressing Burkholderia cenocepacia (negatively correlated with Fv). This suggests host genetic background-dependent recruitment of beneficial microbes that suppress pathogens and that microbe–microbe interactions play a role in FER severity. Quantitative and species/strain-level metagenomic profiles hold promise for identifying robust disease-suppressing microbes and microbes that act in synergy with pathogens and for developing holobiont-aware breeding strategies that account for host–microbiome coevolution and host genotype, microbiome/metagenome, and environment interactions. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Suppression PCR-Based Selective Enrichment Sequencing for Pathogen and Antimicrobial Resistance Detection on Cell-Free DNA in Sepsis-A Targeted, Blood Culture-Independent Approach for Rapid Pathogen and Resistance Diagnostics in Septic Patients.

Sepsis is a life-threatening syndrome triggered by infection and accompanied by high mortality, with antimicrobial resistances (AMRs) further escalating clinical challenges. The rapid and reliable detection of causative pathogens and AMRs are key factors for fast and appropriate treatment, in order to improve outcomes in septic patients. However, current sepsis diagnostics based on blood culture is limited by low sensitivity and specificity while current molecular approaches fail to enter clinical routine. Therefore, we developed a suppression PCR-based selective enrichment sequencing approach (SUPSETS), providing a molecular method combining multiplex suppression PCR with Nanopore sequencing to identify most common sepsis-causative pathogens and AMRs using plasma cell-free DNA. Applying only 1 mL of plasma, we targeted eight pathogens across three kingdoms and ten AMRs in a proof-of-concept study. SUPSETS was successfully tested in an experimental research study on the first ten clinical samples and revealed comparable results to clinical metagenomics while clearly outperforming blood culture. Several clinically relevant AMRs could be additionally detected. Furthermore, SUPSETS provided first pathogen and AMR-specific sequencing reads within minutes of starting sequencing, thereby potentially decreasing time-to-results to 11-13 h and suggesting diagnostic potential in sepsis.

STERNAL WOUND INFECTION AFTER CARDIAC SURGERY: LOOKING FOR INTERACTIONS BETWEEN PERIOPERATIVE VARIABLES AND PATHOGEN AGENTS

Abstract Background Sternal wound infection (SWI) can be a major complication of cardiac surgery. Both the risk factors and the pathogens agents are well known today. Yet, possible interactions between perioperative variables and pathogens have not been explored. Methods Between Jan 1st, 2006 and Nov 30, 2023 (∼18 years), perioperative data of all patients undergoing cardiac operations through median sternotomy at the Cardio–Thoraco–Vascular Department of the University Hospital of Trieste, Italy, were recorded prospectically in a computed database. Every patient developing a sternal complication was followed in a dedicated outpatient clinic and, in the presence of infection, the causative pathogen was always searched for by cultural examination of tissue speciments. Different strategies of unsupervised clustering were applied to find groups of patients with similar clinical, surgical and microbiological profiles. Results Of 9,680 consecutive cardiac surgery patients, 516 (5.3%) experienced a SWI that involved superficial soft tissue in 25.6% of cases, deep soft tissue in 51%, sternal bone in 16.7%, or mediastinum in 6.8% of cases. Staphylococcus aureus, coagulase–negative Staphylococci, Enterobacteriaceae, and Pseudomonas aeruginosa were pathogens in 100 (19.4%), 158 (30.6%), 89 (17.2%) and 36 (7%) patients, respectively; other pathogens, polymicrobial and negative cultures were found in 27 (5.2%), 26 (5%) and 80 (15.5%) patients, respectively. Following statistical analyses, significant associations were found between the Pseudomonas aeruginosa etiology of SWI and some variables such as female gender (p&amp;lt;0.0001) and preoperative renal failure (p=0.008); significant associations between the Pseudomonas aeruginosa etiology were found also with more serious forms of sepsis (p=0.025), as well as with early postoperative death (p=0.02) and complications such as acute kidney injury (p=0.006), multiple transfusion (p=0.003), and prolonged mechanical ventilation (p=0.005). Conclusion For women having renal impairment and undergoing cardiac surgery through median sternotomy, in the case of SWI, the Pseudomonas aeruginosa etiology may be more frequent and a really life–threating complication.

Antifungal Activities of Biogenic Silver Nanoparticles Mediated by Marine Algae: In Vitro and In Vivo Insights of Coating Tomato Fruit to Protect against Penicillium italicum Blue Mold.

In an attempt to reduce such decay induced by pathogenic causes, several studies investigated the effectiveness of nanoparticles (NPs) that play a vital role in saving food products, especially fruits. Current research delves into biogenic silver nanoparticles (using marine alga Turbinaria turbinata (Tt/Ag-NPs) and their characterization using FT-IR, TEM, EDS, and zeta potential. Some pathogenic fungi, which cause fruit spoilage, were isolated. We studied the impact of using Tt/Ag-NPs to protect against isolated fungi in vitro, and the influence of Tt/Ag-NPs as a coating of tomato fruit to protect against blue mold caused by Penicillium italicum (OR770486) over 17 days of storage time. Five treatments were examined: T1, healthy fruits were used as the positive control; T2, healthy fruits sprayed with Tt/Ag-NPs; T3, fruits infected with P. italicum followed by coating with Tt/Ag-NPs (pre-coating); T4, fruits coated with Tt/Ag-NPs followed by infection by P. italicum (post-coating); and T5, the negative control, fruits infected by P. italicum. The results displayed that Tt/Ag-NPs are crystalline, spherical in shape, with size ranges between 14.5 and 39.85 nm, and negative charges. Different concentrations of Tt/Ag-NPs possessed antifungal activities against Botrytis cinerea, Rhodotorula mucilaginosa, Penicillium expansum, Alternaria alternate, and Stemphylium vesicarium. After two days of tomatoes being infected with P. italicum, 55% of the fruits were spoilage. The tomato fruit coated with Tt/Ag-NPs delayed weight loss, increased titratable acidity (TA%), antioxidant%, and polyphenol contents, and decreased pH and total soluble solids (TSSs). There were no significant results between pre-coating and post-coating except in phenol contents increased in pre-coating. A particular focus is placed on the novel and promising approach of utilizing nanoparticles to combat foodborne pathogens and preserve commodities, with a spotlight on the application of nanoparticles in safeguarding tomatoes from decay.

Molecular characterization of human astrovirus infection in children under 5 years of age with acute gastroenteritis in Tehran, Iran, 2021-2022: co-infection with rotavirus.

Human astroviruses (HAstVs) are considered important causative pathogens of acute gastroenteritis (AGE) in children under 5 years of age worldwide, along with group A rotavirus (RVA), norovirus (NoV), and enteric adenovirus (EAdV). The present study was aimed to both detect HAstV and its co-infections and investigate genetic analysis of circulating HAstV and co-infected virus in hospitalized children under 5 years of age with AGE in Iran. Accordingly, a sum of 200 stool specimens were screened by PCR for HAstV during 2021-2022. The HAstV was found in 0.5% of 200 specimens (n = 1) while was co-infected with RVA. The genetic and phylogenetic analysis indicated HAstV1 genotype, which clustered with viruses from lineage 1b, which has not been previously reported in Iran. The detected RVA strain belonged to G1 lineage II/P[8]-lineage III, which has been reported previously in Iran as the most common strain. The further genetic analysis of RVA VP6 and NSP4 demonstrated an atypical genotype pattern G1P[8]-I1-E2, as a mono-reassortant of a Wa-like genogroup, which appeared to be reassorted with the NSP4 gene of E2 genotype of the G2P[4] DS-1 genogroup. Although the clinical outcomes of the AGE-causing viruses co-infection is not yet entirely clear, it seems that future studies will be helpful to merge clinical and epidemiological data of co-infecting viruses for a more accurate medical and clinical relevance in symptomatic children.

Rapid and sensitive diagnosis of live Mycobacterium tuberculosis using clustered regularly interspaced short palindromic repeat‐Cas13a point‐of‐care RNA testing

AbstractMycobacterium tuberculosis (MTB) is the causal pathogen of tuberculosis (TB). Rapid and accurate detection of live MTB is important for transmission control and patient treatment. Here, we described a clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas13a‐based molecular diagnosis approach for rapid and specific detection of live MTB. This detection method, which we termed CRISPR‐Live‐MTB, contained two consecutive reactions including nuclear acid sequence‐based amplification (NASBA) and CRISPR‐Cas13a collateral cleavage reaction. CRISPR‐Live‐MTB could efficiently detect MTB single‐stranded RNA (ssRNA) in 2 hours with high specificity over double‐stranded DNA (dsDNA). Importantly, CRISPR‐Live‐MTB exhibited a limit of detection of 2.4 copies for MTB ssRNA, which was 1000 times lower than that of the clinically used NASBA method. Moreover, lateral flow was integrated into the CRISPR‐Live‐MTB method to enable point‐of‐care testing application with a sensitivity of 95% and a specificity of 100%. Overall, our study demonstrated the feasibility of CRISPR‐Live‐MTB as a rapid, sensitive, and specific approach for live MTB detection.

Pathogenic variations in Colletotrichum spp. causing chilli anthracnose in Peninsular Malaysia

AbstractIn Peninsular Malaysia, Colletotrichum fioriniae, Colletotrichum fructicola, Colletotrichum scovillei, Colletotrichum siamense and Colletotrichum truncatum are the causative pathogens of chilli anthracnose. In the present study, the pathogenic variability and virulence levels of these five Colletotrichum spp. were evaluated. Isolates of C. fioriniae, C. fructicola, C. scovillei, C. siamense and C. truncatum were tested for pathogenicity using red and green Capsicum annuum and red and green Capsicum frutescens. Pathogenicity tests were conducted using spore suspensions and mycelial plugs on wounded and nonwounded chilli fruits. All isolates tested on the wounded chilli fruits caused anthracnose symptoms with variable disease severity and virulence. In nonwounded chilli fruits, only isolates of C. scovillei (n = 7) and C. truncatum (n = 5) caused infection. C. fioriniae, C. fructicola, C. scovillei, C. siamense and C. truncatum showed variability in pathogenicity and virulence levels in red and green chilli fruits of Ca. annuum and Ca. frutescens. Pathogenicity tests on wounded and nonwounded chilli fruits can be used to identify pathotypes within anthracnose pathogen populations. This is important for strategizing the effective disease management of chilli anthracnose.

Well-Established and Traditional Use of Vegetal Extracts as an Approach to the "Deep Roots" of Cough.

Cough is a common presenting symptom for patients in a primary care setting and significantly impacts a patient's quality of life. Cough involves a complex reflex arc beginning with the stimulation of sensory nerves that function as cough receptors that stimulate the cough center in the brain. This "cough center" functions to receive these impulses and produce a cough by activating efferent nervous pathways to the diaphragm and laryngeal, thoracic, and abdominal musculature. Drugs that suppress the neural activity of cough are non-specific as those treatments are not directed toward pathogenic causes such as inflammation and oxidative stress. Moreover, they block a reflex called the watchdog of the lung and have a defense mechanism. Acute respiratory infections of the upper and lower airways most commonly cause acute cough. In contrast, the most common causes of chronic cough are upper airway cough syndrome, asthma, and gastroesophageal reflux disease, all associated with an inflammatory reaction at the level of the cough receptors. The use of natural compounds or herbal drugs such as carob syrup, dry blackcurrant extract, dry extract of caraway fruit, dry extract of ginger rhizome, dry extract of marshmallow root, and dry extract of ivy leaves, to name a few, not only have anti-inflammatory and antioxidant activity, but also act as antimicrobials, bronchial muscle relaxants, and increase gastric motility and empty. For these reasons, these natural substances are widely used to control cough at its deep roots (i.e., contrasting its causes and not inhibiting the arch reflex). With this approach, the lung watchdog is not put to sleep, as with peripheral or central inhibition of the cough reflex, and by contrasting the causes, we may control cough that viruses use at self-advantage to increase transmission.

Evaluation of the ELITe InGenius and Bordetella ELITe MGB Kit for the detection and identification of B. pertussis, B. parapertussis and B. holmesii

Bordetella pertussis is the causative pathogen of whooping cough or pertussis, a contagious respiratory disease. Aside from serodiagnosis, laboratory confirmation of pertussis is done through PCR, as B. pertussis is difficult to culture.The ELITe InGenius instrument (ELITechGroup, France) with accompanying Bordetella ELITe MGB Kit was evaluated against a laboratory-developed assay. Both assays combine two screening (IS481, IS1001) and two confirmation targets (recA, ptxA-Pr or IS1002) for optimal sensitivity and specificity.The company's stated claims on sensitivity and reproducibility were confirmed. Accuracy testing showed full concordance between both assays for the screening targets. Minor discrepancies were seen for the B. pertussis confirmation target. Some cross-reactivity with other Bordetella species was observed for the IS481-target, however, none of these were confirmed in the ptxA-Pr target. These results show the suitability of the Bordetella ELITe MGB Kit for the detection and differentiation of B. pertussis, B. parapertussis and B. holmesii.