Meningococcal Carriage in ‘Men Having Sex With Men’ With Pharyngeal Gonorrhoea

We assessed the characteristics of Neisseria meningitidis pharyngeal carriage in a cohort of ‘men having sex with men’, including patients with pharyngeal Neisseria gonorrhoeae infection. In the period 2017-2019, among all the oropharyngeal samples tested for gonorrhoea from MSM attending a STI Clinic in Bologna (Italy), we randomly selected 244 N. gonorrhoeae-positive samples and 403 negatives (n=647). Pharyngeal specimens were tested for N. meningitidis presence, by the detection of sodC gene. N. meningitidis-positive samples were further grouped by PCR tests for the major invasive genogroups (i.e., A, B, C, W, and Y). A molecular assay, targeting capsule transporter gene, was used to determine meningococcal capsular status. Overall, 75.8% (491/647) of samples tested positive for sodC gene, indicating a pharyngeal meningococcal carriage. Meningococcal colonisation was significantly more frequent in younger subjects (P=0.009), with no association with HIV infection. Non-groupable meningococci represented most of pharyngeal carriages (about 71%). The commonest N. meningitidis serogroup was B (23.6%), followed by C (2.1%), Y (1.8%) and W (1.1%). Meningococci were often characterized by the genetic potential of capsule production. Interestingly, a negative association between N. meningitidis and N. gonorrhoeae was found: pharyngeal gonorrhoea was significantly more present in patients without meningococcal carriage (P=0.03). Although preliminary, our data added knowledge on the epidemiology of meningococcal carriage in MSM communities at high risk of gonococcal infections, gaining new insights into the interactions/dynamics between N. meningitidis and N. gonorrhoeae.

ID-CARD: A clade-specific molecular assay for the detection of 25 Candida spp. causing candidemia based on antifungal susceptibility patterns and in vitro testing of the antifungal activity of a synthetic antimicrobial peptide derived from human lactoferrin (hLF1-11).

Fungal infections are a serious health concern affecting over 1.5 million individuals annually. ID-CARD aims to improve diagnostics taking into account phylogeny and antifungal susceptibility patterns of Candida spp. involved in candidemia.Twenty-five Candida spp. were chosen. Based on ribosomal DNA sequences, clade-specific primers/Taqman probes were designed. Different multiplex panels consisting of four clades that exhibited similar antifungal susceptibility profiles were created. To create the groups, we tested fluconazole and anidulafungin with broth microdilution according to EUCAST against 3-5 isolates/species (n=121), which were also used for specificity testing of the molecular assay. Furthermore, we tested the in vitro activity of hLF(1-11) peptide against isolates that exhibited elevated minimum inhibitory concentrations (MICs) for one or both of the drugs. The groups created are : i. Lodderomyces, Kluyveromyces, Metschnikowiaceae Sensitive, Internal control, (all with low MICs) ii. Pichiaceae, Nakaseomyces, Wickerhamomycetaceae, Debaryomyces & Diutina, (all with high MICs to azoles) and iii. Yarrowia, Wickerhamiella & Meyerozyma, Candida auris, Candida haemulonii complex (all with high MICs to both azoles & echinocandins). The primers/probes showed 100% specificity and capacity for multiplexing. In vitro experiments indicated that hLF(1-11) is fungicidal against various Candidaspp. A synergistic effect of antifungal and hLF(1-11) against various Candida species was shown as combinations of the peptide with antifungals were more effective than these alone ID-CARD will contribute to a fast and reliable molecular detection of yeasts involved in candidiasis. AMPs is a novel way to treat Candida spp. exhibiting high MICs to commonly used antifungal drugs.

Analysis of MTb Rapid Molecular Test Performance Towards Microscopical Acid Fast Bacilli Examination at Labuang Baji General Hospital

Tuberculosis (TB) is a global health problem, which is the third leading cause of death of all infectious diseases aroundthe world, included Indonesia. Acid Fast Bacilli (AFB) smear and rapid molecular assay for Mycobacterium tuberculosis (MTB)are the old and new examinations required for MTB laboratory diagnosis. This study aimed to compare the performance ofMTB rapid molecular assay and AFB smear in diagnosis and screening for TB patients. This observational retrospective studyused a cross-sectional approach, with a purposive sampling technique of 559 patients with suspected TB in Labuang BajiHospital, Makassar. This study was conducted from March 2019 to June 2019 by taking data from medical records fromJanuary 2018 to December 2018 at Labuang Baji Hospital, Makassar. Three hundred and forty-nine subjects were males(62.4%), and 210 subjects were females (37.6%). This study revealed sensitivity and specificity of 98.57% and 84.96%,respectively for MTB rapid molecular assay, and 68.65% and 99.44%, respectively for AFB smear, this shows that MTB rapidmolecular assay was superior to AFB smear in diagnosing TB patients.

Closing the Critical Period Is Required for the Maturation of Binocular Integration in Mouse Primary Visual Cortex

Binocular matching of orientation preference between the two eyes is a common form of binocular integration that is regarded as the basis for stereopsis. How critical period plasticity enables binocular matching under the guidance of normal visual experience has not been fully demonstrated. To investigate how critical period closure affects the binocular matching, a critical period prolonged mouse model was constructed through the administration of bumetanide, an NKCC1 transporter antagonist. Using acute in vivo extracellular recording and molecular assay, we revealed that binocular matching was transiently disrupted due to heightened plasticity after the normal critical period, together with an increase in the density of spines and synapses, and the upregulation of GluA1 expression. Diazepam (DZ)/[(R, S)-3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP)] could reclose the extended critical period, and rescue the deficits in binocular matching. Furthermore, the extended critical period, alone, with normal visual experience is sufficient for the completion of binocular matching in amblyopic mice. Similarly, prolonging the critical period into adulthood by knocking out Nogo-66 receptor can prevent the normal maturation of binocular matching and depth perception. These results suggest that maintaining an optimal plasticity level during adolescence is most beneficial for the systemic maturation. Extending the critical period provides new clues for the maturation of binocular vision and may have critical implications for the treatment of amblyopia.

Performance of the eazyplex® BloodScreen GN as a simple and rapid molecular test for identification of Gram-negative bacteria from positive blood cultures

The LAMP-based eazyplex® BloodScreen GN was evaluated for the detection of frequent Gram-negatives directly from positive blood culture (BC) bottles. A total of 449 BCs were analyzed. Sensitivities and specificities were 100% and 100% for Escherichia coli, 95.7% and 100% for Klebsiella pneumoniae, 100% and 100% for blaCTX-M, 100% and 100% for Klebsiella oxytoca, 100% and 99% for Proteus mirabilis, and 100% and 99.8% for Pseudomonas aeruginosa, respectively. The time to result ranged from 8 to 16 min, plus about 6 min for sample preparation. The eazyplex® BloodScreen GN is a reliable molecular assay for rapid BC testing.

Development and Evaluation of a Novel Fast Broad-Range ITS/LSU DNA PCR and Sequencing Assay (FBR-PCR/S) for Rapid Diagnosis of Invasive Fungal Diseases: Multi-Year Experience in a Large Canadian Healthcare Zone and a Literature Review

BackgroundThis study evaluated the performance of a novel fast broad range PCR and sequencing (FBR-PCR/S) assay for the improved diagnosis of invasive fungal disease (IFD) in high-risk patients in a large Canadian healthcare region.MethodsA total of 114 clinical specimens (CS) including bronchoalveolar lavages (BALs) were prospectively tested from 107 patients over a 2-year period. Contrived BALs (n=33) inoculated with known fungi pathogens were also tested to increase diversity. Patient characteristics, fungal stain and culture results were collected from the laboratory information system. Dual-priming oligonucleotide (DPO) primers targeted to the ITS (~350 bp) and LSU (~550 bp) gene regions were used to perform FBR-PCR/S assays on extracted BALs/CS. The performance of the molecular test was evaluated against results of fungal stains and culture, and where available, histopathology, and clinical review for the presence of IFD.ResultsThe 107 patients were predominantly male (67, 62.6%%) with a mean age of 59 yrs. (range = 0 to 85 yrs.): 74 (69.2%) patients had at least one underlying comorbidity: 19 (34.5%) had confirmed and 12 (21.8%) had probable IFD. Culture recovered 66 fungal isolates from 55 BALs/CS with Candida spp. and Aspergillus spp. being most common. For BALs, the molecular assay vs. fungal culture had sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV), and efficiency of 88.5% vs.100%, 100% vs. 61.1%, 100% vs. 88.5%, 61.1% vs. 100%, and 90.2% for both. For other CS, the molecular assay had similar performance to fungal culture with sensitivity, specificity, PPV, NPV and efficiency of 66.7%, 87.0%, 66.7%, 87.0% and 81.3% for both methods. Both methods also performed similarly, regardless of whether CS stain/microscopy showed yeast/fungal elements. FBR-PCR/S assays results were reported in ~8h compared to fungal cultures that took between 4 to 6 weeks.ConclusionsRapid molecular testing compared to culture has equivalent diagnostic efficiency but improves clinical utility by reporting a rapid species-level identification the same dayshift (~8h).

Antimicrobial Spectrum, Growth/Killing Kinetics, Conventional/Molecular Assay of Characterizing Non-Leguminous Endophytic Bacteria and Fungi from Helianthus annuus, Carica papaya and Lycoperesicum solanum

The aim of this study is to comparative study between conventional and molecular assay of isolation, identification and characterization of non-leguminous endophytic bacteria and fungi in the leguminous root samples. The plant root samples, Helianthus annuus, Carica papaya and Lycoperesicum solanum (Sunflower root and stem, pawpaw root and stem, and tomato root and stem from Adekunle Ajasin University School farm, Akungba Akoko, Ondo state, Nigeria. The isolation of endophytic bacteria were performed using the conventional method of isolation (biochemical test) and characterization were done using both the conventional and molecular method of bacteria characterization. The antibiotic susceptibility test (Antibiogram) was observed using disc diffusion. The four bacteria identified were Bacillus cereus, Enterobacter sp. Actnomycoses sp. and Aeromonas sp. for conventional method and Fusarium solani, Fusarium vortecelium and Bacillus thuringiensis for molecular method as confirmatory point of view. In this study, all isolated organisms tends to be Gram positive using the gram staining technique. Antibiogram shows the zones of inhibition with diameter ranging from 0-20 mm, Enterobacter sp. were more sensitive to the various antibiotics used. Ultraviolet spectrophotometer was also used to determine the growth dynamic as well as the death rate of the isolates, the addition of antibiotics (ciprofloxacin) to the isolates at the 24th hour speed up the death rate of the isolates from non-leguminous endophytic bacteria. After the preliminary identification of the bacteria isolates and the confirmatory identification of both bacteria and fungi isolates of the non-leguminous endophytic microorganism, it was noted that the preliminary identification was only able to achieve the genus level of taxonomic characterization, While the molecular method confirm the molecular sub level identification of isolates depletes the absolute taxonomic identification and characterization to the sub-species level. The results of this study validates the use of molecular sequencing for the assay identification and characterization of non-leguminous endophytic bacteria and fungi as the easy and best mode of identification of both bacteria and fungi isolates as a veritable tools for research purposes.