Rapid high throughput SYBR green assay for identifying the malaria vectors Anophelese arabiensis, Anopheles coluzzii and Anopheles gambia s.s. Giles

The Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding. Several molecular diagnostic techniques for distinguishing between An. coluzzii and An. gambiae are still under improvement. Although, the current SINE method for identification between An. coluzzii and An. gambiae works reliably, this study describes a refinement of the SINE method to increase sensitivity for identification of An. coluzzii, An. gambiae and An. arabiensis based on amplicon dissociation curve characteristics. Field-collected samples, laboratory-reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were sampled from Ghana and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye. The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72˚C for An. arabiensis, 75˚C for An. gambiae and 86˚C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines

Beta-lactam resistance mechanisms in Enterobacter species isolates from Tygerberg Hospital.

Thesis (MScMedSc)--Stellenbosch University, 2018.Background Resistance to carbapenem antibiotics in Gram-negative bacteria is a major public health problem due to limited treatment options. In Enterobacter species, carbapenemase production and reduced membrane permeability, resulting from reduced expression of outer membrane proteins OmpF and OmpC, in combination with extended-spectrum β-lactamase (ESBL) production, hyper-production of chromosomal blaAmpC β-lactamases and / or over-expression of the AcrAB/TolC efflux pump have been speculated to promote carbapenem resistance. This study investigated the mechanisms that promote ertapenem resistance in clinical Enterobacter cloacae isolates from Tygerberg Hospital. Materials and methods Twenty ertapenem non-susceptible clinical E. cloacae isolates, four ertapenem susceptible controls and five wild-type controls were selected based on the VITEK2®AES. Ertapenem MICs were determined using broth microdilution (BMD) and gradient diffusion. Resistance mechanisms were characterized using the phenotypic assays VITEK2®AES, Mastdiscs D68C, D69C and D63C combination sets, Rapidec® Carba NP kit and a synergy assay using disc diffusion in the presence of an efflux pump inhibitor (EPI). Molecular assays included multiplex PCR to identify carbapenemases, multiplex PCR and sequencing to identify ESBLs, SDS-PAGE to characterize OmpC and OmpF abundance and RT-qPCR to quantify expression of blaAmpC, ompC, ompF and acrB. Results and Discussion Seventeen (85%) ertapenem non-susceptible isolates were confirmed non-susceptible by BMD and six (30%) by gradient diffusion, suggesting possible undercalling of ertapenem resistance by gradient diffusion and overcalling by VITEK2®AES. Seven ertapenem non-susceptible isolates were predicted to be carbapenemase producers by the VITEK2®AES and one by the Rapidec® Carba NP kit, however no carbapenemases were detected by PCR. Nineteen ESBL producers were identified by the VITEK2®AES, eight by the D68C combination set and eleven by the D63C combination set. ESBLs were detected in 12 (60%) isolates by PCR and sequencing; of which eight were blaCTX-M, three were blaSHV-12 and one isolate contained both genes. Twelve (60%) ertapenem non-susceptible isolates were predicted to be derepressed blaAmpC producers by the VITEK2®AES, thirteen (65%) by the D68C combination set and six (30%) by the blaAmpC RT-qPCR.Agtergrond Karbapenem-weerstandige Gram-negatiewe bakterieë is 'n alomvattende publieke gesondheidsprobleem weens ‘n beperking van toepaslike mediese behandelings. In Enterobacter spesies word ertapenemweerstandigheid toegeskryf aan karbapenemase-produksie en/of verminderde membraandeurlaatbaarheid, as gevolg van onderdrukking van die OmpF- en OmpC-buitemembraanproteïene, in kombinasie met uitgebreide spektrum β-laktamase (ESBL) produksie, oorproduksie van chromosomale blaAmpC β-laktamases en/of die ooruitdrukking van die AcrAB/TolC efflukspomp. Hierdie studie ondersoek die meganismes wat ertapenemweerstandigheid veroorsaak in kliniese Enterobacter cloacae isolate vanaf Tygerberg hospitaal. Materiale en metodes Twintig ertapenem-nie-vatbare kliniese E. cloacae-isolate, vier ertapenem-vatbare kontrole en vyf wilde-tipe kontrole is ingesluit op grond van VITEK2®AES resultate. Die vlak van ertapenem weerstandigheid is bepaal deur middel van die antibiotiese-mikroverdunning (BMD) metode, asook gradiëntdiffusie. Weerstandigheidsmeganismes is bepaal met verskeie fenotipiese toetse, soos VITEK2®AES, “Mastdiscs” D68C-, D69C- en D63C-kombinasiestelle, Rapidec®Carba NP-kit en 'n sinergie-toets met behulp van skyfdiffusie in die teenwoordigheid van 'n efflukspompinhibeerder (EPI). ‘n “Multiplex” polimerasiekettingreaksie (PKR) is toegepas om ESBLs en karbapenemases te identifiseer. Die hoeveelheid OmpC en OmpF proteïene is met SDS-PAGE bepaal en die vlak van uitdrukking van blaAmpC, ompC, ompF en acrB is met RT-qPKR bepaal. Resultate en Bespreking Sewentien (85%) ertapenem-nie-vatbare isolate is bevestig as ertapenem-nie-vatbaar deur BMD en ses (30%) deur gradientdiffusie. Dit dui aan dat ertapenemweerstandigheid moontlik deur gradientdiffusie onderskat word en oorskat word deur VITEK2® AES. Sewe ertapenem-nie-vatbare isolate is voorspel om karbapenemase-produseerders te wees deur die VITEK2® AES en een volgens die Rapidec Carba NP-kit, maar geen karbapenemases is deur PKR waargeneem nie. Negentien ESBL-produseerders is deur die VITEK2® AES geïdentifiseer, agt deur die D68C-kombinasiestel en elf deur die D63C-kombinasiestel. ESBLs is in 12 (60%) isolate deur middel van PKR en DNA-volgordebepaling geïdentifiseer, waarvan agt blaCTX-M bevat, drie blaSHV-12 bevat en een isolaat beide gene bevat. Twaalf (60%) ertapenem-nie-vatbare isolate is deur VITEK2® AES voorspel om on-onderdrukte blaAmpC-produseerders te wees, dertien (65%) deur die D68C-kombinasiestel en vyf (25%) deur blaAmpC RT-qPKR. Die hoeveelheid OmpC en OmpF kon nie deur SDS-PAGE bepaal word nie, maar verlaagde ompC- en ompF uitdrukking is onderskeidelik in elf (55%) en twaalf (60%) van die ertapenem-nie-vatbare isolate waargeneem. Die uitdrukking van beide gene is verlaag in sewe (35%) van die ertapenem-nie-vatbare isolate. Die sinergie-toets het nie verhoogde antibiotiese vatbaarheid in die teenwoordigheid van die EPI waargeneem nie. Verhoogde effluksaktiwiteit gebaseer op die uitdrukking van acrB is in twee (10%) van die ertapenem nie-vatbare isolate waargeneem. Altesaam het 11 van die 17 BMD bevestigde ertapenem-nie-vatbare isolate ESBL-produksie en ‘n verminderde membraandeurlaatbaarheid getoon; twee hiervan is chromosomale blaAmpC-produseerders, en twee het verhoogde effluksaktiwiteit getoon. Karbapenemaseproduksie is waargeneem in een isolaat, wat ook die oorproduksie van AmpC en verminderde membraandeurlaatbaarheid getoon het. Een isolaat het oorproduksie van chromosomale blaAmpC en verminderde membraandeurlaatbaarheid getoon, terwyl twee slegs verminderde membraandeurlaatbaarheid en een slegs die oorproduksie van chromosomale blaAmpC getoon het. In een ertapenem-nie-vatbare isolaat is geen weerstandsmeganismes opgespoor nie (ERD 13), wat daarop dui dat alternatiewe meganismes bydra tot die weerstandigheid in hierdie isolaat. Gevolgtrekking Ertapenemweerstandigheid in hierdie kliniese E. cloacae isolate is deur middel van 'n kombinasie van meganismes bemiddel; oorwegend verminderde membraandeurlaatbaarheid en ESBL produksie, en nie karbapenemase produksie nie. Onbekende ertapenem-weerstandigheidsmeganismes kan by sommige isolate betrokke wees en daarom is verdere navorsing nodig

Evaluation of neonatal mortality data completeness and accuracy in Ghana.

BackgroundCause-specific mortality data are required to set interventions to reduce neonatal mortality. However, in many developing countries, these data are either lacking or of low quality. We assessed the completeness and accuracy of cause of death (COD) data for neonates in Ghana to assess their usability for monitoring the effectiveness of health system interventions aimed at improving neonatal survival.MethodsA lot quality assurance sampling survey was conducted in 20 hospitals in the public sector across four regions of Ghana. Institutional neonatal deaths (IND) occurring from 2014 through 2017 were divided into lots, defined as neonatal deaths occurring in a selected facility in a calendar year. A total of 52 eligible lots were selected: 10 from Ashanti region, and 14 each from Brong Ahafo, Eastern and Volta region. Nine lots were from 2014, 11 from 2015 and 16 each were from 2016 and 2017. The cause of death (COD) of 20 IND per lot were abstracted from admission and discharge (A&D) registers and validated against the COD recorded in death certificates, clinician's notes or neonatal death audit reports for consistency. With the error threshold set at 5%, ≥ 17 correctly matched diagnoses in a sample of 20 deaths would make the lot accurate for COD diagnosis. Completeness of COD data was measured by calculating the proportion of IND that had death certificates completed.ResultsNineteen out of 52 eligible (36.5%) lots had accurate COD diagnoses recorded in their A&D registers. The regional distribution of lots with accurate COD data is as follows: Ashanti (4, 21.2%), Brong Ahafo (7, 36.8%), Eastern (4, 21.1%) and Volta (4, 21.1%). Majority (9, 47.4%) of lots with accurate data were from 2016, followed by 2015 and 2017 with four (21.1%) lots. Two (10.5%) lots had accurate COD data in 2014. Only 22% (239/1040) of sampled IND had completed death certificates.ConclusionDeath certificates were not reliably completed for IND in a sample of health facilities in Ghana from 2014 through 2017. The accuracy of cause-specific mortality data recorded in A&D registers was also below the desired target. Thus, recorded IND data in public sector health facilities in Ghana are not valid enough for decision-making or planning. Periodic data quality assessments can determine the magnitude of the data quality concerns and guide site-specific improvements in mortality data management

sj-docx-1-smo-10.1177_20503121231225924 – Supplemental material for Health and safety of health workers in the Suame Municipality of Ghana – Lessons learnt from the COVID-19 outbreak in infection prevention and control for future pandemics

Supplemental material, sj-docx-1-smo-10.1177_20503121231225924 for Health and safety of health workers in the Suame Municipality of Ghana – Lessons learnt from the COVID-19 outbreak in infection prevention and control for future pandemics by David Oppong Darko, Douglas Aninng Opoku, Nana Kwame Ayisi-Boateng, Aliyu Mohammed, Jennifer Ashilevi, Obed Kwabena Offe Amponsah, Ayongo Mate-Kole, Dora Egblewogbe, Bridgetta Addai Darko, Ebenezer Agyemang and Paul Okyere in SAGE Open Medicine</p