Use of microarray technology for the detection of a range of RNA viruses in clinical samples

There are many RNA virus pathogens of humans including influenza, parainfluenza, enteroviruses, parechoviruses, coronaviruses, pneumoviruses, and metapneumoviruses. These commonly invade and infect the respiratory and gastrointestinal tracts, giving rise to acute and chronic respiratory tract infections, and some may also reach the central nervous system (CNS) either via haematogenous or neural routes resulting in a variety of clinical presentations, (e.g. meningitis, encephalitis) which may lead to severe irreparable damage such as poliomyelitis especially in young children. Early correct diagnosis of viral infections is indispensable in order to prevent potential outbreaks which threaten the public health worldwide and might lead to high morbidity and some with significant mortality. Several laboratory techniques such as virus isolation, direct visualization of viral particles, detection of viral antigens and/or nucleic acids, and detection of host immune response (e.g. anti-viral antibodies) to infection, are available for diagnosis, but may have significant drawbacks such as time-inefficiency, cost and certain individual limitations. Microarray technology offers one way to overcome some of these limitations. In this study, a microarray chip containing 7967 oligonucleotides (probes) covering the whole genomes of human enteroviruses, rhinoviruses, respiratory syncytial viruses, metapneumoviruses and influenza viruses was designed and constructed using both OligoArray and Agilent eArray software, to allow simultaneous detection of any of the above viruses present in any clinical specimen. This virochip was tested against positive controls and clinical samples known to contain RNA nucleic acids of these viruses. Viral RNA was reverse transcribed, and amplified. Considerable effort was expended in trying to optimise a multiple displacement amplification (MDA) protocol for whole genome amplification, and in addition, long-range PCR was also utilised. Amplification products were fragmented, labelled and loaded into a hybridization reaction with the designed viral probes printed on the virochip. The results revealed (i) a number of technical problems associated with MDA; (ii) that some probes either failed to recognise their intended targets, or produced cross-reactive signals with non-intended targets; (iii) that many of the designed probes hybridized to their relevant viral nucleic acids and generated hybridization signals of high fluorescent intensity offering an opportunity to develop this probe array in order to be used for the identification of a wide variety of virus species up to the serogroup level or beyond (if required) specifically those causing CNS and respiratory tract infections

Use of microarray technology for the detection of a range of RNA viruses in clinical samples

There are many RNA virus pathogens of humans including influenza, parainfluenza, enteroviruses, parechoviruses, coronaviruses, pneumoviruses, and metapneumoviruses. These commonly invade and infect the respiratory and gastrointestinal tracts, giving rise to acute and chronic respiratory tract infections, and some may also reach the central nervous system (CNS) either via haematogenous or neural routes resulting in a variety of clinical presentations, (e.g. meningitis, encephalitis) which may lead to severe irreparable damage such as poliomyelitis especially in young children. Early correct diagnosis of viral infections is indispensable in order to prevent potential outbreaks which threaten the public health worldwide and might lead to high morbidity and some with significant mortality. Several laboratory techniques such as virus isolation, direct visualization of viral particles, detection of viral antigens and/or nucleic acids, and detection of host immune response (e.g. anti-viral antibodies) to infection, are available for diagnosis, but may have significant drawbacks such as time-inefficiency, cost and certain individual limitations. Microarray technology offers one way to overcome some of these limitations. In this study, a microarray chip containing 7967 oligonucleotides (probes) covering the whole genomes of human enteroviruses, rhinoviruses, respiratory syncytial viruses, metapneumoviruses and influenza viruses was designed and constructed using both OligoArray and Agilent eArray software, to allow simultaneous detection of any of the above viruses present in any clinical specimen. This virochip was tested against positive controls and clinical samples known to contain RNA nucleic acids of these viruses. Viral RNA was reverse transcribed, and amplified. Considerable effort was expended in trying to optimise a multiple displacement amplification (MDA) protocol for whole genome amplification, and in addition, long-range PCR was also utilised. Amplification products were fragmented, labelled and loaded into a hybridization reaction with the designed viral probes printed on the virochip. The results revealed (i) a number of technical problems associated with MDA; (ii) that some probes either failed to recognise their intended targets, or produced cross-reactive signals with non-intended targets; (iii) that many of the designed probes hybridized to their relevant viral nucleic acids and generated hybridization signals of high fluorescent intensity offering an opportunity to develop this probe array in order to be used for the identification of a wide variety of virus species up to the serogroup level or beyond (if required) specifically those causing CNS and respiratory tract infections

Serum C - Reactive Protein Level in Diabetic Foot Patients and Their Relation with Bacterial Isolates

Background: Foot infections are one of the major complications of diabetes mellitus and a significant risk factor for lower extremity amputation. C-reactive protein is an acute-phase reactant, rises dramatically in response to infection.   Aim: To determine the microbial isolates of patients with diabetic foot infections and their relation with C-reactive protein level in their sera. Materials and Methods: A prospective study of 90 patients with diabetic foot infections admitted to different public and private hospitals in Erbil city center-Iraq between June 2011 and May 2012 was undertaken. Bacteriological specimens were obtained and processed using standard procedure. The patients serum had been tested for C-reactive protein by high sensitive Enzyme linked Immunosorbent Assay (ELISA). Results: A total of 130 pathogens were isolated from 90 diabetic foot patients 46 (51%) of the patients had polymicrobial infection, 37 (41%) had single organism and 7 (8%) had no growth. Gram positive (G+ve) bacteria 60(53%) were more commonly isolated than Gram negative (G-ve) bacteria 53(47%). Staphylococcus aureus and Escherichia coli were the most frequently among G+ve and G-ve isolates respectively. No significant difference was found between mean serum levels of C-reactive protein in patients infected with G+ve bacteria versus G-ve bacteria, although their concentration was more in the later. However, highly significant differences (P<0.01) were observed between both G+ve and G-ve bacteria versus no bacterial isolate in patients. Conclusion: C-reactive protein serum level was higher in patient with diabetic foot infected by G-ve bacteria, although G+ve bacteria represented a major bacterial isolates.

Evaluation of a task-based community oriented teaching model in family medicine for undergraduate medical students in Iraq

BACKGROUND: The inclusion of family medicine in medical school curricula is essential for producing competent general practitioners. The aim of this study is to evaluate a task-based, community oriented teaching model of family medicine for undergraduate students in Iraqi medical schools. METHODS: An innovative training model in family medicine was developed based upon tasks regularly performed by family physicians providing health care services at the Primary Health Care Centre (PHCC) in Mosul, Iraq. Participants were medical students enrolled in their final clinical year. Students were assigned to one of two groups. The implementation group (28 students) was exposed to the experimental model and the control group (56 students) received the standard teaching curriculum. The study took place at the Mosul College of Medicine and at the Al-Hadba PHCC in Mosul, Iraq, during the academic year 1999–2000. Pre- and post-exposure evaluations comparing the intervention group with the control group were conducted using a variety of assessment tools. RESULTS: The primary endpoints were improvement in knowledge of family medicine and development of essential performance skills. Results showed that the implementation group experienced a significant increase in knowledge and performance skills after exposure to the model and in comparison with the control group. Assessment of the model by participating students revealed a high degree of satisfaction with the planning, organization, and implementation of the intervention activities. Students also highly rated the relevancy of the intervention for future work. CONCLUSION: A model on PHCC training in family medicine is essential for all Iraqi medical schools. The model is to be implemented by various relevant departments until Departments of Family medicine are established

Review of nanomaterials in dentistry: interactions with the oral microenvironment, clinical applications, hazards, and benefits.

Interest in the use of engineered nanomaterials (ENMs) as either nanomedicines or dental materials/devices in clinical dentistry is growing. This review aims to detail the ultrafine structure, chemical composition, and reactivity of dental tissues in the context of interactions with ENMs, including the saliva, pellicle layer, and oral biofilm; then describes the applications of ENMs in dentistry in context with beneficial clinical outcomes versus potential risks. The flow rate and quality of saliva are likely to influence the behavior of ENMs in the oral cavity, but how the protein corona formed on the ENMs will alter bioavailability, or interact with the structure and proteins of the pellicle layer, as well as microbes in the biofilm, remains unclear. The tooth enamel is a dense crystalline structure that is likely to act as a barrier to ENM penetration, but underlying dentinal tubules are not. Consequently, ENMs may be used to strengthen dentine or regenerate pulp tissue. ENMs have dental applications as antibacterials for infection control, as nanofillers to improve the mechanical and bioactive properties of restoration materials, and as novel coatings on dental implants. Dentifrices and some related personal care products are already available for oral health applications. Overall, the clinical benefits generally outweigh the hazards of using ENMs in the oral cavity, and the latter should not prevent the responsible innovation of nanotechnology in dentistry. However, the clinical safety regulations for dental materials have not been specifically updated for ENMs, and some guidance on occupational health for practitioners is also needed. Knowledge gaps for future research include the formation of protein corona in the oral cavity, ENM diffusion through clinically relevant biofilms, and mechanistic investigations on how ENMs strengthen the tooth structure

Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background Disorders affecting the nervous system are diverse and include neurodevelopmental disorders, late-life neurodegeneration, and newly emergent conditions, such as cognitive impairment following COVID-19. Previous publications from the Global Burden of Disease, Injuries, and Risk Factor Study estimated the burden of 15 neurological conditions in 2015 and 2016, but these analyses did not include neurodevelopmental disorders, as defined by the International Classification of Diseases (ICD)-11, or a subset of cases of congenital, neonatal, and infectious conditions that cause neurological damage. Here, we estimate nervous system health loss caused by 37 unique conditions and their associated risk factors globally, regionally, and nationally from 1990 to 2021. Methods We estimated mortality, prevalence, years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs), with corresponding 95% uncertainty intervals (UIs), by age and sex in 204 countries and territories, from 1990 to 2021. We included morbidity and deaths due to neurological conditions, for which health loss is directly due to damage to the CNS or peripheral nervous system. We also isolated neurological health loss from conditions for which nervous system morbidity is a consequence, but not the primary feature, including a subset of congenital conditions (ie, chromosomal anomalies and congenital birth defects), neonatal conditions (ie, jaundice, preterm birth, and sepsis), infectious diseases (ie, COVID-19, cystic echinococcosis, malaria, syphilis, and Zika virus disease), and diabetic neuropathy. By conducting a sequela-level analysis of the health outcomes for these conditions, only cases where nervous system damage occurred were included, and YLDs were recalculated to isolate the non-fatal burden directly attributable to nervous system health loss. A comorbidity correction was used to calculate total prevalence of all conditions that affect the nervous system combined. Findings Globally, the 37 conditions affecting the nervous system were collectively ranked as the leading group cause of DALYs in 2021 (443 million, 95% UI 378–521), affecting 3·40 billion (3·20–3·62) individuals (43·1%, 40·5–45·9 of the global population); global DALY counts attributed to these conditions increased by 18·2% (8·7–26·7) between 1990 and 2021. Age-standardised rates of deaths per 100 000 people attributed to these conditions decreased from 1990 to 2021 by 33·6% (27·6–38·8), and age-standardised rates of DALYs attributed to these conditions decreased by 27·0% (21·5–32·4). Age-standardised prevalence was almost stable, with a change of 1·5% (0·7–2·4). The ten conditions with the highest age-standardised DALYs in 2021 were stroke, neonatal encephalopathy, migraine, Alzheimer's disease and other dementias, diabetic neuropathy, meningitis, epilepsy, neurological complications due to preterm birth, autism spectrum disorder, and nervous system cancer. Interpretation As the leading cause of overall disease burden in the world, with increasing global DALY counts, effective prevention, treatment, and rehabilitation strategies for disorders affecting the nervous system are needed. Funding Bill & Melinda Gates Foundation

Global, regional, and national burden of disorders affecting the nervous system, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

BACKGROUND: Disorders affecting the nervous system are diverse and include neurodevelopmental disorders, late-life neurodegeneration, and newly emergent conditions, such as cognitive impairment following COVID-19. Previous publications from the Global Burden of Disease, Injuries, and Risk Factor Study estimated the burden of 15 neurological conditions in 2015 and 2016, but these analyses did not include neurodevelopmental disorders, as defined by the International Classification of Diseases (ICD)-11, or a subset of cases of congenital, neonatal, and infectious conditions that cause neurological damage. Here, we estimate nervous system health loss caused by 37 unique conditions and their associated risk factors globally, regionally, and nationally from 1990 to 2021. METHODS: We estimated mortality, prevalence, years lived with disability (YLDs), years of life lost (YLLs), and disability-adjusted life-years (DALYs), with corresponding 95% uncertainty intervals (UIs), by age and sex in 204 countries and territories, from 1990 to 2021. We included morbidity and deaths due to neurological conditions, for which health loss is directly due to damage to the CNS or peripheral nervous system. We also isolated neurological health loss from conditions for which nervous system morbidity is a consequence, but not the primary feature, including a subset of congenital conditions (ie, chromosomal anomalies and congenital birth defects), neonatal conditions (ie, jaundice, preterm birth, and sepsis), infectious diseases (ie, COVID-19, cystic echinococcosis, malaria, syphilis, and Zika virus disease), and diabetic neuropathy. By conducting a sequela-level analysis of the health outcomes for these conditions, only cases where nervous system damage occurred were included, and YLDs were recalculated to isolate the non-fatal burden directly attributable to nervous system health loss. A comorbidity correction was used to calculate total prevalence of all conditions that affect the nervous system combined. FINDINGS: Globally, the 37 conditions affecting the nervous system were collectively ranked as the leading group cause of DALYs in 2021 (443 million, 95% UI 378–521), affecting 3·40 billion (3·20–3·62) individuals (43·1%, 40·5–45·9 of the global population); global DALY counts attributed to these conditions increased by 18·2% (8·7–26·7) between 1990 and 2021. Age-standardised rates of deaths per 100 000 people attributed to these conditions decreased from 1990 to 2021 by 33·6% (27·6–38·8), and age-standardised rates of DALYs attributed to these conditions decreased by 27·0% (21·5–32·4). Age-standardised prevalence was almost stable, with a change of 1·5% (0·7–2·4). The ten conditions with the highest age-standardised DALYs in 2021 were stroke, neonatal encephalopathy, migraine, Alzheimer's disease and other dementias, diabetic neuropathy, meningitis, epilepsy, neurological complications due to preterm birth, autism spectrum disorder, and nervous system cancer. INTERPRETATION: As the leading cause of overall disease burden in the world, with increasing global DALY counts, effective prevention, treatment, and rehabilitation strategies for disorders affecting the nervous system are needed

Effects of fluoxetine on functional outcomes after acute stroke (FOCUS): a pragmatic, double-blind, randomised, controlled trial

Background Results of small trials indicate that fluoxetine might improve functional outcomes after stroke. The FOCUS trial aimed to provide a precise estimate of these effects. Methods FOCUS was a pragmatic, multicentre, parallel group, double-blind, randomised, placebo-controlled trial done at 103 hospitals in the UK. Patients were eligible if they were aged 18 years or older, had a clinical stroke diagnosis, were enrolled and randomly assigned between 2 days and 15 days after onset, and had focal neurological deficits. Patients were randomly allocated fluoxetine 20 mg or matching placebo orally once daily for 6 months via a web-based system by use of a minimisation algorithm. The primary outcome was functional status, measured with the modified Rankin Scale (mRS), at 6 months. Patients, carers, health-care staff, and the trial team were masked to treatment allocation. Functional status was assessed at 6 months and 12 months after randomisation. Patients were analysed according to their treatment allocation. This trial is registered with the ISRCTN registry, number ISRCTN83290762. Findings Between Sept 10, 2012, and March 31, 2017, 3127 patients were recruited. 1564 patients were allocated fluoxetine and 1563 allocated placebo. mRS data at 6 months were available for 1553 (99·3%) patients in each treatment group. The distribution across mRS categories at 6 months was similar in the fluoxetine and placebo groups (common odds ratio adjusted for minimisation variables 0·951 [95% CI 0·839–1·079]; p=0·439). Patients allocated fluoxetine were less likely than those allocated placebo to develop new depression by 6 months (210 [13·43%] patients vs 269 [17·21%]; difference 3·78% [95% CI 1·26–6·30]; p=0·0033), but they had more bone fractures (45 [2·88%] vs 23 [1·47%]; difference 1·41% [95% CI 0·38–2·43]; p=0·0070). There were no significant differences in any other event at 6 or 12 months. Interpretation Fluoxetine 20 mg given daily for 6 months after acute stroke does not seem to improve functional outcomes. Although the treatment reduced the occurrence of depression, it increased the frequency of bone fractures. These results do not support the routine use of fluoxetine either for the prevention of post-stroke depression or to promote recovery of function. Funding UK Stroke Association and NIHR Health Technology Assessment Programme