The spread of COVID-19 outbreak in the first 120 days: a comparison between Nigeria and seven other countries.

BACKGROUND: COVID-19 is an emerging public health emergency of international concern. The trajectory of the global spread is worrisome, particularly in heavily populated countries such as Nigeria. The study objective was to assess and compare the pattern of COVID-19 spread in Nigeria and seven other countries during the first 120 days of the outbreak. METHODS: Data was extracted from the World Bank's website. A descriptive analysis was conducted as well as modelling of COVID-19 spread from day one through day 120 in Nigeria and seven other countries. Model fitting was conducted using linear, quadratic, cubic and exponential regression methods (α=0.05). RESULTS: The COVID-19 spread pattern in Nigeria was similar to the patterns in Egypt, Ghana and Cameroun. The daily death distribution in Nigeria was similar to those of six out of the seven countries considered. There was an increasing trend in the daily COVID-19 confirmed cases in Nigeria. During the lockdown, the growth rate in Nigeria was 5.85 (R2=0.728, p< 0.001); however, it was 8.42 (R2=0.625, p< 0.001) after the lockdown was relaxed. The cubic polynomial model (CPM) provided the best fit for predicting COVID-19 cumulative cases across all the countries investigated and there was a clear deviation from the exponential growth model. Using the CPM, the predicted number of cases in Nigeria at 3-month (30 September 2020) was 155,467 (95% CI:151,111-159,824, p< 0.001), all things being equal. CONCLUSIONS: Improvement in COVID-19 control measures and strict compliance with the COVID-19 recommended protocols are essential. A contingency plan is needed to provide care for the active cases in case the predicted target is attained.

Serological and molecular surveys of Anaplasma spp. in Egyptian cattle reveal high A. marginale infection prevalence.

BACKGROUND: Bovine anaplasmosis is an infectious disease with worldwide distribution. It spreads by various routes mainly through tick bites. AIMS: This study aimed to investigate bovine related Anaplasma spp. in cattle from three northern governorates of Egypt by serological and molecular assays, to evaluate the associated risk factors and to analyze the phylogeny of revealed A. marginale isolates. METHODS: During 2020, a total of 650 blood samples were collected from asymptomatic cattle in the governorates of Kafr El-Sheikh (n=240), Menofia (n=230), and Al-Gharbia (n=180). Sera samples were examined using the Anaplasma antibody test kit, cELISA v2. Blood genomic DNA of seropositive cattle was then examined by PCRs specific to A. marginale, A. centrale, and A. bovis. Selected positive samples were subjected to nucleotide sequencing. Risk factors (i.e. geographical area, breed, type of production, sex, age, herd size, season, husbandry system, tick infestation, and application of acaricides) were evaluated by logistic regression approach. RESULTS: In total, 130 cattle (20%, 95% CI: 17.1-23.3) were recorded seropositive for Anaplasma species. Major risk factors associated with seropositivity were being crossbred, dairy cattle, aged more than 5 years, summer season, herd size of below 300, pasture grazing, tick infestation, and not being subjected to regular treatment with acaricides. By using species-specific PCR, only A. marginale was detected. Nucleotide sequencing showed the occurrence of two different msp4 genotypes. CONCLUSION: This study shows the high prevalence of A. marginale in cattle of Kafr El-Sheikh, Al-Gharbia, and Menofia. However, the connection between Anaplasma species and their tick vectors remains unknown in Egypt and merits further investigations. Since these infections primarily spread through ixodid tick bites, effective ectoparasite control strategies, regular examination of cattle and successful chemoprophylaxis are recommended.

Antibiotic resistance profile of Gram-negative bacteria isolated from Lafenwa abattoir effluent and its receiving water (Ogun River) in Abeokuta, Ogun state, Nigeria.

Untreated abattoir effluent constitutes potential reservoir for transmission of pathogenic strains of multiple antibiotic-resistant bacteria by pollution of surface and ground water sources. This study was carried out to determine the antibiotic resistance and extended spectrum ß-lactamase (ESBL) production profiles of Gram-negative bacteria isolated from effluent collected from Lafenwa municipal abattoir and its receiving surface water, Ogun River, in Abeokuta, Ogun state, Nigeria. Twelve effluent and 18 water samples were collected for this study. Total heterotrophic and coliform counts were estimated, bacterial identification was performed using standard culture-based procedures, whilst antibiotic resistance profiles of isolated bacteria against five antibiotics (ceftazidime, cefpodoxime, cefotaxime, ertapenem and amoxicillin-clavulanate) and detection of ESBLs were done using disk diffusion and double-disc synergy tests. A total of 54 Gram-negative bacteria were isolated, including Salmonella spp. (9), Escherichia coli (15), Klebsiella spp. (7), Shigella spp. (5), Pseudomonas spp. (12) and Enterobacter spp. (6). Both Enterobacteriaceae and Pseudomonas isolates (31% and 66.6%, respectively) were resistant to all selected antibiotics except ertapenem (98% susceptibility). Overall, 77% isolates had multiple antibiotic resistance index (MARI) values, but none of the antibiotic-resistant isolates showed evidence of ESBL production. The presence of multiple antibiotic-resistant isolates in the effluent and receiving water of Lafenwa abattoir suggests a major risk to public health and food safety. Current methods of waste disposal at the abattoir are unacceptable and greatly reduce the qualities of the processed meat and contaminate the environment. There is a need for improved abattoir waste management and water treatment strategies.

National Clinical Guidance for the Management of Cardiovascular Intervention in the COVID-19 Pandemic: From Bangladesh Society of Cardiovascular Interventions (BSCI).

Since the first recorded case of SARS-CoV-2 in Bangladesh on 8th March 2020, COVID-19 has spread widely through different regions of the country, resulting in a necessity to re-evaluate the delivery of cardiovascular services, particularly procedures pertaining to interventional cardiology in resource-limited settings. Given its robust capacity for human-to-human transmission and potential of being a nosocomial source of infection, the disease has specific implications on healthcare systems and health care professionals faced with performing essential cardiac procedures in patients with a suspected or confirmed diagnosis of COVID-19. The limited resources in terms of cardiac catheterization laboratories that can be designated to treat only COVID positive patients are further compounded by the additional challenges of unavailability of widespread rapid testing on-site at tertiary cardiac hospitals in Bangladesh. This document prepared for our nation by the Bangladesh Society of Cardiovascular Interventions (BSCI) is intended to serve as a clinical practice guideline for cardiovascular health care professionals, with a focus on modifying standard practice of care during the COVID-19 pandemic, in order to ensure continuation of adequate and timely treatment of cardiovascular emergencies avoiding hospital-based transmission of SARS-COV-2 among healthcare professionals and the patients. This is an evolving document based on currently available global data and is tailored to healthcare systems in Bangladesh with particular focus on, but not limited to, invasive cardiology facilities (cardiac catheterization, electrophysiology & pacing labs). This guideline is limited to the provision of cardiovascular care, and it is expected that specific targeted pharmaco-therapeutics against SARS-CoV-2 be prescribed as stipulated by the National Guidelines on Clinical Management of Corona virus Disease 2019 (COVID-19) published by the Director General of Health Services, Ministry of Health and Family Welfare of Bangladesh.

Molecular epidemiology, risk factors and hematological evaluation of asymptomatic Theileria annulata infected cattle in Odisha, India.

BACKGROUND: Theileria annulata is a tick-borne apicomplexan parasite that affects bovine and causes severe economic losses. Aims: Our study aimed to determine the molecular prevalence of T. annulata infection in asymptomatic carrier cattle in Odisha, India, to study the association of potential risk factors with theileriosis, and to investigate the effect of the parasite infection on hematological parameters in naturally affected animals. METHODS: A total of 226 cattle blood samples were collected from seven districts of Odisha, India. Molecular diagnoses of tropical theileriosis by polymerase chain reaction (PCR), cloning, sequencing, and phylogenetic analysis of isolated parasites were performed. Potential risk factors were investigated by univariable and multivariable logistic regression statistical analysis. Hematological parameters were compared between positive and negative animals. RESULTS: All animals included in our study were clinically normal, however, 54.86% (124/226) of examined animals were positive by PCR for T. annulata. The multivariable logistic regression showed that contact with other cattle from different herds during grazing (P<0.0001; OR: 12.75; 95% CI: 5.21-31.21), previous history of clinical signs (P=0.002; OR: 3.31; 95% CI: 1.53-6.31), and frequency of a ectoparasiticides application pre year (P<0.0001; OR: 9.22; 95% CI: 3.03-28.09) were the potential risk factors for the occurrence of tropical theileriosis. Nucleotide sequence identity data demonstrated that T. annulata strain (MN818858) Odisha shared homology of 99.6%, 99.49%, and 99.36% with Uttar Pradesh, India (MF346035), Bahrain (AF214797), and Hyderabad, India (MK034702), respectively. CONCLUSION: This is the first study to gain insight into the molecular epidemiology, risk factors, phylogeny, and hematological analysis of asymptomatic T. annulata infected cattle from India.

Characterization of Coxiella burnetii strains from ruminants in a Galleria mellonella host-based model.

Coxiella burnetii is a small Gram-negative intracellular bacterium and is the causative agent of Q fever, which is a zoonotic disease with a worldwide distribution. Domesticated ruminants are the main reservoir of the disease, but the bacterium is able to infect a wide range of hosts, including humans, arthropods and invertebrates. Virulence studies of Coxiella strains usually require a suitable animal model. However, mammalian models are costly and are associated with many ethical constraints. An alternative infection model using Galleria mellonella has been used to study the virulence of several bacterial as well as fungal pathogens. Moreover, the G. mellonella larvae model has been used to identify virulence genes using phase II C. burnetii strain Nine Mile mutants. In our study we describe its use for the characterization of C. burnetii strains isolated from ruminants.

Antibacterial activity of silver nanoparticles against field and reference strains of Mycobacterium tuberculosis, Mycobacterium bovis and multiple-drug-resistant tuberculosis strains.

Tuberculosis (TB) is an endemic disease in animals and humans in Egypt. This study aims to investigate the antimycobacterial activity of silver nanoparticles(AgNPs) by determining the minimal inhibitory concentration (MIC) of AgNPs, using the microplate Alamar blue assay. The AgNPs were chemically synthesised and their form and size were characterised by ultraviolet-visible absorption spectrophotometry, transmission electron microscopy and X-ray diffraction.The reference strains of Mycobacterium bovis and Mycobacterium tuberculosisH37Rv, and one multiple-drug-resistant (MDR) strain of M. tuberculosis were tested, as well as clinical isolates of M. bovis and M. tuberculosis. The AgNPs were tetrahydral with a few spherical particles and an average particle size of 50 nm. The mycobacterial strains were varied with MICs of AgNPs. Both reference strains of M. tuberculosis and M. bovis, in addition to the MDR strain of M. tuberculosis, were successfully inhibited by AgNPs at MICs of 1 ?g/ml, 4 ?g/ml and 16 ?g/ml, respectively, whereas clinical isolates of M. bovis and M. tuberculosis were inhibited at MIC values of 4-32 ?g/ml and 1-16 ?g/ml, respectively. The AgNPs showed an in vitro chemotherapeutic effect against Mycobacterium spp.Thus, they can be used to treat TB not only in humans but also in animals, and maybe useful in TB prevention and control strategies worldwide.

En Egypte, la tuberculose est une maladie endémique chez l'homme comme chez l'animal. Les auteurs présentent les résultats d'une étude conduite pour mesurer l'activité antibactérienne des nanoparticules d'argent (NPAg) en déterminant les valeurs de concentration minimale inhibitrice (CMI) des NPAg au moyen du test au bleu Alamar sur microplaques. Les NPAg ont été synthétisées par une méthode chimique et leur forme et taille ont été caractérisées par spectrophotométrie d'absorption dans l'ultra-violet, microscopie électronique à transmission et diffraction des rayons X. L'étude a cible les souches de référence de Mycobacterium bovis et de Mycobacterium tuberculosis H37Rv, ainsi qu'une souche multirésistante de M. tuberculosis et des isolats cliniques de M. bovis et M. tuberculosis. Les NPAg étaient à structure tétraédrique avec quelques particules sphériques ; la taille moyenne des particules etait de 50 nm.La CMI des NPAg variait en fonction des souches. L'inhibition des deux souches de reference de M. tuberculosis et M. bovis et de la souche multirésistante de M. tuberculosis était obtenue avec des CMI de NPAg de 1 ?g/ml, 4 ?g/ml et16 ?g/ml, respectivement, tandis que les isolats cliniques de M. bovis et de M. tuberculosis étaient inhibés en présence de NPAg à des CMI comprises entre 4 et 32 ?g/ml et 1­16 ?g/ml, respectivement. L'efficacité chimiothérapeutique des NPAg contre Mycobacterium spp. a été démontrée in vitro. Ces nanoparticules peuvent donc servir à traiter la tuberculose non seulement chez l'homme mais également chez les animaux et contribuer ainsi aux stratégies de prévention et de lutte contre la tuberculose dans le monde.

En Egipto, la tuberculosis es una enfermedad endemica que afecta a personas y animales. Los autores describen un estudio encaminado a analizar la actividad antimicobacteriana de las nanoparticulas de plata, determinando para ello la concentracion inhibitoria minima de nanoparticulas mediante el ensayo de microtitulacion en placa con azul Alamar. Tras sintetizar quimicamente las nanoparticulas de plata y caracterizar su forma y tamano por espectrometria de absorcion ultravioleta-visible, microscopia electronica de transmision y difraccion de rayos X, se sometieron a prueba las cepas de referencia de Mycobacterium bovis y Mycobacterium tuberculosis H37Rv, asi como una cepa multirresistente de M. tuberculosis y muestras de M. bovis y M. tuberculosis aisladas a partir de casos clinicos. Salvo unas pocas de forma esferica, las nanoparticulas de plataeran tetraedricas. Su tamano era en promedio de 50 nm. Tras someter las cepas de micobacterias a distintas concentraciones de nanoparticulas, se observo que estas inhibian el crecimiento de las cepas de referencia de M. tuberculosis y M. bovis y de la cepa multirresistente de M. tuberculosis a concentraciones minimas de 1 ?g/ml, 4 ?g/ml y 16 ?g/ml, respectivamente, mientras que las muestras clinicas de M. bovis y M. tuberculosis quedaban inhibidas por la presencia de nanoparticulas a valores de concentracion minima de 4­32 ?g/ml y 1­16 ?g/ml, respectivamente. Tambien se observo que, in vitro, las nanoparticulas de plata mostraban actividad farmacologica contra Mycobacterium spp. De ahi se sigue que pueden ser empleadas para tratar la tuberculosis no solo en personas, sino tambien en animales, y que pueden resultar utiles en todo el mundo para las estrategias de prevencion y control de la tuberculosis.

Pathogenicity of the Egyptian A/H5N1 avian influenza viruses in chickens.

Long-term circulation of highly pathogenic avian influenza H5N1 viruses of clade 2.2.1 in Egyptian poultry since February 2006 resulted in the evolution of two distinct clades: 2.2.1.1 represents antigenic-drift variants isolated from vaccinated poultry and 2.2.1.2 that caused the newest upsurge in birds and humans in 2014/2015. In the present study, nine isolates were collected from chickens, ducks and turkeys representing the commercial and backyard sectors during the period 2009-2015. The subtyping was confirmed by hemagglutination inhibition (HI) test, RT-qPCR and sequence analysis. The Mean Death Time (MDT) and Intravenous Pathogenicity Index (IVPI) for all isolates were determined. Sequence analysis of the HA gene sequences of these viruses revealed that two viruses belonged to clade 2.2.1.1 and the rest were clade 2.2.1.2. Antigenic characterisation of the viruses supported the results of the phylogenetic analysis. The MDT of the isolates ranged from 18 to 72 h and the IVPI values ranged from 2.3 to 2.9; viruses of the 2.2.1.1 clade were less virulent than those of the 2.2.1.2 clade. In addition, clade-specific polymorphism in the HA cleavage site was observed. These findings indicate the high and variable pathogenicity of H5N1 viruses of different clades and host-origin in Egypt. The upsurge of outbreaks in poultry in 2014/2015 was probably not due to a shift in virulence from earlier viruses.

Bacteriological and molecular studies of Clostridium perfringens infections in newly born calves.

Clostridium perfringens is considered one of the important causes of calf diarrhea. Two hundred and twenty-seven clinical samples from newly born and dead diarrheic calves were examined bacteriologically and by PCR. Bacterial culture identified C. perfringens in 168 of 227 samples. A total of 144 of these isolates were lecithinase positive, indicating C. perfringens Type A. In addition, 154 isolates were positive by alpha toxin encoding gene-PCR assay. This study showed high agreement between the results of bacteriology and multiplex PCR. The multiplex PCR typed all isolates that were typed as C. perfringens Type A through bacteriologic methods, but ten samples that were lecithinase negative were positive in the multiplex PCR. The study showed the highest occurrence of C. perfringens Type A isolations from calves during the winter and autumn compared with other seasons.

Risk assessment of recent Egyptian H5N1 influenza viruses.

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype are enzootic in poultry populations in different parts of the world, and have caused numerous human infections in recent years, particularly in Egypt. However, no sustained human-to-human transmission of these viruses has yet been reported. We tested nine naturally occurring Egyptian H5N1 viruses (isolated in 2014-2015) in ferrets and found that three of them transmitted via respiratory droplets, causing a fatal infection in one of the exposed animals. All isolates were sensitive to neuraminidase inhibitors. However, these viruses were not transmitted via respiratory droplets in three additional transmission experiments in ferrets. Currently, we do not know if the efficiency of transmission is very low or if subtle differences in experimental parameters contributed to these inconsistent results. Nonetheless, our findings heighten concern regarding the pandemic potential of recent Egyptian H5N1 influenza viruses.

Introduction and enzootic of A/H5N1 in Egypt: Virus evolution, pathogenicity and vaccine efficacy ten years on.

It is almost a decade since the highly pathogenic H5N1 avian influenza virus (A/H5N1) of clade 2.2.1 was introduced to Egypt in 2005, most likely, via wild birds; marking the longest endemic status of influenza viruses in poultry outside Asia. The endemic A/H5N1 in Egypt still compromises the poultry industry, poses serious hazards to public health and threatens to become potentially pandemic. The control strategies adopted for A/H5N1 in Egyptian poultry using diverse vaccines in commercialized poultry neither eliminated the virus nor did they decrease its evolutionary rate. Several virus clades have evolved, a few of them disappeared and others prevailed. Disparate evolutionary traits in both birds and humans were manifested by accumulation of clade-specific mutations across viral genomes driven by a variety of selection pressures. Viruses in vaccinated poultry populations displayed higher mutation rates at the immunogenic epitopes, promoting viral escape and reducing vaccine efficiency. On the other hand, viruses isolated from humans displayed changes in the receptor binding domain, which increased the viral affinity to bind to human-type glycan receptors. Moreover, viral pathogenicity exhibited several patterns in different hosts. This review aims to provide an overview of the viral evolution, pathogenicity and vaccine efficacy of A/H5N1 in Egypt during the last ten years.

Emergence of a novel cluster of influenza A(H5N1) virus clade 2.2.1.2 with putative human health impact in Egypt, 2014/15.

A distinct cluster of highly pathogenic avian influenzaviruses of subtype A(H5N1) has been found to emergewithin clade 2.2.1.2 in poultry in Egypt since summer2014 and appears to have quickly become predominant.Viruses of this cluster may be associated withincreased incidence of human influenza A(H5N1) infectionsin Egypt over the last months.

Evolution of highly pathogenic avian influenza H5N1 viruses in Egypt indicating progressive adaptation.

Highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype was first diagnosed in poultry in Egypt in 2006, and since then the disease became enzootic in poultry throughout the country, affecting the poultry industry and village poultry as well as infecting humans. Vaccination has been used as a part of the control strategy to help to control the disease. Epidemiological data with sequence analysis of H5N1 viruses is important to link the mechanism of virus evolution in Egypt. This study describes the evolutionary pattern of Egyptian H5N1 viruses based on molecular characterization for the isolates collected from commercial poultry farms and village poultry from 2006 to 2011. Genetic analysis of the hemagglutinin (HA) gene was done by sequencing of the full-length H5 gene. The epidemiological pattern of disease outbreaks in Egyptian poultry farms seems to be seasonal with no specific geographic distribution across the country. The molecular epidemiological data revealed that there are two major groups of viruses: the classic group of subclade 2.2.1 and a variant group of 2.2.1.1. The classic group is prevailing mainly in village poultry and had fewer mutations compared to the originally introduced virus in 2006. Since 2009, this group has started to be transmitted back to commercial sectors. The variant group emerged by late 2007, was prevalent mainly in vaccinated commercial poultry, mutated continuously at a higher rate until 2010, and started to decline in 2011. Genetic analysis of the neuraminidase (NA) gene and the other six internal genes indicates a grouping of the Egyptian viruses similar to that obtained using the HA gene, with no obvious reassortments. The results of this study indicate that HPAI-H5N1 viruses are progressively evolving and adapting in Egypt and continue to acquire new mutations every season.

Pathogenicity of two Egyptian H5N1 highly pathogenic avian influenza viruses in domestic ducks.

Domestic ducks have been implicated in the dissemination and evolution of H5N1 highly pathogenic avian influenza (HPAI) viruses. In this study, two H5N1 HPAI viruses belonging to clade 2.2.1 isolated in Egypt in 2007 and 2008 were analyzed for their pathogenicity in domestic Pekin ducks. Both viruses produced clinical signs and mortality, but the 2008 virus was more virulent, inducing early onset of neurological signs and killing all ducks with a mean death time (MDT) of 4.1 days. The 2007 virus killed 3/8 ducks with a MDT of 7 days. Full-genome sequencing and phylogenetic analysis were used to examine differences in the virus genes that might explain the differences observed in pathogenicity. The genomes differed in 49 amino acids, with most of the differences found in the hemagglutinin protein. This increase in pathogenicity in ducks observed with certain H5N1 HPAI viruses has implications for the control of the disease, since vaccinated ducks infected with highly virulent strains shed viruses for longer periods of time, perpetuating the virus in the environment and increasing the possibility of transmission to susceptible birds.

Isolation of highly pathogenic avian influenza H5N1 from table eggs after vaccinal break in commercial layer flock.

In May 2009, during routine monitoring of a commercial layer flock of about 87,000 birds kept in cages in 4 different houses that had been vaccinated 3 times with an inactivated H5N1 vaccine at weeks 1, 7, and 16, highly pathogenic avian influenza (HPAI) virus of subtype H5N1 was isolated and detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) in tracheal and cloacal swabs collected from houses 3 and 4; 7 days after onset of clinical signs, there was an increase in mortality accompanied by a decrease in egg production and egg quality. In addition, using RT-PCR, the viral RNA could be detected from albumin and eggshell as well. Seven days after the onset of the clinical signs, the hemagglutination inhibition (HI) titers in the affected houses were 3.2 and 1.9 log2. In the other two houses, there were no clinical signs, and all tested samples were negative using virus isolation and real-time RT-PCR. The HI titers were 6.6 and 7.0 log2 in nonaffected houses. The isolated virus from egg albumin showed high nucleotides and amino-acid identities and clustered with viruses from recently H5N1-confirmed human infections and poultry from different places in Egypt. Moreover, several amino-acid substitutions of viral H5 protein were observed. The vaccinal break seems to be associated with immune escape mutants and/or improper vaccination. The role of contaminated eggs as a source of infection and as a vehicle for spread of the virus should be considered in area with avian influenza outbreaks.

Avian influenza H5N1 virus infections in vaccinated commercial and backyard poultry in Egypt.

In this paper, we describe results from a high-pathogenic H5N1 avian influenza virus (AIV) surveillance program in previously H5-vaccinated commercial and family-backyard poultry flocks that was conducted from 2007 to 2008 by the Egyptian National Laboratory for Veterinary Quality Control on Poultry Production. The real-time reverse transcription PCR assay was used to detect the influenza A virus matrix gene and detection of the H5 and N1 subtypes was accomplished using a commercially available kit real-time reverse transcription PCR assay. The virus was detected in 35/3,610 (0.97%) and 27/8,682 (0.31%) of examined commercial poultry farms and 246/816 (30%) and 89/1,723 (5.2%) of backyard flocks in 2007 and 2008, respectively. Positive flocks were identified throughout the year, with the highest frequencies occurring during the winter months. Anti-H5 serum antibody titers in selected commercial poultry ranged from <2 (negative) to 9.6 log(2) when determined in the hemagglutination inhibition test using a H5 AIV antigen. In conclusion, despite the nationwide vaccination strategy of poultry in Egypt to combat H5N1 AIV, continuous circulation of the virus in vaccinated commercial and backyard poultry was reported and the efficacy of the vaccination using a challenge model with the current circulating field virus should be revised.

Circulation of avian influenza H5N1 in live bird markets in Egypt.

The poultry meat trade in Egypt depends mainly on live bird markets (LBMs) because of insufficient slaughterhouses, lack of marketing infrastructure, and cultural preference for consumption of freshly slaughtered poultry. There are two types of LBMs in Egypt: retail shops and traditional LBMs where minimal, if any, food safety standards or veterinary inspection are implemented. Before January 2009, LBMs were considered to be a missing link in the epidemiology of avian influenza in Egypt. This incited us to initiate this surveillance to better understand the perpetuation of H5N1 and the risk of infection in poultry markets. Seventy-one out of 573 (12.4%) examined LBMs were positive for the H5N1 subtype by real-time--quantitative polymerase chain reaction (RT-qPCR) from January to April 2009. Where a 70.4% detection rate from LBMs had waterfowl only as a solitary sold species, a 26.8% detection rate from LBMs had waterfowl mixed with chicken and/or turkey, and 2.8% from LBMs had only turkey. Higher incidence, 40.8%, of positive LBMs was recorded during the cold month of February and concentrated mainly in the highly populated Nile Delta. These findings revealed wide circulation of H5N1 avian influenza virus in LBMs in Egypt, which poses a threat to public health and the poultry industry. Long-term control measures are required, and routine surveillance of bird markets should be conducted year-round.

Genetic characterization of variant strains of highly pathogenic avian influenza H5N1 that escaped detection by real-time reverse transcriptase-PCR diagnostic tests.

The highly pathogenic avian influenza virus of subtype H5N1 that caused serious outbreaks in Egypt in 2006 was efficiently detected using a commercially available real-time reverse transcriptase-PCR (RRT-PCR) for the type A specific matrix (M) gene in field samples of cloacal and tracheal swabs. RRT-PCR was also used for subtyping and confirmation of H5 subtype. During late 2007 the National Laboratory for Veterinary Quality Control on Poultry Production detected five field cases that were positive for avian influenza virus (AIV) based on the M gene RRT-PCR. Three different commercial H5 RRT-PCRs were used for identification of the H5 subtype, as well as a published World Organization for Animal Health (OIE) H5 RRT-PCR that had been previously carefully validated. The five cases had positive results for the H5 gene using the published OIE H5 RRT-PCR, but the three commercial H5 RRT-PCRs tests only returned two to four positive results out of the five positive cases. The hemagglutinin gene (HA) sequencing analysis of these five isolates showed multiple nucleotide substitution mutations, suggesting genetic variation that could affect the H5 primer and/or probe binding sequences. These data highlight the importance of continued monitoring of RRT-PCR primers and probes to ensure that sensitivity and specificity are maintained. The use of conventional methods in national and reference AIV laboratories, including virus isolation, serologic subtyping, and alternative RRT-PCR primers, is necessary to detect the newly emerging variant H5N1 strains that affect diagnostic performance.