Direct Fluorescent Antibody Assay Research Articles

Bardoxolone methyl inhibits the infection of rabies virus via Nrf2 pathway activation in vitro

BackgroundRabies is a widespread, fatal, infectious disease. Several antivirals against rabies virus (RABV) infection have been reported, but no approved, RABV-specific antiviral drugs that inhibit RABV infection in the clinic after symptom onset are available. Therefore, more effective drugs to reduce rabies fatalities are urgently needed. Bardoxolone methyl (CDDO-Me), an FDA-approved compound that has long been known as an antioxidant inflammatory modulator and one of the most potent nuclear factor erythroid-derived 2-like 2 (Nrf2) activators, protects myelin, axons, and CNS neurons by Nrf2 activation. Therefore, we investigated the potency of its anti-RABV activity in vitro.MethodsThe mouse neuroblastoma cell line Neuro2a (N2a) and three RABV strains of different virulence were used; the cytotoxicity and anti-RABV activity of CDDO-Me in N2a cells were evaluated by CCK-8 assay and direct fluorescent antibody (DFA) assay. Pathway activation in N2a cells infected with the RABV strains SC16, CVS-11 or CTN upon CDDO-Me treatment was evaluated by western blotting (WB) and DFA assay.ResultsCDDO-Me significantly inhibited infection of the three RABV strains of differing virulence (SC16, CVS-11 and CTN) in N2a cells. We also examined whether CDDO-Me activates the Nrf2-associated pathway upon infection with RABV strains of differing virulence. Nrf2, phosphorylated sequestosome (SQSTM1), SQSTM1, hemoglobin oxygenase (HO-1) and NAD(P)H dehydrogenase quinone 1 (NQO1) expression in N2a cells increased to varying degrees with CDDO-Me treatment, accompanied by Kelch-like ECH-associated protein 1 (Keap1) dissociation, upon infection with SC16, CVS-11 or CTN. The activation of SQSTM1 phosphorylation was significantly associated with the degradation of Keap-1 in CDDO-Me-treated N2a cells upon RABV infection. Furthermore, N2a cells pretreated with the Nrf2-specific inhibitor ATRA showed a significant decrease in HO-1 and NQO1 expression and a decrease in the anti-RABV efficacy of CDDO-Me. These inhibitory effects were observed upon infection with three RABV strains of differing virulence.ConclusionCDDO-Me inhibited RABV infection via Nrf2 activation, promoting a cytoprotective defense response in N2a cells. Our study provides a therapeutic strategy for RABV inhibition and neuroprotection during viral infection.

Epidemiology of Viruses Causing Pediatric Community Acquired Pneumonia in Shanghai During 2010-2020: What Happened Before and After the COVID-19 Outbreak?

IntroductionSince the global outbreak of COVID-19, there has been a significant reduction in pediatric outpatient and emergency visits for infectious diseases. The purpose of this study was to analyze the changes in respiratory viruses in children with community-acquired pneumonia (CAP) in Shanghai in the past 10 years, especially in the first year after COVID-19.MethodsWe conducted a retrospective, observational study; the results for eight common respiratory viruses (respiratory syncytial virus (RSV), influenza virus A and B, parainfluenza virus 1–3 (PIV), adenovirus (ADV) and human metapneumovirus) tested by direct fluorescent antibody assays in hospitalized CAP cases in Children’s Hospital of Fudan University during 2010–2020 were analyzed.ResultsOf the 5544 hospitalized CAP patients included in this study, 20.2% (1125/5544) were positive for the eight respiratory viruses. The top three pathogens were RSV, PIV3 and ADV, detected from 9.8% (543/5544), 5.3% (294/5544) and 2.0% (111/5544) of the samples, respectively. RSV had the highest positive rates among children < 2 years old. In 2020, the detection rate of all viruses showed a sharp decline from February to August compared with the previous 9 years. When the Shanghai community reopened in August 2020, the detection rate of eight viruses rebounded significantly in September.ConclusionsThese eight respiratory viruses, especially RSV and PIV, were important pathogens of CAP in Shanghai children in the past 10 years. The COVID-19 pandemic had a significant impact on the detection rates for eight respiratory viruses in children with CAP in Shanghai.

Development and Evaluation of a Fully Automated Molecular Assay Targeting the Mitochondrial Small Subunit rRNA Gene for the Detection of Pneumocystis jirovecii in Bronchoalveolar Lavage Fluid Specimens

The fungal pathogen Pneumocystis jirovecii causes Pneumocystis pneumonia. Although the mitochondrial large subunit rRNA gene (mtLSU) is commonly used as a PCR target, a mitochondrial small subunit rRNA gene (mtSSU)-targeted MultiCode PCR assay was developed on the fully automated ARIES platform for detection of P. jirovecii in bronchoalveolar lavage fluid specimens in 2.5hours. The assay showed a limit of detection of 800 copies/mL (approximately equal to 22 organisms/mL), with no cross-reactivity with other respiratory pathogens. Compared with the reference Pneumocystis-specific direct fluorescent antibody assay (DFA) and mtLSU-targeted PCR assay, the new assay demonstrated sensitivity of 96.9% (31/32) and specificity of 94.6% (139/147) in detecting P.jirovecii in 180 clinical bronchoalveolar lavage fluid specimens. This assay was concordant with all DFA-positive samples and all but one mtLSU PCR-positive sample, and detected eight positive samples that were negative by DFA and mtLSU PCR. Receiver operating characteristic curve analysis revealed an area under the curve of 0.98 and a threshold cycle (CT) cutoff of 39.1 with sensitivity of 90.9% and specificity of 99.3%. The detection of 39.1 <CT<40.0 indicates the presence of a low load of the organism and needs further determination of either colonization or probable/possible Pneumocystis pneumonia. Overall, the new assay demonstrates excellent analytical and clinical performance and may be more sensitive than mtLSU PCR target for the detection of P. jirovecii.

Rabies viruses in specific wild fur animals in northern China, 2017–2019

In recent years, rabies virus (RABV) has been detected in numerous specific wild fur animals in northern China. Therefore, we performed an epidemiologic investigation of RABV in the main fur animal farming provinces during 2017-2019. The results showed that brain tissue samples from eight animals that presented with central nervous symptoms were positive for rabies virus according to direct fluorescent antibody assays and RT-PCR. The phylogenetic relationships and distributions of the viruses were determined, and the results indicated that they belonged to Cosmopolitan and Arctic-related lineages. Serological investigations revealed a RABV positivity rate of 2.78% (34/1,222) in fur animals. A total of 79 unimmunized breeders were negative for serum antibodies, and 9.62% of 52 immunized breeders (5/52) were not seroconverted. The results emphasize that specific wild fur animals are potential sources of RABV and that the current vaccination programme for animals and breeders is deficient, indicating the need for mandatory rabies vaccination to eliminate rabies transmission from dogs to farmed fur animals.

Molecular evidence of bovine herpesvirus 1 and 5 in cattle with suspected rabies in Rio Grande do Sul state, Brazil

Rabies and herpetic encephalitis are the main viral infections in bovines with neurological symptoms. Bovine rabies has a high prevalence in Central and South America, while bovine encephalitis associated with herpesvirus is especially important in South America. Viral isolation is the classical way to confirm herpesvirus infection, but molecular evidence of the presence of the virus in affected animals is gaining importance in the diagnosis of the disease in the laboratory. This study investigated the presence of herpesvirus type 1 and 5 (BoHV-1 and BoHV-5) in 182 encephalon of rabies-suspected cattle in Rio Grande do Sul state (RS), Brazil using multiplex real-time polymerase chain reaction (mRT-PCR). The rabies virus was investigated by direct fluorescent antibody assay and intracerebral suckling mouse inoculation. The genomes of BoHV-1 and BoHV-5 were detected in 17% of samples. BoHV-5 and BoHV-1 were detected in 100% and 19% of BoHV positive samples, respectively, indicating the circulation of the pathogens in cattle herds in RS. The high Ct values and the absence of isolation suggest viral latency. Coinfection of herpesvirus and the rabies virus was detected in 28% of samples, although no significant association between pathogens was observed. Rabies was detected in 57.7% of suspected samples, confirming the importance of the disease in the state. Concerning the method by which samples were conserved, no significant difference was observed between the number of positive results in frozen and refrigerated samples.

Production of Antibody for Direct Fluorescence Antibody Assay against Avian Influenza H9N2

Production of Antibody for Direct Fluorescence Antibody Assay against Avian Influenza H9N2

A pathological study of Leishmania infantum natural infection in European rabbits (Oryctolagus cuniculus) and Iberian hares (Lepus granatensis).

In this study, we describe the pathology of Leishmania infantum infection in naturally infected wild Leporidae and compare diagnosis of infection using histopathology, direct fluorescent antibody (DFA) assay, immunofluorescence antibody test (IFAT) and quantitative real-time PCR (qPCR). Tissues were analysed from 52 European rabbits (Oryctolagus cuniculus) and 7 Iberian hares (Lepus granatensis) from the Community of Madrid (Spain). Our results show that L. infantum infection is associated with only minimal histopathological lesions and that L. infantum amastigotes can be detected by DFA assay in all tissues types tested, including skin. These results were confirmed by qPCR on fresh frozen tissues in 13% of rabbits and 100% of hares. However, L. infantum DNA could not be detected by qPCR on paraffin-embedded tissue obtained by laser capture microdissection. Using the DFA assay to diagnose L. infantum, infection may provide further insights into this disease in wild animals and may allow the precise tissue localization of L. infantum, thereby guiding follow-up tests with more accurate qPCR.

Human metapneumovirus in Pediatric Patients with Acute Respiratory Tract Infections in the Aseer Region of Saudi Arabia.

Background:Human metapneumovirus (hMPV) is a Paramyxovirus known to cause acute respiratory tract infections in children and young adults. To date, there is no study from the Aseer region of Saudi Arabia determining the proportion and severity of hMPV infection among pediatric hospitalized patients with respiratory infections.Objectives:The objective of this study is to determine the presence of hMPV antigens in the nasopharyngeal secretions of pediatric patients hospitalized with respiratory tract infections in the Aseer region of Saudi Arabia.Materials and Methods:This prospective, serological hospital-based study included all pediatric patients who were admitted to Aseer Central Hospital, Abha, Saudi Arabia, from July 2016 to November 2017 with upper and/or lower respiratory tract infections. Basic demographics of patients and their clinical data on and after admission were recorded. Direct fluorescent antibody assay was used to detect the presence of hMPV antigens in the obtained nasopharyngeal secretion specimens.Results:During the study, 91 pediatric patients were hospitalized due to upper and/or lower respiratory tract infections, of which 9.9% were positive for hMPV. These patients were aged 9 months to 16 years, were from Abha city or its surrounding localities and were mostly (77.8%) hospitalized during autumn or winter. The most common diagnosis on admission was bronchopneumonia (55.5%) and aspiration pneumonia (22.2%), and some patients also had underlying chronic conditions such as chronic heart disease (22.2%) and bronchial asthma (11.1%).Conclusions:The results obtained indicated that hMPV is a potential etiologic factor for the commonly occurring acute respiratory infections in hospitalized children from the Aseer region of Saudi Arabia. hMPV infection was also found to be associated with complicated respiratory conditions such as bronchopneumonia, chronic heart disease and bronchial asthma.

Molecular characterization of a rabies virus isolated from trade dogs in Plateau State, Nigeria

To have a better understanding of the prevalence of rabies virus (RABV) in the dog trading markets in Plateau State of Nigeria, a total of 532 brain samples collected between 2010 and 2011 were subjected to rabies virus antigen detection and molecular characterization. RABV antigen was detected in 92 out of 532 (17.3%) brain samples from two major commercial dog markets in Plateau State of Nigeria by direct fluorescence antibody assay, indicating the possibility of a high prevalence of RABV in the dog trading markets. The complete genomic sequence was obtained from one of these RABV positive samples. The overall organization of this virus (DRV-NG11) was typical of all the other wild-type RABV. Phylogenetic analysis using either the complete or partial genomic sequence of DRV-NG11 demonstrates that this isolate is most closely related to viruses previously shown to circulate in Nigeria, belonging to the Africa 2 Clade. To our knowledge, this is the first report on the molecular characterization of the complete genome of RABV from trade dogs, which provides a better understanding of the molecular epidemiology, pathogenesis and rabies control in this country.Keywords: Genome, Nigeria, Rabies virus, Street strain, Trade dogs

Biofilms Comprise a Component of the Annual Cycle of Vibrio cholerae in the Bay of Bengal Estuary.

ABSTRACTVibrio cholerae, an estuarine bacterium, is the causative agent of cholera, a severe diarrheal disease that demonstrates seasonal incidence in Bangladesh. In an extensive study of V. cholerae occurrence in a natural aquatic environment, water and plankton samples were collected biweekly between December 2005 and November 2006 from Mathbaria, an estuarine village of Bangladesh near the mangrove forests of the Sundarbans. Toxigenic V. cholerae exhibited two seasonal growth peaks, one in spring (March to May) and another in autumn (September to November), corresponding to the two annual seasonal outbreaks of cholera in this region. The total numbers of bacteria determined by heterotrophic plate count (HPC), representing culturable bacteria, accounted for 1% to 2.7% of the total numbers obtained using acridine orange direct counting (AODC). The highest bacterial culture counts, including toxigenic V. cholerae, were recorded in the spring. The direct fluorescent antibody (DFA) assay was used to detect V. cholerae O1 cells throughout the year, as free-living cells, within clusters, or in association with plankton. V. cholerae O1 varied significantly in morphology, appearing as distinctly rod-shaped cells in the spring months, while small coccoid cells within thick clusters of biofilm were observed during interepidemic periods of the year, notably during the winter months. Toxigenic V. cholerae O1 was culturable in natural water during the spring when the temperature rose sharply. The results of this study confirmed biofilms to be a means of persistence for bacteria and an integral component of the annual life cycle of toxigenic V. cholerae in the estuarine environment of Bangladesh.

Lymphopenia is associated with late onset Pneumocystis jirovecii pneumonia in solid organ transplantation.

Pneumocystis jirovecii pneumonia (PJP) affected 5%-15% of solid organ transplant (SOT) recipients prior to universal prophylaxis, classically with trimethoprim-sulfamethoxazole (TMP-SMX). Guidelines generally recommend 6-12months of prophylaxis post-SOT, yet optimal duration and robust PJP risk stratification have not been established. A retrospective, single-center, case-control study of PJP among SOT recipients from January 1998 to December 2013 was conducted. Cases had positive PJ direct fluorescent antibody assay of respiratory specimens. Controls were matched 4:1 by nearest date of SOT. Univariate testing and multivariate logistic regressions were performed. Fifteen cases were identified among 5505 SOT recipients (0.27% rate) and analyzed vs 60 controls. PJP occurred on average 6.1years (range 0.9-13.8) post-SOT; no case was receiving PJP prophylaxis at diagnosis. Most were treated with reduced immunosuppression and TMP-SMX plus steroids (80%). Six patients (40%) required critical care; 3 (20%) died. There were no significant demographic differences, though cases tended to be older at SOT (54 vs 48years, P=.1). In univariate analysis, prior viral infection was more common among cases (67% vs 37%, P=.08). Lower absolute lymphocyte count (ALC) at diagnosis date was strongly associated with PJP (400 vs 1230×106 cells/μL, P<.001); odds of infection were high with ALC ≤ 500×106 cells (OR 18.7, P<.01). Pneumocystis jirovecii pneumonia is a rare, late complication of SOT with significant morbidity and mortality. Severe lymphopenia may be useful in identifying SOT recipients who warrant continued or reinstated PJP prophylaxis.

Bacillus anthracis gamma phage lysis among soil bacteria: an update on test specificity

BackgroundBacillus anthracis, which causes anthrax in humans and animals, is enzootic in parts of the U.S. state of Texas where cases are typically reported in animals annually. The gamma phage lysis assay is a common diagnostic method for identification of B. anthracis and is based on the bacterium’s susceptibility to lysis. This test has been shown to be 97% specific for B. anthracis, as a small number of strains of other Bacillus spp. are known to be susceptible. In this study, we evaluated the performance of a combination of B. anthracis diagnostic assays on 700 aerobic, spore-forming isolates recovered from soil collected in Texas. These assays include phenotypic descriptions, gamma phage susceptibility, and real-time polymerase chain reaction specific for B. anthracis. Gamma phage-susceptible isolates were also tested using cell wall and capsule direct fluorescent-antibody assays specific for B. anthracis. Gamma phage-susceptible isolates that were ruled out as B. anthracis were identified by 16S rRNA gene sequencing.FindingsWe identified 29 gamma phage-susceptible isolates. One was confirmed as B. anthracis, while the other 28 isolates were ruled out for B. anthracis by the other diagnostic tests. Using 16S rRNA gene sequencing results, we identified these isolates as members of the B. cereus group, Bacillus sp. (not within B. cereus group), Lysinibacillus spp., and Solibacillus silvestris. Based on these results, we report a specificity of 96% for gamma phage lysis as a diagnostic test for B. anthracis, and identified susceptible isolates outside the Bacillus genus.ConclusionsIn this study we found gamma phage susceptibility to be consistent with previously reported results. However, we identified non-B. anthracis environmental isolates (including isolates from genera other than Bacillus) that are susceptible to gamma phage lysis. To date, susceptibility to gamma phage lysis has not been reported in genera other than Bacillus. Though these isolates are not of clinical origin, description of unexpected positives is important, especially as new diagnostic assays for B. anthracis are being developed based on gamma phage lysis or gamma phage proteins.

Risk factors of respiratory syncytial virus infection among pediatric influenza-like illness and severe acute respiratory infections in Suzhou, China.

The characteristics and risk factors of respiratory syncytial virus (RSV) infection among children has not yet been fully understood. To address the characteristics of RSV-associated illness and risk factors of RSV infection among children under 5 years of age in Suzhou, China. From April 2011 to March 2014, we conducted a prospective surveillance among children in Suzhou, China. Nasal or throat swabs were collected from outpatients with influenza-like illness (ILI) and inpatients with severe acute respiratory infections (SARI). RSV was detected by reverse-transcriptase polymerase chain reaction and direct fluorescent antibody assay for children with ILI and SARI, respectively. Multivariable logistic-regression models were constructed to explore risk factors and symptoms of RSV infection. Of 3267 ILI and 1838 SARI children enrolled in the study, 192 (5.9%) and 287 (15.6%) tested positive for RSV, respectively. Among ILI patients, children with RSV infections visited clinics more often (P = 0.005) and had longer duration of fever (P = 0.032) than those without RSV infection. All RSV-positive children had an increased risk of having cough (OR = 2.9), rhinorrhea (OR = 1.6), breathing difficulty (OR = 3.4), wheezing (OR = 3.3), and irritability (OR = 2.7). Children aged <2 years, had history of prematurity (OR = 2.0) and recent respiratory infections (OR = 1.3) were more likely to get infected by RSV. Children with SARI had higher positive rate of RSV than those with ILI. Cough, rhinorrhea, and wheezing were the most common symptoms in RSV infection. Children aged <2 years, had history of prematurity and recent respiratory infections were the potential risk factors for RSV infection.

Epidemiology of Rabies in Lesotho: The Importance of Routine Surveillance and Virus Characterization.

Rabies is widespread throughout Africa and Asia, despite the fact that the control and elimination of this disease has been proven to be feasible. Lesotho, a small landlocked country surrounded by South Africa, has been known to be endemic for rabies since the 1980s but the epidemiology of the disease remains poorly understood due to limited sample submission, constrained diagnostic capabilities, and a lack of molecular epidemiological data. Considering the existing challenges experienced in Lesotho, we aimed to evaluate the direct, rapid immunohistochemical test (DRIT) as an alternative to the direct fluorescent antibody (DFA) test for rabies diagnosis in Lesotho. Towards this aim, extensive training on the implementation and interpretation of the DRIT was hosted in Lesotho in April 2016 before both tests were applied to all samples subjected to routine rabies diagnosis at the Central Veterinary Laboratory (CVL). We found agreement between the DFA and DRIT assays in 90/96 samples (93.75%). The samples that produced inconsistent results (n = 6) were re-tested a further two times with both assays before being subjected to a real-time qPCR to confirm the diagnosis. Additionally, a statistically significant three-fold increase in the average number of samples submitted per month was observed after the DRIT implementation started, following continuous rabies awareness initiatives amongst the animal health professionals in the country over a 12-month period (p = 0.0279). Partial G-L intergenic regions of selected rabies-positive samples (n = 21) were amplified, sequenced, and subjected to phylogenetic analyses. Molecular epidemiological analyses, which included viruses from neighbouring provinces in South Africa, suggested that at least three independent rabies cycles within Lesotho were implicated in instances of cross-border transmission. This study has evaluated alternative methods for diagnosing and improving rabies surveillance in Lesotho, as well as providing new information that would be of importance in the planning of future disease intervention campaigns, not only in Lesotho, but also in neighbouring South Africa.

A Pan-Lyssavirus Taqman Real-Time RT-PCR Assay for the Detection of Highly Variable Rabies virus and Other Lyssaviruses

Rabies, resulting from infection by Rabies virus (RABV) and related lyssaviruses, is one of the most deadly zoonotic diseases and is responsible for up to 70,000 estimated human deaths worldwide each year. Rapid and accurate laboratory diagnosis of rabies is essential for timely administration of post-exposure prophylaxis in humans and control of the disease in animals. Currently, only the direct fluorescent antibody (DFA) test is recommended for routine rabies diagnosis. Reverse-transcription polymerase chain reaction (RT-PCR) based diagnostic methods have been widely adapted for the diagnosis of other viral pathogens, but there is currently no widely accepted rapid real-time RT-PCR assay for the detection of all lyssaviruses. In this study, we demonstrate the validation of a newly developed multiplex real-time RT-PCR assay named LN34, which uses a combination of degenerate primers and probes along with probe modifications to achieve superior coverage of the Lyssavirus genus while maintaining sensitivity and specificity. The primers and probes of the LN34 assay target the highly conserved non-coding leader region and part of the nucleoprotein (N) coding sequence of the Lyssavirus genome to maintain assay robustness. The probes were further modified by locked nucleotides to increase their melting temperature to meet the requirements for an optimal real-time RT-PCR assay. The LN34 assay was able to detect all RABV variants and other lyssaviruses in a validation panel that included representative RABV isolates from most regions of the world as well as representatives of 13 additional Lyssavirus species. The LN34 assay was successfully used for both ante-mortem and post-mortem diagnosis of over 200 clinical samples as well as field derived surveillance samples. This assay represents a major improvement over previously published rabies specific RT-PCR and real-time RT-PCR assays because of its ability to universally detect RABV and other lyssaviruses, its high throughput capability and its simplicity of use, which can be quickly adapted in a laboratory to enhance the capacity of rabies molecular diagnostics. The LN34 assay provides an alternative approach for rabies diagnostics, especially in rural areas and rabies endemic regions that lack the conditions and broad experience required to run the standard DFA assay.

Detection of Chlamydia trachomatis infections by polymerase chain reaction in asymptomatic pregnant women with special reference to the utility of the pooling of urine specimens.

Background & objectives:Genital Chlamydia trachomatis (CT) infections are one of the most prevalent sexually transmitted infections across the world. In pregnant women, if not detected and treated early, these may result in poor pregnancy outcomes and complications. The present study was aimed to screen CT infections from first void urine (FVU) samples of asymptomatic pregnant women using molecular methods. The secondary objective was to evaluate cost-effectiveness in pooling FVU samples for their diagnostic application.Methods:FVU samples were collected from 1000 asymptomatic pregnant women over a period of three years. Pooling was done by including five specimens in one pool in the amount of 10 µl and subjected to polymerase chain reaction (PCR) and further confirmed by direct fluorescent antibody assay (DFA).Results:The age of study participants ranged from 18 to 43 yr with the median±standard deviation of 26±3.84 yr. Majority of positive participants were younger than 25 years. A total of 200 pools were prepared and 20 of these were PCR positive. When individual specimen in 20 positive pools was tested, 20 PCR-positive specimens were identified from 19 pools, of which 16 were positive by DFA. Thus, CT was detected in 1.6 per cent asymptomatic pregnant women in India and pooling strategy resulted in 70 per cent reduction in a number of tests performed.Interpretation & conclusions:Our study detected C. trachomatis infection in 1.6 per cent asymptomatic pregnant women, and pooling of FVU specimens for PCR testing was found to be a cost-saving strategy in comparison to testing individual samples. Further evaluation and studies on the bigger sample size are warranted to validate these results.

Evaluation of patients with dry eye disease for conjunctival Chlamydia trachomatis and Ureaplasma urealyticum.

To determine the possibility of the development of dry eye disease (DED) as a result of persistent infection with Chlamydia trachomatis and Ureaplasma urealyticum in the conjunctiva of patients. This study was conducted on 58 patients of age range 20-50y, diagnosed with DED confirmed by Schirmer I test and tear breakup time. The non-dry eye control group included 27 subjects of the same age. Ocular specimens were collected as conjunctival scrapings and swabs divided into three groups: the first used for bacterial culture, the second and third taken to detect Chlamydia trachomatis and Ureaplasma urealyticum by direct fluorescent antibody (DFA) assay and polymerase chain reaction (PCR) method. Chlamydia trachomatis was detected in 65.5% and 76% of DED patients by DFA and PCR methods respectively. Ureaplasma urealyticum was found in 44.8% of DED infected patients using the PCR method. Both organisms were identified in only 37.9% of DED patients found to be infected. Control subjects had a 22% detection rate of Chlamydia trachomatis by DFA assay versus a 7% detection rate by PCR; while Ureaplasma urealyticum was detected in 3.7% of the controls by PCR method. The conjunctival culture revealed that gram positive microorganisms represented 75% of isolates with coagulase negative Staphylococci the most common (50%) followed by Staphylococcus aureus (20%), whereas gram negative microorganisms occurred in 25% of cases, isolating Moraxella spp. as the most frequent organism. Our results tend to point out that Chlamydia trachomatis and Ureaplasma urealyticum were detected in a moderate percentage of patients with DED, and could be a fair possibility for its development. PCR is more reliable in detecting Chlamydia trachomatis than DFA technique. The presence of isolated conjunctival bacterial microflora can be of some potential value.

Human Metapneumovirus Pediatric Respiratory Infections: Comparing Direct Immunofluorescence versus Polymerase Chain Reaction

Background: Estimated as the second or third most prevalent respiratory pathogen in the pediatric population, routine testing for human metapneumovirus (hMPV) can have a pivotal impact on children’s clinical outcome. Objectives: This cross-sectional analytical study aimed to determine the efficiency of direct fluorescent antibody (DFA) assay as a rapid tool for the diagnosis of hMPV infection as compared to real time reverse transcriptase polymerase chain reaction (rRT-PCR). In the meantime, we endeavored to analyze the clinical features in hMPV patients. Methods: A total of 50 children aged ≤ 24 months presenting with manifestations of acute respiratory tract infection (ARTI) at El-Mounira pediatric university hospital, Cairo university were enrolled in the study. Nasopharyngeal aspirates (or endotracheal aspirates in intubated children) were examined with the DFA assay as well as rRT-PCR as a gold standard for the detection and quantification of hMPV. Results and conclusion: Human MPV was detected in two cases by DFA and in four cases by rRT-PCR among hospitalized children with ARTIs. The DFA assay proved to be a highly specific test, yet with low sensitivity when compared to rRT-PCR. Most of hMPV-infected cases presented during the winter season, with January and February exhibiting the highest hMPV activity. Pneumonia was the most common presentation of ARTIs in hMPV-infected patients. Direct evaluation of respiratory specimens by DFA provides rapid results with low cost and a subsequent early medical management. However, its use should be restricted as a first-line approach, and a confirmatory test would be needed for a definite diagnosis.

Potential Ceiling Effect of Healthcare Worker Influenza Vaccination on the Incidence of Nosocomial Influenza Infection.

OBJECTIVE To evaluate the effect of healthcare worker (HCW) influenza vaccination on the incidence of nosocomial influenza DESIGN Retrospective cross-sectional study SETTING A 550-bed tertiary-care academic medical center METHODS All admitted patients with a direct fluorescent antibody (DFA) or polymerase chain reaction (PCR) assay positive for influenza ordered between October 1 and May 31 from 2010 to 2015 were eligible for inclusion. Nosocomial influenza was defined as a positive influenza test collected ≥48 hours after admission in patients without influenza-like illness present within 24 hours of admission. Relative nosocomial influenza frequency was calculated by dividing the number of nosocomial cases by the total number of admitted patients with influenza for each season. A univariate logistic regression was used to determine the association between HCW influenza vaccination coverage and nosocomial influenza. RESULTS Over 5 seasons, 533 patients had positive influenza tests during their hospitalization; 29 of these patients (5.4%) acquired influenza during their hospitalization. HCW vaccination coverage increased over the 5 seasons from 47% to 90% (P<.001). Despite an initial decrease in relative nosocomial influenza frequency during the first year (9% to 4.9%), subsequent seasons failed to show an additional decrease in nosocomial infections (4.3%, 5.2%, and 4.8%, respectively); the overall decrease in nosocomial influenza from the first season to the final season was not significant (P=.282). No association was detected between HCW vaccination coverage and nosocomial influenza (odds ratio [OR], 0.990; 95% confidence interval [CI], 0.970-1.011). CONCLUSION HCW vaccination >50% may not have a significant effect on nosocomial influenza. Infect Control Hosp Epidemiol 2016;37:840-844.

Epidemiological and Genetic Characteristics of Rabies Virus in Ubon Ratchathani Province, Thailand, 2011-2014

Total 105 human rabies cases were reported in Thailand during 2006 to 2012. The objectives of this study were to describe epidemiology of animal rabies and genetic characteristics of the viruses found in Ubon Ratchathani Province where the highest number of animal rabies reported in 2011-2015. All 120 animal brain samples submitted to Veterinary Research and Development Center during 2011 to 2014 were included in this study. Epidemiological information was obtained from the sample submitter interview. The virus was identified with direct fluorescent antibody assay and characterized by neighbor-joining analysis. Muang (30.7%), Khemarat (23.1%) and Trakarn Puetpon (10.3%) districts had the highest incidence. The majority of the positive samples were from dogs (92.3%) and samples from dogs were 3.7 times more likely to be found positive, compared to those of other animals. The genetic analysis revealed close relationship between isolates from this study and other rabies viruses isolated in Asia. Rabies viruses in the province appeared to be localized and there was no evidence of transmission from China or Philippines. Determination of epidemiology and genetic characterization of rabies virus in specific areas or nationwide should be performed continuously to monitor the disease trend.