Polymerase Chain Reaction Confirmation Research Articles

Metagenomic characterization of viruses in the serum of children with newly diagnosed cancer

Background and ObjectivesA large cohort of pediatric patients with various forms of childhood cancer was investigated for the presence of viruses using metagenomics. A total of 476 patient samples, collected between 1989 and 2018, were analyzed, representing various pediatric oncological diagnoses and a control group of non-malignant diagnoses. Study designThe study was carried out using metagenomic sequencing of serum samples. Viruses were identified and analyzed using bioinformatics methods, followed by Polymerase chain reaction (PCR) confirmation ResultsThe results indicate that a wide range of viruses can be detected in the bloodstream of children with newly diagnosed cancer. Nine viral genomes were identified: Human Pegivirus (HPgV), Hepatitis C virus, Parechovirus 1, Rhinovirus C, Human papillomavirus 116, Human polyomavirus 10, Parvovirus B19, and different variants of Torque Teno Virus (TTV). In this study, a previously unknown virus was found belonging to the Iflavirdae family in the order Picornavirales. HPGV was significantly more common in patients with leukemia compared to other conditions. ConclusionsThese results highlight the abundance of systemic virus infections in children, and the value of metagenomic sequencing for hypothesis forming regarding the associations between virus infections and cancer.

Influence of microbiota on the growth and gene expression of Clostridioides difficile in an in vitro coculture model.

Clostridioides difficile is an anaerobic, spore-forming, Gram-positive pathogenic bacterium. This study aimed to analyze the effect of two samples of healthy fecal microbiota on C. difficile gene expression and growth using an in vitro coculture model. The inner compartment was cocultured with spores of the C. difficile polymerase chain reaction (PCR)-ribotype 078, while the outer compartment contained fecal samples from donors to mimic the microbiota (FD1 and FD2). A fecal-free plate served as a control (CT). RNA-Seq and quantitative PCR confirmation were performed on the inner compartment sample. Similarities in gene expression were observed in the presence of the microbiota. After 12 h, the expression of genes associated with germination, sporulation, toxin production, and growth was downregulated in the presence of the microbiota. At 24 h, in an iron-deficient environment, C. difficile activated several genes to counteract iron deficiency. The expression of genes associated with germination and sporulation was upregulated at 24 h compared with 12 h in the presence of microbiota from donor 1 (FD1). This study confirmed previous findings that C. difficile can use ethanolamine as a primary nutrient source. To further investigate this interaction, future studies will use a simplified coculture model with an artificial bacterial consortium instead of fecal samples.

Rapid classification of epidemiologically relevant age categories of the malaria vector, Anopheles funestus

BackgroundAccurately determining the age and survival probabilities of adult mosquitoes is crucial for understanding parasite transmission, evaluating the effectiveness of control interventions and assessing disease risk in communities. This study was aimed at demonstrating the rapid identification of epidemiologically relevant age categories of Anopheles funestus, a major Afro-tropical malaria vector, through the innovative combination of infrared spectroscopy and machine learning, instead of the cumbersome practice of dissecting mosquito ovaries to estimate age based on parity status.MethodsAnopheles funestus larvae were collected in rural south-eastern Tanzania and reared in an insectary. Emerging adult females were sorted by age (1–16 days old) and preserved using silica gel. Polymerase chain reaction (PCR) confirmation was conducted using DNA extracted from mosquito legs to verify the presence of An. funestus and to eliminate undesired mosquitoes. Mid-infrared spectra were obtained by scanning the heads and thoraces of the mosquitoes using an attenuated total reflection–Fourier transform infrared (ATR–FT-IR) spectrometer. The spectra (N = 2084) were divided into two epidemiologically relevant age groups: 1–9 days (young, non-infectious) and 10–16 days (old, potentially infectious). The dimensionality of the spectra was reduced using principal component analysis, and then a set of machine learning and multi-layer perceptron (MLP) models were trained using the spectra to predict the mosquito age categories.ResultsThe best-performing model, XGBoost, achieved overall accuracy of 87%, with classification accuracy of 89% for young and 84% for old An. funestus. When the most important spectral features influencing the model performance were selected to train a new model, the overall accuracy increased slightly to 89%. The MLP model, utilizing the significant spectral features, achieved higher classification accuracy of 95% and 94% for the young and old An. funestus, respectively. After dimensionality reduction, the MLP achieved 93% accuracy for both age categories.ConclusionsThis study shows how machine learning can quickly classify epidemiologically relevant age groups of An. funestus based on their mid-infrared spectra. Having been previously applied to An. gambiae, An. arabiensis and An. coluzzii, this demonstration on An. funestus underscores the potential of this low-cost, reagent-free technique for widespread use on all the major Afro-tropical malaria vectors. Future research should demonstrate how such machine-derived age classifications in field-collected mosquitoes correlate with malaria in human populations.Graphical

Genetic characterisation of canine adenovirus type 1 in an 18‐month‐old mixed breed male dog and its effect on clinico‐pathology, hemato‐biochemistry and serum oxidant–antioxidant status

AbstractAn 18‐month‐old, mixed breed male dog was presented with hypothermia, recumbency, hematemesis, melena, and moribund condition, and screened by rapid antigen detection kit followed by polymerase chain reaction confirmation for canine adenovirus type 1 infection. The phylogenetic analysis of the sequence revealed clustering with Indian, Spanish, Italian, Portuguese, Chinese, and Australian sequences with amino acid homology of 98.81%–100%. There was a mutation (N127D) in the E3 protein encoding sequence, which was hitherto reported only in one Indian (GQ340423) sequence. Haemogram revealed anaemia, neutropenic leukopenia, and thrombocytopenia. Serum biochemistry revealed marked hyperglycaemia, hypoproteinemia, hypoalbuminemia, and an increase in the activities of hepatic enzymes. Serum oxidant antioxidant parameters revealed a marked increase in the level of oxidant and a decreased level of antioxidants. Postmortem examination revealed multifocal necrotic patches in the liver, marked haemorrhage in the stomach and intestines, emphysematous lungs, splenomegaly, and congested kidney. Histopathology revealed necrohaemorrhagic hepatitis and intranuclear inclusion bodies in the hepatocytes and glomerular degeneration.

Charlson comorbidity index and the severity of community-acquired pneumonia caused by SARS-CoV-2: A retrospective analysis.

The Charlson comorbidity index (CCI) has been considered as a valid and reliable tool for predicting poor clinical outcomes and mortality in patients with coronavirus disease 2019 (COVID-19). However, its relationship with the severity of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has not been thoroughly explored. The aim of the present study was to identify the impact of the comorbidity burden, quantitatively assessed by applying CCI, on the severity of inpatient community-acquired pneumonia (CAP) caused by SARS-CoV-2. The study was conducted using the medical records of 208 patients with CAP who had an epidemiological history of a plausible SARS-CoV-2 infection, with positive polymerase chain reaction (PCR) confirmation no later than 1 month before being admitted for inpatient treatment. The CCI was calculated using a custom computer program. The statistical analysis of data was carried out using Statistica, v. 7.0. Our study found a significant correlation between the comorbidity burden and the severity of CAP caused by SARS-CoV-2. Specifically, we observed a low CCI score in the majority of patients in the pneumonia risk class II and III groups, and a high CCI score ≥3 in the majority of patients in the pneumonia risk class IV group. Moreover, a direct correlation between CCI and age was established. The comorbidities most commonly associated with CAP caused by SARS-CoV-2 were congestive heart failure, moderate to severe liver diseases and diabetes mellitus (DM) with chronic complications. The use of CCI to evaluate comorbid pathology in hospitalized patients with CAP caused by SARS-CoV-2 can assist the medical staff in developing timely preventive and therapeutic strategies, leading to improved patient prognosis.

Detection of Human Adenovirus from Clinical Samples of Conjunctivitis by Real-time PCR: A Cross-sectional Descriptive Study

Introduction: Human Adenoviruses (HAdV) have been implicated in a variety of infections, including conjunctivitis, respiratory tract infections, genitourinary infections and gastroenteritis. Epidemic Keratoconjunctivitis (EKC) is a severe ocular surface infection strongly associated with HAdV, known to occur in widespread outbreaks. HAdV species B, D and E are associated with ocular manifestations ranging from simple follicular conjunctivitis (types 3, 4, 7) and pharyngoconjunctival fever (types 3, 7, 14) to the more severe EKC (types 8, 19, 37, 53, 54, 56 and 64). Viral cell cultures of conjunctival specimens help confirm adenovirus infection through the characteristic Cytopathic Effect (CPE) and Polymerase Chain Reaction (PCR) is the best standard method to diagnose viral conjunctivitis due to its sensitivity, accuracy and rapidity. Aim: To diagnose HAdV in clinically suspected cases of viral conjunctivitis using real-time PCR. To isolate HAdV in viral cell culture and confirm it by observing its characteristic CPE and performing adenoviral real-time PCR. Materials and Methods: A cross-sectional descriptive study was conducted from July 2023 to September 2023 at the state-level Virus Research and Diagnostic Laboratory (VRDL) at Bangalore Medical College and Research Institute (BMCRI), Bengaluru, Karnataka, India. A total of 45 conjunctival swab samples from patients with suspected viral conjunctivitis, who attended the Ophthalmology Outpatient Department (OPD) or were admitted to the wards, were included in the study. Conjunctival swab samples collected in Viral Transport Medium (VTM) were centrifuged at 3000 rpm for five minutes at 4°C. Real-time PCR was performed and samples positive for HAdV by real-time PCR were taken up for viral cell culture. Confirmatory PCR was conducted for samples showing CPEs in the cell line. The data collected were analysed using descriptive statistics. Results: The majority of patients in the present study were in the age group of 18-45 years, comprising 25 (56%) of the total patients. Out of 45 conjunctival samples tested, 8 samples were positive in adenovirus real-time PCR. All eight PCR-positive samples showed a CPE in viral cell culture on the A549 cell line. The study found PCR positivity in 8 samples (17.7%), while adenoviral recovery from cell culture was observed in 6 samples (13.3%). Conclusion: Real-time PCR has become the standard diagnostic procedure for detecting adenovirus conjunctivitis. Rapid and accurate diagnosis is key to interrupting the contagious spread of adenoviral conjunctivitis, along with timely treatment.

Isolation and identification of multidrug resistance bacterial agents implicated in duck enteritis with first record of Salmonella enterica subspecies arizonae in Egypt.

Bacterial infections causing digestive problems are among the most serious threats to Egypt's duck industry, owing to their effects on feed utilization and body weight gain. As a result, the goal of this study was to identify bacterial pathogens causing enteritis in ducks as well as testing their antimicrobials resistance capabilities. Forty-two duck flocks from different localities at four Egyptian Governorates (El-Sharkia, El-Gharbia, El-Dakahlia, and El-Qaliobia) have been subjected to clinical and postmortem examination as well as bacterial isolation and identification. The liver samples have been collected aseptically from freshly euthanized ducks for bacterial isolation followed by identification using conventional biochemical tests, VITEK 2 system, and confirmatory polymerase chain reaction (PCR) for detection of the uid A gene (beta-glucuronidase enzyme) of Escherichia coli. In addition, antimicrobial sensitivity testing for the isolates against different antimicrobials by the VITEK 2 system was used. Forty-six positive bacterial isolates were identified using conventional methods and the VITEK 2 system including Staphylococcus spp. (52.17%), E. coli (41.30%), and 2.17% for each of Enterococcus casseli lavus, Salmonella enterica subspecies arizonae, and Enterobacter cloacae. PCR was positive for E. coli uid A gene at 556 bp. The antibiogram patterns of isolated pathogens from naturally infected ducks in our work demonstrated 87% multidrug resistance with varying results against different antimicrobial drugs tested. Such findings supported the fact of the upgrading multidrug resistance of Staphylococci and Enterobacteriacae. The most prevalent bacterial pathogens associated with duck enteritis were Staphylococcus spp. and E. coli with the first report of S. enterica subspecies arizonae causing duck enteritis in Egypt.

A female-specific role for trigeminal dynorphin in orofacial pain comorbidity.

Migraine is commonly reported in patients with temporomandibular disorders (TMDs), but little is known about the mechanisms underlying the comorbid condition. Here, we prepared a mouse model to investigate this comorbidity, in which masseter muscle tendon ligation (MMTL) was performed to induce a myogenic TMD, and the pre-existing TMD enabled a subthreshold dose of nitroglycerin (NTG) to produce migraine-like pain in mice. RNA sequencing followed by real-time quantitative polymerase chain reaction confirmation showed that MMTL plus NTG treatment increased prodynorphin ( Pdyn ) mRNA expression in the spinal trigeminal nucleus caudalis (Sp5C) of female mice but not in male mice. Chemogenetic inhibition of Pdyn -expressing neurons or microinjection of antidynorphin antiserum in the Sp5C alleviated MMTL-induced masseter hypersensitivity and diminished the MMTL-enabled migraine-like pain in female mice but not in male mice. Moreover, chemogenetic activation of Pdyn -expressing neurons or microinjection of dynorphin A (1-17) peptide in the Sp5C enabled a subthreshold dose of NTG to induce migraine-like pain in female mice but not in male mice. Taken together, our results suggest that trigeminal dynorphin has a female-specific role in the modulation of comorbid TMDs and migraine.

Deletion of Endogenous Neuregulin-4 Limits Adaptive Immunity During Interleukin-10 Receptor-Neutralizing Colitis.

Growth factors are essential for maintenance of intestinal health. We previously showed that exogenous neuregulin-4 (NRG4) promotes colonocyte survival during cytokine challenge and is protective against acute models of intestinal inflammation. However, the function(s) of endogenous NRG4 are not well understood. Using NRG4-/- mice, we tested the role of endogenous NRG4 in models of colitis skewed toward either adaptive (interleukin-10 receptor [IL-10R] neutralization) or innate (dextran sulfate sodium [DSS]) immune responses. NRG4-/- and wild-type cage mate mice were subjected to chronic IL-10R neutralization colitis and acute DSS colitis. Disease was assessed by histological examination, inflammatory cytokine levels, fecal lipocalin-2 levels, and single cell mass cytometry immune cell profiling. Homeostatic gene alterations were evaluated by RNA sequencing analysis from colonic homogenates, with real-time quantitative polymerase chain reaction confirmation in both tissue and isolated epithelium. During IL-10R neutralization colitis, NRG4-/- mice had reduced colonic inflammatory cytokine expression, histological damage, and colonic CD8+ T cell numbers vs wild-type cage mates. Conversely, in DSS colitis, NRG4-/- mice had elevated cytokine expression, fecal lipocalin-2 levels, and impaired weight recovery. RNA sequencing showed a loss of St3gal4, a sialyltransferase involved in immune cell trafficking, in NRG4-null colons, which was verified in both tissue and isolated epithelium. The regulation of St3gal4 by NRG4 was confirmed with ex vivo epithelial colon organoid cultures from NRG4-/- mice and by induction of St3gal4 in vivo following NRG4 treatment. NRG4 regulates colonic epithelial ST3GAL4 and thus may allow for robust recruitment of CD8+ T cells during adaptive immune responses in colitis. On the other hand, NRG4 loss exacerbates injury driven by innate immune responses.

Parasitological detection of Theileria parasites in livestock in various regions of the Northern Emirates of the United Arab Emirates.

Theileriosis, caused by tick-borne hemoprotozoans of the genus Theileria, severely impacts the economics of the livestock industry in most tropical and subtropical countries. The aim of the present study was to detect Theileria spp. in domesticated animals (camels, cows, sheep, and goats) using direct microscopy and to determine the infection rate in geographically distinct regions in the northern emirates of the UAE. Blood samples (n = 536) were collected from clinically symptomatic and asymptomatic domesticated animals and subjected to Giemsa staining and examined microscopically for the identification of Theileria. Smears showed an overall rate of positivity for Theileria spp. in 325/536 (60.6%) animals. Different infection rates were recorded across the various animal groups in the different study areas (Middle region 215/386 [55.7%], East region 100/139 [71.9%]). Of the 11 goat samples collected from the North region, 10 (90%) were positive. Infection rates per animal group based on microscopy were as follows: camels, 3/35 (8.5%); cows, 19/36 (52.7%); goats, 200/303 (66%); and sheep, 103/162 (63.5%). Real-time polymerase chain reaction confirmation of all microscopy-positive samples identified 23/325 (7.1%) results as false-positive. This study clarified that Theileria spp. is present in the Middle (Sharjah, Umm Al Quwain, and Ajman), East, and North regions. This report also confirmed the use of direct microscopy with Giemsa-stained blood films as the method of choice for diagnosing acute infections. Further work is needed to molecularly determine the prevalence and species of Theileria spp. circulating in the different parts of the UAE.

Parasitological detection of Theileria parasites in livestock in various regions of the Northern Emirates of the United Arab Emirates

Background and Aim: Theileriosis, caused by tick-borne hemoprotozoans of the genus Theileria, severely impacts the economics of the livestock industry in most tropical and subtropical countries. The aim of the present study was to detect Theileria spp. in domesticated animals (camels, cows, sheep, and goats) using direct microscopy and to determine the infection rate in geographically distinct regions in the northern emirates of the UAE. Materials and Methods: Blood samples (n = 536) were collected from clinically symptomatic and asymptomatic domesticated animals and subjected to Giemsa staining and examined microscopically for the identification of Theileria. Results: Smears showed an overall rate of positivity for Theileria spp. in 325/536 (60.6%) animals. Different infection rates were recorded across the various animal groups in the different study areas (Middle region 215/386 [55.7%], East region 100/139 [71.9%]). Of the 11 goat samples collected from the North region, 10 (90%) were positive. Infection rates per animal group based on microscopy were as follows: camels, 3/35 (8.5%); cows, 19/36 (52.7%); goats, 200/303 (66%); and sheep, 103/162 (63.5%). Reverse transcription polymerase chain reaction confirmation of all microscopy-positive samples identified 23/325 (7.1%) results as false-positive. Conclusion: This study clarified that Theileria spp. is present in the Middle (Sharjah, Umm Al Quwain, and Ajman), East, and North regions. This report also confirmed the use of direct microscopy with Giemsa-stained blood films as the method of choice for diagnosing acute infections. Further work is needed to molecularly determine the prevalence and species of Theileria spp. circulating in the different parts of the UAE. Keywords: microscopic examination, Theileria, UAE.

Hepatitis C screening and detection program in a large population: Epidemiological transition and characterization of the disease.

Chronic hepatitis C (CHC) is considered an important public health challenge. Traditionally identified risk factors have undergone an epidemiological transition where other risk factors have become the main cause of new infections. To describe risk factors associated to hepatitis C positivity through the evaluation of the epidemiological profile in hepatitis-C high-risk populations. Cross-sectional study was conducted as part of an HCV screening program in Mexican population. All participants answered an HCV risk-factor questionnaire and took a rapid test (RT). All patients reactive to the test were subject to HCV PCR (polymerase chain reaction) confirmation. A logistic regression model was used to examine associations between HCV infection and risk factors. The study included 297 631 participants that completed a risk factor questionnaire and underwent an HCV rapid test (RT). In total, 12 840 (4.5%) were reactive to RT and 9257 (3.2% of participants) were confirmed as positives by PCR test. Of these, 72.9% had at least one risk factor and 10.8% were in prison. Most common risk factors were history of acupuncture/tattooing/piercing (21%), intravenous drug use (15%) and high-risk sexual practices (12%). Logistic regressions found that having at least one risk factor increased the probability of having an HCV-positive result by 20% (OR = 1.20, 95% CI: 1.15-1.26), compared to the population without risk factors. We identified 3.2% of HCV-viremic subjects, all associated with risk factors and older age. Screening and diagnosis of HCV in high-risk populations (including underserved populations) should be more efficient.

MONKEY POX DISEASE DETECTING SYSTEM USING DEEP LEARNING

Due to the recent monkeypox outbreak's quick spread to more than 40 nations outside of Africa, public health is now at risk. It is difficult to diagnose clinically in its early stages since it resembles both chickenpox and measles. Computer-assisted monkey pox lesion detection may be helpful for surveillance and quick identification of suspected cases when confirmatory Polymerase Chain Reaction (PCR) assays are not accessible. Under the condition there are enough training examples available, deep learning techniques have been demonstrated to be useful in the automated detection of skin lesions. Such databases are not currently accessible for the monkeypox condition. The majority of the photographs came from news websites, blogs, and publicly available case reports. A 3- fold cross-validation experiment is set up, and the sample size is increased through data augmentation. The second phase is categorizing diseases like monkeypox using several pre-trained deep-learning models including VGG-16, CNN, and Mobile Net. It is an ensemble of the three models created. An online method for screening for monkeypox is also being developed as a prototype web application. While the early results on this tiny dataset are promising. Keywords: Monkeypox image dataset, Skin disease classification, Transfer Learning, image processing, CNN, Mobile Net & VGG19.

Evaluation of the ID NOW among symptomatic individuals during the Omicron wave.

Introduction. Starting in December, 2020, the ID NOW was implemented throughout the province of Alberta, Canada (population 4.4 million) in various settings.Gap statement. ID NOW's test performance with SARS-CoV-2 Omicron variant BA.1 is unknown.Aim. To assess the ID NOW performance among symptomatic individuals during the BA.1 Omicron wave and compare it to previous SARS-CoV-2 variant waves.Methodology. The ID NOW was assessed in two locations among symptomatic individuals: rural hospitals and community assessment centres (AC) during the period 5-18 January 2022. Starting 5 January, Omicron represented >95 % of variants detected in our population. For every individual tested, two swabs were collected: one for ID NOW testing and the other for either reverse-transcriptase polymerase chain reaction (RT-PCR) confirmation of negative ID NOW results or for variant testing of positive ID NOW results.Results. A total of 3041 paired samples were analysed (1139 RT-PCR positive). From this, 1873 samples were from 42 COVID-19 AC and 1168 from 69 rural hospitals. ID NOW sensitivity for symptomatic individuals presenting to community AC and rural hospitals was 96.0 % [95 % confidence interval (CI) 94.5-97.3 %, n=830 RT-PCR positive], and 91.6 % (95 % CI 87.9-94.4 %, n=309 RT-PCR positive), respectively. SARS-CoV-2 positivity rate was very high for both populations (44.3 % at AC, 26.5 % in hospital).Conclusions. Sensitivity of ID NOW SARS-CoV-2, compared to RT-PCR, is very high during the BA.1 Omicron wave, and is significantly higher when compared to previous SARS-CoV-2 variant waves.

Adaptive strategies for the deployment of rapid diagnostic tests for COVID-19: a modelling study

Background: Lateral flow assays (LFAs) for the rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) provide an affordable, rapid and decentralised means for diagnosing coronavirus disease 2019 (COVID-19). Concentrating on urban areas in low- and middle-income countries, we examined whether ‘dynamic’ screening algorithms, that adjust the use of confirmatory polymerase chain reaction (PCR) testing based on epidemiological conditions, could reduce cost without substantially reducing the impact of testing. Methods: Concentrating on a hypothetical ‘second wave’ of COVID-19 in India, we modelled the potential impact of testing 0.5% of the population per day at random with LFA, regardless of symptom status. We considered dynamic testing strategies where LFA positive cases are only confirmed with PCR when LFA positivity rates are below a given threshold (relative to the peak positive rate at the height of the epidemic wave), compared to confirming either all positive LFA results or confirming no results. Benefit was estimated based on cumulative incidence of infection, and resource requirements, based on the cumulative number of PCR tests used and the cumulative number of unnecessary isolations. Results: A dynamic strategy of discontinuing PCR confirmation when LFA positivity exceeded 50% of the peak positivity rate in an unmitigated epidemic would achieve comparable impact to one employing PCR confirmation throughout (9.2% of cumulative cases averted vs 9.8%), while requiring 35% as many PCR tests. However, the dynamic testing strategy would increase the number of false-positive test results substantially, from 0.07% of the population to 1.1%. Conclusions: Dynamic diagnostic strategies that adjust to epidemic conditions could help maximise the impact of testing at a given cost. Generally, dynamic strategies reduce the number of confirmatory PCR tests needed, but increase the number of unnecessary isolations. Optimal strategies will depend on whether greater priority is placed on limiting confirmatory testing or false-positive diagnoses.

Symptomatic and Asymptomatic SARS-CoV-2 Infection and Follow-up of Neutralizing Antibody Levels

ObjectiveTo investigate neutralizing antibody levels in symptomatic and asymptomatic patients with coronavirus disease 2019 (COVID-19) at 6 and 10 months after disease onset. MethodsBlood samples were collected at three different time points from 27 asymptomatic individuals and 69 symptomatic patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Virus-neutralizing antibody titers against SARS-CoV-2 in both groups were measured and statistically analyzed. ResultsThe symptomatic and asymptomatic groups had higher neutralizing antibodies at 3 months and 1–2 months post polymerase chain reaction confirmation, respectively. However, neutralizing antibodies in both groups dropped significantly to lower levels at 6 months post-PCR confirmation. ConclusionContinued monitoring of symptomatic and asymptomatic individuals with COVID-19 is key to controlling the infection.

Detection of Chlamydia sp. by fluorescence in situ hybridization (FISH) in histologic sections of the liver from exotic and native avian species

Chlamydiosis is a zoonotic disease that affects several animal species. Therefore, reliable detection techniques are essential for efficient control of the disease. Fluorescence in situ hybridization (FISH) for Chlamydia sp. was applied to 137 formalin-fixed, paraffin-embedded liver sections from native and exotic birds. The samples were divided into two groups: retrospective (n=57) and prospective (n=80). The probe was designed based on an annealing sequence that targets the Major Outer Membrane Protein coding gene. Livers previously confirmed for Chlamydia psittaci by polymerase chain reaction (PCR) and immunohistochemistry (IHC) were used as positive controls. Also, 47 randomly selected samples from the prospective group were submitted to PCR for Chlamydia psittaci for confirmation. From all 137 samples, 67% (92/137) were positive for Chlamydia sp. through FISH, from which 39% (36/57) and 61% (56/80) were from the retrospective and prospective groups, respectively. From the samples of the prospective group submitted for PCR confirmation, 83% (39/47) of them had a positive correlation with FISH results. When considering the number of targeted microorganisms per 400x field, 42,39% (39/92) of the samples had up to five microorganisms, 14,13% (13/92) had from six to ten, and 43,47% (40/92) of the samples had 11 or more microorganisms per field. FISH is a specific and reliable method to identify Chlamydia sp. in histologic sections of the liver, providing an additional tool for detecting avian chlamydiosis.

The Effectiveness of Platelet and D-Dimer Levels in Predicting Prognosis in Intensive Care Patients Diagnosed With COVID-19

Background: The pathophysiology of coagulopathy in patients with Corona virus disease 2019 (COVID-19) and its clinical manifestations remain unclear. However, several studies have reported abnormal coagulation parameters, notably in patients with COVID-19 associated pneumonia and acute respiratory distress syn-drome. Although the underlying mechanism of COVID-19 coagulopathy remains unknown, it has been sug-gested to be a form of disseminated intravascular coagulation. We aimed to determine the predictive value of platelet count and D-dimer levels in predicting prognosis in intensive care patients with a diagnosis of COVID-19.Materials and Methods: Demographic, clinical, laboratory data and radiological findings were obtained from the hospital electronic patient record using a standard data collection form. Platelet counts and D-dimer data were noted. Intensive care stay, mechanical ventilator duration and hospital stay of the patients were ana-lyzed retrospectively. Clinical data covers also comorbid conditions.Results: The study included 102 intensive care patients with COVID-19 diagnosis. All the patients had Poly-merase Chain Reaction (PCR) confirmation and abnormalities on chest computed tomography (CT) consistent with COVID-19. Bilateral pneumonia proven by chest CT was reported in 91.2% of the patient. The platelet count of patients who died was median 247x109 /L (min-max 192 - 354), D dimer levels was median 7.03 (min-max 3.36-17.7) mg/L. Patients who living were platelet counts median 310 x109/L (min-max 234 – 350), D-dimer levels median 1.59 (min-max 0.82 -2). There was no statistically significant difference when the platelet count of the survived and deceased patients were compared (p=0.193). But the patients who died was D-dimer levels statistically higher (p=0.001).Conclusions: High or non-decreasing D-dimer levels may indicate poor prognosis in patients with COVID-19 pneumonia whereas platelet counts don’t have a predictive value.

Genetic characterization of Staphylococcus aureus isolated from Norway rats in Boston, Massachusetts.

Despite the importance of domesticated animals in the generation and transmission of antibiotic-resistant Staphylococcus aureus, the role of wild animals, specifically rodents, in the ecology of S. aureus remains unclear. We recovered and genotyped S. aureus isolates from wild Norway rats (Rattus norvegicus) in Boston, Massachusetts to examine genetic relationships between common human and animal S. aureus isolates in a large US metropolitan area. We collected and necropsied 63 rats from June 2016 to June 2017. Nasal, foot pad, fur, and fecal swabs were collected. Staphylococcus aureus was isolated using culture-based methods and polymerase chain reaction confirmation. S. aureus isolates were spa typed, tested for antibiotic susceptibility, and whole genome sequenced. Assembled sequences were uploaded to the Comprehensive Antibiotic Resistance Database to identify antibiotic resistance elements. A phylogenetic tree was constructed using the neighbor-joining method with the maximum composite likelihood distance in MEGA7. We recovered 164 Gram-positive bacterial isolates from Norway rats. Nineteen isolates from eight individual rats were confirmed as S. aureus (prevalence: 12.9% (8/63)). All S. aureus isolates were methicillin-susceptible S. aureus (MSSA), pvl-negative, and resistant to penicillin. Two isolates displayed resistance to erythromycin. Four different S. aureus spa types were detected (t933, t10751, t18202, and t189). Thirteen unique antibiotic resistance elements were identified, and all isolates shared genes mepR, mgrA, arlR, and S. aureus norA. Phylogenetic analysis if the 19 S. aureus isolates revealed they were genetically similar to four clades of S. aureus with similar resistance gene profiles isolated from both human- and animal-derived S. aureus, as well as formed a distinct phylogenetic cluster composed only of rat isolates. Wild rodents may serve as a reservoir or vector of antibiotic resistance genes in the urban environment with relevance for human and animal health.

Tracing Some Salivary Immune Elements in Iraqi SARS-2 Patients.

Saliva is one of the most significant components in maintaining oral homeostasis and symbiosis. It contains antimicrobial proteins and peptides, such as mucins, lactoferrin, lysozyme, lactoperoxidase, Catherine, statins, and antibodies (secretory immunoglobin A [sIgA]). Early defenses against respiratory infections rely heavily on mucosal immunity, especially secretory sIgA, which has several features and functions that make it suitable for mucosal defense. Salivary testing has been utilized to define mucosal immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Lysozyme has muramidase, with antimicrobial activity, and high concentrations in body fluids, such as saliva and tear. This research aimed to offer an update on how saliva components suppress viral infection and sustain health. A total of 50 individuals, including 30 SARS-2 patients and 20 non-infected subjects, in the age range of 32-54 years were enrolled in this study. Saliva specimens were obtained from polymerase chain reaction (PCR)-confirmed coronavirus disease 2019 (COVID-19) patients and non-infected participants. To collect saliva, the subjects were advised to swirl water over their lips three times, and 5.0 ml of saliva was collected. Samples were centrifuged at 800 x g for 10 min. Saliva was diluted at 1:2,000 with 1 × Diluent N. The immunoglobulin A (IgA) titer in saliva was detected. A spectrophotometer was used to measure the solution's change in absorbance at 550 nm. Measurements (salivary IgA and lysozyme) were made after 7, 30, and 60 days of confirmatory PCR COVID-19 test. The mean scores of salivary IgA levels were obtained at 17.85, 15.26, and 10.73 mg/dl in patients and 9.53, 10.33, and 9.21 mg/dl in healthy individuals after 7, 30, and 60 days, respectively. The salivary lysozyme activity levels in SARS-2 patients compared to controls were 9.7, 7.3, and 4.2 mg/dl versus 2.9, 3.4, and 3.77 mg/dl, respectively. The salivary IgA level was significantly higher in patients of a confirmatory test for COVID-19 compared to healthy individuals.