Development of a colloidal gold immunochromatographic strip for the simultaneous detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus.

In recent years, porcine diarrhea-associated viruses have caused significant economic losses globally. These viruses present similar clinical symptoms, such as watery diarrhea, dehydration, and vomiting. Co-infections with porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are common. For the rapid and on-site preliminary diagnosis on the pig farms, this study aimed to develop a colloidal gold immunochromatography assay (GICA) strip for the detection of PEDV and TGEV simultaneously. The GICA kit showed that there was no cross-reactivity with the other five common porcine viruses. With visual observation, the lower limits were approximately 104 TCID50/mL and 104 TCID50/mL for PEDV and TGEV, respectively. The GICA strip could be stored at 4°C or 25°C for 12 months without affecting its efficacy. To validate the GICA strip, 121 clinical samples were tested. The positive rates of PEDV and TGEV were 42.9 and 9.9%, respectively, and the co-infection rate of the two viruses was 5.8% based on the duplex GICA strip. Thus, the established GICA strip is a rapid, specific, and stable tool for on-site preliminary diagnosis of PEDV- and TGEV-associated diarrhea.

Assessment of performance and comparison of three commercial HDV RNA detection assays: implications for diagnosis and treatment monitoring.

Objectives: Precise HDV-RNA detection and quantification are pivotal for diagnosis and monitoring of response to newly approved treatment. We evaluate the performance of three HDV RNA detection and quantification assays. Methods: Hepatitis Delta RT-PCR system kit, EurobioPlex HDV assay, and RoboGene HDV RNA Quantification kit 2.0 were used for testing 151 HBsAg-positive samples, 90 HDV-RNA negative and 61 HDV-RNA positive. We also evaluated serial dilutions of the WHO international standard for HDV, PEI 7657/12. All HDV-RNA positive samples were genotyped using a next-generation sequencing strategy. Results: Qualitative results indicated a 100% concordance between tests. Quantitative results correlated well, r2 = 0.703 (Vircell-vs-Eurobio), r2 = 0.833 (Vircell-vs-RoboGene), r2 = 0.835 (Robogene-vs-Eurobio). Bias index was 2.083 (Vircell-vs-Eurobio), -1.283 (Vircell-vs-RoboGene), and -3.36 (Robogene-vs-Eurobio). Using the WHO IS, Vircell overestimated the viral load by 0.98 log IU/mL, Eurobio by 1.46 log IU/mL, and RoboGene underestimated it by 0.98 log IU/mL. Fifty-nine samples were successfully genotyped (Genotype 1, n=52; Genotype 5, n=7; Genotype 6, n=1), with similar results for correlation and bias. Conclusion: This study underscores the necessity of using reliable HDV-RNA detection and quantification assays, as evidenced by the high concordance rates in qualitative detection and the observed variability in quantitative results. These findings highlight the importance of consistent assay use in clinical practice to ensure accurate diagnosis and effective treatment monitoring of HDV infection.

Test accuracy of rapid diagnostic tests and reverse-transcription polymerase chain reaction against virus isolation in cell culture for assessing SARS-CoV-2 infectivity: Systematic review and meta-analysis.

We aimed to assess the performance of Ag-RDT and RT-qPCR with regard to detecting infectious SARS-CoV-2 in cell cultures, as their diagnostic test accuracy (DTA) compared to virus isolation remains largely unknown. We searched three databases up to 15 December 2021 for DTA studies. The bivariate model was used to synthesise the estimates. Risk of bias was assessed using QUADAS-2/C. Twenty studies (2605 respiratory samples) using cell culture and at least one molecular test were identified. All studies were at high or unclear risk of bias in at least one domain. Three comparative DTA studies reported results on Ag-RDT and RT-qPCR against cell culture. Two studies evaluated RT-qPCR against cell culture only. Fifteen studies evaluated Ag-RDT against cell culture as reference standard in RT-qPCR-positive samples. For Ag-RDT, summary sensitivity was 93% (95% CI 78; 98%) and specificity 87% (95% CI 70; 95%). For RT-qPCR, summary sensitivity (continuity-corrected) was 98% (95% CI 95; 99%) and specificity 45% (95% CI 28; 63%). In studies relying on RT-qPCR-positive subsamples (n = 15), the summary sensitivity of Ag-RDT was 93% (95% CI 92; 93%) and specificity 63% (95% CI 63; 63%). Ag-RDT show moderately high sensitivity, detecting most but not all samples demonstrated to be infectious based on virus isolation. Although RT-qPCR exhibits high sensitivity across studies, its low specificity to indicate infectivity raises the question of its general superiority in all clinical settings. Study findings should be interpreted with caution due to the risk of bias, heterogeneity and the imperfect reference standard for infectivity.

Characterization of predominant genotype (QX) of avian infectious bronchitis virus isolated from layer chickens in Bangladesh.

Avian infectious bronchitis (AIB) is a highly transmissible infection that affects the poultry industry globally. This study aims to isolate and characterize emerging strains of infectious bronchitis virus (IBV) from field samples of layer chickens in Bangladesh. A total of 108 samples (trachea, lung, and kidney) were taken from dead and sick layer chickens from 18 farms in 4 areas detecting outbreaks in Bangladesh. The samples were processed and inoculated in embryonated chicken eggs (ECEs) and finally screened by the trypsin-induced hemagglutination (THA) test. Using various techniques such as hemagglutination inhibition (HI), agar gel immuno-diffusion (AGID), virus neutralization test (VNT), reverse transcription-polymerase chain reaction (RT-PCR), and nucleotide sequencing, we were able to identify and confirm the isolated IBV viruses. The study also determined the hemagglutination (HA) pattern of isolated virus using avian and mammalian red blood cells. The pathogenicity of the isolated IBV was determined using embryonated chicken eggs and day-old chicks. The study found that 8 samples were positive for IBV using ECEs, and 4 were positive by the THA test. These isolates were confirmed using HI, AGID, and VN tests. S1 gene-based RT-PCR confirmed all four isolates as IBV, with the recent isolates belonging to the genotype-QX and being similar to IBV isolates from Thailand, Saudi Arabia, and India. The HA pattern of the recent isolates showed that the isolated IBV was virulent. The pathogenicity test also revealed that the four isolates were highly pathogenic. The study indicated that the prevalent genotype (QX) of the IBV strain is present in the layer chicken population of Bangladesh.

Genomic epidemiology of severe acute respiratory syndrome coronavirus 2 from Theni, Tamil Nadu.

Introduction: The coronavirus disease 2019 (COVID-19) is a viral infection characterized by respiratory and gastrointestinal symptoms. The causative agent of this infection is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The genomic study helps in understanding the pathogenesis, epidemiology, and the development of therapeutic and preventive strategies in the combat against COVID-19. Materials and Methods: Nasopharyngeal and oropharyngeal swab samples were collected from asymptomatic and symptomatic patients during the time period of 2021-2022 for the detection of SARS-CoV-2 by employing real-time reverse transcriptase, cDNA synthesis, whole-genome sequencing by next-genome sequencing, analysis of SARS-CoV-2 sequence data and lineage and variant of concern assignment along with phylogenetic analysis. Results: Lineages BA.2.10 and BA.4.1.1 clustered with genomes from Senegal suggested the spread of infections. Similarly, high clustering among delta samples during the second wave showed possible importation and subsequent spread via local transmission. Conclusions: Studies like these are important to understand the characteristics and origins of locally circulating SARS-CoV-2 diversity in order to prevent further spread.

Socio-demographic association with confirmed hepatitis C virus infection: A cross-sectional analysis from a teaching institute.

Introduction: Hepatitis C virus (HCV) infection is the most common chronic blood-borne disease and is more commonly associated with chronic active hepatitis leading to cirrhosis, hepato-cellular carcinoma and end-stage liver disease. Methodology: 160 consecutive screening positive (Enzyme linked immuno sorbent assay positive) Hepatitis C samples were tested by HCV RNA Real Time-PCR for confirmation. Result: Prevalence of confirmed hepatitis C among screening positive patient in the present study was found to be 24.4%. Vaccinated individual with Hepatitis A and Hepatitis B had significant association with PCR positivity in screening positive Hepatitis C patient (p< 0.05). IV drug users and patient having multiple sex partners have significant association with PCR positivity among screening positive Hepatitis C patients (p< 0.05). Conclusion: Due to the lack of an effective vaccine and the increased risk of serious complications, it is important to focus on prevention and early detection of HCV.

Search for biocontrol agents among endophytic lipopeptide-synthesizing bacteria Bacillus spp. to protect wheat plants against Greenbug aphid (Schizaphis graminum).

Beneficial endophytic bacteria can suppress the development of insect pests through direct antagonism, with the help of metabolites, or indirectly by the induction of systemic resistance through the regulation of hormonal signaling pathways. Lipopeptides are bacterial metabolites that exhibit direct antagonistic activity against many organisms, including insects. Also, lipopeptides are able to trigger induced systemic resistance (ISR) in plants against harmful organisms, but the physiological mechanisms of their action are just beginning to be studied. In this work, we studied ten strains of bacteria isolated from the tissues of wheat and potatoes. Sequencing of the 16S rRNA gene showed that all isolates belong to the genus Bacillus and to two species, B. subtilis and B. velezensis. The genes for lipopeptide synthetase - surfactin synthetase (Bs_srf ), iturin synthetase (Bs_ituA, Bs_ituB) and fengycin synthetase (Bs_fenD) - were identified in all bacterial isolates using PCR. All strains had high aphicidal activity against the Greenbug aphid (Schizaphis graminum Rond.) due to the synthesis of lipopeptides, which was proven using lipopeptide-rich fractions (LRFs) isolated from the strains. Endophytic lipopeptide-synthesizing strains of Bacillus spp. indirectly affected the viability of aphids, the endurance of plants against aphids and triggered ISR in plants, which manifested itself in the regulation of oxidative metabolism and the accumulation of transcripts of the Pr1, Pr2, Pr3, Pr6 and Pr9 genes due to the synthesis of lipopeptides, which was proven using LRF isolated from three strains: B. subtilis 26D, B. subtilis 11VM, and B. thuringiensis B-6066. We have for the first time demonstrated the aphicidal effect of fengycin and the ability of the fengycin-synthesizing strains and isolates, B. subtilis Ttl2, Bacillus sp. Stl7 and B. thuringiensis B-6066, to regulate components of the pro-/antioxidant system of aphid-infested plants. In addition, this work is the first to demonstrate an elicitor role of fengycin in triggering a systemic resistance to S. graminum in wheat plants. We have discovered new promising strains and isolates of endophytes of the genus Bacillus, which may be included in the composition of new biocontrol agents against aphids. One of the criteria for searching for new bacteria active against phloem-feeding insects can be the presence of lipopeptide synthetase genes in the bacterial genome.

How does a Saccharomyces cerevisiae extract influence the components of isolated rotavirus particles from stool samples collected in a clinical setting from children?

Human Rotavirus (HRV) is the causative pathogen of severe acute enteric infections that cause mortality among children worldwide. This study focuses on developing a new and effective treatment for rotavirus infection using an extract from Saccharomyces cerevisiae, aiming to make this treatment easily accessible to everyone. 15 antigens and 26 antibodies were detected in serum and stool using ELISA. The titers of HRVq1, HRVq2, HRVC1, and HRVC2 on Vero cells were determined to be 1.2x106, 3.0x106, 4.2x106, and 7.5x105 (Plaque forming unit, PFU/ml) four days after infection, respectively. The HRVq1 isolate induced cytopathic effects, i.e., forming multinucleated, rounded, enlarged, and expanding gigantic cells. RT-PCR identified this isolate, and the accession number 2691714 was assigned to GeneBank. The molecular docking analysis revealed that nonstructural proteins (NSPs) NSP1, NSP2, NSP3, NSP4, NSP5, and NSP6 exhibited significant binding with RNA. NSP2 demonstrated the highest binding affinity and the lowest binding energy (-8.9 kcal/mol). This affinity was maintained via hydrophobic interactions and hydrogen bonds spanning in length from 1.12 Å to 3.11 Å. The ADMET and bioactivity predictions indicated that the yeast extract possessed ideal solubility, was nontoxic, and did not cause cancer. The inhibitory constant values predicted for the S. cerevisiae extract in the presence of HRV vital proteins varied from 5.32 to 7.45 mM, indicating its potential as a viable drug candidate. Saccharomyces cerevisiae extract could be utilized as a dietary supplement to combat HRV as an alternative dietary supplement.

Porcine skeletal muscle typing in histochemical and in-situ RT-PCR analysis.

Currently, there are plenty of histochemical methods to classify pig muscle fibers, which confused the naming and classification of muscle fibers. This study aims to analyze the difference and correlation of 6 different histochemical methods and select the most suitable method for muscle fiber classification at the molecular and histomological levels by in-situ RT-PCR and enzyme histochemical methods. Muscle fiber samples, including psoas (PM), semitendinosus (SM) and trapezius muscle (TM), were collected from Large Spotted (LS), Lantang (LT) and Landrace (LR) pigs at their market-ages (LS at 150 d, LT at 210 d, and LR at 150 d). 6 kinds of histochemical methods combining actomyosin adenosine triphosphatase (AM-ATPase) with succinate dehydrogenase (SDH) enzyme were conducted to differentiate fiber types. 2 types of fibers (I and II) were differentiated by acid 2-fibre (2-AC) or alkaline 2-fibre classification(2-AL), 3 types of fibers (ßR, αR and αW) by 3-AC or 3-AL, and 4 types of fibers (I, IIa, IIx and IIb) by 4-AC, or 4-AL. Results showed that AC and AL muscle-fiber classification were consistent in reflecting the characteristics of muscle fibers(P > 0.05), but the color of each muscle fiber type was just opposite. AC methods may be superior to AL methods because of their clear staining background, the sensitivity to staining condition. But there were breed differences and tissue specificity in the optimal preincubation condition. The optimal acid preincubation condition for classifying muscle fibers was pH4.30 for LT, while pH 4.35 for the LS and LR pigs. Meanwhile the optimal acid preincubation condition was pH4.35 for PM, while pH4.40 for TM or SM. For further selection from 2, 3, 4-AC, in-situ RT-PCR was applied to detect the mRNA distribution of myosin heavy chain I (MyHC-I). By combining in-situ PCR with enzyme histochemistry methods, MyHC-I gene and its product - Type I fibrocytes were directly located in cells at both molecular level and morphological level. Compared with the cross-sectional area (CSA) of different muscle fibers (i.e. I, II, ßR, αR, αW, IIa, IIx and IIb) identified by enzyme histochemistry, it was found that the CSAs with stronger mRNA expression signal of MyHC-Ⅰ were closer to those of the Type I muscle fiber measured by 4-AC enzyme histochemistry (P > 0.05). Therefore, 4-AC may be considered as the most proper muscle typing method to study muscle fiber typing as well as meat quality. And the combination of in-situ RT-PCR and histochemistry may help better understand porcine muscle fiber characteristics and meat quality in pigs.

Molecular Characterization of Lineage-IV Peste Des Petits Ruminants Virus and the Development of In-House Indirect Enzyme-Linked Immunosorbent Assay (IELISA) for its Rapid Detection".

Peste des petits ruminants (PPRV), a highly contagious viral disease, causes significant economic losses concerning sheep and goats. Recently, PPR viruses (PPRVs), have adopted new hosts and lineage IV of PPRVs represents genetic diversity within the same lineage. 350 samples, including blood, swabs, and tissues from sheep/goats, were collected during the 2020-2021 disease outbreaks in Pakistan. These samples were analysed through RT-PCR and three isolates of PPRV with accession numbers, MW600920, MW600921, and MW600922, were submitted to GenBank, based on the partial N-gene sequencing. This analysis provides a better understanding of genetic characterizations and a targeted RT-PCR approach for rapid PPRV diagnosis. An IELISA test was developed using the semi-purified antigen MW600922 isolate grown in Vero cells. The PPRV isolates currently present high divergence with the Turkish strain; conversely, similarities equivalent to 99.73% were observed for isolates collected from Pakistan. The developed indirect ELISA (IELISA) test demonstrated antibody detection rates at dilutions of 1:200 for antibodies (serum) and 1:32 for antigens. In comparison to cELISA, high specificity (85.23%) and sensitivity (90.60%) rates were observed. In contrast to the virus neutralization test (VNT), IELISA was observed to be 100% specific and 82.14% sensitive in its results. Based on these results, serological surveys conducted for PPR antibodies using IELISA can be a more effective strategy on a larger scale. Furthermore, our results demonstrate a significant breakthrough in the research in terms of cost-effectiveness and storage efficiency, and the developed IELISA test is highly recommended for use in developing countries.

Peste des petits ruminants (PPRV) is a transboundary, highly contagious, and economically significant viral disease affecting small ruminants and wildlife. PPRV, a disease that only targets animals, is the focus of the Global Eradication Programme (PPRV GEP), which aims to eradicate the disease by 2030. Following the completion of the first phase of the GEP (2017­2021), Pakistan has initiated the second phase: PPRV presence and the implementation of a control strategy. Rapid and accurate laboratory diagnosis is vital to the disease's effective control and eradication. In the present study, we have improved diagnosis by reverse transcriptase polymerase chain reaction (RT-PCR), which not only can detect low viral concentrations but also contributes to the genetic analysis of lineage-IV viruses. However, the development of cost-effective indirect ELISA (iELISA) may allow for the analysis of serum samples obtained from larger populations of small ruminants.

Electrophysiology of human iPSC-derived vascular smooth muscle cells and cell autonomous consequences of Cantu Syndrome mutations.

OBJECTIVE: Cantu Syndrome (CS), a multisystem disease with a complex cardiovascular phenotype, is caused by GoF variants in the Kir6.1/SUR2 subunits of ATP-sensitive potassium (KATP) channels, and is characterized by low systemic vascular resistance, as well as tortuous, dilated vessels, and decreased pulse-wave velocity. Thus, CS vascular dysfunction is multifactorial, with both hypomyotonic and hyperelastic components. To dissect whether such complexities arise cell-autonomously within vascular smooth muscle cells (VSMCs), or as secondary responses to the pathophysiological milieu, we assessed electrical properties and gene expression in human induced pluripotent stem cell-derived VSMCs (hiPSC-VSMCs), differentiated from control and CS patient-derived hiPSCs, and in native mouse control and CS VSMCs. APPROACH AND RESULTS: Whole-cell voltage-clamp of isolated aortic and mesenteric arterial VSMCs isolated from wild type (WT) and Kir6.1[V65M] (CS) mice revealed no clear differences in voltage-gated K+ (Kv) or Ca2+ currents. Kv and Ca2+ currents were also not different between validated hiPSC-VSMCs differentiated from control and CS patient-derived hiPSCs. While pinacidil-sensitive KATP currents in control hiPSC-VSMCs were consistent with those in WT mouse VSMCs, they were considerably larger in CS hiPSC-VSMCs. Under current-clamp conditions, CS hiPSC-VSMCs were also hyperpolarized, consistent with increased basal K conductance, and providing an explanation for decreased tone and decreased vascular resistance in CS. Increased compliance was observed in isolated CS mouse aortae, and was associated with increased elastin mRNA expression. This was consistent with higher levels of elastin mRNA in CS hiPSC-VSMCs, suggesting that the hyperelastic component of CS vasculopathy is a cell-autonomous consequence of vascular KATP GoF. CONCLUSIONS: The results show that hiPSC-VSMCs reiterate expression of the same major ion currents as primary VSMCs, validating the use of these cells to study vascular disease. Results in hiPSC-VSMCs derived from CS patient cells suggest that both the hypomyotonic and hyperelastic components of CS vasculopathy are cell-autonomous phenomena driven by KATP overactivity within VSMCs.

The Time to Negative Conversion among Adult COVID-19 Patients on Chronic Hemodialysis Admitted at the Philippine General Hospital - A Retrospective Cohort Study.

Objectives: In the Philippines, patients on chronic hemodialysis with COVID-19 remain admitted in hospitals despite clinical recovery because most free-standing dialysis units require proof of negative conversion via Reverse Transcriptase - Polymerase Chain Reaction (RT-PCR). This study aims to determine the time to negative conversion of COVID-19 RT-PCR testing among adult patients on chronic hemodialysis with COVID-19 admitted at the Philippine General Hospital (PGH) and bring insight in using the symptom or time-based procedure as recommended by local guideline, and ultimately, to ensure delivery of adequate hemodialysis despite being infected with COVID-19, shorten isolation period, and conserve resources especially in resource-limited settings. Methods: This is a retrospective cohort study on all adult patients on chronic hemodialysis who were admitted in PGH after the diagnosis of COVID-19 by RT-PCR between March 2020 and February 2021. Descriptive statistics was used in summarizing the data. Results: A total of 90 patients on chronic hemodialysis who tested positive for COVID-19 via RT-PCR admitted at PGH were included in the study. Most of these patients had moderate COVID-19 at 53.3%. The median number of days from onset of symptoms to clinical recovery was 14.5 days. The median time to first negative conversion was 18 days. Most of these patients had negative conversion at the second week. The correlation coefficient between time to clinical recovery and negative conversion was 0.214. Conclusion: Among adult patients on chronic hemodialysis who were admitted in PGH after the diagnosis of COVID-19, the time to negative conversion was longer compared to the time to clinical recovery with a very weak correlation between the two.

Assessing the performance of LumiraDx™ SARS-CoV-2 Ag test in detecting Omicron lineages: 2022-2023 study.

Background: The introduction of rapid antigen tests revolutionized the approach to SARS-CoV-2 diagnosis, offering prompt and accurate results with high sensitivity and specificity. Although it is more cost- and time-saving than the gold standard, real-time polymerase chain reaction (RT-PCR), the efficacy in general population screening in both hospital- and community-based settings remains unknown. Moreover, rapid antigen testing is limited by qualitative results. This study aims to evaluate the diagnostic reliability of the LumiraDx™ rapid antigen test during the Omicron era and to investigate its quantitative (analogue-to-digital converter (ADC)) results in comparison with RT-PCR Ct values. Methods: This prospective study included all adult patients with mild-to-moderate SARS-CoV-2 symptoms who were not hospitalised and did not require oxygen supplementation, consented to participate, and attended the Infectious and Tropical Diseases Unit of Padua University Hospital from July 14th, 2022 to January 3rd, 2023. The patients underwent two different tests simultaneously: a nasal LumiraDx™ swab and a real-time RT-PCR assay performed on a nasopharyngeal swab. Sampling was repeated several times for a subset of subjects. Results: We enrolled 266 consecutive participants and collected 601 pairs of LumiraDx™ and RT-PCR samples. The most prevalent variant was BA.4/BA.5 Omicron (60.2 %). The sensitivity and specificity of LumiraDx™ test when compared to real-time RT-PCR results as the reference standard were 93.1 % and 79.75 %, respectively. No significant differences in diagnostic reliability were found based on the available characteristics, age, sex, symptom status, or COVID-19 variant, except for the days from symptom onset. According to the multilevel logistic regression analysis, the only independent variable significantly associated with test concordance was the Ct value (adjusted odds ratio (OR) = 0.56, p < 0.001). Significant differences in quantitative ADC values were found between false negative (FN) versus true negative (TN), and false positive (FP) and true positive (TP) tests. Conclusions: This study showed that LumiraDx™ test is reliable for SARS-CoV-2 diagnosis in patients with mild-to-moderate SARS-CoV-2 symptoms. This finding confirms the efficacy of rapid antigen tests in monitoring vulnerable individuals during the current post-vaccination era. When compared with the RT-PCR, LumiraDx™ test effectively quantitatively distinguishes between FN and TN cases, as well as FP and true TP tests, despite inaccuracies in qualitative results.

Platelet Indices as Novel Surrogate Markers for the Prognosis of COVID-19 Infection: An Observational Study.

Background  The new severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) causes severe acute respiratory illness accountable for causing the coronavirus disease 2019 (COVID-19) illness. Thrombotic issues, acute respiratory distress syndrome (ARDS), and cytokine storm are significant contributors to morbidity and mortality in patients with COVID-19. Elevated D-dimer levels and prothrombin times are further indicators of abnormal coagulation parameters in COVID-19 patients. This study aimed to study the platelet indices as prognostic markers in COVID-19 infection. Methods In this prospective observational study, 150 real-time reverse transcription-polymerase chain reaction (RT-PCR)-positive COVID-19 patients were enrolled between October 2020 and September 2021. All the subjects were screened and explained the study procedure in their native language. Following enrolment, a detailed history and physical examination were performed. Subsequently, laboratory investigations were performed, and patients were subjected to high-resolution computed tomography (HRCT) examination to classify patients into mild, moderate, and severe according to the severity of the illness. The platelet indices taken into account were plateletcrit (PCT) in percentage, platelet count (PLT) in lakh per microlitre, mean platelet volume (MPV) in femtolitres, and platelet distribution width (PDW) in femtolitres. Results The mean PLT was significantly greater among survivors than non-survivors (2.03 ± 0.72 versus 1.76 ± 0.47; p-value = 0.018). The mean MPV (10.42 ± 0.53 versus 9.22 ± 0.64; p-value <0.0001) and PDW (17.99 ± 1.53 versus 16.54 ± 0.91 fl; p-value <0.0001) were significantly greater among non-survivors than survivors. However, the mean PCT was significantly greater among survivors than non-survivors (0.22 ± 0.03% versus 0.18 ± 0.33%; p-value <0.0001). At a cut-off of 0.213, the sensitivity and specificity of PCT in predicting death were found to be 79.2% and 74.5%, respectively. At a cut-off of 16.75, the sensitivity and specificity of PDW in predicting death were found to be 68.8% and 59.8%, respectively. The findings demonstrated a relationship between elevated MPV and PDW and mortality and severe COVID-19 infection. Increased PCT was connected to higher survival, with a specificity and sensitivity of 87.5% and 75.5%, respectively, and MPV >9.75 may predict death. PDW >16.75 exhibited a specificity and sensitivity of 68.8% and 59.8%, respectively, in predicting death. With comparable sensitivity and specificity of 79.2% and 74.5%, PCT >0.213 may predict death. Conclusion In severely sick COVID-19 patients, platelet indices should be routinely calculated and can be utilized as simple, low-cost prognostic indicators.

The epidemiology of intestinal protozoa in the Israeli population based on molecular stool test: a nationwide study.

Stool examination using microscopy was the traditional method for the diagnosis of intestinal parasites. Recently, the use of molecular tests to identify stool protozoa has become the main tool used in most clinical laboratories in Israel. This study aimed to evaluate the prevalence of intestinal parasites in Israel and to compare this prevalence in laboratories that use molecular tests vs a laboratory that uses microscopy. Samples collected from January to October 2021 at seven laboratories were analyzed by real-time PCR (RT-PCR) or by microscopy. The multiplex panel included the following pathogens: Giardia lamblia, Entamoeba histolytica, Cryptosporidium spp., Cyclospora, Dientamoeba fragilis, and Blastocystis spp. Overall, 138,415 stool samples were tested by RT-PCR and 6,444 by microscopy. At least one protozoa species was identified in 28.4% of the PCR-tested samples compared to 4.6% of the microscopy-tested samples. D. fragilis was the most common PCR-identified species (29%). D. fragilis, G. lamblia, and Cryptosporidium spp. were mainly found in pediatric population, while Blastocystis spp. was most prevalent among adults (P < 0.001). In a sub-cohort of 21,480 samples, co-infection was found in 4,113 (19.15%) samples, with Blastocystis spp. and D. fragilis being the most common (14.9%) pair. Molecular stool testing proved more sensitive compared to microscopy. D. fragilis was the most commonly detected pathogen. The above profile was identified during the COVID pandemic when traveling was highly restricted and most likely represents the locally circulating protozoa. IMPORTANCE: This study sheds light on the prevalence of stool parasites in Israel. Additionally, this study indicates that the shift from microscope analysis to molecular tests improved protozoa diagnosis.

Evaluation of the sensitivity of a measles diagnostic real-time RT-PCR assay incorporating recently observed priming mismatch variants, 2024.

We investigated a variant of measles virus that encodes three mismatches to the reverse priming site for a widely used diagnostic real-time RT-PCR assay; reduction of sensitivity was hypothesised. We examined performance of the assay in context of the variant using in silico data, synthetic RNA templates and clinical specimens. Sensitivity was reduced observed at low copy numbers for templates encoding the variant sequence. We designed and tested an alternate priming strategy, rescuing the sensitivity of the assay.

Reduction of osteoclast formation and survival following suppression of cytokines by diacerein in periodontitis.

Periodontitis causes an increase in several bioactive agents such as interleukins (IL), tumor necrosis factor (TNF)-α and receptor activator of NF-kB ligand (RANKL), which induce the osteoclast formation and activity. Since diacerein exerts anti-TNF-α and anti-IL-1 effects, alleviating bone destruction in osteoarthritis, we investigated whether this drug inhibits the formation and survival of osteoclast in the periodontitis. Rats were distributed into 3 groups: 1) group with periodontitis treated with 100 mg/kg diacerein (PDG), 2) group with periodontitis treated with saline (PSG) and group control (CG) without any treatment. After 7, 15 and 30 days, the maxillae were collected for light and transmission electron microscopy analyses. Gingiva samples were collected to evaluate the mRNA levels for Tnf, Il1b, Tnfsf11 and Tnfrsf11b by RT-qPCR. In PDG, the expression of Tnf and Il1b genes reduced significantly compared to PSG, except for Tnf expression at 7 days. The number of osteoclasts reduced significantly in the PDG in comparison with PSG at 7 and 15 days. In all periods, the IL-6 immunoexpression, RANKL/OPG immunoexpression and mRNA levels of Tnfsf11/Tnfrsf11b ratio were significantly lower in PDG than in PSG. PDG exhibited significantly higher frequency of TUNEL-positive osteoclasts than in PSG and CG at all time points. Osteoclasts with caspase-3-immunolabelled cytoplasm and nuclei with masses of condensed chromatin were observed in PDG, confirming osteoclast apoptosis. Diacerein inhibits osteoclastogenesis by decreasing Tnf and Il1b mRNA levels, resulting in decreased RANKL/OPG ratio, and induces apoptosis in osteoclasts of alveolar process of rat molars with periodontitis.

A one-step low-cost molecular test for SARS-CoV-2 detection suitable for community testing using minimally processed saliva.

The gold standard for coronavirus disease 2019 diagnostic testing relies on RNA extraction from naso/oropharyngeal swab followed by amplification through reverse transcription-polymerase chain reaction (RT-PCR) with fluorogenic probes. While the test is extremely sensitive and specific, its high cost and the potential discomfort associated with specimen collection made it suboptimal for public health screening purposes. In this study, we developed an equally reliable, but cheaper and less invasive alternative test based on a one-step RT-PCR with the DNA-intercalating dye SYBR Green, which enables the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly from saliva samples or RNA isolated from nasopharyngeal (NP) swabs. Importantly, we found that this type of testing can be fine-tuned to discriminate SARS-CoV-2 variants of concern. The saliva RT-PCR SYBR Green test was successfully used in a mass-screening initiative targeting nearly 4500 asymptomatic children under the age of 12. Testing was performed at a reasonable cost, and in some cases, the saliva test outperformed NP rapid antigen tests in identifying infected children. Whole genome sequencing revealed that the antigen testing failure could not be attributed to a specific lineage of SARS-CoV-2. Overall, this work strongly supports the view that RT-PCR saliva tests based on DNA-intercalating dyes represent a powerful strategy for community screening of SARS-CoV-2. The tests can be easily applied to other infectious agents and, therefore, constitute a powerful resource for an effective response to future pandemics.

Time to SARS-CoV-2 clearance in African, Caucasian, and Asian ethnic groups.

BACKGROUND: COVID-19 may become a seasonal disease. SARS-CoV-2 active circulation coupled with vaccination efforts has undoubtedly modified the virus dynamic. It is therefore important investigate SARS-CoV-2 dynamic in different groups of population following the course of spatiotemporal variance and immunization. METHODS: To investigate SARS-CoV-2 clearance in different ethnic groups and the impact of immunization, we recruited 777 SARS-CoV-2-positive patients (570 Africans, 156 Caucasians, and 51 Asians). Participants were followed and regularly tested for 2 months until they had two negative tests. RESULTS: The vaccination rate was 64.6%. African individuals were less symptomatic (2%), Caucasians (41%) and Asians (36.6%). On average, viral clearance occurred after 10.5 days. Viral load at diagnosis was inversely correlated with viral clearance (p < 0.0001). The time of SARS-CoV-2 clearance was higher in Africans and Caucasians than in Asians (Dunn's test p < 0.0001 and p < 0.05, respectively). On average, viral clearance occurred within 9.5 days during the second semester (higher rate of vaccination and SARS-CoV-2 exposition), whereas it took 13.6 days during the first semester (lower rate of vaccination and SARS-CoV-2 exposition) (Mann-Whitney t-test p < 0.0001). CONCLUSION: In conclusion, ethnicity and spatiotemporal changes including SARS-CoV-2 exposition and immunization affect SARS-CoV-2 clearance.

The Development of New Primer Sets for the Amplification and Sequencing of the Envelope Gene of All Dengue Virus Serotypes.

Dengue virus (DENV) poses a significant threat to global health, infecting approximately 390 million people annually. This virus comprises four serotypes capable of causing severe disease. Genetic analyses are crucial for understanding the epidemiology, evolution, and spread of DENV. Although previous studies have focused on the envelope protein-coding (E) gene, only a few primers can efficiently detect and amplify the viral genes from multiple endemic countries simultaneously. In this study, we designed degenerate primer pairs for each DENV serotype to amplify and sequence the entire E gene, using globally representative sequences for each serotype. These primers were validated using DENV isolates from various Asian countries and demonstrated broad-spectrum detection capabilities and high-quality sequences. The primers provide effective tools for genetic analysis in the regions affected by dengue, aiding strain identification and epidemiological studies during outbreaks.