SARS-CoV-2 RT-PCR as a universal screening on planned admission in asymptomatic patients.

Universal screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on admission is reportedly beneficial in preventing nosocomial infections. However, some issues remain, including low positivity rate, cost, and time required for testing. We describe SARS-CoV-2 reverse transcription polymerase chain reaction (PCR) for universal screening in asymptomatic patients on planned admissions. In total, 14,574 patients were included between October 12, 2020, and June 23, 2022. The PCR-positive rate for the period was 0.44 % (64/14,574). The PCR positivity for the epidemic period by strain was 0.28 % (95 % confidence interval [CI] 0.12-0.56 %), 0.16 % (95 % CI 0.05-0.37 %), 0.21 % (95 % CI 0.09-0.41 %), and 0.9 % (95 % CI 0.65-1.2 %) for the wild-type strain, Alpha, Delta, and Omicron variants, respectively. The proportion of Ct values < 30 was higher in the first half of the epidemic (first vs. second, 29.4 % [95 % CI 16.9-44.8 %] vs. 16.7 % [95 % CI 6.0-28.5 %]), whereas that of Ct values ≥ 35 increased significantly in the second half (first vs. second, 32.4 % [95 % CI 19.3-47.8 %] vs. 70.0 % [95 % CI 53.5-83.4 %]). Of all positives, 50 % (32/64) had a coronavirus disease (COVID-19) history before PCR screening, with a median of 28 days (10-105) from COVID-19 onset or positive to PCR screening. PCR screening may help detect positives with high viral loads early in the epidemic for each mutant strain, with an increasing proportion of positives with low viral loads later in the epidemic. PCR testing may be unnecessary for recently diagnosed cases and patients in whom reinfection is unlikely.

Immunohistochemistry and RT-PCR as ancillary tools in the diagnosis of acquired oral syphilis.

OBJECTIVE: To investigate Treponema pallidum detection using immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) assays in acquired oral syphilis (AOS). MATERIALS AND METHODS: Thirty-seven paraffin-embedded tissue specimens of AOS (32 secondary and five primary) were analyzed, integrating double-positive serological results with clinicodemographic and histopathological data. T. pallidum presence was semiquantitatively assessed by IHC, while RT-PCR targeted T. pallidum DNA. Sensitivity, specificity, and the area under the curve (AUC) were calculated with 95% confidence intervals (CI). RESULTS: The study included mostly females (62.2%) with a mean age of 27.1 years. T. pallidum was detected in all samples by IHC, predominantly in the epithelium across all layers (43.2%). RT-PCR identified T. pallidum DNA in 32 cases, with negative results observed in cases of secondary AOS. The AUC for IHC versus disease stage was 62.5% (95% CI: 45.1-77.8), and for RT-PCR, it was 57.8% (95% CI: 40.5-73.8). The AUC comparing IHC to RT-PCR was 83.8% (95% CI: 67.9-93.8). CONCLUSION: This study represents the first attempt to evaluate the proposed direct detection algorithm for AOS. IHC and RT-PCR serve as ancillary tools for detecting T. pallidum in both primary and secondary stages of AOS.

Genomic and proteomic characterization of Philadelphia-like B-lineage acute lymphoblastic leukemia: A report of Indian patients.

BACKGROUND: The gold standard for the identification of Philadelphia (Ph)-like acute lymphoblastic leukemia (ALL) patients is gene expression profiling. Because of its diverse nature, its identification is extremely difficult and expensive. On the genomic and proteomic landscape of Ph-like ALL patients, there is a paucity of published literature from developing countries. METHODS: The authors used digital barcoded nCounter NanoString gene expression profiling for its detection, followed by molecular and proteomic characterization using fluorescence in situ hybridization and liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: The authors found 32.05% Ph-like ALL patients and their median age at presentation was considerably higher than Ph-negative ALL cases (p = .0306). Furthermore, we identified 20% CRLF2 overexpressed cases having 8.33% CRLF2-IGH translocation with concomitant R683S mutation and 8.33% CRLF2-P2RY8 translocation. In 80% of CRLF2 downregulated cases, we identified 10% as having JAK2 rearrangement. Minimal residual disease-positivity was more common in Ph-like ALL cases (55.55% vs. 25% in Ph-negative ALL cases). Immunoglobulin J chain (Jchain), small nuclear ribonucleoprotein SmD1 (SNRPD1), immunoglobulin κ constant (IGKC), NADH dehydrogenase (ubiquinone) 1 α subcomplex subunit 2 (NDUFA2), histone H2AX (H2AFX), charged multivesicular body protein 4b (CHMP4B), and carbonyl reductase (NADPH) (CBR1) proteins were identified to be substantially expressed in Ph-like ALL patients, using LC-MS/MS. Gene enrichment analysis indicated that involvement of spliceosomal mediated messenger RNA splicing pathway and four microRNAs was statistically significant in Ph-like ALL patients. CONCLUSIONS: For the first time, we have described incidence, molecular, and proteomic characterization of Ph-like ALL, in developing nations. PLAIN LANGUAGE SUMMARY: In developing countries, detecting Philadelphia (Ph)-like B-lineage acute lymphoblastic leukemia is complicated and challenging due to its diverse genetic landscape. There is no well-defined and cost-effective methodology for its detection. The incidence of this high-risk subtype is very high in adult cases, and there is an urgent need for its accurate detection. We have developed an online PHi-RACE classifier for its rapid detection, followed by delineating the genomic and proteomic landscape of Ph-like acute lymphoblastic leukemias for the first time in Indian patients.

Prevalence of different virulence factors and their association with antimicrobial resistance among Pseudomonas aeruginosa clinical isolates from Egypt.

BACKGROUND: Emergence of multi-drug resistant Pseudomonas aeruginosa, coupled with the pathogen's versatile virulence factors, lead to high morbidity and mortality rates. The current study investigated the potential association between the antibiotic resistance and the production of virulence factors among P. aeruginosa clinical isolates collected from Alexandria Main University Hospital in Egypt. We also evaluated the potential of the phenotypic detection of virulence factors to reflect virulence as detected by virulence genes presence. The role of alginate in the formation of biofilms and the effect of ambroxol, a mucolytic agent, on the inhibition of biofilm formation were investigated. RESULTS: A multi-drug resistant phenotype was detected among 79.8% of the isolates. The most predominant virulence factor was biofilm formation (89.4%), while DNase was least detected (10.6%). Pigment production was significantly associated with ceftazidime susceptibility, phospholipase C production was significantly linked to sensitivity to cefepime, and DNase production was significantly associated with intermediate resistance to meropenem. Among the tested virulence genes, lasB and algD showed the highest prevalence rates (93.3% and 91.3%, respectively), while toxA and plcN were the least detected ones (46.2% and 53.8%, respectively). Significant association of toxA with ceftazidime susceptibility, exoS with ceftazidime and aztreonam susceptibility, and plcH with piperacillin-tazobactam susceptibility was observed. There was a significant correlation between alkaline protease production and the detection of algD, lasB, exoS, plcH and plcN; pigment production and the presence of algD, lasB, toxA and exoS; and gelatinase production and the existence of lasB, exoS and plcH. Ambroxol showed a high anti-biofilm activity (5% to 92%). Quantitative reverse transcriptase polymerase chain reaction showed that alginate was not an essential matrix component in P. aeruginosa biofilms. CONCLUSIONS: High virulence coupled with the isolates' multi-drug resistance to commonly used antimicrobials would increase morbidity and mortality rates among P. aeruginosa infections. Ambroxol that displayed anti-biofilm action could be suggested as an alternative treatment option, yet in vivo studies are required to confirm these findings. We recommend active surveillance of antimicrobial resistance and virulence determinant prevalence for better understanding of coregulatory mechanisms.

Innate differences in the molecular signature of normal inferior & superior human parathyroid glands: potential implications for parathyroid adenoma.

Primary hyperparathyroidism is a common endocrine disorder. Interestingly, the majority (75%) of parathyroid tumors are localized to the inferior parathyroid glands. To date, the reason for this natural bias has not been investigated. We assessed the global gene expression profile of superior and inferior glands obtained from forensic autopsies. The genes with significant differential expression between superior and inferior parathyroids were further assessed by RT-PCR in 19 pairs. As an iterative approach, additional genes with an established role in parathyroid disorders, i.e., CASR, MAFB, PAX9, TBCE, TBX1, VDR, MEN1, CCND1, and CDC73 were also evaluated by RT-PCR in all 19 pairs of superior and inferior parathyroid glands. Seven homeobox genes, namely HOXA4, HOXA5, HOXBAS3, HOXB4, HOXB6, HOXB9, IRX1, and one encoding for ALDH1A2 showed a lower expression in the inferior parathyroid glands than in the superior. Conversely, SLC6A1 showed a higher expression in the inferior glands. Of the nine genes with significant differential mRNA expression among superior and inferior glands HOXB9, HOXB4 and IRX1 could be detected by western blotting/mass spectrometry. The study is the first to show the differential expression of nine genes HOXA4, HOXA5, HOXBAS3, HOXB4, HOXB6, HOXB9, IRX1, ALDH1A2, and SLC6A1 in inferior versus the superior parathyroid glands. This could have potential implications for the preferential localization of parathyroid tumors to the inferior parathyroid glands as observed in patients with primary hyperparathyroidism.

Estimating the number of probable new SARS-CoV-2 infections among tested subjects from the number of confirmed cases.

OBJECTIVES: In most African countries, confirmed COVID-19 case counts underestimate the number of new SARS-CoV-2 infection cases. We propose a multiplying factor to approximate the number of biologically probable new infections from the number of confirmed cases. METHODS: Each of the first thousand suspect (or alert) cases recorded in South Kivu (DRC) between 29 March and 29 November 2020 underwent a RT-PCR test and an IgM and IgG serology. A latent class model and a Bayesian inference method were used to estimate (i) the incidence proportion of SARS-CoV-2 infection using RT-PCR and IgM test results, (ii) the prevalence using RT-PCR, IgM and IgG test results; and, (iii) the multiplying factor (ratio of the incidence proportion on the proportion of confirmed -RT-PCR+- cases). RESULTS: Among 933 alert cases with complete data, 218 (23%) were RT-PCR+; 434 (47%) IgM+; 464 (~ 50%) RT-PCR+, IgM+, or both; and 647 (69%) either IgG + or IgM+. The incidence proportion of SARS-CoV-2 infection was estimated at 58% (95% credibility interval: 51.8-64), its prevalence at 72.83% (65.68-77.89), and the multiplying factor at 2.42 (1.95-3.01). CONCLUSIONS: In monitoring the pandemic dynamics, the number of biologically probable cases is also useful. The multiplying factor helps approximating it.

Comparison of performance between three SARS-CoV-2 molecular assays (Aptima™, Laboratory Developed Test-Fusion, and R-GENE®) with special attention to turnaround time, a key point in laboratory management.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) highlights the importance of rapid diagnostic testing to identify individuals with SARS-CoV-2 infections and to limit the spread of the virus. Many molecular assays have become commercially available to cope with this surging demand for timely diagnosis of COVID-19 cases, but identifying individuals requires accurate diagnostic tools. We compared the performance of three molecular SARS-CoV-2 assays: Aptima™ SARS-CoV-2 assay running on the Panther system (Hologic), an in-house assay (Laboratory Developed Test, LDT) running on the Fusion module of the Panther Fusion system (LDT-Fusion; Hologic), and the R-GENE® SARS-CoV-2 assay (bioMérieux). In addition, we also evaluated the turnaround time. This parameter is crucial to managing the SARS-CoV-2 diagnosis and represents a key point in the quality management at the laboratory. Aptima™ and LDT-Fusion assays exhibited an excellent positive percent agreement (PPA) (100.0%), while the R-GENE® assay showed a slightly decreased PPA (98.2%). The Hologic assays have a higher throughput with less hands-on time than the R-GENE® assays (24-25 vs. 71 min). Both Hologic assays are used on a fully automated random-access testing system with on-demand testing capabilities that avoid run series, unlike the R-GENE® assay. Automated random-access testing systems should be preferred during periods of high SARS-CoV-2 prevalence.

Transcriptome analysis of cervical cancer exosomes and detection of HPVE6*I transcripts in exosomal RNA.

BACKGROUND: Exosomes play a key role in cell-to-cell communication and are integral component of the tumor microenvironment. Recent observations suggest transfer of RNA through tumor-derived exosomes that can potentially translate into regulatory proteins in the recipient cells. Role of cervical cancer-derived exosomes and their transcript cargo is poorly understood. MATERIALS AND METHODS: The total RNA of exosomes from HPV-positive (SiHa and HeLa) and HPV-negative (C33a) cervical cancer cell lines were extracted and the transcripts were estimated using Illumina HiSeq X. Further, validation of HPV transcripts were performed using RT-PCR. RESULTS: 3099 transcripts were found to be differentially-exported in HPV-positive vs. HPV-negative exosomes (p value <0.05). Analysis of top 10 GO terms and KEGG pathways showed enrichment of transcripts belonging to axon guidance and tumor innervation in HPV-positive exosomes. Among top 20 overexpressed transcripts, EVC2, LUZP1 and ANKS1B were the most notable due to their involvement in Hh signaling, cellular migration and invasion, respectively. Further, low levels of HPV-specific reads were detected. RT-PCR validation revealed presence of E6*I splice variant of HPV18 in exosomal RNA of HeLa cells. The E6*I transcripts were consistently retained in exosomes obtained from HeLa cells undergoing 5-FU and cisplatin-induced oxidative stress. CONCLUSION: Our data suggests the enrichment of poly-A RNA transcripts in the exosomal cargo of cervical cancer cells, which includes pro-tumorigenic cellular RNA and viral transcripts such as HPV E6, which may have clinical utility as potential exosomal biomarkers of cervical cancer.

Current trends in COVID-19 diagnosis and its new variants in physiological fluids: Surface antigens, antibodies, nucleic acids, and RNA sequencing.

Rapid, highly sensitive, and accurate virus circulation monitoring techniques are critical to limit the spread of the virus and reduce the social and economic burden. Therefore, point-of-use diagnostic devices have played a critical role in addressing the outbreak of COVID-19 (SARS-CoV-2) viruses. This review provides a comprehensive overview of the current techniques developed for the detection of SARS-CoV-2 in various body fluids (e.g., blood, urine, feces, saliva, tears, and semen) and considers the mutations (i.e., Alpha, Beta, Gamma, Delta, Omicron). We classify and comprehensively discuss the detection methods depending on the biomarker measured (i.e., surface antigen, antibody, and nucleic acid) and the measurement techniques such as lateral flow immunoassay (LFIA), enzyme-linked immunosorbent assay (ELISA), reverse transcriptase-polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), microarray analysis, clustered regularly interspaced short palindromic repeats (CRISPR) and biosensors. Finally, we addressed the challenges of rapidly identifying emerging variants, detecting the virus in the early stages of infection, the detection sensitivity, selectivity, and specificity, and commented on how these challenges can be overcome in the future.

Multisystem inflammatory syndrome in neonates (MIS-N) associated with SARS-CoV2 infection: a case series.

Multisystem inflammatory syndrome in neonates (MIS-N) is hypothesised to be caused either following transplacental transfer of SARS-CoV2 antibodies or antibodies developed in the neonate after infection with SARS-CoV-2. In this paper, we aim to discuss the clinical manifestations, laboratory features, and management of neonates diagnosed with MIS-N. We collated information from five participating hospitals in western India. A cohort of newborn infants presenting with multi-system involvement, along with the presence of SARS-CoV2 antibodies, was identified. Current proposed international diagnostic criteria for MIS-N were used to group the cases into three categories of Most likely, Possible, and Unlikely MIS-N. A total of 20 cases were reported with a diagnosis of MIS-N, all having high titres of SARS CoV2 IgG antibodies and negative for SARS CoV2 antigens. Most likely MIS (n = 5) cases presented with respiratory distress (4/5), hypotension and shock (4/5), and encephalopathy (2/5). Inflammatory markers like CRP (1/5), Procalcitonin (1/5), Ferritin (3/5), D-dimer (4/5), and LDH (2/5) were found to be elevated, and four of them had significantly high levels of proBNP. The majority of them (4/5) responded to immunomodulators, three neonates were discharged home, and two died. Possible MIS infants (n = 9) presented with fever (7/9), respiratory distress (4/9), refusal to feed (6/9), lethargy (5/9), and tachycardia (3/9). ProBNP as a marker of cardiac dysfunction was noted to be elevated in four (4/9) infants, correlating with abnormal echocardiography findings in two. In the Unlikely MIS (n = 6) category, three (3/6) infants presented with respiratory distress, one (1/6) with shock and cardiac dysfunction, and only one (1/6) with fever. All of them had elevated inflammatory markers. However, there were other potential diagnoses that could have been responsible for the clinical scenarios in these six cases.   Conclusion: MIS-N requires a high index of suspicion and should be considered in a neonate presenting with two or more systems involvement, in the presence of SARS-CoV2 antibodies, along with elevated inflammatory markers, once other common neonatal conditions have been ruled out. What is Known: • Severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) associated multisystem inflammatory syndrome in children (MIS-C) is  widely reported in paediatric population, however only few reports of newborn affection. • MIS-C is known to cause by virus-induced post-infective antibody mediated immune dysregulation with severe multi-system affection. What is New: • MIS-N may present with varied clinical manifestations with multi-system involvement of variable severity with milder disease in term and severe disease with cardiac dysfunction in preterm newborns. • Multisystem inflammatory syndrome in newborns (MIS-N) is postulated to occur following immune dysregulation associated with transplacental transfer of SARS-CoV2 antibodies or antibodies developed in the neonate after infection with SARS-CoV-2.

Mass mortalities associated with viral nervous necrosis in Murray cod in China.

In December 2019, a mass mortality among cultured Murray cod (Maccullochella peelii peelii) fry occurred on a freshwater farm located at Foshan city of Guangdong province, China. The cumulative mortality was up to 45% within 15 days. The diseased fish showed clinical signs, including abnormal swimming behaviour, loss of appetite and dark body colouration before mass mortality. Samples of brain and retina tissues were collected from affected fish and subjected to reverse transcriptase polymerase chain reaction detection and virus isolation in cell culture. Approximately 430 bp product was detected from the brain and retina tissues and culture supernatant of betanodavirus-infected SSN-1 cells. The typical cytopathic effect of betanodavirus infection, which is characterized by vacuolation, was observed in SSN-1 cells at three days after inoculating with the tissue filtrate of diseased Murry cod fry, and the TCID50 of the infected SSN-1 cell supernatant was 107.8 . Histopathological examinations revealed vacuolation and necrosis in the brain and retina of naturally and experimentally infected Murray cod fry. Electron microscopic observation also showed the aggregation of numerous spherical, non-enveloped viral particles measuring 22-28 nm in diameter in the cytoplasm of betanodavirus-infected SSN-1 cells. Sequence and phylogenetic analysis based on RdRp and Cp genes further indicated that the betanodavirus isolated from Murray cod belonged to the RGNNV genotype. Much higher mortality was obtained in challenged Murray cod fry compared with the controls through immersion challenge. This study is the first report of the natural infection of betanodavirus in freshwater fish in China.

PCR to CRISPR: Role of Nucleic Acid Tests (NAT) in detection of COVID-19.

COVID-19 infection has emerged as an unparalleled pandemic with morbidity and mortality tolls challenging diagnostic approaches and therapeutic interventions, and raising serious questions for healthcare policy-makers. From the diagnostic perspective, Reverse transcriptase polymerase chain reaction remains the gold standard. However, issues associated with gene primer variation in different countries, low analytical sensitivity, cross-reactivity with certain human coronaviruses have raised serious concerns within the scientific community. Alongside longer turnaround times, requirements of sophisticated equipment and trained technicians are the other challenges for conventional reverse transcriptase polymerase chain reaction testing. The recent biotechnological boom has now allowed newer nucleic acid testing options for diagnosing severe acute respiratory syndrome Coronovairus 2 (SARS-CoV2) with much better diagnostic efficiency, reduced turnaround times and possible benefit for use as a point-of-care test. Isothermal techniques with simple equipment requirements along with uniform temperature for analysis have emerged to be more sensitive and specific with turnaround times as low as 10-15 minutes. Similarly, Cluster Regularly Interspaced Short Palindromic Repeats have also been seen to play a very decisive role in COVID-19 diagnostics with much superior diagnostic efficiency and feasibility as a point-of-care test and its possible use for sequencing. The current narrative review was planned to consolidate data for all possible nucleic acid testing options under research/clinical use, and to provide a comparative assessment from the perspective of both the clinician and the laboratory.

Comparison of Chest X-rays findings in COVID-19 suspected and confirmed cases at a university teaching hospital: A retrospective comparative study.

OBJECTIVES: To compare Chest X-rays findings in COVID -19 suspected and confirmed patients on RT-PCR, presented at corona filtration center, Benazir Bhutto hospital Rawalpindi. METHODS: In this study, Chest radiographs of 100 COVID-19 RT-PCR positive confirmed patients were compared with 100 RT-PCR negative suspected COVID-19 patients screened at corona filtration center, Benazir Bhutto Hospital Rawalpindi from November 2020 to December 2020. Data on demographics, presenting complaints, co-morbid, lesion characteristic, distribution and attenuation, lobar involvement, pleural effusion and lymphadenopathy were collected. Associations between imaging characteristics and COVID-19 pneumonia were analyzed with univariate and multivariate logistic regression modals. RESULTS: Chest X-rays findings revealed bilateral lung consolidation with peripheral and diffuse distribution, involving middle and lower lobe to be statistically significant (p<0.05) between RT-PCR positives and negative patients. Peripheral distribution was associated with an 11.08-fold risk in COVID-19 positive patients than diffuse distribution. Middle lobe involvement had four folds risk and lower lobe involvement had 11.04 folds risk in COVID-19 cases as compared to upper lobe involvement. Consolidation had 2.6 folds risk in COVID-19 positive cases. CONCLUSIONS: Bilateral, peripheral distribution of middle and lower lobes ground glass haze or consolidation with no pleural effusion is significantly related to COVID-19 pneumonia. Overlapping imaging features of the infectious and non-infectious COVID mimickers can be further excluded by detailed clinical evaluation and further radiological workup.

Serum albumin levels monitoring in ICU in early days and mortality risk association in patients with moderate to severe COVID-19 pneumonia.

Objective: To establish correlation between serum albumin during early days of ICU admission and risk of death in COVID-19 pneumonia. Methods: In this retrospective study, we included 76 patients hospitalized in ICU, who stayed for at least four days with COVID-19 pneumonia, from May 1, 2020 to June 30, 2020 in Lahore Health Care Hospital and Al-Shafi Hospital. Patients were labelled as COVID-19 pneumonia on radiological basis as bilateral 'ground-glass opacity' in lower zones and RT-PCR positive result in nasopharyngeal swab. All patients were oxygen dependent, either on high flow oxygen via non rebreathing mask or invasive positive pressure ventilation support. Serum albumin levels were measured daily from first day to fourth day of ICU admission. The data was analyzed using SPSS version 26 and Microsoft excel 2016. Results: Out of 76 patients of COVID-19 pneumonia admitted in ICU who stayed for more than four days, 38 patients expired. The mean age of all the patients was 58.9±12.56 years, 38(50%) of the patients were ≥60 years and 49 (62%) of them were male. On day four of ICU admission, mean serum albumin of discharged patients was 3.83±0.22 g/dl while mean serum albumin level of expired patients was 2.96±0.46 g/dl. Strong negative correlation (r = -767) was found between decrease in serum albumin level and increase number of deaths from COVID-19 pneumonia. Weak correlation was observed between increase in serum CRP and increase number of deaths in the same patients. Conclusion: Daily monitoring of serum albumin level of COVID-19 pneumonia patients can be used as a biological marker for monitoring of cytokine storm and risk of death in COVID-19 pneumonia.

A comparative study on the efficiency of commercial reverse transcriptase-Polymerase chain reaction kits for the detection of severe acute respiratory syndrome coronavirus 2 infections.

Background: Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) kits have been reliably employed for the diagnosis of coronavirus disease 2019 (COVID-19) by the detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA since the beginning of the disease outbreak. In consideration of reliable diagnosis, apart from RT-PCR, the isothermal nucleic acid amplification-based point-of-care automated kits have also been tagged as a simpler and rapid alternative to the conventional techniques. Currently, the availability of a better diagnostic method for COVID-19 when compared to RT-PCR is nil. The most important step in the detection of SARS-CoV-2 in a RT-PCR diagnostic laboratory is to identify and employ RT-PCR kits with higher sensitivity as well as specificity. Objectives: This study aimed to study commercially available RT-PCR kits for the detection of SARS-CoV-2 infections. Methods: The performance of seven different RT-PCR kits from different manufacturers used for diagnosis of COVID-19 in Govt Theni Medical College and Hospital, Theni, Tamil Nadu were analysed. Nasopharyngeal and oropharyngeal swabs were collected from patients and subjected to RT-PCR using these kits. Results and Conclusion: The sensitivities and batch effects of the assessed kits were slightly different for different targets, for SARS-CoV-2 detection in nasopharyngeal swab specimens. Examination of COVID-19 kits should be done using currently employed kits in routine diagnosis for better efficiency.

Detection of dengue virus serotypes by single-tube multiplex RT-PCR and multiplex real-time PCR assay.

Background: All four dengue serotypes cause infection, with one of them predominantly reported from a particular geographical region. Coinfection by more than one serotype is reported from hyperendemic regions. These coinfections are clinically more severe than infection with a single serotype. This study was carried out to detect the predominant dengue serotype and presence of coinfections. Methods: Acute-phase serum samples of patients suffering from dengue infection were collected. They were screened for the presence of IgM, IgG and NS1Ag by a rapid test. Conventional multiplex reverse transcriptase polymerase chain reaction (RT-PCR) and multiplex real-time RT-PCR assays were carried out for detection and serotyping of the dengue virus. Results: A total of 196 samples were positive by the rapid card test. Of these, 139 were NS1Ag positive, 40 were positive only for IgM, 5 were positive only for IgG and 12 samples were positive for different combinations of antigen and antibodies. All four serotypes were detected in these samples by PCR. DENV-3 was found to be most common circulating serotype. A total of 22 cases were found to have coinfection with more than one dengue serotypes. Samples having only antibodies and no antigen on rapid card test were also positive for virus by PCR. Conclusion: Prevalence of dengue co-infections is increasing. Moreover, it is important to screen for dengue virus in those samples also which do not show NS1Ag on rapid tests and have either one or both the antibodies. Real-time multiplex RT-PCR is found to be more sensitive in detecting coinfection than conventional multiplex RT-PCR.

Adherence of Candida albicans and Malassezia Species to Skin Cells Induces Changes in the Expression of Genes Responsible for Heparan and Chondroitin Sulfate Chain Synthesis. / La unión de Candida albicans y Malassezia spp. a células de piel promueve cambios de expresión en los genes responsables de la síntesis de las cadenas de heparán y condroitín sulfato.

Superficial fungal infections are common in dermatology and are often caused by opportunistic species in the Candida and Malassezia genera. The aim of this study was to analyze changes in the expression of genes coding for enzymes involved in the biosynthesis of glycosaminoglycans (GAGs) chains following the adherence of Candida and Malassezia yeasts to skin cell lines. Gene expression was analyzed using reverse transcriptase-quantitative polymerase chain reaction assays. Interactions between the yeasts and the skin cells induced the following changes in genes involved in the biosynthesis of heparan sulfate and chondroitin sulfate: downregulation of CHPF in keratinocytes and downregulation of EXT1, EXT2, CHSY3, and CHPF in fibroblasts. Adherence to fibroblasts had an even greater effect on GAG biosynthetic enzymes, inducing the downregulation of 13 genes and the upregulation of two (CHST15 and CHST7). Interactions between yeasts and skin cells might affect the binding affinity of GAG chains, possibly changing their ability to function as receptors for pathogens and interfering with a key stage at the start of infection.

COVID-19-associated Guillain-Barre syndrome: Postinfectious alone or neuroinvasive too?

Coronavirus disease 2019 (COVID-19) has been shown to be associated with a lot of neurological complications, of whom Guillain-Barre syndrome (GBS) is an important post-infectious consequentiality. More than 220 patients with GBS have been reported thus far. We intend to share our experience with five patients of GBS where one of them had severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the cerebrospinal fluid (CSF). This is the first-ever report demonstrating the presence of SARS-CoV-2 in the CSF of an adult patient; a similar occurrence has recently been described in a pediatric patient. We wish to emphasize the fact that commonly GBS occurs as a result of a post-infectious process but in a few cases where the symptoms of COVID-19 and GBS occur concurrently, corresponding to the viremic phase, separate pathogenesis needs to be thought of. This para-infectious nature is exemplified by the presence of virus in the cerebrospinal fluid of one of our patients. We review the neuroinvasive potential of SARS-Cov-2 in this regard and draw parallels with Cytomegalovirus, Zika virus, and Human Immunodeficiency virus-associated occurrences of GBS.

Evaluation of SARS-CoV-2 in Tears of Patients with Moderate to Severe COVID-19.

PURPOSE: To investigate the presence of SARS-CoV-2 RNA in tears of patients with moderate to severe coronavirus disease 2019 (COVID-19). DESIGN: Cross-sectional study. PARTICIPANTS: Patients with laboratory-proven moderate to severe COVID-19. METHODS: Tears were collected within 48 hours of laboratory confirmation using 3 methods: conjunctival swab plus Schirmer's test strips (group 1), conjunctival swab (group 2), and Schirmer's test strips (group 3). Samples from both the eyes of each patient were transported in a single viral transport media for real-time RT-PCR. Detailed demographic profiles, systemic symptoms, comorbidities, and ocular manifestations were noted. MAIN OUTCOME MEASURES: Viral load of a sample was determined using cycle threshold (Ct) value of E gene. A specimen was considered to show positive results if the amplification curve for the E gene crossed the threshold line within 35 cycles and if it showed positive results on an RNA-dependent RNA polymerase or open reading frame 1b gene assay. RESULTS: Of the 78 patients enrolled in the study, samples from 3 patients were found to be inadequate for analysis. Thirty-six patients (48%) had moderate disease, whereas 39 patients (52%) had severe disease, with no ocular involvement in any patient. In the 75 patients, RT-PCR analysis of tears showed positive results in 18 patients (24%), and 29 of 225 samples (12.9%) showed positive results. Positive results were found in 11 (14.7%), 11 (14.7%), and 7 (9.3%) patients in groups 1, 2, and 3, respectively (P = 0.3105). Mean Ct values in groups 1, 2, and 3 were 28.36 ± 6.15, 29.00 ± 5.58, and 27.86 ± 6.46 (P = 0.92), respectively. Five patients showed positive RT-PCR results by all 3 methods (mean Ct value, 25.24 ± 6.33), and 12 patients showed positive results by any of the 3 methods (mean Ct value, 32.16 ± 1.94), the difference in Ct values being statistically significant (P = 0.029). The median value of symptomatology in patients with positive RT-PCR results from tears was 5 days (range, 4-9 days). CONCLUSIONS: SARS-CoV-2 RNA was detected in tears of 24% of patients with laboratory-proven moderate to severe COVID-19. Conjunctival swab remains the gold standard of tear collection for RT-PCR assay. A significantly higher possibility of viral transmission exists through tears in patients with moderate to severe COVID-19.

Ruling out COVID-19 by chest CT at emergency admission when prevalence is low: the prospective, observational SCOUT study.

BACKGROUND: It is essential to avoid admission of patients with undetected corona virus disease 2019 (COVID-19) to hospitals' general wards. Even repeated negative reverse transcription polymerase chain reaction (RT-PCR) results do not rule-out COVID-19 with certainty. The study aimed to evaluate a rule-out strategy for COVID-19 using chest computed tomography (CT) in adults being admitted to the emergency department and suspected of COVID-19. METHODS: In this prospective, single centre, diagnostic accuracy cohort study, consecutive adults (≥ 18 years) presenting with symptoms consistent with COVID-19 or previous contact to infected individuals, admitted to the emergency department and supposed to be referred to general ward were included in March and April 2020. All participants underwent low-dose chest CT. RT-PCR- and specific antibody tests were used as reference standard. Main outcome measures were sensitivity and specificity of chest CT. Predictive values were calculated based on the theorem of Bayes using Fagan's nomogram. RESULTS: Of 165 participants (56.4% male, 71 ± 16 years) included in the study, the diagnosis of COVID-19 was confirmed with RT-PCR and AB tests in 13 participants (prevalence 7.9%). Sensitivity and specificity of chest CT were 84.6% (95% confidence interval [CI], 54.6-98.1) and 94.7% (95% CI, 89.9-97.7), respectively. Positive and negative likelihood ratio of chest CT were 16.1 (95% CI, 7.9-32.8) and 0.16 (95% CI, 0.05-0.58) and positive and negative predictive value were 57.9% (95% CI, 40.3-73.7) and 98.6% (95% CI, 95.3-99.6), respectively. CONCLUSION: At a low prevalence of COVID-19, chest CT could be used as a complement to repeated RT-PCR testing for early COVID-19 exclusion in adults with suspected infection before referral to hospital's general wards. Trial registration ClinicalTrials.gov: NCT04357938 April 22, 2020.