Challenges to the effectiveness of next-generation sequencing in formalin-fixed paraffin-embedded tumor samples for non-small cell lung cancer.

INTRODUCTION: Next-generation sequencing (NGS) of Formalin-Fixed and Paraffin-Embedded (FFPE) specimens is routine in precision oncology practice. However, results are not always conclusive, and it is important to identify which factors may influence FFPE tumor sequencing success. MATERIALS AND METHODS: Here, we evaluated the influence of pre-analytical factors on 705 samples of non-small cell lung cancer specimens that underwent NGS testing. Factors such as tumor site, tumor cell percentage, fragment size, primary tumor or metastasis, presence of necrosis, DNA purity, DNA concentration, sample origin and year of testing. RESULTS: The overall NGS success rate was 84.9 % (n = 599). Bone site specimens had a very low success rate (42.1 %), differing from lung samples (79.8 %) (P < 0.05). Samples with tumor percentages <5 % (success rate of 44.4 %) represented 14.1 % of failed sequencings. Moreover, samples with tumor percentages >10 %-20 % (82 %) did not differ from those with >30 % (88.9 %) on sequencing outcomes (P = 0.086). Specimens that provided DNA concentrations >2.0 ng/uL, 1.0-2.0 ng/uL, 0.5-1.0 ng/uL and <0.5 ng/uL had success rates of 92 %, 77.1 %, 61.3 % and 20.4 %, respectively. Small fragments (≤0.2 cm2) had a success rate of 74.7 % and were more prevalent in the unsuccessful group (P < 0.05). CONCLUSIONS: Our results suggest that tumor percentage, fragment size, decalcified bone specimens, and DNA concentration are potential modifiers of NGS success rates. Interestingly, specimens with tumor percentages between 10 % and 20 % have the same sequencing outcome than specimens with >30 %. These results can strengthen the understanding of factors that lead to NGS success variability.

SARS-CoV-2 Variants of Concern: Presumptive Identification via Sanger Sequencing Analysis of the Receptor Binding Domain (RBD) Region of the S Gene.

Variants of concern (VOCs) of SARS-CoV-2 are viral strains that have mutations associated with increased transmissibility and/or increased virulence, and their main mutations are in the receptor binding domain (RBD) region of the viral spike. This study aimed to characterize SARS-CoV-2 VOCs via Sanger sequencing of the RBD region and compare the results with data obtained via whole genome sequencing (WGS). Clinical samples (oro/nasopharyngeal) with positive RT-qPCR results for SARS-CoV-2 were used in this study. The viral RNA from SARS-CoV-2 was extracted and a PCR fragment of 1006 base pairs was submitted for Sanger sequencing. The results of the Sanger sequencing were compared to the lineage assigned by WGS using next-generation sequencing (NGS) techniques. A total of 37 specimens were sequenced via WGS, and classified as: VOC gamma (8); delta (7); omicron (10), with 3 omicron specimens classified as the BQ.1 subvariant and 12 specimens classified as non-VOC variants. The results of the partial Sanger sequencing presented as 100% in agreement with the WGS. The Sanger protocol made it possible to characterize the main SARS-CoV-2 VOCs currently circulating in Brazil through partial Sanger sequencing of the RBD region of the viral spike. Therefore, the sequencing of the RBD region is a fast and cost-effective laboratory tool for clinical and epidemiological use in the genomic surveillance of SARS-CoV-2.

Prolonged SARS-CoV-2 Infection and Intra-Patient Viral Evolution in an Immunodeficient Child.

BACKGROUND: With the progression of the Coronavirus disease pandemic, the number of mutations in the viral genome has increased, showing the adaptive evolution of severe acute respiratory syndrome coronavirus 2 in humans and intensification in transmissibility. Long-term infections also allow the development of viral diversity. In this study, we report the case of a child with severe combined immu presenting a prolonged severe acute respiratory syndrome coronavirus 2 infection. We aimed to analyze 3 naso-oropharyngeal swab samples collected between August and December 2021 to describe the amino acid changes present in the sequence reads that may have a role in the emergence of new viral variants. METHODS: The whole genome from clinical samples was sequenced through high throughput sequencing and analyzed using a workflow to map reads and then find variations/single-nucleotide polymorphisms. In addition, the samples were isolated in cell culture, and a plaque forming units assay was performed, which indicates the presence of viable viral particles. RESULTS: The results obtained showed that the virus present in all samples is infectious. Also, there were 20 common mutations among the 3 sequence reads, found in the ORF1ab and ORF10 proteins. As well, a considerable number of uncommon mutations were found. CONCLUSIONS: In conclusion, we emphasize that genomic surveillance can be a useful tool to assess possible evolution signals in long-term patients.

SARS-CoV-2 nucleic acid shedding is variable for every person.

INTRODUCTION: Considering the persistent positivity on RT-qPCR tests, the results of SARS-CoV-2 were monitored to evaluate the viral RNA shedding period. METHODS: Between March and June 2020, the sequential results of 29 healthcare workers' were monitored using RT-qPCR. RESULTS: More than 50% of the individuals remained RT-qPCR positive after 14 days. Furthermore, this is the first study to describe positive RT-qPCR for SARS-CoV-2 in a healthcare worker with mild symptoms 95 days after the first positive test. CONCLUSIONS: Sequential RT-qPCR results were heterogeneous, and the viral RNA shedding period is unique for each person.

Detection of SARS-CoV-2 lineage P.1 in patients from a region with exponentially increasing hospitalisation rate, February 2021, Rio Grande do Sul, Southern Brazil.

The emergence of SARS-CoV-2 P.1 lineage coincided with a surge in hospitalisations in the North region of Brazil. In the South region's Rio Grande do Sul state, severe COVID-19 case numbers rose 3.8 fold in February 2021. During that month, at a COVID-19 referral hospital in this state, whole-genome sequencing of a subset of cases' specimens (n = 27) revealed P.1 lineage SARS-CoV-2 in most (n = 24). Findings raise concerns regarding a possible association between lineage P.1 and rapid case and hospitalisation increases.

Pooling of samples to optimize SARS-CoV-2 diagnosis by RT-qPCR: comparative analysis of two protocols.

This study has aimed to evaluate the use pool of samples as a strategy to optimize the diagnostic of SARS-CoV-2 by RT-qPCR. A total of 220 naso/orofaryngeal swab samples were collected and tested using two different protocols of sample pooling. Results from protocol A were identical with the individual results. However, for results from protocol B, reduced agreement (91%) was observed in relation to individual testing. Inconsistencies observed were related to RT-qPCR results with higher cycle thresholds. These results suggest that pooling of samples before RNA extraction is preferable in terms of diagnostic for SARS-CoV-2.

Comparison of a direct immunofluorescence assay (Oxoid IMAGEN®) and a multiplex RT-PCR DNA microarray assay (CLART® PneumoVir) for the detection of respiratory viruses in hospitalized children.

The objective of this study was to compare the positive detection rates obtained using the Oxoid IMAGEN® direct immunofluorescence assay (designated as IF) with those obtained using the CLART® PneumoVir multiplex RT-PCR DNA microarray assay (designated as RT-PCR) in the diagnosis of respiratory viruses in hospitalized children. This was a retrospective study of 62 individuals < 18 years old who had nasopharyngeal aspirates collected for virus identification in a tertiary university hospital in south Brazil between January 1st, 2014 and December 31st, 2014. All 62 nasopharingeal aspirates were analyzed using both assay methods. The main outcome to be measured was the difference in the proportion of test samples returning a positive virus detection result between the IF and the RT-PCR. The McNemar test was used for data analysis and the results showed that the RT-PCR and the IF methods produced 55 (88.7 %) and 17 (27.4 %) virus-positive samples, respectively (p < 0.001). The most prevalent virus was rhinovirus (45.5 % of the RT-PCR positive samples). The RT-PCR method increased the detection rates of human respiratory syncytial virus, influenza A virus and parainfluenza 3 virus. The RT-PCR and IF had concordant results in 19 samples (30.6 %) and discordant results in 43 samples (69.4 %). It is concluded that in comparison to the Oxoid IMAGEN® IF method, the CLART® PneumoVir multiplex RT-PCR method had a greater potential to contribute to the clinical management of hospitalized children due its greater ability in detecting respiratory viruses than the IF method.

Analysis of Predictive Biomarkers in Patients With Lung Adenocarcinoma From Southern Brazil Reveals a Distinct Profile From Other Regions of the Country.

PURPOSE: Adenocarcinoma is the most common histologic subtype of non-small-cell lung cancer, representing 40% of all diagnoses. Several biomarkers are currently used to determine patient eligibility for targeted treatments, including analysis of molecular alterations in EGFR and ALK, as well as programmed death-ligand 1 (PD-L1) protein expression. Epidemiologic data reporting the frequency of these biomarkers in Brazilian patients with lung adenocarcinoma (LUAD) are limited, and existing studies predominantly included patients from the southeast region of the country. MATERIALS AND METHODS: The goal of this study was to investigate the frequency of somatic mutations in the EGFR, KRAS, NRAS, and BRAF genes, ALK, and PD-L1 expression in a series of Brazilian patients diagnosed with LUAD predominantly recruited from centers in southern Brazil. Molecular analysis of the EGFR, KRAS, NRAS, and BRAF genes was performed by next-generation sequencing using DNA extracted from tumor tissue. Immunohistochemistry was used to detect ALK and PD-L1 expression. RESULTS: Analysis of 619 tumors identified KRAS mutations in 189 (30.2%), EGFR mutations in 120 (19.16%), and BRAF mutations in 19 (3%). Immunohistochemistry demonstrated ALK and PD-L1 expression in 4% and 35.1% of patients, respectively. CONCLUSION: To our knowledge, this is the first study investigating the molecular epidemiology of patients with LUAD from southern Brazil and the largest assessing the frequency of multiple predictive biomarkers for this tumor in the country. The study also reveals a distinct mutation profile compared with data originating from other regions of Brazil.

Assessment of Changes in Body Composition During the First Postoperative Year After Bariatric Surgery.

BACKGROUND: The potential effect of bariatric surgery on weight reduction and improvement of associated comorbidities is known, but the ratio obtained between the components of body weight, including lean body mass, body fat mass, and bone mass, is still not determined. This study aims to verify the changes in body composition during the first year after bariatric surgery. METHODS: We conducted a prospective observational cohort study. Fifty patients who underwent bariatric surgery and maintained follow-ups were selected. Patients were assessed preoperatively and postoperatively for periods of 1, 3, 6, and 12 months using tetrapolar bioelectrical impedance analysis and laboratory testing of lipids and serum albumin levels. Data were statistically analyzed. RESULTS: Statistically significant differences (p < 0.001) were obtained between the preoperative and 12-month evaluation respectively, for body mass index (BMI) (45.8 ± 7.5 to 30.0 ± 4.8 kg/m2), FM (64.7 ± 15.5 to 30.6 ± 9.8 kg), PFM (51.6 ± 4.17 to 37.3 ± 7.6%), and total cholesterol levels (197.1 ± 49.8 to 169.8 ± 31.0 mg/dL). The decrease in PFM shows a better proportion between the body components. PFM showed significantly higher decrease in males than in females (p = 0.012). Lean body mass (p = 0.000) reduction was highest for patients operated by the Unified Health System (SUS, Government of Brazil) probably because of its few financial resources to maintain postoperative care. CONCLUSION: The change in body composition of patients who underwent Roux-en-Y gastric bypass was statistically significant for all variables examined during the first year postoperatively. This shows the effectiveness of the surgical procedure and clinical protocol set, which tends to favor a better health prognosis and weight maintenance in the long term.

Assessment of conventional PCR and real-time PCR compared to the gold standard method for screening Streptococcus agalactiae in pregnant women.

Group B Streptococcus is a causative agent of invasive neonatal infections. Maternal colonization by Streptococcus agalactiae is a necessary condition for vertical transmission, with efficient screening of pregnant women playing an essential role in the prevention of neonatal infections. In this study, we aimed to compare the performance of conventional polymerase chain reaction and real-time PCR assays as screening methods for S. agalactiae in pregnant women against the microbiological culture method considered as the gold-standard. A total of 130 samples from pregnant women were analyzed for sensitivity, specificity, positive predictive value, and negative predictive value. Statistical analysis was performed using the SPSS software, version 20.0. The verified colonization rate was 3.8% with the gold-standard, 17.7% with conventional PCR assay, and 29.2% with the real-time PCR test. The trials with conventional PCR and real-time PCR had a sensitivity of 100% and a specificity of 85.6% and 73.6%, respectively. The real-time PCR assay had a better performance compared to the gold-standard and a greater detection rate of colonization by S. agalactiae compared to conventional PCR assay. With its quick results, it would be suitable for using in routine screenings, contributing to the optimization of preventive approaches to neonatal S. agalactiae infection.

Assessment of conventional PCR and real-time PCR compared to the gold standard method for screening Streptococcus agalactiae in pregnant women

ABSTRACT Group B Streptococcus is a causative agent of invasive neonatal infections. Maternal colonization by Streptococcus agalactiae is a necessary condition for vertical transmission, with efficient screening of pregnant women playing an essential role in the prevention of neonatal infections. In this study, we aimed to compare the performance of conventional polymerase chain reaction and real-time PCR assays as screening methods for S. agalactiae in pregnant women against the microbiological culture method considered as the gold-standard. A total of 130 samples from pregnant women were analyzed for sensitivity, specificity, positive predictive value, and negative predictive value. Statistical analysis was performed using the SPSS software, version 20.0. The verified colonization rate was 3.8% with the gold-standard, 17.7% with conventional PCR assay, and 29.2% with the real-time PCR test. The trials with conventional PCR and real-time PCR had a sensitivity of 100% and a specificity of 85.6% and 73.6%, respectively. The real-time PCR assay had a better performance compared to the gold-standard and a greater detection rate of colonization by S. agalactiae compared to conventional PCR assay. With its quick results, it would be suitable for using in routine screenings, contributing to the optimization of preventive approaches to neonatal S. agalactiae infection.

Global respiratory syncytial virus-associated mortality in young children (RSV GOLD): a retrospective case series.

BACKGROUND: Respiratory syncytial virus (RSV) infection is an important cause of pneumonia mortality in young children. However, clinical data for fatal RSV infection are scarce. We aimed to identify clinical and socioeconomic characteristics of children aged younger than 5 years with RSV-related mortality using individual patient data. METHODS: In this retrospective case series, we developed an online questionnaire to obtain individual patient data for clinical and socioeconomic characteristics of children aged younger than 5 years who died with community-acquired RSV infection between Jan 1, 1995, and Oct 31, 2015, through leading research groups for child pneumonia identified through a comprehensive literature search and existing research networks. For the literature search, we searched PubMed for articles published up to Feb 3, 2015, using the key terms "RSV", "respiratory syncytial virus", or "respiratory syncytial viral" combined with "mortality", "fatality", "death", "died", "deaths", or "CFR" for articles published in English. We invited researchers and clinicians identified to participate between Nov 1, 2014, and Oct 31, 2015. We calculated descriptive statistics for all variables. FINDINGS: We studied 358 children with RSV-related in-hospital death from 23 countries across the world, with data contributed from 31 research groups. 117 (33%) children were from low-income or lower middle-income countries, 77 (22%) were from upper middle-income countries, and 164 (46%) were from high-income countries. 190 (53%) were male. Data for comorbidities were missing for some children in low-income and middle-income countries. Available data showed that comorbidities were present in at least 33 (28%) children from low-income or lower middle-income countries, 36 (47%) from upper middle-income countries, and 114 (70%) from high-income countries. Median age for RSV-related deaths was 5·0 months (IQR 2·3-11·0) in low-income or lower middle-income countries, 4·0 years (2·0-10·0) in upper middle-income countries, and 7·0 years (3·6-16·8) in high-income countries. INTERPRETATION: This study is the first large case series of children who died with community-acquired RSV infection. A substantial proportion of children with RSV-related death had comorbidities. Our results show that perinatal immunisation strategies for children aged younger than 6 months could have a substantial impact on RSV-related child mortality in low-income and middle-income countries. FUNDING: Bill & Melinda Gates Foundation.

Effects of Lactobacillus rhamnosus GG on hepatic and serum lipid profiles in zebrafish exposed to ethanol.

Zebrafish is a powerful tool in pharmacological research and useful to identify new therapies. Probiotics can offer therapeutic options in alcoholic liver disease. This study was done in two independent experiments: first, we confirmed the intestinal colonization of probiotic Lactobacillus rhamnosus GG (LGG) after ethanol exposure. Second, four groups were performed: control (C), probiotic (P), ethanol (E), and probiotic+ethanol (P+E). Liver histology, hepatocytes morphometry, hepatic and serum lipid quantifications were conducted in second experiment. During 4 weeks, P and P+E groups were fed with LGG supplemented feed; E and C unsupplemented. E and P+E groups received 0.5% of ethanol added into tank water. Zebrafish exposed to ethanol (E group) presented intense liver steatosis after 28 days in contrast to the almost normalized liver histology of P+E group at the same period. Liver morphometry showed a significant enlargement of hepatocytes of E group after 4 weeks (p<0.0001). Serum triglycerides decreased in P+E group compared with C, P (p<0.001), and E (p=0.004), after 14 and 28 days similarly. Serum cholesterol was also decreased by LGG; P group decreased compared with C and E after 14 days (p=0.002 and p=0.007, respectively) and P+E group decreased significantly compared with E and C groups (p<0.0001) after 28 days. Hepatic triglycerides were reduced in P+E group after 28 days compared to E (p=0.006). The persistence of LGG in zebrafish intestines was demonstrated. LGG decreased serum levels of triglycerides and cholesterol and improved hepatic steatosis.

Outbreaks due to Mycobacterium abscessus subsp. bolletii in southern Brazil: persistence of a single clone from 2007 to 2011.

Outbreaks associated with rapidly growing mycobacteria (RGM) have been increasingly reported worldwide, including in Brazil. Among the RGM, the Mycobacterium abscessus complex is the most pathogenic and related to multidrug resistance. The aim of this study was to evaluate the antimicrobial susceptibility and molecular profile of RGM isolates involved in new postsurgical infection outbreaks in Brazil since 2007. Of the 109 cases reported in the state of Rio Grande do Sul between 2007 and 2011, 43 (39 %) had confirmed mycobacterial growth in culture. Clinical isolates were obtained from biopsy specimens or abscess aspirates. PRA-hsp65 restriction pattern identified the isolates as M. abscessus type 2, and partial rpoB sequencing confirmed the identification as M. abscessus subsp. bolletii. All isolates were susceptible to amikacin and resistant to ciprofloxacin, doxycycline, sulfamethoxazole, moxifloxacin and tobramycin. Most isolates (72 %) were fully susceptible to cefoxitin but six isolates (14 %) were fully resistant to clarithromycin. The latter differed from the susceptibility profiles of the previously described BRA100 clone from other Brazilian regions. Nevertheless, pulsed-field gel electrophoresis analysis revealed that these isolates belonged to a single BRA100 clone. In conclusion, our study reports the persistence of an emergent single and highly resistant clone of M. abscessus subsp. bolletii for several years even after national implementation of infection control measures.

Viral epidemiology of respiratory infections among children at a tertiary hospital in Southern Brazil.

INTRODUCTION: This study reports the pediatric epidemiology of respiratory syncytial virus (RSV), influenza (IF), parainfluenza (PIV), and adenovirus (ADV) at Hospital de Clínicas de Porto Alegre. METHODS: Cases of infection, hospitalizations in intensive care units (ICUs), nosocomial infections, and lethality rates were collected from 2007 to 2010. RESULTS: RSV accounted for most nosocomial infections. Intensive care units admission rates for ADV and RSV infections were highest in 2007 and 2010. During 2008-2009, H1N1 and ADV had the highest ICU admission rates. ADV had the highest fatality rate during 2007-2009. CONCLUSIONS: Each virus exhibited distinct behavior, causing hospitalization, outbreaks, or lethality.

Evaluation of respiratory syncytial virus group A and B genotypes among nosocomial and community-acquired pediatric infections in Southern Brazil.

BACKGROUND: Respiratory syncytial virus (RSV) is the main cause of lower respiratory tract illness in children worldwide. Molecular analyses show two distinct RSV groups (A and B) that comprise different genotypes. This variability contributes to the capacity of RSV to cause yearly outbreaks. These RSV genotypes circulate within the community and within hospital wards. RSV is currently the leading cause of nosocomial respiratory tract infections in pediatric populations. The aim of this study was to evaluate the G protein gene diversity of RSV amplicons. METHODS: Nasopharyngeal aspirate samples were collected from children with nosocomial or community-acquired infections. Sixty-three RSV samples (21 nosocomial and 42 community-acquired) were evaluated and classified as RSV-A or RSV-B by real-time PCR. Sequencing of the second variable region of the G protein gene was performed to establish RSV phylogenetics. RESULTS: We observed co-circulation of RSV-A and RSV-B, with RSV-A as the predominant group. All nosocomial and community-acquired RSV-A samples were from the same phylogenetic group, comprising the NA1 genotype, and all RSV-B samples (nosocomial and community-acquired) were of the BA4 genotype. Therefore, in both RSV groups (nosocomial and community-acquired), the isolates belonged to only one genotype in circulation. CONCLUSIONS: This is the first study to describe circulation of the NA1 RSV genotype in Brazil. Furthermore, this study showed that the BA4 genotype remains in circulation. Deciphering worldwide RSV genetic variability will aid vaccine design and development.

Optimization of one-step real-time PCR for the X-tail target of HCV as a diagnostic test

Infection with hepatitis C virus (HCV) is a global public health issue. The bloodborne nature of HCV transmission poses a substantial risk to healthcare workers, due to occupational exposure to needlestick injuries and blood and other body fluids containing the virus. Undiagnosed HCV infection, including in healthcare workers, represents a growing problem worldwide as the infected population ages, and HCV-related mortality and morbidity is expected to rise substantially over the coming decades. Consequently, diagnostic tests for HCV play an important role in this scenario. The aim of this study was to standardize a one-step RT-PCR assay for detection of HCV. The test demonstrated reproducibility, sensibility (100%), and the limit of detection was set at 100IU/mL. Our study indicates that this assay can be used as a diagnostic tool to follow up healthcare workers after occupational exposure

Minimum detection limit of an in-house nested-PCR assay for herpes simplex virus and varicella zoster virus.

INTRODUCTION: Herpes simplex virus (HSV) and varicella zoster virus (VZV) are responsible for a variety of human diseases, including central nervous system diseases. The use of polymerase chain reaction (PCR) techniques on cerebrospinal fluid samples has allowed the detection of viral DNA with high sensitivity and specificity. METHODS: Serial dilutions of quantified commercial controls of each virus were subjected to an in-house nested-PCR technique. RESULTS: The minimum detection limits for HSV and VZV were 5 and 10 copies/µL, respectively. CONCLUSIONS: The detection limit of nested-PCR for HSV and VZV in this study was similar to the limits found in previous studies.

Minimum detection limit of an in-house nested-PCR assay for herpes simplex virus and varicella zoster virus

Introduction Herpes simplex virus (HSV) and varicella zoster virus (VZV) are responsible for a variety of human diseases, including central nervous system diseases. The use of polymerase chain reaction (PCR) techniques on cerebrospinal fluid samples has allowed the detection of viral DNA with high sensitivity and specificity. Methods Serial dilutions of quantified commercial controls of each virus were subjected to an in-house nested-PCR technique. Results The minimum detection limits for HSV and VZV were 5 and 10 copies/µL, respectively. Conclusions The detection limit of nested-PCR for HSV and VZV in this study was similar to the limits found in previous studies. .

Genetic similarity of Burkholderia cenocepacia from cystic fibrosis patients.

Burkholderia cenocepacia may cause serious infections in patients with cystic fibrosis, and this microorganism can be highly transmissible. Pulsed-field gel electrophoresis is widely used to study the dynamics of strain spread in cystic fibrosis patients. The aim of this work was to perform pulsed-field gel electrophoresis-based molecular typing of B. cenocepacia isolates to evaluate the epidemiology of this species at our hospital. A total of 28 isolates from 23 cystic fibrosis patients were analyzed. Initially, we compared isolates obtained from the same patient at different periods of time. We then compared the pulsed-field gel electrophoresis profiles of 15 IIIA isolates, and in a third analysis, evaluated the genetic profile of 8 IIIB isolates from different patients. The pulsed-field gel electrophoresis profiles of isolates from the same patient indicated that they are genetically indistinguishable. Analysis of isolates from different patients revealed the presence of multiple clonal groups. These results do not indicate cross-transmission of a unique clone of B. cenocepacia among cystic fibrosis patients, although this has been observed in some patients. Our findings highlight the importance of adequate patient follow-up at cystic fibrosis centers and adherence to management and segregation measures in cystic fibrosis patients colonized with B. cenocepacia.