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.

RT-qPCR half-reaction optimization for the detection of SARS-CoV-2.

INTRODUCTION: The main laboratory test for the diagnosis of coronavirus disease 2019 (COVID-19) is the reverse transcription real-time polymerase chain reaction (RT-qPCR). However, RT-qPCR is expensive because of the number of tests required. This study aimed to evaluate an alternative to the RT-qPCR approach for the detection of sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is half of the total volume currently recommended by the US Centers for Disease Control and Prevention. METHODS: The analytical limit of detection (LoD) and the reaction efficiency using half volumes of the RT-qPCR assay were evaluated for the N1 and N2 regions using a synthetic control RNA. A panel of 76 SARS-CoV-2-positive and 26 SARS-CoV-2-negative clinical samples was evaluated to establish clinical sensitivity and specificity. RESULTS: The RT-qPCR assay efficiency was 105% for the half and standard reactions considering the N2 target and 84% (standard) and 101% (half) for N1. The RT-qPCR half-reaction LoD for N1 and N2 were 20 and 80 copies/µL, respectively. The clinical sensitivity and specificity were 100%. The half reaction presented a decrease of up to 5.5 cycle thresholds compared with standard RT-qPCR. CONCLUSIONS: The use of the RT-qPCR half-reaction proved feasible and economic for the detection of SARS-CoV-2 RNA.

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.

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.

Enhancing tuberculosis diagnosis by polymerase chain reaction: an experience at a tertiary hospital

Introduction: Tuberculosis (TB) persists as a severe global public health issue. The aim of the present study was to evaluate the performance of an in-house TB PCR (polymerase chain reaction) in sputum. Methods: DNA from sputum specimens were submitted to a nested-PCR protocol for the IS6110 region detection. PCR results were compared to those of the traditional methods for TB diagnosis, i.e., acid-fast bacilli (AFB) smear microscopy and culture. We analyzed sputum samples obtained from 133 patients. Results: A total of 48 (36%) cultures yielded indeterminate results due to contamination. This high contamination rate may be explained by the fact that samples from fibrocystic patients were included in this study. Additionally, other five samples were positive for nontuberculous mycobacteria (NTM). Therefore, it was possible to compare 80 patients for M. tuberculosis detection. We found 14 positive samples: five presented positive results in the three methods (5/14; 35.7%), two were positive in culture and PCR (2/14; 14.3%), one was positive in AFB and PCR (1/14; 7.1%), five were positive only in PCR (5/14; 35.7%) and 1 was positive only in culture (1/14; 7.1%). Thus, positivity rates for each technique were: 7.5% for AFB (6/80), 10% for culture (8/80) and 16.25% for PCR (13/80). Among the 48 patients who had indeterminate results in sputum culture, two samples were positive in PCR. Conclusion: Considering the limitations of the traditional methods, the use of PCR as a molecular technique could be advantageous for TB diagnosis.

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.

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. .

The impact of serum vancomycin levels and minimum inhibitory concentrations of methicillin-resistant Staphylococcus aureus on mortality in patients with nosocomial pneumonia.

BACKGROUND: Vancomycin is the treatment of choice for methicillin-resistant Staphylococcus aureus (MRSA) infections; however, treatment failure is not uncommon, even when the minimum inhibitory concentration (MIC) of the MRSA strain is within the susceptible range for vancomycin. OBJECTIVE: To describe the relationship between molecular markers such as the mecA and agrII genes, serum vancomycin levels and vancomycin MICs, and the 30-day mortality rate of patients with nosocomial MRSA pneumonia in an intensive care unit (ICU). METHODS: The present study was a prospective cohort study including all patients with MRSA hospital-acquired pneumonia or ventilator-associated pneumonia who were admitted to the ICU of a tertiary care hospital between June 2009 and December 2011. The MIC for vancomycin was determined using the E-test and broth microdilution methods. Variables analyzed included age, sex, comorbid conditions, serum vancomycin trough concentration, the Acute Physiology and Chronic Health Evaluation II (APACHE) score and the presence of the agrII gene. The primary outcome was mortality at 30 days. RESULTS: Thirty-six (42.4%) patients died within 30 days of the index MRSA culture. A multiple regression analysis that included the variables of MIC (determined using the E-test or broth microdilution methods), APACHE II score, serum vancomycin level and the presence of agrII revealed that only the APACHE II score was related to the 30-day mortality rate (P=0.03). Seven patients (9.0%) with isolates exhibiting an MIC ≥1.5 µg/mL according to the E-test method died, and nine patients (11.6%) survived (P=0.76). Of the patients for whom MICs were determined using the broth microdilution method, 11 (14.1%) patients with MICs of 1.0 µg/mL died, and 16 (20.5%) survived (P=0.92). The median APACHE II score of survivors was 22.5, and the median score of nonsurvivors was 25.0 (P=0.03). The presence of the agrII gene was not related to the 30-day mortality rate. CONCLUSIONS: Patients with severe hospital-acquired pneumonia presented with MRSA isolates with low to intermediate vancomycin MICs in the ICU setting. At the Hospital de Clínicas de Porto Alegre (Porto Alegre, Brazil), the 30-day mortality rate was high, and was similar among patients with severe hospital-acquired pneumonia infected with MRSA isolates that exhibited MICs of ≤1.5 µg/mL determined using the E-test method and ≤1.0 µg/mL determined using the broth microdilution method in those who achieved optimal serum vancomycin levels. The APACHE II scores which provides an overall estimate of ICU mortality were independently associated with mortality in the present study, regardless of the MICs determined. Molecular markers, such as the agrII gene, were not associated with higher mortality in the present study.

HISTORIQUE: La vancomycine est le traitement de première intention des infections par le Staphylococcus aureus résistant à la méthicilline (SARM), mais les échecs thérapeutiques ne sont pas rares, même lorsque la concentration minimale inhibitrice (CMI) de la souche de SARM se situe dans la plage susceptible de vancomycine. OBJECTIF: Décrire le lien entre les marqueurs moléculaires comme les gènes mecA et agrII, le taux de vancomycine sérique et la CMI de vancomycine, et le taux de mortalité au bout de 30 jours des patients atteints d'une pneumonie à SARM d'origine nosocomiale à l'unité de soins intensifs (USI). MÉTHODOLOGIE: La présente étude prospective de cohorte incluait tous les patients ayant une pneumonie à SARM d'origine nosocomiale ou d'une pneumonie acquise sous ventilation mécanique qui ont été hospitalisés à l'USI d'un hôpital de soins tertiaires entre juin 2009 et décembre 2011. Les chercheurs ont déterminé la CMI de la vancomycine au moyen des méthodes d'E-test et de microdilution en bouillon. Les variables qu'ils ont analysées sont l'âge, le sexe, les états comorbides, la concentration minimale de vancomycine sérique, le score APACHE (évaluation de physiologie aiguë et de maladie chronique II) et la présence du gène agrII. La mortalité au bout de 30 jours était l'issue primaire. RÉSULTATS: Trente-six patients (42,4 %)sont décédés dans les 30 jours suivant la culture de référence du SARM. Une analyse de régression multiple qui incluait les variables de la CMI (déterminée au moyen des méthodes d'E-test ou de microdilution en bouillon, le score APACHE II, le taux de vancomycine sérique et la présence du gène f agrII a révélé que seul le score APACHE II était lié au taux de mortalité au bout de 30 jours (P=0,03). Sept patients (9,0 %) dont les isolats présentaient une CMI d'au moins 1,5 µg/mL d'après la méthode d'E-test sont décédés, et neuf patients (11,6 %) ont survécu (P=0,76). Chez les patients dont la CMI a été déterminée au moyen de la méthode de microdilution en bouillon, 11 (14,1 %) ayant une CMI de 1,0 µg/mL sont décédés et 16 (20,5 %) ont survécu (P=0,92). Les survivants avaient un score APACHE II médian de 22,5, et les non-survivants, de 25,0 (P=0,03). La présence du gène agrII n'était pas liée au taux de décès au bout de 30 jours. CONCLUSIONS: Les patients ayant une grave pneumonie d'origine nosocomiale présentaient des isolats de SARM à la CMI faible à intermédiaire à la vancomycine à l'USI. Au Hospital de Clínicas de Porto Alegre (Porto Alegre, Brésil), le taux de mortalité au bout de 30 jours était élevé, tout comme chez les patients atteints d'une grave pneumonie d'origine nosocomiale infectés par des isolats du SARM dont la CMI était égale ou inférieure à 1,5 µg/mL d'après par la méthode d'E-test (ou égale ou inférieure à 1,0 µg/mL d'après la méthode de microdilution en bouillon) qui ont atteint des taux optimaux de vancomycine sérique. Les scores APACHE II qui procurent une évaluation globale de la mortalité à l'USI s'associaient de manière indépendante avec la mortalité dans la présente étude, quelle que soit la CMI établie. De plus, les marqueurs moléculaires, tels que le gène agrII, n'étaient pas liés à un taux de mortalité plus élevé y.

Macrolides decrease the minimal inhibitory concentration of anti-pseudomonal agents against Pseudomonas aeruginosa from cystic fibrosis patients in biofilm.

BACKGROUND: Biofilm production is an important mechanism for bacterial survival and its association with antimicrobial resistance represents a challenge for the patient treatment. In this study we evaluated the in vitro action of macrolides in combination with anti-pseudomonal agents on biofilm-grown Pseudomonas aeruginosa recovered from cystic fibrosis (CF) patients. RESULTS: A total of 64 isolates were analysed. The biofilm inhibitory concentration (BIC) results were consistently higher than those obtained by the conventional method, minimal inhibitory concentration, (MIC) for most anti-pseudomonal agents tested (ceftazidime: P = 0.001, tobramycin: P = 0.001, imipenem: P < 0.001, meropenem: P = 0.005). When macrolides were associated with the anti-pseudomonal agents, the BIC values were reduced significantly for ceftazidime (P < 0.001) and tobramycin (P < 0.001), regardless the concentration of macrolides. Strong inhibitory quotient was observed when azithromycin at 8 mg/L was associated with all anti-pseudomonal agents tested in biofilm conditions. CONCLUSIONS: P. aeruginosa from CF patients within biofilms are highly resistant to antibiotics but macrolides proved to augment the in vitro activity of anti-pseudomonal agents.

Optimization of one-step duplex real-time RT-PCR for detection of influenza and respiratory syncytial virus in nasopharyngeal aspirates.

Viruses are major contributors to acute respiratory infection-related morbidity and mortality worldwide. The influenza (IF) viruses and human respiratory syncytial virus (RSV) play a particularly important role in the etiology of acute respiratory infections. This study sought to standardize a one-step duplex real-time RT-PCR technique to optimize diagnosis of IFA/IFB and RSVA/RSVB infection. Viral RNA was extracted with the commercially available QIAamp Mini Kit according to manufacturer instructions. RT-PCR was performed with primers to the matrix protein gene of IFA, the hemagglutinin gene of IFB and the N gene of RSVA and RSVB. The limits of detection were 1 copy/µL for IFA, 10 copies/µL for IFB, 5 copies/µL for RSVA, and 250 copies/µL for RSVB. The specificity of RT-PCR was determined by comparison against a panel of several respiratory pathogens. RT-PCR and indirect immunofluorescence (IIF) were compared in a sample of 250 nasopharyngeal aspirates (NPAs) collected during the year 2010. RT-PCR was more sensitive than IIF and able to detect viral co-infections. In summary, RT-PCR optimized for IFA/IFB and RSVA/RSVB is sensitive and specific for these viral agents and is therefore useful for assessment of the etiology of respiratory infections, whether for clinical or epidemiological purposes.

Distribution of erm genes and low prevalence of inducible resistance to clindamycin among staphylococci isolates

INTRODUCTION: Resistance to macrolides, lincosamides and streptogramins B (MLS B antibiotics) in staphylococci may be due to modification in ribosomal target methylase encoded by erm genes. The expression of MLS B resistance lead to three phenotypes, namely constitutive resistance (cMLS B), inducible resistance (iMLS B), and resistance only to macrolides and streptogramins B (MS B). The iMLS B resistance is the most difficult to detect in the clinical laboratory. OBJECTIVE: This study investigated the expression of MLS B resistance and the prevalence of the erm genes among 152 clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus (CNS) from Hospital de Clínicas de Porto Alegre. METHODS: Primary MLS B resistance was detected by the disk diffusion method. Isolates with iMLS B phenotype were tested by double-disk induction method. All isolates were tested by a genotypic assay, PCR with specific primers. RESULTS: A total of 46.7 percent of staphylococci were positive for cMLS B; 3.3 percent for iMLS B and 3.3 percent for MS B. One or more erm genes were present in 50.1 percent of isolates. The gene ermA was detected in 49 isolates, ermC in 29 and ermB in 3. CONCLUSION: The prevalence of the ermA, ermB and ermC genes were 29.6 percent, 17.1 percent and 0.66 percent respectively, and constitutive resistance was the most frequent as compared to the other two phenotypes.

Distribution of erm genes and low prevalence of inducible resistance to clindamycin among staphylococci isolates.

INTRODUCTION: Resistance to macrolides, lincosamides and streptogramins B (MLS(B) antibiotics) in staphylococci may be due to modification in ribosomal target methylase encoded by erm genes. The expression of MLS(B) resistance lead to three phenotypes, namely constitutive resistance (cMLS(B)), inducible resistance (iMLS(B)), and resistance only to macrolides and streptogramins B (MS(B)). The iMLS(B) resistance is the most difficult to detect in the clinical laboratory. OBJECTIVE: This study investigated the expression of MLS(B) resistance and the prevalence of the erm genes among 152 clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus (CNS) from Hospital de Clínicas de Porto Alegre. METHODS: Primary MLS(B) resistance was detected by the disk diffusion method. Isolates with iMLS(B) phenotype were tested by double-disk induction method. All isolates were tested by a genotypic assay, PCR with specific primers. RESULTS: A total of 46.7% of staphylococci were positive for cMLS(B); 3.3% for iMLS(B) and 3.3% for MS(B). One or more erm genes were present in 50.1% of isolates. The gene ermA was detected in 49 isolates, ermC in 29 and ermB in 3. CONCLUSION: The prevalence of the ermA, ermB and ermC genes were 29.6%, 17.1% and 0.66% respectively, and constitutive resistance was the most frequent as compared to the other two phenotypes.

Distribution of staphylococcal cassette chromosome mec (SCCmec) types I, II, III and IV in coagulase-negative staphylococci from patients attending a tertiary hospital in southern Brazil.

Coagulase-negative staphylococci (CoNS) are now recognized as the aetiological agents of an important range of infections in humans. Most developed countries have reported an increase in CoNS infections in hospitalized patients that are resistant to meticillin and other antibiotics. Staphylococcal cassette chromosome mec (SCCmec) typing is essential for understanding the molecular epidemiology of meticillin-resistant Staphylococcus strains. SCCmec elements are currently classified into types I to VI based on the characteristics of the mec and ccr gene complexes and are further classified into subtypes according to their 'junkyard DNA' region. We evaluated the distribution of SCCmec types in CoNS from patients attending the Hospital de Clínicas de Porto Alegre over the period August 2004-December 2005. Among the 129 bloodstream isolates, 36 (27.9 %) harboured SCCmec type I, 4 (3.0 %) harboured SCCmec type II, 67 (52 %) harboured SCCmec type III, 1 (0.8 %) harboured SCCmec type IV and 4 (3.0 %) harboured SCCmec types I and III. Seventeen isolates were not typable. Identification of CoNS at the species level indicated that Staphylococcus epidermidis was the most common species, with 87 isolates, followed by Staphylococcus haemolyticus (15), Staphylococcus hominis (13), Staphylococcus capitis (12) and Staphylococcus sciuri (1). SCCmec type III was the most prevalent among isolates of S. epidermidis (52 %). Among these strains, 30 (23 %) harboured a modified SCCmec type III which contained an additional dcs region in comparison with regular type III. SCCmec type III was also highly prevalent (75 %) among S. capitis isolates. The predominant SCCmec type found among S. haemolyticus isolates was type I. However, all four isolates harbouring SCCmec type II belonged to S. haemolyticus. Our results indicate that SCCmec type III was the most prevalent among the CoNS. Isolates with SCCmec type III were more resistant to non-beta-lactam antimicrobials than isolates harbouring SCCmec types I, II and IV, although the increase in resistance was statistically significant only for clindamycin (P=0.021), rifampicin (P=0.010) and levofloxacin (P=0.005).