- Volume 69, Issue 9, 2020
Volume 69, Issue 9, 2020
- Antimicrobial Resistance
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High frequency of colonization by diverse clones of beta-lactam-resistant Gram-negative bacilli in haemodialysis: different sources of transmission outside the renal unit?
Introduction. While colonization by Staphylococcus aureus in haemodialysis patients has been assessed, knowledge about colonization by beta-lactam-resistant Gram-negative bacilli is still limited.
Aim. To describe clinical and molecular characteristics in haemodialysis patients colonized by S. aureus (MSSA-MRSA) and beta-lactam-resistant Gram-negative bacilli in an ambulatory renal unit.
Methodology. The study included patients with central venous catheters in an outpatient haemodialysis facility in Medellín, Colombia (October 2017–October 2018). Swab specimens were collected from the nostrils and skin around vascular access to assess colonization by S. aureus (MSSA-MRSA). Stool samples were collected from each patient to evaluate beta-lactam-resistant Gram-negative bacilli colonization. Molecular typing included PFGE, multilocus sequence typing (MLST), spa typing and enterobacterial repetitive intergenic consensus-PCR (ERIC). Clinical information was obtained from medical records and personal interview.
Results. A total of 210 patients were included in the study. S. aureus colonization was observed in 33.8 % (n=71) of the patients, 4.8 % (n=10) of which were colonized by methicillin-resistant S. aureus . Stool samples were collected from 165 patients and of these 41.2 % (n=68) and 11.5 % (n=19) were colonized by extended-spectrum-beta-lactamase-producing (ESBL) and carbapenem-resistant bacilli, respectively. Typing methods revealed high genetic diversity among S. aureus and ESBL-producing Gram-negative bacilli (ESBL-GNB). Antibiotic use and hospitalization in the previous 6 months were observed in more than half of the studied population.
Conclusion. The high colonization by ESBL-GNB in haemodialysis patients shows evidence for the need for stronger surveillance, not only for S. aureus but also for multidrug-resistant bacilli in order to avoid their spread. Additionally, the high genetic diversity suggests other sources of transmission outside the renal unit instead of horizontal transmission between patients.
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- Clinical Microbiology
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Nontuberculous mycobacterial and Nocardia infections mimicking pulmonary tuberculosis: a retrospective study of a general hospital patient population in China
Guowei Dong, Ping Chu, Jie Guo, Yuduan Xie and Jie LuPurpose. In this study, we differentiated between tuberculosis (TB) and infections caused by nontuberculous mycobacteria (NTM) or Nocardia in a tertiary general hospital in China. Differences in clinical manifestations and factors between respiratory infections associated with these organisms were also investigated.
Methodology. A retrospective analysis was conducted for suspected pulmonary TB patients with positive bacterial culture results under treatment at Wangjing Hospital, a tertiary general hospital, between January 2014 and June 2017. Sputum samples were submitted for liquid culture and species identification by mass spectrometry.
Results. Between January 2014 and June 2017, a total of 3981 suspected TB cases were analysed, of which 151 (3.8 %) exhibited positive mycobacterial culture results. Using mass spectrometry, the 151 isolates were classified into three groups: Mycobacterium tuberculosis (MTB) (n=112; 74.2 %), NTM (n=21 13.9 %) and Nocardia (n=18; 11.9 %). The NTM and Nocardia prevalence rates were significantly higher amongst elderly patients [aged ≥65 years; odds ratio (95 % confidence interval): 3.89 (1.05–14.38) for NTM; odds ratio (95 % confidence interval): 5.10 (1.09–23.91) for Nocardia ]. In addition, treatment with immunosuppressive therapy [odds ratio (95 % confidence interval): 3.92 (1.16–13.27)] was identified as a risk factor for Nocardia infection in these patients.
Conclusion. Our results demonstrated that a quarter of culture-positive ‘suspected TB patients’ harboured NTM or Nocardia infections. Notably, nearly all patients with non-TB infections presented with clinical syndromes mimicking pulmonary TB. Individuals receiving immunosuppressive therapy were at greater risk of acquiring Nocardia infections.
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Characterization of φEf-vB1 prophage infecting oral Enterococcus faecalis and enhancing bacterial biofilm formation
More LessIntroduction. Enterococcus faecalis is a facultative, anaerobic, opportunistic pathogen associated with medical and dental diseases. Bacterial phenotypic traits and pathogenesis are often influenced by lysogeny.
Aim. The aim of this study was to characterize both the morphology and complete genome sequences of induced prophages purified from E. faecalis clinical isolates.
Methodology. E. faecalis isolates were recovered from the roots of teeth of patients attending an endodontic clinic. The morphological features of isolated phage were characterized using transmission electron microscopy (TEM). DNA sequencing was performed using the Illumina MiSeq platform.
Results. TEM indicated that the isolated φEf-vB1 prophage belongs to the family Siphoviridae. The φEf-vB1 prophage was stable over a wide range of temperatures and pH. Sequencing of φEf-vB1 DNA revealed that the phage genome is 37 561 bp in length with a G+C content of 37.6mol% and contained 53 ORFs. Comparison with previously predicted prophage genomes using blast revealed that φEf-vB1 has a high sequence similarity to previously characterized phage genomes. The lysogenic E. faecalis strain exhibited a higher biofilm formation capacity relative to the non-lysogenic strain.
Conclusion. The current findings highlight the role of lysogeny in modification of E. faecalis properties and reveal the potential importance of prophages in E. faecalis biology and pathogenesis.
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- Disease, Diagnosis and Diagnostics
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Validation of a single-step, single-tube reverse transcription loop-mediated isothermal amplification assay for rapid detection of SARS-CoV-2 RNA
Jean Y. H. Lee, Nickala Best, Julie McAuley, Jessica L. Porter, Torsten Seemann, Mark B. Schultz, Michelle Sait, Nicole Orlando, Karolina Mercoulia, Susan A. Ballard, Julian Druce, Thomas Tran, Mike G. Catton, Melinda J. Pryor, Huanhuan L. Cui, Angela Luttick, Sean McDonald, Arran Greenhalgh, Jason C. Kwong, Norelle L. Sherry, Maryza Graham, Tuyet Hoang, Marion Herisse, Sacha J. Pidot, Deborah A. Williamson, Benjamin P. Howden, Ian R. Monk and Timothy P. StinearIntroduction. The SARS-CoV-2 pandemic of 2020 has resulted in unparalleled requirements for RNA extraction kits and enzymes required for virus detection, leading to global shortages. This has necessitated the exploration of alternative diagnostic options to alleviate supply chain issues.
Aim. To establish and validate a reverse transcription loop-mediated isothermal amplification (RT- LAMP) assay for the detection of SARS-CoV-2 from nasopharyngeal swabs.
Methodology. We used a commercial RT-LAMP mastermix from OptiGene in combination with a primer set designed to detect the CDC N1 region of the SARS-CoV-2 nucleocapsid (N) gene. A single-tube, single-step fluorescence assay was implemented whereby 1 µl of universal transport medium (UTM) directly from a nasopharyngeal swab could be used as template, bypassing the requirement for RNA purification. Amplification and detection could be conducted in any thermocycler capable of holding 65 °C for 30 min and measure fluorescence in the FAM channel at 1 min intervals.
Results. Assay evaluation by assessment of 157 clinical specimens previously screened by E-gene RT-qPCR revealed assay sensitivity and specificity of 87 and 100%, respectively. Results were fast, with an average time-to-positive (Tp) for 93 clinical samples of 14 min (sd±7 min). Using dilutions of SARS-CoV-2 virus spiked into UTM, we also evaluated assay performance against FDA guidelines for implementation of emergency-use diagnostics and established a limit-of-detection of 54 Tissue Culture Infectious Dose 50 per ml (TCID50 ml−1), with satisfactory assay sensitivity and specificity. A comparison of 20 clinical specimens between four laboratories showed excellent interlaboratory concordance; performing equally well on three different, commonly used thermocyclers, pointing to the robustness of the assay.
Conclusion. With a simplified workflow, The N1 gene Single Tube Optigene LAMP assay (N1-STOP-LAMP) is a powerful, scalable option for specific and rapid detection of SARS-CoV-2 and an additional resource in the diagnostic armamentarium against COVID-19.
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Molecular detection of Mycobacterium tuberculosis in poor-quality cough specimens
More LessClinical specimens unfit for laboratory processing represent missed opportunities for diagnosing tuberculosis. Poor-quality cough specimens (n=61) from presumptive tuberculosis cases were cultured and GeneXpert MTB/RIF (Xpert) successfully performed on samples transferred by flocked swab into PrimeStore molecular transport medium (PS-MTM). Mycobacterium tuberculosis was grown in culture from 13 (21.3 %) and Xpert reported 15 (24.2 %) positive, of which 10 concordant. RT-PCR of PS-MTM samples showed enhanced sensitivity; three positives were missed by Xpert, five by culture and three more detected for a total of 21 positives (34.4 %).
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Establishment of a Rep′ protein antibody detection method to distinguish natural infection with PCV2 from subunit vaccine immunization
More LessIntroduction. PCV2 is a DNA virus that exists widely in pigs and has caused great economic losses to the pig industry worldwide. In the existing commercial PCV2 enzyme-linked immunosorbent assay (ELISA) kits both natural infection with PCV2 and vaccine immunization produce results that are positive for PCV2 Cap antibodies and therefore they cannot diagnose PCV2 infection in immunized pig farms.
Aim. To establish a PCV2 non-structural protein antibody detection method that distinguishes between antibodies resulting from natural prior exposure (infection) and those induced by subunit vaccine immunization.
Methodology. Based on the non-structural Rep′ protein, we established an indirect ELISA (iELISA) using sera from guinea pigs and piglets.
Results. The results for iELISA for guinea pig serum showed that animals vaccinated with a whole-virus inactivated PCV2 vaccine had 100 % (10/10) Cap antibody positivity and 100 % (10/10) Rep′ antibody positivity. Guinea pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (10/10) Cap antibody positivity, while no (0/10) guinea pigs were Rep′ antibody-positive. The combined detection results for the Rep′ iELISA and a PCV2 Antibody Test kit (Commercial) showed that pigs vaccinated with a whole-virus inactivated PCV2 vaccine or PCV2 SD/2017 had 100 % (5/5) Cap antibody positivity and 100 % (5/5) Rep′ antibody positivity. Pigs vaccinated with a recombinant subunit PCV2 vaccine had 100 % (5/5) Cap antibody positivity, while no (0/10) pigs were Rep′ antibody-positive.
Conclusion. This paper describes an effective iELISA method that can distinguish natural infection with PCV2 (Cap and Rep positive) or inoculation with a whole-virus inactivated vaccine (Cap and Rep positive) from subunit vaccine immunization (Cap-positive, Rep-negative). These comparative assays could be very useful in the control of PCV2 in pig herds.
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Detection and phylogenetic analysis of Human bocavirus in children diagnosed with acute respiratory tract infection
More LessIntroduction. Human bocavirus (HBoV) is a recently discovered parvovirus; it has been shown to be a common cause of respiratory infections and gastroenteritis in children. Since its identification, HBoV has been detected worldwide in nasopharyngeal swabs, serum and stool samples particularly those obtained from young children suffering from respiratory or gastrointestinal tract infections.
Aim. The aim of this work was to determine HBoV prevalence among children with acute respiratory tract infection in Egypt, to detect the most prevalent HBoV genotype and to compare PCR and ELISA as diagnostic techniques for HBoV infection.
Methods. Nasopharyngeal swabs and blood samples were obtained within the first day of admission from 75 children diagnosed with acute respiratory tract infection in El-Shatby University Hospital for Children in Alexandria, Egypt from October 2018 to March 2019. Conventional PCR was used to detect HBoV DNA, ELISA was used to detect HBoV IgM antibodies and sequencing of the VP1/2 genes was used for genotyping.
Results. Seven (9.3%) of the 75 nasopharyngeal swabs obtained from patients with acute respiratory tract infection were positive for HBoV by PCR, while 5 (6.7 %) of the 75 serum samples were positive for HBoV IgM antibodies using ELISA. The correlation between PCR and ELISA results showed a highly significant association between PCR and ELISA techniques (X 2=52.041, P<0.01) and a highly significant agreement between the two methods (Kappa=81.9 %, P<0.01). Phylogenetic analysis showed that all positive samples were related to the HBoV-1 genotype.
Conclusion. Human bocavirus was detected at 9.3 % prevalence in nasopharyngeal swabs obtained from children with acute respiratory tract infection. The HBoV-1 genotype was the only genotype detected, suggesting that a single genetic lineage of HBoV is circulating in Egypt. PCR and ELISA are two reliable methods for detection and diagnosis of HBoV.
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- Molecular and Microbial Epidemiology
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Molecular characterization of respiratory syncytial virus circulating in Tunisia between 2015 and 2018
Introduction. Respiratory syncytial virus (RSV) is the most frequently identified viral agent in children with lower respiratory tract infection (LRTI). No data are available to date regarding RSV genotypes circulating in Tunisia.
Aim. The aim of the present study was to investigate the genetic variability of the glycoprotein G gene in Tunisian RSV strains.
Methodology. Nasopharyngeal aspirates were collected from infants hospitalized for LRTI in five Tunisian hospitals. All specimens were screened for RSV by a direct immunofluorescence assay (DIFA). To molecularly characterize Tunisian RSV strains, a phylogenetic analysis was conducted. Randomly selected positive samples were subjected to reverse transcription PCR amplifying the second hyper-variable region (HVR2) of the G gene.
Results. Among a total of 1417 samples collected between 2015 and 2018, 394 (27.8 %) were positive for RSV by DIFA. Analysis of 61 randomly selected RSV strains revealed that group A RSV (78.7 %) predominated during the period of study as compared to group B RSV (21.3 %). The phylogenetic analysis showed that two genotypes of RSV-A were co-circulating: the ON1 genotype with a 72-nt duplication in HVR2 of the G gene was predominant (98.0 % of RSV-A strains), while one RSV-A strain clustered with the NA1 genotype (2.0 %). Concerning Tunisian group B RSV strains, all sequences contained a 60-nt insertion in HVR2 and a clustered BA10 genotype.
Conclusion. These data suggest that RSV-A genotype ON1 and RSV-B genotype BA10, both with duplications in the G gene, were widely circulating in the Central coastal region of Tunisia between 2015 and 2018.
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Volumes and issues
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 71 (2022)
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Volume 70 (2021)
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Volume 69 (2020)
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Volume 68 (2019)
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Volume 67 (2018)
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Volume 66 (2017)
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Volume 65 (2016)
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Volume 64 (2015)
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Volume 63 (2014)
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Volume 61 (2012)
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