Browsing by Author "Robberts, Lourens"

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    Comparison of a real-time multiplex PCR and sequetyping assay for pneumococcal serotyping
    (Public Library of Science, 2015) Dube, Felix S; van Mens, Suzan P; Robberts, Lourens; Wolter, Nicole; Nicol, Paul; Mafofo, Joseph; Africa, Samantha; Zar, Heather J; Nicol, Mark P
    BACKGROUND: Pneumococcal serotype identification is essential to monitor pneumococcal vaccine effectiveness and serotype replacement. Serotyping by conventional serological methods are costly, labour-intensive, and require significant technical expertise. We compared two different molecular methods to serotype pneumococci isolated from the nasopharynx of South African infants participating in a birth cohort study, the Drakenstein Child Health Study, in an area with high 13-valent pneumococcal conjugate vaccine (PCV13) coverage. METHODS: A real-time multiplex PCR (rmPCR) assay detecting 21 different serotypes/-groups and a sequetyping assay, based on the sequence of the wzh gene within the pneumococcal capsular locus, were compared. Forty pneumococcal control isolates, with serotypes determined by the Quellung reaction, were tested. In addition, 135 pneumococcal isolates obtained from the nasopharynx of healthy children were tested by both serotyping assays and confirmed by Quellung testing. Discordant results were further investigated by whole genome sequencing of four isolates. RESULTS: Of the 40 control isolates tested, 25 had a serotype covered by the rmPCR assay. These were all correctly serotyped/-grouped. Sequetyping PCR failed in 7/40 (18%) isolates. For the remaining isolates, sequetyping assigned the correct serotype/-group to 29/33 (88%) control isolates. Of the 132/135 (98%) nasopharyngeal pneumococcal isolates that could be typed, 69/132 (52%) and 112/132 (85%) were assigned the correct serotype/-group by rmPCR and sequetyping respectively. The serotypes of 63/132 (48%) isolates were not included in the rmPCR panel. All except three isolates (serotype 25A and 38) were theoretically amplified and differentiated into the correct serotype/-group with some strains giving ambigous results (serotype 13/20, 17F/33C, and 11A/D/1818F). Of the pneumococcal serotypes detected in this study, 69/91 (76%) were not included in the current PCV13. The most frequently identified serotypes were 11A, 13, 15B/15C, 16F and 10A. CONCLUSION: The rmPCR assay performed well for the 21 serotypes/-groups included in the assay. However, in our study setting, a large proportion of serotypes were not detected by rmPCR. The sequetyping assay performed well, but did misassign specific serotypes. It may be useful for regions where vaccine serotypes are less common, however confirmatory testing is advisable.
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    Nasopharyngeal carriage with Staphylococcus aureus in healthy children during the first year of life - The Drakenstein Child Health study
    (2016) Abdulgader, Shima Mohammed Ahmed Algalaa; Nicol, Mark Patrick; Robberts, Lourens
    Background: Staphylococcus aureus carriage is a risk factor for subsequent infections. Data on the prevalence, determinants, population structure and molecular epidemiology of S. aureus nasal carriage among healthy African populations are scarce, especially during infancy. The different S. aureus nasal carriage patterns (intermittent and persistent) were defined in the adult population, however, were ill defined during infancy. A consensus definition for these patterns is still lacking since different approaches were used to define carriage patterns. In addition, few studies used strain genotype to support the intermittent and persistent carriage patterns. This thesis describes the prevalence, determinants, population structure and carriage patterns of S. aureus nasopharyngeal carriage among healthy South African infants and their mothers participating in an intensively sampled cohort. Methods: Nasopharyngeal swabs (NP) were collected on the day of birth from 137 mother-infant pairs and two-weekly thereafter from infants during the first year of life. S. aureus isolates were characterized by antibiotic susceptibility testing, PCR detection of the mecA and Panton-Valentine Leuckocidin (PVL) genes, and typed by targeting the Staphylococcal protein A locus. All genetically related spa types were clustered into spa-clonal complexes using the 'based upon repeat pattern clustering analysis. The Logistic regression model for binomial outcomes, Cox proportional hazards model, and Pearson's Chi-square and correlation coefficient tests were used to determine S. aureus NP carriage dynamics and determinants. Median permutation test was performed to determine the difference in the median carriage duration for each genotype. The NP carriage dynamics i.e. incidence, acquisition, and carriage patterns were analysed at the species and the genotype levels. Genotype diversity (number of spa-clonal complexes carried by the infant and the alpha diversity) were incorporated with the carrier index to define the carriage patterns. Results: S. aureus was identified in 21% (725/3292) of the NP swabs; 704 isolates from infants and 21 from mothers. S. aureus NP carriage occurred from birth, peaked by four weeks of age and declined over the following 11 months. Male gender, higher socioeconomic status, maternal carriage, large family size and hospitalization were risk factors for S. aureus NP carriage. The prevalence of methicillin-resistant S. aureus (MRSA) was 1.7% among infants and none of the mothers carried MRSA at birth. Genotyping of 725 S. aureus isolates by targeting the spa gene resulted in 85 spa types. BURP analysis clustered 71 spa types (n=578) into 11 spa-clonal complexes (spa-CCs). Eleven spa types (n=116) were singletons and three spa types (n=27) were excluded from the cluster analysis due to the small number of repeats. The PVL prevalence was 21% (155/725) consisted mainly on MSSA. Eighty three percent of S. aureus strains were resistant to penicillin, 9.1% to gentamicin, 3.4% to tetracycline, and 3.1% and 2.8% to cotrimoxazole and rifampicin, respectively. Constitutive erythromycin resistance was identified in 1.7% (n=12), whereas the inducible MLSB phenotype (ICR) was observed in 2% (n=18) of isolates. All isolates were susceptible to tigecycline, linezolid and mupirocin. A strong relationship between the spa-clonal complexes and antimicrobial resistance phenotypes was noted. We also documented shifts in the resistance patterns over time within the same genotype carried by the same infant. This study demonstrates the importance of strain genotyping to fully describe carriage dynamics; the incidence of acquisition was higher (0.65 episodes per 100 child days) at the genotype level compared to (0.24 episodes) the species level. During the first year of life, the acquisition rate was 1.8 acquisitions per 137 child-year at the species level compared to 2.4 acquisitions per 137 child-year. At the level of the individual child, a positive correlation (r=0.6; 95% CI 0.46 – 0.70, p < 0.0001) was observed between genotype diversity and the proportion of samples testing positive for S. aureus. Using the genotype diversity measure we found that true persistent carriage with a single strain is rare (2%) during infancy. Conclusion: This study provide baseline data on the prevalence, determinants, population structure and dynamics of S. aureus NP carriage among South African infants. A low prevalence of MRSA was observed in this cohort. A diverse MSSA population with relatively high PVL prevalence was observed. Persistent carriage with a single strain was uncommon during the first year of life. The detailed phenotypic and genotypic analysis of S. aureus in this intensively sampled birth cohort has extended our knowledge of the nature and determinants of NP carriage during infancy
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    Prevalence and antibiotic susceptibility patterns of enteric bacterial pathogens in human and non-human sources in an urban informal settlement in Cape Town, South Africa
    (2019-11-06) Kalule, John B; Smith, Anthony M; Vulindhlu, Mjikisile; Tau, Nomsa P; Nicol, Mark P; Keddy, Karen H; Robberts, Lourens
    Abstract Background In light of rampant childhood diarrhoea, this study investigated bacterial pathogens from human and non-human sources in an urban informal settlement. Meat from informal abattoirs (n = 85), river water (n = 64), and diarrheic stool (n = 66) were collected between September 2015 and May 2016. A duplex real-time PCR, gel-based PCR, and CHROMagar™STEC were used to screen Tryptic Soy Broth (TSB) for diarrheic E. coli. Standard methods were used to screen for other selected food and waterborne bacterial pathogens. Results Pathogens isolated from stool, meat, and surface water included Salmonella enterica (6, 5, 0%), Plesiomonas shigelloides (9, 0, 17%), Aeromonas sobria (3, 3, 0%), Campylobacter jejuni (5, 5, 0%), Shigella flexneri (17, 5, 0%), Vibrio vulnificus (0, 0, 9%), and diarrheic E. coli (21, 3, 7%) respectively. All the isolates were resistant to trimethoprim–sulphamethoxazole. Conclusions There was a high burden of drug resistant diarrheal pathogens in the stool, surface water and meat from informal slaughter. Integrated control measures are needed to ensure food safety and to prevent the spread of drug resistant pathogens in similar settings.