OpenUCT :: Browsing by Author "Nicol, Mark P"

Browsing by Author "Nicol, Mark P"

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Open Access
Azithromycin versus placebo for the treatment of HIV-associated chronic lung disease in children and adolescents (BREATHE trial): study protocol for a randomised controlled trial
(BioMed Central, 2017-12-28) Gonzalez-Martinez, Carmen; Kranzer, Katharina; McHugh, Grace; Corbett, Elizabeth L; Mujuru, Hilda; Nicol, Mark P; Rowland-Jones, Sarah; Rehman, Andrea M; Gutteberg, Tore J; Flaegstad, Trond; Odland, Jon O; Ferrand, Rashida A
Background: Human immunodeficiency virus (HIV)-related chronic lung disease (CLD) among children is associated with substantial morbidity, despite antiretroviral therapy. This may be a consequence of repeated respiratory tract infections and/or dysregulated immune activation that accompanies HIV infection. Macrolides have anti-inflammatory and antimicrobial properties, and we hypothesised that azithromycin would reduce decline in lung function and morbidity through preventing respiratory tract infections and controlling systemic inflammation. Methods/design: We are conducting a multicentre (Malawi and Zimbabwe), double-blind, randomised controlled trial of a 12-month course of weekly azithromycin versus placebo. The primary outcome is the mean change in forced expiratory volume in 1 second (FEV1) z-score at 12 months. Participants are followed up to 18 months to explore the durability of effect. Secondary outcomes are FEV1 z-score at 18 months, time to death, time to first acute respiratory exacerbation, number of exacerbations, number of hospitalisations, weight for age z-score at 12 and 18 months, number of adverse events, number of malaria episodes, number of bloodstream Salmonella typhi infections and number of gastroenteritis episodes. Participants will be followed up 3-monthly, and lung function will be assessed every 6 months. Laboratory substudies will be done to investigate the impact of azithromycin on systemic inflammation and on development of antimicrobial resistance as well as impact on the nasopharyngeal, lung and gut microbiome. Discussion: The results of this trial will be of clinical relevance because there are no established guidelines on the treatment and management of HIV-associated CLD in children in sub-Saharan Africa, where 80% of the world’s HIVinfected children live and where HIV-associated CLD is highly prevalent. Trial registration: ClinicalTrials.gov, NCT02426112. Registered on 21 April 2015.
Open Access
Characterisation of STEC and other diarrheic E. coli isolated on CHROMagar™STEC at a tertiary referral hospital, Cape Town
(BioMed Central, 2018-06-08) Kalule, John B; Keddy, Karen H; Nicol, Mark P
Abstract Background Shiga toxin producing E. coli (STEC) is an emerging zoonotic pathogen that can cause acute renal failure, especially in children. Clinical microbiology laboratories may fail to detect STEC and other diarrhoeic E. coli unless purposive rigorous screening procedures are followed using appropriate diagnostic technology; CHROMagar™STEC has rarely been used for isolation of African diarrhoeic E. coli hence characteristics of isolates on this medium are not yet fully understood. This study aimed to determine the prevalence and characteristics of STEC and other diarrhoeic E. coli isolated on CHROMagar™STEC from stool samples submitted to the microbiology laboratory of a South African public sector tertiary care hospital. Results In total, 733 stool samples were tested. Of these, 4.5% (33/733) possessed diarrhoeic E. coli. Of the diarrheic E. coli, 5/33 (15.2%) were STEC, 15/33 (45.5%) EAggEC, 6/33 (18.2%) atypical EPEC, 5/33 (15.2%) typical EPEC, and 1/33 (3%) DAEC. None of the STEC isolates had been identified by routine testing (based on using sorbitol media to test for E. coli O157: H7 strains and not the other STEC) in the laboratory. Of the 33 strains, 55% (95% CI = 40.8–72.7) showed resistance to ampicillin. Conclusions CHROMagar™STEC enabled detection of tellurite - resistant diarrhoeic E. coli that would be missed using routine methods. Further studies are needed to determine the proportion and characteristics of those which might have been missed using this approach.
Open Access
Characterization of Mycobacterium tuberculosis isolates with discordant rifampicin susceptibility test results
(2018) Ghebrekristos, Yonas; Beylis, Natalie; Nicol, Mark P
Background: The Xpert MTB/RIF assay was adopted as the initial diagnostic test for patients with presumptive tuberculosis (TB) by the South African National TB Control programme in December 2010. Rifampicin (RIF) resistance detected by the Xpert MTB/RIF (Xpert) is confirmed by a line probe assay (LPA) (GenoType MTBDRplus) and/or phenotypic (culture-based) drug susceptibility testing (DST) by MGIT (Mycobacterial Growth Indicator Tube) on the culture isolate from a 2nd specimen. Although both the Xpert and LPA target the rifampicin resistance determining region (RRDR) of the rpoB gene, discordant RIF results (Xpert RIF resistant (RIFR ), LPA RIF susceptible (RIFS )) have been reported. In addition, in cases where genotypic tests detect an rpoB mutation, inferring RIF resistance, routine phenotypic DST may report a RIF susceptible result. This is usually due to disputed rpoB mutations. Aim: The aims of this study are to determine 1) whether the discordance between Xpert and LPA is due to false RIFR by Xpert or false RIFS by LPA and to elucidate the causes of false results and 2) the frequency and types of rpoB mutations expected to test susceptible on routine phenotypic DST and their corresponding RIF MIC (minimum inhibitory concentration). Methods: Consecutive isolates with discordant Xpert RIFR and LPA RIFS results were selected during routine review. For the Xpert, parameters including bacterial DNA load and cycle threshold (Ct) of the probes were evaluated. In addition, isolates with a pattern of any absent rpoB WT band and absent MUT band on the LPA strip (“miscellaneous rpoB mutations”) were selected for MIC testing using the MGIT 960 system and EpiCenter TB eXiST software. Sanger sequencing of the rpoB gene from codon 462 to 591 was performed on all selected isolates. Results and discussion: Discordant Xpert/LPA results: From the total of 1542 patients with RIFR results by Xpert, 106 (6.9%) had a discordant LPA RIFS result. Sequencing results were available for 101 isolates of which 78 (77.2%) had no rpoB mutation detected and these were categorized as false RIFR by Xpert. Mutations were detected by sequencing in the remaining 23 (22.8%); these were categorized as false RIFS by LPA. Probe delay occurred in 56/76 (73.7%) cases compared with 104/1436 (7.2%) controls (p 4 and there is a Very Low bacterial load has a positive predictive value (PPV) of 64.2 % of being false and where the Ct max is between 4.1 and 4.9 with Very Low bacterial load, the PPV of a false result increases to 85.7%. For the false RIFS results by LPA, the majority 11/23 (47.8%) were due to technical errors. In 6/23 (26.1%) it was due to mixed infection and in 2/23 (8.7%) there was laboratory mix up. In the remaining 4/23 (17.4%) the cause could not be determined and mixed infection or a laboratory mix up could not be excluded. Discordant genotypic/phenotypic results: RIF resistance was detected in 1502 patients by LPA, of which 169 (11.3%) had a miscellaneous mutation. In addition, a further 21 isolates were selected from “Part 1” of the study, where sequencing confirmed that the rpoB mutation was not one of the high level / high confidence rpoB mutations. A total of 178 isolates had both MIC and rpoB sequencing results. In our study 140/178 (78.7%) isolates with miscellaneous rpoB mutations (n=158) or previously described disputed rpoB mutations (n=20) had MIC values ranging from ≤0.0625 µg/ml to 1.0 µg/ml. An MIC >1.0 µg/m was determined for 38/178 (21.3%) that would have tested RIFR by MGIT DST. Conclusion: Arising from this study is a laboratory based guideline that is now used within NHLS TB laboratories detailing steps on how to detect possible false RIFR results by Xpert MTB/RIF and on how to troubleshoot discordant Xpert RIFR and LPA RIFS results. A database has been created from the results obtained in this study that lists specific rpoB mutations and their corresponding MIC value and has the potential to assist clinicians in individualizing the patient TB treatment regimen.
Open Access
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.
Open Access
Correction to: Diagnostic accuracy of the Xpert MTB/Rif Ultra for tuberculosis adenitis
(2020-03-02) Antel, Katherine; Oosthuizen, Jenna; Malherbe, Francois; Louw, Vernon J; Nicol, Mark P; Maartens, Gary; Verburgh, Estelle
After publication of the original article [1], we were notified that there is a mistake in the article note.
Open Access
Detection of Streptococcus pneumoniae from different types of nasopharyngeal swabs in children
(Public Library of Science, 2013) Dube, Felix S; Kaba, Mamadou; Whittaker, Elizabeth; Zar, Heather J; Nicol, Mark P
BACKGROUND: A better understanding of the epidemiology of nasopharyngeal carriage of Streptococcus pneumoniae is important to assess the impact of vaccination and the pathogenesis of pneumococcal disease. We compared the recovery of S. pneumoniae from nylon flocked, Dacron and rayon swabs. METHODS: The recovery of S. pneumoniae from mocked specimens using flocked, Dacron and rayon swabs were compared by culture. The yield from paired nasopharyngeal (NP) samples obtained from healthy children sampled with flocked and Dacron swabs was also determined using culture and lytA -targeted real-time polymerase chain reaction (qPCR). RESULTS: Using mock specimen, the percentage recovery of S. pneumoniae ATCC 49619 (serotype 19F) strain from the flocked swabs was 100%, while it was 41% from Dacron swabs and 7% from rayon swabs. Similar results were observed for S. pneumoniae serotypes 1 and 5. S. pneumoniae was cultured from 18 of 42 (43%) paired NP samples from the healthy children (median age 8 [interquartile range (IQR) 5-16] months). The median number of colony-forming units (CFU) recovered from flocked swabs was two-fold higher (8.8×10 4 CFU/mL [IQR, 2.0×10 2 - 4.0×10 5 CFU/mL]) than Dacron swabs (3.7×10 4 CFU/mL [IQR, 4.0×10 2 -3.2×10 5 CFU/mL], p = 0.17). Using lytA -targeted qPCR from paired NP samples, the median copy number of S. pneumoniae detected from flocked swabs was significantly higher than from Dacron swabs (3.0×10 5 genome copies/mL [IQR, 1.3×10 2 −1.8×10 6 ] vs. 9.3×10 4 genome copies/mL [IQR, 7.0×10 1 −1.1×10 6 ]; p = 0.005). CONCLUSION: Flocked swabs released more S. pneumoniae compared to both Dacron and rayon swabs from mock specimens. Similarly, higher bacterial loads were detected by qPCR from flocked swabs compared with Dacron swabs from healthy children.
Open Access
Diagnostic accuracy of the Xpert MTB/Rif Ultra for tuberculosis adenitis
(2020-01-13) Antel, Katherine; Oosthuizen, Jenna; Malherbe, Francois; Louw, Vernon J; Nicol, Mark P; Maartens, Gary; Verburgh, Estelle
Abstract Background The WHO recently recommended the new Xpert MTB/RIF Ultra assay (Ultra) instead of the Xpert MTB/RIF assay because Ultra has improved sensitivity. We report the diagnostic accuracy of Ultra for tuberculous adenitis in a tuberculosis and HIV endemic setting. Methods We obtained fine-needle aspirates (FNA) and lymph node tissue by core-needle biopsy in adult patients with peripheral lymphadenopathy of >20 mm. Ultra and mycobacterial culture were performed on FNA and tissue specimens, with histological examination of tissue specimens. We assessed the diagnostic accuracy of Ultra against a composite reference standard of ‘definite tuberculosis’ (microbiological criteria) or ‘probable tuberculosis’ (histological and clinical criteria). Results We prospectively evaluated 99 participants of whom 50 were HIV positive: 21 had ‘definite tuberculosis’, 15 ‘probable tuberculosis’ and 63 did not have tuberculosis (of whom 38% had lymphoma and 19% disseminated malignancy). Using the composite reference standard the Ultra sensitivity on FNA was 70% (95% CI 51–85; 21 of 30), and on tissue was 67% (45–84; 16/24) these were far superior to the detection of acid-fast bacilli on an FNA (26%; 7/27); AFB on tissue (33%; 8/24); or tissue culture (39%; 9/23). The detection of granulomas on histology had high senstivity (83%) but the lowest specficity. When compared with culture the Ultra on FNA had a sensitvity of 78% (40-97; 7/9) and tissue 90% (55-100; 9/10). Conclusions Ultra performed on FNA or tissue of a lymph node had good sensitivity and high specificity. Ultra had a higher yield than culture and has the advantage of being a rapid test. Ultra on FNA would be an appropriate initial investigation for lymphadenopathy in tuberculosis endemic areas followed by a core biopsy for histopathology with a repeat Ultra on tissue if granulomas are present.
Open Access
Impact of Xpert MTB/RIF for TB diagnosis in a primary care clinic with high TB and HIV prevalence in South Africa: a pragmatic randomised trial
(Public Library of Science, 2014) Cox, Helen S; Mbhele, Slindile; Mohess, Neisha; Whitelaw, Andrew; Muller, Odelia; Zemanay, Widaad; Little, Francesca; Azevedo, Virginia; Simpson, John; Boehme, Catharina C; Nicol, Mark P
Background: Xpert MTB/RIF is approved for use in tuberculosis (TB) and rifampicin-resistance diagnosis. However, data are limited on the impact of Xpert under routine conditions in settings with high TB burden. Methods and Findings: A pragmatic prospective cluster-randomised trial of Xpert for all individuals with presumptive (symptomatic) TB compared to the routine diagnostic algorithm of sputum microscopy and limited use of culture was conducted in a large TB/HIV primary care clinic. The primary outcome was the proportion of bacteriologically confirmed TB cases not initiating TB treatment by 3 mo after presentation. Secondary outcomes included time to TB treatment and mortality. Unblinded randomisation occurred on a weekly basis. Xpert and smear microscopy were performed on site. Analysis was both by intention to treat (ITT) and per protocol. Between 7 September 2010 and 28 October 2011, 1,985 participants were assigned to the Xpert (n = 982) and routine (n = 1,003) diagnostic algorithms (ITT analysis); 882 received Xpert and 1,063 routine (per protocol analysis). 13% (32/257) of individuals with bacteriologically confirmed TB (smear, culture, or Xpert) did not initiate treatment by 3 mo after presentation in the Xpert arm, compared to 25% (41/167) in the routine arm (ITT analysis, risk ratio 0.51, 95% CI 0.33–0.77, p = 0.0052). The yield of bacteriologically confirmed TB cases among patients with presumptive TB was 17% (167/1,003) with routine diagnosis and 26% (257/982) with Xpert diagnosis (ITT analysis, risk ratio 1.57, 95% CI 1.32–1.87, p<0.001). This difference in diagnosis rates resulted in a higher rate of treatment initiation in the Xpert arm: 23% (229/1,003) and 28% (277/982) in the routine and Xpert arms, respectively (ITT analysis, risk ratio 1.24, 95% CI 1.06–1.44, p = 0.013). Time to treatment initiation was improved overall (ITT analysis, hazard ratio 0.76, 95% CI 0.63–0.92, p = 0.005) and among HIV-infected participants (ITT analysis, hazard ratio 0.67, 95% CI 0.53–0.85, p = 0.001). There was no difference in 6-mo mortality with Xpert versus routine diagnosis. Study limitations included incorrect intervention allocation for a high proportion of participants and that the study was conducted in a single clinic. Conclusions: These data suggest that in this routine primary care setting, use of Xpert to diagnose TB increased the number of individuals with bacteriologically confirmed TB who were treated by 3 mo and reduced time to treatment initiation, particularly among HIV-infected participants.
Open Access
Improving point-of-care diagnosis of tuberculosis: development and evaluation of novel technologies
(2017) Moodley, Vineshree Mischka; Nicol, Mark P; Dorman, Susan E
With an estimated third of all tuberculosis (TB) cases being missed, the need to develop rapid, simple and accurate diagnostic tests is critical. The last five years has seen an unprecedented activity in the development of a range of new tests. However, a major concern is that not all marketed TB tests have been assessed rigorously, particularly in terms of diagnostic accuracy, robustness under operational conditions in the field, and practical usefulness. This dissertation comprises a compilation of diagnostic clinical studies of novel point-of-care tests, namely a chemiresistive "TB breath-analyser"; a lipoarabinomannan (LAM) urine dipstick, and an adaptation of the Xpert®MTB/RIF assay for use on blood. Lastly, there is a modification of the sputum collection device (SCD) to enable specimen processing without the requirement of a biosafety cabinet. The chemiresistive sensor, which detects volatile organic compounds released by Mycobacterium tuberculosis in a patient's breath, demonstrated a high sensitivity (100%) and specificity (92%) for distinguishing patients with active TB from healthy controls. However, sensitivity (74%) and specificity (63%) were lower when the culture-negative participant group was compared to the culture-positive participants. The test shows potential as a useful screening test for TB with further refinement of the sensor technology. The LAM dipstick was shown to be useful in hospitalised HIV-infected patients with CD4 T-cell counts <200 cells/μL reinforcing the data from other studies. Although the blood Xpert®MTB/RIF assay showed some utility in diagnosis of TB in hospitalised patients with very advanced HIV, given the poor sensitivity and specificity, and the requirement for specialised equipment as well as a large volume of blood for testing, it is unlikely that Xpert®MTB/RIF testing on blood will contribute much over other existing diagnostics in resource-limited settings. Finally, the redesigned SCD offers a solution to biosafety concerns with minimal impact on patient acceptability and clinical care.
Open Access
Modern lineages of Mycobacterium tuberculosis exhibit lineage-specific patterns of growth and cytokine induction in human monocyte-derived macrophages
(Public Library of Science, 2012) Sarkar, Rajesh; Lenders, Laura; Wilkinson, Katalin A; Wilkinson, Robert J; Nicol, Mark P
BACKGROUND: Strains of Mycobacterium tuberculosis vary in virulence. Strains that have caused outbreaks in the United States and United Kingdom have been shown to subvert the innate immune response as a potential immune evasion mechanism. There is, however, little information available as to whether these patterns of immune subversion are features of individual strains or characteristic of broad clonal lineages of M. tuberculosis . METHODS: Strains from two major modern lineages (lineage 2 [East-Asian] and lineage 4 [Euro-American]) circulating in the Western Cape in South Africa as well as a comparator modern lineage (lineage 3 [CAS/Delhi]) were identified. We assessed two virulence associated characteristics: mycobacterial growth (in liquid broth and monocyte derived macrophages) and early pro-inflammatory cytokine induction. RESULTS: In liquid culture, Lineage 4 strains grew more rapidly and reached higher plateau levels than other strains (lineage 4 vs. lineage 2 p = 0.0024; lineage 4 vs. lineage 3 p = 0.0005). Lineage 3 strains were characterized by low and early plateau levels, while lineage 2 strains showed an intermediate growth phenotype. In monocyte-derived macrophages, lineage 2 strains grew faster than lineage 3 strains (p<0.01) with lineage 4 strains having an intermediate phenotype. Lineage 2 strains induced the lowest levels of pro-inflammatory TNF and IL-12p40 as compared to other lineages (lineage 2: median TNF 362 pg/ml, IL-12p40 91 pg/ml; lineage 3: median TNF 1818 pg/ml, IL-12p40 123 pg/ml; lineage 4: median TNF 1207 pg/ml, IL-12p40 205 pg/ml;). In contrast, lineage 4 strains induced high levels of IL-12p40 and intermediate level of TNF. Lineage 3 strains induced high levels of TNF and intermediate levels of IL-12p40. CONCLUSIONS: Strains of M. tuberculosis from the three major modern strain lineages possess distinct patterns of growth and cytokine induction. Rapid growth and immune subversion may be key characteristics to the success of these strains in different human populations.
Open Access
Molecular epidemiology of Staphylococcus aureus in African children from rural and urban communities with atopic dermatitis
(2021-04-13) Ndhlovu, Gillian O N; Abotsi, Regina E; Shittu, Adebayo O; Abdulgader, Shima M; Jamrozy, Dorota; Dupont, Christopher L; Mankahla, Avumile; Nicol, Mark P; Hlela, Carol; Levin, Michael E; Lunjani, Nonhlanhla; Dube, Felix S
Abstract Background Staphylococcus aureus has been associated with the exacerbation and severity of atopic dermatitis (AD). Studies have not investigated the colonisation dynamics of S. aureus lineages in African toddlers with AD. We determined the prevalence and population structure of S. aureus in toddlers with and without AD from rural and urban South African settings. Methods We conducted a study of AD-affected and non-atopic AmaXhosa toddlers from rural Umtata and urban Cape Town, South Africa. S. aureus was screened from skin and nasal specimens using established microbiological methods and clonal lineages were determined by spa typing. Logistic regression analyses were employed to assess risk factors associated with S. aureus colonisation. Results S. aureus colonisation was higher in cases compared to controls independent of geographic location (54% vs. 13%, p < 0.001 and 70% vs. 35%, p = 0.005 in Umtata [rural] and Cape Town [urban], respectively). Severe AD was associated with higher colonisation compared with moderate AD (86% vs. 52%, p = 0.015) among urban cases. Having AD was associated with colonisation in both rural (odds ratio [OR] 7.54, 95% CI 2.92–19.47) and urban (OR 4.2, 95% CI 1.57–11.2) toddlers. In rural toddlers, living in an electrified house that uses gas (OR 4.08, 95% CI 1.59–10.44) or utilises kerosene and paraffin (OR 2.88, 95% CI 1.22–6.77) for heating and cooking were associated with increased S. aureus colonisation. However, exposure to farm animals (OR 0.3, 95% CI 0.11–0.83) as well as living in a house that uses wood and coal (OR 0.14, 95% CI 0.04–0.49) or outdoor fire (OR 0.31, 95% CI 0.13–0.73) were protective. Spa types t174 and t1476, and t272 and t1476 were dominant among urban and rural cases, respectively, but no main spa type was observed among controls, independent of geographic location. In urban cases, spa type t002 and t442 isolates were only identified in severe AD, t174 was more frequent in moderate AD, and t1476 in severe AD. Conclusion The strain genotype of S. aureus differed by AD phenotypes and rural-urban settings. Continued surveillance of colonising S. aureus lineages is key in understanding alterations in skin microbial composition associated with AD pathogenesis and exacerbation.
Open Access
One Health-One City; the extent of Shiga-toxin producing Escherichia coli in Cape Town
(2017) Kalule, John Bosco; Nicol, Mark P; Keddy, Karen H
The estimated global burden of STEC (Shiga toxin producing Escherichia coli) is 2,481,511 illnesses, 269 deaths, and 26,827 DALYs with 48% of these being foodborne. This thesis provides information on STEC diagnostic strategy, undetected STEC in a tertiary referral hospital in Cape Town, and the virulence and antimicrobial resistance properties of tellurite resistant diarrheic E. coli isolated on CHROMagar(TM)STEC (CHROMagar Microbiology, Paris, France). Deploying the One - Health surveillance approach to study selected diarrheic bacterial pathogens in an informal settlement setting, this study sheds light on the extent of bacterial foodborne pathogens in human and non-human sources.
Open Access
Optimizing 16S rRNA gene profile analysis from low biomass nasopharyngeal and induced sputum specimens
(2020-05-12) Claassen-Weitz, Shantelle; Gardner-Lubbe, Sugnet; Mwaikono, Kilaza S; du Toit, Elloise; Zar, Heather J; Nicol, Mark P
Careful consideration of experimental artefacts is required in order to successfully apply high-throughput 16S ribosomal ribonucleic acid (rRNA) gene sequencing technology. Here we introduce experimental design, quality control and “denoising” approaches for sequencing low biomass specimens. Results We found that bacterial biomass is a key driver of 16S rRNA gene sequencing profiles generated from bacterial mock communities and that the use of different deoxyribonucleic acid (DNA) extraction methods [DSP Virus/Pathogen Mini Kit® (Kit-QS) and ZymoBIOMICS DNA Miniprep Kit (Kit-ZB)] and storage buffers [PrimeStore® Molecular Transport medium (Primestore) and Skim-milk, Tryptone, Glucose and Glycerol (STGG)] further influence these profiles. Kit-QS better represented hard-to-lyse bacteria from bacterial mock communities compared to Kit-ZB. Primestore storage buffer yielded lower levels of background operational taxonomic units (OTUs) from low biomass bacterial mock community controls compared to STGG. In addition to bacterial mock community controls, we used technical repeats (nasopharyngeal and induced sputum processed in duplicate, triplicate or quadruplicate) to further evaluate the effect of specimen biomass and participant age at specimen collection on resultant sequencing profiles. We observed a positive correlation (r = 0.16) between specimen biomass and participant age at specimen collection: low biomass technical repeats (represented by < 500 16S rRNA gene copies/μl) were primarily collected at < 14 days of age. We found that low biomass technical repeats also produced higher alpha diversities (r = − 0.28); 16S rRNA gene profiles similar to no template controls (Primestore); and reduced sequencing reproducibility. Finally, we show that the use of statistical tools for in silico contaminant identification, as implemented through the decontam package in R, provides better representations of indigenous bacteria following decontamination. Conclusions We provide insight into experimental design, quality control steps and “denoising” approaches for 16S rRNA gene high-throughput sequencing of low biomass specimens. We highlight the need for careful assessment of DNA extraction methods and storage buffers; sequence quality and reproducibility; and in silico identification of contaminant profiles in order to avoid spurious results.
Open Access
Outbreak of multi-drug resistant Pseudomonas aeruginosa bloodstream infection in the haematology unit of a South African academic hospital
(Public Library of Science, 2013) Mudau, Maanda; Jacobson, Rachael; Minenza, Nadia; Kuonza, Lazarus; Morris, Vida; Engelbrecht, Heather; Nicol, Mark P; Bamford, Colleen
Objective: To describe an outbreak of multi-resistant Pseudomonas aeruginosa bloodstream infections (MRPA-BSI) that occurred in the haematology ward of a tertiary academic hospital in Cape Town, South Africa, and determine risk factors for acquisition of MRPA-BSI. METHODS: The outbreak investigation included a search for additional cases, review of patient records, environmental and staff screening, molecular typing using pulsed-field gel electrophoresis (PFGE) and Multi-locus sequencing (MLST) and a retrospective case-control study. RESULTS: Ten MRPA-BSI cases occurred in the haematology ward between January 2010 and January 2011. The case fatality rate was 80%. Staff screening specimens were negative for MRPA and an environmental source was not identified. PFGE showed that 9/10 isolates were related. MLST showed that 3 of these 9 isolates belonged to Sequence type (ST) 233 while the unrelated isolate belonged to ST260. CONCLUSION: We have described an outbreak of MRPA-BSI occurring over an extended period of time among neutropenic haematology patients. Molecular typing confirms that the outbreak was predominantly due to a single strain. The source of the outbreak was not identified, but the outbreak appears to have been controlled following intensive infection control measures.
Open Access
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.
Open Access
Prevalence and antimicrobial resistance profiles of respiratory microbial flora in African children with HIV-associated chronic lung disease
(2021-02-25) Abotsi, Regina E; Nicol, Mark P; McHugh, Grace; Simms, Victoria; Rehman, Andrea M; Barthus, Charmaine; Mbhele, Slindile; Moyo, Brewster W; Ngwira, Lucky G; Mujuru, Hilda; Makamure, Beauty; Mayini, Justin; Odland, Jon Ø; Ferrand, Rashida A; Dube, Felix S
Background HIV-associated chronic lung disease (CLD) is common among children living with HIV (CLWH) in sub-Saharan Africa, including those on antiretroviral therapy (ART). However, the pathogenesis of CLD and its possible association with microbial determinants remain poorly understood. We investigated the prevalence, and antibiotic susceptibility of Streptococcus pneumoniae (SP), Staphylococcus aureus (SA), Haemophilus influenzae (HI), and Moraxella catarrhalis (MC) among CLWH (established on ART) who had CLD (CLD+), or not (CLD-) in Zimbabwe and Malawi. Methods Nasopharyngeal swabs (NP) and sputa were collected from CLD+ CLWH (defined as forced-expiratory volume per second z-score < − 1 without reversibility post-bronchodilation with salbutamol), at enrolment as part of a randomised, placebo-controlled trial of azithromycin (BREATHE trial - NCT02426112 ), and from age- and sex-matched CLD- CLWH. Samples were cultured, and antibiotic susceptibility testing was conducted using disk diffusion. Risk factors for bacterial carriage were identified using questionnaires and analysed using multivariate logistic regression. Results A total of 410 participants (336 CLD+, 74 CLD-) were enrolled (median age, 15 years [IQR = 13–18]). SP and MC carriage in NP were higher in CLD+ than in CLD- children: 46% (154/336) vs. 26% (19/74), p = 0.008; and 14% (49/336) vs. 3% (2/74), p = 0.012, respectively. SP isolates from the NP of CLD+ children were more likely to be non-susceptible to penicillin than those from CLD- children (36% [53/144] vs 11% [2/18], p = 0.036). Methicillin-resistant SA was uncommon [4% (7/195)]. In multivariate analysis, key factors associated with NP bacterial carriage included having CLD (SP: adjusted odds ratio (aOR) 2 [95% CI 1.1–3.9]), younger age (SP: aOR 3.2 [1.8–5.8]), viral load suppression (SP: aOR 0.6 [0.4–1.0], SA: 0.5 [0.3–0.9]), stunting (SP: aOR 1.6 [1.1–2.6]) and male sex (SA: aOR 1.7 [1.0–2.9]). Sputum bacterial carriage was similar in both groups (50%) and was associated with Zimbabwean site (SP: aOR 3.1 [1.4–7.3], SA: 2.1 [1.1–4.2]), being on ART for a longer period (SP: aOR 0.3 [0.1–0.8]), and hot compared to rainy season (SP: aOR 2.3 [1.2–4.4]). Conclusions CLD+ CLWH were more likely to be colonised by MC and SP, including penicillin-non-susceptible SP strains, than CLD- CLWH. The role of these bacteria in CLD pathogenesis, including the risk of acute exacerbations, should be further studied.
Open Access
Rapid diagnosis of tuberculosis with the Xpert MTB/RIF assay in high burden countries: a cost-effectiveness analysis
(Public Library of Science, 2011) Vassall, Anna; van Kampen, Sanne; Sohn, Hojoon; Michael, Joy S; John, K R; den Boon, Saskia; Davis, J Lucian; Whitelaw, Andrew; Nicol, Mark P; Gler, Maria Tarcela; Khaliqov, Anar; Zamudio, Carlos; Perkins, Mark D; Boehme, Catharina C; Cobelens, Frank
Background: Xpert MTB/RIF (Xpert) is a promising new rapid diagnostic technology for tuberculosis (TB) that has characteristics that suggest large-scale roll-out. However, because the test is expensive, there are concerns among TB program managers and policy makers regarding its affordability for low- and middle-income settings. Methods and Findings: We estimate the impact of the introduction of Xpert on the costs and cost-effectiveness of TB care using decision analytic modelling, comparing the introduction of Xpert to a base case of smear microscopy and clinical diagnosis in India, South Africa, and Uganda. The introduction of Xpert increases TB case finding in all three settings; from 72%–85% to 95%–99% of the cohort of individuals with suspected TB, compared to the base case. Diagnostic costs (including the costs of testing all individuals with suspected TB) also increase: from US$28–US$49 to US$133–US$146 and US$137–US$151 per TB case detected when Xpert is used "in addition to" and "as a replacement of" smear microscopy, respectively. The incremental cost effectiveness ratios (ICERs) for using Xpert "in addition to" smear microscopy, compared to the base case, range from US$41–$110 per disability adjusted life year (DALY) averted. Likewise the ICERS for using Xpert "as a replacement of" smear microscopy range from US$52–$138 per DALY averted. These ICERs are below the World Health Organization (WHO) willingness to pay threshold. Conclusions: Our results suggest that Xpert is a cost-effective method of TB diagnosis, compared to a base case of smear microscopy and clinical diagnosis of smear-negative TB in low- and middle-income settings where, with its ability to substantially increase case finding, it has important potential for improving TB diagnosis and control. The extent of cost-effectiveness gain to TB programmes from deploying Xpert is primarily dependent on current TB diagnostic practices. Further work is required during scale-up to validate these findings.
Open Access
Rapid microbiological screening for tuberculosis in HIV-positive patients on the first day of acute hospital admission by systematic testing of urine samples using Xpert MTB/RIF: a prospective cohort in South Africa
(2015-08-14) Lawn, Stephen D; Kerkhoff, Andrew D; Burton, Rosie; Schutz, Charlotte; van Wyk, Gavin; Vogt, Monica; Pahlana, Pearl; Nicol, Mark P; Meintjes, Graeme
Abstract Background Autopsy studies of HIV/AIDS-related hospital deaths in sub-Saharan Africa reveal frequent failure of pre-mortem diagnosis of tuberculosis (TB), which is found in 34–64 % of adult cadavers. We determined the overall prevalence and predictors of TB among consecutive unselected HIV-positive adults requiring acute hospital admission and the comparative diagnostic yield obtained by screening urine and sputum samples obtained on day 1 of admission with Xpert MTB/RIF (Xpert). Methods To determine overall TB prevalence accurately, comprehensive clinical sampling (sputum, urine, blood plus other relevant samples) was done and TB was defined by detection of Mycobacterium tuberculosis in any sample using Xpert and/or mycobacterial liquid culture. To evaluate a rapid screening strategy, we compared the diagnostic yield of Xpert testing sputum samples and urine samples obtained with assistance from a respiratory study nurse in the first 24 h of admission. Results Unselected HIV-positive acute adult new medical admissions (n = 427) who were not receiving TB treatment were enrolled irrespective of clinical presentation or symptom profile. From 2,391 cultures and Xpert tests done (mean, 5.6 tests/patient) on 1,745 samples (mean, 4.1 samples/patient), TB was diagnosed in 139 patients (median CD4 cell count, 80 cells/μL). TB prevalence was very high (32.6 %; 95 % CI, 28.1–37.2 %; 139/427). However, patient symptoms and risk factors were poorly predictive for TB. Overall, ≥1 non-respiratory sample(s) tested positive in 115/139 (83 %) of all TB cases, including positive blood cultures in 41/139 (29.5 %) of TB cases. In the first 24 h of admission, sputum (spot and/or induced samples) and urine were obtainable from 37.0 % and 99.5 % of patients, respectively (P <0.001). From these, the proportions of total TB cases (n = 139) that were diagnosed by Xpert testing sputum, urine or both sputum and urine combined within the first 24 h were 39/139 (28.1 %), 89/139 (64.0 %) and 108/139 (77.7 %) cases, respectively (P <0.001). Conclusions The very high prevalence of active TB and its non-specific presentation strongly suggest the need for routine microbiological screening for TB in all HIV-positive medical admissions in high-burden settings. The incremental diagnostic yield from Xpert testing urine was very high and this strategy might be used to rapidly screen new admissions, especially if sputum is difficult to obtain.
Open Access
Respiratory microbes present in the nasopharynx of children hospitalised with suspected pulmonary tuberculosis in Cape Town, South Africa
(BioMed Central, 2016-10-24) Dube, Felix S; Kaba, Mamadou; Robberts, F J Lourens; Tow, Lemese A; Lubbe, Sugnet; Zar, Heather J; Nicol, Mark P
Background: Lower respiratory tract infection in children is increasingly thought to be polymicrobial in origin. Children with symptoms suggestive of pulmonary tuberculosis (PTB) may have tuberculosis, other respiratory tract infections or co-infection with Mycobacterium tuberculosis and other pathogens. We aimed to identify the presence of potential respiratory pathogens in nasopharyngeal (NP) samples from children with suspected PTB. Method: NP samples collected from consecutive children presenting with suspected PTB at Red Cross Children’s Hospital (Cape Town, South Africa) were tested by multiplex real-time RT-PCR. Mycobacterial liquid culture and Xpert MTB/RIF was performed on 2 induced sputa obtained from each participant. Children were categorised as definite-TB (culture or qPCR [Xpert MTB/RIF] confirmed), unlikely-TB (improvement of symptoms without TB treatment on follow-up) and unconfirmed-TB (all other children). Results: Amongst 214 children with a median age of 36 months (interquartile range, [IQR] 19–66 months), 34 (16 %) had definite-TB, 86 (40 %) had unconfirmed-TB and 94 (44 %) were classified as unlikely-TB. Moraxella catarrhalis (64 %), Streptococcus pneumoniae (42 %), Haemophilus influenzae spp (29 %) and Staphylococcus aureus (22 %) were the most common bacteria detected in NP samples. Other bacteria detected included Mycoplasma pneumoniae (9 %), Bordetella pertussis (7 %) and Chlamydophila pneumoniae (4 %). The most common viruses detected included metapneumovirus (19 %), rhinovirus (15 %), influenza virus C (9 %), adenovirus (7 %), cytomegalovirus (7 %) and coronavirus O43 (5.6 %). Both bacteria and viruses were detected in 73, 55 and 56 % of the definite, unconfirmed and unlikely-TB groups, respectively. There were no significant differences in the distribution of respiratory microbes between children with and without TB. Using quadratic discriminant analysis, human metapneumovirus, C. pneumoniae, coronavirus 043, influenza virus C virus, rhinovirus and cytomegalovirus best discriminated children with definite-TB from the other groups of children. Conclusions: A broad range of potential respiratory pathogens was detected in children with suspected TB. There was no clear association between TB categorisation and detection of a specific pathogen. Further work is needed to explore potential pathogen interactions and their role in the pathogenesis of PTB.
Open Access
Screening for HIV-associated tuberculosis and rifampicin resistance before antiretroviral therapy using the Xpert MTB/RIF assay: a prospective study
(Public Library of Science, 2011) Lawn, Stephen D; Brooks, Sophie V; Kranzer, Katharina; Nicol, Mark P; Whitelaw, Andrew; Vogt, Monica; Bekker, Linda-Gail; Wood, Robin
Background: The World Health Organization has endorsed the Xpert MTB/RIF assay for investigation of patients suspected of having tuberculosis (TB). However, its utility for routine TB screening and detection of rifampicin resistance among HIV-infected patients with advanced immunodeficiency enrolling in antiretroviral therapy (ART) services is unknown. Methods and Findings: Consecutive adult HIV-infected patients with no current TB diagnosis enrolling in an ART clinic in a South African township were recruited regardless of symptoms. They were clinically characterised and invited to provide two sputum samples at a single visit. The accuracy of the Xpert MTB/RIF assay for diagnosing TB and drug resistance was assessed in comparison with other tests, including fluorescence smear microscopy and automated liquid culture (gold standard) and drug susceptibility testing. Of 515 patients enrolled, 468 patients (median CD4 cell count, 171 cells/µl; interquartile range, 102–236) produced at least one sputum sample, yielding complete sets of results from 839 samples. Mycobacterium tuberculosis was cultured from 81 patients (TB prevalence, 17.3%). The overall sensitivity of the Xpert MTB/RIF assay for culture-positive TB was 73.3% (specificity, 99.2%) compared to 28.0% (specificity, 100%) using smear microscopy. All smear-positive, culture-positive disease was detected by Xpert MTB/RIF from a single sample (sensitivity, 100%), whereas the sensitivity for smear-negative, culture-positive TB was 43.4% from one sputum sample and 62.3% from two samples. Xpert correctly identified rifampicin resistance in all four cases of multidrug-resistant TB but incorrectly identified resistance in three other patients whose disease was confirmed to be drug sensitive by gene sequencing (specificity, 94.1%; positive predictive value, 57%). Conclusions: In this population of individuals at high risk of TB, intensive screening using the Xpert MTB/RIF assay increased case detection by 45% compared with smear microscopy, strongly supporting replacement of microscopy for this indication. However, despite the ability of the assay to rapidly detect rifampicin-resistant disease, the specificity for drug-resistant TB was sub-optimal.