No description available.

The overall aim of this study was to determine whether host genetic factors are associated with resistance to HIV-1 infection in a group of highly exposed persistently seronegative sex workers from KwaZulu/Natal, South Africa. A cohort of 17 African highly exposed but persistently seronegative (HEPS) commercial sex workers (CSW) were identified who had been in sex work for more than four years (range between 4-26 years). The women had been followed monthly for at least four years as part of HIV-1 prevention programmes (Ramjee, et al., 1998). The overall aim of this study was to identify the frequency of polymorphisms and mutations in chemokine genes, chemokine receptors and chemokine receptor promoter region which ma y be associated with HIV-1 resistance and prolonged disease progression. Secondly, to determine if the chemokine receptors on CD4 T-cells are sufficiently expressed and functional to enable infection. This information will shed light on correlates of immunity as influenced by these polymorphisms and this knowledge will help in the bigger objective of determining factors influencing disease progression as well as the development of an effective HIV-1 vaccine in South Africa.

The determination of phagocytosis (P) and oxidative burst (OB) in unfractionated blood is a rapid and sensitive flow cytometric method for quantifying neutrophil activation, and was modified for single laser systems by using propidium iodide labeled Staphylococcus aureus (S.aureus) as a quantitative measure of phagocytosis and simultaneously the green fluorescence of oxidized 2'7' dichlorofluorescein diacetate was used to measure oxidative burst. Propidium-iodide labels dead organisms by intercalation with the DNA of the dead organism. This assay was characterized with respect to the stimulatory activity of bacterial lipopolysaccharide (LPS) on OB and P and to determine the effects of lipopolysaccharide and different antibiotics on oxidative burst and phagocytosis by polymorphonuclear leukocytes (PMNL) using a FACS 440 flow cytometer. Blood from healthy donors was pre-incubated with log doses of bacterial LPS (0.1 ng/ml - 1000 ng/ml) or sterile pyrogen-free saline at 37 °c from 0-120 minutes. LPS increased both phagocytosis and oxidative burst in a dose-dependent manner (up to 62 and 121 percent respectively) at all time points tested, and this effect on P and OB could be detected even with no pre- incubation. This LPS - induced phagocytic activity could be blocked by the addition of polymyxin B (10 μg/ml) during preincubation. The priming effect of LPS was maximal at 45 minutes. P and OB were inhibited by pre-incubation with EDTA at doses greater than 1000 μg/ml (60 and 80 percent inhibition) respectively. These observations are consistent with the exquisite sensitivity of the neutrophil to endotoxin. Blood from healthy subjects was subjected to different concentrations of antibiotics, or sterile pyrogenfree saline at 37 °c from O to 120 minutes. The antibiotics used were the following: Ceftriaxone, ciprofloxacin, clindamycin, doxycycline, enoxacin, imipenem, norfloxacin, pefloxacin, teicoplanin, tetracycline and vancomycin. Blood from healthy subjects was exposed to concentrations of the above antibiotics ranging from 0.1 to 200 μg/ml for 60 minutes. EDTA and bacterial LPS used as system controls demonstrated dose-dependent inhibition and increase respectively. Doxycycline showed inhibition of both parameters, while pefloxacin enhanced and tetracycline inhibited oxidative burst. The remaining antibiotics showed no doserelated modulation of either oxidative burst or phagocytosis. The method described provides an environment that mimics physiological conditions; is a rapid and sensitive assay not requiring separation of white cells; and simultaneously measures two neutrophil functions. It can evaluate neutrophil response to immunomodulatory and chemotherapeutic agents in a physiological milieu. The necessity of using pyrogen - free reagents in any study of neutrophil function is re-emphasized.

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.

Pneumocystis pneumonia (PCP) is an important opportunistic infection caused by thefungus Pneumocystis jirovecii. The incidence of PCP in sub-Saharan Africa is on theincrease. This is due to the progression of the HIV-pandemic and limited access to healthcare facilities, specific highly active anti-retroviral therapy and chemoprophylaxis. It is a major cause of hospitalization and mortality in HIV-infected children with in-hospital case-fatality rates ranging from 20-63%.

Includes bibliographical references. / A previous study of 100 methicillin-resistant Staphylococcus aureus (MRSA) isolates, collected between January 2007 and December 2008 from five Cape Town hospitals, identified ST612-MRSA-IV as the predominant MRSA. This raised the question of whether methicillin-susceptible S. aureus (MSSA) isolates with the same sequence type (ST) were present in hospitals in this city. Nineteen MSSA isolates, collected during the same period, and from four of the hospitals in Cape Town, as the previously characterized MRSA isolates, were screened for the presence of ST612-MSSA. spa typing and multi-locus sequence typing (MLST) identified one isolate, MS14, as ST612-MSSA, spa type t064.

Includes abstract. Includes bibliographical references (leaves 116-123).

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

Since the 1970s, relatively uncommon species of non-enteric gram-negative bacilli have emerged as nosocomial colonizers and pathogens. Among them, one of the most significant genera is Acinetobacter, which has given rise to an increasing number of reports of nosocomial infections. The introduction of new broad-spectrum antimicrobials in the hospitals has been one of the main factors responsible for this development.In addition leading to a move from more susceptible towards more resistant pathogens. This occurred both between and within genera. Owing to the unpredictable antimicrobial susceptibility of Acinetobacter spp., it is prudent to test each isolate for its susceptibility profile to guide in the proper treatment of infections caused by Acinetobacter.The disk diffusion method is the technique most widely used by microbiology laboratories for the routine assessment of antimicrobial susceptibility patterns.The zone diameters obtained by this technique for individual antimicrobials are reported as susceptible, intermediate or resistant by referring to an interpretative chart.Most laboratories use interpretative charts (zone size criteria) supplied by the National Committee for Clinical Laboratory Standards (NCCLS) for this purpose.In our laboratory we have noted discrepancies between sensitivities as determined by the NCCLS zone size criteria and the local minimum inhibitory concentration (MIC) results obtained for Acinetobacter species, especially for cefepime (CPIM) and ceftazidime (CT AZ). Since Acinetobacter species contribute significantly to the increased morbidity and mortality of debilitated patients ( especially v entilated ICU patients, and the fact that clinicians rely on the antimicrobial susceptibility results in the management and treatment of these patients, it was felt necessary to investigate the appropriateness/validity of the NCCLS zone size criteria used to interpret the susceptibility test results of cefepime (CPIM), ceftazidime (CT AZ), trimethoprim-sulfamethoxazole (TMP-SULF A), and piperacillin-tazobactam (PIP-T AZO) against Acinetobacter spp.

Background: The prevalence of extended-spectrum beta-lactamase (ESBL) - and carbapenemase-producing Enterobacteriaceae in healthy humans in the community is largely unknown. We aimed to determine the prevalence and genetic characteristics of ESBL- and carbapenemase-producing Enterobacteriaceae in stools from healthy infants and their mothers, and to determine the risk factors associated with their carriage. Methods: This study was nested within the Drakenstein Child Health Study, a birth cohort in a semi-rural region of Western Cape Province, South Africa. Maternal and infants faecal samples (including the meconium) were collected at birth and at two additional time-points (5-12 and 20-28 weeks) from the infants only. Samples were screened for ESBLs and carbapenemase-producing organisms using ChromID ESBL and ChromID CARBA media, respectively. Identification of suspect ESBL/carbapenemase-producing isolates and antibiotic susceptibility were determined using the Vitek 2 system. ESBL production was confirmed using the combination disc test, and that of carbapenemase using the modified hodge test. Selected ESBL and carbapenemase genes were evaluated by the singleplex conventional polymerase chain reaction and Sanger sequencing. Risk factors were assessed by univariate analysis using the EPI Info version 7 software.