Marleen M. Kock

Molecular identification and genotyping of MRSA isolates

The aim of this study was to identify and characterize 97 methicillin-resistant Staphylococcus aureus (MRSA) isolates. Two conventional multiplex PCR assays, a real-time PCR assay and two PCR-based genotyping techniques including the spa- and hypervariable region (HVR)-typing methods were used to identify and characterize 97 MRSA strains isolated between April 2006 to September 2007 from the Steve Biko Academic Hospital. All MRSA isolates were positive for 16S rRNA gene, 99% were positive for the mecA gene and 4% positive for the Panton-Valentine leukocidin (PVL) gene. Staphylococcal cassette chromosome mec (SCCmec) typing showed 67% of isolates were SCCmec II [health-care-associated MRSA (HA-MRSA)], 14% were SCCmec III (HA-MRSA) and 4% were SCCmec IVd [community-associated MRSA (CA-MRSA)]. These CA-MRSA isolates showed a prevalence of 100% for the PVL gene. Using spa typing, three distinct clusters could be identified while HVR typing revealed six different clusters. CA-MRSA isolates were clustered together using spa and HVR typing. This study showed the prevalence of the CA-MRSA strains, PVL genes, the SCCmec types and the clonality of the MRSA strains. The high prevalence of the PVL gene in CA-MRSA isolates already residing in intensive care units was alarming and indicated the emergence of new MRSA lineages with a particular fitness for community and hospital transmission.

Antimicrobial susceptibility patterns of Ureaplasma species and Mycoplasma hominis in pregnant women

BackgroundGenital mycoplasmas colonise up to 80% of sexually mature women and may invade the amniotic cavity during pregnancy and cause complications. Tetracyclines and fluoroquinolones are contraindicated in pregnancy and erythromycin is often used to treat patients. However, increasing resistance to common antimicrobial agents is widely reported. The purpose of this study was to investigate antimicrobial susceptibility patterns of genital mycoplasmas in pregnant women.MethodsSelf-collected vaginal swabs were obtained from 96 pregnant women attending an antenatal clinic in Gauteng, South Africa. Specimens were screened with the Mycofast Revolution assay for the presence of Ureaplasma species and Mycoplasma hominis. The antimicrobial susceptibility to levofloxacin, moxifloxacin, erythromycin, clindamycin and tetracycline were determined at various breakpoints. A multiplex polymerase chain reaction assay was used to speciate Ureaplasma positive specimens as either U. parvum or U. urealyticum.ResultsSeventy-six percent (73/96) of specimens contained Ureaplasma spp., while 39.7% (29/73) of Ureaplasma positive specimens were also positive for M. hominis. Susceptibilities of Ureaplasma spp. to levofloxacin and moxifloxacin were 59% (26/44) and 98% (43/44) respectively. Mixed isolates (Ureaplasma species and M. hominis) were highly resistant to erythromycin and tetracycline (both 97% resistance). Resistance of Ureaplasma spp. to erythromycin was 80% (35/44) and tetracycline resistance was detected in 73% (32/44) of Ureaplasma spp. Speciation indicated that U. parvum was the predominant Ureaplasma spp. conferring antimicrobial resistance.ConclusionsTreatment options for genital mycoplasma infections are becoming limited. More elaborative studies are needed to elucidate the diverse antimicrobial susceptibility patterns found in this study when compared to similar studies. To prevent complications in pregnant women, the foetus and the neonate, routine screening for the presence of genital mycoplasmas is recommended. In addition, it is recommended that antimicrobial susceptibility patterns are determined.

Evaluation of the GenoType® MTBDRsl assay for susceptibility testing of second-line anti-tuberculosis drugs.

BACKGROUND The GenoType® MTBDRsl assay is a new rapid assay for the detection of resistance to second-line anti-tuberculosis drugs. OBJECTIVE To evaluate the MTBDRsl assay on 342 multidrug-resistant tuberculosis isolates for resistance to ofloxacin (OFX), kanamycin (KM), capreomycin (CPM) and ethambutol (EMB), to compare the results to the agar proportion method, and to test discrepant results using DNA sequencing. RESULT The sensitivity and specificity of the MTBDRsl assay were respectively 70.3% and 97.7% for OFX, 25.0% and 98.7% for KM, 21.2% and 98.7% for CPM and 56.3% and 56.0% for EMB. DNA sequencing identified mutations that were not detected by the MTBDRsl assay. The 8/11 phenotypically OFX-resistant isolates had mutations in gyrA (2/8 had an additional mutation in the gyrB gene), 1/11 had mutations only in the gyrB gene, 6/21 phenotypically KM-resistant isolates had mutations in the rrs gene, and 7/26 and 20/26 phenotypically CPM-resistant isolates had mutations in the rrs and tlyA genes. CONCLUSION The MTBDRsl assay showed lower sensitivity than previous studies. The assay performed favourably for OFX; however, it was less sensitive in the detection of KM/CPM resistance and demonstrated low sensitivity and specificity for EMB resistance. It is recommended that the MTBDRsl assay include additional genes to achieve better sensitivity for all the drugs tested.

A broad profile of co-dominant epitopes shapes the peripheral Mycobacterium tuberculosis specific CD8+ T-Cell immune response in South African patients with active tuberculosis

We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins (ESAT-6 [Rv3875], Ag85B [Rv1886c], and TB10.4 [Rv0288], including amino acid variations for Rv0288, i.e., A10T, G13D, S27N, and A71S for MHC allotypes common in a South African population (e.g., human leukocyte antigen [HLA]-A*30, B*58, and C*07). Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition.

Molecular characterization and second-line antituberculosis drug resistance patterns of multidrug-resistant mycobacterium tuberculosis isolates from the Northern Region of South Africa

ABSTRACT Despite South Africa being one of the high-burden multidrug-resistant tuberculosis (MDR-TB) countries, information regarding the population structure of drug-resistant Mycobacterium tuberculosis strains is limited from many regions of South Africa. This study investigated the population structure and transmission patterns of drug-resistant M. tuberculosis isolates in a high-burden setting of South Africa as well as the possible association of genotypes with drug resistance and demographic characteristics. A total of 336 consecutive MDR-TB isolates from four provinces of South Africa were genotyped using spoligotyping and mycobacterial interspersed repetitive-unit–variable number tandem repeat (MIRU-VNTR) typing. Drug susceptibility testing for ofloxacin, kanamycin, and capreomycin was performed using the agar proportion method. The results showed that 4.8% of MDR-TB isolates were resistant to ofloxacin, 2.7% were resistant to kanamycin, and 4.5% were resistant to capreomycin, while 7.1% were extensively drug resistant (XDR), and the remaining 83.6% were susceptible to all of the second-line drugs tested. Spoligotyping grouped 90.8% of the isolates into 25 clusters, while 9.2% isolates were unclustered. Ninety-one percent of the 336 isolates were assigned to 21 previously described shared types, with the Beijing family being the predominant genotype in the North-West and Limpopo Provinces, while the EAI1_SOM family was the predominant genotype in the Gauteng and Mpumalanga Provinces. No association was found between genotypes and specific drug resistance patterns or demographic information. The high level of diversity and the geographical distribution of the drug-resistant M. tuberculosis isolates in this study suggest that the transmission of TB in the study settings is not caused by the clonal spread of a specific M. tuberculosis strain.

Comparison between the BACTEC MGIT 960 system and the agar proportion method for susceptibility testing of multidrug resistant tuberculosis strains in a high burden setting of South Africa

BackgroundThe increasing problem of multi-drug-resistant (MDR) tuberculosis (TB) [ie resistant to at least isoniazid (INH) and rifampicin (RIF)] is becoming a global problem. Successful treatment outcome for MDR-TB depends on reliable and accurate drug susceptibility testing of first-line and second-line anti-TB drugs.MethodConsecutive M. tuberculosis isolates identified as MDR-TB during August 2007 to January 2008 using the BACTEC MGIT 960 systems and the agar proportion method were included in this study. Susceptibility testing of MDR-TB isolates against ethambutol (EMB) and streptomycin (STR) as well as two second-line anti-TB drugs, kanamycin (KAN) and ofloxacin (OFX) was performed using the BACTEC MGIT 960 systems at a routine diagnostic laboratory. The results were compared to those obtained by the agar proportion method.ResultThe agreement between the BACTEC MGIT 960 system and the agar proportion method was 44% for EMB, 61% for STR and 89% for both KAN and OFX. The sensitivity and specificity of the BACTEC MGIT 960 system using the agar proportion method as a gold standard was 92% and 37% for EMB, 95% and 37% for STR, 27% and 97% for KAN and 84% and 90% for OFX, respectively.ConclusionsThe BACTEC MGIT 960 system showed acceptable sensitivity for EMB, STR, and OFX; however, the BACTEC MGIT 960 system was less specific for EMB and STR and demonstrated a low sensitivity for KAN. The lower agreement found between the two methods suggests the unreliability of the BACTEC MGIT 960 system for the drugs tested. The reasons for the lower agreement between the two methods need to be investigated and further studies are needed in this setting to confirm the study finding.

Detection of blaSHV, blaTEM and blaCTX‐M antibiotic resistance genes in randomly selected bacterial pathogens from the Steve Biko Academic Hospital

Extended-spectrum beta-lactamases (ESBLs) are considered to be one of the most important antibiotic resistance mechanisms. This study reported the ESBL-producing genes in 53 randomly selected clinical bacterial isolates from the Steve Biko Academic Hospital. The presence of the bla(SHV), bla(TEM) and bla(CTX-M) genes was determined, and the overall prevalence of these genes detected in this study was 87% (46/53) in comparison with the literature; these results were higher when compared with 33% for Escherichia coli in Europe and 0.8% in Denmark for similar pathogens. These research findings indicated that it is crucial to routinely monitor the prevalence of these resistance genes.

Diversity and antimicrobial susceptibility profiling of staphylococci isolated from bovine mastitis cases and close human contacts

The objectives of this study were to examine the diversity of Staphylococcus spp. recovered from bovine intramammary infections and humans working in close contact with the animals and to evaluate the susceptibility of the staphylococcal isolates to different antimicrobials. A total of 3,387 milk samples and 79 human nasal swabs were collected from 13 sampling sites in the KwaZulu-Natal province of South Africa. In total, 146 Staph. aureus isolates and 102 coagulase-negative staphylococci (CNS) were recovered from clinical and subclinical milk samples. Staphylococcusaureus was isolated from 12 (15.2%) of the human nasal swabs and 95 representative CNS were recovered for further characterization. The CNS were identified using multiplex-PCR assays, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and tuf gene sequencing. Seven Staphylococcus spp. were identified among the CNS of bovine origin, with Staph.chromogenes (78.4%) predominating. The predominant CNS species recovered from the human nasal swabs was Staph.epidermidis (80%) followed by Staph.chromogenes (6.3%). The antimicrobial susceptibility of all staphylococcal isolates was evaluated using disk diffusion and was supplemented by screening for specific antimicrobial resistance genes. Ninety-eight (67.1%) Staph.aureus isolates of bovine origin were pansusceptible; 39 (26.7%) isolates were resistant to a single class, and 7 (4.8%) isolates were resistant to 2 classes of antimicrobials. Two Staph. aureus (1.4%) isolates were multidrug-resistant. Resistance to penicillin was common, with 28.8% of the bovine and 75% of the human Staph. aureus isolates exhibiting resistance. A similar observation was made with the CNS, where 37.3% of the bovine and 89.5% of the human isolates were resistant to penicillin. Multidrug-resistance was common among the human CNS, with 39% of the isolates exhibiting resistance to 3 or more classes of antimicrobials. The antimicrobial susceptibility results suggest that resistance among staphylococci causing bovine intramammary infections in South Africa is uncommon and not a significant cause for concern. In contrast, antimicrobial resistance was frequently observed in staphylococcal isolates of human origin, highlighting a possible reservoir of resistance genes. Continued monitoring of staphylococcal isolates is warranted to monitor changes in the susceptibility of isolates to different classes of antimicrobials.

Human Leukocyte Antigens A*3001 and A*3002 Show Distinct Peptide-Binding Patterns of the Mycobacterium tuberculosis Protein TB10.4: Consequences for Immune Recognition

ABSTRACT High-tuberculosis (TB)-burden countries are located in sub-Saharan Africa. We examined the frequency of human leukocyte antigen (HLA) alleles, followed by recombinant expression of the most frequent HLA-A alleles, i.e., HLA-A*3001 and HLA-A*3002, to study differences in mycobacterial peptide presentation and CD8+ T-cell recognition. We screened a peptide library (9-mer peptides with an 8-amino-acid overlap) for binding, affinity, and off-rate of the Mycobacterium tuberculosis-associated antigen TB10.4 and identified only three TB10.4 peptides with considerable binding to HLA-A*3001. In contrast, 22 peptides bound to HLA-A*3002. This reflects a marked difference in the binding preference between the two alleles, with A*3002 tolerating a more promiscuous peptide-binding pattern and A*3001 accommodating only a very selective peptide repertoire. Subsequent analysis of the affinity and off-rate of the binding peptides revealed a strong affinity (8 nM to 7 μM) and moderate off-rate (20 min to 3 h) for both alleles. Construction of HLA-A*3001 and HLA-A*3002 tetramers containing selected binding peptides from TB10.4, including a peptide which was shared among both alleles, QIMYNYPAM (TB10.43-11), allowed us to enumerate epitope-specific T cells in HLA-A*3001- and HLA-A*3002-typed patients with active TB. HLA-A*3001 and HLA-A*3002 major histocompatibility complex-peptide complexes were recognized in individuals with active TB, irrespective of their homozygous HLA-A*3001 or HLA-A*3002 genetic background. The antigen-specific T cells exhibited the CD45RA+ CCR7+ precursor phenotype and the interleukin-7 receptor (CD127), which were different from the phenotype and receptor exhibited by the parental CD8+ T-cell population.

QuantiFERON-TB GOLD ELISA assay for the detection of Mycobacterium tuberculosis-specific antigens in blood specimens of HIV-positive patients in a high-burden country

Tuberculosis is a life-threatening infection worldwide. Despite improvements in therapy, it results in 2 million deaths and 9 million new cases annually. This study evaluated the use of the QuantiFERON-TB GOLD enzyme-linked immunosorbent assay in a high HIV/TB burden setting in an ARV clinic at the Tshwane District Hospital, South Africa. The sensitivity and specificity of the QF assay in the clinic were 30% (9/30) and 63% (19/30), respectively, when compared with the gold standard culture results. Analysis also suggested that the sensitivity of the QuantiFERON assay is determined by a limiting patient CD4 value between 150 and 200.