Anwar Ahmed Hoosen

Next-Generation Ion Torrent Sequencing of Drug Resistance Mutations in Mycobacterium tuberculosis Strains

ABSTRACT A novel protocol for full-length Mycobacterium tuberculosis gene analysis of first- and second-line drug resistance was developed using the Ion Torrent Personal Genome Machine (PGM). Five genes—rpoB (rifampin), katG (isoniazid), pncA (pyrazinamide), gyrA (ofloxacin/fluoroquinolone), and rrs (aminoglycosides)—were amplified and sequenced, and results were compared to those obtained by genotypic Hain line probe assay (LPA) and phenotypic Bactec MGIT 960 analysis using 26 geographically diverse South African clinical isolates collected between July and November 2011. Ion Torrent sequencing exhibited 100% (26/26) concordance to phenotypic resistance obtained by MGIT 960 culture and genotypic rpoB and katG results by LPA. In several rifampin-resistant isolates, Ion Torrent sequencing revealed uncommon substitutions (H526R and D516G) that did not have a defined mutation by LPA. Importantly, previously uncharacterized mutations in rpoB (V194I), rrs (G878A), and pncA (Q122Stop) genes were observed. Ion Torrent sequencing may facilitate tracking and monitoring geographically diverse multidrug-resistant and extensively drug-resistant strains and could potentially be integrated into selected regional and reference settings throughout Africa, India, and China.

Real-time quantitative PCR in the diagnosis of tuberculosis in formalin-fixed paraffin-embedded pleural tissue in patients from a high HIV endemic area.

The aim of the study was to improve the diagnosis of pleural tuberculosis (TB) based on formalin-fixed biopsies from patients living in high TB and human immunodeficiency virus (HIV) endemic areas. A real-time polymerase chain reaction (real-time PCR) assay targeting a segment of the gene for mycobacterial 65-kd heat shock protein was developed and evaluated on pleural biopsies from 25 patients clinically diagnosed as having TB, on the basis of the good response to treatment, and from 11 controls. A nested polymerase chain reaction (N-PCR) assay for the repetitive genetic sequence insert IS6110, common to Mycobacterium tuberculosis complex organisms, was performed for comparison. When compared with N-PCR, the real-time PCR assay gave a sensitivity and specificity of 83% and 72%, respectively. When compared with clinical diagnosis, the sensitivity and specificity of real-time PCR (68% and 73%, respectively) was comparable with the sensitivity and specificity of the N-PCR assay (64% and 82%, respectively). There were no major differences in the diagnostic validity for the confirmed TB/HIV coinfected patients compared with the results from the whole TB group. In conclusion, the overall accuracy of the real-time PCR assay was comparable with that of the N-PCR and both were equally useful as diagnostic tools in the setting of a HIV coinfection. The real-time PCR has the additional advantage of a short turn-around time, low risk of sample contamination, and offers the possibility to quantify bacterial load, making it a powerful tool for the rapid diagnosis of TB pleuritis.

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.

Systemic Shigellosis in South Africa

BACKGROUND Systemic disease due to shigellae is associated with human immunodeficiency virus (HIV), malnutrition, and other immunosuppressed states. We examined the clinical and microbiologic characteristics of systemic shigellosis in South Africa, where rates of HIV infection are high. METHODS From 2003 to 2009, 429 cases of invasive shigellosis were identified through national laboratory-based surveillance. At selected sites, additional information was captured on HIV serostatus and outcome. Isolates were serotyped and antimicrobial susceptibility testing performed. RESULTS Most cases of systemic shigellosis were diagnosed on blood culture (408 of 429 cases; 95%). HIV prevalence was 67% (80 of 120 cases), highest in patients aged 5-54 years, and higher among females (55 of 70 cases; 79%) compared with males (25 of 48 cases; 52%; P = .002). HIV-infected people were 4.1 times more likely to die than HIV-uninfected cases (case-fatality ratio, 29 of 78 HIV-infected people [37%] vs 5 of 40 HIV-uninfected people [13%]; P = .008; 95% confidence interval [CI], 1.5-11.8). The commonest serotype was Shigella flexneri 2a (89 of 292 serotypes [30.5%]). Pentavalent resistance occurred in 120 of 292 isolates (41.1%). There was no difference in multidrug resistance between HIV-infected patients (33 of 71 [46%]) and uninfected patients (12 of 33 [36%]; 95% CI, .65--3.55). CONCLUSIONS Systemic shigellosis is associated with HIV-infected patients, primarily in older girls and women, potentially due to the burden of caring for sick children in the home; interventions need to be targeted here. Death rates are higher in HIV-infected versus uninfected individuals.

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.

Rapid and specific diagnosis of tuberculous pleuritis with immunohistochemistry by detecting Mycobacterium tuberculosis complex specific antigen MPT64 in patients from a HIV endemic area.

AimThe aim of the study was to evaluate the diagnostic potential of immunohistochemistry using an antibody to the secreted mycobacterial antigen MPT64, specific for Mycobacterium tuberculosis complex organisms, on formalin-fixed biopsies from patients with pleural tuberculosis (TB) from a high TB and HIV endemic area. Methods and ResultsPleural biopsies from 25 TB cases and 11 non-TB cases were studied. Ziehl-Neelsen staining for acid-fast bacilli and immunohistochemistry with anti-MPT64 and anti-Bacille Calmette-Guérin (BCG) antibodies was performed. Nested polymerase chain reaction (N-PCR) for IS6110 was performed for comparison. Acid-fast bacilli were detected in only 2 cases and 3 biopsies showed granulomas with caseous necrosis. Immunostaining with anti-MPT64, anti-BCG, and N-PCR were positive in 20 (80%), 12 (48%), and 16 (64%) of the cases, and 0, 3 (27%), and 2 (18%) of the non-TB controls, respectively. The diagnostic validity of immunohistochemistry was calculated by comparison with N-PCR–positive TB cases and N-PCR–negative non-TB controls. The sensitivity of immunohistochemistry with anti-MPT64 and anti-BCG were 81% and 56% respectively, and the corresponding specificities were 100% and 78%. ConclusionsDetection of the MPT64 antigen by immunohistochemistry improves the diagnosis of TB pleuritis caused by M. tuberculosis complex organisms in patients living in HIV-endemic areas with atypical histology and negative staining for acid-fast bacilli.

Hospital and community isolates of uropathogens at a tertiary hospital in South Africa

AIM To investigate the profile of common uropathogens isolated from urine specimens submitted to the diagnostic microbiology laboratory at a tertiary teaching hospital and assess their antimicrobial susceptibility patterns to commonly used antimicrobial agents. METHODS We conducted a retrospective analysis of laboratory reports for all urine specimens submitted for investigations over a 1-year period. Isolates were tested by means of the Kirby-Bauer disc diffusion method for susceptibility to amoxicillin, ciprofloxacin, gentamicin, co-trimoxazole and nitrofurantoin, and for extended-spectrum beta-lactamase (ESBL) production. RESULTS Out of the total specimens (N=2,203) received over the 1-year study period, 51.1% (1,126) of the urine samples were culture-positive, the majority (65.4%) having come from females. The most common isolate was Escherichia coli (39.0%) followed by Klebsiella species (20.8%) and Enterococcus faecalis (8.2%). The Gram-negative isolates displayed a very high level of resistance to amoxicillin (range 43 - 100%) and co-trimoxazole (range 29 - 90%), whereas resistance to gentamicin (range 0 - 50%) and ciprofloxacin (range 0 - 33%) was lower. E. coli isolates were susceptible to nitrofurantoin (94%), and ESBL production was significantly higher (p=0.01) in the hospital isolates, compared with those from the community referral sites. CONCLUSIONS The culture-positive rate for uropathogens was high, with a greater incidence among females. E. coli was the most common aetiological agent identified, and remained susceptible to nitrofurantoin. Resistance levels to amoxicillin and co-trimoxazole were very high for all Gram-negative isolates, and it is recommended that these antibiotics should not be used for the empiric treatment of urinary tract infections.

HPV genotypes in women with squamous intraepithelial lesions and normal cervixes participating in a community-based microbicide study in Pretoria, South Africa

BACKGROUND Little is known regarding the human papillomaviruses (HPV) genotypes prevalent in women in South Africa, a country with a high incidence of cervical cancer. OBJECTIVE To determine the prevalence and HPV genotypes in women with squamous abnormalities and normal cervixes participating in a community-based microbicide study. STUDY DESIGN A total of 159 cervical specimens, including 56 specimens from women with abnormal cytology (cases) and 103 randomly selected specimens from women with normal cytology (controls), were collected. HPV was detected by consensus PCR primers and HPV genotypes were determined by Roche Linear Array HPV genotyping assay. RESULTS HPV genotypes were found in 91% of cases and 40% of controls (p<0.005). High-risk HPV was detected in all high-grade squamous intraepithelial lesions (HSILs), 69% of low-grade squamous intraepithelial lesions (LSILs), 57% of atypical squamous cells of undetermined significance (ASCUS), and 86% of ASCUS in which HSIL could not be excluded (ASCUS-H), and 73% of HPV positive controls. HPV-35 was the predominant genotype in HSILs; HPV-18 in ASCUS; HPV-58 in ASCUS-H and HPV-16 in LSILs and controls. CONCLUSION High-risk HPV prevalence was high in both cases and controls. HPV genotype distribution in HSILs was different from that reported worldwide and from other studies in South Africa.

The microbial aetiology of genital ulcers in black men in Durban, South Africa.

The microbial aetiology of genital ulcers was assessed in 100 black men attending a sexually transmitted disease (STD) clinic in Durban, South Africa. Forty patients harboured Haemophilus ducreyi, one hepes simplex virus, and one Neisseria gonorrhoeae. Syphilis was diagnosed in 44 patients on the basis of dark field microscopy or positive syphilis serology test results, or both. Of these 44 patients, eight also harboured N ducreyi, one herpes simplex virus. Lymphogranuloma venereum was diagnosed in one patient. No cause of ulceration could be found in the remaining 16 patients.

In vitro activity of medicinal plants of the Venda region,South Africa, against Trichomonas vaginalis

Trichomonas vaginalis is an important and common cause of urogenital infections in both developed and in developing countries. In view of the high prevalence, increase in resistance to drug therapy and associated risk of acquisition and transmission of HIV, we screened the aqueous extracts of 29 plants. These plants are used to treat venereal diseases and infections in the Venda region. Extracts of four plants showed trichomonicidal activity: Securidaca longepedunculata Fresen. (Polygalaceae; 0.10 mg/ml), Solanum aculeastrum Dun. (Solanaceae; 1.06 mg/ml), Piper capense L.f. (Piperaceae; 11.19 mg/ml) and Cassine transvaalensis (Burtt. Davy) Codd (Celastraceae; 9.69 mg/ml). Further investigations are required to determine whether these plants possess the potential to be developed as new drugs for the treatment of trichomoniasis.