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.

Evaluation of TBc identification immunochromatographic assay for rapid identification of Mycobacterium tuberculosis complex in samples from broth cultures.

Tuberculosis (TB) is a disease of major public health concern worldwide, especially in developing countries. In addition, the human immunodeficiency virus (HIV) epidemic has increased the incidence of infection with nontuberculous mycobacteria (NTM). Rapid, accurate, and simple methods for differentiation of Mycobacterium tuberculosis complex (MTBC) isolates from NTM is greatly needed for successful control of the TB epidemic. This study was done to evaluate the clinical performance of the BD MGIT TBc identification test (TBc ID) for rapid identification of MTBC in samples from broth cultures. A total of 229 Ziehl-Neelsen (ZN) stain-positive MGIT cultures were tested using the TBc ID test, and the results were compared with those of the AccuProbe MTBC identification test (GenProbe, San Diego, CA). The agreement between the TBc ID test and the AccuProbe assay was 96% (kappa = 0.92; confidence interval [CI] = 0.869 to 0.971). The sensitivity, specificity, and negative and positive predictive values of the TBc ID test compared to the AccuProbe assay were 100%, 92.4%, 100%, and 92.2%, respectively. After additional molecular testing, the agreement between the two methods increased to 97.8% (kappa = 0.96; CI = 0.917 to 0.994), and the specificity and positive predictive value increased to 95.6% and 95.7%, respectively. The TBc ID test is a simple, sensitive, and specific test for identification of MTBC in samples from acid-fast bacillus (AFB) smear-positive cultures. The TBc ID test could be a good alternative to the AccuProbe test in TB diagnostic laboratories.

Meningococcal infections in hospitalised patients in Pretoria.

We report on 13 patients diagnosed with meningococcal infections in patients attending state-owned hospitals serving an indigent population in Pretoria in 2009. The case fatality rate was 27%. Ceftriaxone was the main antibiotic (9 out of 13 patients) for therapy. Five isolates (39%) were serogroup B and 4 (31%) serogroup W135. Most isolates (12/13) were fully susceptible to penicillin (MIC range 0.016 - 0.047 µg/ml). A single isolate was intermediately resistant to penicillin (MIC, 0.125 µg/ml) while all isolates were uniformly susceptible to ceftriaxone, ciprofloxacin and rifampicin. This pattern reveals a shift in serogroups with an increase of serogroup B disease in the Pretoria region, and the need for ongoing monitoring of antimicrobial susceptibility profiles and the value of ceftriaxone for favourable therapeutic outcome.

Aetiological agents of urethritis in symptomatic South African men attending a family practice.

The objective was to determine the occurrence of four urethral pathogens in urine specimens from symptomatic men using transcription mediated amplification (TMA) assay. Urethral swab and urine specimens from 300 men presenting to a family practitioner were Gram stained and tested for Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis and Mycoplasma genitalium using three TMA assays respectively. Except for T. vaginalis, the other recognized pathogens viz. N. gonorrhoeae, C. trachomatis and M. genitalium were detected in significantly larger numbers of patients with urethral discharge than in those with burning on micturition (BOM). The overall prevalences were 16.7% for N. gonorrhoeae, 12.3% C. trachomatis, 8.0% T. vaginalis and 17.3% M. genitalium. With regard to microscopic evidence of urethritis, significant associations were found for N. gonorrhoeae and C. trachomatis, but not for M. genitalium and T. vaginalis. This study demonstrated that in symptomatic men attending family practice, M. genitalium and T. vaginalis are also important aetiological agents of urethritis and hence treatment strategies be they syndromic management or laboratory directed should cover for these causative agents. The microscopic diagnosis of urethritis may not be important for treatment strategies. The current syndromic treatment guidelines for developing countries including South Africa need modification.

Risk of having a sexually transmitted infection in women presenting at a termination of pregnancy clinic in Pretoria, South Africa.

This study was undertaken to assess the risk of being infected with a known sexually transmitted pathogen at the time of presentation for termination of pregnancy. Endocervical and vaginal swabs were collected for the diagnosis of Neisseria gonorrhoeae, Chlamydia trachomatis and Trichomonas vaginalis. Single infections were found in 21.5% of the women, with C. trachomatis being the commonest (10.0%). Mixed infections were found in nine women, with trichomoniasis and chlamydial infections in six. During speculum examination, vaginal discharge was observed in 73% of the women. The commonest organism detected in patients with vaginal discharge was C. trachomatis (11.6%), while T. vaginalis (11.1%) was the most common in women without visible vaginal discharge. No significant differences were found when comparing symptomatic and non-symptomatic women. This study strongly recommends that women presenting for termination of pregnancy be screened for STIs and receive relevant sexual health education.

Drug-susceptibility patterns of Mycobacterium tuberculosis in Mpumalanga province, South Africa: possible guiding design of retreatment regimen.

Multidrug-resistant tuberculosis (MDR-TB) has been a cause of concern in both developed and developing countries. The prevalence of drug resistance in Mycobacterium tuberculosis (MTB) isolates (n=692) from Mpumalanga province was assessed. In total, 692 (64%) MTB strains from cases with pulmonary TB were tested for susceptibility against rifampicin, isoniazid, ethambutol, and streptomycin using the MGIT 960 instrument. Two hundred and nine (30.2%) strains were resistant to one or more drugs. Resistance to one drug ranged from 1.4% for ethambutol to 17.7% for rifampicin. The prevalence of MDR-TB ranged from 6.7% for three drugs to 34% for four drugs, with significant predictors being patients' age-groups of 25-54 years (p=0.0012) and >55 years (p=0.007). The result showed a high level (58.4%) of MDR-TB from cases in Mpumalanga province. To achieve a higher cure rate in this province, drug-susceptibility tests must be done for every case.

Antimicrobial Susceptibility Patterns of Gonococcal Isolates in Pretoria; South Africa; over a 20-Year Period (1984-2004)

This paper reviews the susceptibility profiles of Neisseria gonorrhoeae over a 20-year period in the Pretoria region. Endourethral specimens were collected from adult men with symptoms of urethritis attending primary health care clinics and private medical practitioners. These swabs were plated on enriched media for isolation of N. gonorrhoeae. Antimicrobial susceptibility of the organisms was performed using the disc diffusion and agar dilution methods. Plasmid analyses were performed on beta-lactamase-producing isolates. Penicillase-producing N. gonorrhoeae strains increased from 4to 16; whilst chromosomally mediated penicillin-resistant strains increased dramatically from 0to 16from 1984 to 2004. There was an equal distribution of the 3.2 MDa African and 4.4 MDa Asian plasmids. High-level tetracycline-resistant strains (36) were detected for the first time in 2004. Ciprofloxacin resistance emerged at 7in the same year. Gonococcal isolates remained susceptible to cefoxitin; ceftriaxone; cefpodoxime; and spectinomycin. However; the minimum inhibitory concentration values for spectinomycin were very close to the breakpoint. We have shown a continuing increase in resistance to penicillin (plasmid and chromosomal); the emergence of high-level tetracycline resistance and an emergence of resistance to ciprofloxacin. Susceptibility testing is essential for successful therapeutic outcomes and needs to be performed in an ongoing basis

Antimicrobial Susceptibility Patterns of Gonococcal Isolates in Pretoria; South Africa; Over a 20-Year Period (1984-2004)

Abstract:This paper reviews the susceptibility profiles of Neisseria gonorrhoeae over a 20-year period in the Pretoria region. Endourethral specimens were collected from adult men with symptoms of urethritis attending primary health care clinics and private medical practitioners. These swabs were plated on enriched media for isolation of N. gonorrhoeae. Antimicrobial susceptibility of the organisms was performed using the disc diffusion and agar dilution methods. Plasmid analyses were performed on beta-lactamase-producing isolates. Penicillase-producing N. gonorrhoeae strains increased from 4 to 16; whilst chromosomally mediated penicillin-resistant strains increased dramatically from 0 to 16 from 1984 to 2004. There was an equal distribution of the 3.2 MDa African and 4.4 MDa Asian plasmids. High-level tetracycline-resistant strains (36) were detected for the first time in 2004. Ciprofloxacin resistance emerged at 7 in the same year. Gonococcal isolates remained susceptible to cefoxitin; ceftriaxone; cefpodoxime; and spectinomycin. However; the minimum inhibitory concentration values for spectinomycin were very close to the breakpoint. We have shown a continuing increase in resistance to penicillin (plasmid and chromosomal); the emergence of high-level tetracycline resistance and an emergence of resistance to ciprofloxacin. Susceptibility testing is essential for successful therapeutic outcomes and needs to be performed in an ongoing basis

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.

Use of an immunochromatographic kit for the rapid detection of Mycobacterium tuberculosis from broth cultures.

In settings of high human immunodeficiency virus (HIV) prevalence, culture confirmation, preferably by liquid culture, is required for the diagnosis of tuberculosis (TB). However, long delays with phenotypic identification offsets the short turnaround time of liquid cultures. We report here the advantages of using a commercial immunochromatographic (ICT) assay targeting the Mycobacterium tuberculosis protein 64 (MPT-64) Ag and compare it with the Accuprobe MTB complex molecular probe assay. The performance of the ICT kit was excellent, with sensitivity, specificity, positive and negative predictive values of respectively 97%, 100%, 100%, and 92%. The kit requires a 15-min assay time, is easy to perform and is a good method for simplifying the diagnosis of TB.

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.

Antimicrobial Susceptibility Patterns of Selected Invasive Pathogens from Public Sector Hospitals in South Africa; 2007

Retrospective antibiotic surveillance data of selected invasive pathogens isolated from blood and cerebrospinal fluid at public sector hospitals in South Africa in 2007 are presented. Antimicrobial susceptibilities were determined according to the 2007 Clinical and Laboratory Standards Institute criteria. Klebsiella pneumoniae remains a highly resistant pathogen; with approximately half of all strains producing extended-spectrum beta-lactamases. All laboratories reported considerable resistance among Acinetobacter spp. Approximately 50-60of Staphylococcus aureus isolates from blood were resistant to cloxacillin. Among Streptococcus pneumoniae isolates from blood and cerebrospinal intermediate resistance to penicillin. Resistance to ceftriaxone in S. pneumoniae was rare

A retrospective review of malaria cases seen in a non-endemic area of South Africa.

BACKGROUND: Malaria is a risk for travelers to endemic areas. We describe the diagnosis and treatment of malaria in Pretoria, a non-endemic area in South Africa. METHODS: Records of specimens submitted to the medical microbiology laboratory for malaria investigations over 3 years were reviewed with follow up of hospital records for positive specimens for clinical data. The laboratory performs malaria smears and uses HRP2-Ag testing for rapid diagnosis of Plasmodium falciparum. RESULTS: A total of 516 specimens were received, with a 211/516 (41%) malaria smear positive rate. The number of malaria positive specimens has been increasing overtime and this increase was statistically significant in children [p=0.005]. HRP2-Ag testing was done on 430 specimens with124/430 (29%) being positive, of which 10/124 (8%) were smear negative, giving 98% sensitivity. Hospital records for 198/211 (94%) smear positive cases showed that 190/198 (96%) of the patients had a travel history with 170/190 (71%) having traveled to Mozambique, a malaria endemic country. Most patients presented with uncomplicated malaria; the CFR was 4/198 (2%). Treatment mainly followed South African national guidelines. CONCLUSION: Imported malaria is increasingly being diagnosed in returning travelers, especially from Mozambique. Rapid antigen tests remain useful for the diagnosis of malaria in non-endemic areas.

Salmonella typhimurium meningitis in an adult patient with AIDS.

Salmonella meningitis is an unusual complication of Salmonella sepsis and occurs mainly in children. A rare case of Salmonella typhimurium meningitis occurring in an adult HIV positive man who presented with a history of fever and diarrhoea is reported. On examination he was dehydrated, and had oral thrush, weakness of lower limbs and neck stiffness. A septic diagnostic screen was performed and he was commenced on empiric intravenous cefotaxime therapy for meningitis. S typhimurium was cultured from cerebrospinal fluid and blood culture specimens. It was non-lactose fermenting, oxidase negative, H(2)S positive and motile. Cefotaxime was continued for 14 days and the patient responded without neurological sequelae.

Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) of ciprofloxacin resistant isolates of Pretoria, South Africa.

Gonococcal isolates resistant to ciprofloxacin collected from 2004 to 2005, in the Pretoria region, were characterised using Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST). The isolates were obtained from men presenting with urethritis to primary healthcare clinics and general practitioners. All isolates were tested for susceptibility to ciprofloxacin by Etest, disc diffusion and agar dilution methods. Sequence-based typing, directed at the por and tbpB genes, and compared with international isolates using the NG-MAST database on the internet, was done for 18 isolates (10 ciprofloxacin-resistant isolates and 8 susceptible controls). There was one cluster (four isolates) of known sequence type (ST) similar to the pattern seen among strains from Scotland, England, and Durban, South Africa. Two other known STs were identified, while the remaining STs were unique.

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

Increased exposure to hepatitis B virus infection in HIV-positive South African antenatal women.

The prevalence of markers for hepatitis B virus (HBV) exposure and active infection in HIV-positive (n=710) and HIV-negative (n=710) pregnant South African women was investigated. The following statistically significant increases in the HIV-positive group were found: anti-hepatitis B core antigen (anti-HBc) (37.3% versus 28.6%; odds ratio [OR]: 1.49); anti-hepatitis B surface antigen (anti-HBs) (29.5% versus 20.1%; OR: 1.66); exposure based on hepatitis B surface antigen (HBsAg) and anti-HBc (39.2% versus 30.1%; OR: 1.49); and exposure based on anti-HBs, anti-HBc and HBsAg (37.1% versus 24.5%; OR: 1.82). However, there was no increase in active HBV infections, with 2.4% of the HIV positives and 2.2% of the HIV negatives being HBV DNA positive. Although the impact that HIV has had on the prevalence of HBV in this population group is not as pronounced as that found in areas of low endemicity (where up to seven-fold increases have been reported), there is a statistically significant increased exposure to HBV.

Molecular typing of Treponema pallidum strains from patients with neurosyphilis in Pretoria, South Africa.

OBJECTIVE: To evaluate the molecular typing system for Treponema pallidum using cerebrospinal fluid (CSF) specimens obtained from patients with neurosyphilis in Pretoria, South Africa. METHODS: CSF specimens were collected from 32 men and 18 women with suspected late neurosyphilis. Typing of T pallidum involved PCR amplification and restriction analysis of the tprE, G and J genes and determination of the number of 60 base pair tandem repeats within the arp gene by PCR amplification. RESULTS: Of 13 typeable specimens, 4 strain types were identified: 2i, 3e, 14a and 17e. Subtype 14a was identified in 7 specimens (53.8%), subtype 3e in 4 specimens (30.7%) and subtypes 17e and 2i in 1 specimen (7.6%) each. CONCLUSIONS: This study shows that the typing system can be applied to specimens which may contain low numbers of spirochaetes such as CSF.

Hepatitis B virus and human immunodeficiency virus co-infection in sub-Saharan Africa: a call for further investigation.

A growing body of evidence indicates that human immunodeficiency virus (HIV)-positive individuals are more likely to be infected with hepatitis B virus (HBV) than HIV-negative individuals, possibly as a result of shared risk factors. There is also evidence that HIV-positive individuals who are subsequently infected with HBV are more likely to become HBV chronic carriers, have a high HBV replication rate, and remain hepatitis Be antigen positive for a much longer period. In addition, it is evident that immunosuppression brought about by HIV infection may cause reactivation or reinfection in those previously exposed to HBV. Furthermore, HIV infection exacerbates liver disease in HBV co-infected individuals, and there is an even greater risk of liver disease when HIV and HBV co-infected patients are treated with highly active anti-retroviral therapy (HAART). Complicating matters further, there have been several reports linking HIV infection to 'sero-silent' HBV infections, which presents serious problems for diagnosis, prevention, and control. In sub-Saharan Africa, where both HIV and HBV are endemic, little is known about the burden of co-infection and the interaction between these two viruses. This paper reviews studies that have investigated HIV and HBV co-infection in sub-Saharan Africa, against a backdrop of what is currently known about the interactions between these two viruses.