The diagnosis of skeletal tuberculosis by polymerase chain reaction.

OBJECTIVE: To determine the potential of the polymerase chain reaction (PCR) for detecting Mycobacterium tuberculosis in skeletal samples by comparing results obtained by 1) Ziehl Neelsen staining, Lowenstein-Jensen and Bactec culture, 2) histopathology and clinical findings at the level of agreement, sensitivity and specificity. DESIGN: Cross sectional study. SETTING: Department of Medical Microbiology, Orthopaedics and Anatomical pathology, University of the Orange Free State Bloemfontein, South Africa. SUBJECTS: 45 consecutive patients were extensively investigated, 30 patients with clinical presumptive active tuberculosis and 15 with other pathology. RESULTS: Detection using culture could confirm only three of the 26 clinically diagnosed tuberculosis cases while PCR detection confirmed disease in 15 cases. The use of PCR increased the confirmation of clinically probable tuberculosis from 14 using standard laboratory techniques and histology to 18 of 26 cases. Calculated sensitivity and specificity for PCR employing culture as the "gold standard" were 100% (with 95% CI 29.2; 100.0) and 71.4% (55.4; 84.3), which due to low detection levels, basically excludes culture as a standard for statistical analysis. Sensitivity and specificity for PCR using histology as the "gold standard" were 78.6% (49.2; 95.3) and 87.1% (70.2; 96.4) respectively with positive and negative predictive values of 73.3% (44.9; 92.2) and 90% (73.5; 97.9) respectively. Positive agreement between PCR and histology was 0.64 (0.4; 0.9) indicating fair agreement. CONCLUSION: Although numbers in the study were too low to effectively draw statistically valid conclusions the importance of the relevance of PCR for rapid detection of low numbers of acid-fast bacilli and confirmation of mycobacterial infection in spinal biopsies has been established.

Comparative activity of eighteen antimicrobial agents against anaerobic bacteria isolated in South Africa.

The in vitro activity of 18 antimicrobial agents was determined against 378 anaerobic bacteria isolated in Bloemfontein, South Africa, during 1996/97. Against the gram-positive isolates, MICs of penicillin and cefoxitin were >0.5 microg/ml and >16 microg/ml, respectively, for five and three strains of non-perfringens Clostridium spp. Seventeen Peptostreptococcus anaerobius strains were resistant to penicillin (MIC > or = 2 microg/ml). All gram-positive anaerobes tested except one Peptostreptococcus sp. and one Clostridium sp. were susceptible to dalfopristin-quinupristin (MICs < or = 1 microg/ml). The carbapenems exhibited excellent activity against the gram-positive isolates and were effective against most gram-negative anaerobes, with the exception of the fusobacteria. Only seven strains exhibited decreased susceptibility to trovafloxacin (MICs > 2 microg/ml). In mixed anaerobic/aerobic infections, carbapenems and the fourth-generation quinolone trovafloxacin were the agents most suitable for us as broad-spectrum monotherapy.

In vitro activities of 15 antimicrobial agents against clinical isolates of South African enterococci.

The activities of a panel of currently available antibiotics and the investigational agents LY 333328, linezolid, CL 331,002, CL 329,998, moxifloxacin (BAY 12-8039), trovafloxacin, and quinupristin-dalfopristin against 274 clinical isolates of enterococci were determined. No vancomycin resistance or beta-lactamase production was observed. Except for 12 isolates (all non-Enterococcus faecalis) showing reduced susceptibility to quinupristin-dalfopristin (MIC, >/=4 microg/ml), the new agents exhibited promising in vitro antienterococcal activity.

Enterococcal endocarditis--a case treated with teicoplanin and amoxycillin.

The study aimed to determine the antibacterial therapy effective in the cure of endocarditis caused by Enterococcus faecalis resistant to clinically achievable levels of vancomycin. Isolation of the causative enterococcus had been achieved by direct inoculation of the resected valve into the culture medium in theatre. The patient was known to have had an aortic valve defect since childhood and had recently undergone splenectomy following trauma. Blood cultures were negative prior to valve replacement. A perivalvular abscess was noted at operation. In vitro minimal bactericidal results and serum activity were the basis of the postoperative choice of drugs. The minimal bactericidal level of teicoplanin was 250 micrograms/ml and that of amoxycillin 64 micrograms/ml. Neither is achievable with the advocated dosage. A combination of these two cell-wall-active agents successfully eliminated the infection. Acting at two different sites in the synthesis of the bacterial cell wall, teicoplanin and amoxycillin were found to be bactericidal in vitro at the trough levels of the antibiotics in the serum. The patient recovered fully.

Plasmid analysis of Neisseria gonorrhoeae isolates and dissemination of tetM genes in southern Africa 1993-1995.

One group (145 isolates) of Neisseria gonorrhoeae was collected from municipal clinics in Bloemfontein in 1994 and a second group (65 isolates) in 1995. Penicillin and tetracycline MICs were determined and plasmid analysis performed to monitor antimicrobial susceptibilities in conjunction with the occurrence of plasmids in these isolates. The prevalence of penicillin resistance caused by beta-lactamase plasmids remained constant at 9% during the study period. Three high-level tetracycline-resistant strains (MICs 16 mg/L), the first to be detected in South Africa, were isolated in 1994. Although there was a reduction in the percentage of isolates harbouring 24.5 MDa conjugative plasmids (from 79% in 1994 to 46% in 1995), this was partially counteracted by an increase in TetM-encoding conjugative plasmids (25.2 MDa) from 2% to 18.5%. The tetM genes of 13 isolates shown to exhibit high-level tetracycline resistance were characterized as the American type. The American-type tetracycline resistance plasmid was demonstrated in 11 isolates. Digestion with Bg/l showed that two isolates harboured tetM-encoding plasmids that differed from the American- and Dutch-type plasmids described previously: one isolate contained a plasmid that produced two fragments of different sizes from those of the American-type plasmid and the second isolate possessed an American/Dutch hybrid plasmid. Auxotyping/serotyping and random amplified polymorphic DNA analysis revealed a predominant tetracycline-resistant family (NR/IA-6, genomic group I) in Bloemfontein. As there is a high incidence of chlamydial infections in southern Africa requiring tetracycline therapy, selective pressures exist in the environment for the maintenance and rapid spread of high-level tetracycline-resistant N. gonorrhoeae. It is possible that tetM genes may have emanated from Botswana and/or Namibia to Bloemfontein. The establishment of high-level tetracycline-resistant N. gonorrhoeae in Bloemfontein was seen to be complex as a related group of strains was identified, plasmid dissemination was evident and two new TetM-encoding plasmids were demonstrated. The appearance of these TetM-encoding plasmids indicates either that the American- and Dutch-type plasmids are continuing to evolve or that tetM genes are being introduced into different families of 24.5 MDa conjugative plasmids.

Characterisation of penA and tetM resistance genes of Neisseria gonorrhoeae isolated in southern Africa--epidemiological monitoring and resistance development.

OBJECTIVE: To investigate penA and tetM resistance gene variation of Neisseria gonorrhoeae in order to define gene types for epidemiological monitoring and resistance development. DESIGN: Isolates of N. gonorrhoeae which were susceptible and resistant to penicillin and/or tetracycline were selected. Strains comprised South African isolates (22 from Bloemfontein, 13 from Transvaal, 20 from the Cape) and 15 Botswana and 4 Namibia isolates. The penA genes (2 kb) of all strains and tetM genes (765 bp) of 11 high-level tetracycline-resistant strains were amplified and restricted with HpaII. RESULTS AND CONCLUSIONS: Twelve different HpaII fingerprint patterns were obtained from the 74 isolates analysed for penicillin-binding protein (PBP) 2 gene (penA) alterations. Focusing on the transpeptidase domain, 25 isolates (3 whole gene patterns, minimal inhibitory concentrations (MICs) < or = 0.03-0.125 micrograms/ml) had restriction sites equivalent to those previously described for a susceptible strain. Of the remaining 9 PBP 2 gene groups, 25 strains fell into a designated group E. Penicillin/ penicillin + clavulanic acid MICs determined on these group E isolates gave a range of 0.125-2.0 micrograms/ml, although MICs against 4 strains were < or = 0.03 micrograms/ml. MICs of penicillin/penicillin + clavulanic acid for the 24 isolates that contained altered PBP 2 transpeptidase gene regions not designated group E were only < or = 0.03-0.125 micrograms/ml. The lack of a HpaII restriction site at nucleotide 1934 in the PBP 2 gene of group E strains was indicative of a small terminal region of N. cinerea DNA. This gene block, which was found in all the southern African areas studied, appears to predispose isolates to increased penicillin resistance. The 25.2 MDa conjugative plasmid carrying the tetM resistance determinant was readily demonstrated in 11 Botswana/Namibia isolates exhibiting high-level resistance to tetracycline (MICs > or = 16 micrograms/ml). The tetM gene was shown to be of the American type.

The incidence of hospital fungal infections--yeast fungaemia.

In order to determine the incidence and the causative agents of fungaemia, a survey was undertaken of blood culture specimens received from the Bloemfontein academic hospitals. Over a period of 1 year, 5,017 successive blood cultures were examined; 1,030 (20.5%) had growth of which 106 (2.1%) yielded yeasts. Candida albicans (42%), C. tropicalis (26%) and C. parapsilosis (20%) were the species most frequently isolated. Fungaemia occurred most often after broad-spectrum antimicrobial therapy and abdominal disorders.

[Rapid identification methods of yeasts during fungaemia]. / Vinnige identifikasiemetodes van giste tydens fungemie.

A wet preparation method, modified serum test and a modified disc diffusion method were evaluated in order to identify yeasts more rapidly during fungaemia. A total of 2,932 blood cultures were processed, of which 54 (1.8%) yielded yeasts. By using these methods, Candida albicans was identified within 3 hours, after yeast growth in the blood culture was confirmed by Gram stain. The wet preparation examinations were accurate in 60% of cases and the serum test in 95% of cases. After growth was detected, yeast species other than C. albicans were identified within 24 hours using the modified disc diffusion method. C. albicans (50%), C. tropicalis (22%) and C. parapsilosis (22%) were the species most frequently isolated. Fungaemia occurred most often after antimicrobial therapy and abdominal conditions.

In vitro activity of a new 'higher-lactam' antibacterial agent LY 193239.

In vitro activity of the new bicyclic pyrazolidinone LY 193239 (Eli Lilly) was evaluated against 52 clinical isolates of Haemophilus influenzae (4 were beta-lactamase producers), 32 Enterococcus faecalis, 14 Neisseria gonorrhoeae (1 beta-lactamase-positive) and 19 Neisseria meningitidis. Activity was best against Neisseria spp. and H. influenzae, including penicillinase-producing strains. Results of the time-kill study against a non-enzyme-mediated penicillin resistant strain of N. meningitidis indicate that exposure to an antibacterial concentration four times the minimal inhibitory concentration was bactericidal. E. faecalis was insensitive.

A microbiological study of acute maxillary sinusitis in Bloemfontein.

Microbiological analyses and antibiotic sensitivity tests were done on 26 patients with acute maxillary sinusitis during the first 9 months of 1986. Positive cultures were obtained in 23 of the patients, with anaerobes cultured in 13 (50%). Haemophilus influenzae was cultured in all non-producers of beta-lactamase. Therapy with erythromycin and chloromycetin appeared to be equally effective in aerobic cases and metronidazole was effective in all anaerobic cases.

In vitro evaluation of the cyclic lipopeptide LY 146032 (daptomycin).

In vitro activity of the new cyclic lipopeptide LY 146032 (daptomycin, Eli Lilly) was evaluated against 67 clinical isolates of Staphylococcus aureus, 15 clinical isolates of S. epidermidis and 80 clinical isolates of Streptococcus faecalis. Activity was best against methicillin-resistant S. epidermidis (minimal inhibitory concentrations of LY 146032 0.25-0.5 mg/l). Minimal inhibitory concentrations and minimal bactericidal concentrations were of similar value. LY 146032 requires physiological concentrations of Ca++ ion for the expression of antibacterial activity. Results of the time-kill curves indicated that exposure to a concentration of the antibiotic equivalent to the minimal inhibitory concentration abolished recovery in the strains of S. aureus and S. epidermidis investigated. Response by S. faecalis was less predictable although most S. faecalis strains were killed in vitro by LY 146032 concentrations of less than or equal to 8 mg/l which is within the predicted clinically achievable range.

Aminoglycoside resistance among isolates of nosocomial Enterobacteriaceae.

Fifty-seven gentamicin-resistant isolates of Enterobacteriaceae, obtained from patients attending hospital, were examined for the production of aminoglycoside-modifying enzymes. Of the 51 strains producing such enzymes, 34 were presumptively plasmid-mediated as indicated by conjugation experiments.

Incidence of aerobic and anaerobic infection in patients with incomplete abortion.

The incidence of infection in 100 cases of abortion was studied. Intra-uterine swabs and products of conception were cultured both aerobically and anaerobically. Significant potentially-pathogenic organisms were cultured from 94 of 100 intra-uterine swabs and from all 100 samples of the products of conception. Anaerobes were cultured from 70 intra-uterine swabs and from 56 products of conception. The spectrum and relative proportion of organisms isolated resemble those found in the normal vagina and cervical os. In addition, positive blood cultures were obtained from 24 patients, 8 on admission and 17 after evacuation. With this high incidence of infection, routine antimicrobial therapy for aerobic and anaerobic organisms should be given to all patients with abortion.

Isolation of Campylobacter fetus from recent cases of human vibriosis.

Campylobacter fetus was isolated from five recent cases of human vibriosis, of which two were adults and three were children. One adult presented with pericarditis and the other with recurrent pyrexia. Campylobacter fetus subsp. intestinalis which resembled cattle strains serologically, was isolated under CO2 or anaerobic conditions from blood cultures of these patients. Two of the three children had kwashiorkor and the third was only 8 days old. Isolates identified as Campylobacter fetus subsp. jejuni were cultured from blood of these patients, two of whom had diarrhoea. Three patients succumbed, despite adequate antibiotic therapy. The epidemiology of the disease is discussed and it is suggested that infection may have been from the patients' own flora.