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BACKGROUND: Despite the breadth of data supporting evidence-based practice for sepsis care in high-resource settings, there are relatively few data to guide the management of sepsis in low-resource settings, particularly in areas where HIV and tuberculosis (TB) are prevalent. Furthermore, few studies had broadened sepsis parameters to include all patients with acute infectious illness or followed patients up after hospital discharge. Understanding the epidemiology and outcomes of acute infections in a local context is the critical first step to developing locally informed targeted management strategies. OBJECTIVES: To quantify and describe the incidence of and risk factors for mortality in a cohort of patients with undifferentiated acute infectious illnesses who presented to an emergency department (ED) in the Eastern Cape region of South Africa (SA). METHODS: In this prospective cohort study, patients with suspected acute infectious illness were enrolled at a district casualty ward in Mthatha, SA, between 1 July and 1 September 2017. Demographic data, interventions, diagnostic studies and disposition were prospectively collected during the initial encounter and during the hospital stay. Follow-up was conducted both in hospital and via phone interviews 30 days after the index visit. RESULTS: A total of 301 patients presented to the ED with acute infectious illness during the study period, of whom 54.8% had complete 30-day follow-up. Of the study population, only 5.7% had a complete set of vital signs (heart rate, respiratory rate, blood pressure and temperature) documented. Of the cohort, 51.8% had HIV and 32.9% active or treated TB; 25.2% of patients died within 30 days. Accounting for medical history, diagnosis and ED interventions, risk of mortality was independently associated with age (odds ratio (OR) 1.03; 95% confidence interval (CI) 1.00 - 1.06), HIV-positive status (OR 4.10; 95% CI 1.44 - 11.67) and Quick Sequential (Sepsis-Related) Organ Failure Assessment (qSOFA) score (OR 1.90; 95% CI 1.14 - 3.19) in an adjusted model. No ED interventions were protective for mortality, with intravenous fluid administration associated with increased 30-day mortality in this cohort (OR 3.65; 95% CI 1.38 - 9.62). CONCLUSIONS: Among adults with suspected acute infectious illness in Mthatha, SA, 30-day mortality was concerningly high. Mortality was highest in patients with concomitant HIV infection. In particular, vital sign assessment to identify possible sepsis in this cohort is crucial, as it affects mortality to a meaningful extent, yet is often unavailable. Future research is needed on the management of sepsis in low-resource settings, particularly in HIV-positive individuals.

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BACKGROUND: Symptomatic hyperlactataemia and lactic acidosis (SHLA) are potentially life-threatening complications associated with stavudine (d4T), an antiretroviral therapy (ART) drug widely used in developing countries. METHODS: Cases comprised all symptomatic patients with measured lactates >or= 5 mmol/L referred to a South African hospital between August 2003 and November 2005. Matched controls were selected according to facility and duration on ART. RESULTS: Seventy-one cases and 142 controls were included in the study. The majority of cases presented between 6 and 18 months on ART. Female sex [adjusted odds ratio (AOR) 23.4; 95% confidence interval (CI) 4.0-136.6], a baseline weight between 60 and 75 kg (AOR 4.5; 95% CI 1.4-14.1) or, in particular, >or= 75 kg (AOR 19.4; 95% CI 4.1-82.5) at ART initiation and gaining >or= 6 kg in the first 3 months on therapy (AOR 3.5; 95% CI 1.3-9.5) were independent risk factors identifying patients who may subsequently develop SHLA. Weight loss of >or= 2 kg (AOR 6.1; 95% CI 2.0-18.3), a rise in alanine aminotransferase (ALT) >or= 10 U/L (AOR 3.1; 95% CI 1.1-8.9), the presence of at least one of three major symptoms (vomiting, nausea and abdominal pains) of SHLA (AOR 12.6; 95% CI 3.3-47.2) and peripheral neuropathy (AOR 3.4; 95% CI 1.1-9.8) were the clinical parameters that were most able to identify patients with early manifestations of SHLA. CONCLUSIONS: This is the first case-control study for SHLA in Southern Africa. Given these findings, we advise that stavudine is avoided in overweight women. Weight loss, a rise in ALT, peripheral neuropathy and/or gastrointestinal symptoms should prompt healthcare workers to assess for SHLA, especially at between 6 and 18 months on ART.

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Root mat of cucumbers and tomatoes has previously been shown to be caused by Agrobacterium radiobacter strains harboring a root-inducing Ri plasmid (pRi). Nine other pRi-harboring alpha-Proteobacteria have subsequently been isolated from root mat-infected crops. Fatty acid profiling and partial 16S rRNA sequence analysis identified three of these strains as being in the genus Ochrobactrum, five as being in the genus Rhizobium, and one as being in the genus Sinorhizobium: An in vitro pathogenicity test involving inoculation of cucumber cotyledons was developed. All pRi-harboring alpha-Proteobacteria induced typical root mat symptoms from the cotyledons. Average transformation rates for rhizogenic Ochrobactrum (46%) and Rhizobium (44%) strains were lower than those observed for rhizogenic A. radiobacter strains (64%). However, individual strains from these three genera all had transformation rates comparable to those observed from cotyledons inoculated with a rhizogenic Sinorhizobium strain (75%).

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The genus Burkholderia comprises over 28 species and species-specific, recA-based polymerase chain reaction (PCR) tests are available for several species, but not for some soil-inhabiting species including B. fungorum. Previous analysis of several novel rhizospheric, environmental isolates belonging to the B. cepacia complex suggested they may be closely related to B. fungorum. To discover any relationship between these isolates and B. fungorum we set out to clone and sequence a portion of the B. fungorum recA gene in order to design species-specific primer pairs for use in a recA-based PCR assay. Using a similar procedure we extended the recA-based PCR assay to identify B. sacchari and B. caledonica, two additional soil-inhabiting Burkholderia spp.

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AIMS: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon. METHODS AND RESULTS: Five methods were evaluated and fine-tuned for extraction of RNA from Cms. The most efficient non-commercial RNA extraction method included an enzymatic breakdown of the cell wall followed by a phenol extraction. AmpliDet RNA enabled detection of 10,000 molecules of purified rRNA per reaction and 100 cfu of Cms per reaction in more complex samples. Two primer pairs were tested with DNA and RNA purified from Cms. One primer pair was able to distinguish live from dead cells. CONCLUSIONS: An AmpliDet RNA was developed which enabled fast and specific detection of viable cells of Cms in complex substrates at a detection limit of 100 cfu per reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel AmpliDet RNA is carried out in sealed tubes, thus reducing the risk of carry-over contamination. The method will be particularly suitable for studies on the epidemiology of Cms in which viable cells should be exclusively detected.

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AIMS: Determination of genetic diversity among UK Burkholderia cepacia isolates from various environmental niches, principally woodland tree rhizospheres and onions. METHODS AND RESULTS: Genus determination was made using polymerase chain reaction (PCR) amplification and fatty acid methyl ester profiling. Genetic diversity was investigated by repetitive sequence genetic PCR fingerprinting. Several onion isolates were similar to clinical isolates but others were diverse. Some environmental isolates were possibly synonymous with B. cepacia and B. gladioli but most from woodland rhizospheres were distinct and clustered together. The 16S rRNA genes of representatives from these clusters were PCR amplified, sequenced and phylogenetically compared with all known Burkholderia and related species. This revealed that the rhizospheric isolates had closest affinity with Burkholderia spp. with known bioremediative and biocontrol capabilities and were unrelated to taxa comprising plant or human pathogenic strains. CONCLUSIONS: All of the analyses investigated revealed that environmental and onion isolates of B. cepacia complex bacteria are genetically diverse but that woodland rhizospheric isolates are related to each other and unrelated to plant or human pathogenic strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Woodland rhizospheric isolates of B. cepacia are potentially good candidates for use in bioremediation and biocontrol, as they appear distinct from plant or human pathogenic strains.

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AIMS: The development of a fluorogenic, 5' nuclease, TaqMan PCR assay for the detection of Ri-plasmids from root mat inducing Agrobacterium biovar 1 strains. METHODS AND RESULTS: A TaqMan probe and primer set were designed within the T-DNA sequence of a known root mat inducing Agrobacterium strain. One hundred and ten Agrobacterium and closely related bacteria were tested using this novel PCR and compared with results from a conventional PCR which detects Ti and Ri-plasmids. The Agrobacterium selective media, Medium 1A was modified into broth form for use as an enrichment of the pathogen from samples prior to the TaqMan PCR. CONCLUSIONS: The root mat pathogen was detected successfully from a range of sample types using the enriched fluorogenic PCR assay, negating the need for complex DNA extraction procedures and post-PCR processing techniques such as gel electrophoresis. The technique is therefore a rapid and cost-effective detection method. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first known report of a fluorogenic, 5' nuclease, TaqMan assay designed to detect an Agrobacterium plant pathogen. The method can be used as a model system for the detection of other Agrobacterium pathogens.

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PCR-amplification has been used to screen 75 isolates of the Burkholderia cepacia complex for the cblA pilin gene. PCR-amplified products of the correct size (664 bp) were cloned and sequenced and the sequences compared. Apart from in the control, epidemic cystic fibrosis (CF)-associated B. cepacia lineage we also identified, for the first time, cblA genes in a unique, non-CF clinical isolate from France and a plant (onion) pathogenic isolate from Italy. The sequence of the cblA gene amplified from the clinical isolate was more diverged from the epidemic lineage than that amplified from the onion pathogenic isolate.

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ABSTRACT In a recent Letter to the Editor of Phytopathology, proposals were made for endorsement and for rejection of selected names of plant pathogenic Pseudomonas spp. and Xanthomonas spp. We believe that support for, and rejection of, several names was based on misconceptions concerning the Approved Lists of Bacterial Names and entails misinterpretations of several Rules of the International Code of Nomenclature of Bacteria. This letter aims to clarify those misconceptions and misinterpretations.

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The aminoglycosides are a large and diverse class of antibiotics that characteristically contain two or more aminosugars linked by glycosidic bonds to an aminocyclitol component. Structures are presented for over 30 of the most important members of this family of compounds. The use of aminoglycosides in clinical and veterinary medicine and in agriculture is described. Qualitative methods for aminoglycoside analysis include X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The major part of this article comprises a comprehensive review of quantitative methods for the determination of aminoglycosides. These are microbiological assay, radiochemical assay, radioimmunoassay, enzyme immunoassay, fluoroimmunoassay and other immunoassays, spectrophotometric and other non-separative methods, gas chromatography (GC), thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and capillary electrophoresis (CE). Simple spectrophotometric methods may be adequate for the assay of bulk pharmaceuticals and their formulations. Microbiological assays make useful semi-quantitative screening tests for the analysis of veterinary drug residues in food, but rapid enzyme immunoassays are more suitable for accurate measurements of aminoglycosides in complex matrices. Automated immunoassays are the most appropriate methods for serum aminoglycoside determinations during therapeutic drug monitoring. HPLC techniques provide the specificity and sensitivity required for pharmacokinetic and other research studies, while HPLC-MS is employed for the confirmation of veterinary drug residues. The potential for further development of chromatographic and CE methods for the analysis of biological samples is outlined.

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Fatty acid profiling was used to study variation amongst strains of Xanthomonas campestris pv. vasculorum (Xcv). They could be divided into five groups using cellular fatty acid profiles. Group A strains represent a new and little known taxon and all came from plants of broom bamboo (Thysanolaena maxima) from Mauritius. Group B strains included the Xcv pathotype reference strain and were from palms, broom bamboo and sugarcane from Mauritius, Reunion and Australia. Group C contained southern African and Malagasy strains from sugarcane and maize, together with X. campestris pv. holcicola strain. No Mascarene strains fell into this group. Group D strains isolated from sugarcane, maize and royal palm (Roystonea regia) were from Mauritius and Reunion, the earliest known strains coming from Réunion. These groups represented in the Mascarene Islands possibly belong to three different Xanthomonas species. A further Group E comprised one Xcv strain (NCPPB 182) from Puerto Rico, one X. vasicola pv. holcicola strain plus 6 other unclassified Xanthomonas strains causing red stripe disease symptoms in sugarcane. Three of these groups occur on Mauritius and two occur on Réunion. Group B strains originally caused serious problems in noble canes. As resistant interspecific hybrids were introduced, group D strains appeared in Mauritius possibly being introduced from Reunion but having similar host ranges within the Gramineae and Palmae. The findings that 3 of these groups (A, B, D) can cause gumming disease in a grass species (T. maxima) and that 2 of them (B, D) also cause gumming disease in sugar cane (Gramineae) and palms (Palmae) is unusual.

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We demonstrated that oxidative stress plays a role in freeze-thaw-induced killing of Campylobacter coli following analysis of mutants deficient in key antioxidant functions. Superoxide anions, but not H(2)O(2), were formed during the freeze-thaw process. However, a failure to detoxify superoxide anions may lead to spontaneous disproportionation of the radicals to H(2)O(2).

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A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5' nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to >/=10(2) cells ml(-1) was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.

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A reversed-phase HPLC method previously developed for the analysis of progesterone and its major metabolites has been transferred successfully to a capillary electrochromatography (CEC) system. Procedures for fabricating packed capillaries and the modifications made to the capillary electropherograph which allow operation in the CEC mode without pressurisation are described. The dependence of electroosmotic flow on electric field strength, pH and organic modifier content is discussed. Direct comparison with HPLC shows that CEC provides useful gains in efficiency and speed of analysis and requires vastly reduced amounts of both chromatographic phases and material for analysis. On-line concentration is described which allows the lower sensitivity of CEC to be offset by injecting analytes from a non-eluting solution. Examination of steroids in plasma demonstrates that the superior separation by CEC is maintained in a complex biological matrix.

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Following the development of a sensitive high-performance liquid chromatographic (HPLC) assay for gentamicin in biological matrices, the utility of this assay for the determination of other clinically important aminoglycosides (neomycin, netilmicin and sisomicin) in bacterial culture media or plasma is demonstrated. The high sensitivity of the assay enables direct measurement of the aminoglycoside content of bacterial cells cultured in the presence of unlabelled drug.

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Two hundred and thirty-eight patients with femoral neck fractures were entered into a randomised pilot study comparing the use of sequential treatment by 'Flowtron DVT' garments in the perioperative period followed by Enoxaparin (Clexane-Rhône-Poulenc Rorer), and Enoxaparin alone. One hundred and ninety-three patients were excluded indicating the difficulty of achieving pure comparisons in this population. The remaining 44 were randomised: 21 received Enoxaparin from the time of admission, and 23 had sequential treatment. There was no statistically significant difference in the incidence of thromboembolism. Patient preference did not indicate a favoured treatment subjectively. The operation field was drier in the sequential group, although this did not reach significance. Sequential treatment was not shown to be better or worse than treatment with Enoxaparin, but the trends favoured sequential treatment rather than drug treatment alone. The technique allows the operation to be carried out without the problems produced by low dose heparins and mobilisation is not hindered by compression garments.

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A prospective randomised trial was carried comparing the use of Enoxaparin with intermittent pneumatic calf compression garments for thromboprophylaxis in total hip replacement. Fifty consecutive patients were studied and randomised to evaluate these two methods. There were 2 deep vein thromboses, one in each group, and no cases of pulmonary embolism. The operative field was judged to be drier in the compression group, but the mean fall in the postoperative haemoglobin level was the same in each. In the peroperative period, 6 patients in the Enoxaparin group needed 2 units of blood and one 3 units. In the compression group, 3 patients needed transfusions of 2 units. Intermittent calf compression has fewer problems than the use of Enoxaparin and has no contraindications.

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A high-performance liquid chromatographic method is described for the determination of gentamicin in bacterial culture medium or plasma with increased sensitivity and improved separation of the C1 component. Gentamicin was extracted from the biological matrix with high efficiency using carboxypropyl (CBA)-bonded silica. Derivatization with 9-fluorenylmethyl chloroformate (FMOC-Cl) followed by C18 reversed-phase chromatography allowed the fluorimetric detection of gentamicins C1, C1a and C2. A fourth component, considered to be gentamicin C2a, was partially resolved from the C2 peak. Optimal conditions for the extraction and derivatization of gentamicin are described. The detection limit was below 50 micrograms/l, the assay was linear to 5 mg/l and showed good reproducibility. It is concluded that pre-column derivatization with FMOC-Cl substantially improves the analysis of gentamicin compared with present methods based on reaction with o-phthaldialdehyde.

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Hydroxycinnamic acids and their derivatives occur widely in plants, fruits and wine. The effect of the common hydroxycinnamic acids (caffeic, coumaric and ferulic acids), at concentrations of 100 and 500 mg l-1, on growth of 11 strains of spoilage yeasts was measured spectrophotometrically and compared with that of potassium sorbate. Ferulic acid was the most generally inhibitory hydroxycinnamic acid. At 500 mg l-1 it appreciably inhibited Pichia anomala, Debaryomyces hansenii and Saccharomyces cerevisiae and prevented detectable growth of one strain each of P. anomala and D. hansenii. Caffeic acid was the least inhibitory compound and coumaric acid had an intermediate effect. The more resistant strains of yeast were P. membranaefaciens, Saccharomycodes ludwigii and Zygosaccharomyces bailii. Sensitivity to hydroxycinnamic acid was, in general, associated with sensitivity to potassium sorbate; at a given concentration potassium sorbate was more inhibitory than were any of the hydroxycinnamic acids.

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