A cost-effective protocol for screening patients for methicillin-resistant Staphylococcus aureus.

The incidence of hospital-acquired infection with methicillin-resistant Staphylococcus aureus (MRSA) is rising worldwide. Rapid identification of MRSA carriers is an important step in reducing the risk of transmission to other patients. Molecular methods are increasingly popular but are technically demanding and expensive. This study assesses the modification of one of the commercially available latex agglutination tests (Mastalex-MRSA) for the identification of penicillin-binding protein 2' on known strains of MRSA as well as other organisms identified from chromogenic agar plates. A total of 3050 patients with unknown MRSA status were processed through the routine laboratory during the investigation period and 73 of these were presumptive positive following overnight incubation. Of 70 patients who could be evaluated, 32 (43.8%) specimens would be suitable for use with the kit directly from overnight incubation on chromogenic agar, and the other 38 (52.1%) would be suitable following four hours' incubation on blood agar. The cost of one positive MRSA test with the inclusion of this test is Euro 15.15 compared with published reports of Euro 35.00 for a commercial polymerase chain reaction (PCR) test. This protocol would allow the reporting of presumptive positive MRSA results approximately 24 hours earlier than currently achieved.

Mitochondrial DNA background modifies the bioenergetics of NARP/MILS ATP6 mutant cells.

Mutations in the mitochondrial DNA (mtDNA) encoded subunit 6 of ATPase (ATP6) are associated with variable disease expression, ranging from adult onset neuropathy, ataxia and retinitis pigmentosa (NARP) to fatal childhood maternally inherited Leigh's syndrome (MILS). Phenotypical variations have largely been attributed to mtDNA heteroplasmy. However, there is often a discrepancy between the levels of mutant mtDNA and disease severity. Therefore, the correlation among genetic defect, bioenergetic impairment and clinical outcome in NARP/MILS remains to be elucidated. We investigated the bioenergetics of cybrids from five patients carrying different ATP6 mutations: three harboring the T8993G, one with the T8993C and one with the T9176G mutation. The bioenergetic defects varied dramatically, not only among different ATP6 mutants, but also among lines carrying the same T8993G mutation. Mutants with the most severe ATP synthesis impairment showed defective respiration and disassembly of respiratory chain complexes. This indicates that respiratory chain defects modulate the bioenergetic impairment in NARP/MILS cells. Sequencing of the entire mtDNA from the different mutant cell lines identified variations in structural genes, resulting in amino acid changes that destabilize the respiratory chain. Taken together, these results indicate that the mtDNA background plays an important role in modulating the biochemical defects and clinical outcome in NARP/MILS.

Pyridine compounds increase thrips (Thysanoptera: Thripidae) trap capture in an onion crop.

The effect of the thrips (Thysanoptera: Thripidae) lures ethyl isonicotinate, methyl isonicotinate, and ethyl nicotinate on numbers of thrips captured in white water traps in an onion (Allium spp.) crop, in New Zealand, was examined. Female onion thrips, Thrips tabaci Lindeman (69.0%), and Thrips obscuratus Crawford (27.8%) (males and females) were the most common species found in traps in the onion crop. Ethyl isonicotinate, methyl isonicotinate, and ethyl nicotinate caught 18, 12, and 4x more onion thrips, respectively, than controls (no-lure). In contrast, traps with ethyl isonicotinate set up in a grass field at the same time as the onion crop trial caught 84x more onion thrips than traps without this lure. For T. obscuratus, traps with ethyl isonicotinate, methyl isonicotinate, and ethyl nicotinate caught 10, 13, and 12x more thrips, respectively, than controls in the onion crop.

Testing by Sysmex UF-100 flow cytometer and with bacterial culture in a diagnostic laboratory: a comparison.

A large proportion of the samples tested in routine diagnostic microbiology laboratory are urine samples. The gold standard is bacterial culture, but a high proportion of samples cultured are negative. Unnecessary testing can be reduced and an improved service provided by an effective screening test. The Sysmex UF-100 flow cytometer has been developed to count cells and casts accurately in urine samples. Its performance in a screening test was compared with bacterial culture by using 1005 consecutive urine samples, and cut-off criteria were established. Cut-off values of 3000 bacteria/microl and 111 WBC/microl provided the best discrimination. Of 1005 samples, 606 (60%) would be cultured. Sixteen samples that were not selected according to these criteria were culture positive. This was considered acceptable for our routine use. The use of a testing algorithm incorporating the Sysmex UF-100 flow cytometer has improved the quality and efficiency of urine testing within the routine microbiology laboratory.

Resistance conferred against bean common mosaic virus by the incompletely dominant I locus of Phaseolus vulgaris is active at the single cell level.

A protoplast transfection system was used in Phaseolus vulgaris to study the incompletely dominant resistance locus I. The genetic materials in the study were cultivar 'Black Turtle Soup' lines nearly isogenic for I and their F1. Accumulation of bean common mosaic virus (BCMV; genus Potyvirus) RNA and virions was assayed following BCMV RNA electrotransfection of protoplasts from each genotype. BCMV RNA and virions accumulated in all genotypes tested but the relative rates of RNA accumulation differed. This suggests that the I allele is active at the single cell level and in a dosage-dependent fashion and supports previous work in this area.

Evaluation of transgenic approaches for controlling tuber moth in potatoes.

A wide spectrum of strategies to genetically engineer potato plants resistant to potato tuber moth, Phthorimaea operculella (Zeller), have been investigated. The potato cv Iwa was transformed with a range of genes under the transcriptional control of the CaMV 35S promoter using Agrobacterium-mediated gene transfer. The transferred genes encode protease inhibitors (spleen inhibitor and alpha1-antitrypsin inhibitor), biotin-binding proteins (avidin and streptavidin) and Cry proteins (crylAc9, cry1Ba1, crylCa5 and cry9Aa2). Of these three transgenic approaches, cry genes have proved the most useful. In order to control the expression of the cry genes in foliage and not in the tubers a light-inducible Lhca3 promoter from potato was also used. The interaction of different cry genes was investigated using an experimental approach to simulate gene pyramiding in potato. Potato plants transgenic for both the crylAc9 and cryAa2 genes were developed and evaluated to help provide a more durable resistance to potato tuber moth.

Lyme disease--what is the cost for Scotland?

This paper analyses likely Lyme disease costs incurred by patients tested in the authors' laboratory over an 18 month period, based on patient histories and test results relating to 2110 samples submitted from laboratories serving 59% of the Scottish population. Cost analysis takes account of the direct costs of consultation, laboratory tests, antibiotic treatment and management of any sequelae, as well as indirect costs of the loss of healthy time through illness. Standard costs for each element are derived from published information, and the proportions applied to each patient category are estimated from studies described elsewhere in the literature. Of the sample, 295 patients had evidence of early Lyme disease and 31 had late Lyme disease symptoms. Based on these figures, the total annual cost for Lyme disease, when projected to the whole of Scotland, is estimated to be significant at 331,000 Pounds (range 47,000-615,000 Pounds). The range is inevitably wide because it was not possible to document complete clinical and management histories on individual patients. In addition, some late Lyme disease sequelae will require management for more than 1 year, and costs are also identified that could justifiably be included for all the other patients who tested negative for Lyme disease. These data raise the question of whether there is sufficient focus on prevention and the best management of this disease.

Morphoea and Borrelia burgdorferi: results from the Scottish Highlands in the context of the world literature.

AIMS: Previous studies investigating the link between infection with Borrelia burgdorferi and morphoea have produced conflicting results. Often, these studies have been undertaken in patients from different regions or countries, and using methods of varying sensitivity for detecting Borrelia burgdorferi infection. This study aimed to establish whether a relation could be demonstrated in the Highlands of Scotland, an area with endemic Lyme disease, with the use of a sensitive method for detecting the organism. METHODS: The study was performed on biopsies of lesional skin taken from 16 patients from the Highlands of Scotland with typical clinical features of morphoea. After histological confirmation of the diagnosis, a nested polymerase chain reaction (PCR) using primers to a unique conserved region of the Borrelia burgdorferi flagellin gene was performed on DNA extracts from each biopsy. A literature search was also performed for comparable studies. RESULTS: None of the 16 patients had documented clinical evidence of previous infection with B burgdorferi. DNA was successfully extracted from 14 of the 16 cases but all of these were negative using PCR for B burgdorferi specific DNA, despite successful amplification of appropriate positive controls in every test. The results were compared with those of other documented studies. CONCLUSIONS: Examination of the literature suggests that there is a strong geographical relation between B burgdorferi and morphoea. These results, in which no such association was found, indicate that morphoea may not be associated with the subspecies of B burgdorferi found in the Highlands of Scotland.

Borrelia burgdorferi-associated cutaneous marginal zone lymphoma: a clinicopathological study of two cases illustrating the temporal progression of B. burgdorferi-associated B-cell proliferation in the skin.

AIMS: A relationship between Borrelia burgdorferi and primary cutaneous B-cell lymphoma (PCBCL) has recently been confirmed following demonstration of the organism in lesional skin of patients with PCBCL. We report herein two cases of B. burgdorferi-associated PCBCL which strengthen this association by demonstrating the organism in cutaneous B-cell infiltrates present at sites in which PCBCL subsequently developed. METHODS AND RESULTS: All studies were performed on formalin-fixed paraffin-embedded tissues. These were examined by routine light microscopy and immunohistochemically by a standard streptavidin-biotin-complex technique. Genotypic studies were also undertaken using semi-nested polymerase chain reaction (PCR) for immunoglobulin heavy chain gene rearrangement, and nested PCR for B. burgdorferi flagellin gene. Both patients presented with erythematous skin lesions, biopsy of which showed dense perivascular infiltrates comprising small T-lymphocytes and collections of B-blasts. Primary cutaneous marginal zone lymphoma (MZL) developed subsequently in both cases at the same site. PCR for B. burgdorferi flagellin gene was positive in the perivascular lymphocytic infiltrates and the succeeding lymphomas in both patients. CONCLUSIONS: These results show that, at least in some instances, PCBCL arises from chronically stimulated lymphoid tissue acquired in the skin in response to B. burgdorferi infection. This may have significant therapeutic implications and warrant further studies on the extent of this association.

Recurrent myoglobinuria due to a nonsense mutation in the COX I gene of mitochondrial DNA.

OBJECTIVE: To elucidate the molecular basis of a mitochondrial myopathy associated with recurrent myoglobinuria and cytochrome c oxidase (COX) deficiency in muscle. BACKGROUND: Recurrent myoglobinuria is typically seen in patients with inborn errors of carbohydrate or lipid metabolism, the main sources of energy for muscle contraction. Relatively little attention has been directed to defects of the mitochondrial respiratory chain in patients with otherwise unexplained recurrent myoglobinuria. METHODS: Having documented COX deficiency histochemically and biochemically in the muscle biopsy from a patient with exercise-induced recurrent myoglobinuria, the authors sequenced the three mitochondrial DNA (mtDNA)-encoded COX genes, and performed restriction fragment length polymorphism analysis and single-fiber PCR. RESULTS: The authors identified a nonsense mutation (G5920A) in the COX I gene in muscle mtDNA. The mutation was heteroplasmic and abundantly present in COX-negative fibers, but less abundant or absent in COX-positive fibers; it was not found in blood or fibroblasts from the patient or in blood samples from the patient's asymptomatic mother and sister. CONCLUSIONS: The G5920A mutation caused COX deficiency in muscle, explaining the exercise intolerance and the low muscle capacity for oxidative phosphorylation documented by cycle ergometry. The sporadic occurrence of this mutation in muscle alone suggests that it arose de novo in myogenic stem cells after germ-layer differentiation. Mutations in mtDNA-encoded COX genes should be considered in patients with recurrent myoglobinuria.

Primary cutaneous B-cell lymphoma and Borrelia burgdorferi infection in patients from the Highlands of Scotland.

Although a link between primary cutaneous B-cell lymphoma (PCBCL) and Borrelia burgdorferi infection has long been suspected, previous studies have not demonstrated a significant association. The authors looked for evidence of B. burgdorferi in 20 cases of PCBCL from the Scottish Highlands, an area with endemic Lyme disease, and compared their findings with those in 40 control patients (20 undergoing wide reexcision at sites of malignant melanoma and 20 biopsies of inflammatory dermatoses). All studies were performed on formalin-fixed, paraffin-embedded tissues. The cases of PCBCL were classified according to criteria described by the European Organization for Research and Treatment of Cancer Cutaneous Lymphoma Project Group using a combination of morphology, immunohistochemistry, and seminested polymerase chain reaction (PCR) for immunoglobulin heavy chain gene rearrangement. A nested PCR was performed on deoxyribonucleic acid (DNA) extracts from the lymphoma and control cases using primers to a unique conserved region of the B. burgdorferi flagellin gene. B. burgdorferi-specific DNA was detected in seven of 20 lymphoma cases (five of 12 marginal zone lymphomas, one of five primary cutaneous follicle center cell lymphomas, one of three diffuse, large B-cell lymphomas of the leg) and in one melanoma reexcision patient of 40 control subjects. The relationship between B. burgdorferi and PCBCL was significant when compared with the control groups separately (p <0.05) or in combination (p <0.01). These results provide strong evidence to support the concept of B. burgdorferi-driven lymphomagenesis in the skin.

Identification of different Borrelia burgdorferi genomic groups from Scottish ticks.

AIM: To characterise 12 Borrelia burgdorferi sensu lato isolates cultured from ticks collected in the Highlands of Scotland. METHODS: Three molecular methods were used: an outer surface membrane protein A (OSP A) gene polymerase chain reaction (PCR) designed to give different molecular weight products with different genomic groups, randomly amplified polymorphic DNA (RAPD) analysis, and ribosomal RNA (rRNA) gene PCRs using genomic group specific primers. RESULTS: All of the molecular methods used were quick and easy to perform and capable of differentiating between the different genomic groups of B burgdorferi sensu lato. All 12 tick isolates were characterised successfully with each method: five were characterised as B afzelii and seven were characterised as B burgdorferi sensu stricto. RAPD also identified differences within these genomic groups. CONCLUSIONS: From this study, it is now known that at least two different B burgdorferi sensu lato genomic groups are present in the Highlands of Scotland: B afzelii and B burgdorferi sensu stricto. This information can now be used to develop appropriate serological tests, which should improve the diagnosis and management of patients with Lyme disease in Scotland. The molecular methods chosen were found to be useful typing tools and will allow rapid identification of any future isolates.

Cellular and molecular studies in muscle and cultures from patients with multiple mitochondrial DNA deletions.

In the last decade, several mitochondrial encephalomyopathies have been pathogenically associated with large-scale mitochondrial DNA deletions that are sporadic, or with point mutations that are maternally inherited. The mutations were also demonstrated in cultures of muscle satellite cells obtained from the patients. Subsequently, multiple deletions in mitochondrial DNA were found in several families. The affected members had progressive external ophthalmoplegia, cataracts and limb weakness, inherited as an autosomal dominant trait, or progressive external ophthalmoplegia with neurogastrointestinal encephalomyopathy or with cardiomyopathy, inherited as an autosomal recessive trait. To better understand the developmental pathobiology and localization of the multiple deletions, we performed comparative molecular genetic studies in muscle and cultures from patients. Whereas multiple deletions were found in muscle fragments from which muscle satellite cells were removed by enzymatic digestion, no deletions were found in the satellite cells or their cultured progeny. Our results suggest that multiple mitochondrial DNA deletions arise as somatic mutations during later stages of muscle development, or in terminally differentiated myofibers.

Long-term analysis of differentiation in human myoblasts repopulated with mitochondria harboring mtDNA mutations.

Short-term analysis of myogenesis in respiration-deficient myoblasts demonstrated that respiratory chain dysfunction impairs muscle differentiation. To investigate long-term consequences of a deficiency in oxidative phosphorylation on myogenesis, we quantitated myoblast fusion and expression of sarcomeric myosin in respiration-deficient myogenic cybrids. We produced viable myoblasts harboring exclusively mtDNA with large-scale deletions by treating wild-type myoblasts with rhodamine 6G and fusing them with cytoplasts homoplasmic for two different mutated mtDNAs. Recovery of growth in transmitochondrial myoblasts demonstrated that respiratory chain function is not required for recovery of rhodamine 6G-treated cells. Both transmitochondrial respiration-deficient cultures exhibited impaired myoblast fusion. Expression of sarcomeric myosin was also delayed in deficient myoblasts. However, 4 weeks after induction of differentiation, one cell line was able to quantitatively recover its capacity to form postmitotic muscle cells. This indicates that while oxidative phosphorylation is an important source of ATP for muscle development, myoblast differentiation can be supported entirely by glycolysis.

Fatal infantile cardioencephalomyopathy with COX deficiency and mutations in SCO2, a COX assembly gene.

Mammalian cytochrome c oxidase (COX) catalyses the transfer of reducing equivalents from cytochrome c to molecular oxygen and pumps protons across the inner mitochondrial membrane. Mitochondrial DNA (mtDNA) encodes three COX subunits (I-III) and nuclear DNA (nDNA) encodes ten. In addition, ancillary proteins are required for the correct assembly and function of COX (refs 2, 3, 4, 5, 6). Although pathogenic mutations in mtDNA-encoded COX subunits have been described, no mutations in the nDNA-encoded subunits have been uncovered in any mendelian-inherited COX deficiency disorder. In yeast, two related COX assembly genes, SCO1 and SCO2 (for synthesis of cytochrome c oxidase), enable subunits I and II to be incorporated into the holoprotein. Here we have identified mutations in the human homologue, SCO2, in three unrelated infants with a newly recognized fatal cardioencephalomyopathy and COX deficiency. Immunohistochemical studies implied that the enzymatic deficiency, which was most severe in cardiac and skeletal muscle, was due to the loss of mtDNA-encoded COX subunits. The clinical phenotype caused by mutations in human SCO2 differs from that caused by mutations in SURF1, the only other known COX assembly gene associated with a human disease, Leigh syndrome.

Evidence-based diagnosis of Lyme disease.

The aim of this study was to make an evidence-based comparison of four commercial enzyme immunoassays (EIAs) (Serion Classics, Sigma Diagnostics, Cambridge Biotech and ICN Diagnostics) and an in-house enzyme immunoassay (EIA) in order to select the most appropriate screening assay for diagnosis of Lyme disease. Borrelia burgdorferi sensu stricto cultured in BSK-H medium was used to develop the in-house assay. Escherichia coli antigen (0.9 mg/ml) was included in the serum diluent to reduce non-specific background. Comparison of the number of tests needed to diagnose (i.e. to indicate a positive result) and the cost per positive diagnosis for the five assays was made using a panel of 176 Western blot-characterised sera. The Cambridge Biotech and Sigma assays had the highest sensitivity but poorer specificity, whereas the Serion and ICN assays had highest specificity but poorer sensitivity. The in-house assay had average sensitivity and specificity, the number of tests needed to diagnose being 2.32 compared to 1.92 for Serion, 2.17 for ICN, 2.5 for Sigma and 2.7 for Cambridge Biotech. In a diagnostic protocol that uses an EIA as screening test, with confirmation by Western blot, a good balance of sensitivity and specificity is essential. The in-house assay was the most cost-effective (lowest cost per positive diagnosis), and is probably the best option for specialist laboratories in Europe.

Isolation of Borrelia burgdorferi from ticks in the Highlands of Scotland.

Borrelia burgdorferi, the causative agent of Lyme disease, was first isolated in 1982 and since then has been regularly isolated from ticks and clinical material in both Continental Europe and the USA. However, only three isolations have been reported in Britain. During the summer of 1997, 128 ticks were collected from two sites in the Highlands of Scotland and examined by the polymerase chain reaction (PCR) and culture. Eleven fresh isolates were obtained from culture and passed up to 22 times. Seven of the tick emulsions were also positive by flagellin gene PCR, and a further one was positive by PCR but negative on culture. All 11 isolate cultures were positive by the flagellin gene PCR. Further studies on four of these isolates confirmed their identity by immunofluorescence, but also detected possible differences between them and B. burgdorferi ACA-1 by enzyme profiles and by PCR with OspA gene primers. Culture of these new strains provides antigens that should improve diagnostic serological tests in Britain.

Improved diagnosis of reactivated toxoplasmosis.

AIMS: To identify antigens detected by western blotting in primary Toxoplasma gondii infection and determine their role in diagnosis of reactivated toxoplasmosis. METHODS: Twenty three immunocompromised patients were tested by IgG western blotting. Patients were grouped retrospectively. Group 1 comprised 15 human immunodeficiency (HIV)/AIDS patients and included: group 1A (six patients with clinical and/or serological evidence of reactivation), group 1B (five patients with clinical evidence only), and group 1C (four asymptomatic patients). Group 2 comprised eight non-HIV/AIDS immunocompromised patients with clinical and/or serological evidence of reactivation. Immunocompetent patients (n = 23) with primary toxoplasmosis were a control group used to determine the progression of the antigens detected. RESULTS: In primary toxoplasmosis, antibodies against 6, 20, 22, 23, 25, 28, 29, and 36 kDa antigens predominated. Detection of four or more of the 6, 20, 22, 23, 25, and 36 kDa antigens was considered to be western blot positive. In two group 1A patients, western blotting indicated past infection. During reactivation, this reverted to being western blot positive. Three other group 1A patients were western blot positive. In three of five group 1B patients, western blot positive results improved serological diagnosis of reactivated toxoplasmosis (p < 0.05). In two of five group 1B patients and all four group 1C patients, western blot indicated past infection. In group 2, two of eight patients reverted from a pattern of past infection to western blot positive. Five other patients from group 2 were western blot positive. CONCLUSIONS: Detection of some low molecular weight antigens is diagnostic of reactivated toxoplasmosis. These antigens can be detected even with normal dye test titres and their detection improves the diagnosis of reactivated toxoplasmosis. They might be the result of the release of bradyzoites from ruptured tissue cysts.