Biomedical subjects

M S Hughes

Publications and source records attributed to M S Hughes.

At least 19 recordsLinked to original sources

PubMed2006

Obstructive sleep apnoea syndrome in morbidly obese children with tibia vara.

Morbid obesity and its association with obstructive sleep apnoea syndrome have been increasingly recognised in children. Orthopaedic surgeons are often the primary medical contact for older children with tibia vara, which has long been associated with obesity, but are unfamiliar with the evaluation and treatment of sleep apnoea in children. We reviewed all children with tibia vara treated surgically at one of our institutions over a period of five years. Thirty-seven patients were identified; 18 were nine years of age or older and 13 of these (72%) had morbid obesity and a history of snoring. Eleven children were diagnosed as having sleep apnoea on polysomnography. The incidence of this syndrome in the 18 children aged nine years or older with tibia vara, was 61%. All these patients required pre-operative non-invasive positive-pressure ventilation; tonsillectomy and adenoidectomy were necessary in five (45%). No peri-operative complications related to the airway occurred. There is a high incidence of sleep apnoea in morbidly obese patients with tibia vara. These patients should be screened for snoring and, if present, should be further evaluated for sleep apnoea before corrective surgery is undertaken.

Adenoidectomy↗

PubMed2005

Molecular analyses of mycobacteria other than the M. tuberculosis complex isolated from Northern Ireland cattle.

Mycobacteria other than the Mycobacterium tuberculosis complex (MOTT), isolated from Northern Ireland cattle, were identified by PCR amplification of the 16S rRNA gene, and subsequent reverse cross blot hybridisation and sequence analyses. Elucidation of the MOTT species was to facilitate specificity testing of new and existing diagnostic test reagents for bovine tuberculosis. The presence of the genes for potential diagnostic antigens: MPB70, MPB64, ESAT-6 and CFP-10 in the isolated MOTT species was investigated. Molecular analyses of cultured isolates from bovine lymph node specimens of 48 cattle identified a wide variety of mycobacterial species including Mycobacterium nonchromogenicum, Mycobacterium malmoense, Mycobacterium bohemicum, Mycobacterium paratuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium holsaticum, Mycobacterium palustre, Mycobacterium sp. IWGMT 90210, Mycobacterium sp. LIV-2129, a potentially novel mycobacterial species (EMBL/GenBank/DDBJ Accession Number AJ617495) and Rhodococcus equi. Apart from M. kansasii, the results of traditional (standard phenotypic and biochemical) and molecular identification methods did not correlate well, with traditional methods identifying fewer species. Most of the species identified were either recognised pathogenic or potential pathogenic species. The genes for ESAT-6, CFP-10 and, unusually, MPB64 were detected in M. kansasii only. The MPB70 gene was not detected in any of the species. This study supported restricted species distribution of these genes as well as identifying a different range of MOTT species that could be included in specificity testing of new diagnostic reagents for bovine tuberculosis.

Animals↗

PubMed2004

Mycobacterium nonchromogenicum in nasal mucus from cattle in a herd infected with bovine tuberculosis.

Skin test negative cattle from a herd containing an unusually high proportion (194/382) of tuberculin skin test positive cattle were investigated for remaining Mycobacterium bovis infected animals. Blood samples from the skin test negative cattle, analysed by an antibody ELISA and an interferon-gamma assay, were mostly test negative for M. bovis. Radiometric culture of nasal mucus samples from 48 of the cattle yielded 22 culture positives with acid-fast bacilli and cording in 6 of these. Subculture on solid media was successful for 7, including 2 with cording of the 22 radiometric culture positives. Mycobacterium tuberculosis complex DNA probe testing using the Accuprobe (Gen-Probe, Inc.) and M. tuberculosis complex-specific PCR amplification, performed on the solid media subcultures, were negative. 16S rRNA PCR and sequence analysis were successful for 6 of the 7 solid media subcultures obtained and revealed the presence of Mycobacterium nonchromogenicum in all 6 subcultures. This is the first report of M. nonchromogenicum in nasal mucus of cattle. The observation highlights the importance of integrating definitive tests such as the PCR for diagnosis of bovine tuberculosis and indicates a possible zoonotic risk.

Animals↗

PubMed2004

PCR studies of feline leprosy cases.

16S rRNA gene sequence analysis provided evidence for two different mycobacterial species, Mycobacterium lepraemurium and a potentially novel species, as causative agents of 'feline leprosy'. Comparison of 16S rRNA gene sequence data obtained for M. lepraemurium and the potentially novel species indicated 12 nucleotide differences over a 446 bp region encompassing the V2 and V3 hypervariable regions. From available 16S rRNA gene sequence data, M. lepraemurium shared greatest nucleotide identity with M. avium subsp paratuberculosis and M. avium. The novel species had a long helix 18 in the V3 region and shared greatest nucleotide identity with M. leprae, M. haemophilum and M. malmoense. The novel species had an additional 'A' nucleotide at position 105 of the aligned 16S rRNA gene sequence, the only other mycobacterial database sequence having this same extra nucleotide being M. leprae. This nucleotide variation was exploited to develop specific PCR assays for the two species. These were found to be effective and specific when tested against a panel of mycobacteria including species found in feline leprosy lesions and closely related mycobacteria and also when applied directly to formalin-fixed, paraffin-embedded tissues from feline leprosy cases.

Animals↗

PubMed2003

Heterotopic ossification prophylaxis with indomethacin increases the risk of long-bone nonunion.

Indomethacin is commonly administered for the prophylaxis of heterotopic ossification (HO) after the surgical treatment of acetabular fractures. Non-steroidal anti-inflammatory drugs such as indomethacin, have been associated with delayed healing of fractures and mechanically weaker callus. Our aim was to determine if patients with an acetabular fracture, who received indomethacin for prophylaxis against HO, were at risk of delayed healing or nonunion of any associated fractures of long bones. We reviewed 282 patients who had had open reduction and internal fixation of an acetabular fracture. Patients at risk of HO were randomised to receive either radiation therapy (XRT) or indomethacin. Of these patients, 112 had sustained at least one concomitant fracture of a long bone; 36 needed no prophylaxis, 38 received focal radiation and 38 received indomethacin. Fifteen patients developed 16 nonunions. When comparing patients who received indomethacin with those who did not, a significant difference was noted in the rate of nonunion (26% v 7%; p = 0.004). Patients with concurrent fractures of the acetabulum and long bones who receive indomethacin have a significantly greater risk of nonunion of the fractures of the long bones when compared with those who receive XRT or no prophylaxis.

Acetabulum↗

PubMed2002

Feline leprosy: two different clinical syndromes.

Feline leprosy refers to a condition in which cats develop granulomas of the subcutis and skin in association with intracellular acid-fast bacilli that do not grow on routine laboratory media. In this study, the definition was extended to include cases not cultured, but in which the polymerase chain reaction (PCR) identified amplicons characteristic of mycobacteria. Tissue specimens from 13 such cases from eastern Australia were obtained between 1988 and 2000. This cohort of cats could be divided into two groups on the basis of the patients' age, histology of lesions, clinical course and the sequence of 16S rRNA PCR amplicons. One group consisted of four young cats (less than 4 years) which initially developed localised nodular disease affecting the limbs. Lesions progressed rapidly and sometimes ulcerated. Sparse to moderate numbers of acid-fast bacilli were identified using cytology and/or histology, typically in areas of caseous necrosis and surrounded by pyogranulomatous inflammation. Organisms did not stain with haematoxylin and ranged from 2 to 6 microm (usually 2 to 4 microm). Mycobacterium lepraemurium was diagnosed in two cases based on the sequence of a 446 bp fragment encompassing the V2 and V3 hypervariable regions of the 16S rRNA gene a different sequence was obtained from one additional case, while no PCR product could be obtained from the remaining case. The clinical course was considered aggressive, with a tendency towards local spread, recurrence following surgery and development of widespread lesions over several weeks. The cats resided in suburban or rural environments. A second group consisted of nine old cats (greater than 9 years) with generalised skin involvement, multibacillary histology and a slowly progressive clinical course. Seven cats initially had localised disease which subsequently became widespread, while two cats allegedly had generalised disease from the outset. Disease progression was protracted (compared to the first group of cats), typically taking months to years, and skin nodules did not ulcerate. Microscopically, lesions consisted of sheets of epithelioid cells containing large to enormous numbers of acid-fast bacilli 2 to 8 microm (mostly 4 to 6 microm) which stained also with haematoxylin. A single unique sequence spanning a 557 bp fragment of the 16S rRNA gene was identified in six of seven cases in which it was attempted. Formalin-fixed paraffin-embedded material was utilised by one laboratory, while fresh tissue was used in another. The same unique sequence was identified despite the use of different primers and PCR methodologies in the two laboratories. A very slow, pure growth of a mycobacteria species was observed on Lowenstein-Jensen medium (supplemented with iron) and semi-solid agar in one of three cases in which culture was attempted at a reference laboratory. Affected cats were domicile in rural or semi-rural environments. These infections could generally be cured using two or three of rifampicin (10-15 mg/kg once a day), clofazimine (25 to 50 mg once a day or 50 mg every other day) and clarithromycin (62.5 mg per cat every 12 h). These findings suggest that feline leprosy comprises two different clinical syndromes, one tending to occur in young cats and caused typically by M lepraemurium and another in old cats caused by a single novel mycobacterial species.

Animals↗

PubMed2001

Treatment of canine leproid granuloma syndrome: preliminary findings in seven dogs.

OBJECTIVE: To determine effective treatment strategies for patients with refractory canine leproid granuloma syndrome. DESIGN: Multi-institutional retrospective/prospective case series using client-owned dogs. PROCEDURE: Seven dogs (four Boxers, one Dobermann, one Bullmastiff and one Bullmastiff cross-bred; ages 3 to 11 years) with leproid granulomas were treated successfully using a variety of treatment regimens. These cases were recruited because: lesions were either widely distributed over the dog; progressive, despite routine therapy, or were associated with particularly disfiguring lesions. The treatment regimen evolved during the course of the clinical study. RESULTS: Combination therapy using rifampicin (5 to 15 mg/kg p.o., every 24 h) and clarithromycin (8 to 24 mg/kg p.o. daily; dose divided every 8 or every 12 h) was used most frequently and proved to be effective and free from side effects. Total daily doses of clarithromycin in excess of 14 mg/kg were considered optimal and long treatment courses, in the order of 1 to 3 months, were used. Combination therapy using rifampicin (25 mg/kg; that is, higher than the recommended dose) and clofazimine was effective in one case, but resulted in hepatotoxicity. A topical formulation of clofazimine in petroleum jelly was used as an adjunct to oral rifampicin and doxycycline in another patient treated successfully. CONCLUSION: Based on our evolving clinical experience, a combination of rifampicin (10 to 15 mg/kg p.o., every 24 h) and clarithromycin (15 to 25 mg/kg p.o. total daily dose; given divided every 8 to 12 h) is currently recommended for treating severe or refractory cases of canine leproid granuloma syndrome. Treatment should be continued (typically for 4 to 8 weeks) until lesions are substantially reduced in size and ideally until lesions have resolved completely. A topical formulation, containing clofazimine in petroleum jelly may be used as an adjunct to systemic drug therapy. Further work is required to determine the most cost effective treatment regimen for this condition.

Animals↗

PubMed2001

Detection of Mycobacterium bovis in bovine clinical specimens using real-time fluorescence and fluorescence resonance energy transfer probe rapid-cycle PCR.

Nucleic acid sequence capture extraction was coupled with LightCycler PCR amplification and product detection using real-time fluorescence for rapid, definitive detection of Mycobacterium bovis in lymph node specimens from 38 cattle with bovine tuberculosis lesions. PCR amplification of sequence-captured DNA using both a conventional heating block thermocycler and a LightCycler thermocycler was compared with culture and histopathological analyses. Conventional PCR enabled detection of 26 of 28 culture-positive specimens (93%) in approximately 9 h, and the LightCycler PCR detected 20 of 28 culture-positive specimens (71%) in only 30 min. Specific confirmation of Mycobacterium tuberculosis complex DNA was achieved by LightCycler PCR amplification using Syb Green 1 and an M. tuberculosis complex-specific Cy5-labeled fluorescence resonance energy transfer probe. The system described here enabled rapid and specific laboratory confirmation of bovine tuberculosis, and this is the first report of the detection of M. bovis in tissues using LightCycler PCR. The fluorescence technology used in the study has potential to allow development of a high-throughput molecular diagnostic test for bovine tuberculosis.

Animals↗

PubMed2000

Simultaneous detection and strain differentiation of Mycobacterium bovis directly from bovine tissue specimens by spoligotyping.

Culture of Mycobacterium bovis is used routinely to support field diagnosis of bovine tuberculosis; however, this method is slow. Rapid detection and strain-typing of M. bovis directly from 37 lesioned bovine lymph node specimens was performed by the polymerase chain reaction (PCR) based method, spoligotyping. Mycobacterial DNA was extracted from the specimens using a nucleic acid sequence capture technique. Two sets of specimens were tested, the first set comprising 16 decontaminated tissue homogenates from lesioned lymph node specimens which had been processed for BACTEC culture and a second set of 21 non-decontaminated lesioned lymph node specimens. Both sets of specimens had been frozen before analysis. Sequence capture PCR enabled detection and strain-typing of M. bovis directly from 15 of the 16 decontaminated homogenates and all 21 of the non-decontaminated tissues. Four spoligotype (ST) patterns were obtained from each set; ST1, ST2, ST3 and ST16 were detected in the decontaminated specimens and ST1, ST2, ST11 and ST14 in the non-decontaminated specimens. For both sets of specimens, ST1 was the predominant strain type detected. ST patterns obtained from the BACTEC cultures of the decontaminated specimens were in agreement with those obtained directly from the tissue. The sensitivity of detection by sequence capture-PCR compared very favourably with that of BACTEC culture. ST patterns were obtained directly from tissues of 34 of the 35 culture positive specimens and the two culture negative specimens. DNA extraction from the 21 non-decontaminated specimens involved an initial stomaching treatment. An assessment of sequence capture on both liquid alone and liquid and tissue homogenate combined, following stomaching, indicated that PCR was less successful on the liquid component alone.

Animals↗

PubMed2000

Mycobacterium genavense infection in two aged ferrets with conjunctival lesions.

Mycobacterium genavense infection was diagnosed in two adult ferrets. Disseminated mycobacteriosis was diagnosed in a castrated 5-year-old sable ferret with generalised peripheral lymph node enlargement and a proliferative lesion of the conjunctiva of the nictitating membrane. The diagnosis was based on characteristic cytology and sequence analysis of the 16S rRNA gene amplified using the polymerase chain reaction from fresh biopsy material. Therapy with rifampicin, clofazimine and clarithromycin probably cured the infection. An entire 4-year-old female ferret with conjunctival swelling, serous ocular discharge and swelling of the subcutaneous tissues of the nasal bridge was diagnosed as having M genavense infection on the basis of typical cytology, histopathology and sequence analysis of 16S rRNA amplicons from formalin-fixed paraffin-embedded tissue. This patient was treated successfully using rifampicin. Both ferrets subsequently died as a result of other disease conditions, 10 and 4 months following initiation of therapy, respectively. This is the first report documenting M genavense as a cause of disseminated mycobacterial disease in ferrets. Conjunctival involvement may be a feature of disseminated mycobacteriosis in the ferret. The possibility that these infections were the consequence of a ferret retrovirus infection should be considered further.

Animals↗

PubMed2000

Broadband time-domain reflectometry measurement of attenuation and phase velocity in highly attenuating suspensions with application to the ultrasound contrast medium Albunex.

We describe a technique for broadband measurements of the attenuation coefficient and phase velocity of highly attenuating liquid suspensions. To validate the technique we apply it to the ultrasound contrast agent Albunex at concentrations ranging from 0.69 x 10(6) particles/mL to 364 x 10(6) particles/mL. These longitudinal wave measurements were performed on Albunex suspensions maintained at 37 degrees C in a special time-domain reflectometer designed and constructed in our laboratory. The frequency-dependent attenuation coefficients and phase velocities obtained in the reflectometer are compared to broadband through-transmission measurements of these same quantities, which were also performed in our laboratory. Although comparison data between the two techniques are only available at lower concentrations, the agreement is quite good and serves to validate the methods described in this paper.

Albumins↗

PubMed2000

Identification by 16S rRNA gene analyses of a potential novel mycobacterial species as an etiological agent of canine leproid granuloma syndrome.

PCR amplifications of the 16S rRNA gene were performed on 46 specimens obtained from 43 dogs with canine leproid granuloma syndrome to help determine its etiology. Sequence capture PCR was applied to 37 paraffin-embedded specimens from 37 dogs, and nested PCR was attempted on DNA from 9 fresh tissue specimens derived from 3 of the 37 aforementioned dogs and from an additional 6 dogs. Molecular analyses of the paraffin-embedded tissues and fresh tissue specimen analyses were performed at separate institutions. PCR products with identical sequences over a 350-bp region encompassing variable regions 2 and 3 of the 16S rRNA gene were obtained from 4 of 37 paraffin-embedded specimens and from all 9 specimens of fresh tissue originating from 12 of the 43 dogs. Identical sequences were determined from amplicons obtained from paraffin-embedded and fresh specimens from one dog. The consensus DNA sequence, amplified from paraffin-embedded tissue and represented by GenBank accession no. AF144747, shared highest nucleotide identity (99.4% over 519 bp) with mycobacterial strain IWGMT 90413 but did not correspond exactly to any EMBL or GenBank database sequence. With a probe derived from the V2 region of the novel canine sequence, reverse cross blot hybridization identified an additional four paraffin-embedded specimens containing the same novel sequence. In total, molecular methodologies identified the proposed novel mycobacterial sequence in 16 of 43 dogs with canine leproid granuloma syndrome, indicating that the species represented by this sequence may be the principal etiological agent of canine leproid granuloma syndrome.

Animals↗

PubMed1999

Targeting and ultrasound imaging of microbubble-based contrast agents.

Preparation and characterization of targeted microbubbles (ultrasound contrast agents) is described. Specific ligands were attached to the microbubble shell, and ligand-coated microbubbles were selectively attached to various targets, using either an avidin biotin model system or an antigen-antibody system for targeting to live activated endothelial cells. Firm attachment of microbubbles to the target was achieved. Forces necessary to detach microbubbles from the target were estimated to exceed dozens of pN. Microbubbles were bound to the target even in the rapidly moving stream of the aqueous medium. Down to 20 ng of the ultrasound contrast material on the target surface could be detected by the ultrasound imaging with a commercial medical imaging system. At high bubble density on the target surface, strong ultrasound image attenuation was observed.

Antigen-Antibody Reactions↗

PubMed1999

Broadband measurement of the scattering-to-attenuation ratio for Albunex at 37 degrees C.

The attenuation coefficient and backscatter coefficient of Albunex were measured over a wide range of concentrations and frequencies (at a temperature of 37 degrees C), and were used to calculate the scattering-to-attenuation ratio (STAR) value. Each of these quantities exhibited concentration dependence in agreement with predictions from simple scattering theory: the backscatter coefficient grew linearly with concentration, the power of the transmitted signal decreased exponentially with concentration due to attenuation, and the STAR was independent of concentration scaling. Because of the markedly differing concentration dependence, it is necessary to consider all of these quantities (not just the STAR value alone) when evaluating and comparing the potential efficacy of ultrasonic contrast agents.

Albumins↗

PubMed1999

Disseminated Mycobacterium genavense infection in a FIV-positive cat.

An 8-year-old FIV-positive Australian cat was presented with coughing, periocular alopecia, pyrexia and inappetence. Skin scrapings demonstrated Demodex cati mites. Antibiotics were administered and it was treated successfully for periocular demodectic mange, but the cat continued to exhibit respiratory signs and lose weight. Further investigation revealed an ascarid infection and active chronic inflammation of undetected cause affecting the lower airways. Repetitive treatment with pyrantel failed to eradicate the ascarid infection. The cat became cachectic and developed moist ulcerative dermatitis of the neck, severe non-regenerative anaemia, leucopenia and thrombocytopenia. Necropsy and histopathology revealed mycobacteriosis affecting skin, lungs, spleen, lymph nodes, liver and kidney. Attempted culture of frozen tissues at a mycobacteria reference laboratory was unsuccessful. Paraffin-embedded, formalin-fixed tissue was retrieved and examined using PCR to amplify part of the 16S rRNA gene. A diagnosis of disseminated Mycobacterium genavense infection was made based on the presence of acid fast bacteria in many tissues and partial sequence of the 16S rRNA gene. Although M genavense has been identified previously as a cause of disseminated disease in AIDS patients, this is the first report of infection in a cat. It was suspected that the demodecosis, recurrent ascarid infections and disseminated M genavense infection resulted from an immune deficiency syndrome consequent to longstanding FIV infection.

Animals↗

PubMed1999

Effects of transducer frequency and output power on the ultrasonographic contrast produced by Optison using fundamental and harmonic imaging techniques.

This in vivo study demonstrated the effects of transducer frequency and output power on the sonographic contrast produced by Optison during continuous ultrasonographic imaging in both fundamental and harmonic modes. During fundamental imaging, Optison produced greater contrast intensity while imaging at higher transducer frequencies. However, in the harmonic mode, lower imaging frequencies caused Optison to produce more intense myocardial enhancement. Regardless of imaging mode, increases in transducer output power caused decreases in both contrast intensity and contrast duration produced by Optison. Proper selection of transducer frequency and output power will help optimize the quality and duration of contrast produced by Optison during continuous ultrasonographic imaging.

Albumins↗

PubMed1998

Rapid diagnosis and strain differentiation of Mycobacterium bovis in radiometric culture by spoligotyping.

An assessment of spoligotyping for rapid detection and strain typing of Mycobacterium bovis isolates in radiometric culture was made. Spoligotyping was applied to BACTEC 12B broth cultures of 54 lesioned bovine lymph node specimens from 44 herds in Northern Ireland. A nucleic acid sequence capture technique was performed on BACTEC cultures at growth index points of approximately (approximately) 60, approximately 200, and 999. Definitive spoligotype patterns were obtained for 90.4% and 94.2% of all 52 BACTEC culture-positives at growth indexes approximately 60 and approximately 200, respectively. Within 10 days, definitive spoligotype patterns were obtained for 84.6% of the culture-positives. This technique, therefore, allowed earlier and more accurate diagnosis of M. bovis than traditional methodologies, as well as simultaneous strain differentiation. Application of this molecular tool to BACTEC cultures would be a significant advance in bovine tuberculosis eradication programmes. Seven distinct spoligotype patterns were identified in this study, 2 of which (ST21 and ST25), had not been identified previously in cattle from Northern Ireland. Two spoligotype patterns (ST1 and ST2) accounted for 80.7% of the culture-positives. These were found to have widespread geographic distribution, whereas 1 spoligotype pattern (ST14) had limited geographical distribution.

Animals↗

PubMed1998

Spacer oligotyping of Mycobacterium bovis isolates compared to typing by restriction fragment length polymorphism using PGRS, DR and IS6110 probes.

Ninety-two Mycobacterium bovis isolates from cattle, deer and badgers in Northern Ireland and the Republic of Ireland were genotyped by spacer-oligotyping (spoligotyping) and 67 of these were analysed by restriction fragment length polymorphism (RFLP). RFLP analysis was performed using three DNA probes, PGRS, DR and IS6110. Forty-seven of the M. bovis isolates were from 45 different sources; these were typed using both RFLP and spoligotyping. These 47 isolates could be differentiated into 24 different RFLP types and 15 distinct spoligotypes. Although RFLP was found to be more discriminatory compared to the present spoligotyping technique, spoligotyping was able to differentiate 21 RFLP type 'ACA' isolates into three different patterns. The remaining 45 M. bovis isolates were from a small case study, involving infected cattle, deer and badgers from the same geographic region. All these isolates were analysed by spoligotyping and a selection of 20 isolates were RFLP typed. All the isolates in the case study had the same spoligotype pattern with the exception of one cervine isolate. Similarly all the isolates typed by RFLP had the same pattern. Consequently, the predominant strain in the case study was not host restricted. The consistency between the results obtained using the two techniques indicates the potential value of both techniques for epidemiological studies. Spoligotyping was found to be a much more rapid technique and easier to perform, requiring less sophisticated computer software for strain typing. Spoligotyping results were more readily documented and analysed and the technique was also more suitable than RFLP analysis for large-scale screening studies.

Animals↗