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Biomedical subjects

T D McHugh

Publications and source records attributed to T D McHugh.

At least 19 recordsLinked to original sources
PubMed2006

Prevalences of Pneumocystis jiroveci, Mycobacterium tuberculosis and Streptococcus pneumoniae infection in children with severe pneumonia, in a tertiary referral hospital in northern Tanzania.

At the Kilimanjaro Christian Medical Centre, a tertiary referral hospital in northern Tanzania, both the number of paediatric cases of lower respiratory-tract infection (LRTI) and the associated mortality increased between 2000 and 2001. Molecular diagnostic tools were used to enhance the identification of the pathogens responsible for this perceived increase. All 72 children aged between 2 and 60 months who were admitted with LRTI over a 3-month period were enrolled in the study. Induced sputum was collected from each child and, if the parents consented, the subjects were also tested for HIV. The sputum samples were each checked for bacteria by culture and, in amplification assays, for the DNA of Pneumocystis jiroveci, Mycobacterium tuberculosis and Streptococcus pneumoniae. Twenty-two (50%) of the 44 children tested for HIV had HIV-1 antibodies. Although only two children, both aged <6 months, were found PCR-positive for P. jiroveci, and only one was found positive for M. tuberculosis, 46 (including one of those found positive for P. jiroveci and the child found positive for M. tuberculosis) were found PCR-positive for S. pneumoniae. It therefore appears that most paediatric cases of LRTI who present at the hospital are attributable to S. pneumoniae, and that infections with this pathogen are entirely responsible for the observed increase in the incidence of LTRI in the local children. The increase seen in LRTI-associated mortality among the children may be the result of pneumococcal antibiotic resistance.

Anti-Bacterial Agents↗
PubMed2005

Validation and clinical application of molecular methods for the identification of molds in tissue.

BACKGROUND: Invasive fungal infections due to less-common molds are an increasing problem, and accurate diagnosis is difficult. METHODS: We used our previously established molecular method, which allows species identification of molds in histological tissue sections, to test sequential specimens from 56 patients with invasive fungal infections who were treated at our institution from 1982 to 2000. RESULTS: The validity of the method was demonstrated with the establishment of a molecular diagnosis in 52 cases (93%). Confirmation of the causative organism was made in all cases in which a mold had been cultured from the tissue specimen. Less-common molds were identified in 7% of cases and appear to be an increasing problem. CONCLUSIONS: Our previously established method has proven to be of value in determining the incidence of invasive infection caused by less-common molds. Institutions should continue to pursue diagnosis of invasive fungal infections by means of tissue culture and microbiologic analysis.

Aspergillosis↗
PubMed2005

Aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (LRTI) in primary care.

BACKGROUND: Lower respiratory tract infections (LRTI) are a common reason for consulting general practitioners (GPs). In most cases the aetiology is unknown, yet most result in an antibiotic prescription. The aetiology of LRTI was investigated in a prospective controlled study. METHODS: Eighty adults presenting to GPs with acute LRTI were recruited together with 49 controls over 12 months. Throat swabs, nasal aspirates (patients and controls), and sputum (patients) were obtained and polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR) assays were used to detect Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, influenza viruses (AH1, AH3 and B), parainfluenza viruses 1-3, coronaviruses, respiratory syncytial virus, adenoviruses, rhinoviruses, and enteroviruses. Standard sputum bacteriology was also performed. Outcome was recorded at a follow up visit. RESULTS: Potential pathogens were identified in 55 patients with LRTI (69%) and seven controls (14%; p<0.0001). The identification rate was 63% (viruses) and 26% (bacteria) for patients and 12% (p<0.0001) and 6% (p = 0.013), respectively, for controls. The most common organisms identified in the patients were rhinoviruses (33%), influenza viruses (24%), and Streptococcus pneumoniae (19%) compared with 2% (p<0.001), 6% (p = 0.013), and 4% (p = 0.034), respectively, in controls. Multiple pathogens were identified in 18 of the 80 LRTI patients (22.5%) and in two of the 49 controls (4%; p = 0.011). Atypical organisms were rarely identified. Cases with bacterial aetiology were clinically indistinguishable from those with viral aetiology. CONCLUSION: Patients presenting to GPs with acute adult LRTI predominantly have a viral illness which is most commonly caused by rhinoviruses and influenza viruses.

Acute Disease↗
PubMed2005

Mycobacterium tuberculosis lineage: a naming of the parts.

There have been many reports of groups of related Mycobacterium tuberculosis strains described variously as lineages, families or clades. There is no objective definition of these groupings, making it impossible to define relationships between those groups with biological advantages. Here we describe two groups of related strains obtained from an epidemiological study in Tanzania, which we define as the Kilimanjaro and Meru lineages on the basis of IS6110 restriction fragment length polymorphism (RFLP), polymorphic GC rich sequence (PGRS) RFLP and mycobacterial interspersed repeat unit (MIRU) typing. We investigated the concordance between each of the typing techniques and the dispersal of the typing profiles from a core pattern. The Meru lineage is more dispersed than the Kilimanjaro lineage and we speculate that the Meru lineage is older. We suggest that this approach provides an objective definition that proves robust in this epidemiological study. Such a framework will permit associations between a lineage and clinical or bacterial phenomenon to be tested objectively. This definition will also enable new putative lineages to be objectively tested.

AIDS-Related Opportunistic Infections↗
PubMed2004

Prospective evaluation of BDProbeTec strand displacement amplification (SDA) system for diagnosis of tuberculosis in non-respiratory and respiratory samples.

Nucleic acid amplification techniques (NAATs) have been demonstrated to make significant improvements in the diagnosis of tuberculosis (TB), particularly in the time to diagnosis and the diagnosis of smear-negative TB. The BD ProbeTec strand displacement amplification (SDA) system for the diagnosis of pulmonary and non-pulmonary tuberculosis was evaluated. A total of 689 samples were analysed from patients with clinically suspected TB. Compared with culture, the sensitivity and specificity for pulmonary samples were 98 and 89 %, and against final clinical diagnosis 93 and 92 %, respectively. This assay has undergone limited evaluation for non-respiratory samples and so 331 non-respiratory samples were tested, identifying those specimens that were likely to yield a useful result. These were CSF (n = 104), fine needle aspirates (n = 64) and pus (n = 41). Pleural fluid (n = 47) was identified as a poor specimen. A concern in using the SDA assay was that low-positive samples were difficult to interpret; 7.8 % of specimens fell into this category. Indeed, 64 % of the discrepant results, when compared to final clinical diagnosis, could be assigned as low-positive samples. Specimen type did not predict likelihood of a sample being in the low-positive zone. Although the manufacturers do not describe the concept of a low-positive zone, we have found that it aids clinical diagnosis.

Ascitic Fluid↗
PubMed2003

Molecular methods for Mycobacterium tuberculosis strain typing: a users guide.

There are now a wide range of techniques available to type Mycobacterium tuberculosis, the problem is to choose the correct technique. For large scale epidemiological studies the portability and standardization of IS6110 restriction fragment length polymorphism (RFLP) means that this remains the gold standard technique. In the next few years the internationally standard mycobacterial interspersed repetitive unit (MIRU) may come to challenge this primacy. Low copy number stains remain a problem and these can be typed by either polymorphic Guanine cytosine-rich repetitive sequence (PGRS) or MIRU-variable numbers of tandem repeat (VNTR). To confirm whether strains are part of a true cluster PGRS remains the method of choice. For local outbreaks and investigations of laboratory cross contamination where speed is of greatest importance suspect strains should be initially investigated using a PCR-based method. The superior reproducibility and discrimination of MIRU-VNTR means that these methods should be favoured. If matches are found, then further confirmation of identity can be achieved using IS6110 RFLP or PGRS if the strains prove to have a low IS6110 copy number.

Bacterial Typing Techniques↗
PubMed2002

Estimation of the rate of unrecognized cross-contamination with mycobacterium tuberculosis in London microbiology laboratories.

Isolates from patients with confirmed tuberculosis from London were collected over 2.5 years between 1995 and 1997. Restriction fragment length polymorphism (RFLP) analysis was performed by the international standard technique as part of a multicenter epidemiological study. A total of 2,779 samples representing 2,500 individual patients from 56 laboratories were examined. Analysis of these samples revealed a laboratory cross-contamination rate of between 0.54%, when only presumed cases of cross-contamination were considered, and 0.93%, when presumed and possible cases were counted. Previous studies suggest an extremely wide range of laboratory cross-contamination rates of between 0.1 and 65%. These data indicate that laboratory cross-contamination has not been a common problem in routine practice in the London area, but in several incidents patients did receive full courses of therapy that were probably unnecessary.

Bacterial Typing Techniques↗
PubMed2002

Molecular epidemiology of tuberculosis in London 1995-7 showing low rate of active transmission.

BACKGROUND: Tuberculosis notification rates for London have risen dramatically in recent years. Molecular typing of Mycobacterium tuberculosis has contributed to our understanding of the epidemiology of tuberculosis throughout the world. This study aimed to assess the degree of recent transmission of M tuberculosis in London and subpopulations of the community with high rates of recent transmission. METHODS: M tuberculosis isolates from all persons from Greater London diagnosed with culture positive tuberculosis between 1 July 1995 and 31 December 1997 were genetically fingerprinted using IS6110 restriction fragment length polymorphism (RFLP) typing. A structured proforma was used during record review of cases of culture confirmed tuberculosis. Cluster analysis was performed and risk factors for clustering were examined in a univariate analysis followed by a logistic regression analysis with membership of a cluster as the outcome variable. RESULTS: RFLP patterns were obtained for 2042 isolates with more than four copies of IS6110; 463 (22.7%) belonged to 169 molecular clusters, which ranged in size from two (65% of clusters) to 12 persons. The estimated rate of recent transmission was 14.4%. Young age (0-19 years) (odds ratio (OR) 2.65, 95% confidence interval (CI) 1.59 to 4.44), birth in the UK (OR 1.55, 95% CI 1.04 to 2.03), black Caribbean ethnic group (OR 2.19, 95% CI 1.15 to 4.16), alcohol dependence (OR 2.33, 95% CI 1.46 to 3.72), and streptomycin resistance (OR 1.82, 95% CI 1.15 to 2.88) were independently associated with an increased risk of clustering. CONCLUSIONS: Tuberculosis in London is largely caused by reactivation or importation of infection by recent immigrants. Newly acquired infection is also common among people with recognised risk factors. Preventative interventions and early diagnosis of immigrants from areas with a high incidence of tuberculosis, together with thorough contact tracing and monitoring of treatment outcome among all cases of tuberculosis (especially in groups at higher risk of recent infection), remains most important.

Adolescent↗
PubMed2000

Comparison of fitness of two isolates of Mycobacterium tuberculosis, one of which had developed multi-drug resistance during the course of treatment.

OBJECTIVES: We report the cases of two patients, brother and sister, both with pulmonary tuberculosis. Both patients complied poorly with treatment. One developed multi-drug resistant disease, whilst the other did not. We aimed to show that the two infecting strains were the same, and then to compare the fitness of the resistant strain to that of the sensitive strain. METHODS: The isolates were typed by RFLP. The fitness of the multi-drug resistant tuberculosis strain was determined by calculating the ratio of generation produced by the drug-resistant and a drug-susceptible strain in a mixed culture. The number of bacteria present in this broth culture was estimated using the Miles and Misra technique. The number of drug-resistant bacteria present was determined by inoculating aliquots of broth onto Middlebrook 7H10 agar with 5mg/l rifampicin. RESULTS: The infecting strain of Mycobacterium tuberculosis was shown to be the same on RFLP typing in both cases. It was found that the multi-drug resistant organism had decreased fitness compared to the sensitive organism. CONCLUSION: The decreased relative fitness of the resistant strain implies a physiologic cal cost for the development of drug resistance.

Antitubercular Agents↗
PubMed2000

Comparison of phenotypic and genotypic methods for pyrazinamide susceptibility testing with Mycobacterium tuberculosis.

Mycobacterium tuberculosis converts pyrazinamide to its active form by using the enzyme pyrazinamidase. This enzyme is coded for on the pncA gene, and mutations in the pncA gene result in absence of active enzyme, conferring resistance to the drug pyrazinamide. We investigated 27 strains of Mycobacterium tuberculosis suspected of being multidrug resistant. Each isolate was tested for susceptibility to pyrazinamide by the BACTEC 460TB method, and 19 were pyrazinamide resistant. The presence of active pyrazinamidase enzyme was sought by using the Wayne assay, which was positive in all of the sensitive isolates and four of the resistant isolates. The pncA gene was amplified by PCR, and mutations were sought by single-strand conformation polymorphism (SSCP) analysis. We identified four isolates which were phenotypically resistant to pyrazinamide, but which had active pyrazinamide enzyme on the Wayne assay and normal pncA gene SSCP. MICs measured by BACTEC 460TB and susceptibility testing at a lower pH of 5.5 confirmed genuine resistance. The pncA gene was sequenced in these four isolates and found not to have any mutations. This implies that an alternative mechanism of resistance exists in these strains. We conclude that genotypic assessment of pyrazinamide resistance is unreliable, because it depends on the identification of a single resistance mechanism. Phenotypic methods such as the BACTEC 460TB technique remain the best methods for pyrazinamide susceptibility testing.

Amidohydrolases↗
PubMed2000

False molecular clusters due to nonrandom association of IS6110 with Mycobacterium tuberculosis.

We sought to determine whether nonrandom association of IS6110 with Mycobacterium tuberculosis could result in false-positive clustering in unselected collections of isolates. We typed 196 strains of M. tuberculosis from an unselected community-based study in northern Tanzania by IS6110 and polymorphic GC-rich repetitive-sequence (PGRS) methodologies. The strains were analyzed by Gelcompar computer software. Analysis of 13 out of 25 groups showed that isolates with identical IS6110 and PGRS patterns were likely to be the same strain. Some IS6110 groups containing strains with identical PGRS patterns had similar IS6110 patterns that differed only by movement of the element. Isolates assigned to a single group (i.e., group 11) on the basis of sharing an identical IS6110 fingerprint pattern did not share identical PGRS fingerprint patterns. Six out of the nine bands in these isolates were in hot-spot locations, as previously defined. This indicates that nonrandom association may result in false-positive clustering in unselected community-based studies. Only strains with identical PGRS and IS6110 patterns are likely to be recently transmitted.

Bacterial Typing Techniques↗
PubMed1999

Physiological cost of rifampin resistance induced in vitro in Mycobacterium tuberculosis.

Drug-resistant Mycobacterium tuberculosis is a major threat to public health. In clinical practice, a limited number of resistance mutations in a short sequence of the beta subunit of RNA polymerase (encoded by rpoB) have been described. Spontaneous resistance to rifampin was induced in vitro in M. tuberculosis H37Rv (ATCC 9360). Only three resistance patterns could be detected by PCR-single-strand conformation polymorphism analysis. Sequence analysis revealed that Ser(531)-->Leu arose most frequently, followed by His(526)-->Arg and then either His(526)-->Tyr or His(526)-->Asp. The relative Darwinian fitness of all but one of the mutant genotypes was less than that of the susceptible parent and, for these mutations, there was a significant correlation between fitness and clinical isolation rate (regression analysis P = 0.026). The fitness deficit in some mutants was small, suggesting that there is little likelihood of a spontaneous reversion to susceptibility.

Antibiotics, Antitubercular↗
PubMed1998

Nonrandom association of IS6110 and Mycobacterium tuberculosis: implications for molecular epidemiological studies.

IS6110 restriction fragment length polymorphism typing is now established as the primary typing method for Mycobacterium tuberculosis. It has been assumed that the position of bands is random. Thus, the discrimination of the technique increases in proportion to the copy number. Two collections of M. tuberculosis were investigated to test this hypothesis. We identified 33 positions in isolates from a Tanzanian collection and 25 positions in isolates from a London, United Kingdom, collection where bands were significantly more likely to be present than would be expected by chance. These data suggest that band position is not random, and this possibility may have an impact on the interpretation of molecular epidemiological studies of M. tuberculosis.

DNA Transposable Elements↗