Usefulness of resistant gene markers for predicting treatment outcome on second-line anti-tuberculosis drugs.

AIM: Mutations in rrs [nucleotide (nt) 1401], gyrA gene (codons 90, 91 or 94), tlyA, ethA and thyA genes of Mycobacterium tuberculosis (MTB) were evaluated for their usefulness in predicting treatment outcome of kanamycin (KM), capreomycin (CPM), ofloxacin (OFX), ethionamide (ETH) and para-aminosalicylic acid (PAS). METHODS AND RESULTS: DNA sequence analyses of these genes were performed against 188 MTB isolates obtained from patients put on second-line anti-TB drugs (SLDs) with well-documented clinical history and treatment outcome. Mutations in rrs and gyrA have 100% positive predictive value (PPV) in predicting treatment failure for KM and OFX, while 88·9 and 80% were obtained, respectively, when tlyA and rrs mutations were considered in CPM. For ETH and PAS, the PPV of using ethA and thyA mutations to predict treatment failure was 82·5 and 89·3%, respectively. CONCLUSIONS: Our study demonstrated high specificities of gene mutations in predicting poor treatment outcome; however, further technical advancement is required to make the molecular detection of resistances to other SLDs feasible in clinical laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to correlate different polymorphisms of major SLD resistance gene markers with predicted treatment outcome, using an international set of well-documented clinical MTB strains.

Variations in quality of carbol fuchsin stains collected from routine tuberculosis laboratories.

SETTING: In-use carbol fuchsin stains were collected from 10 different routine acid-fast bacilli smear microscopy laboratories. OBJECTIVE: To examine the variations in the composition of carbol fuchsin stains. METHOD: Carbol fuchsin concentrations were first determined spectrophotometrically by measuring absorbance at 547 nm. High-performance liquid chromatography (HPLC) separated and quantified the four basic fuchsin homologues: para-rosaniline, rosaniline, magenta II and new fuchsin, and identity was confirmed by mass spectrometry (MS). RESULTS: Absorbance measurement showed that three of 10 (30%) samples contained insufficient carbol fuchsin (<70%). Wide variations in relative proportions of fuchsin homologues were found. CONCLUSION: The relative abundance of rosaniline + new fuchsin was quite stable among the different laboratories. Spectrophotometry and HPLC/MS are necessary and sensitive tools for monitoring fuchsin quality.

Development of a simple and low-cost real-time PCR method for the identification of commonly encountered mycobacteria in a high throughput laboratory.

AIMS: To facilitate efficient identification of commonly encountered mycobacteria species (Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium fortuitum complex, Mycobacterium chelonae/abscessus, Mycobacterium kansasii, Mycobacterium gordonae) in high throughput laboratories, a 16s rDNA sequence based real-time PCR assay was developed and evaluated. METHODS AND RESULTS: Oligonucleotide primers and hybridization probes were designed based on sequence differences of the mycobacterial 16S rDNA gene. This assay was evaluated with 1649 suspected non-tuberculosis mycobacterial isolates. Apart from 3 out of 40 M. avium isolates that showed false signal with M. intracellulare specific probe, 100% specificity was obtained for all tested probes. Assay sensitivity varied from 88.9 to 100% depending on species. Average cost for obtaining a definite identification was only USD 1.1 with an average turn around time of less than 3 days. CONCLUSIONS: A rapid, simple and inexpensive real-time PCR assay was developed for the identification of common encountered mycobacteria in a high throughput laboratory setting. SIGNIFICANCE AND IMPACT OF THE STUDY: With this assay, more than 80% of the clinically isolated nontuberculous mycobacteria could be identified in a highly cost effective manner. This helped to save resources for other laboratory activities especially in high throughput mycobacterial laboratories.

Cloning and characterization of chicken growth hormone binding protein (cGHBP).

Growth hormone (GH) is indispensable for the growth of animals and its biological activity is mediated by binding to the growth hormone receptor (GHR) [Harvey S, Scanes CG, Daughaday WH. Growth hormone. Boca Raton: CRC Press; 1995]. GHR is a transmembrane protein responsible for signal transduction upon GH binding. GH also binds to the growth hormone binding protein (GHBP) which is the soluble form of GHR extracellular domain existing in circulation. Actions of GHBP include prolongation of GH bioavailability and prevention of GH signaling system from over-stimulation. To date, little is known about the mechanisms generating the chicken GHBP (cGHBP). Elucidating the genomic structure of cGHR will provide insights into such underlying mechanisms. Using polymerase chain reaction and library screening methods, we have characterized the genomic organization of chicken GHR (cGHR). The full-length coding region of the cGHR transcript is composed of eight exons (exons 2-10), lacking a human homolog exon 3 and spans at least 71 kb on the genome. A novel transcript of size 1.2kb was isolated from chicken liver total RNA using 5' and 3' rapid cDNA ends amplification (RACE). It was generated by utilizing a previously unknown polyadenylation signal located at the intron 6. Semi-quantitative reverse transcription polymerase chain reaction showed that this transcript is widely expressed in a variety of tissues. This transcript has an open reading frame comprising 203 amino acids. In vitro binding assay using ELISA demonstrated that Escherichia coli expressed recombinant protein encoded by this transcript was able to bind with chicken GH. Hence, this transcript is a potential candidate for cGHBP.

Denaturing HPLC for high-throughput screening of rifampicin-resistant Mycobacterium tuberculosis isolates.

OBJECTIVE: To evaluate the use of denaturation high-performance liquid chromatography (dHPLC) as a rapid method to detect rifampicin (RMP) resistance based on mutations in the rpoB gene in a high-volume laboratory setting. METHODS: A total of 132 RMP-resistant Mycobacterium tuberculosis strains with different rpoB mutation were used to optimise the running condition of dHPLC as a pilot study. A blind correlation study was subsequently done between dHPLC and in vitro RMP susceptibility tests on 3167 M. tuberculosis strains in a high-throughput clinical setting. RESULTS: In the pilot study, rpoB mutation could be detected on 116/132 (87.9%) RMP-resistant strains by dHPLC. In the second phase of the study, 84/3107 (2.7%) clinical M. tuberculosis isolates were RMP-resistant. The sensitivity and specificity of dHPLC in the prediction of RMP resistance were 70/84 (83.3%) and 70/77 (91.0%), respectively. The specificity became 100% when 511 Leu to Pro mutation was excluded from the RMP resistance-related genetic changes. CONCLUSION: In the detection of RMP resistance in a high-throughput laboratory setting, dHPLC has been demonstrated to be rapid, simple, workable, automatable and inexpensive in terms of running costs and the labour involved.

Mycoplasma pneumoniae infection complicated by empyema: a rare presentation.

Mycoplasma pneumoniae is a common causative agent for childhood pneumonia. However, empyema is a rare presentation. We report a case of a previously well child who presented with a right-sided empyema. M. pneumoniae was confirmed serologically with evidence of a four-fold rise in Mycoplasma IgM titre. The empyema required drainage procedures for more than two weeks. The infection resolved with a course of six weeks of treatment with erythromycin.

IS6110 dot blot hybridization for the identification of Mycobacterium tuberculosis complex.

To explore a simple, rapid, and inexpensive way to identify Mycobacterium tuberculosis complex culture, dot blot hybridization using IS6110 as the marker was performed against 2788 known clinical isolates of mycobacteria including M. tuberculosis (n = 721), M. kansasii (177), M. marinum (10), M. avium complex (700), M. terrae complex (203), M. fortuitum (476), M. chelonae (439), and other nonpigmented Runyon's Group IV mycobacteria (62). We found that the sensitivity and specificity of the test were 94.3% and 100%, respectively. When this method was evaluated in a laboratory blind study of 1253 initially unknown clinical isolates, its sensitivity and specificity were 91.2% and 100%, respectively. Because this identification test is technically simple, rapid, and can be done in batches, together with its high sensitivity and specificity, it is a cost-effective method for routine identification of M. tuberculosis complex in laboratories of areas with a high incidence of tuberculosis.

Characterization of Mycobacterium fortuitum isolates from sternotomy wounds by antimicrobial susceptibilities, plasmid profiles, and ribosomal ribonucleic acid gene restriction patterns.

An outbreak of sternotomy infections due to Mycobacterium fortuitum in patients who had received cardiovascular surgery occurred in a cardiothoracic hospital in Hong Kong, and 21 such isolates from different patients had antimicrobial susceptibility studies against 14 drugs in vitro. These isolates were also studied for plasmid profiles and ribosomal ribonucleic acid gene restriction patterns. The latter method proved valuable in categorization of these isolates into two groups (comprising of nine and seven isolates, respectively) and five other sporadic strains. When the plasmid profiles and ribotyping are matched against the clinical and epidemiologic data, multisource contamination is suspected to be responsible for the outbreak. The organisms were probably derived from the environment rather than contaminated surgical equipments and materials.

Surveillance of Mycobacterium tuberculosis susceptibility to second-line drugs in Hong Kong, 1995-2002, after the implementation of DOTS-plus.

OBJECTIVE: To determine the trend in changes in susceptibility of Mycobacterium tuberculosis strains, including to second-line drugs, from patients with a history of previous anti-tuberculosis (TB) treatment in a 'DOTS-Plus' programme. METHODS: A retrospective survey of centralised M. tuberculosis laboratory records of all culture-positive cases over an 8-year period. The drug susceptibility of the isolates was determined using the absolute concentration method. Isolates obtained from patients with a history of previous treatment were further analysed for trends of changes in susceptibility to first- and second-line drugs. RESULTS: Of 1921 patients with a previous history of treatment and positive cultures, 1425 (74.2%) had isolates susceptible to all four first-line drugs, while 176 (9.2%) were multidrug-resistant (MDR-TB). For the MDR-TB group, 101 (57.4%) isolates were sensitive to all second-line drugs, while 30 (17.0%) were resistant to three or more second-line drugs. CONCLUSION: In a DOTS-Plus programme environment where there is strict control on use of second-line drugs, the prevalence of MDR-TB is low amongst retreatment cases and the prudent use of second-line drugs in a population with well functioning DOTS-Plus programme does not generate super-resistant strains. In circumstances where most retreatment strains are still susceptible and good laboratory support for detection of MDR cases is available, retreatment using first-line drugs is feasible.

Surveillance of Mycobacterium tuberculosis drug resistance in Hong Kong, 1986-1999, after the implementation of directly observed treatment.

OBJECTIVE: To study changing trends in TB epidemiology with emphasis on drug resistance rates in various age groups from 1986-1999. DESIGN: Laboratory-based data on drug susceptibility testing against streptomycin (SM), isoniazid (NH), rifampicin (RMP) and ethambutol (EMB) had been collected continuously in a centralised TB laboratory in Hong Kong. Epidemiological parameters such as sex, age and drug resistance rates in new and retreatment cases were measured and analysed for longitudinal trends. RESULTS: Of 48 924 non-duplicate isolates from new TB cases, 7045 (14.4%) were resistant to one or more drugs, 5773 (11.8%) were resistant to SM and/or INH while 881 (1.8%) were multidrug-resistant (MDR-TB). Of 3857 isolates from retreatment patients, 1176 (30.5%) were resistant to one or more drugs, 616 (16.0%) were resistant to SM and/or [NH, and 467 (12.1%) were MDR-TB. For isolates from new cases, significant declines were observed in the resistance rates against any drug, SM alone, INH alone, SM+INH and INH+RMP. For retreatment isolates, significant declines were also observed in resistance to any drug and INH+RMP. In both new and retreatment cases, isolates from patients aged over 65 years showed significantly lower drug resistance (any drug and INH+RMP) compared with other age groups (16-34 years and 35-65 years). CONCLUSION: With successful implementation of DOTS over a 14-year period, laboratory-based surveillance data showed significant declines in drug resistance, including MDR-TB. This has occurred amidst demographic changes associated with a generally ageing population as well as highly mobile sectors that are in constant exchange with highly endemic areas.

Mathematical modeling of PCB bioaccumulation in Perna viridis.

In the present work, we built a mathematical model of polychlorinated biphenyl (PCB) bioaccumulation in Perna viridis, namely, a one-compartment model with a time dependent incorporation rate R (microg g(-1) lipid per ppb water per day), with positive substrate cooperativity as the underlying physical mechanism. The temporal change of the PCB concentration Q (microg g(-1) lipid) in the soft tissues of the mussel depends on the competition of the input rate R Wand the output rate kQ, where W is the concentration of PCB in water (ppb water) and k is the elimination rate (per day). From our experimental data, k = 0.181 +/- 0.017 d(-1). The critical concentration in water Wc for positive substrate cooperativity was found to be approximately 2.4 ppb. Below Wc, R is a constant. For a water concentration of 0.5 ppb Aroclor 1254, R = 24.0 +/- 2.4 microg g(-1) lipid ppb(-1) d(-1). Above Wc, positive substrate cooperativity comes into effect and R becomes a function of time and dependent on the concentration Q in a form R = gammaQ/(Q + delta). This is the case for a water concentration of 5 ppb Aroclor 1254, where gamma = 15.1 microg g(-1) lipid ppb(-1) d(1) and delta approximately 200 microg g(-1) lipid. From this model, the uptake is exponentially increasing when the PCB concentration in the mussel is small compared to 200 microg g(-1) lipid, and hyperbolically increasing when the concentration is large compared to 200 microg g(-1) lipid, which are consistent with the experimental data. The model is useful for understanding the true processes taking place during the bioaccumulation and for risk assessment with higher confidence. Future experimental data which challenge the present model are anticipated and in fact desirable for improvement and perfection of

Deposition fractions of 218Po in diffusion chambers.

After radon gas diffuses into a diffusion chamber, 218Po will be formed. Due to its short half-life, a fraction f of 218Po decays before deposition onto available inner surfaces of the chamber, and the deposition fraction (1-f) represents the part which decays after deposition. In the present work, f has been experimentally determined for six diffusion chambers with different materials and dimensions using the radial distribution of track density on the LR115 detectors inside the diffusion chambers. For all the six studied diffusion chambers, f was found to be approximately 0.4. Therefore, the deposition fraction does not depend on the shape and dimensions of the diffusion chambers, the surface to volume ratios or the internal surface materials of the diffusion chambers.

Sensitivity of LR115 detector in diffusion chamber to 222Rn in the presence of 220Rn.

Determination has been made of the sensitivity of LR115 type 2-track detectors (in units of m) to 222Rn, measured in the presence of 220Rn. Measurements have been made by means of a widely used diffusion chamber while Monte Carlo simulations have also been conducted. The experimentally derived sensitivities for 222Rn and 220Rn were found to be 0.470+/-0.022 and 0.486+/-0.042 m, respectively. For Monte Carlo simulations, the sensitivities to 222Rn gas were found to range from 0.618 x 10(-2) m (assuming that all 218Po progeny decay before deposition onto the internal walls of the diffusion chamber) to 0.405 x 10(-2) m (assuming that all 215Po progeny are deposited on the internal walls of the same containment vessel before decaying). The sensitivity to 220Rn gas of 0.465 x 10(-2) m found from Monte Carlo simulations agrees to within uncertainty with experimental findings. The experimentally derived sensitivity value for 222Rn indicates that 30% of the 218Po progeny decay before deposition onto the internal walls of the diffusion chamber.

Measurement of parameters of tracks in CR-39 detector from replicas.

In determining the etched track rate in solid-state nuclear track detectors, track lengths should be determined accurately. A method based on surface profilometry is proposed to determine the track lengths in CR-39 detectors through measurements of their replicas. Tracks from alpha particles with an incident energy of 4 MeV have been chosen to demonstrate the method. After irradiation and chemical etching, resin replicas were made from the tracks, of which the heights were measured by the Form Talysurf PGI Profilometer. The results showed that the surface of the replicas were smooth and the heights of the replicas were uniform, so the replicating fluid should have filled the tracks completely and the replicas truly reflected the dimensions of the tracks. The heights of the replicas were conveniently determined from the lateral view of the replicas generated by the Form Talysurf PGI Profilometer.

Central ocular motor disorders, including gaze palsy and nystagmus.

An impairment of eye movements, or nystagmus, is seen in many diseases of the central nervous system, in particular those affecting the brainstem and cerebellum, as well as in those of the vestibular system. The key to diagnosis is a systematic clinical examination of the different types of eye movements, including: eye position, range of eye movements, smooth pursuit, saccades, gaze-holding function and optokinetic nystagmus, as well as testing for the different types of nystagmus (e.g., central fixation nystagmus or peripheral vestibular nystagmus). Depending on the time course of the signs and symptoms, eye movements often indicate a specific underlying cause (e.g., stroke or neurodegenerative or metabolic disorders). A detailed knowledge of the anatomy and physiology of eye movements enables the physician to localize the disturbance to a specific area in the brainstem (midbrain, pons or medulla) or cerebellum (in particular the flocculus). For example, isolated dysfunction of vertical eye movements is due to a midbrain lesion affecting the rostral interstitial nucleus of the medial longitudinal fascicle, with impaired vertical saccades only, the interstitial nucleus of Cajal or the posterior commissure; common causes with an acute onset are an infarction or bleeding in the upper midbrain or in patients with chronic progressive supranuclear palsy (PSP) and Niemann-Pick type C (NP-C). Isolated dysfunction of horizontal saccades is due to a pontine lesion affecting the paramedian pontine reticular formation due, for instance, to brainstem bleeding, glioma or Gaucher disease type 3; an impairment of horizontal and vertical saccades is found in later stages of PSP, NP-C and Gaucher disease type 3. Gaze-evoked nystagmus (GEN) in all directions indicates a cerebellar dysfunction and can have multiple causes such as drugs, in particular antiepileptics, chronic alcohol abuse, neurodegenerative cerebellar disorders or cerebellar ataxias; purely vertical GEN is due to a midbrain lesion, while purely horizontal GEN is due to a pontomedullary lesion. The pathognomonic clinical sign of internuclear ophthalmoplegia is an impaired adduction while testing horizontal saccades on the side of the lesion in the ipsilateral medial longitudinal fascicule. The most common pathological types of central nystagmus are downbeat nystagmus (DBN) and upbeat nystagmus (UBN). DBN is generally due to cerebellar dysfunction affecting the flocculus bilaterally (e.g., due to a neurodegenerative disease). Treatment options exist for a few disorders: miglustat for NP-C and aminopyridines for DBN and UBN. It is therefore particularly important to identify treatable cases with these conditions.

A prospective pilot study of repetitive transcranial magnetic stimulation for gait dysfunction in vascular parkinsonism.

OBJECTIVE: Vascular Parkinsonism (VP) causes significant gait dysfunction in patients who otherwise have good lower limb strength. Its pathophysiology is not clearly understood, and current treatment with physical therapy remains unsatisfactory. The study explores repetitive transcranial magnetic stimulation (rTMS) as a potential new and safe therapy for VP. MATERIALS AND METHODS: We prospectively applied 5 Hz rTMS treatment to 5 patients who satisfied all the criteria for VP. Repetitive TMS was performed on 5 consecutive days and patients were assessed on (1) timed 10 m walk (T10MW), (2) Unified Parkinson's Disease Rating Scale (UPDRS) motor subsection, (3) Clinician's Global Impression of Change (CGIC), and (4) Patient's Global Impression of Change (PGIC), for up to 6 weeks post-rTMS. RESULTS: All the outcome measures were found to have improved ratings post-rTMS when compared with baseline, and were statistically significant. The T10MW showed significant improvement at 4 weeks post-rTMS with a trend towards improvement at 2 weeks post-rTMS. The UPDRS motor subscores was significantly reduced at 2 weeks, 4 weeks and 6 weeks post-rTMS. The PGIC and CGIC scores were significantly better post-rTMS. The treatment was well-tolerated and all patients completed the study. CONCLUSION: This study demonstrated for the first time that 5 sessions of rTMS could improve gait in a measurable way for up to 6 weeks without any significant side-effects. Repetitive TMS could be a potentially useful adjunct in rehabilitation of VP patients and further research is warranted.

A pilot external quality assurance programme for line-probe assay detection of anti-tuberculosis drug resistance.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB; resistance to isoniazid and rifampicin) is difficult to detect and control. Line-probe assays (LiPA) are widely used for the rapid detection of MDR-TB. OBJECTIVE: To ensure the quality of the test, a pilot external quality assurance (EQA) programme was initiated to assess the feasibility of running such a programme and the possibility of improving the proficiency of TB laboratories in performing the test. DESIGN: Prepared filter-paper-based Mycobacterium tuberculosis DNA samples were shipped to participant laboratories for LiPA EQA. The tests were performed blind, and the results were returned to the organising laboratory for comparison and analysis. RESULTS: A total of four rounds of EQA samples were dispatched to five laboratories in four countries. Overall inter- and intra-laboratory reproducibility was respectively 97% and 96%. The strengths and weaknesses of the participant laboratories in performing the test were discussed. CONCLUSION: A LiPA EQA programme can ensure quality and improve the performance of TB laboratories. This is a critical step during the initial stages at the time of setting up this method of testing.

Random blinded rechecking of sputum acid-fast bacilli smear using fluorescence microscopy: 8 years' experience.

BACKGROUND: The Hong Kong TB Reference Laboratory is a high volume laboratory examining around 400 sputum acid-fast bacilli smears daily using fluorescence microscopy (FM). OBJECTIVE: To assess the effectiveness of blinded rechecking applied to FM in a high-throughput laboratory. METHOD: From 2003, 2.5% (5% in 2003 and 2004) of all smears were randomly selected, relabelled and assigned to each technician (rechecker) in turn. These smears were restained and re-examined. Discordance between initial screener and rechecker was resolved by a controller. RESULTS: From 2003 to 2010, low false-negative (LFN) errors (0.10-0.27%) were within the critical values, at 85% (1 year) and 90% (7 years) sensitivity. However, LFN error (0.28-0.62%) among recheckers was prominent. There were also low false-positive (LFP) cases (0.13-0.75%), but subsequent cultures showed these to be mycobacteria culture-positive. This relatively poor performance among the recheckers might be due to background fluorescence increase after restaining and/or inefficiency of the rechecking procedure. CONCLUSION: In a high-throughput laboratory, blind rechecking is a good means of quality assurance. To minimise false LFP, problems due to restaining should be resolved before blinded rechecking can be generally applied in the field for FM where mycobacterial cultures are not routinely performed.