Molecular screening of multidrug-resistance tuberculosis by a designated public health laboratory in Taiwan.

This manuscript describes our experience in early identifying MDR-TB cases in high-risk populations by setting up a single-referral molecular diagnosis laboratory in Taiwan. Taiwan Centers for Disease Control designated a single-referral laboratory to provide the GenoType MTBDRplus test for screening high-risk MDR-TB populations nationwide in 2012-2015. A total of 5,838 sputum specimens from 3,308 patients were tested within 3 days turnaround time. Compared with the conventional culture and drug susceptibility testing, the overall performance of the GenoType MTBDRplus test for detecting TB infection showed accuracy of 70.7%, sensitivity of 85.9%, specificity of 65.7%, positive predictive value of 45.5%, and negative predictive value of 93.3%. And the accuracy of detecting rifampin (RIF) resistance, isoniazid (INH) resistance, and MDR-TB (resistant to at least RIF and INH) were 96.5%, 95.2%, and 97.7%, respectively. MDR-TB contacts presented a higher rate of mutated codons 513-519, GenoType MTBDRplus banding pattern: rpoB WT3(-), and rpoB WT4(-) than the treatment failure group. The MDR-TB contact group also had a higher rate of inhA C15T mutation, banding pattern: inhA WT1(-), and inhA MUT1(+) than the recurrent group. Resistance profiles of MDR-TB isolates also varied geographically. The referral molecular diagnosis system contributed to rapid detection and initiation of appropriate therapy.

A pulmonary tuberculosis outbreak in a long-term care facility.

In long-term care facilities (LTCFs), the elderly are apt to be infected because those with latent tuberculosis infections (LTBIs) are at an increased risk for reactivation and post-primary TB disease. We report an outbreak of TB in staff and residents in a LTCF. An outbreak investigation was conducted after two TB cases were reported from the LTCF. A tuberculin skin test (TST), bacteriological examination and chest radiograph were administered to all facility staff and residents. An outbreak is defined as at least two epidemiologically linked cases that have identical Mycobacterium tuberculosis genotype isolates. This outbreak infected eight residents and one staff member, who were confirmed to have TB in a LTCF between September 2011 and October 2012. Based on the Becker method, the latent and infectious periods were estimated at 223·6 and 55·9 days. Two initial TST-negative resident contacts were diagnosed as TB cases through comprehensive TB screening. Observing elderly people who have a negative TST after TB screening appears to be necessary, given the long latent period for controlling a TB outbreak in a LTCF. It is important to consider providing LTBI treatment for elderly contacts.

Rapid and accurate detection of rifampin and isoniazid-resistant Mycobacterium tuberculosis using an oligonucleotide array.

To rapidly detect rifampin, isoniazid and multidrug resistance in Mycobacterium tuberculosis isolates, a new system (BluePoint MtbDR, Bio Concept Inc., Taichung, Taiwan) including an oligonucleotide array and an automatic reader was evaluated. The array simultaneously identifies M. tuberculosis and predominant mutations in the rpoB, katG and inhA upstream regulatory region (inhA-r) genes. The system was assessed with 324 clinical M. tuberculosis isolates, including 210 multidrug-resistant, 41 rifampin mono-resistant, 34 isoniazid mono-resistant and 39 fully susceptible isolates. The results were compared with those obtained using the GenoType MTBDRplus test, drug-resistant gene sequencing and conventional drug susceptibility testing. The detection limit of the array was 25 pg DNA. The array and the GenoType MTBDRplus test detected 179 (85.2%) and 182 (86.7%) multidrug-resistant M. tuberculosis strains, respectively. The sensitivities of the array for detecting rifampin and isoniazid resistance were 98.4% and 87.7%, respectively, whereas the sensitivities of the GenoType MTBDRplus test for detecting rifampin and isoniazid resistance were 98.8% and 88.9%, respectively. No significant difference was found between the tests with respect to their sensitivities to detect multidrug resistance (p 0.66), rifampin resistance (p 0.69) or isoniazid resistance (p 0.68). The discrepancies were mainly attributed to rare mutations in inhA-r, which were not included in the array. The array can directly reveal transmission-associated mutations, which are useful for epidemiological investigations. The turnaround time of the array test was 6-7 h. This study confirms the feasibility of using this system for rapid and accurate diagnosis of isoniazid and rifampin resistance in M. tuberculosis.

Proficiency of drug susceptibility testing for Mycobacterium tuberculosis in Taiwan, 2007-2011.

SETTING: Authorised clinical mycobacteriology laboratories in Taiwan. OBJECTIVE: To evaluate the impact of external quality assessment (EQA) on the quality of drug susceptibility testing (DST) in 2007-2011. DESIGN: Panels consisting of 20-30 Mycobacterium tuberculosis strains were used. Efficiency of 95% in detecting resistance to both isoniazid (INH) and rifampicin (RMP), and of 90% to ethambutol (EMB) and streptomycin (SM) was used to define a competent laboratory. RESULTS: The proportion of laboratories that fulfilled the competency criteria for all first-line drugs was 16.7% in 2007, increasing to 85.7% in 2008, 86.1% in 2009, 82.4% in 2010, and to 96.8% in 2011 (P < 0.01). The mean efficiency in detecting resistance to INH and RMP reached >99% during 2008-2011 (P = 0.90 for INH and P = 0.82 for RMP), and for EMB it increased from 82.0% in 2007 to 92.2% in 2008 and 99.5% in 2011 (P < 0.01), while that for resistance to SM increased from 82.0% in 2007 to 98.1% in 2008 and 99.5% in 2011 (P < 0.01). Preparations of inoculum for DST and detection of EMB resistance were the main reasons for non-competence. CONCLUSION: The EQA programme was effective in improving the competency of clinical laboratories in performing DST for tuberculosis.

Structural neural phenotype of autism: preliminary evidence from a diffusion tensor imaging study using tract-based spatial statistics.

BACKGROUND AND PURPOSE: There is mounting evidence suggesting widespread aberrations in neural connectivity as the underlying neurobiology of autism. Using DTI to assess white matter abnormalities, this study implemented a voxelwise analysis and tract-labeling strategy to test for a structural neural phenotype in autism. MATERIALS AND METHODS: Subjects included 15 boys with autism and 8 controls, group-matched on age, cognitive functioning, sex, and handedness. DTI data were obtained by using a 3T scanner. FSL, including TBSS, was used to process and analyze DTI data where FA was chosen as the primary measure of fiber tract integrity. Affected voxels were labeled by using an integrated white matter tractography atlas. Post hoc correlation analyses were performed between FA of each affected fiber tract and scores on the Social Responsiveness Scale. RESULTS: The autism group exhibited bilateral reductions in FA involving numerous association, commissural, and projection tracts, with the most severely affected being the forceps minor. The most affected association tracts were the inferior fronto-occipital fasciculus and superior longitudinal fasciculus. There were no areas of increased FA in the autism group. All post hoc correlation analyses became nonsignificant after controlling for multiple comparisons. CONCLUSIONS: This study provides preliminary evidence of reduced FA along many long-range fiber tracts in autism, suggesting aberrant long-range corticocortical connectivity. Although the spatial distribution of these findings suggests widespread abnormalities, there are major differences in the degree to which different tracts are affected, suggesting a more specific neural phenotype in autism.

Proficiency of drug susceptibility testing for Mycobacterium tuberculosis in Taiwan.

OBJECTIVE: To evaluate the impact of external quality assessment on the quality of drug susceptibility testing (DST) in clinical mycobacteriology laboratories. DESIGN: A pilot evaluation of DST proficiency was conducted in 2006 and scaled up in 2007. A panel consisting of 20 Mycobacterium tuberculosis isolates was used. Accuracy of 95% in detecting resistance to both isoniazid (INH) and rifampicin (RMP), and 90% to both ethambutol (EMB) and streptomycin (SM), was used to define a competent laboratory. RESULTS: Nine laboratories participated in 2006 and 30 in 2007. In 2006, the mean accuracy in detecting resistance to INH was 91.6%, for RMP it was 96.1%, for EMB it was 90.5% and for SM it was 93.9%. In 2007, the mean accuracy in detecting resistance to INH increased to 95.7% and that for RMP to 97.2%, while the accuracy of EMB resistance detection decreased to 82.0% and that for SM resistance to 86.8%. Quality improvement was observed in those laboratories that had adopted standardised methods. Overall, only five (17%) laboratories fulfilled the competency criteria for all four drugs in 2007. CONCLUSION: The majority of the laboratories that participated in 2006 demonstrated an improvement in DST performance in 2007. It is essential to continue external quality assessment to strengthen the quality of DST.

External quality assessment of sputum smear microscopy in Taiwan.

OBJECTIVE: To evaluate the quality of sputum smear microscopy in nine Taiwan Centers for Disease Control contract laboratories, an external quality assessment (EQA) programme has been implemented since 2005. DESIGN: A sampling strategy based on the lot quality assurance system was applied to select slides for rechecking. Supervisory visits and technical training were conducted to determine the causes of errors and to take corrective action. RESULTS: Of the 1017 slides sampled in 2005, 637 (63%) had proper smear size, 492 (48%) proper thickness and 884 (87%) proper staining; the corresponding figures were 972 (100%), 748 (77%) and 809 (99.6%) for the 972 slides rechecked in 2006. After training, the quality of size and staining of smear preparation had significantly improved (P < 0.001) in 2006. Rechecking of 981 readable slides in 2005 identified 3 (0.3%) high false-negatives, 3 (16.7%) low false-positives and 26 (2.8%) low false-negatives; the corresponding errors were 3 (0.3%), 8 (28.6%) and 12 (1.3%) for the 972 slides rechecked in 2006. Of the eight laboratories, two (25%) and four (50%) reached 80% sensitivity in 2005 and 2006, respectively. CONCLUSION: Technical training and EQA improved the quality of sputum smear microscopy services.

Brainstem volumetric alterations in children with autism.

BACKGROUND: Although several studies have examined brainstem volume in autism, results have been mixed and no investigation has specifically measured gray- and white-matter structures. The aim of this investigation was to assess gray- and white-matter volumes in children with autism. METHOD: Subjects included 22 right-handed, non-mentally retarded boys with autism and 22 gender- and age-matched controls. Magnetic resonance imaging (MRI) scans were obtained using a 1.5-T scanner and volumetric measurements were performed using the BRAINS2 software package. Gray- and white-matter volumes were measured using a semi-automated segmentation process. RESULTS: There were no significant differences in age and total brain volume (TBV) between the two groups but full-scale IQ was higher in controls. A decrease in brainstem gray-matter volume was observed in the autism group before and after controlling for TBV. No significant differences were observed in white-matter volume. A significant relationship was observed between brainstem gray-matter volume and oral sensory sensitivity as measured by the Sensory Profile Questionnaire (SPQ). CONCLUSIONS: Findings from this study are suggestive of brainstem abnormalities in autism involving gray-matter structures with evidence supporting the existence of a relationship between these alterations and sensory deficits. These results are consistent with previous investigations and support the existence of disturbances in brainstem circuitry thought to be implicated in the sensory dysfunction observed in autism.

Screening of protective antigens of Japanese encephalitis virus by DNA immunization: a comparative study with conventional viral vaccines.

In this study, we evaluated the relative role of the structural and nonstructural proteins of the Japanese encephalitis virus (JEV) in inducing protective immunities and compared the results with those induced by the inactivated JEV vaccine. Several inbred and outbred mouse strains immunized with a plasmid (pE) encoding the JEV envelope protein elicited a high level of protection against a lethal JEV challenge similar to that achieved by the inactivated vaccine, whereas all the other genes tested, including those encoding the capsid protein and the nonstructural proteins NS1-2A, NS3, and NS5, were ineffective. Moreover, plasmid pE delivered by intramuscular or gene gun injections produced much stronger and longer-lasting JEV envelope-specific antibody responses than immunization of mice with the inactivated JEV vaccine did. Interestingly, intramuscular immunization of plasmid pE generated high-avidity antienvelope antibodies predominated by the immunoglobulin G2a (IgG2a) isotype similar to a sublethal live virus immunization, while gene gun DNA immunization and inactivated JEV vaccination produced antienvelope antibodies of significantly lower avidity accompanied by a higher IgG1-to-IgG2a ratio. Taken together, these results demonstrate that the JEV envelope protein represents the most critical antigen in providing protective immunity.

Synthesis and cytotoxic evaluation of substituted sulfonyl-N-hydroxyguanidine derivatives as potential antitumor agents.

A series of sulfonyl-N-hydroxyguanidine derivatives was designed and synthesized for cytotoxic evaluation as potential anticancer agents on the basis of the lead compound LY-181984. Replacement of the ureido moiety of the lead compound with hydroxyguanidine provided a stable cytotoxic agent. The conformation of sulfonyl-N-hydroxyguanidine derivatives, such as N-(4-chlorophenyl)-N'-[(benzo[2,1,3]thiadiazol-4-yl)sulfonyl]-N"- hydroxyguanidine (4g), investigated utilizing HMBC NMR, theoretical calculations, and X-ray crystallography, indicated stacking of the two aromatic rings. The derivatives were evaluated for in vitro cytoxicity against five human tumor cell lines, including HepG2, TSGH 8302, COLO 205, KB, and MOLT-4. The cytotoxic activities of the derived compounds against the human tumor cell lines were equal to or greater than that of the lead compound. N-(4-Chlorophenyl)-N'-[[3,5-dichloro-4-(4-nitrophenoxy)phenyl]sulfonyl]- N"- hydroxyguanidine (4n) and N-(4-chlorophenyl)-N'-[[3,5-dichloro-4-(2-chloro-4-nitrophenoxy)phenyl] sulfonyl]-N"-hydroxyguanidine (4o) exhibited the greatest growth inhibition of solid tumor cell lines. Compound 4o was found to possess antitumor activity against murine K1735/M2 melanoma xenografts.

[Study on the stability of Japanese encephalitis vaccine--development of freeze-dry dosage form].

In order to prolong shelf-life and improve the quality of the vaccine product, not only an effective stabilizer but also a more proper dosage form has been sought. The stability of a Japanese encephalitis (JE) vaccine produced from mouse brain along with a variety of stabilizers and lyophilization protocols was evaluated. Without any stabilizers added, almost 90% of the antigenicity would vanish after freeze-drying process. Comparative studies of various compounds, including carbohydrates, amino acids, peptides and medium 199, on both antigenicity preservation and thermostability of the vaccine were carried out. The results indicated that the best reconstituted vaccines were prepared with two stabilizer formulations, sucrose and sucrose/gelatin. They were further examined by accelerated stability test at room and higher temperatures. The sucrose-added lyophilized vaccine can retain its original antigenicity for more than 60 days both at 37 degrees C and 45 degrees C. We conclude the thermostability efficiency of each of the stabilizers tested is as that follows: sucrose > sucrose/gelatin > gelatin/medium > gelatin.