The role of chronic viral hepatitis on tuberculosis treatment interruption.

BACKGROUND: Tuberculosis (TB) drug-induced liver injury (TB-DILI) usually occurs within 8 weeks of anti-tuberculosis drug initiation. In Singapore, we suspected that the onset of TB drug-induced transaminitis may be confounded with hepatitis C virus (HCV) and hepatitis B (HBV) virus co-infection. OBJECTIVE: To determine the impact of HCV/HBV co-infection on the course of treatment in patients with TB treatment interrupted due to transaminitis. DESIGN: TB patients with treatment interruption during 2013-2014 were identified through the Singapore national TB registry. Case notes of those with transaminitis were perused. RESULTS: Of 3860 TB patients notified, 140 had suspected TB-DILI. Of these, respectively 20/140 (14.3%) and 16/140 (11.4%) were HCV- or HBV-positive. The median time to treatment interruption/transaminitis was 5 weeks vs. 9.9 weeks and 9.6 weeks for transaminitis patients without chronic liver disease and with HCV/HBV co-infection (P < 0.01). Multivariate logistic regression analysis revealed that having HCV/HBV co-infection was associated with treatment interruption occurring beyond 8 weeks (adjusted OR [aOR] 4.06, 95%CI 1.28-12.85); HCV transaminitis patients were more likely to take 10 months to complete anti-tuberculosis treatment (aOR 5.11, 95%CI 1.21-21.67) than those without chronic liver disease. CONCLUSION: TB treatment interruption due to transaminitis in HCV/HBV co-infected patients occurred later than in those without liver disease. Most had completed 2 months of pyrazinamide-containing intensive phase treatment before the onset of transaminitis.

Highly sensitive and scalable AAO-based nano-fibre SERS substrate for sensing application.

Well-ordered periodic nanostructures are excellent substrates for many surface-enhanced Raman spectroscopy (SERS) applications. Conventional fabrication approaches such as high precision electron beam lithography or focused ion beam produce high resolution nano-features with great reproducibility at the expense of low throughput. In this work, a highly sensitive and scalable AAO-nano-fibre (ANF) SERS substrate is demonstrated by optimising the second anodisation time of the standard two-step anodisation of aluminium and performing an additional wet etching step on the resulting AAO substrate. The optimised ANF substrate exhibits SERS sensitivity that surpasses the AAO nanoholes and the metal-film-on-nanoparticles substrates. A detection limit of 0.1 nM is achieved with a signal-to-noise ratio of 2.6-3 using a low excitation power of 0.1 mW. The ANF substrate exhibits an enhancement factor of 9.28 × 106 and a standard deviation of no more than 8%. The results indicate that the highly sensitive and scalable ANF substrate is a promising substrate for commercial SERS application.

Surveillance of tuberculosis treatment outcomes of Singapore citizens and permanent residents, 2002-2011.

SETTING: Singapore, which had a tuberculosis (TB) incidence rate of 41 per 100,000 resident population in 2011. OBJECTIVE: To report the outcomes of Singapore citizens and permanent residents treated for TB from 2002 to 2011. METHODS: A computerised treatment surveillance module (TSM) was launched in 2001 to track the progress and outcome of TB patients nationally. Physicians were required to submit an electronic or paper return for every patient at each clinic visit. Treatment adherence, drugs prescribed, treatment delivery mode and final outcome, specified as 'completed treatment', 'lost to follow-up', 'death', 'transferred out', 'permanent cessation of treatment' and 'still on treatment/no final outcome', were captured. Quarterly cohort outcomes at 12-15 months after starting treatment were combined to generate annual treatment outcomes. RESULTS: Treatment completion rates increased from 73.4% to 82.8%. The proportion of patients lost to follow-up decreased from 3.4% to 1.7%, while that of patients still on treatment or with no final outcome decreased from 10.5% to 4.4%. The death rate ranged between 10.2% and 11.7%; the majority were not attributed to TB. CONCLUSION: TB treatment completion among Singapore citizens and permanent residents has improved since 2002 as the likely result of the TSM and other initiatives introduced over the past decade.

Notified tuberculosis among Singapore residents by ethnicity, 2002-2011.

SETTING: The National Tuberculosis Programme in Singapore where, among resident cases, higher tuberculosis (TB) rates have been reported in ethnic Malays. OBJECTIVE: To describe the socio-demographic and clinical characteristics of resident TB cases by ethnicity, and to assess whether Malays differ from other groups in terms of the above parameters. DESIGN: Cross-sectional review of records from the tuberculosis registry's electronic database. RESULTS: Among 15 622 resident cases notified, 72.2% were Chinese, 18.7% Malay, 5.8% Indian and 2.9% were from other minorities. Compared to other ethnicities, Malays were more likely to be incarcerated at the time of notification (odds ratio [OR] 3.70, 95%CI 3.03-4.52) and clustered at the same residential address (OR 1.65, 95%CI 1.44-1.89), but were less likely to be aged ≥65 years (OR 0.61, 95%CI 0.54-0.70) or to reside in high-cost housing (OR 0.11, 95%CI 0.07-0.17). In terms of disease characteristics, more Malays had diabetes mellitus (OR 1.54, 1.37-1.73), a highly-positive acid-fast bacilli smear (OR 1.64, 95%CI 1.47-1.83) and cavitary disease on chest X-ray (OR 1.41, 95%CI 1.28-1.55). CONCLUSION: Compared to other ethnicities, reported TB cases among Malays were more severe and were likely to be more infectious. Increased vigilance in case management and contact investigations, as well as an improvement in the socio-economic conditions of this community, are required to reduce TB rates in this ethnic group.

Use of a T cell interferon-gamma release assay to evaluate tuberculosis risk in newly qualified physicians in Singapore healthcare institutions.

BACKGROUND: Surveillance for latent tuberculosis in high-risk groups such as healthcare workers is limited by the nonspecificity of the tuberculin skin test (TST) in BCG-vaccinated individuals. The Mycobacterium tuberculosis antigen-specific interferon-gamma release assays (IGRAs) show promise for more accurate latent tuberculosis detection in such groups. OBJECTIVE: To compare the utility of an IGRA, the T-SPOT.TB assay, with that of the TST in healthcare workers with a high rate of BCG vaccination. METHODS: Two hundred seven medical students from 2 consecutive cohorts underwent the T-SPOT.TB test and the TST in their final year of study. Subjects with negative baseline test results underwent repeat testing after working for 1 year as junior physicians in Singapore's public hospitals. RESULTS: The baseline TST result was an induration 10 mm or greater in diameter in 177 of the 205 students who returned to have their TST results evaluated (86.3%), while the baseline T-SPOT.TB assay result was positive in 9 (4.3%) of the students. Repeat T-SPOT.TB testing in 182 baseline-negative subjects showed conversion in 9 (4.9%). A repeat TST in 18 subjects with baseline-negative TST results did not reveal any TST result conversion. CONCLUSIONS: The high rate of positive baseline TST results in our BCG-vaccinated healthcare workers renders the TST unsuitable as a surveillance tool in this tuberculosis risk group. Use of an IGRA has enabled the detection and treatment of latent tuberculosis in this group. Our T-SPOT.TB conversion rate highlights the need for greater tuberculosis awareness and improved infection control practices in our healthcare institutions.

Monitoring of dengue viruses in field-caught Aedes aegypti and Aedes albopictus mosquitoes by a type-specific polymerase chain reaction and cycle sequencing.

Virologic surveillance for dengue through the detection of the prevalent serotype(s) circulating in the human population during inter- and intra-epidemic periods constitutes a reliable sentinel system for dengue outbreaks. We have applied a rapid and sensitive, semi-nested, reverse transcription-polymerase chain reaction (RT-PCR) assay using nonstructural protein 3 gene primers for the type-specific-detection of dengue viruses in artificially infected and in field-caught adult Aedes mosquitoes. In laboratory experiments, the assay was sensitive enough to detect one virus-infected mosquito head in pools of up to 59 uninfected heads. In a prospective field study conducted from April 1995 to July 1996, female adult Ae. aegypti and Ae. albopictus mosquitoes were caught from selected dengue-sensitive areas in Singapore and assayed by RT-PCR. Approximately 20% of 309 mosquito pools were positive for dengue viruses. Of the 23 RT-PCR-positive Ae. aegypti pools (containing 1-17 mosquitoes each), 18 pools (78.3%) were positive for dengue 1 virus. There were 40 RT-PCR-positive Ae. albopictus pools (containing 1-33 mosquitoes each) of which 31 (77.5%) were positive for dengue 1 virus. The predominant virus type responsible for the current dengue epidemic since 1995 was also dengue 1. The geographic locations of the virus-infected mosquitoes correlated with the residences or workplaces of patients within dengue outbreak areas. A total of 43.5% of the positive Ae. aegypti pools and 25.0% of the positive Ae. albopictus pools contained only a single mosquito. Both Aedes species showed similar overall minimum infection rates of 57.6 and 50 per 1,000 mosquitoes. Infected Ae. aegypti were detected as early as six weeks before the start of the dengue outbreaks in 1995 and 1996. However, infected Ae. albopictus appeared later, when the number of cases was increasing. Virologic surveillance by RT-PCR for detecting dengue virus-infected Aedes mosquitoes in the field may serve as an early warning monitoring system for dengue outbreaks.

Syntaxin 12, a member of the syntaxin family localized to the endosome.

We have cloned a new member of the syntaxin family of proteins. The open reading frame encodes a polypeptide of 272 amino acids with potential coiled-coil domains and a C-terminal hydrophobic tail. Northern blot analysis showed that the transcript is fairly ubiquitous. A soluble recombinant form of the polypeptide without the hydrophobic region binds to alpha-SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) and syndet/SNAP-23 in vitro. Polyclonal antibody raised against the recombinant protein recognized a 39-kDa protein in the membrane fraction of cell lysates. Indirect immunofluorescence studies using the polyclonal antibody showed that the protein is localized to intracellular membrane structures. Selective permeabilization studies with digitonin and saponin indicate that the epitope(s) recognized by the antibody is expose to the cytoplasm, consistent with the predicted orientation characteristic of SNAP receptor molecules. Morphological alterations of the staining pattern of the protein with brefeldin A and wortmannin treatment indicate that the protein is localize to the endosome. The cDNA we have cloned apparently corresponded to three previously described expressed sequence tags named as syntaxins 12, 13, and 14, respectively. We therefore propose to retain the name syntaxin 12 for this protein.

Structure-activity analysis of the activation of pertussis toxin.

Bordetella pertussis, the causative agent of whooping cough, releases pertussis toxin in an inactive form. The toxin consists of an A protomer containing one S1 peptide subunit and a B oligomer containing several other peptide subunits. The toxin binds to cells via the B oligomer, and the S1 subunit is activated and expresses ADP-ribosyltransferase and NAD glycohydrolase activities. Treatment of purified toxin with dithiothreitol (DTT) in vitro increases both activities. ATP and the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) synergistically reduce the A0.5 (activation constant) for DTT from greater than 100 mM to 200 microM. We studied the structure-activity relationships of activators of the toxin. In the presence of CHAPS (1%) and DTT (10 mM) the following compounds increased the NAD glycohydrolase activity of the toxin with the following A0.5's in microM and fraction of the ATP effect in parentheses: ATP, 0.2 (1.0); ADP, 6 (0.8); UTP, 15 (0.7); GTP, 35 (0.6); pyrophosphate, 45 (0.7); triphosphate, 60 (0.6); tetraphosphate, greater than or equal to 170 (greater than or equal to 0.4). Thus, the polyphosphate moiety is sufficient to stimulate the toxin, and the adenosine moiety confers upon ATP its extraordinary affinity for the toxin. Phospholipid and detergents could substitute for CHAPS in the activation of the toxin. Glutathione substituted for DTT with an A0.5 of 2 mM, a concentration within the range found in eucaryotic cells. Thus, membrane lipids and cellular concentrations of glutathione and ATP are sufficient to activate pertussis toxin without the need for a eucaryotic enzymatic process.

Therapeutic ultrasound and the liver in vivo: action and possible mechanisms.

This paper reports on the effects of therapeutic ultrasound upon the liver in vivo. Exposure of the liver of rats to therapeutic ultrasound at a frequency of 0.87 MHz results in intravascular haemolysis which is dependent upon the product of the intensity and the exposure duration (energy deposited within the liver). The resulting structural change (noted when a spatial peak intensity of 1.25 W/cm2 was applied for a duration of 10 min) is a lesion which has four zones. The severity of the injury increased across the four zones when scanning towards the ischaemic area. The biochemical changes observed included an increase in aspartate transaminase, alanine transferase, creatinine kinase and cholesterol and a decrease in total protein and albumin. Healing processes reversed the structural changes after 72 h which was also reflected in the plasma biochemical profile. Exposure of the liver to infrared radiation resulted in haemolysis but not the biochemical or structural alterations observed following sonication suggesting a mechanism for ultrasound action other than heating alone.

Reactive-oxygen formation and its relationship to prostaglandin and cyclic AMP production by zymosan-treated rat peritoneal macrophages.

Addition of zymosan (20 particles/cell) to suspensions of resident rat peritoneal macrophages caused an increase in the concns of prostaglandins and cyclic AMP. Preincubation of the cells with inhibitors of arachidonate metabolism led to inhibition of prostaglandin, but not of cyclic AMP, formation, which suggested that the two processes may occur independently of each other in phagocytosing cells. The luminol-dependent chemiluminescence associated with the addition of zymosan to the cells consisted of a minor, Ca2+-dependent, glucose-independent component and a major, glucose-dependent, Ca2+-independent component. Only the minor, Ca2+-dependent component appeared to be related to the lipoxygenation of arachidonic acid. Close examination of the production of prostaglandins and cyclic AMP and of chemiluminescence after zymosan addition, indicated that the expanded pool of endogenous cyclic AMP was probably not a negative modulator of the other two processes, although they remained susceptible to inhibition by exogenously-added cyclic AMP analogues or PGE2. The events induced by zymosan may be relevant to the physiological roles of prostaglandins during the inflammatory response.

Adenine nucleotides directly stimulate pertussis toxin.

Both cholera toxin and pertussis toxin catalyzed ADP-ribosylation of purified bovine brain tubulin. The effect of cholera toxin was evident in the absence or presence of nucleotides. In contrast, pertussis toxin required adenine nucleotides for its ADP-ribosylating activity. ATP, ATP gamma S, App(NH)p, deoxy-ATP, and ADP all supported pertussis toxin-catalyzed ADP-ribosylations in the absence or presence of EDTA, suggesting that nucleotide hydrolysis was not involved. Adenine nucleotides also promoted pertussis toxin-catalyzed ADP-ribosylation of heat-treated bovine serum albumin. This result suggests that adenine nucleotides directly affect pertussis toxin. ATP stimulation of pertussis toxin-catalyzed hydrolysis of NAD to ADP-ribose supports this hypothesis.

Activation of cyclic AMP-dependent protein kinase in macrophages.

The activity of cyclic AMP-dependent protein kinase (cyclic AMP-PK) was significantly higher (P less than 0.001) in thioglycollate-elicited than in resident rat peritoneal macrophages. The activity ratio of the enzyme (its activity in the absence of added cyclic AMP divided by that in the presence of 5 microM cyclic AMP) was similar in the two cell types. The divalent ion ionophore A23187 induced a rapid increase in the activity ratio of cyclic AMP-PK in both macrophage types. This effect was blocked by pretreating the cells with indomethacin or aspirin (inhibitors of cyclo-oxygenase) and bromo-phenacyl bromide (an inhibitor of phospholipase A2), implicating the synthesis of a prostanoid as an intermediary step. Prostaglandin (PG) E2, 8-bromo cyclic AMP and cholera toxin, all of which inhibit chemiluminescence and/or PG formation in macrophages, increased the activity ratio of cyclic AMP-PK in these cells. We propose that the activation of cyclic AMP-PK plays a central role in the response of macrophages to both endogenously-generated and exogenously added PGE.

Cyclic AMP and the regulation of prostaglandin production by macrophages.

The ionophore A23187 (6.7 microM) increased the rates of formation of prostaglandins and cyclic AMP in suspensions of thioglycollate-elicited rat peritoneal macrophages. Both effects were inhibited by the calmodulin blocker trifluoperazine (50 microM) and the calcium channel blocker verapamil (500 microM). Inhibitors of phospholipase A2 and cyclo-oxygenase also blocked both actions of A23187. The stimulated prostaglandin formation was markedly reduced when the cells were preincubated with 8-bromo-cyclic AMP (1mM), dibutyryl cyclic AMP (1mM) or cholera toxin (500ng/ml). Addition of exogenous arachidonic acid (30 microM) alleviated this inhibition. We propose that the effect of A23187 on macrophages includes a 'self-limiting' mechanism whereby newly-synthesized prostaglandins can inhibit, via cyclic AMP, a step(s) prior to the transformation of arachidonic acid and thus modulate their own production.

Reactive oxygen production, arachidonate metabolism and cyclic AMP in macrophages.

Chemiluminescence was used as an indicator of the production of reactive oxygen species by thioglycollate-elicited rat peritoneal macrophages stimulated by A23187. This action of the ionophore was inhibited by bromophenacyl bromide and nordihydroguaiaretic acid, inhibitors of the phospholipase A2 and lipoxygenase enzymes, respectively. The cyclo-oxygenase inhibitors, indomethacin and aspirin, did not diminish the light output. Preincubation of the cells with the 8-bromo- or dibutyryl analogues of cyclic AMP or with the cyclic AMP-phosphodiesterase inhibitors theophylline and RO-20-1724, or with PGE2, inhibited the A23187-evoked chemiluminescence. The results suggest that he lipoxygenase pathway of arachidonic acid metabolism may make a significant contribution to reactive oxygen production. This process may be modulated, and its duration limited, by cyclic AMP-mediated actions of prostaglandins, which are products of the cyclo-oxygenation of arachidonate.

Glycogen synthesis in resident and thioglycollate-elicited rat peritoneal exudate cells.

The values of the A0.5 for glucose-6-P, apparent Km for UDPglucose and -/+glucose-6-P activity ratio are similar for glycogen synthase derived from rat resident and thioglycollate-elicited peritoneal macrophages; the specific activity is 7-fold higher for the enzyme from thioglycollate-elicited macrophages. The rate of incorporation of [14C]glucose into macrophage glycogen is 7-fold greater in the elicited population that that in the resident one; the values of the S0.5 for glucose are similar. The in vitro activation of glycogen synthase proceeds at a greater rate and extent for the enzyme from elicited macrophages; thus, phosphatase activity may be reduced in resident macrophages relative to that in thioglycollate-elicited ones.

Evidence that in chick embryos destruction of hepatic microsomal cytochrome P-450 haem is a general mechanism of induction of delta-aminolaevulinate synthase by porphyria-causing drugs.

A variety of prophyrinogenic compounds were tested for their effect in ovo on chick-embryo liver microsomal cytochrome P-450 haem concentration and mitochondrial delta-aminolaevulinate synthase activity. With all drugs tested, there was a 30--50% decrease in cytochrome P-450 haem concentration within 1 h of treatment, and this was closely followed by an increase in delta-aminolaevulinate synthase activity. The relationship was independent of the extent of enzyme induction and is consistent with the proposal that drug-mediated destruction of cytochrome P-450 haem is the primary mechanism of induction of delta-aminolaevulinate synthase. After induction, synthesis of delta-aminolaevulinate synthase could be maintained by inhibiting further haem synthesis. These studies suggest that induction of porphyria is a combination of two distinct processes: (a) induction of delta-aminolaevulinate synthase synthesis by destruction of cytochrome P-450 haem and consequent depletion of cellular free haem; (b) maintenance of continued delta-aminolaevulinate synthase synthesis by preventing replenishment of cellular haem either by inhibiting haem synthesis and/or by promoting continuous removal of newly synthesized haem.