Sputum smear microscopy result: a predictor for drug-resistant tuberculosis?

Until countries establish capacity for continuous surveillance systems, representative surveys of tuberculosis (TB) patients continue to improve our understanding of the burden of drug-resistant TB and help ensure appropriate allocation of resources. Although the available data are limited, the current recommendation of restricting surveys to sputum smear-positive patients is justified, given the greatly simplified logistics and only limited evidence in specific settings of an association between drug resistance and sputum smear status. Nonetheless, the relationship between drug resistance and sputum smear microscopy results may vary according to the setting and population under study. With the increasing availability and use of molecular diagnostics and the drive for universal drug susceptibility testing under the World Health Organization's End TB Strategy, substantially more data on drug resistance in the whole TB patient population should become available in the near future.

Western Pacific Regional Green Light Committee: progress and way forward.

The Western Pacific Regional Green Light Committee (rGLC WPR) was established in 2011 to promote the rational scale-up of programmatic management of drug-resistant tuberculosis (PMDT). We reflect on its achievements, consider the challenges faced, and explore its potential future role. Achievements include the supervision and support of national PMDT action plans, increased local ownership, contextualized guidance, and a strong focus on regional capacity building, as well as a greater awareness of regional challenges. Future rGLC activities should include (1) advocacy for high-level political commitment; (2) monitoring, evaluation, and supervision; (3) technical support and contextualized guidance; and (4) training, capacity building, and operational research. Regional activities require close collaboration with both national and global efforts, and should be an important component of the new Global Drug-resistant TB Initiative.

Practical biosafety in the tuberculosis laboratory: containment at the source is what truly counts.

In industrialised countries, sufficient resources for establishing and maintaining fully equipped biosafety level 3 (BSL-3) laboratories according to international standards are generally available. BSL-3 laboratories are designed to provide several layers of containment to protect the laboratory worker as well as the outside environment and community from risk of exposure in case of local contamination. However, such facilities are scarce in high-burden settings, primarily due to the high financial burden and complexity of the initial construction and/or regular maintenance. Measures to prevent unintended exposure to Mycobacterium tuberculosis during laboratory manipulation of specimens and cultures is the first, and by far the most important, aspect of containment. This paper focuses on the need for risk containment at source. Assuming that in many settings the establishment of BSL-3 laboratories with all the required features is not achievable, this paper also discusses the minimum requirements necessary to mitigate risks associated with particular laboratory procedures. The term 'TB containment laboratory' is used throughout this paper to describe the minimum requirements for a laboratory suitable for high-risk procedures. The TB containment laboratory has many, but not all, of the features of a BSL-3 laboratory.

A systematic review of abusive visceral injuries in childhood--their range and recognition.

OBJECTIVES: To define what abusive visceral injuries occur, including their clinical features and the value of screening tests for abdominal injury among abused children. METHODS: We searched 12 databases, with snowballing techniques, for the period 1950-2011, with all identified studies undergoing two independent reviews by trained reviewers, drawn from pediatrics, radiology, pediatric surgery and pathology. Of 5802 studies identified, 188 were reviewed. We included studies of children aged 0-18, with confirmed abusive etiology, whose injury was defined by computed tomography, contrast studies or at surgery/post mortem. We excluded injuries due to sexual abuse, or those exclusively addressing management or outcome. RESULTS: Of 88 included studies (64 addressing abdominal injuries), only five were comparative. Every organ in the body has been injured, intra-thoracic injuries were commoner in those aged less than five years. Children with abusive abdominal injuries were younger (2.5-3.7 years vs. 7.6-10.3 years) than accidentally injured children. Duodenal injuries were commonly recorded in abused children, particularly involving the third or fourth part, and were not reported in accidentally injured children less than four years old. Liver and pancreatic injuries were frequently recorded, with potential pancreatic pseudocyst formation. Abdominal bruising was absent in up to 80% of those with abdominal injuries, and co-existent injuries included fractures, burns and head injury. Post mortem studies revealed that a number of the children had sustained previous, unrecognized, abdominal injuries. The mortality from abusive abdominal injuries was significantly higher than accidental injuries (53% vs. 21%). Only three studies addressed screening for abdominal injury among abused children, and were unsuitable for meta-analysis due to lack of standardized investigations, in particular those with 'negative' screening tests were not consistently investigated. CONCLUSIONS: Visceral injuries may affect any organ of the body, predominantly abdominal viscera. A non-motor vehicle related duodenal trauma in a child aged

Rifampicin-resistant Mycobacterium tuberculosis: susceptibility to isoniazid and other anti-tuberculosis drugs.

Based on data from 14 Supranational Tuberculosis (TB) Reference Laboratories worldwide, the proportion of rifampicin (RMP) resistant isolates that were isoniazid (INH) susceptible by phenotypic drug susceptibility testing varied widely (0.5-11.6%). RMP-resistant isolates that were INH-susceptible had significantly lower rates of resistance to other first- and second-line anti-tuberculosis drugs (except rifabutin) compared to multidrug-resistant isolates. RMP resistance is not always a good proxy for a presumptive diagnosis of multidrug-resistant TB, which has implications for use of molecular assays that identify only RMP resistance-associated DNA mutations.

Liquid culture for Mycobacterium tuberculosis: proceed, but with caution.

Attempts to improve the diagnosis of tuberculosis (TB) in high-burden countries has resulted in significant funding and initiatives to change the method of diagnosis of TB from light microscopy supplemented with X-ray to a sophisticated diagnostic algorithm based on the latest technological innovations. Such activities are overdue and should be welcomed, but the lack of skills and support available to interpret and use the results represents a danger. The introduction of new diagnostic methods, particularly liquid culture, should be carefully structured according to the local situation, failing which frustration and the disruption of previously underdeveloped but adequately functioning laboratories may result.

Evaluating the performance of basic fuchsin for the Ziehl-Neelsen stain.

SETTING: An investigation of commercially available basic fuchsin (BF) dye powders used for the detection of acid-fast bacilli (AFB). OBJECTIVE: To determine whether single, or multiple, assays may predict the performance of BF in the Ziehl-Neelsen (ZN) method. DESIGN: The composition and staining properties of six BF dye samples were assessed using continuous recording spectrophotometry, reverse phase thin-layer chromatography (RPTLC) and a standardised ZN biological staining test. RESULTS: Variable proportions of BF homologues could be demonstrated in the samples, but neither spectroscopy nor RPTLC was fully predictive of their staining quality. ZN staining of standard smears was needed to identify five powders that yielded satisfactory results and one powder with unsatisfactory performance. Increasing the BF concentration did not always result in improved staining. CONCLUSIONS: Simple analytical methods, such as spectrophotometry and RPTLC, should be complemented by biological staining of control smears to assess the quality of BF dye powders. This allows tuberculosis control programmes to avoid procurement of BF dyes that would fail to detect AFB even when strictly adhering to current international guidelines for ZN staining.

Critical appraisal of current recommendations and practices for tuberculosis sputum smear microscopy.

This report is the consensus-based, agreed position of the participants in a workshop for experts in sputum smear microscopy organised in August 2005 by the International Union Against Tuberculosis and Lung Disease (The Union), and as such reflects their views, but not necessarily those of their respective organisations. The group reviewed current practices in sputum smear microscopy for tuberculosis (TB) and suggests considering certain changes in standard guidelines for the sputum collection strategy and the definition of sputum smear-positive TB. The Ziehl-Neelsen staining guidelines need to incorporate a wider error margin for widespread application under field conditions. Quality assurance is of utmost importance, and needs more commitment from National Tuberculosis Programmes and other health authorities. In particular, allocation of sufficient resources for rechecking and integration of laboratory supervision must be ensured. Countries must make better investments in the purchase of high quality microscopes and laboratory supplies. To address the human resource crisis, personnel without specific laboratory schooling can, in principle, be trained to respond to immediate needs for TB diagnostic microscopy services. Periodic reporting on acid-fast smear examinations is highly desirable for regular monitoring and a more balanced provision of supplies.

A community-based TB drug susceptibility study in Mimika District, Papua Province, Indonesia.

SETTING: A district level tuberculosis (TB) control programme in Papua Province, Indonesia. OBJECTIVE: To determine the nature and extent of drug-resistant TB in newly diagnosed sputum smear-positive patients. METHODS: Sputum was collected from previously untreated smear-positive pulmonary TB patients diagnosed in the district over a 10-month period. Sputum specimens were processed and inoculated into a BACTEC MGIT960 tube. Isolates were identified by Ziehl-Neelsen staining, hybridisation with nucleic acid probes and biochemical investigations. Susceptibility testing was performed using the radiometric proportion method. Pyrazinamide testing was performed using the Wayne indirect method. RESULTS: One hundred and seven patients had sputum sent to a reference laboratory; 101 (94.4%) were culture-positive for Mycobacterium tuberculosis, with 87 (86.1%) fully sensitive to first-line anti-tuberculosis drugs. Two per cent were multidrug-resistant (MDR-TB) and 12 (11.9%) had other drug resistance. Each of the MDR-TB isolates was susceptible to amikacin, capreomycin, ciprofloxacin and para-aminosalicylic acid (PAS), but were resistant to rifabutin. One isolate was also resistant to ethionamide. CONCLUSIONS: MDR-TB is present in Indonesia but is not a major problem for TB control in this district. Generalisability to other districts in Indonesia, particularly large urban areas, needs to be confirmed by future studies.

An alternative method for sputum storage and transport for mycobacterium tuberculosis drug resistance surveys.

SETTING: A district level tuberculosis (TB) programme in Indonesia. OBJECTIVE: To evaluate whether a single sputum specimen could be stored by refrigeration for an extended period of time, then transported to a reference laboratory and successfully cultured for Mycobacterium tuberculosis. METHODS: Single sputum specimens were collected from newly diagnosed smear-positive pulmonary TB patients, refrigerated at the study site without addition of 1% cetylpyridinium chloride, batched and sent to the reference laboratory, where they were decontaminated and inoculated into BACTEC MGIT 960 liquid media. RESULTS: One hundred and seven patients were enrolled. The median specimen storage time was 12 days (range 1-38) and median transportation time was 4 days (2-12). The median time from specimen collection until processing was 18 days (4-42). Only 4 (3.7%) specimens failed to grow Mycobacterium species and M. tuberculosis was isolated from 101 (94.4%) specimens. Six specimens with breakthrough contamination successfully grew M. tuberculosis after a second decontamination procedure. CONCLUSIONS: Single sputum specimens collected at a remote setting, refrigerated for relatively long periods without preservatives and transported without refrigeration to a reference laboratory can yield a high positive culture rate. These findings offer potential logistic simplification and cost savings for drug resistance surveys in low-resource countries.

Not all microscopes are equal.

A functional microscope is the most important and most expensive piece of equipment required for smear microscopy. Global Fund monies have provided a unique opportunity to equip laboratories in low-resource countries, but these funds must be spent wisely. Cheap microscopes are superficially appealing. However, higher-quality microscopes generally have better optics and last longer. International agencies should consider establishing a purchasing service to assist countries in their microscope procurement. Such a system would provide quality assurance and increased purchasing power.

Analysis of Mycobacterium tuberculosis isolates from treatment failure patients living in East Timor.

SETTING: In the first 2 years of the East Timor National TB Control Programme, 7960 new patients were treated (2RHZE/6HE) and 224 received a retreatment regimen (2SRHZE/1RHZE/4R3H3Z3E3). OBJECTIVE: To determine the nature and extent of drug-resistant tuberculosis (TB) in treatment failure cases in East Timor. METHODS: Sputum specimens from retreatment failure cases were processed and inoculated into a BACTEC MGIT960 tube and onto Löwenstein-Jensen media. Isolates were identified by Ziehl-Neelsen staining, hybridisation with nucleic acid probes and biochemical investigations. Susceptibility testing was performed using the radiometric proportion method. Pyrazinamide testing was performed using the Wayne indirect method. RESULTS: Eighteen patients failed retreatment (0.7% of new cases) and 14 were available for analysis. Mycobacterium tuberculosis was cultured from all specimens, despite considerable transport delays. Nine (64.3%) had multiple drug resistance (MDR-TB) and four (28.6%) had other drug resistance. All MDR-TB isolates were susceptible to amikacin, capreomycin and ethionamide, with most also susceptible to ciprofloxacin and para-aminosalicylic acid (PAS). CONCLUSIONS: An excellent TB control programme has been established in East Timor. If funds are available, East Timor provides an ideal setting for a small-scale DOTS-Plus programme to treat prevalent cases of MDR-TB, and this study could inform the second-line drug regimen.

Commensal ocular bacteria degrade mucins.

BACKGROUND/AIMS: Antimicrobial activity in tears prevents infection while maintaining a commensal bacterial population. The relation between mucin and commensal bacteria was assessed to determine whether commensals possess mucinolytic activity, how degradation depends on mucin integrity, and whether mucins affect bacterial replication. METHODS: Bacteria were sampled from healthy eyes and contact lenses from asymptomatic wearers. Intracellular mucins were extracted and purified from cadaver conjunctivas, and surface mucins from extended wear contact lenses. After exposure to bacteria, changes in mucin hydrodynamic volume (proteolytic cleavage) and subunit charge (oligosaccharide degradation) were assayed by size exclusion and ion exchange chromatography. The effect of mucin on bacterial replication was followed for up to 24 hours from the end of incubation with purified ocular mucins. RESULTS: Ocular bacteria decreased the hydrodynamic volume of intracellular and contact lens adherent mucins, irrespective of glycosylation density. A decrease in mucin sialylation was observed after exposure to commensal bacteria. Subunit charge distributions were generally shifted to lesser negative charge, consistent with loss of charged epitopes. Subunits with high negative charge, observed after digesting lightly adhering contact lens mucins with bacteria, suggest preferential cleavage sites in the mucin molecule. The presence of purified ocular mucin in the medium inhibited bacterial growth. CONCLUSION: Bacteria in the healthy ocular surface possess mucinolytic activity on both intact and surface processed mucins, targeted to discrete sites in the mucin molecule. Inhibition of bacterial growth by ocular mucins can be seen as part of the mucosal control of microbiota.

Nocardia asteroides isolated from three patients with cystic fibrosis.

Over a 7-year period, three patients with cystic fibrosis had multiple sputum specimens that were smear- and culture-positive for Nocardia asteroides. Two of the patients had received long-term, low-dose inhalational corticosteroid therapy. Although all three patients were treated with cotrimoxazole, resulting in eradication of the organism from the sputum, there was no change in their clinical state, radiological findings, or pulmonary function. The isolation of Nocardia asteroides from the respiratory tract of cystic fibrosis patients is an unusual finding. Its presence does not necessarily imply disease, and in these three cases, it most likely represented colonisation. The clinical significance of Nocardia spp. isolated from the respiratory tract of cystic fibrosis patients needs to be considered in the context of the individual clinical picture.

Quality control in the endoplasmic reticulum: PDI mediates the ER retention of unassembled procollagen C-propeptides.

Quality control within the endoplasmic reticulum (ER) is thought to be mediated by the interaction of a folding protein with one or several resident ER proteins [1]. Protein disulphide isomerase (PDI) is one such ER resident protein that has been previously shown to interact with proteins during their folding and assembly pathways [2, 3]. It has been assumed that, as a consequence of this interaction, unassembled proteins are retained within the ER. Here, we experimentally show that this is indeed the case. We have taken advantage of our previous finding that PDI interacts with procollagen chains early on in their assembly pathway [2] to address the role of this protein in directly retaining unassembled chains within the ER. Our experimental approach involved expressing individual C-propeptide domains from different procollagen chains in mammalian cells and determining the ability of these domains to interact with PDI and to be secreted. The C-propeptide from the proalpha2(I) chain was retained within the cell, where it formed a complex with PDI. Conversely, the C-propeptide from the proalpha1(III) chain did not form a complex with PDI and was secreted. Both domains were secreted, however, from a stable cell line expressing a secreted form of PDI lacking its ER retrieval signal. Hence, we have demonstrated directly that the intracellular retention of one substrate for ER quality control is due to an interaction with PDI.

Screening for refractive errors with the Topcon PR2000 Pediatric Refractometer.

PURPOSE: The PR2000 (Topcon, Tokyo, Japan) is a photorefractor that has been used in a population study comparing different methods of screening preschool children. The present study was conducted to determine the accuracy of the device in a largely clinical population. METHODS: Two hundred twenty-two children less than 8 years of age were included. All children were examined by an orthoptist using the PR2000 without inducing cycloplegia. All children then underwent retinoscopy with cycloplegia by an examiner who was unaware of the results from the PR2000 examination. RESULTS: The PR2000 gave a numerical reading for 90% of the children's right eyes and the message "Out of range" for a further 5%. The readings underestimated the amount of hypermetropic or astigmatic refractive error found on retinoscopy by an amount proportional to the magnitude of the refractive error. Agreement with retinoscopy for the axis of astigmatism more than 0.75 D was moderately good (intraclass correlation coefficient [ICC] = 0.63). The PR2000 was more useful as a screener, especially for anisometropia for which it was 91% sensitive and 92% specific. The repeatability was good for sphere (ICC = 0.74), less so for astigmatism (ICC = 0.59), and better than the optometrist for anisometropia (ICC = 0.38). The presence of nonrefractive diagnoses and the age of the children examined made little difference in the screening results. CONCLUSIONS: The PR2000 underestimated hypermetropic refractive errors when used without cycloplegia. However, it was at least as good a screening device as other similar instruments, especially when judged by its ability to detect anisometropia and the repeatability of the results.

Polyunsaturated fatty acids enhance the heat induced stress response in rainbow trout (Oncorhynchus mykiss) leukocytes.

The heat shock response has been studied extensively, yet the molecular signals that trigger the response remain elusive. The dogma of the heat shock response contends that denatured proteins initiate the response, but evidence is accumulating to point to a more complex system in which at least more than one signal is involved in this process. Thermal stress initiates changes in cellular phospholipid membrane physical state, which when acted upon by phospholipases may release lipid mediators that could serve as triggering signals during the heat shock response. We have examined the heat shock response in freshly isolated leukocytes from the pronephros of rainbow trout (Oncorhynchus mykiss). In this study, we show that leukocytes isolated from rainbow trout acclimated to 5 or 19 degrees C express elevated levels of heat shock protein 70 (hsp70) mRNA when heat shocked at 5 degrees C above their respective acclimation temperature and supplementation with exogenous docosahexaenoic acid or arachidonic acid followed by heat shock enhanced levels of hsp70 mRNA. The time course for docosahexaenoic acid induced enhancement of hsp70 mRNA was accelerated compared with heat shock alone, and staurosporine inhibited the docosahexaenoic acid induced increase of hsp70 mRNA. We also provide evidence that phospholipase A2 is involved in the heat shock response.

Multicenter evaluation of the Abbott LCx Mycobacterium tuberculosis ligase chain reaction assay.

Four Australian hospital laboratories evaluated the performance of the Abbott LCx Mycobacterium tuberculosis assay with 2,347 specimens (2,083 respiratory and 264 nonrespiratory specimens) obtained from 1, 411 patients. A total of 152 specimens (6.5%) were culture positive for Mycobacterium tuberculosis complex (MTBC); of these, 79 (52%) were smear positive. After resolution of discrepant data, the overall sensitivity, specificity, and positive and negative predictive values for the LCx assay were 69.7, 99.9, 99.1, and 97.7% respectively. For smear-positive respiratory specimens that were culture positive for MTBC, the values were 98.5, 100, 100, and 98.4%, respectively, while the values for smear-negative respiratory specimens were 41.5, 99.9, 96.4, and 98%, respectively. Relative operating characteristic curves were constructed to demonstrate the relationship between sensitivity and specificity for a range of possible cutoff values in the LCx assay. These graphs suggested that the assay sensitivity for respiratory samples could be increased from 70.2 to 78.6%, while the specificity would be reduced from 99.9 to 99.4% by inclusion of a grey zone (i.e., LCx assay values of between 0.2 and 0.99). An algorithm is presented for the handling of specimens with LCx assay values within this grey zone.

Subcellular localization of enzyme activities involved in the metabolism of platelet-activating factor in rainbow trout leukocytes.

The subcellular distribution of an alkyllyso-GPC: acetyl-CoA acetyltransferase (EC 2.3.1.67) and transacylase, two important enzyme activities involved in the remodeling pathway for the biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) have been examined in leukocytes isolated from the pronephros of the rainbow trout, Oncorhynchus mykiss. Contrary to mammalian systems, in which the acetyltransferase is localized to intracellular membranes, the subcellular distribution of an acetyltransferase activity in rainbow trout leukocytes was localized to the plasma membrane. Analysis of the acetyltransferase products by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) confirmed synthesis of two subclasses of PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. The transacylase activity in this study was detected in membrane fractions in two domains of the intermediate density region which also contained the NADH dehydrogenase activity, a marker enzyme for the endoplasmic reticulum. Acylation of lysoPAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) exhibited approximately 95% specificity for omega-3 fatty acids. Acylation patterns were not significantly different in either domain of the endoplasmic reticulum. A model is proposed herein for the metabolism of PAF in rainbow trout leukocytes.