Implementing the Xpert? MTB/RIF Diagnostic Test for Tuberculosis and Rifampicin Resistance: Outcomes and Lessons Learned in 18 Countries

Background

The Xpert^® MTB/RIF (Xpert) is an automated molecular test for simultaneous detection of tuberculosis (TB) and rifampicin resistance, recommended by the World Health Organization as the preferred diagnostic method for individuals presumed to have multi-drug resistant TB (MDR-TB) or HIV-associated TB. We describe the performance of Xpert and key lessons learned during two years of implementation under routine conditions in 33 projects located in 18 countries supported by Médecins Sans Frontières across varied geographic, epidemiological and clinical settings.

Methods

Xpert was used following three strategies: the first being as the initial test, with microscopy in parallel, for all presumptive TB cases; the second being only for patients at risk of MDR-TB, or with HIV- associated TB, or presumptive paediatric TB; and the third being as the initial test for these high-risk patients plus as an add-on test to microscopy in others. Routine laboratory data were collected, using laboratory registers. Qualitative data such as logistic aspects, human resources, and tool acceptance were collected using a questionnaire.

Findings

In total, 52,863 samples underwent Xpert testing from April 2011 to December 2012. The average MTB detection rate was 18.5%, 22.3%, and 11.6% for the three different strategies respectively. Analysis of the results on samples tested in parallel showed that using Xpert as add-on test to microscopy would have increased laboratory TB confirmation by 49.7%, versus 42.3% for Xpert replacing microscopy. The main limitation of the test was the high rate of inconclusive results, which correlated with factors such as defective modules, cartridge version (G3 vs. G4) and staff experience. Operational and logistical hurdles included infrastructure renovation, basic computer training, regular instrument troubleshooting and maintenance, all of which required substantial and continuous support.

Conclusion

The implementation of Xpert was feasible and significantly increased TB detection compared to microscopy, despite the high rate of inconclusive results. Xpert implementation was accompanied by considerable operational and logistical challenges. To further decentralize diagnosis, simpler, low-cost TB technologies well-suited to low-resource settings are still urgently needed.     

Hollow fiber-based liquid phase microextraction with factorial design optimization and gas chromatography–tandem mass spectrometry for determination of cannabinoids in human hair

A new method, based on hollow fiber liquid-phase microextraction (HF-LPME) and gas chromatography–tandem mass spectrometry (GC–MSMS), was developed for determination of Δ⁹-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in samples of human hair. Since hair is a solid matrix, the samples were subjected to alkaline digestion using NaOH. The aqueous solutions obtained were extracted using a 6 cm polypropylene fiber (600 μm i.d., 200 μm wall thickness, 0.2 μm pore size) for each extraction. A 2⁵⁻¹ fractional factorial design for screening, and a central composite design for optimization of significant variables, was applied during development of the extraction method. The variables evaluated were the type of extraction solvent, pH, stirring speed, extraction time, and acceptor phase volume. The optimized conditions for the proposed extraction procedure were 10 mg of hair sample; 20 μL of butyl acetate; aqueous (pH 14) donor phase containing 6.8% NaCl; 600 rpm stirring speed; 20 min extraction time. A linear response was obtained in the ranges 1–500 pg mg⁻¹ (CBD and CBN) and 20–500 pg mg⁻¹ (THC), with regression coefficients >0.99. Precision, determined as the relative standard deviation, was 3.3–8.9% (intra-day) and 4.4–13.7% (inter-day). Absolute recoveries varied in the ranges 4.4–4.8% (CBD), 7.6–8.9% (THC) and 7.7–8.2% (CBN). Limits of detection (LOD, S/N = 3) and quantification (LOQ, S/N = 10) were 0.5–15 pg mg⁻¹ and 1–20 pg mg⁻¹, respectively. The method was successfully used to determine CBD, THC and CBN in hair samples from patients in a drug dependency rehabilitation center. Concentrations varied in the ranges 1–18 pg mg⁻¹ (CBD), 20–232 pg mg⁻¹ (THC) and 9–107 pg mg⁻¹ (CBN), confirming the suitability of the method for monitoring studies.     

Molecular characterization of Lactobacillus curvatus and Lact. sake isolated from sauerkraut and their application in sausage fermentations

Lactobacillus curvatus and Lact. sake are best adapted to meat fermentations and dominate the flora during the whole process. In fermenting sauerkraut, Leuconostoc mesenteroides subsp. mesenteroides is the major organism only during the early phase. In this environment Lact. curvatus and Lact. sake provide up to 50% of the microbial flora especially of the later phase, depending on the process conditions. Strains of Lact. curvatus and Lact. sake isolated from fermenting sauerkraut were identified by hybridization with species specific 23S rRNA-targeted oligonucleotide probes and further characterized. In 59 of 72 strains, plasmid DNA was detected. Small cryptic plasmids of 20 strains were found to be homologous with pLc2, a 2·6 kb plasmid from Lact. curvatus LTH683, which was originally isolated from meat. The ability to compete was investigated in fermenting sausages of two strains each of Lact. curvatus and Lact. sake isolated from sauerkraut. One strain each of Lact. curvatus and Lact sake was found to outnumber the meat-borne flora and govern the process.     

Direct trade-off between cyanogenesis and resistance to a fungal pathogen in lima bean (Phaseolus lunatus L.)

1.  Plants are simultaneously attacked by multiple herbivores and pathogens. While some plant defences act synergistically, others trade-off against each other. Such trade-offs among resistances to herbivores and pathogens are usually explained by the costs of resistance, i.e. resource limitations compromising a plant's overall defence.
2.  Here, we demonstrate that trade-offs can also result from direct negative interactions among defensive traits. We studied cyanogenesis (release of HCN) of lima bean (Fabaceae: Phaseolus lunatus ) and effects of this efficient anti-herbivore defence on resistance to a fungal pathogen (Melanconiaceae: Colletotrichum gloeosporioides ).
3.  Leaf tissue destruction by fungal growth was significantly higher on high cyanogenic (HC) lima bean accessions than on low cyanogenic (LC) plants. The susceptibility of HC accessions to the fungal pathogen was strongly correlated to reduced activity of resistance-associated polyphenol oxidases (PPOs) in leaves of these plants. LC accessions, in contrast, showed high PPO activity, which was correlated with distinct resistance to C. gloeosporioides .
4.  Experimentally applied, gaseous HCN reduced PPO activity and significantly increased the size of lesions caused by C. gloeosporioides in LC leaves.
5.  Field observations of a wild lima bean population in Mexico revealed a higher infection rate of HC compared to LC plant individuals. The types of lesions observed on the different cyanogenic plants in nature were similar to those observed on HC and LC plants in the laboratory.
6.   Synthesis. We suggest that cyanogenesis of lima bean directly trades off with plant defence against fungal pathogens and that the causal mechanism is the inhibition of PPOs by HCN. Our findings provide a functional explanation for the observed phenomenon of the low resistance of HC lima beans in nature.     

Cytosolic and chloroplastic glutamine synthetase of sugarbeet (Beta vulgaris) respond differently to organ ontogeny and nitrogen source

Changes in the activity and subunit composition of cytosolic glutamine synthetase (GS 1; EC 6.3.1.2) and chloroplastic GS (GS 2) were studied in response to an internal (organ ontogeny) and external signal (N source: NO₃⁻ or NH₄⁺). Maximum GS 1 activity of all organs examined was measured in the fibre roots, irrespective of the N source. The response of GS 1 to the N source was, however, organ specific. In the fibre roots, NH₄⁺ nutrition resulted in a 2- to 7-fold (based on protein or freshweight, respectively) increase of GS 1 activity compared to NO₃⁻-grown plants. In contrast to the roots, GS 1 activity in the leaf blades was 2-fold lower with NH₄⁺ nutrition, whereas only minor changes occurred in the petioles. GS 2 activity was highest in the mature and senescing leaf blade; activity was 2-fold higher with NH₄⁺ than with NO₃⁻ nutrition. Not only activity, but also subunit composition of GS 1 changed during organ ontogeny as well as in response to the N source. In contrast to GS 1, only minor changes were evident in GS 2 subunit composition, despite significant changes in GS 2 activity. Up to 5 different GS 1 subunits of ≈41–43 kDa were separated; they were identical in all organs examined. GS 2 was composed of 4 different subunits of ≈48 kDa.     

Screening of some Romanian plants for their activity against medically important insects

The effects of the action of extracts from 82 plant species included in 39 families of indigenous flora against the larvae and adults of house fly (Musca domestica), the mosquitoes (Aedes aegypti and Anopheles atroparvus) and the German cockroach (Blattella germanica) are presented. Some of the extracts were prepared from air-dried and ground plant material which was exhaustively extracted with successive solvents of different polarity: ethyl ether, ethyl alcohol and water. Other extracts were prepared either by distillation of the whole water extracts of 24 hours macerated plants or by water extraction from residues of plants remained after distillation. These plant extracts act as toxicants, growth and development and reproduction inhibitors and repellents. The differential responses induced by these plant extracts on assessed insects were influenced by several factors such as the plant species, the solvents used for extractions, the species and the stages of insect life and also the methods employed for evaluation. Some of the tested plants appear to have a great potential for providing safer insect control agents.     

Scavenging of superoxide and hydrogen peroxide in blue‐green algae (cyanobacteria)

Blue‐green algae (cyanobacteria) have evolved as the most primitive, oxygenic, plant‐type photosynthetic organisms. Within a single prokaryotic cell, they have uniquely accommodated both oxygenic photosynthesis and aerobic respiration, which are known to produce superoxide and hydrogen peroxide as inevitable byproducts. Two types of superoxide dismutase have been characterized in both N₂‐fixing and non‐N₂‐fixing cyanobacteria, namely cytosolic iron‐containing superoxide dismutase and thylakoid‐bound manganese‐containing superoxide dismutase. No qualitative differences between various cell types (vegetative cells, heterocysts) were found. In contrast to chloroplasts, most of the cyanobacterial species show catalatic activity. From two species the corresponding enzymes have been characterized as typical prokaryotic (bifunctional) catalase‐peroxidases with homologies to cytochrome c peroxidases and ascorbate peroxidases. In addition to catalatic activity, some strains exhibit ascorbate peroxidase activity, but to date there are no reports detailing purification and characterization.
Cyanobacteria were found to contain low intracellular ascorbate concentrations (30‐100 µ M ) and 2‐5 m M glutathione. Both monodehydroascorbate and glutathione reductase activities were detected in most species examined, whereas dehydroascorbate reductase activity was absent. The question as to whether a glutathione‐ascorbate cycle exists in cyanobacteria cannot be answered at present.     

Bradykinin Stimulates Arachidonic Acid Release Through the Sequential Actions of an sn-1 Diacylglycerol Lipase and a Monoacylglycerol Lipase

In cultured dorsal root ganglion (DRG) neurons prelabeled with [3H]arachidonic acid [( 3H]AA), bradykinin (BK) stimulation resulted in increased levels of radioactive diacylglycerol, monoacylglycerol, and free AA. The transient increases in content of radioactive diacylglycerol and monoacylglycerol preceded the increase in level of free AA, suggesting the contribution of a diacylglycerol lipase pathway to AA release. An analysis of the molecular species of diacylglycerols in unstimulated cultures revealed the presence of two primary [3H]AA-containing species, 1-palmitoyl-2-arachidonoyl and 1-stearoyl-2-arachidonoyl diacylglycerol. BK stimulation resulted in a preferential increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. When DRG cultures were labeled with [3H]stearic acid, treatment with BK increased the amount of label in diacylglycerol and free stearic acid, but not in monoacylglycerol. This result suggested that AA release occurred through the successive actions of an sn-1 diacylglycerol lipase and monoacylglycerol lipase. Other data supporting a diacylglycerol lipase pathway was the significant inhibition of [3H]AA release and consequent accumulation of diacylglycerol by RG 80267, which preferentially inhibits diacylglycerol lipase. Analysis of the molecular species profiles of individual phospholipids in DRG neurons indicated that phosphoinositide hydrolysis may account for a significant portion of the rapid increase in content of 1-stearoyl-2-arachidonoyl diacylglycerol. We were unable to obtain evidence that the phospholipase A2 pathway makes a significant contribution to BK-stimulated AA release in DRG cultures. Under our assay conditions there were no BK-stimulated increases in levels of radioactive lysophosphatidylinositol, lysophosphatidylcholine, or lysophosphatidylethanolamine in cultures prelabeled with [3H]inositol, [3H]choline, or [3H]-ethanolamine, respectively.     

In Saccharomyces cerevisiae a short amino acid sequence facilitates excretion in the growth medium of periplasmic proteins

In Saccharomyces cerevisiae the cell wall is a barrier to excretion of proteins in the growth medium. Although small proteins are more easily released than bigger ones, other factors besides molecular sieving may play a role in partitioning of periplasmic proteins. By using several complementary approaches including enzyme-activity assays, quantitative immunoblotting on subcellular fractions and growth media, as well as a novel approach involving the use of flow cytometry and specific antibodies, we show that residues 1–8 of mature glucoamylase greatly enhance excretion of both glucoamylase and β-galactosidase in vivo and facilitate extraction of periplasmic proteins in vitro . Immunological data obtained by flow cytometry on whole cells indicate that this amino acid sequence increases the fraction of enzyme reaching the outer cell-wall layers. This amino acid sequence may define a novel type of topogenic sequence, facilitating the crossing of the yeast cell wall in vivo and facilitating extraction of periplasmic proteins by non-disruptive means in vitro .