Photoinactivation of Mycobacterium tuberculosis and Mycobacterium smegmatis by Near-Infrared Radiation Using a Trehalose-Conjugated Heptamethine Cyanine.

The spread of multidrug-resistant mycobacterium strains requires the development of new approaches to combat diseases caused by these pathogens. For that, photodynamic inactivation (PDI) is a promising approach. In this study, a tricarbocyanine (TCC) is used for the first time as a near-infrared (740 nm) activatable PDI photosensitizer to kill mycobacteria with deep light penetration. For better targeting, a novel tricarbocyanine dye functionalized with two trehalose units (TCC2Tre) is developed. The photodynamic effect of the conjugates against mycobacteria, including Mycobacterium tuberculosis, is evaluated. Under irradiation, TCC2Tre causes more effective killing of mycobacteria compared to the photosensitizer without trehalose conjugation, with 99.99% dead vegetative cells of M. tuberculosis and M. smegmatis. In addition, effective photoinactivation of dormant forms of M. smegmatis is observed after incubation with TCC2Tre. Mycobacteria treated with TCC2Tre are more sensitive to 740 nm light than the Gram-positive Micrococcus luteus and the Gram-negative Escherichia coli. For the first time, this study demonstrates the proof of principle of in vitro PDI of mycobacteria including the fast-growing M. smegmatis and the slow-growing M. tuberculosis using near-infrared activatable photosensitizers conjugated with trehalose. These findings are useful for the development of new efficient alternatives to antibiotic therapy.

Mycobacterium tuberculosis-specific CD4+ and CD8+ T cells differ in their capacity to recognize infected macrophages.

Containment of Mycobacterium tuberculosis (Mtb) infection requires T cell recognition of infected macrophages. Mtb has evolved to tolerate, evade, and subvert host immunity. Despite a vigorous and sustained CD8+ T cell response during Mtb infection, CD8+ T cells make limited contribution to protection. Here, we ask whether the ability of Mtb-specific T cells to restrict Mtb growth is related to their capacity to recognize Mtb-infected macrophages. We derived CD8+ T cell lines that recognized the Mtb immunodominant epitope TB10.44-11 and compared them to CD4+ T cell lines that recognized Ag85b240-254 or ESAT63-17. While the CD4+ T cells recognized Mtb-infected macrophages and inhibited Mtb growth in vitro, the TB10.4-specific CD8+ T cells neither recognized Mtb-infected macrophages nor restricted Mtb growth. TB10.4-specific CD8+ T cells recognized macrophages infected with Listeria monocytogenes expressing TB10.4. However, over-expression of TB10.4 in Mtb did not confer recognition by TB10.4-specific CD8+ T cells. CD8+ T cells recognized macrophages pulsed with irradiated Mtb, indicating that macrophages can efficiently cross-present the TB10.4 protein and raising the possibility that viable bacilli might suppress cross-presentation. Importantly, polyclonal CD8+ T cells specific for Mtb antigens other than TB10.4 recognized Mtb-infected macrophages in a MHC-restricted manner. As TB10.4 elicits a dominant CD8+ T cell response that poorly recognizes Mtb-infected macrophages, we propose that TB10.4 acts as a decoy antigen. Moreover, it appears that this response overshadows subdominant CD8+ T cell response that can recognize Mtb-infected macrophages. The ability of Mtb to subvert the CD8+ T cell response may explain why CD8+ T cells make a disproportionately small contribution to host defense compared to CD4+ T cells. The selection of Mtb antigens for vaccines has focused on antigens that generate immunodominant responses. We propose that establishing whether vaccine-elicited, Mtb-specific T cells recognize Mtb-infected macrophages could be a useful criterion for preclinical vaccine development.

The efficacy of vacuum-ultraviolet light disinfection of some common environmental pathogens.

BACKGROUND: This study is to elucidate the disinfection effect of ozone producing low-pressure Hg vapor lamps against human pathogens. Ozone producing low-pressure Hg vapor lamps emit mainly 254 nm ultraviolet light C (UVC) with about 10% power of Vacuum-ultraviolet (VUV) light at 185 nm. The combination of UVC and VUV can inactivate airborne pathogens by disrupting the genetic materials or generation of reactive oxygen species, respectively. In this study, inactivation of common bacteria including Escherichia coli ATCC25922 (E. coli), Extended Spectrum Beta-Lactamase-producing E. coli (ESBL), Methicillin-resistant Staphylococcus aureus (MRSA) and Mycobacterium tuberculosis (MTB), and that of influenza A viruses H1N1 and H3N2 under the radiation from ozone producing low-pressure Hg vapor lamps was examined. Log reduction values at different treatment durations were determined. METHODS: In vitro tests were carried out. Various bacterium and virus suspensions were added onto nitrocellulose filter papers and subjected to the illumination from ozone producing low-pressure Hg vapor lamps. The extents of pathogen inactivation at different illumination times were investigated by conducting a series of experiments with increasing duration of illumination. log10 reduction in CFU/ml and reduction at log10(TCID50) were respectively measured for bacteria and viruses. The disinfection effectiveness of this type of lamps against the pathogens under the environment with a moderate barrier to light was therefore evaluated. RESULTS: Ozone producing low-pressure Hg vapor lamp successfully inactivated these human pathogens. Nevertheless, among these pathogens, disinfection of MTB required more intense treatment. In the best tested situation, 3-log10 inactivation of pathogens can be achieved with ≤10 min of VUV treatment except MTB which needed about 20 min. This demonstrated the high resistance against UV disinfection of MTB. CONCLUSIONS: Following the criteria that valid germicidal results can be reflected with 3-log10 inactivation for bacteria, 4-log10 inactivation for viruses and 5-log10 inactivation for MTB, most of the bacteria required ≤10 min of VUV treatment, 20 min for the influenza viruses while MTB needed about 30 min VUV treatment. This indicated that VUV light is an effective approach against different environmental microorganisms.

Increased vitamin D receptor expression from macrophages after stimulation with M. tuberculosis among persons who have recovered from extrapulmonary tuberculosis.

BACKGROUND: Independent of HIV infection, extrapulmonary TB (EPTB) risk is increased in women, persons of black race or foreign birth, and by genetic variants in vitamin D receptor (VDR), interleukin-1 beta (IL-1ß), and toll-like receptor (TLR)-2; functional correlates are unclear. We evaluated macrophage expression of VDR, TLR2, cathelicidin, and TNF-α, and production of IL-1ß in HIV-seronegative persons with previous EPTB, previous pulmonary TB, latent M. tuberculosis infection, and uninfected TB contacts. Persons with previous pleural TB were excluded due to enhanced immune responses at the site of disease. METHODS: Macrophages were stimulated with TLR-2 agonist M. tuberculosis lipoprotein (LpqH), live and gamma-irradiated M. tuberculosis. RESULTS: M. tuberculosis - infected macrophages from persons with previous EPTB had increased VDR expression (29.17 relative value unit increase in median expression vs. uninfected contacts, after adjusting for foreign-born status; P = 0.02). Macrophages from persons with previous EPTB had a 38.88 µg/mL increase in median IL-1ß production after stimulation with LpqH compared to uninfected contacts (P = 0.01); the effect was similar (44.99 µg/mL) but not statistically significant after controlling for foreign-born status. Median 25-hydroxyvitamin D levels were low but not significantly different between groups. CONCLUSIONS: There was increased macrophage expression of VDR after stimulation with live M. tuberculosis in persons with previous extrapulmonary TB. If post-treatment VDR expression reflects expression prior to disease, it may identify persons at risk for extrapulmonary TB.

Mycobacterium tuberculosis RuvX is a Holliday junction resolvase formed by dimerisation of the monomeric YqgF nuclease domain.

The Mycobacterium tuberculosis genome possesses homologues of the ruvC and yqgF genes that encode putative Holliday junction (HJ) resolvases. However, their gene expression profiles and enzymatic properties have not been experimentally defined. Here we report that expression of ruvC and yqgF is induced in response to DNA damage. Protein-DNA interaction assays with purified M. tuberculosis RuvC (MtRuvC) and YqgF (MtRuvX) revealed that both associate preferentially with HJ DNA, albeit with differing affinities. Although both MtRuvC and MtRuvX cleaved HJ DNA in vitro, the latter displayed robust HJ resolution activity by symmetrically related, paired incisions. MtRuvX showed a higher binding affinity for the HJ structure over other branched recombination and replication intermediates. An MtRuvX(D28N) mutation, eliminating one of the highly conserved catalytic residues in this class of endonucleases, dramatically reduced its ability to cleave HJ DNA. Furthermore, a unique cysteine (C38) fulfils a crucial role in HJ cleavage, consistent with disulfide-bond mediated dimerization being essential for MtRuvX activity. In contrast, E. coli YqgF is monomeric and exhibits no branched DNA binding or cleavage activity. These results fit with a functional modification of YqgF in M. tuberculosis so that it can act as a dimeric HJ resolvase analogous to that of RuvC.

A transcriptional co-repressor regulatory circuit controlling the heat-shock response of Mycobacterium tuberculosis.

The co-ordinated regulation of heat shock proteins is critically important for the stress response of M. tuberculosis, failure of which results in enhanced immune recognition of the tubercle bacilli with reduced survival during chronic infections. In this study, we show that PhoP regulates the transcription of α-crystallin 2 (acr2), expression of which increases more than any other gene of M. tuberculosis during heat-shock or following macrophage infection. We also show that regulation of acr2 by PhoP is attributable to direct regulator-promoter interactions at specific sites proximal to a sequence motif comprising the target site of another virulence factor, HspR. While both these regulators, on their own, are capable of influencing acr2 expression, remarkably our results show that the two virulence regulators PhoP and HspR interact with each other to influence their in vivo recruitment at the acr2 regulatory region, and in turn, contribute to stress-specific regulation of acr2 expression. We propose a model to suggest how protein-protein interactions between PhoP and HspR influence the regulation of α-crystallin 2, an essential pathogenic determinant of M. tuberculosis.

Cryopreservation of Mycobacterium tuberculosis complex cells.

Successful long-term preservation of Mycobacterium tuberculosis cells is important for sample transport, research, biobanking, and the development of new drugs, vaccines, biomarkers, and diagnostics. In this report, Mycobacterium bovis bacillus Calmette-Guérin and M. tuberculosis H37Ra were used as models of M. tuberculosis complex strains to study cryopreservation of M. tuberculosis complex cells in diverse sample matrices at different cooling rates. Cells were cryopreserved in diverse sample matrices, namely, phosphate-buffered saline (PBS), Middlebrook 7H9 medium with or without added glycerol, and human sputum. The efficacy of cryopreservation was quantified by microbiological culture and microscopy with BacLight LIVE/DEAD staining. In all sample matrices examined, the microbiological culture results showed that the cooling rate was the most critical factor influencing cell viability. Slow cooling (a few degrees Celsius per minute) resulted in much higher M. tuberculosis complex recovery rates than rapid cooling (direct immersion in liquid nitrogen) (P < 0.05). Among the three defined cryopreservation media (PBS, 7H9, and 7H9 plus glycerol), there was no significant differential effect on viability (P = 0.06 to 0.87). Preincubation of thawed M. tuberculosis complex cells in 7H9 broth for 20 h before culture on solid Middlebrook 7H10 plates did not help the recovery of the cells from cryoinjury (P = 0.14 to 0.71). The BacLight LIVE/DEAD staining kit, based on Syto 9 and propidium iodide (PI), was also applied to assess cell envelope integrity after cryopreservation. Using the kit, similar percentages of "live" cells with intact envelopes were observed for samples cryopreserved under different conditions, which was inconsistent with the microbiological culture results. This implies that suboptimal cryopreservation might not cause severe damage to the cell wall and/or membrane but instead cause intracellular injury, which leads to the loss of cell viability.

Bactericidal effects of 405 nm light exposure demonstrated by inactivation of Escherichia, Salmonella, Shigella, Listeria, and Mycobacterium species in liquid suspensions and on exposed surfaces.

The bactericidal effect of 405 nm light was investigated on taxonomically diverse bacterial pathogens from the genera Salmonella, Shigella, Escherichia, Listeria, and Mycobacterium. High-intensity 405 nm light, generated from an array of 405-nm light-emitting diodes (LEDs), was used to inactivate bacteria in liquid suspension and on exposed surfaces. L. monocytogenes was most readily inactivated in suspension, whereas S. enterica was most resistant. In surface exposure tests, L. monocytogenes was more susceptible than Gram-negative enteric bacteria to 405 nm light when exposed on an agar surface but interestingly less susceptible than S. enterica after drying onto PVC and acrylic surfaces. The study findings, that 405 nm light inactivates diverse types of bacteria in liquids and on surfaces, in addition to the safety advantages of this visible (non-UV wavelength) light, indicate the potential of this technology for a range of decontamination applications.

Mycobacterium tuberculosis uvrC essentiality in response to UV-induced cell damage.

Control of tuberculosis depends both on an effective, accurate, and rapid diagnosis and an effective treatment and management. Antituberculous drugs have been used for more than 50 years and are likely ineffective against multidrug-resistant strains, leading to an urgent need for new drugs. Comparative genome analysis has indicated that Mycobacterium tuberculosis uvrC, a component of nucleotide excision repair (NER) system, is an essential gene without any human homolog. This raises the possibility to use this gene as a new drug target. This study investigated the essential role of uvrC on growth of M. tuberculosis in the presence of DNA damaging agents, UV light and hydrogen peroxide (generator of reactive oxygen species). Results revealed that the M. tuberculosis uvrC mutant was more sensitive to UV than the control strain (p < 0.01), but was not more sensitive to hydrogen peroxide. These results showed that uvrC is essential for M. tuberculosis DNA repair system, particularly in response to DNA damage caused by UV irradiation.

Identification of a chemical that inhibits the mycobacterial UvrABC complex in nucleotide excision repair.

Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen's ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb's Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.

Mycobacterium tuberculosis MT2816 encodes a key stress-response regulator.

BACKGROUND: Sigma (sigma) factors are transcription initiation factors that modulate the response of Mycobacterium tuberculosis to changes in extracellular milieu, allowing it to survive stress. METHODS: We analyzed the expression of MT2816/Rv2745c under various stress conditions that mimic the intracellular environment faced by M. tuberculosis. RESULTS: MT2816/Rv2745c expression was induced in M. tuberculosis following redox stress, heat shock and acid shock and intracellular replication. Its expression was also induced by SDS and thioridazine, agents that impact M. tuberculosis cell-envelope. However, exposure to isoniazid or ethambutol, front-line antituberculosis drugs which also target the cell envelope, did not induce the expression of MT2816/Rv2745c. Studies using Delta-sigma(H) and Delta-sigma(E) mutants showed that sigma(H) was required for the induction of MT2816/Rv2745c. Conditional expression of the MT2816/Rv2745c in M. tuberculosis showed that apart from regulating proteolysis, this gene may control the expression of trehalose biosynthesis and impact the maintenance of cellular redox potential and energy generation. CONCLUSIONS: The protein encoded by MT2816/Rv2745c is important for the pathogen's response to stress conditions that mimic in vivo growth and it is subject to complex regulation. The MT2816/Rv2745c encoded protein likely functions by protecting intracellular redox potential and by inducing the expression of trehalose, a constituent of M. tuberculosis cell walls that is important for defense against cell-surface and oxidative stress.

On-Slide Heat Sterilization Enables Mass Spectrometry Imaging of Tissue Infected with High-Threat Pathogens Outside of Biocontainment: A Study Directed at Mycobacterium tuberculosis.

Mass spectrometry imaging investigations of tissues infected with agents that require high-security biocontainment, such as Mycobacterium tuberculosis, have been limited due to incompatible sterilization techniques. Here we describe an on-slide heat sterilization method that enables mass spectrometry imaging investigations of pharmaceuticals, lipids, and metabolites in infected tissue samples outside of biocontainment. An evaluation of different temperatures and incubation times determined that 100 °C for 1 h was essential to sterilize 5 times the bacterial burden observed in tuberculosis (TB) cavity sections. Laser-capture microdissection combined with liquid chromatography with tandem mass spectrometry quantitation, in addition to mass spectrometry imaging, showed that no degradation was observed following the on-slide heat sterilization protocol for a variety of drug classes covering a range of physicochemical properties. Utilizing the tissue mimetic model, we demonstrated that the detection of lipid and metabolite ions was not impacted by heat sterilization and that, for several metabolites, the on-slide heat sterilization method improved the sensitivity when compared to control samples. An application of the on-slide heat sterilization to M. tuberculosis infected tissue enabled the first detection and spatial distribution of lipids indicative of a lysosomal storage disease phenotype within TB granuloma macrophages, in addition to the differential distribution of metabolites central to the fatty acid oxidation pathway. These initial investigations detected a pronounced heterogeneity within the cellular regions and necrotic cores of individual TB granulomas and across different evolving granulomas. This study provides the framework for mass spectrometry imaging investigations of high-threat pathogens outside of biocontainment.

Development of a novel self-sanitizing mask prototype to combat the spread of infectious disease and reduce unnecessary waste.

With the spread of COVID-19, significant emphasis has been placed on mitigation techniques such as mask wearing to slow infectious disease transmission. Widespread use of face coverings has revealed challenges such as mask contamination and waste, presenting an opportunity to improve the current technologies. In response, we have developed the Auto-sanitizing Retractable Mask Optimized for Reusability (ARMOR). ARMOR is a novel, reusable face covering that can be quickly disinfected using an array of ultraviolet C lamps contained within a wearable case. A nanomembrane UVC sensor was used to quantify the intensity of germicidal radiation at 18 different locations on the face covering and determine the necessary exposure time to inactivate SARS-CoV-2 in addition to other viruses and bacteria. After experimentation, it was found that ARMOR successfully provided germicidal radiation to all areas of the mask and will inactivate SARS-CoV-2 in approximately 180 s, H1N1 Influenza in 130 s, and Mycobacterium tuberculosis in 113 s, proving that this design is effective at eliminating a variety of pathogens and can serve as an alternative to traditional waste-producing disposable face masks. The accessibility, ease of use, and speed of sanitization supports the wide application of ARMOR in both clinical and public settings.

The intrinsic ATPase activity of Mycobacterium tuberculosis UvrC is crucial for its damage-specific DNA incision function.

To ensure genome stability, bacteria have evolved a network of DNA repair mechanisms; among them, the UvrABC-dependent nucleotide excision repair (NER) pathway is essential for the incision of a variety of bulky adducts generated by exogenous chemicals, UV radiation and by-products of cellular metabolism. However, very little is known about the enzymatic properties of Mycobacterium tuberculosis UvrABC excinuclease complex. Furthermore, the biochemical properties of Escherichia coli UvrC (EcUvrC) are not well understood (compared to UvrA and UvrB), perhaps due to its limited availability and/or activity instability in vitro. In addition, homology modelling of M. tuberculosis UvrC (MtUvrC) revealed the presence of a putative ATP-binding pocket, although its function remains unknown. To elucidate the biochemical properties of UvrC, we constructed and purified wild-type MtUvrC and its eight variants harbouring mutations within the ATP-binding pocket. The data from DNA-binding studies suggest that MtUvrC exhibits high-affinity for duplex DNA containing a bubble or fluorescein-dT moiety, over fluorescein-adducted single-stranded DNA. Most notably, MtUvrC has an intrinsic UvrB-independent ATPase activity, which drives dual incision of the damaged DNA strand. In contrast, EcUvrC is devoid of ATPase activity; however, it retains the ability to bind ATP at levels comparable to that of MtUvrC. The ATPase-deficient variants map to residues lining the MtUvrC ATP-binding pocket. Further analysis of these variants revealed separation of function between ATPase and DNA-binding activities in MtUvrC. Altogether, these findings reveal functional diversity of the bacterial NER machinery and a paradigm for the evolution of a catalytic scaffold in UvrC.

Upper-room ultraviolet light and negative air ionization to prevent tuberculosis transmission.

BACKGROUND: Institutional tuberculosis (TB) transmission is an important public health problem highlighted by the HIV/AIDS pandemic and the emergence of multidrug- and extensively drug-resistant TB. Effective TB infection control measures are urgently needed. We evaluated the efficacy of upper-room ultraviolet (UV) lights and negative air ionization for preventing airborne TB transmission using a guinea pig air-sampling model to measure the TB infectiousness of ward air. METHODS AND FINDINGS: For 535 consecutive days, exhaust air from an HIV-TB ward in Lima, Perú, was passed through three guinea pig air-sampling enclosures each housing approximately 150 guinea pigs, using a 2-d cycle. On UV-off days, ward air passed in parallel through a control animal enclosure and a similar enclosure containing negative ionizers. On UV-on days, UV lights and mixing fans were turned on in the ward, and a third animal enclosure alone received ward air. TB infection in guinea pigs was defined by monthly tuberculin skin tests. All guinea pigs underwent autopsy to test for TB disease, defined by characteristic autopsy changes or by the culture of Mycobacterium tuberculosis from organs. 35% (106/304) of guinea pigs in the control group developed TB infection, and this was reduced to 14% (43/303) by ionizers, and to 9.5% (29/307) by UV lights (both p < 0.0001 compared with the control group). TB disease was confirmed in 8.6% (26/304) of control group animals, and this was reduced to 4.3% (13/303) by ionizers, and to 3.6% (11/307) by UV lights (both p < 0.03 compared with the control group). Time-to-event analysis demonstrated that TB infection was prevented by ionizers (log-rank 27; p < 0.0001) and by UV lights (log-rank 46; p < 0.0001). Time-to-event analysis also demonstrated that TB disease was prevented by ionizers (log-rank 3.7; p = 0.055) and by UV lights (log-rank 5.4; p = 0.02). An alternative analysis using an airborne infection model demonstrated that ionizers prevented 60% of TB infection and 51% of TB disease, and that UV lights prevented 70% of TB infection and 54% of TB disease. In all analysis strategies, UV lights tended to be more protective than ionizers. CONCLUSIONS: Upper-room UV lights and negative air ionization each prevented most airborne TB transmission detectable by guinea pig air sampling. Provided there is adequate mixing of room air, upper-room UV light is an effective, low-cost intervention for use in TB infection control in high-risk clinical settings.

Safety of upper-room ultraviolet germicidal air disinfection for room occupants: results from the Tuberculosis Ultraviolet Shelter Study.

OBJECTIVES: We evaluated the safety of room occupants in the Tuberculosis Ultraviolet Shelter Study (TUSS), a double-blind, placebo-controlled field trial of upper-room ultraviolet germicidal irradiation (UVGI) at 14 homeless shelters in six U.S. cities from 1997 to 2004. METHODS: Data collection involved administering questionnaires regarding eye and skin irritation to a total of 3,611 staff and homeless study subjects. RESULTS: Among these subjects, there were 223 reports of eye or skin symptoms. During the active UV period, 95 questionnaires (6%) noted such symptoms, and during the placebo period, 92 questionnaires (6%) did so. In the 36 remaining cases, either the UV period when symptoms took place was unknown or the symptoms spanned both periods. There was no statistically significant difference in the number of reports of symptoms between the active and placebo periods. One definite instance of UV-related keratoconjunctivitis occurred, resulting from a placement of a bunk bed in a dormitory where a single bed had been used when the UV fixtures were first installed. CONCLUSIONS: These findings demonstrate that careful application of upper-room UVGI can be achieved without an apparent increase in the incidence of the most common side effects of accidental UV overexposure.

Thermal lysis and isothermal amplification of Mycobacterium tuberculosis H37Rv in one tube.

Tuberculosis (TB) is a leading cause of high mortality rates in developing countries. Sample preparation is one of the major challenges in developing an inexpensive point-of-care device for rapid and confirmed detection of tuberculosis. Existing chemical and mechanical lysis methods are unsuitable for field applications, as they require intermediate wash steps, manual intervention or separate lysis equipment. We report a one-step reaction protocol (65°C and 60min) for the H37Rv strain of Mycobacterium tuberculosis that (i) completely disinfects the mycobacteria culture, (ii) lyses the cells and (iii) performs helicase dependent amplification on the extracted DNA. Our assay combines multiple functions in a single step, uses a dry heat bath and does not require any intermediate user intervention, which makes it suitable for use by minimally trained health workers at the point of care.

Primary macrophages and J774 cells respond differently to infection with Mycobacterium tuberculosis.

Macrophages play an essential role in the early immune response to Mycobacterium tuberculosis and are the cell type preferentially infected in vivo. Primary macrophages and macrophage-like cell lines are commonly used as infection models, although the physiological relevance of cell lines, particularly for host-pathogen interaction studies, is debatable. Here we use high-throughput RNA-sequencing to analyse transcriptome dynamics of two macrophage models in response to M. tuberculosis infection. Specifically, we study the early response of bone marrow-derived mouse macrophages and cell line J774 to infection with live and γ-irradiated (killed) M. tuberculosis. We show that infection with live bacilli specifically alters the expression of host genes such as Rsad2, Ifit1/2/3 and Rig-I, whose potential roles in resistance to M. tuberculosis infection have not yet been investigated. In addition, the response of primary macrophages is faster and more intense than that of J774 cells in terms of number of differentially expressed genes and magnitude of induction/repression. Our results point to potentially novel processes leading to immune containment early during M. tuberculosis infection, and support the idea that important differences exist between primary macrophages and cell lines, which should be taken into account when choosing a macrophage model to study host-pathogen interactions.

Use of Gen-Probe and Bactec for rapid isolation and identification of mycobacteria. Correlation of probe results with growth index.

Gen-Probe culture confirmation tests (Gen-Probe, San Diego, CA) for Mycobacterium tuberculosis complex and Mycobacterium avium complex were performed on 276 mycobacterial isolates. All 138 M. tuberculosis complex isolates and 79 of 80 M. avium complex isolates were identified correctly. No falsely positive test results were obtained; 58 nontuberculous mycobacteria other than M. avium complex were negative by Gen-Probe. In a second phase of testing, Gen-Probe tests were performed using concentrates from 101 patient Bactec 12B cultures. Positive results by Gen-Probe tests were correlated with the growth index (GI) reading on the day of processing as well as the accumulated GI readings. For those 51 with high (greater than or equal to 999) final GIs, 40/40 (100%) M. tuberculosis complex isolates and 9/11 M. avium complex isolates were positive by Gen-Probe, and six other mycobacteria were negative. Of the 25 with moderate final readings (400 less than or equal to GI less than 999), 12/17 M. tuberculosis complex isolates and 1/1 M. avium complex isolates were correctly identified by Gen-Probe; seven other mycobacteria were negative. Of 25 with low readings (GI less than 400), 8/24 M. tuberculosis isolates were correctly identified by Gen-Probe, and no falsely positive test results were obtained with the other probes. All true negative tests on seven other mycobacteria (not M. tuberculosis complex or M. avium complex) had less than 2% hybridization. Of the 24 falsely negative tests on M. tuberculosis complex isolates or M. avium complex isolates, 22 had greater than 2% hybridization with their respective probes. Thus, percent hybridization greater than 2% may be a useful indicator of the need for retesting.

[Prognosis of mono- and polydrug resistant pulmonary tuberculosis in the city of Santa Cruz, Bolivia]. / Pronóstico de la tuberculosis pulmonar mono y polirresistente en la ciudad de Santa Cruz (Bolivia).

OBJECTIVE: To evaluate the results of treatment in patients with pulmonary tuberculosis (TB) resistant to one antituberculosis drug (single drug resistance [SDR]) or to more than one drug (polyresistance [PDR]), excluding patients with resistance to the combination of isoniazid and rifampicin (multidrug resistance). PATIENTS AND METHOD: Retrospective review of all the records of patients with pulmonary TB diagnosed in an outpatient clinic in Santa Cruz, Bolivia, from 1983 through 1993 whose first cultures were SDR or PDR and who were started on treatment in the clinic. RESULTS: We identified 368 patients: 276 (75%) with SDR TB and 92 (25%) with PDR TB. There were 164 new patients among the SDR cases (59%) and 41 (45%) among the PDR cases (P<.05). The mean (SD) age of PDR patients was 31.5 (14.2) years and there were 165 (68%) males. The mean age of SDR patients was 29.4 (13.4) years and there were 50 (54%) males. Eleven patients (3%) experienced a change in the type of resistance (SDR: 7 [3%]; PDR: 4 [4%]) and in 119 cases (32%) the initial treatment regimen was changed (SDR: 84 [30%]; PDR: 35 [38%]). One hundred ninety-six SDR patients (71%) and 56 PDR patients (61%) were cured (P>.05). There were no significant differences in the cure rates in either of the 2 groups between new patients and patients with a history of previous treatment. Seventy-three SDR (26%) and 35 PDR patients (38%) failed to complete treatment (P<.05). CONCLUSIONS: The cure rates among patients who presented with SDR TB and PDR TB were similar to those of drug-sensitive patients; there were no significant differences between new and previously treated patients. The low cure rates were due to the high number of patients who abandoned treatment. Unless a system of therapy administered under supervision is set up it is not likely that these figures can be improved in Santa Cruz, Bolivia.