Evaluation of Gamma Interferon (IFN-γ)-Induced Protein 10 Responses for Detection of Cattle Infected with Mycobacterium bovis: Comparisons to IFN-γ Responses

Gamma interferon (IFN-γ)-induced protein 10 (IP-10) has recently shown promise as a diagnostic biomarker of Mycobacterium tuberculosis infection of humans. The aim of the current study was to compare IP-10 and IFN-γ responses upon Mycobacterium bovis infection in cattle by using archived samples from two aerosol inoculation studies. In the first study (10⁴ CFU M. bovis by aerosol, n = 7), M. bovis purified protein derivative (PPDb)-specific IP-10 and IFN-γ gene expression was detected as early as 29 days after challenge. PPDb-specific IP-10 and IFN-γ mRNA responses followed a similar pattern of expression over the course of this study and were highly correlated (r = 0.87). In the second study (10⁵ CFU M. bovis by aerosol, n = 5), IP-10 and IFN-γ (protein) responses to mycobacterial antigens were compared following challenge. IFN-γ responses to mycobacterial antigens were detected at 29 days after challenge and were sustained during the remainder of the study. IFN-γ responses to mycobacterial antigens exceeded corresponding responses in nonstimulated cultures. IP-10 responses to mycobacterial antigens exceeded preinfection responses at 7, 29, and 63 days after challenge. In contrast to IFN-γ responses, IP-10 responses to mycobacterial antigens generally did not exceed the respective responses in nonstimulated cultures. IP-10 responses to medium alone and to mycobacterial antigens followed a similar pattern of response. Correlations between IP-10 and IFN-γ (protein) responses were modest (r ≈ 0.50 to 0.65). Taken together, these findings do not support the use of IP-10 protein as a biomarker for bovine tuberculosis using the current testing protocol and reagents; however, mRNA-based assays may be considered for further analysis.     

Accuracy of interferon-γ-induced protein 10 for diagnosing latent tuberculosis infection: a systematic review and meta-analysis

BackgroundEffective diagnostic methods for detecting latent tuberculosis infection (LTBI) are important for its eradication. A number of studies have evaluated the use of interferon-γ-induced protein 10 (IP-10), which is elevated after tuberculosis infection, as a biomarker for LTBI, but conclusive results regarding its effectiveness have not been reported.ObjectivesOur objective was to assess the diagnostic value of IP-10 for LTBI.Data sourcesWe searched the PubMed, Embase, the Cochrane Library and Web of Science databases to find eligible studies.Study eligibility criteriaWe included cohort, case–control and cross-sectional studies that evaluated IP-10 in LTBI participants in comparison with tuberculin skin tests (TST) and interferon-γ release assays (IGRA).ParticipantsIndividuals with LTBI and uninfected participants.InterventionsIP-10 (index test) compared with TST and IGRA (reference standard) for diagnosing LTBI.MethodsPubMed, Embase, the Cochrane Library, and Web of Science databases were searched up to June 2018. A hierarchical summary receiver operating characteristic (HSROC) model was used to evaluate the pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and HSROC curve for the diagnostic efficiency of IP-10.ResultsTwelve studies including 1023 participants and 1122 samples were included. The overall pooled sensitivity was 0.85 (95% CI 0.80–0.88), specificity was 0.89 (95% CI 0.84–0.92), PLR was 7.55 (95% CI 5.20–10.97), NLR was 0.17 (95% CI 0.13–0.22) and DOR was 44.23 (95% CI 28.86–67.79), indicating a high accuracy for diagnosing LTBI. Based on a meta-regression analysis, high-burden countries, study design, IP-10 method, reference standard and the IP-10 cut-off could not explain the heterogeneity (p >0.05).ConclusionsOur results suggested that IP-10 is a promising biomarker for the diagnosis of LTBI.     

Serum IFN-γ-induced protein 10 (IP-10) as a biomarker for severity of acute respiratory infection in healthy adults

BackgroundThe inflammatory chemokine, interferon-gamma inducible protein of 10 kDa (IP-10), is a biomarker associated with several conditions.ObjectivesThis study investigated serum concentrations of IP-10 in healthy individuals who developed acute respiratory infection (ARI). The hypothesis is that serum IP-10 concentrations correlate with ARI severity and detection of viral pathogens.Study designData come from a randomized controlled trial measuring the effects of mindfulness meditation or exercise on ARI (Clinical Trials ID: NCT01654289). Healthy adults ages 30–69 were followed for a single season for ARI incidence and severity. This trial is ongoing, and the investigators are still blinded. When a participant reported ARI symptoms, nasal swab and lavage for PCR-based viral identification and blood samples were collected within the first 72 h of ARI symptoms. Serum IP-10 concentrations were measured by ELISA (R&D Systems, Inc., Quantikine ELISA, Minneapolis, MN). ARI severity was measured using the validated Wisconsin Upper Respiratory Symptom Survey (WURSS-24) until the ARI episode resolved.ResultsSerum IP-10 concentrations from 225 ARI episodes correlated with ARI global severity (rho 0.28 [95% CI: 0.15–0.39]; p < 0.001). IP-10 concentrations were higher with an ARI in which a viral pathogen was detected compared to no viral pathogen detected (median 366 pg/ml [IQR: 227–486] vs 163 pg/ml [IQR: 127–295], p < 0.0001). Influenza infections had higher IP-10 concentrations than coronavirus, enterovirus or rhinovirus, and paramyxovirus.ConclusionSerum IP-10 concentration correlates with ARI global severity. Also, IP-10 concentration measured early in the course of the ARI correlates with the daily severity, duration, and illness symptoms.     

Ex-vivo whole blood secretion of interferon (IFN)-γ and IFN-γ-inducible protein-10 measured by enzyme-linked immunosorbent assay are as sensitive as IFN-γ enzyme-linked immunospot for the detection of gluten-reactive T cells in human leucocyte antigen (HLA)-DQ2·5+-associated coeliac disease

T cell cytokine release assays are used to diagnose infectious diseases, but not autoimmune or allergic disease. Coeliac disease (CD) is a common T cell-mediated disease diagnosed by the presence of gluten-dependent intestinal inflammation and serology. Many patients cannot be diagnosed with CD because they reduce dietary gluten before medical workup. Oral gluten challenge in CD patients treated with gluten-free diet (GFD) mobilizes gluten-reactive T cells measurable by interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) or major histocompatibility complex (MHC) class II tetramers. Immunodominant peptides are quite consistent in the 90% of patients who possess HLA-DQ2·5. We aimed to develop whole blood assays to detect gluten-specific T cells. Blood was collected before and after gluten challenge from GFD donors confirmed to have CD (n = 27, all HLA-DQ2·5⁺), GFD donors confirmed not to have CD (n = 6 HLA-DQ2·5⁺, 11 HLA-DQ2·5⁻) and donors with CD not following GFD (n = 4, all HLA-DQ2·5⁺). Plasma IFN-γ and IFN-γ inducible protein-10 (IP-10) were measured by enzyme-linked immunosorbent assay (ELISA) after whole blood incubation with peptides or gliadin, and correlated with IFN-γ ELISPOT. No T cell assay could distinguish between CD patients and controls prior to gluten challenge, but after gluten challenge the whole blood IFN-γ ELISA and the ELISPOT were both 85% sensitive and 100% specific for HLA-DQ2·5⁺ CD patients; the whole blood IP-10 ELISA was 94% sensitive and 100% specific. We conclude that whole blood cytokine release assays are sensitive and specific for detection of gluten-reactive T cells in CD; further clinical studies addressing the utility of these tests in patients with an uncertain diagnosis of CD is warranted.     

Association of the level of IFN-γ produced by T cells in response to Mycobacterium tuberculosis-specific antigens with the size of skin test indurations among individuals with latent tuberculosis in a highly tuberculosis-endemic setting

There is growing evidence showing the potential of T-cell-based gamma interferon (IFN-γ) release assays (IGRAs) for predicting the risk of progression of Mycobacterium tuberculosis (Mtb) infection, though there is little information from tuberculosis (TB)-endemic settings. In this study, we assessed the association between the level of IFN-γ produced by T cells in response to Mtb-specific antigens and the size of skin test indurations in 505 adult individuals who were screened for latent tuberculosis infection (LTBI) using the QuantiFERON-TB Gold In Tube (QFTGIT) assay and tuberculin skin test (TST). There was a strong positive correlation between the level of IFN-γ induced by the specific antigens and the diameter of the skin indurations (Spearman's rho = 0.6, P < 0.001). Body mass index and parasitic infection were not associated with the level of IFN-γ production or the TST reaction. In linear regression analysis, the size of the skin test indurations was significantly associated with the mean level of IFN-γ [coefficient, 0.65; 95% confidence interval (CI), 0.47 to 0.82, P < 0.001]. Similarly, results from logistic regression analysis demonstrated that individuals who had skin test indurations ≥ 10 mm were 6.82 times more likely than individuals who had skin test indurations < 10 mm to have high levels of IFN-γ (i.e. positive QFTGIT result) (adjusted odd ratio = 6.82; 95% CI, 3.67 to 12.69, P < 0.001). In conclusion, the results of this study could provide indirect evidence for the prognostic use of the QFTGIT assay for progression of Mtb infection, though prospective follow-up studies are needed to provide direct evidence.     

Physiological and metabolic responses of female games and endurance athletes to prolonged, intermittent, high-intensity running at 30° and 16°C ambient temperatures

Eight female games players (GP) and eight female endurance athletes (EA) ran intermittently at high-intensity and for prolonged periods in hot (30°C) and moderate (16°C) ambient temperatures. The subjects performed a two-part (A, B) test based on repeated 20-m shuttle runs. Part A comprised 60 m of walking, a maximal 15-m sprint, 60 m of cruising (90% maximal oxygen uptake, V˙O_2max) and 60 m of jogging (45% V˙O_2max) repeated for 75 min with a 3-min rest every 15 min. Part B involved an exercise and rest pattern of 60-s running at 100% V˙O_2max and 60-s rest which was continued until fatigue. Although the GP and EA did not respond differently in terms of distances completed, performance was 25 (SEM 4)% less (main effect trial, P < 0.01) in the hot (HT) compared with the moderate trial (MT). Sprints of 15 m took longer to complete in the heat (main effect, trial, P < 0.01), and sprint performance declined during HT but not MT (interaction, trial × time, P < 0.01). A very high correlation was found between the rate of rise in rectal temperature in HT and the distance completed [GP, r =−0.94, P < 0.01; EA (n = 7), r = −0.93, P < 0.01]. Blood lactate [La⁻ ]_b and plasma ammonia [NH₃]_p1 concentrations were higher for GP than EA, but were similar in HT and MT [La⁻ ]_b, HT: GP vs EA, 8.0 (SEM 0.9) vs 4.9 (SEM 1.1) mmol · l⁻¹; MT: GP vs EA, 8.0 (SEM 1.3) vs 4.4 (SEM 1.2) mmol · l⁻¹; interaction, group × time, P < 0.01; [NH₃]_p1, HT: GP vs EA, 70.1 (SEM 12.7) vs 43.2 (SEM 6.1) mmol · l⁻¹; MT: GP vs EA, 76.8 (SEM 8.8) vs 32.5 (SEM 3.8) μmol · l⁻¹; interaction, group × time, P < 0.01. Ad libitum water consumption was higher in HT [HT: GP vs EA, 18.9 (SEM 2.9) vs 13.5 (SEM 1.7) ml · kg⁻¹ · h⁻¹; MT: GP vs EA, 12.7 (SEM 3.7) vs 8.5 (SEM 1.5) ml · kg⁻¹ · h⁻¹; main effect, group, n.s.; main effect, trial, P < 0.01]. These results would suggest that elevated body temperature is probably the key factor limiting performance of prolonged, intermittent, high-intensity running when the ambient temperature is high, but not because of its effect on the metabolic responses to exercise. Accepted: 19 July 1999     

Ameliorated course of glucose-6-phosphate isomerase (G6PI)-induced arthritis in IFN-γ receptor knockout mice exposes an arthritis-promoting role of IFN-γ

The absence of IFN-γ signaling leads to an increased inflammatory response in many murine models of autoimmune diseases induced by a CFA-assisted immunization schedule. We investigated the role of endogenous IFN-γ in arthritis induced by immunization with glucose-6-phosphate isomerase (G6PI) in CFA in DBA/1 mice. Surprisingly, and in contrast to our previous findings in collagen-induced arthritis (CIA), G6PI-induced arthritis was found to be reduced in IFN-γ receptor-deficient (IFN-γR KO) mice, demonstrating a proinflammatory role for IFN-γ in this model. Milder disease in IFN-γR KO mice was associated with less vigorous innate and adaptive immune responses early (day 9) after immunization: less proliferation of myeloid cells in the spleen, less osteoclast formation, less G6PI-reactive Th cells (as measured by ex vivo stimulation and flow cytometry and by in vivo skin reactivity to G6PI) and lower G6PI-specific immunoglobulin serum levels. Surprisingly, on day 21, despite continued milder disease in IFN-γR KO mice, their Th cell responses were no longer diminished but augmented as compared to wild-type mice, and their numbers of immature myeloid splenocytes were also more increased. These data reveal that IFN-γ signaling is critical for the induction of the early immune responses which trigger G6PI-induced arthritis. The strikingly different clinical consequences of absent IFN-γ signaling in G6PI-induced arthritis compared with the very similarly induced CIA emphasize that the role of a single cytokine in experimentally induced arthritis depends critically on the very nature of the inciting (auto)antigen and in particular on the kinetics of the disease manifestation elicited by the antigen.     

Evaluation of IFN-γ secretion after stimulation with C. neoformans and C. gattii antigens in individuals with frequent exposure to the fungus

In this study we produced antigenic extracts from prototypical strains of C. neoformans (VNI-VNIV) and C. gattii (VGI-VGIV) and tested IFN-γ secretion by Elispot. Antigens from the eight Cryptococcus molecular types (VNI –VNIV and VGI - VGIV) were obtained after capsule reduction. IFN-γ secretion by Elispot method were stimulated with C. neoformans and C. gattii antigens. Peripheral blood mononuclear cells of fourteen healthy control subjects, being: five ecotourists, two mycologists, three poultry keepers, and four individuals without reports of exposure to the fungus. We observed a significant increase in IFN-γ secretion in the group of ecotourists, mycologists and bird keepers in relation to the group of individuals without reports of occupational exposures to these agents. Our results suggest the significant increase in IFN-γ secretion may be related to the continuous exposure of these groups of individuals to the fungus, as well as to the specific antigen memory immune response developed during exposure to Cryptococcus.     

Effect of lower body compression garments on submaximal and maximal running performance in cold (10°C) and hot (32°C) environments

No previous studies have investigated the effect of lower body compression garments (CG) on running performance in the heat. This study tested the hypothesis that CG would negatively affect running performance in the heat by comparing CG and non-CG conditions for running performance and physiological responses in hot and cold conditions. Ten male recreational runners (29.0 ± 10.0 years, [(V)\dot]\textO ₂ \dot{V}{\text{O}}_{ 2} max: 58.7 ± 2.7 ml kg⁻¹ min⁻¹) performed four treadmill tests consisting of 20-min running at first ventilatory threshold followed by a run to exhaustion at [(V)\dot]\textO ₂ \dot{V}{\text{O}}_{ 2} max velocity in four conditions: 10°C with CG, 10°C without CG, 32°C with CG, and 32°C without CG (randomised, counterbalanced order). Time to exhaustion (TTE), skin and rectal temperature, [(V)\dot]\textO ₂ \dot{V}{\text{O}}_{ 2} , heart rate and rating of perceived exertion (RPE) were compared between CG and non-CG conditions at each environmental temperature. TTE was not significantly different between the CG and non-CG conditions at 10°C (158 ± 74 vs. 148 ± 73 s) and 32°C (115 ± 40 vs. 97 ± 33 s); however, there was a small (0.15) and moderate effect size (0.48), respectively, suggestive of an improvement in TTE with CG. Lower limb skin temperature was 1.5°C higher at 10°C with CG (P < 0.05), but no significant differences in other physiological variables, including rectal temperature, were observed between garment conditions. Interestingly, RPE was lower (P < 0.05) during submaximal running at 32°C with CG (13.8 ± 2.0) compared with non-CG (14.5 ± 2.7). It was concluded that CG had no adverse effects on running performance in hot conditions.     

Could a loss of memory T cells limit responses to hepatitis C virus (HCV) antigens in blood leucocytes from patients chronically infected with HCV before and during pegylated interferon-α and ribavirin therapy?

The proportions and activation status of T cells may influence responses to hepatitis C virus (HCV) and treatment outcome in patients receiving pegylated interferon (IFN)‐α/ribavirin therapy. We confirmed that IFN‐γ enzyme‐linked immunospot (ELISPOT) responses to HCV are poor in HCV patients and showed that responses to HCV and cytomegalovirus (CMV) antigens decrease during therapy. This was most apparent in patients with sustained virological response (SVR). Baseline frequencies of CD4⁺ effector memory (T_EM) T cells were lower in SVR than non‐SVR. Proportions of CD4⁺ and CD8⁺ T_EM and terminally differentiated effector memory (T_EMRA) T cells declined on therapy in SVR, as did proportions of Fas⁺ CD8⁺ T_EMRA T cells. Baseline frequencies of programmed death (PD)‐1‐expressing CD4⁺ T_EM and T_EMRA T‐cells were higher in SVR. Therapy increased percentages of PD‐1⁺ CD4⁺ central memory (T_CM) T cells and PD‐1⁺ CD8⁺ T_EM and T_EMRA T cells in SVR. We conclude that successful therapy depletes circulating antigen‐specific CD4⁺ T cell responses. This paralleled decreases in proportions of effector memory T cells and higher percentages of CD4⁺ T_CM T cells expressing PD‐1.     

Polymorphisms of IFN-γ (+874A/T) and IL-12 (+1188A/C) in tuberculosis patients and their household contacts in Hyderabad,India

Several cytokine gene variants have shown to be associated with host susceptibility to infectious diseases including tuberculosis (TB). High rates of transmission were identified within household members of TB patients. In this study, we examined whether single nucleotide polymorphisms of IFN-γ +874A/T and IL-12 +1188A/C affect susceptibility to TB. Genomic DNA from patients with active disease, their household contacts HHC and healthy controls HC was genotyped for IFN-γ +874A/T and IL-12 +1188A/C SNPs by amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). IFN-γ +874 AA and AT genotypes were significantly with different frequencies in patients and total HHC as compared to HC (p < 0.0001). In patients IL-12 +1188 AC and CC genotypes were associated with TB (p < 0.003, p < 0.008). In total HHC AC, CC genotypes and both alleles (A&C) were significantly different as compared to HC (p < 0.004, p < 0.001, p < 0.034) and the same result was obtained when HHC were stratified into related (p < 0.02, p < 0.001) and unrelated (p < 0.009, p < 0.017) individuals. Allelic frequencies, however, were significant only in related contacts (p < 0.021). Generalized multifactor dimensionality reduction method (GMDR) testing revealed high risk combinations of several genotypes in IFN-γ & IL-12 genes. Our findings suggest an important role of genetic variations of IFN-γ and IL-12 for susceptibility to TB.     

Study of immunological aspects of aspergillosis in mice and effect of polyene macrolide antibiotic (SJA-95) and IFN-γ: a possible role of IFN-γ as an adjunct in antifungal therapy

New polyene macrolide antibiotic SJA-95 in free as well as liposomal (lip.) forms, with and without interferon-γ (IFN-γ) was studied in mice model of aspergillosis using biological and biochemical parameters viz. colony forming units (CFU) in liver, spleen, kidney, lung and brain, and serum IgG, and interleukin-4 (IL-4). Treatment with free and lip SJA-95 along with IFN-γ prolonged the survival time, reduced CFU in vital organs, decreased serum IgG and IL-4 levels. SJA-95 lip form showed greater antifungal activity as compared to free form. The combined treatment of lip SJA-95 with IFN-γ showed further enhancement in antifungal activity of SJA-95 (lip). The present experimental findings demonstrated IFN-γ might act as a potent modulator in immune reaction during fungal infection and can be a useful adjunctive in antifungal therapy in the management of deep seated systemic mycoses.     

Putative roles of a proline–glutamic acid-rich protein (PE3) in intracellular survival and as a candidate for subunit vaccine against Mycobacterium tuberculosis

The proline–glutamic acid (PE) protein family of Mycobacterium tuberculosis (Mtb) plays diverse roles in the pathogenesis and modulation of host immune responses. The uniqueness of conserved regions of PE proteins may be useful to test and validate their corresponding functions. Hence, the present study has been undertaken to demonstrate the role of PE3 (Rv0159c) for persistence, host immune response and immunoprophylaxis. We have expressed Mtb-specific PE3 gene in M. smegmatis (MS) and used the strain to infect J774A.1 macrophage cells and BALB/c mice. It was observed that during the infection, the MS expressing PE3 showed higher bacterial load when compared to infection with wild-type MS. In hypoxic condition, the expression level of PE3 gene was induced in Mtb, which further showed its relevance in the cell survival during hypoxia-induced persistence. The expression level of PE3 in Mtb was markedly induced during chronic stage of murine infection, which reiterated its importance in mycobacterial persistence in the host. The immunization of mice with recombinant PE3 protein stimulated the secretion of TNF, IL-6 and IL-2 cytokines and generated strong protective immunity against challenge with live mycobacteria, which was evidenced by decreased viable bacilli in the lungs, histopathological changes and increased survival of PE3 immunized mice. Conclusively, the results indicated that PE3 plays significant roles in mycobacterial persistence during infection, modulate host immune response and hence could be a prospective candidate for the development of subunit vaccine against tuberculosis.     

Deletion of the D domain of the human parainfluenza virus type 3 (HPIV3) PD protein results in decreased viral RNA synthesis and beta interferon (IFN-β) expression

Parvoviridae is a family of small non-enveloped viruses and divided into two subfamilies. The family members infect a wide range of organisms from insects to humans and some of the members (e.g., nonpathogenic adeno-associated viruses) are effective gene therapy delivery vectors. We detailed the synonymous codon usage pattern of Parvoviridae family from the available 58 sequenced genomes through multivariate statistical methods. Our results revealed that nine viruses showed some degree of strong codon bias, and the others possessed a general weak trend of codon bias. ENc-plot and neutrality plot results showed that selective pressure dominated over mutation in shapes coding sequence’s composition. The overall GC content and GC content at the third synonymous codon position were the principal determinants behind the variations within the codon usage patterns, as they both significantly correlated with the first axis of correspondence analysis. In addition, gene length had no direct influence on the codon usage pattern. Densovirinae subfamily and Parvovirinae subfamily possessed nine identical preferred codons, though most of the two subfamilies codon usage frequencies were significantly different. The result of cluster analysis based on synonymous codon usage was discordant with that of taxonomic classification. Adeno-associated viruses formed a separated clade far from other Parvoviridae members in the dendrogram. Thus, we concluded that natural selection rather than mutation pressure accounts for the main factor that affects the codon bias in Parvoviridae family.     

Phagocytic activity of neutrophils and lymphocytes at various periods of storage in the state of cold anabiosis (-40°C)

We studied changes in phagocytic activity of neutrophils and lymphocytes subjected to cold anabiosis. The proposed effective method for introduction of human blood leukocytes into cold anabiosis at moderately low temperature (-40°C) in the presence of a cryopreserving agent does not require washout of the biological material after defrosting.Translated from Byulleten Eksperimentalnoi Biologii i Meditsiny, Vol. 138, No. 10, pp. 400–402, October, 2004     

Effect of deworming on human T cell responses to mycobacterial antigens in helminth-exposed individuals before and after bacille Calmette-Guérin (BCG) vaccination

The protective efficacy of BCG vaccination against pulmonary tuberculosis (TB) is highly variable in different populations. The reason remains to be elucidated. This study aims to investigate the possible effect of intestinal helminths on the immune response to PPD in naturally immunized or BCG-vaccinated humans. The study population was assessed for helminthic infection and those found to be positive were randomly assigned to either an albendazole treatment group or a control group who received a placebo. The immune response to PPD was compared between the two groups. In addition, subjects who were tuberculin skin test-negative in both groups were BCG vaccinated and later on tested for PPD-specific responses. Albendazole induced elimination/or reduction in intestinal worms resulting in a significant improvement in T cell proliferation and in interferon-gamma production by peripheral blood mononuclear cells (PBMC) stimulated with PPD. Moreover, BCG vaccination significantly improved PPD-specific immune responses in the treated group but not in the placebo group. The differences in the in vivo skin test responses were not significant. The data show that cellular immune responses to PPD are reduced in persons with concurrent helminthic infections, perhaps reflecting a lowered resistance to mycobacterial infections. This could explain, at least in part, the reduced efficacy of BCG against TB in helminth-endemic areas of the world.     

φ(2)GFP10, a high-intensity fluorophage, enables detection and rapid drug susceptibility testing of Mycobacterium tuberculosis directly from sputum samples

The difficulty of diagnosing active tuberculosis (TB) and lack of rapid drug susceptibility testing (DST) at the point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterium-specific-fluorophage (φ(2)GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis in clinical sputum samples. Engineered features distinguishing φ(2)GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. φ(2)GFP10 produces a 100-fold increase in fluorescence per cell compared to existing reporter phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. Use of φ(2)GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampin and kanamycin from sputum samples yielded results after 12 h of incubation with φ(2)GFP10. Fluorophage φ(2)GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for M. tuberculosis infection and for rapid DST.