Inoculum size and traits of the infecting clinical strain define the protection level against Mycobacterium tuberculosis infection in a rabbit model.

Host protective immunity against pathogenic Mycobacterium tuberculosis (Mtb) infection is variable and poorly understood. Both prior Mtb infection and BCG vaccination have been reported to confer some protection against subsequent infection and/or disease. However, the immune correlates of host protection with or without BCG vaccination remain poorly understood. Similarly, the host response to concomitant infection with mixed Mtb strains is unclear. In this study, we used the rabbit model to examine the host response to various infectious doses of virulent Mtb HN878 with and without prior BCG vaccination, as well as simultaneous infection with a mixture of two Mtb clinical isolates HN878 and CDC1551. We demonstrate that both the ability of host immunity to control pulmonary Mtb infection and the protective efficacy of BCG vaccination against the progression of Mtb infection to disease is dependent on the infectious inoculum. The host response to infection with mixed Mtb strains mirrors the differential responses seen during infection with each of the strains alone. The protective response mounted against a hyperimmunogenic Mtb strain has a limited impact on the control of disease caused by a hypervirulent strain. This preclinical study will aid in predicting the success of any vaccination strategy and in optimizing TB vaccines.

Evaluation of Semiautomated IS6110-Based Restriction Fragment Length Polymorphism Typing for Mycobacterium tuberculosis in a High-Burden Setting.

The manual IS6110-based restriction fragment length polymorphism (RFLP) typing method is highly discriminatory; however, it is laborious and technically demanding, and data exchange remains a challenge. In an effort to improve IS6110-based RFLP to make it a faster format, DuPont Molecular Diagnostics recently introduced the IS6110-PvuII kit for semiautomated typing of Mycobacterium tuberculosis using the RiboPrinter microbial characterization system. This study aimed to evaluate the semiautomated RFLP typing against the standard manual method. A total of 112 isolates collected between 2013 and 2014 were included. All isolates were genotyped using manual and semiautomated RFLP typing methods. Clustering rates and discriminatory indexes were compared between methods. The overall performance of semiautomated RFLP compared to manual typing was excellent, with high discriminatory index (0.990 versus 0.995, respectively) and similar numbers of unique profiles (72 versus 74, respectively), numbers of clustered isolates (33 versus 31, respectively), cluster sizes (2 to 6 and 2 to 5 isolates, respectively), and clustering rates (21.9% and 17.1%, respectively). The semiautomated RFLP system is technically simple and significantly faster than the manual RFLP method (8 h versus 5 days). The analysis is fully automated and generates easily manageable databases of standardized fingerprints that can be easily exchanged between laboratories. Based on its high-throughput processing with minimal human effort, the semiautomated RFLP can be a very useful tool as a first-line method for routine typing of M. tuberculosis isolates, especially where Beijing strains are highly prevalent, followed by manual RFLP typing if resolution is not achieved, thereby saving time and labor.

Epidemiologic Correlates of Pyrazinamide-Resistant Mycobacterium tuberculosis in New York City.

Pyrazinamide (PZA) has important sterilizing activity in tuberculosis (TB) chemotherapy. We describe trends, risk factors, and molecular epidemiology associated with PZA-resistant (PZA(r)) Mycobacterium tuberculosis in New York City (NYC). From 2001 to 2008, all incident culture-positive TB cases reported by the NYC Department of Health and Mental Hygiene (DOHMH) were genotyped by IS6110-based restriction fragment length polymorphism and spoligotype. Multidrug-resistant (MDR) isolates underwent DNA sequencing of resistance-determining regions of pncA, rpoB, katG, and fabG1. Demographic and clinical information were extracted from the NYC DOHMH TB registry. During this period, PZA(r) doubled (1.6% to 3.6%) overall, accounting for 44% (70/159) of the MDR population and 1.4% (75/5511) of the non-MDR population. Molecular genotyping revealed strong microbial phylogenetic associations with PZA(r). Clustered isolates and those from acid-fast bacillus (AFB) smear-positive cases had 2.7 (95% confidence interval [CI] = 1.71 to 4.36) and 2.0 (95% CI = 1.19 to 3.43) times higher odds of being PZA(r), respectively, indicating a strong likelihood of recent transmission. Among the MDR population, PZA(r) was acquired somewhat more frequently via primary transmission than by independent pathways. Our molecular analysis also revealed that several historic M. tuberculosis strains responsible for MDR TB outbreaks in the early 1990s were continuing to circulate in NYC. We conclude that the increasing incidence of PZA(r), with clear microbial risk factors, underscores the importance of routine PZA drug susceptibility testing and M. tuberculosis genotyping for the identification, control, and prevention of increasingly resistant organisms.

Phosphodiesterase-4 inhibition alters gene expression and improves isoniazid-mediated clearance of Mycobacterium tuberculosis in rabbit lungs.

Tuberculosis (TB) treatment is hampered by the long duration of antibiotic therapy required to achieve cure. This indolent response has been partly attributed to the ability of subpopulations of less metabolically active Mycobacterium tuberculosis (Mtb) to withstand killing by current anti-TB drugs. We have used immune modulation with a phosphodiesterase-4 (PDE4) inhibitor, CC-3052, that reduces tumor necrosis factor alpha (TNF-α) production by increasing intracellular cAMP in macrophages, to examine the crosstalk between host and pathogen in rabbits with pulmonary TB during treatment with isoniazid (INH). Based on DNA microarray, changes in host gene expression during CC-3052 treatment of Mtb infected rabbits support a link between PDE4 inhibition and specific down-regulation of the innate immune response. The overall pattern of host gene expression in the lungs of infected rabbits treated with CC-3052, compared to untreated rabbits, was similar to that described in vitro in resting Mtb infected macrophages, suggesting suboptimal macrophage activation. These alterations in host immunity were associated with corresponding down-regulation of a number of Mtb genes that have been associated with a metabolic shift towards dormancy. Moreover, treatment with CC-3052 and INH resulted in reduced expression of those genes associated with the bacterial response to INH. Importantly, CC-3052 treatment of infected rabbits was associated with reduced ability of Mtb to withstand INH killing, shown by improved bacillary clearance, from the lungs of co-treated animals compared to rabbits treated with INH alone. The results of our study suggest that changes in Mtb gene expression, in response to changes in the host immune response, can alter the responsiveness of the bacteria to antimicrobial agents. These findings provide a basis for exploring the potential use of adjunctive immune modulation with PDE4 inhibitors to enhance the efficacy of existing anti-TB treatment.

Spontaneous latency in a rabbit model of pulmonary tuberculosis.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is an exquisitely adapted human pathogen capable of surviving for decades in the lungs of immune-competent individuals in the absence of disease. The World Health Organization estimates that 2 billion people have latent TB infection (LTBI), defined by a positive immunological response to Mtb antigens, with no clinical signs of disease. A better understanding of host and pathogen determinants of LTBI and subsequent reactivation would benefit TB control efforts. Animal models of LTBI have been hampered generally by an inability to achieve complete bacillary clearance. Herein, we have characterized a rabbit model of LTBI in which, similar to most humans, complete clearance of pulmonary Mtb infection and pathological characteristics occurs spontaneously. The evidence that Mtb-CDC1551-infected rabbits achieve LTBI, rather than sterilization, is based on the ability of the bacilli to be reactivated after immune suppression. These rabbits showed early activation of T cells and macrophages and an early peak in the TNFα level, which decreased in association with clearance of bacilli from the lungs. In the absence of sustained tumor necrosis factor-α production, no necrosis was seen in the evolving lung granulomas. In addition, bacillary control was associated with down-regulation of several metalloprotease genes and an absence of lung fibrosis. This model will be used to characterize molecular markers of protective immunity and reactivation.

Molecular immunologic correlates of spontaneous latency in a rabbit model of pulmonary tuberculosis.

BACKGROUND: Infection of humans with Mycobacterium tuberculosis (Mtb) results in latent tuberculosis infection (LTBI) in 90-95% of immune competent individuals, with no symptoms of active disease. The World Health Organization estimates that 1.5 billion people have LTBI, which can reactivate in the setting of waning host immunity, posing a threat to global TB control. Various animal models have been used to study the pathogenesis of TB. However, besides nonhuman primates, rabbits are the only animal model that fully recapitulates the pathological features of human TB, including progressive disease with necrosis and cavitation or establishment of spontaneous latency. RESULTS: We defined the molecular immunological correlates of LTBI establishment in a rabbit model of pulmonary infection with Mtb CDC1551. After aerosol infection, exponential bacterial growth was noted in the lungs for 4 weeks, followed by a significant decline by 12 weeks, resulting in the absence of cultivable bacilli by 24 weeks. We used rabbit whole genome microarrays to profile the lung transcriptome during the course of infection. At 2 weeks post-infection, gene networks involved in natural killer (NK) and dendritic cell (DC) activation and macrophage antimicrobial activities were highly upregulated. This was followed by upregulation of gene networks involved in macrophage and T cell activation and autophagy, peaking at 4 to 8 weeks. Concomitantly, host Th1, but not Th2 or inflammatory, immune response genes were significantly upregulated. Thus, the expression kinetics of genes involved in cross-talk between innate and adaptive immunity over the first 8 weeks post-infection were consistent with early efficient control of infection in the lungs. Interestingly, expression of many genes of the host innate and adaptive immune response pathways was downregulated at 12 weeks, suggesting that immune activation did not persist once bacilli began to clear from the infected lungs. CONCLUSIONS: Our results suggest that early activation of host innate immunity prior to efficient activation of T cell-mediated adaptive immunity but not inflammation is essential for establishment of LTBI in Mtb CDC1551-infected rabbits. We also show that T cell activation and the host adaptive immune response networks are dampened once bacterial growth is controlled, ultimately resulting in spontaneous LTBI.

Early innate immunity determines outcome of Mycobacterium tuberculosis pulmonary infection in rabbits.

BACKGROUND: Pulmonary infection of humans by Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), results in active disease in 5-10% of individuals, while asymptomatic latent Mtb infection (LTBI) is established in the remainder. The host immune responses that determine this differential outcome following Mtb infection are not fully understood. Using a rabbit model of pulmonary TB, we have shown that infection with the Mtb clinical isolate HN878 (a hyper-virulent W-Beijing lineage strain) leads to progressive cavitary disease similar to what is seen in humans with active TB. In contrast, infection with Mtb CDC1551 (a hyper-immunogenic clinical isolate) is efficiently controlled in rabbit lungs, with establishment of LTBI, which can be reactivated upon treatment with immune-suppressive drugs. We hypothesize that the initial interaction of Mtb with the cells of the host response in the lungs determine later outcome of infection. RESULTS: To test this hypothesis, we used our rabbit model of pulmonary TB and infected the animals with Mtb HN878 or CDC1551. At 3 hours, with similar lung bacillary loads, HN878 infection caused greater accumulation of mononuclear and polymorphonuclear leukocytes (PMN) in the lungs, compared to animals infected with CDC1551. Using whole-genome microarray gene expression analysis, we delineated the early transcriptional changes in the lungs of HN878- or CDC1551-infected rabbits at this time and compared them to the differential response at 4 weeks of Mtb-infection. Our gene network and pathway analysis showed that the most significantly differentially expressed genes involved in the host response to HN878, compared to CDC1551, at 3 hours of infection, were components of the inflammatory response and STAT1 activation, recruitment and activation of macrophages, PMN, and fMLP (N-formyl-Methionyl-Leucyl-Phenylalanine)-stimulation. At 4 weeks, the CDC1551 bacillary load was significantly lower and the granulomatous response reduced compared to HN878 infection. Moreover, although inflammation was dampened in both Mtb infections at 4 weeks, the majority of the differentially expressed gene networks were similar to those seen at 3 hours. CONCLUSIONS: We propose that differential regulation of the inflammation-associated innate immune response and related gene expression changes seen at 3 hours determine the long term outcome of Mtb infection in rabbit lungs.

Epidemiologic consequences of microvariation in Mycobacterium tuberculosis.

BACKGROUND: Evidence from genotype-phenotype studies suggests that genetic diversity in pathogens have clinically relevant manifestations that can impact outcome of infection and epidemiologic success. We studied 5 closely related Mycobacterium tuberculosis strains that collectively caused extensive disease (n = 862), particularly among US-born tuberculosis patients. METHODS: Representative isolates were selected using population-based genotyping data from New York City and New Jersey. Growth and cytokine/chemokine response were measured in infected human monocytes. Survival was determined in aerosol-infected guinea pigs. RESULTS: Multiple genotyping methods and phylogenetically informative synonymous single nucleotide polymorphisms showed that all strains were related by descent. In axenic culture, all strains grew similarly. However, infection of monocytes revealed 2 growth phenotypes, slower (doubling ∼55 hours) and faster (∼25 hours). The faster growing strains elicited more tumor necrosis factor α and interleukin 1ß than the slower growing strains, even after heat killing, and caused accelerated death of infected guinea pigs (∼9 weeks vs 24 weeks) associated with increased lung inflammation/pathology. Epidemiologically, the faster growing strains were associated with human immunodeficiency virus and more limited in spread, possibly related to their inherent ability to induce a strong protective innate immune response in immune competent hosts. CONCLUSIONS: Natural variation, with detectable phenotypic changes, among closely related clinical isolates of M. tuberculosis may alter epidemiologic patterns in human populations.

Phosphodiesterase-4 inhibition combined with isoniazid treatment of rabbits with pulmonary tuberculosis reduces macrophage activation and lung pathology.

Tuberculosis (TB) is responsible for significant morbidity and mortality worldwide. Even after successful microbiological cure of TB, many patients are left with residual pulmonary damage that can lead to chronic respiratory impairment and greater risk of additional TB episodes due to reinfection with Mycobacterium tuberculosis. Elevated levels of the proinflammatory cytokine tumor necrosis factor-α and several other markers of inflammation, together with expression of matrix metalloproteinases, have been associated with increased risk of pulmonary fibrosis, tissue damage, and poor treatment outcomes in TB patients. In this study, we used a rabbit model of pulmonary TB to evaluate the impact of adjunctive immune modulation, using a phosphodiesterase-4 inhibitor that dampens the innate immune response, on the outcome of treatment with the antibiotic isoniazid. Our data show that cotreatment of M. tuberculosis infected rabbits with the phosphodiesterase-4 inhibitor CC-3052 plus isoniazid significantly reduced the extent of immune pathogenesis, compared with antibiotic alone, as determined by histologic analysis of infected tissues and the expression of genes involved in inflammation, fibrosis, and wound healing in the lungs. Combined treatment with an antibiotic and CC-3052 not only lessened disease but also improved bacterial clearance from the lungs. These findings support the potential for adjunctive immune modulation to improve the treatment of pulmonary TB and reduce the risk of chronic respiratory impairment.

Mycobacterium leprae actively modulates the cytokine response in naive human monocytes.

Leprosy is a chronic but treatable infectious disease caused by the intracellular pathogen Mycobacterium leprae. Host immunity to M. leprae determines the diversity of clinical manifestations seen in patients, from tuberculoid leprosy with robust production of Th1-type cytokines to lepromatous disease, characterized by elevated levels of Th2-type cytokines and a suboptimal proinflammatory response. Previous reports have indicated that M. leprae is a poor activator of macrophages and dendritic cells in vitro. To understand whether M. leprae fails to elicit an optimal Th1 immune response or actively interferes with its induction, we have examined the early interactions between M. leprae and monocytes from healthy human donors. We found that, in naïve monocytes, M. leprae induced high levels of the negative regulatory molecules MCP-1 and interleukin-1 (IL-1) receptor antagonist (IL-1Ra), while suppressing IL-6 production through phosphoinositide-3 kinase (PI3K)-dependent mechanisms. In addition, low levels of proinflammatory cytokines were observed in association with reduced activation of nuclear factor-kappaB (NF-kappaB) and delayed activation of IL-1beta-converting enzyme, ICE (caspase-1), in monocytes stimulated with M. leprae compared with Mycobacterium bovis BCG stimulation. Interestingly, although in itself a weak stimulator of cytokines, M. leprae primed the cells for increased production of tumor necrosis factor alpha and IL-10 in response to a strongly inducing secondary stimulus. Taken together, our results suggest that M. leprae plays an active role to control the release of cytokines from monocytes by providing both positive and negative regulatory signals via multiple signaling pathways involving PI3K, NF-kappaB, and caspase-1.

Molecular epidemiology of Mycobacterium tuberculosis in a South African community with high HIV prevalence.

To explore the relationship between human immunodeficiency virus (HIV) and Mycobacterium tuberculosis genotypes, we performed IS6110-based restriction fragment-length polymorphism analysis on M. tuberculosis culture specimens from patients with smear-positive tuberculosis in a periurban community in South Africa from 2001 through 2005. Among 151 isolates, 95 strains were identified within 26 families, with 54% clustering. HIV status was associated with W-Beijing strains (P = .009) but not with clustering per se. The high frequency of clustering suggests ongoing transmission in both HIV-negative and HIV-positive individuals in this community. The strong association between W-Beijing and HIV infection may have important implications for tuberculosis control.

Lessons from molecular epidemiology and comparative genomics.

Molecular biology has revolutionized the field of tuberculosis (TB) research. Comparative genomics and molecular epidemiology are providing revelations about the evolutionary origins of MYCOBACTERIUM TUBERCULOSIS and phylogenetic relationships between different strains and strain families. Accumulating evidence indicates that distinct strains of M. TUBERCULOSIS (genotype) may be associated with differential transmissibility, virulence, and/or clinical manifestations (phenotype). As advances in our understanding of the relationships between genotype and phenotype progress, this knowledge will have important ramifications for TB control and the development of novel vaccines and improved diagnostics. Some of the greatest advantages of molecular epidemiological methods include our abilities to follow transmission of particular strains within communities, track epidemics, and recognize the presence of historic outbreaks. Moreover, there are critical questions about TB that are essentially unanswerable in the absence of molecular techniques. These include our capacity to distinguish exogenous reinfection from endogenous reactivation in recurrent TB cases and to recognize primary transmission of drug resistant strains versus the acquisition of drug resistance via de novo mutations. Finally, an elucidation of the phylogenetic structure and evolutionary history of M. TUBERCULOSIS provides a necessary background for understanding the underlying mechanisms responsible for the continued success of this deadly pathogen.

Frequency of Circulating CD4+Ki67+HLA-DR- T Regulatory Cells Prior to Treatment for Multidrug Resistant Tuberculosis Can Differentiate the Severity of Disease and Predict Time to Culture Conversion.

Identifying a blood circulating cellular biomarker that can be used to assess severity of disease and predict the time to culture conversion (TCC) in patients with multidrug resistant tuberculosis (MDR-TB) would facilitate monitoring response to treatment and may be of value in the design of future drug trials. We report on the frequency of blood Ki67+HLA-DR- CD4+ T regulatory (Treg) cells in predicting microbiological outcome before initiating second-line treatment for MDR-TB. Fifty-one patients with MDR-TB were enrolled and followed over 18 months; a subset of patients was sputum culture (SC) negative at baseline (n = 9). SC positive patients were divided into two groups, based on median TCC: rapid responders (≤71 days TCC; n = 21) and slow responders (>71 days TCC; n = 21). Whole blood at baseline, months 2 and 6 was stimulated with M tuberculosis (Mtb) antigens and Treg cells were then identified as CD3+CD4+CD25hiFoxP3+CD127-CD69- and further delineated as Ki67+HLA-DR- Treg. The frequency of these cells was significantly enlarged at baseline in SC positive relative to SC negative and smear positive relative to smear negative patients and in those with lung cavitation. This difference was further supported by unsupervised hierarchical clustering showing a significant grouping at baseline of total and early differentiated memory Treg cells in slow responders. Conversely, there was a clustering of a lower proportion of Treg cells and activated IFNγ-expressing T cells at baseline in the rapid responders. Examining changes over time revealed a more gradual reduction of Treg cells in slow responders relative to rapid responders to treatment. Receiver operating curve analysis showed that baseline Mtb-stimulated Ki67+HLA-DR- Treg cells could predict the TCC of MDR-TB treatment response with 81.2% sensitivity and 85% specificity (AUC of 0.87, p < 0.0001), but this was not the case after 2 months of treatment. In conclusion, our data show that the frequency of a highly defined Mtb-stimulated blood Treg cell population at baseline can discriminate MDR-TB disease severity and predict time to culture clearance.

A combination of baseline plasma immune markers can predict therapeutic response in multidrug resistant tuberculosis.

OBJECTIVE: To identify plasma markers predictive of therapeutic response in patients with multidrug resistant tuberculosis (MDR-TB). METHODS: Fifty HIV-negative patients with active pulmonary MDR-TB were analysed for six soluble analytes in plasma at the time of initiating treatment (baseline) and over six months thereafter. Patients were identified as sputum culture positive or negative at baseline. Culture positive patients were further stratified by the median time to sputum culture conversion (SCC) as fast responders (< 76 days) or slow responders (≥ 76 days). Chest X-ray scores, body mass index, and sputum smear microscopy results were obtained at baseline. RESULTS: Unsupervised hierarchical clustering revealed that baseline plasma levels of IP-10/CXCL10, VEGF-A, SAA and CRP could distinguish sputum culture and cavitation status of patients. Among patients who were culture positive at baseline, there were significant positive correlations between plasma levels of CRP, SAA, VEGF-A, sIL-2Rα/CD40, and IP-10 and delayed SCC. Using linear discriminant analysis (LDA) and Receiver Operating Curves (ROC), we showed that a combination of MCP-1/CCL2, IP-10, sIL-2Rα, SAA, CRP and AFB smear could distinguish fast from slow responders and were predictive of delayed SCC with high sensitivity and specificity. CONCLUSION: Plasma levels of specific chemokines and inflammatory markers measured before MDR-TB treatment are candidate predictive markers of delayed SCC. These findings require validation in a larger study.

Mycobacterium leprae alters classical activation of human monocytes in vitro.

BACKGROUND: Macrophages play a central role in the pathogenesis of leprosy, caused by Mycobacterium leprae. The polarized clinical presentations in leprosy are associated with differential immune activation. In tuberculoid leprosy, macrophages show a classical activation phenotype (M1), while macrophages in lepromatous disease display characteristics of alternative activation (M2). Bacille Calmette-Guérin (BCG) vaccination, which protects against leprosy, can promote sustained changes in monocyte response to unrelated pathogens and may preferentially direct monocytes towards an M1 protective phenotype. We previously reported that M. leprae can dampen the response of naïve human monocytes to a strong inducer of pro-inflammatory cytokines, such as BCG. Here, we investigated the ability of the pathogen to alter the direction of macrophage polarization and the impact of BCG vaccination on the monocyte response to M. leprae. FINDINGS: We show that in vitro exposure of monocytes from healthy donors to M. leprae interferes with subsequent M1 polarization, indicated by lower levels of M1-associated cytokine/chemokines released and reduced expression of M1 cell surface markers. Exposure to M. leprae phenolic glycolipid (PGL) 1, instead of whole bacteria, demonstrated a similar effect on M1 cytokine/chemokine release. In addition, we found that monocytes from 10-week old BCG-vaccinated infants released higher levels of the pro-inflammatory cytokines TNF-α and IL-1ß in response to M. leprae compared to those from unvaccinated infants. CONCLUSION: Exposure to M. leprae has an inhibitory effect on M1 macrophage polarization, likely mediated through PGL-1. By directing monocyte/macrophages preferentially towards M1 activation, BCG vaccination may render the cells more refractory to the inhibitory effects of subsequent M. leprae infection.

Pharmacologic Inhibition of Host Phosphodiesterase-4 Improves Isoniazid-Mediated Clearance of Mycobacterium tuberculosis.

The lengthy duration of multidrug therapy needed to cure tuberculosis (TB) poses significant challenges for global control of the disease. Moreover, chronic inflammation associated with TB leads to pulmonary damage that can remain even after successful cure. Thus, there is a great need for the development of effective shorter drug regimens to improve clinical outcome and strengthen TB control. Host-directed therapy (HDT) is emerging as a novel adjunctive strategy to enhance the efficacy and shorten the duration of TB treatment. Previously, we showed that the administration of CC-3052, a phosphodiesterase-4 inhibitor (PDE4i), reduced the host inflammatory response during Mycobacterium tuberculosis (Mtb) infection and improved the antimicrobial efficacy of isoniazid (INH) in both the mouse and rabbit models. In the present study, we evaluated the pharmacokinetics and explored the mechanism underlying the efficacy of a more potent PDE4i, CC-11050, as adjunct to INH treatment in a mouse model of pulmonary Mtb infection. Genome-wide lung transcriptome analysis confirmed the dampening of inflammation and associated network genes that we previously reported with CC-3052. Consistent with the reduction in inflammation, a significant improvement in Mtb control and pathology was observed in the lungs of mice treated with CC-11050 plus INH, compared to INH alone. This important confirmatory study will be used to help design upcoming human clinical trials with CC-11050 as an HDT for TB treatment.

Correlation of rpoB Mutations with Minimal Inhibitory Concentration of Rifampin and Rifabutin in Mycobacterium tuberculosis in an HIV/AIDS Endemic Setting, South Africa.

Treatment of tuberculosis (TB) and HIV co-infections is often complicated by drug-to-drug interactions between anti-mycobacterial and anti-retroviral agents. Rifabutin (RFB) is an alternative to rifampin (RIF) for TB regimens and is recommended for HIV patients concurrently receiving protease inhibitors because of reduced induction of CYP3A4. This study sought to determine the proportion of RFB susceptible isolates among RIF-resistant strains in a high HIV prevalence setting in South Africa. In addition, the study explored the association between rpoB mutations and minimum inhibitory concentrations (MIC) of RIF and RFB. A total of 189 multidrug resistant (MDR) Mycobacterium tuberculosis isolates from the Centre for Tuberculosis repository were analyzed. The MICs were determined using a MYCOTB Sensititre plate method and the rpoB gene was sequenced. Of the 189 MDR isolates, 138 (73%) showed resistance to both RIF and RFB, while 51 (27%) isolates were resistant to RIF but retained susceptibility to RFB. The S531L was the most frequent rpoB point mutation in 105/189 (56%) isolates, followed by H526Y in 27/189 (14%) isolates. Resistance to both RIF and RFB was found predominantly in association with mutations S531L (91/105, 87%), H526Y (20/27, 74%), and H526D (15/19, 79%), while D516V (15/17, 88%), and L533P (3/4, 75%) were found in RIF-resistant, RFB-susceptible isolates. This study has shown that up to 27% of MDR-TB patients in South Africa may benefit from a treatment regimen that includes RFB.

A Novel Molecular Strategy for Surveillance of Multidrug Resistant Tuberculosis in High Burden Settings.

BACKGROUND: In South Africa and other high prevalence countries, transmission is a significant contributor to rising rates of multidrug resistant tuberculosis (MDR-TB). Thus, there is a need to develop an early detection system for transmission clusters suitable for high burden settings. We have evaluated the discriminatory power and clustering concordance of a novel and simple genotyping approach, combining spoligotyping with pncA sequencing (SpoNC), against two well-established methods: IS6110-RFLP and 24-loci MIRU-VNTR. METHODS: A total of 216 MDR-TB isolates collected from January to June 2010 from the NHLS Central TB referral laboratory in Braamfontein, Johannesburg, representing a diversity of strains from South Africa, were included. The isolates were submitted for genotyping, pncA sequencing and analysis to the Centre for Tuberculosis in South Africa and the Public Health Research Institute Tuberculosis Center at Rutgers University in the United States. Clustering rates, Hunter-Gaston Discriminatory Indexes (HGI) and Wallace coefficients were compared between the methods. RESULTS: Overall clustering rates were high by both IS6110-RFLP (52.8%) and MIRU-VNTR (45.8%), indicative of on-going transmission. Both 24-loci MIRU-VNTR and IS6110-RFLP had similar HGI (0.972 and 0.973, respectively), with close numbers of unique profiles (87 vs. 70), clustered isolates (129 vs. 146), and cluster sizes (2 to 26 vs. 2 to 25 isolates). Spoligotyping alone was the least discriminatory (80.1% clustering, HGI 0.903), with 28 unique types. However, the discriminatory power of spoligotyping was improved when combined with pncA sequencing using the SpoNC approach (61.8% clustering, HGI 0.958). A high proportion of MDR-TB isolates had mutations in pncA (68%, n = 145), and pncA mutations were significantly associated with clustering (p = 0.007 and p = 0.0013 by 24-loci MIRU-VNTR and IS6110-RFLP, respectively), suggesting high rates of resistance to pyrazinamide among all MDR-TB cases and particularly among clustered cases. CONCLUSION: We conclude that SpoNC provides good discrimination for MDR-TB surveillance and early identification of outbreaks in South Africa, with 24-loci MIRU-VNTR applied for pncA wild-type strains as needed.

Etanercept exacerbates inflammation and pathology in a rabbit model of active pulmonary tuberculosis.

Treatment of chronic inflammatory diseases with tumor necrosis factor alpha (TNF-α) antagonists has been associated with increased risk of tuberculosis (TB). We examined the usefulness of the rabbit model of active pulmonary TB for studying the impact of the human immune modulatory reagent etanercept on the host immune response. Control of Mycobacterium tuberculosis (Mtb) infection, disease pathology, and the global transcriptional response in Mtb-infected lungs of rabbits were studied. Etanercept treatment exacerbated disease pathology and reduced bacillary control in the lungs, compared with infected untreated animals. Reduced collagen and fibrin deposition in the granulomas was associated with significant downregulation of the collagen metabolism and fibrosis network genes and upregulation of genes in the inflammatory response and cell recruitment networks in the lungs of etanercept treated, compared with untreated rabbits. Our results suggest that targeting the TNF-α signaling pathway disrupts the tissue remodeling process, which is required for the formation and maintenance of well-differentiated granulomas and for control of Mtb growth in the lungs. These results validate the use of the rabbit model for investigating the impact of selected human immune modulatory drugs, such as a TNF-α antagonist, on the host immune response and pathogenesis in TB.

A subset of circulating blood mycobacteria-specific CD4 T cells can predict the time to Mycobacterium tuberculosis sputum culture conversion.

We investigated 18 HIV-negative patients with MDR-TB for M. tuberculosis (Mtb)- and PPD-specific CD4 T cell responses and followed them over 6 months of drug therapy. Twelve of these patients were sputum culture (SC) positive and six patients were SC negative upon enrollment. Our aim was to identify a subset of mycobacteria-specific CD4 T cells that would predict time to culture conversion. The total frequency of mycobacteria-specific CD4 T cells at baseline could not distinguish patients showing positive or negative SC. However, a greater proportion of late-differentiated (LD) Mtb- and PPD-specific memory CD4 T cells was found in SC positive patients than in those who were SC negative (p = 0.004 and p = 0.0012, respectively). Similarly, a higher co-expression of HLA-DR+ Ki67+ on Mtb- and PPD-specific CD4 T cells could also discriminate between sputum SC positive versus SC negative (p = 0.004 and p = 0.001, respectively). Receiver operating characteristic (ROC) analysis revealed that baseline levels of Ki67+ HLA-DR+ Mtb- and PPD-specific CD4 T cells were predictive of the time to sputum culture conversion, with area-under-the-curve of 0.8 (p = 0.027). Upon treatment, there was a significant decline of these Ki67+ HLA-DR+ T cell populations in the first 2 months, with a progressive increase in mycobacteria-specific polyfunctional IFNγ+ IL2+ TNFα+ CD4 T cells over 6 months. Thus, a subset of activated and proliferating mycobacterial-specific CD4 T cells (Ki67+ HLA-DR+) may provide a valuable marker in peripheral blood that predicts time to sputum culture conversion in TB patients at the start of treatment.