Multiple Introductions of Yersinia pestis during Urban Pneumonic Plague Epidemic, Madagascar, 2017.

Pneumonic plague (PP) is characterized by high infection rate, person-to-person transmission, and rapid progression to severe disease. In 2017, a PP epidemic occurred in 2 Madagascar urban areas, Antananarivo and Toamasina. We used epidemiologic data and Yersinia pestis genomic characterization to determine the sources of this epidemic. Human plague emerged independently from environmental reservoirs in rural endemic foci >20 times during August-November 2017. Confirmed cases from 5 emergences, including 4 PP cases, were documented in urban areas. Epidemiologic and genetic analyses of cases associated with the first emergence event to reach urban areas confirmed that transmission started in August; spread to Antananarivo, Toamasina, and other locations; and persisted in Antananarivo until at least mid-November. Two other Y. pestis lineages may have caused persistent PP transmission chains in Antananarivo. Multiple Y. pestis lineages were independently introduced to urban areas from several rural foci via travel of infected persons during the epidemic.

Using a multiplex serological assay to estimate time since SARS-CoV-2 infection and past clinical presentation in malagasy patients.

Background: The world is facing a 2019 coronavirus (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this context, efficient serological assays are needed to accurately describe the humoral responses against the virus. These tools could potentially provide temporal and clinical characteristics and are thus paramount in developing-countries lacking sufficient ongoing COVID-19 epidemic descriptions. Methods: We developed and validated a Luminex xMAP® multiplex serological assay targeting specific IgM and IgG antibodies against the SARS-CoV-2 Spike subunit 1 (S1), Spike subunit 2 (S2), Spike Receptor Binding Domain (RBD) and the Nucleocapsid protein (N). Blood samples collected periodically for 12 months from 43 patients diagnosed with COVID-19 in Madagascar were tested for these antibodies. A random forest algorithm was used to build a predictive model of time since infection and symptom presentation. Findings: The performance of the multiplex serological assay was evaluated for the detection of SARS-CoV-2 anti-IgG and anti-IgM antibodies. Both sensitivity and specificity were equal to 100% (89.85-100) for S1, RBD and N (S2 had a lower specificity = 95%) for IgG at day 14 after enrolment. This multiplex assay compared with two commercialized ELISA kits, showed a higher sensitivity. Principal Component Analysis was performed on serologic data to group patients according to time of sample collection and clinical presentations. The random forest algorithm built by this approach predicted symptom presentation and time since infection with an accuracy of 87.1% (95% CI = 70.17-96.37, p-value = 0.0016), and 80% (95% CI = 61.43-92.29, p-value = 0.0001) respectively. Interpretation: This study demonstrates that the statistical model predicts time since infection and previous symptom presentation using IgM and IgG response to SARS-CoV2. This tool may be useful for global surveillance, discriminating recent- and past- SARS-CoV-2 infection, and assessing disease severity. Fundings: This study was funded by the French Ministry for Europe and Foreign Affairs through the REPAIR COVID-19-Africa project coordinated by the Pasteur International Network association. WANTAI reagents were provided by WHO AFRO as part of a Sero-epidemiological "Unity" Study Grant/Award Number: 2020/1,019,828-0 P·O 202546047 and Initiative 5% grant n°AP-5PC-2018-03-RO.

Plasma host protein signatures correlating with Mycobacterium tuberculosis activity prior to and during antituberculosis treatment.

There is a need for rapid non-sputum-based tests to identify and treat patients infected with Mycobacterium tuberculosis (Mtb). The overall objective of this study was to measure and compare the expression of a selected panel of human plasma proteins in patients with active pulmonary tuberculosis (ATB) throughout anti-TB treatment (from baseline to the end of treatment), in Mtb-infected individuals (TBI) and healthy donors (HD) to identify a putative host-protein signature useful for both TB diagnosis and treatment monitoring. A panel of seven human host proteins CLEC3B, SELL, IGFBP3, IP10, CD14, ECM1 and C1Q were measured in the plasma isolated from an HIV-negative prospective cohort of 37 ATB, 24 TBI and 23 HD. The protein signatures were assessed using a Luminex xMAP® to quantify the plasmatic levels in unstimulated blood of the different clinical group as well as the protein levels at baseline and at three timepoints during the 6-months ATB treatment, to compare the plasma protein levels between culture slow and fast converters that may contribute to monitor the TB treatment outcome. Protein signatures were defined using the CombiROC algorithm and multivariate models. The studied plasma host proteins showed different levels between the clinical groups and during the TB treatment. Six of the plasma proteins (CLEC3B, SELL, IGFBP3, IP10, CD14 and C1Q) showed significant differences in normalised median fluorescence intensities when comparing ATB vs HD or TBI groups while ECM1 revealed a significant difference between fast and slow sputum culture converters after 2 months following treatment (p = 0.006). The expression of a four-host protein markers (CLEC3B-ECM1-IP10-SELL) was significantly different between ATB from HD or TBI groups (respectively, p < 0.05). The expression of the same signature was significantly different between the slow vs the fast sputum culture converters after 2 months of treatment (p < 0.05). The results suggest a promising 4 host-plasma marker signature that would be associated with both TB diagnostic and treatment monitoring.

Analytical framework to evaluate and optimize the use of imperfect diagnostics to inform outbreak response: Application to the 2017 plague epidemic in Madagascar.

During outbreaks, the lack of diagnostic "gold standard" can mask the true burden of infection in the population and hamper the allocation of resources required for control. Here, we present an analytical framework to evaluate and optimize the use of diagnostics when multiple yet imperfect diagnostic tests are available. We apply it to laboratory results of 2,136 samples, analyzed with 3 diagnostic tests (based on up to 7 diagnostic outcomes), collected during the 2017 pneumonic (PP) and bubonic plague (BP) outbreak in Madagascar, which was unprecedented both in the number of notified cases, clinical presentation, and spatial distribution. The extent of these outbreaks has however remained unclear due to nonoptimal assays. Using latent class methods, we estimate that 7% to 15% of notified cases were Yersinia pestis-infected. Overreporting was highest during the peak of the outbreak and lowest in the rural settings endemic to Y. pestis. Molecular biology methods offered the best compromise between sensitivity and specificity. The specificity of the rapid diagnostic test was relatively low (PP: 82%, BP: 85%), particularly for use in contexts with large quantities of misclassified cases. Comparison with data from a subsequent seasonal Y. pestis outbreak in 2018 reveal better test performance (BP: specificity 99%, sensitivity: 91%), indicating that factors related to the response to a large, explosive outbreak may well have affected test performance. We used our framework to optimize the case classification and derive consolidated epidemic trends. Our approach may help reduce uncertainties in other outbreaks where diagnostics are imperfect.

Genotype Diversity and Spread of White Spot Syndrome Virus (WSSV) in Madagascar (2012-2016).

White Spot Disease (WSD) caused by the White Spot Syndrome Virus (WSSV) is the most devastating viral disease threatening the shrimp culture industry worldwide, including Madagascar. WDS was first reported on the island in 2012; however, little is known about the circulation of the virus and its genetic diversity. Our study aimed at describing the molecular diversity and the spread of WSSV in the populations of Madagascan crustaceans. Farmed and wild shrimps were collected from various locations in Madagascar from 2012 to 2016 and were tested for WSSV. Amplicons from positive specimens targeting five molecular markers (ORF75, ORF94, ORF125, VR14/15 and VR23/24) were sequenced for genotyping characterizations. Four genotypes were found in Madagascar. The type-I genotype was observed in the south-west of Madagascar in April 2012, causing a disastrous epidemic, then spread to the North-West coast. Type-II strains were detected in October 2012 causing an outbreak in another Penaeus monodon farm. In 2014 and 2015, types II and III were observed in shrimp farms. Finally, in 2016, types II and IV were found in wild species including Fenneropenaeus indicus, Metapenaeus monoceros, Marsupenaeus japonicus and Macrobrachium rosenbergii. Considering the economic importance of the shrimp industry for Madagascar, our study highlights the need to maintain WSSV surveillance to quickly take appropriate countermeasures in case of outbreak and to sustain this industry.

Multi-country evaluation of RISK6, a 6-gene blood transcriptomic signature, for tuberculosis diagnosis and treatment monitoring.

There is a crucial need for non-sputum-based TB tests. Here, we evaluate the performance of RISK6, a human-blood transcriptomic signature, for TB screening, triage and treatment monitoring. RISK6 performance was also compared to that of two IGRAs: one based on RD1 antigens (QuantiFERON-TB Gold Plus, QFT-P, Qiagen) and one on recombinant M. tuberculosis HBHA expressed in Mycobacterium smegmatis (IGRA-rmsHBHA). In this multicenter prospective nested case-control study conducted in Bangladesh, Georgia, Lebanon and Madagascar, adult non-immunocompromised patients with bacteriologically confirmed active pulmonary TB (ATB), latent TB infection (LTBI) and healthy donors (HD) were enrolled. ATB patients were followed-up during and after treatment. Blood RISK6 scores were assessed using quantitative real-time PCR and evaluated by area under the receiver-operating characteristic curve (ROC AUC). RISK6 performance to discriminate ATB from HD reached an AUC of 0.94 (95% CI 0.89-0.99), with 90.9% sensitivity and 87.8% specificity, thus achieving the minimal WHO target product profile for a non-sputum-based TB screening test. Besides, RISK6 yielded an AUC of 0.93 (95% CI 0.85-1) with 90.9% sensitivity and 88.5% specificity for discriminating ATB from LTBI. Moreover, RISK6 showed higher performance (AUC 0.90, 95% CI 0.85-0.94) than IGRA-rmsHBHA (AUC 0.75, 95% CI 0.69-0.82) to differentiate TB infection stages. Finally, RISK6 signature scores significantly decreased after 2 months of TB treatment and continued to decrease gradually until the end of treatment reaching scores obtained in HD. We confirmed the performance of RISK6 signature as a triage TB test and its utility for treatment monitoring.

SARS-CoV-2 antibody seroprevalence follow-up in Malagasy blood donors during the 2020 COVID-19 Epidemic.

BACKGROUND: The incidence of the 2020 COVID-19 epidemic in Africa seems to be different from that of the rest of the world, however its true extent is probably underestimated. Conducting population based sero-surveys during the epidemic has moreover been extremely challenging, driving our group and others to study blood donor samples. METHODS: We collected regional epidemiological COVID-19 surveillance data, and simultaneously monitored anti-SARS-CoV-2 antibody seroprevalences monthly throughout the epidemic in 5 major Region-associated Blood Transfusion Centres of Madagascar over a period of 9 months. FINDINGS: Soon after attaining the first epidemic peaks between May and August 2020, both crude and population-weighted test-performance-adjusted seroprevalences of anti-SARS-CoV-2 antibodies was in Malagasy blood donors rapidly increased up to over 40% positivity. INTERPRETATION: These findings suggest a high cumulative incidence of infection and seroconversion, which may have contributed to the observed deceleration of infection rates, but was not sufficient to prevent the second epidemic wave that struck Madagascar in Spring 2021. FUNDING: This project was funded by the United States Agency for International Development.

Local adaptation in populations of Mycobacterium tuberculosis endemic to the Indian Ocean Rim.

Background: Lineage 1 (L1) and 3 (L3) are two lineages of the Mycobacterium tuberculosis complex (MTBC) causing tuberculosis (TB) in humans. L1 and L3 are prevalent around the rim of the Indian Ocean, the region that accounts for most of the world's new TB cases. Despite their relevance for this region, L1 and L3 remain understudied. Methods: We analyzed 2,938 L1 and 2,030 L3 whole genome sequences originating from 69 countries. We reconstructed the evolutionary history of these two lineages and identified genes under positive selection. Results: We found a strongly asymmetric pattern of migration from South Asia toward neighboring regions, highlighting the historical role of South Asia in the dispersion of L1 and L3. Moreover, we found that several genes were under positive selection, including genes involved in virulence and resistance to antibiotics. For L1 we identified signatures of local adaptation at the esxH locus, a gene coding for a secreted effector that targets the human endosomal sorting complex, and is included in several vaccine candidates. Conclusions: Our study highlights the importance of genetic diversity in the MTBC, and sheds new light on two of the most important MTBC lineages affecting humans.

Different PPD-stimulated cytokine responses from patients infected with genetically distinct Mycobacterium tuberculosis complex lineages.

OBJECTIVES: The genetic diversity of Mycobacterium tuberculosis complex (MTBC) influences the immune response of the host, which may affect the immunodiagnostic tests and biomarker assessment studies used for tuberculosis (TB). This study aimed to determine whether the mycobacterial-antigen-stimulated cytokine responses vary with the genotype of the MTBC infecting the patient. METHODS: Eighty-one patients with confirmed active pulmonary TB were recruited, and MTBC clinical strains were isolated from their sputum for bacterial lineage single-nucleotide polymorphism typing. Whole blood was drawn from the patients to measure the purified protein derivative (PPD)-stimulated cytokine responses (GM-CSF, IFN-γ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, TNF-α, IFN-α, IL-12, eotaxin, IL-13, IL-15, IL-17, MIP1-α, MIP1-ß, MCP1, IL1RA, IP10, IL2R, MIG) with the Luminex multiplex immunoassay. RESULTS: Of the 24 cytokines studied, three were produced differentially in whole blood dependent on the infecting lineage of MTBC. Decreased production of IL-17 was observed in patients infected with modern lineages compared with patients infected with ancestral lineages (P < 0.01), and production of IFN-γ and IL-2 was significantly decreased in patients infected with lineage 4 strains compared with patients infected with lineage 3 strains (P < 0.05). CONCLUSION: MTBC strains belonging to lineage 4 induced a decreased whole-blood PPD-stimulated pro-inflammatory cytokine response.

Multidrug-resistant tuberculosis surveillance and cascade of care in Madagascar: a five-year (2012-2017) retrospective study.

BACKGROUND: In Madagascar, the multidrug-resistant tuberculosis (MDR-TB) surveillance programme was launched in late 2012 wherein previously treated TB cases and symptomatic MDR-TB contacts (hereafter called presumptive MDR-TB cases) undergo drug susceptibility testing. This retrospective review had per aim to provide an update on the national MDR-TB epidemiology, assess and enhance programmatic performance and assess Madagascar's MDR-TB cascade of care. METHODS: For 2012-2017, national TB control programme notification, clinical management data and reference laboratory data were gathered. The development and coverage of the surveillance programme, the MDR-TB epidemiology and programmatic performance indicators were assessed using descriptive, logistic and spatial statistical analyses. Data for 2017 was further used to map Madagascar's TB and MDR-TB cascade of care. RESULTS: The geographical coverage and diagnostic and referral capacities of the MDR-TB surveillance programme were gradually expanded whereas regional variations persist with regard to coverage, referral rates and sample referral delays. Overall, the rate of MDR-TB among presumptive MDR-TB cases remained relatively stable, ranging between 3.9% in 2013 and 4.4% in 2017. Most MDR-TB patients were lost in the second gap of the cascade pertaining to MDR-TB cases reaching diagnostic centres but failing to be accurately diagnosed (59.0%). This poor success in diagnosis of MDR-TB is due to both the current use of low-sensitivity smear microscopy as a first-line diagnostic assay for TB and the limited access to any form of drug susceptibility testing. Presumptive MDR-TB patients' sample referral took a mean delay of 28 days before testing. Seventy-five percent of diagnosed MDR-TB patients were appropriately initiated on treatment, and 33% reached long-term recurrence-free survival. CONCLUSIONS: An expansion of the coverage and strengthening of MDR-TB diagnostic and management capacities are indicated across all regions of Madagascar. With current limitations, the surveillance programme data is likely to underestimate the true MDR-TB burden in the country and an updated national MDR-TB prevalence survey is warranted. In absence of multiple drivers of an MDR-TB epidemic, including high MDR-TB rates, high HIV infection rates and inter-country migration, Madagascar is in a favourable starting position for MDR-TB control and elimination.

Performance and impact of GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit® assays on tuberculosis case detection in Madagascar.

BACKGROUND: Tuberculosis rapid molecular assays, including GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit®, are highly sensitive and specific. Such performance does not automatically translate in improved disease control and highly depends on their use, local epidemiology and the diagnostic algorithms they're implemented within. We evaluate the performance of both assays and assess their impact on additional cases notification when implemented within WHO recommended tuberculosis diagnostic algorithms in Madagascar. METHODS: Five hundred forty eight presumptive pulmonary tuberculosis patients were prospectively recruited between November 2013 and December 2014 in Antananarivo, Madagascar, a high TB incidence sub-Saharan African urban setting. Both molecular assays were evaluated as first line or add-on testing following negative smear microscopy. Based on locally defined assay performance characteristics we measure the impact of both assays and WHO-recommended diagnostic algorithms on additional tuberculosis case notifications. RESULTS: High sensitivity and specificity was confirmed for both GeneXpert MTB/RIF® (86.6% (95% CI 81.1-90.7%) and 97.4% (95% CI 94.9-98.8%)) and Loopamp MTBC Detection Kit® (84.6% (95% CI 78.9-89.0%) and 98.4% (95% CI 96.2-99.4%)). Implementation of GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit® increased tuberculosis diagnostic algorithms sensitivity from 73.6% (95% CI 67.1-79.3%) up to 88.1% (95% CI 82.8-91.9%). This increase was highest when molecular assays were used as add-on testing following negative smear microscopy. As add-on testing, GeneXpert MTB/RIF® and Loopamp MTBC Detection Kit® respectively improved case detection by 23.8 and 21.2% (p < 0.05). CONCLUSION: Including GeneXpert MTB/RIF® or Loopamp MTBC Detection Kit® molecular assays for TB detection on sputum samples from presumptive TB cases can significantly increase case notification in TB diagnostic centers. The TB case detection rate is further increased when those tests are use as second-line follow-on testing following negative smear microscopy results. A country wide scale-up and digital integration of molecular-based TB diagnosis assays shows promises for TB control in Madagascar.

Epidemiological characteristics of an urban plague epidemic in Madagascar, August-November, 2017: an outbreak report.

BACKGROUND: Madagascar accounts for 75% of global plague cases reported to WHO, with an annual incidence of 200-700 suspected cases (mainly bubonic plague). In 2017, a pneumonic plague epidemic of unusual size occurred. The extent of this epidemic provides a unique opportunity to better understand the epidemiology of pneumonic plagues, particularly in urban settings. METHODS: Clinically suspected plague cases were notified to the Central Laboratory for Plague at Institut Pasteur de Madagascar (Antananarivo, Madagascar), where biological samples were tested. Based on cases recorded between Aug 1, and Nov 26, 2017, we assessed the epidemiological characteristics of this epidemic. Cases were classified as suspected, probable, or confirmed based on the results of three types of diagnostic tests (rapid diagnostic test, molecular methods, and culture) according to 2006 WHO recommendations. FINDINGS: 2414 clinically suspected plague cases were reported, including 1878 (78%) pneumonic plague cases, 395 (16%) bubonic plague cases, one (<1%) septicaemic case, and 140 (6%) cases with unspecified clinical form. 386 (21%) of 1878 notified pneumonic plague cases were probable and 32 (2%) were confirmed. 73 (18%) of 395 notified bubonic plague cases were probable and 66 (17%) were confirmed. The case fatality ratio was higher among confirmed cases (eight [25%] of 32 cases) than probable (27 [8%] of 360 cases) or suspected pneumonic plague cases (74 [5%] of 1358 cases) and a similar trend was seen for bubonic plague cases (16 [24%] of 66 confirmed cases, four [6%] of 68 probable cases, and six [2%] of 243 suspected cases). 351 (84%) of 418 confirmed or probable pneumonic plague cases were concentrated in Antananarivo, the capital city, and Toamasina, the main seaport. All 50 isolated Yersinia pestis strains were susceptible to the tested antibiotics. INTERPRETATION: This predominantly urban plague epidemic was characterised by a large number of notifications in two major urban areas and an unusually high proportion of pneumonic forms, with only 23% having one or more positive laboratory tests. Lessons about clinical and biological diagnosis, case definition, surveillance, and the logistical management of the response identified in this epidemic are crucial to improve the response to future plague outbreaks. FUNDING: US Agency for International Development, WHO, Institut Pasteur, US Department of Health and Human Services, Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases, Models of Infectious Disease Agent Study of the National Institute of General Medical Sciences, AXA Research Fund, and the INCEPTION programme.

Usefulness of environmental DNA for detecting Schistosoma mansoni occurrence sites in Madagascar.

OBJECTIVES: Schistosomiasis is an important disease in Madagascar, and several studies on the disease have focused on the occurrence of the parasite in humans. However, the range of the pathogen in the environment and its impact on human infection is difficult to predict. An environmental DNA (eDNA) detection system for Schistosoma mansoni was developed to improve schistosomiasis eco-epidemiology studies. METHODS: Primers and probes were designed and tested in experimental biotopes. The field study was conducted in Maevatanana District of Madagascar. Seven water sources with human use were sampled, with a total of 21 water samples collected. Snails were collected, and patients were examined by ultrasound to determine the occurrence of schistosomiasis in the study area. RESULTS: One water source with active transmission was identified through the detection of S. mansoni eDNA in the water and the intermediate host Biomphalaria pfeifferi collected from the same water source. People with clinical schistosomiasis were found in the area, reinforcing the findings. CONCLUSIONS: The application of eDNA in eco-epidemiology enables the determination of hot spots and safe spots in endemic areas, constituting an alternative ecological tool for follow-up and monitoring of control programs for schistosomiasis, and contributing information on water safety for improving the standard of living of the people in endemic areas.

Study on causes of fever in primary healthcare center uncovers pathogens of public health concern in Madagascar.

BACKGROUND: The increasing use of malaria diagnostic tests reveals a growing proportion of patients with fever but no malaria. Clinicians and health care workers in low-income countries have few tests to diagnose causes of fever other than malaria although several diseases share common symptoms. We propose here to assess etiologies of fever in Madagascar to ultimately improve management of febrile cases. METHODOLOGY: Consenting febrile outpatients aged 6 months and older were recruited in 21 selected sentinel sites throughout Madagascar from April 2014 to September 2015. Standard clinical examinations were performed, and blood and upper respiratory specimens were taken for rapid diagnostic tests and molecular assays for 36 pathogens of interest for Madagascar in terms of public health, regardless of clinical status. PRINCIPAL FINDINGS: A total of 682 febrile patients were enrolled. We detected at least one pathogen in 40.5% (276/682) of patients and 6.2% (42/682) with co-infections. Among all tested patients, 26.5% (181/682) had at least one viral infection, 17.0% (116/682) had malaria and 1.0% (7/682) presented a bacterial or a mycobacterial infection. None or very few of the highly prevalent infectious agents in Eastern Africa and Asia were detected in this study, such as zoonotic bacteria or arboviral infections. CONCLUSIONS: These results raise questions about etiologies of fever in Malagasy communities. Nevertheless, we noted that viral infections and malaria still represent a significant proportion of causes of febrile illnesses. Interestingly our study allowed the detection of pathogens of public health interest such as Rift Valley Fever Virus but also the first case of laboratory-confirmed leptospirosis infection in Madagascar.

Cytokine Biomarkers Associated with Human Extra-Pulmonary Tuberculosis Clinical Strains and Symptoms.

Background: The primary site of infection for Mycobacterium tuberculosis (Mtb) is the alveolar macrophages. However, Mtb can disseminate into other organs and causes extrapulmonary tuberculosis (EPTB). The diagnosis of EPTB is challenging due to relatively inaccessible infectious sites that may be paucibacillary and with clinical symptoms varying by site that are similar to those seen in other diseases. Hence, we sought to identify the expression patterns of a variety of cytokines that may be specific to EPTB from in vitro infections and in the plasma of TB patients. Methods: To define those cytokine secretions associated with EPTB, human THP-1 derived macrophages were first infected with Mtb clinical isolates from pulmonary and EPTB. Infected macrophages supernatants were harvested at different time points and cytokines known to play key roles in TB immune responses including TNF-α, IL-6, IL-10, IFN-γ, and VEGF-A were measured by ELISA. Those cytokines that were in vitro associated to EPTB were also measured in the plasma from patients with PTB, EPTB, non-EPTB-confirmed-like symptoms and healthy controls. Results: While all of the studied cytokine secretions varied after in vitro infection, higher levels of TNF-α and VEGF secretions were observed in vitro in the infected macrophages respectively in the PTB and EPTB infecting clinical isolates. Similar trends were observed from the plasma of patients where patients with PTB showed significantly higher level of TNF-α compared to EPTB and healthy control groups. The patients with EPTB showed higher plasma level of VEGF compared to those patients with the non-EPTB (p < 0.01) and to healthy controls group (p < 0.0001). Using Receiver Operating Curves (ROC), we showed that TNF-α and VEGF concentrations could distinguish EPTB from non-confirmed EPTB with high sensitivity and specificity. Conclusion: Pulmonary and extrapulmonary Mtb clinical isolates showed different cytokine induction pattern in human macrophages that is also found in the plasma level of the EPTB patients. Further investigations are needed to define cytokine secretions that can lead to the definition of bio-signatures to differentiate EPTB from other pathologies with confusing symptoms that hampered the diagnosis of TB.

Optimizing of a protein extraction method for Mycobacterium tuberculosis proteome analysis using mass spectrometry.

A critical step in proteomic analyses comprises the implementation of a reliable cell lysis method with high yields of qualitative proteins. In Mycobacteria, the protein extraction step is often hampered by the thick waxy cell wall which is rich in mycolic acids. Harsh disruption techniques to release proteins from the cells are thus required. Here, we demonstrate an optimized protein extraction procedure for Mycobacterium tuberculosis (Mbt) that results in protein extracts that are useful for all currently used proteomics platforms, including gel and LC-MS based strategies. We compared the effectiveness of using both thiourea and urea and/or SDS and DTT in the solubilization buffer, in combination or not with sonication and/or bead beating. After some preliminary optimization steps on fast-growing Mbt-like organisms, namely Mycobacterium smegmatis and Mycobacterium fortuitum, the final protein extraction protocol was tested on M. tuberculosis. Based on the concentrations of the proteins recovered from each of the tested methods and on the quality of the extracted proteins as evaluated by SDS PAGE, we propose a lysis buffer that contains both thiourea and urea, in combination with two mechanical cell disruption methods: sonication and bead beating. The optimized protocol results in protein extracts that are useful in M. tuberculosis proteomics studies based on any proteomics strategy or platform.

Mycobacterium tuberculosis exploits the formation of new blood vessels for its dissemination.

The mechanisms by which the airborne pathogen Mycobacterium tuberculosis spreads within the lung and leaves its primary niche to colonize other organs, thus inducing extrapulmonary forms of tuberculosis (TB) in humans, remains poorly understood. Herein, we used a transcriptomic approach to investigate the host cell gene expression profile in M. tuberculosis-infected human macrophages (ΜΦ). We identified 33 genes, encoding proteins involved in angiogenesis, for which the expression was significantly modified during infection, and we show that the potent angiogenic factor VEGF is secreted by M. tuberculosis-infected ΜΦ, in an RD1-dependent manner. In vivo these factors promote the formation of blood vessels in murine models of the disease. Inhibiting angiogenesis, via VEGF inactivation, abolished mycobacterial spread from the infection site. In accordance with our in vitro and in vivo results, we show that the level of VEGF in TB patients is elevated and that endothelial progenitor cells are mobilized from the bone marrow. These results strongly strengthen the most recent data suggesting that mycobacteria take advantage of the formation of new blood vessels to disseminate.

Tuberculosis is associated with expansion of a motile, permissive and immunomodulatory CD16(+) monocyte population via the IL-10/STAT3 axis.

The human CD14(+) monocyte compartment is composed by two subsets based on CD16 expression. We previously reported that this compartment is perturbed in tuberculosis (TB) patients, as reflected by the expansion of CD16(+) monocytes along with disease severity. Whether this unbalance is beneficial or detrimental to host defense remains to be elucidated. Here in the context of active TB, we demonstrate that human monocytes are predisposed to differentiate towards an anti-inflammatory (M2-like) macrophage activation program characterized by the CD16(+)CD163(+)MerTK(+)pSTAT3(+) phenotype and functional properties such as enhanced protease-dependent motility, pathogen permissivity and immunomodulation. This process is dependent on STAT3 activation, and loss-of-function experiments point towards a detrimental role in host defense against TB. Importantly, we provide a critical correlation between the abundance of the CD16(+)CD163(+)MerTK(+)pSTAT3(+) cells and the progression of the disease either at the local level in a non-human primate tuberculous granuloma context, or at the systemic level through the detection of the soluble form of CD163 in human sera. Collectively, this study argues for the pathogenic role of the CD16(+)CD163(+)MerTK(+)pSTAT3(+) monocyte-to-macrophage differentiation program and its potential as a target for TB therapy, and promotes the detection of circulating CD163 as a potential biomarker for disease progression and monitoring of treatment efficacy.

Performance of Four Transport and Storage Systems for Molecular Detection of Multidrug-Resistant Tuberculosis.

BACKGROUND: Detection of drug-resistant tuberculosis is essential for the control of the disease but it is often hampered by the limitation of transport and storage of samples from remote locations to the reference laboratory. We performed a retrospective field study to evaluate the performance of four supports enabling the transport and storage of samples to be used for molecular detection of drug resistance using the GenoType MTBDRplus. METHODS: Two hundred Mycobacterium tuberculosis strains were selected and spotted on slides, FTA cards, GenoCards, and in ethanol. GenoType MTBDRplus was subsequently performed with the DNA extracted from these supports. Sensitivity and specificity were calculated and compared to the results obtained by drug susceptibility testing. RESULTS: For all supports, the overall sensitivity and specificity for detection of resistance to RIF was between 95% and 100%, and for INH between 95% and 98%. CONCLUSION: The four transport and storage supports showed a good sensitivity and specificity for the detection of resistance to RIF and INH in M. tuberculosis strains using the GenoType MTBDRplus. These supports can be maintained at room temperature and could represent an important alternative cost-effective method useful for rapid molecular detection of drug-resistant TB in low-resource settings.