Publications by authors named "Anandi Martin"

92 Publications

Integrative transnational analysis to dissect tuberculosis transmission events along the migratory route from Africa to Europe.

J Travel Med 2021 Jun;28(4)

Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain.

Background: Growing international migration has increased the complexity of tuberculosis transmission patterns. Italy's decision to close its borders in 2018 made of Spain the new European porte entrée for migration from the Horn of Africa (HA). In one of the first rescues of migrants from this region at the end of 2018, tuberculosis was diagnosed in eight subjects, mainly unaccompanied minors.

Methods: Mycobacterium tuberculosis isolates from these recently arrived migrants were analysed by Mycobacterial Interspersed Repetitive-Unit/Variable-Number of Tandem Repeat (MIRU-VNTR) and subsequent whole genome sequencing (WGS) analysis. Data were compared with those from collections from other European countries receiving migrants from the HA and a strain-specific PCR was applied for a fast searching of common strains. Infections in a cellular model were performed to assess strain virulence.

Results: MIRU-VNTR analysis allowed identifying an epidemiological cluster involving three of the eight cases from Somalia (0 single-nucleotide polymorphisms between isolates, HA cluster). Following detailed interviews revealed that two of these cases had shared the same migratory route in most of the trip and had spent a long time at a detention camp in Libya. To confirm potential en route transmission for the three cases, we searched the same strain in collections from other European countries receiving migrants from the HA. MIRU-VNTR, WGS and a strain-specific PCR for the HA strain were applied. The same strain was identified in 12 cases from Eritrea diagnosed soon after their arrival in 2018 to the Netherlands, Belgium and Italy. Intracellular replication rate of the strain did not reveal abnormal virulence.

Conclusions: Our study suggests a potential en route transmission of a pan-susceptible strain, which caused at least 15 tuberculosis cases in Somalian and Eritrean migrants diagnosed in four different European countries.
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http://dx.doi.org/10.1093/jtm/taab054DOI Listing
June 2021

Targeting bedaquiline mycobacterial efflux pump to potentially enhance therapy in abscessus.

Int J Mycobacteriol 2020 Jan-Mar;9(1):71-75

University Hospital Saint-Luc, Brussels, Belgium.

Background: Mycobacterium abscessus is notorious for being intrinsically resistant to most antibiotics. Antibiotic efflux is one of the mechanisms used by M. abscessus to pump out antibiotics from their cells. Inhibiting efflux pumps (EPs) can be an attractive strategy to enhance the activity of drugs. The objective of this study is to determine the activity of EP inhibitors (EPIs) to enhance the efficacy of the new drug bedaquiline against M. abscessus clinical isolates.

Methods: A total of 31 phenotypically and genotypically identified M. abscessus subsp. abscessus, M. abscesss subsp. massiliense, and M. abscessus subsp. bolletii clinical isolates were studied. The contribution of EPs was determined by investigating the minimum inhibitory concentration (MIC) levels of bedaquiline reduction in the absence and presence of EPIs verapamil and reserpine using the resazurin microtiter assay.

Results: The observed bedaquiline MIC reduction by verapamil was observed in 100% isolates and by reserpine in 54.8% isolates. Bedaquiline MIC was 4-32-fold using verapamil with M. abscessus subsp. bolletii showing the highest fold change and between 2- and 4-fold using reserpine.

Conclusions: The results obtained in this study confirm that bedaquiline MIC decreased in the presence of EPIs verapamil and reserpine in clinical isolates of M. abscessus. Verapamil was the most effective EPI. As shown in previous studies, verapamil may have clinical potential as adjunctive therapy to enhance the effect of bedaquiline.
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http://dx.doi.org/10.4103/ijmy.ijmy_181_19DOI Listing
April 2021

Coinfection of Mycobacterium malmoense and Mycobacterium chimaera in a kidney transplant recipient: A case report and review of the literature.

Transpl Infect Dis 2020 Apr 10;22(2):e13241. Epub 2020 Jan 10.

Division of Infectious Disease, Cliniques Universitaires Saint-Luc, Université Catholique de Louvain, Brussels, Belgium.

Nontuberculous mycobacteria (NTM) are ubiquitous organisms found in soil and water. Solid organ recipients are at increased risk of NTM infections due to impaired immunity. Although the NTM infections rate is low, it increases morbidity and the risk of mortality. Diagnosis is often delayed because of the lack of specific clinical symptoms and requires a high index of suspicion. Management may be challenging: long-term treatment with risks of side effects and interactions with immunosuppressive regimen; reduction of immunosuppression; and risk of allograft rejection. Prognosis is widely variable. We report the first case of Mycobacterium malmoense chest infection with concomitant Mycobacterium chimaera urinary tract infection in a kidney transplant recipient. The evolution was marked by poor tolerance of the treatment with severe adverse events and disabled functional status.
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http://dx.doi.org/10.1111/tid.13241DOI Listing
April 2020

In vitro activity of bedaquiline against slow-growing nontuberculous mycobacteria.

J Med Microbiol 2019 Aug 18;68(8):1137-1139. Epub 2019 Jun 18.

Laboratory of Medical Microbiology, Institute of Experimental and Clinical Research, Université catholique de Louvain (UClouvain), Brussels, Belgium.

Bedaquiline (BDQ) is a recently approved antibiotic for the treatment of multidrug-resistant tuberculosis, but its potential against slow-growing mycobacteria (SGM) is still unknown. The objective of this study was to determine the in vitro activity of BDQ on SGM by assessing their MIC and minimal bactericidal concentration (MBC). The MIC of BDQ against 17 clinical isolates including Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium kansasii and Mycobacterium simiae species was determined by the resazurin microtitre assay and the MBC by the c.f.u. determination on 7H10 agar plates. BDQ has a bacteriostatic activity on all SGM tested with a MIC range from 0.03 to 0.007 µg ml and surprisingly a good bactericidal activity on the majority of the isolates tested with an MBC of 1-2 µg ml . Based on these preliminary results BDQ seems to be very promising for treatment of diseases caused by SGM.
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http://dx.doi.org/10.1099/jmm.0.001025DOI Listing
August 2019

Laboratory diagnosis of nontuberculous mycobacteria in a Belgium Hospital.

Int J Mycobacteriol 2019 Apr-Jun;8(2):157-161

Department of Microbiology, Université Catholique de Louvain, Cliniques Universitaires Saint-Luc, Brussels, Belgium.

Background: Nontuberculous mycobacteria (NTM) have been identified in human pulmonary and extrapulmonary infections and are increasing globally, which makes it challenging to identify them. This article reports our experience with the laboratory identification of NTM in clinical practice among pulmonary and extrapulmonary samples received in our routine work.

Methods: The study was conducted at the Université Catholique de Louvain at the Cliniques Universitaires Saint-Luc, Brussels, Belgium, from 2015 to 2018. A total of 386 clinical samples were collected from patients suspected of having pulmonary or extrapulmonary mycobacterial infections. Routine laboratory methods phenotypic and molecular tests were performed.

Results: The majority of NTM species were isolated from pulmonary samples (68%). The most prevalent species identified were Mycobacterium chimaera_intracellulare group (32%), followed by Mycobacterium avium complex (21%), Mycobacterium abscessus complex (18%), Mycobacterium gordonae (9%), and Mycobacterium chelonae (4%). In extrapulmonary samples, M. avium and M. chimaera_intracellulare were the most frequently isolated.

Conclusion: The species diversity of NTM found in our setting suggests the importance of the use of new modern methods for accurate identification of NTM at species level and in some case at subspecies level for the proper treatment and management of patients.
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http://dx.doi.org/10.4103/ijmy.ijmy_40_19DOI Listing
November 2019

Double prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents.

Bioorg Med Chem Lett 2019 05 7;29(10):1232-1235. Epub 2019 Mar 7.

Laboratory for Medicinal Chemistry, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium. Electronic address:

A series of eleven double prodrug derivatives of a fosmidomycin surrogate were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. A pivaloyloxymethyl (POM) phosphonate prodrug modification was combined with various prodrug derivatisations of the hydroxamate moiety. The majority of compounds showed activity comparable with or inferior to fosmidomycin against P. falciparum. N-benzyl substituted carbamate prodrug 6f was the most active antimalarial analog with an IC value of 0.64 µM. Contrary to fosmidomycin and parent POM-prodrug 5, 2-nitrofuran and 2-nitrothiophene prodrugs 6i and 6j displayed promising antitubercular activities.
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http://dx.doi.org/10.1016/j.bmcl.2019.03.009DOI Listing
May 2019

Phosphonodiamidate prodrugs of N-alkoxy analogs of a fosmidomycin surrogate as antimalarial and antitubercular agents.

Bioorg Med Chem Lett 2019 05 7;29(9):1051-1053. Epub 2019 Mar 7.

Laboratory for Medicinal Chemistry, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium. Electronic address:

A series of N-alkoxy analogs of a l-leucine ethyl ester phosphonodiamidate prodrug of a fosmidomycin surrogate were synthesized and investigated for their ability to inhibit in vitro growth of P. falciparum and M. tuberculosis. These compounds originate by merging a previously reported successful phosphonate derivatisation with favorable modifications of the hydroxamate moiety. None of the synthesized compounds showed enhanced activity against either P. falciparum or M. tuberculosis in comparison with the parent free hydroxamate analog.
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http://dx.doi.org/10.1016/j.bmcl.2019.03.008DOI Listing
May 2019

Amino acid based prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents.

Bioorg Med Chem 2019 03 17;27(5):729-747. Epub 2019 Jan 17.

Laboratory for Medicinal Chemistry, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium. Electronic address:

Fosmidomycin is a natural antibiotic with promising IspC (DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase) inhibitory activity. This enzyme catalyzes the first committed step of the non-mevalonate isoprenoid biosynthesis pathway, which is essential in Plasmodium falciparum and Mycobacterium tuberculosis. Mainly as a result of its high polarity, fosmidomycin displays suboptimal pharmacokinetic properties. Furthermore, fosmidomycin is inactive against M. tuberculosis as a result of its inability to penetrate the bacterial cell wall. Temporarily masking the phosphonate moiety as a prodrug has the potential to solve both issues. We report the application of two amino acid based prodrug approaches on a fosmidomycin surrogate. Conversion of the phosphonate moiety into tyrosine-derived esters increases the in vitro activity against asexual blood stages of P. falciparum, while phosphonodiamidate prodrugs display promising antitubercular activities. Selected prodrugs were tested in vivo in a P. berghei malaria mouse model. These results indicate good in vivo antiplasmodial potential.
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http://dx.doi.org/10.1016/j.bmc.2019.01.016DOI Listing
March 2019

First evaluation of the Next-Generation Sequencing platform for the detection of HIV-1 drug resistance mutations in Belgium.

PLoS One 2018 31;13(12):e0209561. Epub 2018 Dec 31.

Université catholique de Louvain (UCLouvain), Institut de Recherche Expérimentale et Clinique (IREC), Medical Microbiology Unit (MBLG), AIDS Reference Laboratory, Brussels, Belgium.

Introduction: The WHO urges action against the threat posed by HIV drug resistance. It is well known that the sensitivity of Next-Generation Sequencing (NGS) is greater than that of Sanger Sequencing (SS). The objective of this study was to evaluate the performance of the novel NGS HIV-1 drug resistance monitoring system.

Materials & Methods: NGS analyses were performed on 67 plasma samples from HIV-1 infected patients using the Sentosa SQ HIV Genotyping Assay from Vela-Dx. This kit was used on a semi-automated Ion Torrent-based platform. Sequences were compared to those obtained by SS. Samples were analysed in the same and in separate runs. Quality controls (QC) were added to control sequencing processes of protease (PRO), reverse transcriptase (RT) and integrase (INT) regions.

Results: Of the 41 analysed samples, 33 (80.5%) had identical drug resistance interpretation reports. Discrepant results were observed for eight samples. Five of them were only detected by NGS and had drug resistance mutations (DRMs) with an allelic frequency below the limit of detection of the SS method (between 6.3 to 20.5%). Two DRMs were only identified using the SS method. The sequences were similar in 98.2% of cases (counting variants as mismatches) and homologous in 99.9% if missed variants. Duplicated samples in a single run were similar in 95.7% (99.9%) of cases. Duplicated samples in two different runs were 98% (100%) homologous. QC results were manually assessed with a score of 340/340 for detection of DRMs in PRO and RT and 100% for INT sequencing.

Conclusions: This is the first preliminary evaluation in Belgium employing the Sentosa SQ HIV Genotyping Assay. The NGS appears to be a promising tool for the detection of DRMs in HIV-1 patients and showed a higher sensitivity compared to SS. Large studies assessing the clinical relevance of low frequency DRMs are needed.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209561PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312258PMC
December 2018

Bedaquiline and linezolid MIC distributions and epidemiological cut-off values for Mycobacterium tuberculosis in the Latin American region.

J Antimicrob Chemother 2019 02;74(2):373-379

Université catholique de Louvain, Institute of Experimental and Clinical Research, Laboratory of Medical Microbiology, Brussels, Belgium.

Objectives: To describe the distributions of bedaquiline and linezolid MIC values for the Mycobacterium tuberculosis WT population and to define the corresponding epidemiological cut-offs (ECOFFs) in three Latin American countries.

Methods: MICs of bedaquiline and linezolid were determined by the resazurin microtitre assay (REMA). In phase 1, interlaboratory reproducibility was assessed using a panel of 10 fully susceptible M. tuberculosis strains. Phase 2 involved MIC determination for 248 clinical isolates from Argentina (n = 58), Brazil (n = 100) and Peru (n = 90) from patients who were treatment-naive for bedaquiline and linezolid. We then determined the ECOFFs for bedaquiline and linezolid by the eyeball method and the ECOFFinder statistical calculator.

Results: Phase 1: REMA MIC values in the three sites were either identical to each other or differed by one 2-fold dilution from the consensus value with the exception of a single value. Phase 2: the bedaquiline MIC range was 0.0039-0.25 mg/L for pan-susceptible and drug-resistant isolates combined. The linezolid MIC range was 0.062-0.5 mg/L for pan-susceptible isolates and 0.031-4 mg/L for drug-resistant isolates. ECOFFs were 0.125 mg/L for bedaquiline and 0.50 mg/L for linezolid.

Conclusions: REMA is reproducible and robust for the determination of bedaquiline and linezolid MIC distributions and ECOFF values when applied in laboratories of medium/low-resource countries. We suggest that WT MIC distributions for both drugs should be used as a monitoring tool to control the possible rapid emergence of resistance.
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http://dx.doi.org/10.1093/jac/dky414DOI Listing
February 2019

Short Communication: An Insertion of Seven Amino Acids in the Envelope Cytoplasmic Tail of HIV-2 Selected During Disease Progression Enhances Viral Replication.

AIDS Res Hum Retroviruses 2019 02 16;35(2):185-190. Epub 2018 Oct 16.

1 Microbiology Unit (MBLG), AIDS Reference Laboratory (ARL), Institute of Experimental and Clinical Research (IREC), Université Catholique de Louvain, Brussels, Belgium.

The cytoplasmic tail (CT) of the HIV-2 envelope glycoprotein (Env) includes amino acid (aa) sequences that are similar to lentiviral lytic peptides (LLP) described in other lentiviruses. Within the putative LLP-2 region, we previously observed insertions of 3 or 7 aa in sequences deduced from plasma viral RNA of symptomatic HIV-2-infected individuals. Based on these observations, we reproduced the insertions in a molecular clone to assess their impact on replicative fitness and cell death in vitro. Using a molecular clone of the HIV-2 reference strain, site-directed mutagenesis experiments allowed the generation of plasmids with the insertion LTAI or LQRALTAI in the Env protein. The clone with 7 aa insertion enhanced viral release 8 to 11 times in infected T cells and cell viability was impaired by more than 20%, compared with the wild-type HIV-2 virus. The effect of the 3 aa insertion was milder, with a nonsignificant trend to enhance viral replication and cell death compared with the wild-type virus. Interestingly, the insertions in the Env proteins did not induce a significant increase of viral infectivity, as revealed by the infectivity assay using TZM-bl cells. The insertions in the Env CT observed in vivo from disease progressors may, therefore, be involved in the higher viral load observed in these individuals. This study may open the way to the development of a prognostic marker related to the HIV-2 infection progression.
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http://dx.doi.org/10.1089/AID.2018.0129DOI Listing
February 2019

String test: A new tool for tuberculosis diagnosis and drug-resistance detection in children.

Int J Mycobacteriol 2018 Apr-Jun;7(2):162-166

Université catholique de Louvain (UCL), Institute of Experimental and Clinical Research, Laboratory of Medical Microbiology, Brussels, Belgium.

Background: There is a critical need to improve the diagnostic accuracy of tuberculosis (TB) in children. Several techniques have been developed to improve the quality of sputum samples; however, these procedures are very unpleasant and invasive and require hospitalization and trained personnel. This study aims to explore the potential use of a new and noninvasive tool, "string test," for TB diagnosis in children and in adults not able to render sputum samples and at risk of developing multidrug-resistant TB (MDR-TB).

Methods: Children with clinical suspicion of TB attending the pediatric consultation at the Cetrangolo or Cordero Hospitals and adults suspected of MDR-TB and unable to produce sputum attending the Infectious Disease Unit of Cetrangolo Hospital were included in this study.

Subjects And Methods: The "string test" is a string that is swallowed by the patients and exposed to gastrointestinal secretions that were late analyzed for TB diagnosis and drug-resistance detection by GenoType MTBDRplus. MedCalc software was used to perform statistical analysis.

Results: This technique could be applied on 62.1% of selected children. About 11 (30.6%) children were diagnosed as TB cases, 8 (22.2%) from gastric aspirate and using the "string test." Six out of 19 adults were also diagnosed. Genotype directly on the string specimen detected two MDR-TB in adults and two isoniazid-resistant cases before obtaining the isolate.

Conclusion: This test was safe, cheap, and easily implemented without requiring hospitalization. This research could represent a significant step forward to diagnose and rapidly detect drug-resistant TB in children.
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http://dx.doi.org/10.4103/ijmy.ijmy_54_18DOI Listing
March 2019

Antimicrobial activity against Mycobacterium tuberculosis under in vitro lipid-rich dormancy conditions.

J Med Microbiol 2018 Mar 15;67(3):282-285. Epub 2018 Jan 15.

Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, 11340, Mexico.

Although tuberculosis treatment is dependent on drug-susceptibility testing (DST) and molecular drug-resistance detection, treatment failure and relapse remain a challenge. This could be partially due to the emergence of antibiotic-tolerant dormant mycobacteria, where host lipids have been shown to play an important role. This study evaluated the susceptibility of Mycobacterium tuberculosis to two antibiotic combinations - rifampicin, moxifloxacin, amikacin and metronidazole (RIF-MXF-AMK-MTZ), and rifampicin, moxifloxacin, amikacin and pretomanid (RIF-MXF-AMK-PA) - in a lipid-rich dormancy model. Although their effectiveness in in vitro cultures with dextrose as a carbon source has been proved, we observed that none of the antibiotic mixtures were bactericidal in the presence of lipids. The presence of lipids may confer tolerance to M. tuberculosis against the mixture of antibiotics tested and such tolerance could be even higher during the dormant stages. The implementation of lipids in DST on clinical isolates could potentially lead to a better treatment strategy.
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http://dx.doi.org/10.1099/jmm.0.000681DOI Listing
March 2018

Performance of a highly successful outbreak strain of Mycobacterium tuberculosis in a multifaceted approach to bacterial fitness assessment.

Int J Med Microbiol 2018 Apr 31;308(3):349-357. Epub 2018 Jan 31.

Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2290, C1425FQB, Buenos Aires, Argentina; Instituto Nacional de Enfermedades Infecciosas (INEI), ANLIS "Carlos G. Malbrán". Vélez Sarsfield 563, C1282AFF, Buenos Aires, Argentina. Electronic address:

Determining bacterial fitness represents a major challenge and no single parameter can accurately predict the ability of a certain pathogen to succeed. The M strain of Mycobacterium tuberculosis managed to spread and establish in the community and caused the largest multidrug-resistant tuberculosis outbreak in Latin America. We have previously shown that the M strain can manipulate the host immune response, but we still have no direct evidence, other than epidemiology, that can account for the enhanced fitness of the M strain. Our objective was to further characterize the performance of the outbreak strain M in different fitness assays. Two main aspects were evaluated: (1) molecular characterization of selected isolates from the M outbreak and related strains and (2) comparative fitness and in vivo performance of representative M strain isolates vs. the non-prosperous M strain variant 410. Our approach confirmed the multifaceted nature of fitness. Altogether, we conclude that the epidemiologically abortive strain 410 was vulnerable to drug-driven pressure, a weak competitor, and a stronger inductor of protective response in vivo. Conversely, the isolate 6548, representative of the M outbreak peak, had a growth disadvantage but performed very well in competition and induced lung damage at advanced stages in spite of reaching relatively low CFU counts. Integration of these observations supports the idea that the M strain managed to find a unique path to success.
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http://dx.doi.org/10.1016/j.ijmm.2018.01.006DOI Listing
April 2018

The transcriptome of Mycobacterium tuberculosis in a lipid-rich dormancy model through RNAseq analysis.

Sci Rep 2017 12 15;7(1):17665. Epub 2017 Dec 15.

Laboratory of Microbiology, Faculty of Science, Ghent University, Gent, Belgium.

Tuberculosis (TB) is currently the number one killer among infectious diseases worldwide. Lipids are abundant molecules during the infectious cycle of Mycobacterium tuberculosis (Mtb) and studies better mimicking its actual metabolic state during pathogenesis are needed. Though most studies have focused on the mycobacterial lipid metabolism under standard culture conditions, little is known about the transcriptome of Mtb in a lipid environment. Here we determined the transcriptome of Mtb H37Rv in a lipid-rich environment (cholesterol and fatty acid) under aerobic and hypoxic conditions, using RNAseq. Lipids significantly induced the expression of 368 genes. A main core lipid response was observed involving efflux systems, iron caption and sulfur reduction. In co-expression with ncRNAs and other genes discussed below, may act coordinately to prepare the machinery conferring drug tolerance and increasing a persistent population. Our findings could be useful to tag relevant pathways for the development of new drugs, vaccines and new strategies to control TB.
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http://dx.doi.org/10.1038/s41598-017-17751-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732278PMC
December 2017

Modulation of the NF-κB signaling pathway by the HIV-2 envelope glycoprotein and its incomplete BST-2 antagonism.

Virology 2018 01 11;513:11-16. Epub 2017 Oct 11.

Université catholique de Louvain, Experimental and Clinical Research Institute (IREC), Medical Microbiology Unit (MBLG), AIDS Reference Laboratory, Avenue Hippocrate 54, B-1200 Brussels, Belgium. Electronic address:

The HIVs have evolved by selecting means to hijack numerous host cellular factors. HIVs exploit the transcription factor NF-κB to ensure efficient LTR-driven gene transcription. However, NF-κB is primarily known to act as a key regulator of the proinflammatory and antiviral responses. Interestingly, retroviruses activate NF-κB during early stages of infection to initiate proviral genome expression while suppressing it at later stages to restrain expression of antiviral genes. During HIV-1 infection, diverse viral proteins such as Env, Nef and Vpr have been proposed to activate NF-κB activity, whereas Vpu has been shown to inhibit NF-κB activation. It is still unclear how HIV-2 regulates NF-κB signaling pathway during its replication cycle. Here we confirm that human BST-2 and HIV-1 Env proteins can trigger potent activation of NF-κB. Importantly, we demonstrate for the first time that the HIV-2 Env induces NF-κB activation in HEΚ293T cells. Furthermore, the anti-BST-2 activity of the HIV-2 Env is not sufficient to completely inhibit NF-κB activity.
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http://dx.doi.org/10.1016/j.virol.2017.09.024DOI Listing
January 2018

In vitro activity of bedaquiline against rapidly growing nontuberculous mycobacteria.

J Med Microbiol 2017 Aug 28;66(8):1140-1143. Epub 2017 Jul 28.

Laboratory of Microbiology, Ghent University, Gent, Belgium.

Bedaquiline (BDQ) has been proven to be effective in the treatment of multidrug-resistant tuberculosis. We hypothesized that BDQ could be a potential agent to treat nontuberculous mycobacterial (NTM) infection. The objective of this study was to evaluate the in vitro activity of BDQ against rapidly growing mycobacteria by assessing the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against 18 NTM strains. For MIC determination we performed the resazurin microtitre assay broth dilution, and for the MBC the c.f.u. was determined. BDQ exhibited a strong inhibitory effect against most NTM tested; however, for some NTM strains the MBC was significantly higher than the MIC. A new finding is that Mycobacterium flavescens has a mutation in the gene atpE associated with natural resistance to BDQ. These preliminary promising results demonstrate that BDQ could be potentially useful for the treatment of NTM.
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http://dx.doi.org/10.1099/jmm.0.000537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817190PMC
August 2017

Characterization of -Like Strains by Comparative Genomics.

Front Microbiol 2017 8;8:789. Epub 2017 May 8.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São PauloSão Paulo, Brazil.

Isolates of the - complex are subdivided into four clusters (CHI to CHIV) in the INNO-LiPA® spp DNA strip assay. A considerable phenotypic variability was observed among isolates of the CHII cluster. In this study, we examined the diversity of 26 CHII cluster isolates by phenotypic analysis, drug susceptibility testing, whole genome sequencing and single-gene analysis. Pairwise genome comparisons were performed using several approaches, including average nucleotide identity (ANI) and genome-to-genome distance (GGD) among others. Based on ANI and GGD the isolates were identified as (14 isolates), (2 isolates) and (1 isolate). The remaining 9 isolates were subdivided into three novel putative genomospecies. Phenotypic analyses including drug susceptibility testing, as well as whole genome comparison by TETRA and delta differences, were not helpful in separating the groups revealed by ANI and GGD. The analysis of standard four conserved genomic regions showed that alone and the concatenated sequences clearly distinguished the taxonomic groups delimited by whole genome analyses. In conclusion, the CHII INNO-LiPa is not a homogeneous cluster; on the contrary, it is composed of closely related different species belonging to the complex and also several unidentified isolates. The detection of these isolates, putatively novel species, indicates a wider inner variability than the presently known in this complex.
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http://dx.doi.org/10.3389/fmicb.2017.00789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5420552PMC
May 2017

Isoniazid-resistant tuberculosis treatment with first-line drugs.

Lancet Infect Dis 2017 03 23;17(3):258-259. Epub 2017 Feb 23.

Pôle de Microbiologie Médicale, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels B-1200, Belgium.

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http://dx.doi.org/10.1016/S1473-3099(17)30075-0DOI Listing
March 2017

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

J Microbiol Methods 2016 12 27;131:144-147. Epub 2016 Oct 27.

Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Gent, Belgium.

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.
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http://dx.doi.org/10.1016/j.mimet.2016.10.021DOI Listing
December 2016

Exploring tuberculosis by molecular tests on DNA isolated from smear microscopy slides.

Int J Infect Dis 2017 Mar 12;56:248-252. Epub 2016 Dec 12.

Unité des Mycobactéries, Institut Pasteur de Madagascar, Antananarivo, Madagascar.

Tuberculosis (TB) is an infectious disease of global public health importance caused by Mycobacterium tuberculosis complex. The disease has worsened with the emergence of multidrug-resistant (MDR)-TB strains. The timely diagnosis and treatment of TB remains a key public health priority, and laboratories have a critical role in the rapid and accurate detection of TB and drug resistance. Molecular assays based on nucleic acid amplification techniques have been developed for the rapid, sensitive, and specific diagnosis of TB, with the ability to determine the drug sensitivity status. These molecular techniques are now available or are being implemented in developing countries. However, traditional microscopy and culture methods cannot yet be replaced; the molecular assays can be applied in parallel with these tests for the diagnosis of TB or for drug susceptibility testing. Performing such molecular tests is often restricted by constraints with regard to sputum sample storage and safe transportation from remote health centres to central laboratories. Since smear slides are performed routinely for the diagnosis of TB in most TB diagnostic laboratories, they are readily available and could be the ideal tool to transport sputum for further molecular tests. The aim of this review was to provide a comprehensive survey on the use of smear slides for both TB diagnosis and the molecular test approach. Based on the literature, stained smear microscopy slides can be a safe system for the transportation of sputum specimens from remote health centres to reference TB laboratories for further molecular TB or MDR-TB detection, and could help in the rapid diagnosis and therefore timely management of TB patients.
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http://dx.doi.org/10.1016/j.ijid.2016.12.005DOI Listing
March 2017

"Genetic regulation of Mycobacterium tuberculosis in a lipid-rich environment".

Infect Genet Evol 2017 11 19;55:392-402. Epub 2016 Oct 19.

Departamento de Microbiologia, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Prolongacion de Carpio y Plan de Ayala S/N, Mexico City, Mexico; Red Multidisciplinaria de Investigación en Tuberculosis, Mexico. Electronic address:

Tuberculosis (TB) remains as one of the leading causes of morbidity and mortality among infectious diseases worldwide. Although lipids (mainly fatty acids and cholesterol) have been reported to play an important role during active and latent infection of M. tuberculosis, there are other molecular aspects of bacterial response to those substrates that are not fully understood, involving gene regulation background. This review highlights recent insights on pathogen gene expression: regulation during its active growth, during survival in presence of lipids and under variable hostile host microenvironments. We also propose several application options of this knowledge that may contribute for improved TB control.
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http://dx.doi.org/10.1016/j.meegid.2016.10.015DOI Listing
November 2017

The potential role of trimethoprim-sulfamethoxazole in the treatment of drug-resistant tuberculosis.

Future Microbiol 2016 12;11(4):539-47. Epub 2016 Apr 12.

Laboratory of Microbiology, Department of Biochemistry & Microbiology, Ghent University, Ghent, Belgium.

Tuberculosis (TB) remains a serious public health threat worsened by emerging drug resistance. Mycobacterium tuberculosis has become resistant not only to front-line drugs but also to second-line antimicrobials directed at drug-resistant TB. Renewed efforts are devoted for the development of new antibiotics active against TB. Also, repurposing of other antibiotics is being explored to shorten the time to develop new drugs against M. tuberculosis. As a result, trimethoprim-sulfamethoxazole (SXT) has emerged as a potential new option to treat drug-resistant TB. SXT has been found to be surprisingly active against drug-resistant M. tuberculosis, not only in vitro but also in vivo. The potential role of SXT for the treatment of multidrug resistant/extensively drug resistant TB might be explored in further clinical evaluations.
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http://dx.doi.org/10.2217/fmb.16.2DOI Listing
December 2016

Resazurin microtiter assay for isoniazid, rifampicin, ethambutol and streptomycin resistance detection in Mycobacterium tuberculosis: Updated meta-analysis.

Int J Mycobacteriol 2014 Dec 29;3(4):230-41. Epub 2014 Sep 29.

Laboratory of Microbiology, Faculty of Sciences, Department of Biochemistry and Microbiology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium.

Aims: The present meta-analysis aims to assess the evidence regarding the diagnostic accuracy and performance characteristics of the colorimetric redox indicator (CRI) assay with a special emphasis on the use of the resazurin microtiter assay (REMA) for determination of primary anti-tuberculosis drug resistance.

Subject And Methods: By updating previous literature searches in Medline PubMed, ISI Web, Web of Science and Google academic databases of the REMA test for determination of primary anti-tuberculosis drug resistance, this meta-analysis includes 14 studies for isoniazid (INH); 15 studies for rifampicin (RIF); 6 studies for streptomycin (STR); and 5 studies for ethambutol (EMB). SROC curve analysis was performed for meta-analysis and diagnostic accuracy was summarized.

Results: Pooled sensitivity was 96% (94-98%) for INH, 97% (95-98%) for RIF, 92% (87-96%) for EMB and 92% (88-95%) for STR. Pooled specificity for INH, RIF, EMB and STR was 96% (95-98%), 99% (98-99%), 86% (81-89%) and 90% (87-93%), respectively. Susceptibility testing results had been obtained in 8-9days.

Conclusion: In conclusion, REMA seems to be a reliable test for the determination of multi-drug resistant (MDR) isolates in laboratories with limited resources. However, few studies for STR and EMB have been found, and cost-effectiveness studies need to be determined to recommend its widespread use.
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http://dx.doi.org/10.1016/j.ijmyco.2014.09.002DOI Listing
December 2014

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

PLoS One 2015 2;10(10):e0139382. Epub 2015 Oct 2.

Laboratory of Microbiology, Department of Biochemistry and Microbiology, Ghent University, Gent, Belgium.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139382PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4591989PMC
June 2016

Mycobacterium saopaulense sp. nov., a rapidly growing mycobacterium closely related to members of the Mycobacterium chelonae--Mycobacterium abscessus group.

Int J Syst Evol Microbiol 2015 Dec;65(12):4403-4409

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.

Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and,in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae-Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae–M.abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1-2 nt differences in rpoB and internal transcribed spacer (ITS) sequences.Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA–DNA hybridization values .70 % confirmed that the five isolates belong to the same species, while values ,70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA–DNA hybridization results,demonstrated that they share characteristics with M. chelonae–M. abscessus members, butconstitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM10906T (5CCUG 66554T5LMG 28586T5INCQS 0733T).
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http://dx.doi.org/10.1099/ijsem.0.000590DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4772522PMC
December 2015

Beninese Medicinal Plants as a Source of Antimycobacterial Agents: Bioguided Fractionation and In Vitro Activity of Alkaloids Isolated from Holarrhena floribunda Used in Traditional Treatment of Buruli Ulcer.

Biomed Res Int 2015 28;2015:835767. Epub 2015 May 28.

Pharmacognosy Research Group, Louvain Drug Research Institute (LDRI), Université Catholique de Louvain (UCL), B1 7203 Avenue E. Mounier 72, 1200 Bruxelles, Belgium.

Buruli ulcer (BU) imposes a serious economic burden on affected households and on health systems that are involved in diagnosing the disease and treating patients. Research is needed to find cost-effective therapies for this costly disease. Plants have always been an important source of new pharmacologically active molecules. Consequently we decided to undertake the study of plants used in traditional treatment of BU in Benin and investigate their antimycobacterial activity as well as their chemical composition. Extracts from forty-four (44) plant species were selected on account of reported traditional uses for the treatment of BU in Benin and were assayed for antimycobacterial activities. Crude hydroethanolic extract from aerial parts of Holarrhena floribunda (G. Don) T. Durand and Schinz was found to have significant antimycobacterial activity against M. ulcerans (MIC = 125 µg/mL). We describe here the identification of four steroidal alkaloids from Mycobacterium ulcerans growth-inhibiting fractions of the alkaloidal extract of the aerial parts of Holarrhena floribunda. Holadysamine was purified in sufficient amount to allow the determination of its MCI (=50 µg/mL). These results give some support to the use of this plant in traditional medicine.
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http://dx.doi.org/10.1155/2015/835767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477427PMC
April 2016

Sensitivity Pattern of Second Line Anti-Tuberculosis Drugs against Clinical Isolates of Multidrug Resistant Mycobacterium tuberculosis.

J Coll Physicians Surg Pak 2015 Apr;25(4):250-3

Department of Biochemistry and Laboratory of Microbiology, University of Ghent, Belgium.

Objective: To determine the current sensitivity pattern of second line anti-tuberculosis drugs against clinical isolates of Multidrug Resistant Mycobacterium tuberculosis (MDR-TB).

Study Design: A cross-sectional study.

Place And Duration Of Study: Department of Microbiology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from November 2011 to April 2013.

Methodology: Samples received during the study period were processed on BACTEC MGIT 960 system for Mycobacterium tuberculosis (MTB) culture followed by first line drugs susceptibility testing of culture proven MTB isolates. On the basis of resistance to rifampicin and isoniazid, 100 clinical isolates of MDR-TB were further subjected to susceptibility testing against amikacin (AMK), capreomycin (CAP), ofloxacin (OFL) and ethionamide (ETH) as per standard BACTEC MGIT 960 instructions.

Results: Out of 100 MDR-TB isolates, 62% were from male patients and 38% from female patients. 97% were sensitive to AMK, 53% to OFL, 87% to CAP; and 87% were sensitive to ETH.

Conclusion: The majority of the MDR-TB isolates showed excellent sensitivity against AMK, CAP and ETH. However, sensitivity of MDR-TB isolates against fluoroquinolones like OFL was not encouraging.
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http://dx.doi.org/04.2015/JCPSP.250253DOI Listing
April 2015

Mycobacterium franklinii sp. nov., a species closely related to members of the Mycobacterium chelonae-Mycobacterium abscessus group.

Int J Syst Evol Microbiol 2015 Jul 9;65(7):2148-2153. Epub 2015 Apr 9.

Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP, Brazil.

Two isolates from water, D16Q19 and D16R27, were shown to be highly similar in their 16S rRNA, 16S-23S internal transcribed spacer (ITS), hsp65 and rpoB gene sequences to 'Mycobacterium franklinii' DSM 45524, described in 2011 but with the name not validly published. They are all nonpigmented rapid growers and are related phenotypically and genetically to the Mycobacterium chelonae-Mycobacterium abscessus group. Extensive characterization by phenotypic analysis, biochemical tests, drug susceptibility testing, PCR restriction enzyme analysis of the hsp65 gene and ITS, DNA sequencing of housekeeping genes and DNA-DNA hybridization demonstrated that 'M. franklinii' DSM 45524, D16Q19 and D16R27 belong to a single species that is separated from other members of the M. chelonae-M. abscessus group. On the basis of these results we propose the formal recognition of Mycobacterium franklinii sp. nov. Strain DSM 45524(T) ( = ATCC BAA-2149(T)) is the type strain.
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http://dx.doi.org/10.1099/ijs.0.000234DOI Listing
July 2015
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