Deciphering Morphological determinants of the helix-shaped Leptospira

Leptospira spp. are thin, highly motile, slow-growing spirochetes that can be distinguished from other bacteria on the basis of their unique helical shape. Defining the mechanisms by which these bacteria generate and maintain this atypical morphology should greatly enhance our understanding of the fundamental physiology of these pathogens. In this study, we showed that peptidoglycan sacculi from Leptospira spp. retain the helical shape of intact cells. Interestingly, the distribution of muropeptides was different from that in the Escherichia coli model, indicating that specific enzymes might be active on the peptidoglycan macromolecule. We could alter the shape of Leptospira biflexa with the broad-spectrum β-lactam antibiotic penicillin G and with amdinocillin and aztreonam, which are β-lactams that preferentially target penicillin-binding protein 2 (PBP2) and PBP3, respectively, in some species. Although genetic manipulations of Leptospira spp. are scarce, we were able to obtain mutants with alterations in genes encoding PBPs, including PBP3. Loss of this protein resulted in cell elongation. We also generated an L. biflexa strain that conditionally expresses MreB. Loss of the MreB function was correlated with morphological abnormalities such as a localized increased diameter and heterogeneous length. A prolonged depletion of MreB resulted in cell lysis, suggesting that this protein is essential. These findings indicate that important aspects of leptospiral cell morphology are determined by the cytoskeleton and the murein layer, thus providing a starting point for a better understanding of the morphogenesis in these atypical bacteria.This work was supported by the Institut Pasteur, Paris, France, and the French Ministry of Research ANR-08-MIE-018. M.A.D.P. was supported by Ministry of Education and Science, Spain (MEC, BFU2006-04574) and Fundación Ramón Areces.Peer Reviewe

Etude des résistances du VIH-1 au traitement antirétroviral et amélioration du suivi virologique des patients vivant avec le VIH dans les pays du Sud

Fifteen years after the introduction and expanded access to antiretroviral therapy (ART) in resource limited countries (RLC), viral suppression, a key objective of its effectiveness is not achieved in many patients infected with HIV. Unlike developed countries, where often more powerful and robust ART coupled with a routine virological monitoring limit the emergence of resistance, the current situation of RLC is more worrying. Operational research conducted here aims to improve care and monitoring of patients living in these countries, and receiving ART according to the WHO public health approach. While recommended routinely since 2013, HIV viral load (VL) is rarely available and monitoring remains primarily clinical or immunological.In several countries in Central Africa and West, we documented high rates of HIV drug resistance (HIVDR) (99%) and multi-drug resistance (97%) in patients with virologic failure after long-term first-line ART, consisting of 2 nucleoside reverse transcriptase inhibitors (NRTI, 3TC+AZT/d4T) associated with 1 non-NRTI (NNRTI, EFV/NVP). This multi-drug resistance also reduces significantly the effectiveness of second-generation NNRTIs and of the NRTI, TDF, which is now currently recommended in 2nd line by the WHO. More unusually, we also reported that high VLs (> 5 log 10 copies / ml) are associated with multi-resistance rather than an insufficient adherence in these patients who were largely asymptomatic. Without improvement of patient monitoring, through expanded access to VL testing to enable early diagnosis of virological failure, a potential new epidemic caused by HIVDR strains could emerge in sub-Saharan Africa. This could jeopardize efforts to achieve the "90-90-90" objective (90% of diagnosed infections, 90% of diagnosed patients on ART, 90% of patients treated durable virologic success) of UNAIDS and WHO in 2020. Moreover, our second study in a district hospital in Cameroon illustrates that these ambitious targets will probably be even more difficult to achieve in decentralized areas, often having inadequate laboratory infrastructure to acquire complex VL platforms.We then successfully transferred an open and polyvalent universal VL assay in a national reference laboratory in Cameroon. This assay can detect HIV-1 group M, N, O and P and their simian ancestors. With this RT-qPCR HIV-1/SIVcpz/SIVgor assay, HIV-1 M variants were better quantified (+0.47 log10 copies / mL) compared to Abbott RealTime HIV-1 assay which is approved by the FDA. At the VL threshold of virological failure defined by the WHO (1000 copies/mL), the correlation of the results between the two tests was 95%, with a higher sensitivity for the new test.We finally explored how to adapt this open test at optimal large-scale application for dried blood spot (DBS), to make VL testing available in decentralized areas, given the current lack of point of care test for VL. We compared different extraction methods of nucleic acids that could potentially reduce the contribution of the pro-viral DNA present in DBS, with the aim to improve their specificity without success. Instead, we have shown that with most of the RT-qPCR VL assays, many patients would be inappropriately eligible to perform a repeated VL testing (or switched to a costly 2nd line ART), which increase significantly costs of national programs.In conclusion, the studies from this thesis have contributed to improve current knowledge about the effectiveness of ART programs and services and also on the use of virological monitoring tools adapted to patients living with HIV in RLCs.Quinze ans après la mise en place et l’accès élargi au traitement antirétroviral (TAR) dans les pays du Sud, la suppression virale, objectif clé de son efficacité, n’est pas atteinte chez de nombreux patients infectés par le VIH. Contrairement aux pays du Nord, où un TAR souvent plus puissant et plus robuste couplé à un suivi virologique de routine limite l’émergence de la résistance, la situation des patients vivant actuellement dans les pays du Sud est beaucoup plus préoccupante. La recherche opérationnelle menée ici vise à améliorer leur prise en charge basée sur l’approche globale de santé publique de l’OMS, et aussi leur suivi. Alors qu’elle est recommandée en routine depuis 2013, la charge virale (CV) du VIH est peu accessible et le suivi des patients reste principalement clinique voire immunologique.Dans plusieurs pays d’Afrique Centrale et de l’Ouest, nous avons documenté une multi-résistance chez presque la totalité des patients en échec virologique après une longue durée de TAR de 1ère ligne, composé de 2 inhibiteurs nucléosidiques de la transcriptase inverse (INTI, 3TC+AZT/d4T) associés à 1 inhibiteur non nucléosidique de la transcriptase inverse (INNTI, EFV/NVP). Cette résistance compromet aussi l’efficacité des INNTIs de 2ème génération, de façon importante, et aussi celle du TDF qui est actuellement recommandé en 2ème ligne. De façon plus inhabituelle, nous avons aussi rapporté que les CV élevées(>5 log10 copies/mL) étaient associées à une multi-résistance plutôt qu’à un défaut d’observance chez ces patients largement asymptomatiques. Sans véritable amélioration du suivi, au travers d’un accès élargi au test de CV permettant un diagnostic précoce de l’échec virologique, une nouvelle épidémie potentielle causée par des souches résistantes pourrait émerger en Afrique subsaharienne. Elle pourrait compromettre les efforts à réaliser pour atteindre l’objectif « 90-90-90 » (90% des infections diagnostiquées, 90% des patients diagnostiqués sous TAR, 90% des patients traités en succès virologique durable) de l’ONUSIDA et de l’OMS en 2020. En outre, notre deuxième étude menée au sein d’un hôpital de district camerounais illustre le fait que ces objectifs ambitieux seront probablement encore plus difficiles à atteindre en zones décentralisées, disposant souvent d’infrastructures de laboratoires inadaptées pour accueillir des plateformes de CV complexes.Nous avons ensuite transféré avec succès un test de CV ouvert et polyvalent universel au sein d’un laboratoire national de référence au Cameroun, capable de détecter les VIH-1 M, N, O et P, ainsi que leurs ancêtres simiens. Avec ce test RT-qPCR VIH-1/SIVcpz/SIVgor, nous avons mesuré une meilleure quantification des variants du VIH-1 M (+0.47 log10 copies/mL), comparé au test Abbott RealTime HIV-1 approuvé par la FDA. Au seuil d’échec virologique de l’OMS (1 000 copies/mL), la concordance des résultats entre les deux tests était de 95%, avec une meilleure sensibilité pour le nouveau test.Nous avons finalement cherché à savoir comment adapter ce test ouvert à une application optimale à grande échelle sur du sang séché sur papier buvard (DBS, dried blood spot en anglais), pour rendre la CV accessible en zones décentralisées, en l’absence actuelle de tests de CV simples (POC, point of care en anglais). Nous avons comparé différentes méthodes d’extraction des acides nucléiques pour réduire l’impact de l’ADN pro-viral afin d’améliorer en vain la spécificité du test sur DBS. Nous avons plutôt montré qu’avec la plupart des tests de CV par RT-qPCR, de nombreux patients pourraient être candidats pour refaire une CV de façon inappropriée (voire changer de TAR pour une 2ème ligne), ce qui induirait un surcoût programmatique.En conclusion, ces travaux de thèse ont contribué à améliorer les connaissances sur l’efficacité réelle des programmes et services de TAR, ainsi que sur l’utilisation d’outils de suivi virologique adaptés aux patients vivant avec le VIH dans les pays du Sud

Study of HIV-1 antiretroviral drug resistance and virological monitoring improvement in patients living in ressource limited setting

Quinze ans après la mise en place et l’accès élargi au traitement antirétroviral (TAR) dans les pays du Sud, la suppression virale, objectif clé de son efficacité, n’est pas atteinte chez de nombreux patients infectés par le VIH. Contrairement aux pays du Nord, où un TAR souvent plus puissant et plus robuste couplé à un suivi virologique de routine limite l’émergence de la résistance, la situation des patients vivant actuellement dans les pays du Sud est beaucoup plus préoccupante. La recherche opérationnelle menée ici vise à améliorer leur prise en charge basée sur l’approche globale de santé publique de l’OMS, et aussi leur suivi. Alors qu’elle est recommandée en routine depuis 2013, la charge virale (CV) du VIH est peu accessible et le suivi des patients reste principalement clinique voire immunologique.Dans plusieurs pays d’Afrique Centrale et de l’Ouest, nous avons documenté une multi-résistance chez presque la totalité des patients en échec virologique après une longue durée de TAR de 1ère ligne, composé de 2 inhibiteurs nucléosidiques de la transcriptase inverse (INTI, 3TC+AZT/d4T) associés à 1 inhibiteur non nucléosidique de la transcriptase inverse (INNTI, EFV/NVP). Cette résistance compromet aussi l’efficacité des INNTIs de 2ème génération, de façon importante, et aussi celle du TDF qui est actuellement recommandé en 2ème ligne. De façon plus inhabituelle, nous avons aussi rapporté que les CV élevées(>5 log10 copies/mL) étaient associées à une multi-résistance plutôt qu’à un défaut d’observance chez ces patients largement asymptomatiques. Sans véritable amélioration du suivi, au travers d’un accès élargi au test de CV permettant un diagnostic précoce de l’échec virologique, une nouvelle épidémie potentielle causée par des souches résistantes pourrait émerger en Afrique subsaharienne. Elle pourrait compromettre les efforts à réaliser pour atteindre l’objectif « 90-90-90 » (90% des infections diagnostiquées, 90% des patients diagnostiqués sous TAR, 90% des patients traités en succès virologique durable) de l’ONUSIDA et de l’OMS en 2020. En outre, notre deuxième étude menée au sein d’un hôpital de district camerounais illustre le fait que ces objectifs ambitieux seront probablement encore plus difficiles à atteindre en zones décentralisées, disposant souvent d’infrastructures de laboratoires inadaptées pour accueillir des plateformes de CV complexes.Nous avons ensuite transféré avec succès un test de CV ouvert et polyvalent universel au sein d’un laboratoire national de référence au Cameroun, capable de détecter les VIH-1 M, N, O et P, ainsi que leurs ancêtres simiens. Avec ce test RT-qPCR VIH-1/SIVcpz/SIVgor, nous avons mesuré une meilleure quantification des variants du VIH-1 M (+0.47 log10 copies/mL), comparé au test Abbott RealTime HIV-1 approuvé par la FDA. Au seuil d’échec virologique de l’OMS (1 000 copies/mL), la concordance des résultats entre les deux tests était de 95%, avec une meilleure sensibilité pour le nouveau test.Nous avons finalement cherché à savoir comment adapter ce test ouvert à une application optimale à grande échelle sur du sang séché sur papier buvard (DBS, dried blood spot en anglais), pour rendre la CV accessible en zones décentralisées, en l’absence actuelle de tests de CV simples (POC, point of care en anglais). Nous avons comparé différentes méthodes d’extraction des acides nucléiques pour réduire l’impact de l’ADN pro-viral afin d’améliorer en vain la spécificité du test sur DBS. Nous avons plutôt montré qu’avec la plupart des tests de CV par RT-qPCR, de nombreux patients pourraient être candidats pour refaire une CV de façon inappropriée (voire changer de TAR pour une 2ème ligne), ce qui induirait un surcoût programmatique.En conclusion, ces travaux de thèse ont contribué à améliorer les connaissances sur l’efficacité réelle des programmes et services de TAR, ainsi que sur l’utilisation d’outils de suivi virologique adaptés aux patients vivant avec le VIH dans les pays du Sud.Fifteen years after the introduction and expanded access to antiretroviral therapy (ART) in resource limited countries (RLC), viral suppression, a key objective of its effectiveness is not achieved in many patients infected with HIV. Unlike developed countries, where often more powerful and robust ART coupled with a routine virological monitoring limit the emergence of resistance, the current situation of RLC is more worrying. Operational research conducted here aims to improve care and monitoring of patients living in these countries, and receiving ART according to the WHO public health approach. While recommended routinely since 2013, HIV viral load (VL) is rarely available and monitoring remains primarily clinical or immunological.In several countries in Central Africa and West, we documented high rates of HIV drug resistance (HIVDR) (99%) and multi-drug resistance (97%) in patients with virologic failure after long-term first-line ART, consisting of 2 nucleoside reverse transcriptase inhibitors (NRTI, 3TC+AZT/d4T) associated with 1 non-NRTI (NNRTI, EFV/NVP). This multi-drug resistance also reduces significantly the effectiveness of second-generation NNRTIs and of the NRTI, TDF, which is now currently recommended in 2nd line by the WHO. More unusually, we also reported that high VLs (> 5 log 10 copies / ml) are associated with multi-resistance rather than an insufficient adherence in these patients who were largely asymptomatic. Without improvement of patient monitoring, through expanded access to VL testing to enable early diagnosis of virological failure, a potential new epidemic caused by HIVDR strains could emerge in sub-Saharan Africa. This could jeopardize efforts to achieve the "90-90-90" objective (90% of diagnosed infections, 90% of diagnosed patients on ART, 90% of patients treated durable virologic success) of UNAIDS and WHO in 2020. Moreover, our second study in a district hospital in Cameroon illustrates that these ambitious targets will probably be even more difficult to achieve in decentralized areas, often having inadequate laboratory infrastructure to acquire complex VL platforms.We then successfully transferred an open and polyvalent universal VL assay in a national reference laboratory in Cameroon. This assay can detect HIV-1 group M, N, O and P and their simian ancestors. With this RT-qPCR HIV-1/SIVcpz/SIVgor assay, HIV-1 M variants were better quantified (+0.47 log10 copies / mL) compared to Abbott RealTime HIV-1 assay which is approved by the FDA. At the VL threshold of virological failure defined by the WHO (1000 copies/mL), the correlation of the results between the two tests was 95%, with a higher sensitivity for the new test.We finally explored how to adapt this open test at optimal large-scale application for dried blood spot (DBS), to make VL testing available in decentralized areas, given the current lack of point of care test for VL. We compared different extraction methods of nucleic acids that could potentially reduce the contribution of the pro-viral DNA present in DBS, with the aim to improve their specificity without success. Instead, we have shown that with most of the RT-qPCR VL assays, many patients would be inappropriately eligible to perform a repeated VL testing (or switched to a costly 2nd line ART), which increase significantly costs of national programs.In conclusion, the studies from this thesis have contributed to improve current knowledge about the effectiveness of ART programs and services and also on the use of virological monitoring tools adapted to patients living with HIV in RLCs

Comparison of different nucleic acid preparation methods to improve specific HIV-1 RNA isolation for viral load testing on dried blood spots

International audienceIn resource-limited countries (RLCs), WHO recommends HIV viral load (VL) on dried blood spots (DBS) for antiretroviral therapy (ART) monitoring of patients living in non-urban settings where plasma VL is not available. In order to reduce the impact of proviral DNA interference, leading to false positive results in samples with low plasma VL, we compared three different nucleic acid preparation methods with the NucliSens (Biomérieux) extraction, known for its high recovery of nucleic acids on DBS. Paired plasma-DBS samples (n = 151) with predominantly low plasma VL (≤ 10,000 copies/ml; 74%) were used. At the threshold of 1,000 copies/ml on DBS, 51% and 10% were misclassified as false positives or false negatives, respectively with NucliSens, versus 41% and 20% with m2000sp (Abbott), described as more specific for RNA recovery. DNase treatments of nucleic acid extracts and free virus elution (FVE) protocol before nucleic acid extraction, reduced the proportion of false positives to 0% and 19%, but increased the proportion of false negatives to 40% and 73%. More efforts are thus still needed to improve performance of VL assays on DBS to monitor patients on ART in RLCs and allow timely switch to more costly second or third line ART regimes

Short Communication: High Viral Load and Multidrug Resistance Due to Late Switch to Second-Line Regimens Could Be a Major Obstacle to Reach the 90-90-90 UNAIDS Objectives in Sub-Saharan Africa

International audienceIn the context of lifelong antiretroviral treatment (ART) as early as possible and to end the HIV/AIDS epidemic as a public health treat by 2030, it is important to evaluate the potential risk of transmission of HIV-1 drug resistance (HIVDR) in resource-limited countries (RLCs). Since HIV transmission is driven by HIV-1 RNA viral load (VL), we studied the association between plasma VL and HIVDR profiles in 451 adults failing first-line ART from the 2LADY-ANRS12169/EDCTP trial in Burkina Faso, Cameroon, and Senegal. Median duration on first-line ART was 49 months (IQR: 33-69) and 91% patients were asymptomatic. Genotypic drug resistance testing was successful for 446 patients and 98.7% of them were resistant to at least one of the first-line drugs; 40.6% and 55.8% were resistant to two or three drugs of their ongoing first-line ART, respectively. The median VL was higher in patients with HIVDR to all ongoing first-line drugs than in those still susceptible to at least one drug; 4.7 log10 copies/ml (IQR: 4.3-5.2) versus 4.2 log10 copies/ml (IQR: 3.7-4.7), respectively (p 5.0 log10 copies/ml. High rates of cross-resistance to other nucleoside reverse-transcriptase inhibitors were observed and were also highest in patients with high VL. Without improvement of patient monitoring to avoid late switch to second-line regimens, a potential new epidemic caused by HIVDR strains could emerge in sub-Saharan Africa and compromise all efforts to reach 90-90-90 UNAIDS objective by 2020

Short Communication: High Viral Load and Multidrug Resistance Due to Late Switch to Second-Line Regimens Could Be a Major Obstacle to Reach the 90-90-90 UNAIDS Objectives in Sub-Saharan Africa

International audienceIn the context of lifelong antiretroviral treatment (ART) as early as possible and to end the HIV/AIDS epidemic as a public health treat by 2030, it is important to evaluate the potential risk of transmission of HIV-1 drug resistance (HIVDR) in resource-limited countries (RLCs). Since HIV transmission is driven by HIV-1 RNA viral load (VL), we studied the association between plasma VL and HIVDR profiles in 451 adults failing first-line ART from the 2LADY-ANRS12169/EDCTP trial in Burkina Faso, Cameroon, and Senegal. Median duration on first-line ART was 49 months (IQR: 33-69) and 91% patients were asymptomatic. Genotypic drug resistance testing was successful for 446 patients and 98.7% of them were resistant to at least one of the first-line drugs; 40.6% and 55.8% were resistant to two or three drugs of their ongoing first-line ART, respectively. The median VL was higher in patients with HIVDR to all ongoing first-line drugs than in those still susceptible to at least one drug; 4.7 log10 copies/ml (IQR: 4.3-5.2) versus 4.2 log10 copies/ml (IQR: 3.7-4.7), respectively (p 5.0 log10 copies/ml. High rates of cross-resistance to other nucleoside reverse-transcriptase inhibitors were observed and were also highest in patients with high VL. Without improvement of patient monitoring to avoid late switch to second-line regimens, a potential new epidemic caused by HIVDR strains could emerge in sub-Saharan Africa and compromise all efforts to reach 90-90-90 UNAIDS objective by 2020

Short Communication: Nucleoside Reverse Transcriptase Inhibitors with Reduced Predicted Activity Do Not Impair Second-Line Therapy with Lopinavir/Ritonavir or Darunavir/Ritonavir

International audienceSecond-line therapy randomized trials with lopinavir/ritonavir question the value of resistance testing to guide nucleoside reverse transcriptase inhibitor (NRTI) selection. In this study, we investigated the association between baseline drug resistance and treatment outcome after 104 weeks of second-line therapy with NRTIs and either darunavir/ritonavir or lopinavir/ritonavir in West-central Africa. We did an observational analysis of data from 387 individuals in a randomized, open-label 2LADY trial in Burkina Faso, Cameroon, and Senegal. We modeled the association between RTI drug resistance mutations (DRMs) and virological failure (VF) (viral load [VL] \textless50 copies/mL) at week 104 using logistic regressions. Covariates included baseline VL and CD4+ count, demographic, and adherence data. Overall, 193 (49.9%), 150 (38.8%), and 44 (11.4%) individuals had, respectively, low/none (genotypic susceptibility score [GSS] \textless1), intermediate (GSS = 1), and high predicted NRTI activity (GSS \textgreater1) in their prescribed second-line regimen. The average number of DRMs by drug class, the proportion of individuals by GSS category, and the duration of first-line therapy were not associated with VF (p \textgreater .05). High VL at switch was the only consistent prognostic factor for VF after multivariate adjustment (p \textless .01). Suboptimal adherence, high predicted RTI activity, or low NRTI mutations were associated with VF (p \textless .05) when using higher end points for VF or in the intention-to-treat analysis. In conclusion, the use of RTIs with predicted reduced activity does not impair second-line protease inhibitor-based therapy. Therefore, HIV care in resource-limited settings should prioritize strategies to improve adherence and targeted VL testing over drug resistance testing for selecting NRTIs during a protease-based second-line switch

Virologic Failure and Human Immunodeficiency Virus Drug Resistance in Rural Cameroon With Regard to the UNAIDS 90-90-90 Treatment Targets

International audienceBACKGROUND:In rural Africa, data on virologic effectiveness of antiretroviral treatment (ART) are not sufficient to assess the gap with the UNAIDS 90-90-90 treatment targets. We investigated the prevalences of unsuppressed viral load and antiretroviral drug resistance and the profile of genotypic resistance mutations among patients routinely treated in rural Cameroon.METHODS:A cross-sectional study was performed in 2013-2014 among patients ≥15 years and on first-line ART for ≥6 months in a district hospital. Patients were offered free access to human immunodeficiency virus viral load testing. Genotypic drug resistance testing was done when the viral load was >1000 copies/mL. Multivariate logistic regression models were used to assess the relationship of unsuppressed viral load or antiretroviral drug resistance with sociodemographic and medical characteristics.RESULTS:Of 407 patients (women 74.9%, median age 41.8 years, median time on ART 29.2 months), 96 (23.6%; 95% confidence interval [CI], 19.5-28.0) had unsuppressed viral load and 74 (18.2%; 95% CI, 14.6-22.3) had antiretroviral drug resistance. The prevalences of unsuppressed viral load and resistance increased with time on ART, from 12.0% and 8.0% in the 6- to 12-month group to 31.3% and 27.1% in the >72-month group, respectively. All 74 patients with antiretroviral drug resistance were resistant to nonnucleoside reverse-transcriptase inhibitors, and 57 of them were also resistant to nucleoside reverse-transcriptase inhibitors.CONCLUSIONS:Our estimations were among the highest observed in the west and central African region. The proportion of patients with virologic failure should be divided at least by 2 to reach the UNAIDS 90-90-90 treatment targets