Publications by authors named "Ana Ibarra-Meneses"

13 Publications

  • Page 1 of 1

Leishmaniasis: A new method for confirming cure and detecting asymptomatic infection in patients receiving immunosuppressive treatment for autoimmune disease.

PLoS Negl Trop Dis 2021 Aug 2;15(8):e0009662. Epub 2021 Aug 2.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda (Madrid), Spain.

Visceral leishmaniasis (VL) in patients receiving immunosuppressant drugs for autoimmune disease has been on the rise. It is important-but difficult-to know when cure has been achieved in these patients since the withdrawal of immunosuppressants during antileishmania treatment is commonly required, and there is a risk of relapse when immunosuppression is restored. The prevalence of asymptomatic infection among those immunosuppressed for autoimmune disease is also uncertain. The present work describes how cytokine release assays can be used to confirm the cure of VL, and to determine the prevalence of asymptomatic infection, in such patients. After collection of blood from volunteers (n = 108), SLA-stimulation of peripheral blood mononuclear cell cultures and of whole blood was found to induce the production of different combinations of cytokines that served to confirm recovery from VL, and asymptomatic Leishmania infection. Indeed, cure was confirmed in 14 patients, all of whom showed a specific Th1 immune response against Leishmania, and the prevalence of asymptomatic infection was determined as 21.27%. Cytokine profiles could be used to manage VL in patients with autoimmune disease, and to identify and better protect those with asymptomatic infection who are at risk of developing this disease.
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http://dx.doi.org/10.1371/journal.pntd.0009662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8360552PMC
August 2021

Loop-Mediated Isothermal Amplification Allows Rapid, Simple and Accurate Molecular Diagnosis of Human Cutaneous and Visceral Leishmaniasis Caused by When Compared to PCR.

Microorganisms 2021 Mar 16;9(3). Epub 2021 Mar 16.

WHO Collaborating Centre for Leishmaniasis, National Center for Microbiology, Instituto de Salud Carlos III, 28220 Majadahonda, Spain.

Loop-mediated isothermal amplification allows the rapid, sensitive and specific amplification of DNA without complex and expensive equipment. We compared the diagnostic performance of Loopamp™ Detection Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) with conventional and real-time polymerase chain reaction (PCR) for human cutaneous and visceral leishmaniasis caused by . A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by nested PCR (LnPCR) were tested by: (i) the Loopamp™ Detection Kit (Loopamp), run on Genie III real-time fluorimeter (OptiGene, UK); and (ii) real-time quantitative PCR (qPCR). The Loopamp test returned 98.8% (95% confidence interval-CI: 96.0-100.00) sensitivity and specificity of 97.7% (95% CI: 92.2-100) on VL samples, and 100% (95% CI: 99.1-100) sensitivity and 100.0% (95% CI: 98.8-100.0) specificity on CL samples. The Loopamp time-to-positivity () obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR's cycle threshold (). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to . The excellent correlation between the and should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.
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http://dx.doi.org/10.3390/microorganisms9030610DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999953PMC
March 2021

Whole Blood Stimulation Assay as a Treatment Outcome Monitoring Tool for VL Patients in Ethiopia: A Pilot Evaluation.

J Immunol Res 2020 23;2020:8385672. Epub 2020 Jan 23.

Unit of Neglected Tropical Diseases, Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.

Visceral leishmaniasis (VL) is a lethal disease if left untreated. Current treatments produce variable rates of treatment failure and toxicity without sterile cure, rendering treatment efficacy monitoring essential. To avoid repeated invasive tissue aspirates as well as empirical treatment, there is a need for new tools that allow a less-invasive and early assessment of treatment efficacy in the field. Cross-sectional studies have suggested levels of cytokines/chemokines after whole blood stimulation as good markers of cure, but longitudinal studies are lacking. In this study, we followed 13 active VL cases in an endemic area in Ethiopia by measuring the production of IFN-, TNF-, IP-10, IL-2, IL-10, MCP-1, and MIG before, during, and at the end of treatment. After 24 hours of stimulation of whole blood with soluble antigen, we observed an early, robust, and incremental increase of IFN-, TNF-, and IP-10 levels in all patients during treatment. Moreover, based on the IFN- levels that showed an average 13-fold increase from the time of diagnosis until the end of treatment, we could almost perfectly discriminate active from cured status. Similar concentrations and patterns were found in stimulation assays with the two main species. The levels of IFN-, IP-10, or TNF- also seemed to be inversely associated with the parasite load at baseline. Despite a 1/10 drop in concentrations, similar patterns were observed in IFN- and IP-10 levels when dried plasma spots were stored at 4°C for an average of 225 days. All the above evidence suggests a detectable restoration of cell-mediated immunity in VL and its association with parasite clearance. With a potential application in rural settings by means of dried plasma spots, we recommend to further explore the early diagnostic value of such assays for treatment efficacy monitoring in large cohort studies including treatment failure cases.
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http://dx.doi.org/10.1155/2020/8385672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7193677PMC
February 2021

New Strategies and Biomarkers for the Control of Visceral Leishmaniasis.

Trends Parasitol 2020 01 9;36(1):29-38. Epub 2019 Nov 9.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda (Madrid), Spain.

Effective diagnosis and treatment of visceral leishmaniasis, together with the study of vectors and reservoirs, can lead to a better understanding of the parasite transmission dynamics and the development of more efficient control measures. Recent studies have applied new methodologies and biomarkers, and these have contributed to the early and rapid diagnosis of the disease; assessment of success of pharmacological treatments; efficient monitoring of immunosuppressed individuals; and to population screening for field trials of vaccine efficacy. This opinion article proposes an update to the diagnostic tools for visceral leishmaniasis and their rational and combined use to establish the real prevalence of infection or of exposure to Leishmania in endemic areas.
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http://dx.doi.org/10.1016/j.pt.2019.10.005DOI Listing
January 2020

Prevalence of asymptomatic infection and associated risk factors, after an outbreak in the south-western Madrid region, Spain, 2015.

Euro Surveill 2019 May;24(22)

WHO Collaborating Centre for Leishmaniasis, National Center for Microbiology, Instituto de Salud Carlos III, Majadahonda, Madrid, Spain.

BackgroundA large outbreak of leishmaniasis with 758 cutaneous and visceral leishmaniasis cases occurred in 2009 in Fuenlabrada, in the south-west of the Madrid region of Spain.AimWe aimed to determine the prevalence of asymptomatic infection after this outbreak, and its associated risk factors.MethodsA cross-sectional study of 804 healthy individuals living in Fuenlabrada who had no history of leishmaniasis, was conducted between January and July 2015. Asymptomatic infections were sought by either a combination of PCR, immunofluorescent antibody titre, and direct agglutination tests, or by whole blood stimulation assay (WBA) with interleukin-2 (IL-2) quantification.ResultsUsing the first approach, prevalence of asymptomatic individuals was 1.1% (9/804), while the second returned a value of 20.7% (143/804). Older age, being male, proximity to the park where the focus of infection was identified, and living in a detached house, were all strongly associated with the prevalence of asymptomatic infection.ConclusionsThe true number of infected individuals may be underestimated if only serological methods are used. The combination of WBA with IL-2 quantification may allow to better determine the prevalence of asymptomatic infection, which would be useful in establishing control measures and in quantifying their impact. In our study, the use of WBA with IL-2 quantification also helped establish the risk factors that influence exposure to and infection by .
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http://dx.doi.org/10.2807/1560-7917.ES.2019.24.22.1800379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549460PMC
May 2019

Asymptomatic immune responders to Leishmania among HIV positive patients.

PLoS Negl Trop Dis 2019 06 3;13(6):e0007461. Epub 2019 Jun 3.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Majadahonda (Madrid), Spain.

Concomitant infection with human immunodeficiency virus (HIV) and the Leishmania parasite is a growing public health problem, the result of the former spreading to areas where the latter is endemic. Leishmania infection is usually asymptomatic in immunocompetent individuals, but the proportion of HIV+ individuals in contact with the parasite who remain asymptomatic is not known. The aim of the present work was to examine the use of cytokine release assays in the detection of asymptomatic immune responders to Leishmania among HIV+ patients with no previous leishmaniasis or current symptomatology. Eighty two HIV+ patients (all from Fuenlabrada, Madrid, Spain, where a leishmaniasis outbreak occurred in 2009) were examined for Leishmania infantum infection using molecular and humoral response-based methods. None returned a positive molecular or serological result for the parasite. Thirteen subjects showed a positive lymphoproliferative response to soluble Leishmania antigen (SLA), although the mean CD4+ T lymphocyte counts of these patients was below the normal range. Stimulation of peripheral blood mononuclear cells (PBMC) or whole blood with SLA (the lymphoproliferative assay and whole blood assay respectively), led to the production of specific cytokines and chemokines. Thus, despite being immunocompromised, HIV+ patients can maintain a Th1-type cellular response to Leishmania. In addition, cytokine release assays would appear to be useful tools for detecting these individuals via the identification of IFN-γ in the supernatants of SLA-stimulated PBMC, and of IFN-γ, MIG and IL-2 in SLA-stimulated whole blood. These biomarkers appear to be 100% reliable for detecting asymptomatic immune responders to Leishmania among HIV+ patients.
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http://dx.doi.org/10.1371/journal.pntd.0007461DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6564048PMC
June 2019

Cellular Markers of Active Disease and Cure in Different Forms of -Induced Disease.

Front Cell Infect Microbiol 2018 13;8:381. Epub 2018 Nov 13.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.

Increased numbers of peripheral blood mononucleocytes (PBMC) and increased IFN-γ secretion following challenge of blood samples with soluble antigen (SLA), have been proposed as biomarkers of specific cell-mediated immunity, indicating that treatment of visceral leishmaniasis (VL) has been successful. However, infection may manifest as cutaneous leishmaniasis (CL), and less commonly as localized leishmanial lymphadenopathy (LLL) or mucosal leishmaniasis (ML). The present work examines the value of these biomarkers as indicators of cured leishmaniasis presenting in these different forms. Blood samples were collected before and after treatment from patients living in Fuenlabrada (Madrid, Spain), an endemic area recently the center of a leishmaniasis outbreak. All samples were subjected to -specific PCR, serological tests (IFAT and rK39-ICT), and the SLA-cell proliferation assay (SLA-CPA), recording PBMC proliferation and the associated changes in IFN-γ production. Differences in the results recorded for the active and cured conditions were only significant for VL. PCR returned positive results in 67% of patients with active VL and in 3% of those with cured leishmaniasis. Similarly, rK39-ICT returned a positive result in 77% of active VL samples . 52% in cured VL samples, and IFAT in 90% . 56%; in the SLA-CPA, PBMC proliferation was seen in 16% . 90%, and an associated increase in IFN-γ production of 14 and 84%, respectively. The present findings reinforce the idea that PBMC proliferation and increased IFN-γ production in SLA-stimulated PBMC provide biomarkers of clinical cure in VL. Other tests are urgently needed to distinguish between the cured and active forms of the other types of clinical leishmaniasis caused by .
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http://dx.doi.org/10.3389/fcimb.2018.00381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6243388PMC
September 2019

Antigenicity of -Activated C-Kinase Antigen (LACK) in Human Peripheral Blood Mononuclear Cells, and Protective Effect of Prime-Boost Vaccination With pCI-neo-LACK Plus Attenuated LACK-Expressing Vaccinia Viruses in Hamsters.

Front Immunol 2018 23;9:843. Epub 2018 Apr 23.

WHO Collaborating Center for Leishmaniasis, National Center of Microbiology, Instituto de Salud Carlos III, Madrid, Spain.

-activated C-kinase antigen (LACK) is a highly conserved protein among species and is considered a viable vaccine candidate for human leishmaniasis. In animal models, prime-boost vaccination with LACK-expressing plasmids plus attenuated vaccinia viruses (modified vaccinia Ankara [MVA] and mutant M65) expressing LACK, has been shown to protect against cutaneous leishmaniasis (CL). Further, LACK demonstrated to induce the production of protective cytokines in patients with active CL or cured visceral leishmaniasis, as well as in asymptomatic individuals from endemic areas. However, whether LACK is capable to trigger cytokine release by peripheral blood mononuclear cells from patients cured of CL due to () or induce protection in -infected hamsters [visceral leishmaniasis (VL) model], has not yet been analyzed. The present work examines the immunogenicity of LACK in cured VL and CL patients, and asymptomatic subjects from an area. It also evaluates the vaccine potential of LACK against infection in hamsters, in a protocol of priming with plasmid pCI-neo-LACK (DNA-LACK) followed by a booster with the poxvirus vectors MVA-LACK or M65-LACK. LACK-stimulated PBMC from both asymptomatic and cured subjects responded by producing IFN-γ, TNF-α, and granzyme B (Th1-type response). Further, 78% of PBMC samples that responded to soluble antigen showed IFN-γ secretion following stimulation with LACK. In hamsters, the protocol of DNA-LACK prime/MVA-LACK or M65-LACK virus boost vaccination significantly reduced the amount of DNA in the liver and bone marrow, with no differences recorded between the use of MVA or M65 virus vector options. In summary, the Th1-type and cytotoxic responses elicited by LACK in PBMC from human subjects infected with , and the parasite protective effect of prime/boost vaccination in hamsters with DNA-LACK/MVA-LACK and DNA-LACK/M65-LACK, revealed the significance of LACK in activating human and hamster immune responses and support LACK to be a valuable candidate for inclusion in a vaccine against human VL.
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http://dx.doi.org/10.3389/fimmu.2018.00843DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924775PMC
June 2019

Evaluation of fluorimetry and direct visualization to interpret results of a loop-mediated isothermal amplification kit to detect Leishmania DNA.

Parasit Vectors 2018 04 17;11(1):250. Epub 2018 Apr 17.

WHO Collaborating Centre for Leishmaniasis, National Centre for Microbiology, Instituto de Salud Carlos III, Madrid, Spain.

Background: Nucleic acid amplification tests (NAATs) have proven to be advantageous in the diagnosis of leishmaniases, allowing sensitive diagnosis of: (i) cutaneous leishmaniasis in long duration lesions and (ii) visceral leishmaniasis using a less-invasive sample like peripheral blood, in opposition to tissue aspiration required for parasite demonstration by microscopy. Despite their benefits, the implementation of NAATs for leishmaniasis diagnosis at the point-of-care has not been achieved yet, mostly due to the complexity and logistical issues associated with PCR-based methods.

Methods: In this work, we have evaluated the performance of a ready-to-use loop-mediated isothermal amplification (LAMP) kit using two real time fluorimeters to amplify leishmanial DNA obtained by silica column-based and Boil & Spin protocols.

Results: The different approaches used to run and interpret the LAMP reactions showed a performance equivalent to PCR and real-time PCR, using spiked and clinical samples. The time to positivity obtained with real-time fluorimetry showed an excellent correlation with both Ct values and parasite load from real-time quantitative PCR.

Conclusions: The results obtained open the possibility of using a highly stable, ready-to-use LAMP kit for the accurate diagnosis of leishmaniasis at the point-of-care. Furthermore, the feasibility of relating time to positivity, determined with a portable real-time fluorimeter, with the parasite burden could have a wider application in the management of leishmaniasis, such as in treatment efficacy monitoring or as a pharmacodynamics tool in clinical trials.
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http://dx.doi.org/10.1186/s13071-018-2836-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905109PMC
April 2018

Monocyte Chemotactic Protein 1 in Plasma from Soluble Antigen-Stimulated Whole Blood as a Potential Biomarker of the Cellular Immune Response to .

Front Immunol 2017 29;8:1208. Epub 2017 Sep 29.

WHO Collaborating Centre for Leishmaniasis, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.

New biomarkers are needed to identify asymptomatic infection as well as immunity following vaccination or treatment. With the aim of finding a robust biomarker to assess an effective cellular immune response, monocyte chemotactic protein 1 (MCP-1) was examined in plasma from soluble antigen (SLA)-stimulated whole blood collected from subjects living in a -endemic area. MCP-1, expressed 110 times more strongly than IL-2, identified 87.5% of asymptomatic subjects and verified some asymptomatic subjects close to the cutoff. MCP-1 was also significantly elevated in all patients cured of visceral leishmaniasis (VL), unlike IL-2, indicating the specific memory response generated against . These results show MCP-1 to be a robust candidate biomarker of immunity that could be used as a marker of cure and to both select and follow the population in vaccine phase I-III human clinical trials with developed rapid, easy-to-use field tools.
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http://dx.doi.org/10.3389/fimmu.2017.01208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5626820PMC
September 2017

F1 Domain of the Nucleoside Hydrolase Promotes a Th1 Response in Cured Patients and in Asymptomatic Individuals Living in an Endemic Area of Leishmaniasis.

Front Immunol 2017 12;8:750. Epub 2017 Jul 12.

Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

The nucleoside hydrolase NH36 is the main antigen of the Leishmune vaccine and one of the promising candidates for vaccination against visceral leishmaniasis. The antigenicity of the N-terminal (F1), the central (F2), or the C-terminal recombinant domain (F3) of NH36 was evaluated using peripheral blood mononuclear cells (PBMC) from individuals infected with from an endemic area of visceral leishmaniasis of Spain. Both NH36 and F1 domains significantly increased the PBMC proliferation stimulation index of cured patients and infected asymptomatic individuals compared to healthy controls. Moreover, F1 induced a 19% higher proliferative response than NH36 in asymptomatic exposed subjects. In addition, in patients cured from visceral leishmaniasis, proliferation in response to NH36 and F1 was accompanied by a significant increase of IFN-γ and TNF-α secretion, which was 42-43% higher, in response to F1 than to NH36. The interleukin 17 (IL-17) secretion was stronger in asymptomatic subjects, in response to F1, as well as in cured cutaneous leishmaniasis after NH36 stimulation. While no IL-10 secretion was determined by F1, a granzyme B increase was detected in supernatants from cured patients after stimulation with either NH36 or F1. These data demonstrate that F1 is the domain of NH36 that induces a recall cellular response in individuals with acquired resistance to the infection by . In addition, F1 and NH36 discriminated the IgG3 humoral response in patients with active visceral leishmaniasis due to (Ethiopia) and (Spain) from that of endemic and non-endemic area controls. NH36 showed higher reactivity with sera from -infected individuals, indicating species specificity. We conclude that the F1 domain, previously characterized as an inducer of the Th1 and Th17 responses in cured/exposed patients infected with , may also be involved in the generation of a protective response against and represents a potential vaccine candidate for the control of human leishmaniasis alone, or in combination with other HLA epitopes/antigens.
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http://dx.doi.org/10.3389/fimmu.2017.00750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5506215PMC
July 2017

IFN-γ, IL-2, IP-10, and MIG as Biomarkers of Exposure to spp., and of Cure in Human Visceral Leishmaniasis.

Front Cell Infect Microbiol 2017 31;7:200. Epub 2017 May 31.

WHO Collaborating Centre for Leishmaniasis, Centro Nacional de Microbiologia, Instituto de Salud CarlosMadrid, Spain.

New biomarkers are needed for monitoring the effectiveness of treatment for visceral leishmaniasis (VL). They might also improve the detection of the asymptomatic population in endemic areas. This paper examines the IL-2, IFN-γ, IFN-γ-induced protein 10 (IP-10), and monokine-induced-by-IFN-γ (MIG) levels in whole blood-stimulated with soluble antigen (SLA)-taken from asymptomatic individuals and patients treated for VL living in a post-outbreak () area in Spain, and in an endemic () area of Bangladesh. IP-10 was found to be an accurate global marker of asymptomatic subjects with positive cellular/humoral tests, while MIG was found to be a better marker of contact with than IL-2 but no for those with . Determining IP-10, MIG, and IFN-γ levels proved useful in monitoring the cellular immune response following treatment for active disease caused by .
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http://dx.doi.org/10.3389/fcimb.2017.00200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5449718PMC
February 2018

Lymphoproliferative response after stimulation with soluble leishmania antigen (SLA) as a predictor of visceral leishmaniasis (VL) relapse in HIV+ patients.

Acta Trop 2016 Dec 28;164:345-351. Epub 2016 Sep 28.

WHO Collaborating Centre for Leishmaniasis, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.

The introduction of HAART resulted in the decrease of Leishmania/HIV co-infection cases; nevertheless, the number of relapses remains high and secondary prophylaxis is recommended. However, secondary prophylaxis is not necessary in all patients, and presents a high risk of toxicity and an elevated cost. Our aim was to study whether specific cellular response to Leishmania infantum (measured by cell proliferation response after stimulation with soluble Leishmania antigen (SLA)), could be a useful tool to attempt a secondary prophylaxis withdrawal. In June 2009 an outbreak of leishmaniasis by Leishmania infantum was declared in the southeast of Madrid, and since January 2013, we recruited 10 HIV+ patients that had been treated for visceral leishmaniasis. 6 patients had positive SLA-cell proliferation test. The mean CD4 cell counts of those patients with positive SLA were 140 cel/mm3 and 40 cel/mm3 in those with negative SLA test. 3 patients with positive SLA-cell proliferation test (CD4 count: 336, 307, 625) were not on prophylaxis, and the other 3 patients (CD4 count: 152, 189, 359) were on secondary prophylaxis that was withdrawn after the positive SLA-cell proliferation test with no posterior relapses (mean follow up 60 weeks). From the 4 patients, which had negative SLA-cell proliferation test and continued on prophylaxis, 3 had positive PCR for Leishmania at the end of the follow-up and 2 presented clinical relapses. The performance of SLA-cell proliferation test can be a useful tool that can permit us to try withdrawal of the prophylaxis in Leishmania/HIV co-infected patients with low CD4 counts under clinical supervision, diminishing risk of toxicity and cost.
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http://dx.doi.org/10.1016/j.actatropica.2016.09.026DOI Listing
December 2016
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