Multicenter analysis of sputum microbiota in tuberculosis patients.

The impact of tuberculosis and of anti-tuberculosis therapy on composition and modification of human lung microbiota has been the object of several investigations. However, no clear outcome has been presented so far and the relationship between M. tuberculosis pulmonary infection and the resident lung microbiota remains vague. In this work we describe the results obtained from a multicenter study of the microbiota of sputum samples from patients with tuberculosis or unrelated lung diseases and healthy donors recruited in Switzerland, Italy and Bangladesh, with the ultimate goal of discovering a microbiota-based biomarker associated with tuberculosis. Bacterial 16S rDNA amplification, high-throughput sequencing and extensive bioinformatic analyses revealed patient-specific flora and high variability in taxon abundance. No common signature could be identified among the individuals enrolled except for minor differences which were not consistent among the different geographical settings. Moreover, anti-tuberculosis therapy did not cause any important variation in microbiota diversity, thus precluding its exploitation as a biomarker for the follow up of tuberculosis patients undergoing treatment

Severe acute malnutrition and antiretroviral treatment in children with HIV.

Doctor of Philosophy in Paediatrics and Child Health. University of KwaZulu-Natal. Durban, 2016.Background: Childhood malnutrition remains a common problem in many parts of the world and is a contributing factor in 45% of the 5.9 million annual deaths in children under 5 years. HIV-infected children have a disproportionately higher prevalence of malnutrition and higher mortality associated with malnutrition as compared to non-infected children. Physiological changes associated with malnutrition and re-nutrition complicate antiretroviral treatment in these children. This thesis explores aspects related to antiretroviral treatment (ART) in severely malnourished HIV-infected children, including the timing of ART initiation, pharmacokinetics of antiretroviral drugs, co-infections with bacterial and mycobacterial infections and the effect of microbial translocation on immune restoration. Methods: Eight-two patients were enrolled in this randomized controlled trial, where HIV-infected children admitted with severe acute malnutrition (SAM) were initiated on ART either early (within 14 days of admission) or delayed (after 14 days with evidence of nutritional recovery). Clinical and laboratory parameters were collected during the admission and patients were followed up at 4, 8, 12, 24 and 48 weeks post admission. A pharmacokinetic evaluation of lopinavir (LPV) was conducted on Day1 and 14 of ART initiation. Samples for evaluation of microbial translocation and immune restoration were collected in 32 study patients and 75 additional patients in 3 control groups. Results/Discussion: There were no significant differences in immunologic, virologic or anthropometric responses at 48 weeks between the early and delayed arms. However, significantly improved rates in the changes in viral load, WAZ (weight-for-age Z score) and HAZ (height-for-age Z score) favoured the delayed arm. Pharmacokinetic (pk) evaluation of the LPV, displayed significant pk variability, reduced bioavailability and consequently greater apparent clearance (CL/F) estimates in comparison to other pk studies of LPV in non-malnourished children. Fat-free Mass (FFM) was shown to affect LPV variability; however delay in ART initiation and “super-boosted” LPV/rtv did not affect LPV variability. Bacterial pathogens were identified in 51% of patients. Of the hospital acquired infections (HAI), 41% were extended spectrum beta-lactamase (ESBL)-producing gram-negative infections. Tuberculosis (TB) co-infection was common (25.6%), with bacteriological confirmation in 38% of treated cases. Malnutrition was associated with increased microbial translocation, immune activation and immune exhaustion, with a negative impact on immune recovery in HIV-infected children on ART. Conclusions: Delaying ART initiation to at least 14 days after starting nutritional support is associated with improved rates of clinical (changes in WAZ and HAZ) and virologic outcomes. However this delay did not improve LPV exposures and dose adjustment of LPV during nutritional recovery needs to be further evaluated. These results can be used to inform changes in clinical practice and national and international guidelines for the management of severely malnourished HIV-infected children

The University of Zimbabwe College of Health Sciences (UZ-CHS) BIRTH COHORT study: rationale, design and methods.

BACKGROUND Commencing lifelong antiretroviral therapy (ART) immediately following HIV diagnosis (Option B+), has greatly improved maternal-infant health. Thus, large and increasing numbers of HIV-infected women are on ART during pregnancy, a situation concurrently increasing numbers of HIV-exposed-uninfected (HEU) infants. Compared to their HIV-unexposed-uninfected (HUU) counterparts, HEU infants show higher rates of adverse birth outcomes, mortality, infectious/non-communicable diseases including impaired growth and neurocognitive development. There is an urgent need to understand the impact of HIV and early life ART exposures, immune-metabolic dysregulation, comorbidities and environmental confounders on adverse paediatric outcomes. METHODS Six hundred (600) HIV-infected and 600 HIV-uninfected pregnant women ≥20 weeks of gestation will be enrolled from four primary health centres in high density residential areas of Harare. Participants will be followed up as mother-infant-pairs at delivery, week(s) 1, 6, 10, 14, 24, 36, 48, 72 and 96 after birth. Clinical, socio-economic, nutritional and environmental data will be assessed for adverse birth outcomes, impaired growth, immune/neurodevelopment, vertical transmission of HIV, hepatitis-B/C viruses, cytomegalovirus and syphilis. Maternal urine, stool, plasma, cord blood, amniotic fluid, placenta and milk including infant plasma, dried blood spot and stool will be collected at enrolment and follow-up visits. The composite primary endpoint is stillbirth and infant mortality within the first two years of life in HEU versus HUU infants. Maternal mortality in HIV-infected versus -uninfected women is another primary outcome. Secondary endpoints include a range of maternal and infant outcomes. Sub-studies will address maternal stress and malnutrition, maternal-infant latent tuberculosis, Helicobacter pylori infections, immune-metabolomic dysregulation including gut, breast milk and amniotic fluid dysbiosis. DISCUSSION The University of Zimbabwe-College of Health-Sciences-Birth-Cohort study will provide a comprehensive assessment of risk factors and biomarkers for HEU infants' adverse outcomes. This will ultimately help developing strategies to mitigate effects of maternal HIV, early-life ART exposures and comorbidities on infants' mortality and morbidity. TRIAL REGISTRATION ClinicalTrial.gov Identifier: NCT04087239 . Registered 12 September 2019

ABSTRACT BOOK 50th World Conference on Lung Health of the International Union Against Tuberculosis and Lung Disease (The Union)

The International Journal of Tuberculosis and Lung Disease is an official journal of The Union. The Journal’s main aim is the continuing education of physicians and other health personnel, and the dissemination of the most up-to-date infor mation in the field of tuberculosis and lung health. It publishes original articles and commissioned reviews not only on the clinical and biological and epidemiological aspects, but also—and more importantly—on community aspects: fundamental research and the elaboration, implementation and assessment of field projects and action programmes for tuberculosis control and the promo tion of lung health. The Journal welcomes articles submitted on all aspects of lung health, including public health-related issues such as training programmes, cost-benefit analysis, legislation, epidemiology, intervention studies and health systems research

The University of Zimbabwe College of Health Sciences (UZ-CHS) BIRTH COHORT study: rationale, design and methods

Background: Commencing lifelong antiretroviral therapy (ART) immediately following HIV diagnosis (Option B+), has greatly improved maternal-infant health. Thus, large and increasing numbers of HIV-infected women are on ART during pregnancy, a situation concurrently increasing numbers of HIV-exposed-uninfected (HEU) infants. Compared to their HIV-unexposed-uninfected (HUU) counterparts, HEU infants show higher rates of adverse birth outcomes, mortality, infectious/non-communicable diseases including impaired growth and neurocognitive development. There is an urgent need to understand the impact of HIV and early life ART exposures, immune-metabolic dysregulation, comorbidities and environmental confounders on adverse paediatric outcomes. Methods: Six hundred (600) HIV-infected and 600 HIV-uninfected pregnant women ≥20 weeks of gestation will be enrolled from four primary health centres in high density residential areas of Harare. Participants will be followed up as mother-infant-pairs at delivery, week(s) 1, 6, 10, 14, 24, 36, 48, 72 and 96 after birth. Clinical, socio-economic, nutritional and environmental data will be assessed for adverse birth outcomes, impaired growth, immune/neurodevelopment, vertical transmission of HIV, hepatitis-B/C viruses, cytomegalovirus and syphilis. Maternal urine, stool, plasma, cord blood, amniotic fluid, placenta and milk including infant plasma, dried blood spot and stool will be collected at enrolment and follow-up visits. The composite primary endpoint is stillbirth and infant mortality within the first two years of life in HEU versus HUU infants. Maternal mortality in HIV-infected versus -uninfected women is another primary outcome. Secondary endpoints include a range of maternal and infant outcomes. Sub-studies will address maternal stress and malnutrition, maternal-infant latent tuberculosis, Helicobacter pylori infections, immune-metabolomic dysregulation including gut, breast milk and amniotic fluid dysbiosis. Discussion: The University of Zimbabwe-College of Health-Sciences-Birth-Cohort study will provide a comprehensive assessment of risk factors and biomarkers for HEU infants’ adverse outcomes. This will ultimately help developing strategies to mitigate effects of maternal HIV, early-life ART exposures and comorbidities on infants’ mortality and morbidity. Trial registration: ClinicalTrial.gov Identifier: NCT04087239. Registered 12 September 2019

Guidelines for the prevention and treatment of opportunistic infections in adults and adolescents with HIV

Developed by the National Institutes of Health, the Centers for Disease Control and Prevention, and the HIV Medicine Association of the Infectious Diseases Society of America Panel on Guidelines for the Prevention and Treatment of Opportunistic Infections in Adults and Adolescents with HIV\u2014A Working Group of the NIH Office of AIDS Research Advisory Council (OARAC).The Guidelines for the Prevention and Treatment of Opportunistic Infections in Adults and Adolescents with HIV document is published in an electronic format and updated as relevant changes in prevention and treatment recommendations occur.All changes are developed by the subject-matter groups listed in the document. (Changes in group composition also are posted promptly.) These changes are reviewed by the editors and by relevant outside reviewers before the document is altered.How to Cite the Adult and Adolescent Opportunistic Infection Guidelines: Panel on Guidelines for the Prevention and Treatment of Opportunistic Infections in Adults and Adolescents with HIV. Guidelines for the Prevention and Treatment of Opportunistic Infections in Adults and Adolescents with HIV. National Institutes of Health, Centers for Disease Control and Prevention, HIV Medicine Association, and Infectious Diseases Society of America. Available at https://clinicalinfo.hiv.gov/en/guidelines/adult-and- adolescent-opportunistic-infection. Accessed (insert date) [include page numbers, table number, etc., if applicable].Publication date from document properties.guidelines-adult-adolescent-oi.pd

Effects of intestinal worms on skin immunity and control of co-infection

Intestinal helminth infections remain a major health concern in developing areas of the world. Consequences of infection range from gastrointestinal discomfort to systemic manifestations. It has been suggested that individuals infected with intestinal helminths are more susceptible to other infections and mount weaker immunity to vaccination. Regions heavily burdened by intestinal helminths geographically coincide with areas plagued by infections with mycobacteria and the protozoan parasite Leishmania spp, causing tuberculosis (TB) and leishmaniasis, respectively. Further, it has been reported that people carrying intestinal worms mount weaker immune responses to the intradermally administered tuberculosis vaccine Mycobacterium bovis Bacillus Calmette Guérin (BCG). Intestinal helminth infections induce type 2 responses that aid in the expulsion of the worms, but also regulatory responses that facilitate chronicity of the worm infection. Both type 2 and regulatory responses are known to counteract type 1 immune responses required for protection against the intracellular pathogens mycobacteria and Leishmania, suggesting that worm infection may dampen protection to these infections. Yet, little is known about the systemic implications of intestinal worm infection, and the aim of the work in this thesis was to investigate the effects of intestinal helminth infection on skin immunity and control of co-infection. To this end, mice were infected with the strictly intestinal nematode Heligmosomoides polygyrus, and at various time points after infection subjected to secondary infection, immunization, or were culled. At the end of each experiment, composition and function of immune cells in skin, skin-draining lymph nodes (LNs), liver, spleen and other tissues involved in responses to secondary infection, were analysed. In Paper I, we found that mice infected with H. polygyrus were more susceptible to systemic infection with BCG and skin infection with Leishmania major. Increased susceptibility to BCG was accompanied by weaker IFN-γ production and fewer mycobacteria-specific transgenic p25 cells in spleen, and less inos expression and granuloma formation in livers. Delayed type hypersensitivity responses (DTH) induced in ears to BCG and L. major-derived antigens were dampened. Dendritic cell (DC) migration from footpad skin to the draining LN was reduced in worm-infected mice, as well as in mice where the footpad skin had been preconditioned with either H. polygyrus excretory-secretory (HES) products or recombinant human transforming growth factor β (TGF-β). In vitro, BCG-induced IFN-γ production by mycobacteria-specific T cells was reduced by HES, soluble worm antigens, or by TGF-β. This led us to hypothesize that H. polygurus-induced reduction of immunity to the T helper cell type 1(TH1)-controlled organisms mycobacterium BCG and L. major was mediated by enhanced TGF-β production in worm-infected mice. In Paper II, we saw that (similar to the situation with H. polygyrus – BCG co-infected animals) worm-infected mice were more susceptible to systemic infection with the TH1- controlled pathogen Leishmania donovani. Reduced protection was accompanied by lower inos levels and granuloma formation in livers and higher il10 levels in spleens. In Paper III, we sought for the explanation to the weaker skin immunity seen in worminfected mice (in Paper I). We found that mice infected with H. polygyrus had substantially smaller skin-draining LNs compared to worm-free animals. Both T cell and B cells were fewer, whereas no significant difference was observed in myeloid and stromal cell populations. As mentioned, numbers of DCs migrating from BCG-injected skin as well as p25 cells were less in skin-draining LNs of worm-infected mice. Notably however, numbers were directly proportional to the total number of cells in that particular LN. This led us to hypothesize that cells enter or are retained in an LN dependent on the original size of that node. As oppose to the atrophic skin draining LNs, the gut-draining mesenteric LNs were instead (as expected) dramatically increased in size. The lymphocyte pool cannot expand without limitation, and we suggested that worm-induced expansion of one LN occurred at the expense of other LN. Removal of worms restored the sizes of the non-draining nodes. However, this took time, since (according to out hypothesis) the atrophy of skin draining LNs and hyperplasia of mesenteric LN in itself decreased or increased infiltration or retention of cells into the respective nodes, maintaining this new “homeostasis”. In the last paper, Paper IV, we proceeded by investigating immune cells in the skin itself after H. polygyrus infection. We found that mice infected with H. polygyrus had fewer CD4+ cells producing IFN-γ in ear skin injected with whole cell lysate (WCL) from Mycobacterium tuberculosis in response to mycobacteria-specific ex-vivo re-stimulation, compared to wormfree mice. IFN-γ production was also lower in the contralateral, untouched ear. Interestingly however, the total number of CD4+ cells were higher in ear skin of worm-infected mice. CD4+ T cell numbers were also higher when comparing H. polygyrus-infected and noninfected animals without any skin stimulation, indicating that the intestinal infection, in itself, caused accumulation of CD4+ T cells in the skin. We found that the accumulated CD4+ T cells responded to H. polygyrus antigen by producing TH2 associated cytokines and that they remained in the skin for several weeks after removal of worms from the intestine. In accordance, skin-homing chemokine receptors were up-regulated on CD4+ T cells in the mesenteric LNs and blood. We hypothesized that the increased number of TH2 cells in the skin, in concert with the atrophy of skin draining LNs, were responsible for the lower protection to TH1-controlled organisms in the skin. In conclusion, mice chronically infected with the strictly intestinal nematode H. polygyrus were more susceptible to systemic and skin infection by TH1-controlled organisms compared to worm-free mice. We suggest that less inos and granuloma formation contributed to lower protection to systemic infection and that a combination of atrophic skin-draining LNs and increased numbers of TH2 cells in the skin caused weaker skin immunity. Taken together, this indicates that deworming may increase protection against secondary infection and increase beneficial effects of BCG vaccination

Investigating the role of the Dendritic Cell Immunoactivating Receptor in the Immune Response during Pneumocystis murina

Pneumocystis jirovecii causes fungal pneumonia in immunocompromised patients and can be fatal if left untreated. The global mortality rate is estimated to be over 200 000 in AIDS patients. In non-AIDS patients there is an estimated mortality rate of 50 000 cases. This rate is increasing in developed countries, attributed to an increase in disorders which require immunotherapy. These include hematologic malignancies, organ transplant, inflammatory disorders and pre-existing lung disease. Immediate immunity is initiated by receptors that recognize pathogen associated molecular patterns on the surface of pathogenic fungi. Specifically, C-type lectin receptors (CLRs) have been shown to be the principal initiators of innate immune response during fungal infection. Limited studies have focused on the role of CLRs in Pneumocystis infection. Dectin1and Mincle have been shown to recognise Pneumocystis surface antigens with Dectin-1 recognizing β-glucans on the Pneumocystis cell wall leading to an effective immune response. However, the role of a newly described CLR, the Dendritic Cell Immunoactivating Receptor (DCAR) remains undefined. For this reason, we investigated the potential role of this receptor in a mouse model of Pneumocystis murina infection. Wild type and DCAR-deficient C57BL/6 mice were infected with P. murina organisms via intratracheal instillation. Fungal burden was measured in the lung using quantitative Polymerase Chain Reaction. DCAR-deficient mice had a significantly reduced burden compared to wild type mice at Day 7 and 14 post-infection. To identify the immune components involved in pathogen clearance in these mice we measured cellular recruitment and cytokine production at both early (48 hours) and late (7, 14 and 21 days) time points. Flow cytometry analysis showed an increase in alveolar macrophage, dendritic cells, inflammatory monocytes, eosinophils and T cell recruitment to the lung. While ELISA showed increased levels of IL-1β and IFN-γ at 48 hours, and later on in infection IL-1β and IL-12p40 levels were also elevated. Histology analysis determined the localization of the recruited cells, and v interestingly showed an increase in mucus production at day 21 in DCARdeficient mice. In conclusion, we have identified DCAR deficiency as a potential driver of protective immunity in mice during P. murina infection. This may be associated with increased levels of IL-1β in DCAR-deficient mice. Furthermore, DCAR may also be important in adaptive inflammatory response regulation, as DCAR-deficient mice have increased cellular recruitment and mucus production later in infection. The mechanism by which the deletion of this receptor affords these mice the ability to efficiently clear P. murina remains to be determined

Regulatory, pro-inflammatory and inhibitory human T-cell responses to M. bovis BCG : opposing T-cell forces in TB-vaccination

There is no effective vaccine against tuberculosis (TB). The only available TB-vaccine, M. bovis BCG, induces only limited, and highly variable protection. TB-vaccine efficacy would have to include protection against active pulmonary TB, since this is the transmissible form of the disease, in the adult population; an effective vaccine would have an enormous impact on the TB-epidemic. This thesis has aimed to characterize the M. bovis BCG-reactive human T-cell response, in order to identify cellular responses that may account for the variable and poorly understood protective efficacy of BCG-vaccination. The studies presented in this thesis describe three BCG-induced cellular immune responses in adults: (i) the induction of CD8+ regulatory T-cells (Tregs), that suppress immunity partly via the ectoenzyme CD39, (ii) a dichotomous pro-inflammatory response, consisting of either induction of polyfunctional CD4+ T-cells in vaccinees with high skin inflammation of the vaccine lesion, or virtually no induction of cytokines with concomitant induction of CD8+ Tregs in vaccinees with low skin inflammation, and (iii) induction of inhibitory KLRG1+ CD8+ T-cells. This network of inter-related and partly opposing regulatory, pro-inflammatory and inhibitory immune responses may impact vaccine-induced protective immunity against TB and this could assist in guiding future TB-vaccine design.UBL - phd migration 201

Genomic insights into Mycobacterium tuberculosis and its interaction with the microbiota

Tuberculosis (TB) is the leading cause of death from a bacterial infection in humans. Despite its impact throughout history on humans across the globe, it remains challenging to diagnose and treat. This work used molecular biology and next generation sequencing to explore these issues. First, in a study to identify potential biomarkers of TB infection, the interaction between Mycobacterium tuberculosis (the causative agent of TB), the mouse immune system, and the murine gut microbiota was examined. The murine gut microbiota was observed to respond specifically to M. tuberculosis infection in several host genotypes, and these changes were most likely mediated by the adaptive immune system. Together, these data confirm that the response of the gut microbiota can be further explored for TB diagnostics. A second study was aimed at understanding the genetic mechanisms of resistance to a novel anti-mycobacterial compound. Resistance was mediated through loss of function of Rv2887, a previously unannotated gene that was found to be a multiple antibiotic resistance repressor (MarR) transcriptional regulator. Analysis of the function of Rv2887 led to the identification of a gene regulation mechanism that could be a potential new drug target. Finally, in a third study the genetic basis of geographic restriction of M. africanum, a mycobacterial species that causes similar disease to human TB but is usually only found in West Africa, was elucidated. Despite conventional dogma, analysis of M. africanum using new bioinformatics tools revealed that it is not a separate species from M. tuberculosis. Furthermore, M. africanum is unimpaired in transmission or virulence compared to M. tuberculosis, thus suggesting that the geographic restriction may be due to host factors. Taken together, this work explores the host-pathogen interactions and genetics of mycobacteria and provides novel insights into how these bacteria cause TB