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Children living with HIV are at risk for iron deficiency, yet optimal strategies for prevention and treatment remain unclear. Here, we investigate iron absorption, losses, and the efficacy and safety of oral iron supplementation with versus without prebiotics in three prospective studies in children with virally suppressed HIV in South Africa (NCT03572010, NCT04931641). In the first study, using stable iron isotopes, we show that iron absorption from iron-fortified maize porridge, a lipid-based nutrient supplement, and an oral iron supplement is comparable between children with HIV ( n  = 43) and without HIV ( n  = 45). In the second study, we use a stable iron isotope dilution method over a 6-month period to demonstrate that children with HIV ( n  = 29) absorb significantly less iron from their habitual diet than their uninfected peers ( n  = 36), while basal iron losses are similar. In the third study, a 12-week randomised, placebo-controlled, double-blind trial, iron-deficient children with HIV receiving iron with prebiotic galacto-oligosaccharides ( n  = 41) exhibit a 39% greater relative increase in serum ferritin (primary outcome) compared to those receiving iron with placebo ( n  = 42) ( p  = 0.053). They also report significantly fewer infection-related symptoms, with no significant differences in gut inflammation or enteropathogen carriage (secondary outcomes). Collectively, these findings indicate that while dietary iron absorption is reduced in children with virally suppressed HIV, supplemental and fortificant iron are well absorbed, and co-administration of iron supplements with prebiotics may improve efficacy and safety. Children living with HIV face a heightened risk of iron deficiency, yet optimal prevention and treatment strategies remain elusive. In these three prospective studies in South African children living with HIV, the authors show reduced dietary iron absorption but adequate absorption from iron supplements. Prebiotics may enhance efficacy and safety of oral iron supplementation in iron-deficient children living with HIV.

PurposeBoth HIV and oral iron interventions may alter gut microbiota composition and increase gut inflammation. We determined the effect of oral iron supplementation on gut microbiota composition, gut inflammation, and iron status in iron-depleted South Africa school-aged children living with HIV (HIV+) but virally suppressed on antiretroviral therapy and children without HIV (HIV-ve).MethodsIn this before-after intervention study with case–control comparisons, we provided 55 mg elemental iron from ferrous sulphate, once daily for 3 months, to 33 virally suppressed (< 50 HIV RNA copies/mL) HIV+ and 31 HIV-ve children. At baseline and endpoint, we assessed microbial composition of faecal samples (16S rRNA sequencing), and markers of gut inflammation (faecal calprotectin), anaemia (haemoglobin) and iron status (plasma ferritin, soluble transferrin receptor). This study was nested within a larger trial registered at clinicaltrials.gov as NCT03572010.ResultsHIV+ (11.3y SD ± 1.8, 46% male) and HIV−ve (11.1y SD ± 1.7, 52% male) groups did not significantly differ in age or sex ratio. Following iron supplementation, improvements were observed in haemoglobin (HIV+ : 118 to 124 g/L, P = 0.003; HIV−ve: 120 to 124 g/L, P = 0.003), plasma ferritin (HIV+ : 15 to 34 µg/L, P < 0.001; HIV−ve: 18 to 37 µg/L, P < 0.001), and soluble transferrin receptor (HIV+ : 7.1 to 5.9 mg/L, P < 0.001; HIV−ve: 6.6 to 5.7 mg/L, P < 0.001), with no significant change in the relative abundance of any genera, alpha diversity of the gut microbiota (HIV+ : P = 0.37; HIV−ve: P = 0.77), or faecal calprotectin (HIV+ : P = 0.42; HIV−ve: P = 0.80).ConclusionOur findings suggest that oral iron supplementation can significantly improve haemoglobin and iron status without increasing pathogenic gut microbial taxa or gut inflammation in iron-depleted virally suppressed HIV+ and HIV−ve school-age children.

Background/Aims: Globally children carry the highest burden of anaemia with at least 60% of anaemia attributed to iron deficiency [1]. Knowledge of usual food intake is fundamental in dietary interventions for iron deficiency anaemia, especially in lowincome settings where staple diets are plant-based [2]. Obtaining dietary intake information from children is challenging since the process is cumbersome and children may disengage, leading to poor quality data [3,4]. School-aged children spend a substantial amount of time away from home which necessitates self-reporting of food consumption during this period. While caregivers can assist with dietary intake reporting, they cannot solely act as a proxy for reporting complete intake. This study was nested within a clinical trial and aimed to assess dietary iron intake in 8 to 13-year old children from low-income areas in Cape Town, South Africa. Considering the potential pitfalls of dietary assessment in this age group, we regarded participant engagement and active participation as key drivers to determine the most appropriate dietary assessment method, and in the subsequent develop process of an abbreviated quantified food frequency questionnaire (QFFQ). Methods: The three main considerations for determining the most appropriate dietary assessment method were the study setting, the nutrients of interest and the age of the study population [4–6]. The clinical trial was performed at the Family Centre for Research with Ubuntu (FAMCRU) in Tygerberg Hospital, Cape Town and had a predetermined study schedule and time frame. The key nutrients of interest encompassed iron and nutrients involved with iron absorption and metabolism, namely animal protein, calcium, vitamin A, vitamin C, zinc and fibre. For this age group, a succinct method with appropriate strategies to improve engagement and active participation were deemed ideal. Given the single interaction advantage, epidemiological appropriateness [5–7], and potential for adaptation to a context-specific abbreviated questionnaire, we identified an interviewer-administered iron QFFQ with the child participant-caregiver pair as the best method to obtain the required data within our study setting. A well-developed QFFQ reflects the nutrients of interest and study population's habitual intake [5]. In addition to these characteristics, we prioritized careful planning of the questionnaire structure and a variety of interesting portion size estimation aids in support of participant engagement and active participation [5,7]. In the absence of an appropriate QFFQ validated for the specific study population and considering the timeline of the clinical trial, we followed a methodical approach driven by one experienced key worker. We used a multiphase approach to develop an abbreviated iron OFFQ and to establish its face and content validity. We engaged individually with key informants from the community, facilitated a larger group discussion with community representatives, performed pilot interviews, and consulted with senior diet methodology researchers. Results: Following the implementation of the QFFQ in the clinical trial (n=180), we reflected on the chosen strategies aimed at collectively supporting participant engagement and active participation (Figure 1). For this age group, interviewing the child participant and caregiver together provided comprehensive and accurate reporting and created a comfortable space for the child to engage with the interviewer. The QFFQ followed a logical but strategic order of categorised items. Careful positioning of foods received from the School Nutrition Program throughout the questionnaire and listing relevant but less affordable products between frequently eaten foods successfully supported continuous engagement and interview momentum. The first category of the QFFQ included frequently eaten staple foods that required active participation to indicate portion sizes. The children were interested in the different dietary intake assessment aids and eagerly engaged when prompted to use them to describe their portion sizes. For example, if a child reported cereal consumption, we asked him or her to indicate which of a selection of bowls best resembled the bowl used for cereal consumption. The interviewer would then empty a weighed portion of the reported cereal into the bowl with further comparison and quantification as needed. Similarly, weighted food portion photographs and drawings elicited interest and the children enjoyed using them to indicate which of a series of portion sizes best resembled their portion size. Using one experienced key worker to drive the development and implementation of the QFFQ proved to be invaluable in obtaining rounded knowledge and understanding of the study population's eating habits and food nuances. We used the development phase to learn the local terms for popular foods and to gain insights into their purchasing preferences when they received money to buy their own food or snacks. This led to better prompting ability and increased relatability, good interaction between the child participants and interviewer and greater interview success. Conclusions: Using one experienced key worker and a methodical approach for developing an abbreviated QFFQ reflecting the nutrients of interest and study population's habitual intake, together with strategies aimed at optimizing participant engagement and active participation, contributed to the successful collection of dietary iron intake information from school-aged children from a low-income setting.

The etiology of multifactorial morbidities such as undernutrition and anemia in children living with the human immunodeficiency virus (HIV) (HIV+) on antiretroviral therapy (ART) is poorly understood. Our objective was to examine associations of HIV and iron status with nutritional and inflammatory status, anemia, and dietary intake in school-aged South African children. Using a two-way factorial case-control design, we compared four groups of 8 to 13-year-old South African schoolchildren: (1) HIV+ and low iron stores (inflammation-unadjusted serum ferritin ≤ 40 µg/L), n = 43; (2) HIV+ and iron sufficient non-anemic (inflammation-unadjusted serum ferritin > 40 µg/L, hemoglobin ≥ 115 g/L), n = 41; (3) children without HIV (HIV-ve) and low iron stores, n = 45; and (4) HIV-ve and iron sufficient non-anemic, n = 45. We assessed height, weight, plasma ferritin (PF), soluble transferrin receptor (sTfR), plasma retinol-binding protein, plasma zinc, C-reactive protein (CRP), α-1-acid glycoprotein (AGP), hemoglobin, mean corpuscular volume, and selected nutrient intakes. Both HIV and low iron stores were associated with lower height-for-age Z-scores (HAZ, p < 0.001 and p = 0.02, respectively), while both HIV and sufficient iron stores were associated with significantly higher CRP and AGP concentrations. HIV+ children with low iron stores had significantly lower HAZ, significantly higher sTfR concentrations, and significantly higher prevalence of subclinical inflammation (CRP 0.05 to 4.99 mg/L) (54%) than both HIV-ve groups. HIV was associated with 2.5-fold higher odds of iron deficient erythropoiesis (sTfR > 8.3 mg/L) (95% CI: 1.03–5.8, p = 0.04), 2.7-fold higher odds of subclinical inflammation (95% CI: 1.4–5.3, p = 0.004), and 12-fold higher odds of macrocytosis (95% CI: 6–27, p < 0.001). Compared to HIV-ve counterparts, HIV+ children reported significantly lower daily intake of animal protein, muscle protein, heme iron, calcium, riboflavin, and vitamin B12, and significantly higher proportions of HIV+ children did not meet vitamin A and fiber requirements. Compared to iron sufficient non-anemic counterparts, children with low iron stores reported significantly higher daily intake of plant protein, lower daily intake of vitamin A, and lower proportions of inadequate fiber intake. Along with best treatment practices for HIV, optimizing dietary intake in HIV+ children could improve nutritional status and anemia in this vulnerable population. This study was registered at clinicaltrials.gov as NCT03572010.

Mycobacterium bovis ( M. bovis ), a member of the Mycobacterium tuberculosis complex (MTBC), is the causative agent of bovine TB (bTB) in animals. Spread occurs through inhalation or ingestion of bacilli transmitted from infected individuals. Early and accurate detection of infected African buffaloes shedding M. bovis is essential for interrupting transmission. In this pilot study, we determined if MTBC DNA could be detected in M. bovis infected buffalo oronasal secretions using a molecular transport media (PrimeStore MTM) with oronasal swabs and a rapid qPCR assay (Xpert MTB/RIF Ultra). Bovine TB test-positive buffaloes were culled, then tissue samples and oronasal swabs collected post-mortem for mycobacterial culture and Ultra testing, respectively. The Ultra detected MTBC DNA in 5/12 swabs from M. bovis culture-confirmed buffaloes. Oronasal swabs from M. bovis negative buffaloes (n = 20) were negative on Ultra, indicating the high specificity of this test. This study showed that MTM can successfully preserve MTBC DNA in oronasal swabs. The proportion of MTBC positive oronasal swabs was higher than expected and suggests that the Ultra may be an additional method for identifying infected buffaloes. Further studies are needed to confirm the utility of the Ultra assay with oronasal swabs as an assay to evaluate possible MTBC shedding in buffaloes.

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB) in wildlife. Confirmation of M. bovis infection relies on mycobacterial culture, which is time-consuming. Collection and transportation of infectious material also pose a human health risk. PrimeStore Molecular Transport Medium (MTM) has been shown to effectively inactivate infectious organisms, making it a safe method for handling infectious samples. This study investigated an in-field sampling technique for rapid, safe detection of M. bovis in buffalo tissues. Potentially infected tissues from bTB test-positive buffaloes were swabbed at post-mortem examination and stored in PrimeStore MTM at ambient temperature until Xpert MTB/RIF Ultra testing was performed. Additionally, tissue samples were frozen and transported before homogenisation for culture and Ultra testing. Oral swabs were collected from M. bovis- unexposed buffaloes as a negative control cohort. Mycobacterium tuberculosis complex (MTBC) DNA was detected by Ultra in 13/16 tissue swabs and 9/16 matched tissue homogenates from culture-confirmed M. bovis- positive buffalo tissues. MTBC DNA was not detected in swabs from M. bovis- unexposed animals, showing the potentially high specificity of Ultra with PrimeStore swabs. PrimeStore MTM sample processing, in combination with the Ultra assay, has the potential to provide a safe, rapid post-mortem screening test for M. bovis in buffaloes.

Since certain Mycobacterium tuberculosis complex (MTBC) members, such as M. bovis, are endemic in specific South African wildlife reserves and zoos, cases of clinically important nontuberculous mycobacteria (NTM) in wildlife may be neglected. Additionally, due to the inability of tests to differentiate between the host responses to MTBC and NTM, the diagnosis of MTBC may be confounded by the presence of NTMs. This may hinder control efforts. These constraints highlight the need for enhanced rapid detection and differentiation methods for MTBC and NTM, especially in high MTBC burden areas. We evaluated the use of the GeneXpert MTB/RIF Ultra, the Hain CMdirect V1.0 line probe assay, and novel amplicon sequencing PCRs targeting the mycobacterial rpoB and ku gene targets, directly on antemortem African elephant (n = 26) bronchoalveolar lavage fluid (BALF) (n = 22) and trunk washes (n = 21) and rhinoceros (n = 23) BALF (n = 23), with known MTBC culture-positive and NTM culture-positive results. Our findings suggest that the Ultra is the most sensitive diagnostic test for MTBC DNA detection directly in raw antemortem respiratory specimens and that the rpoB PCR is ideal for Mycobacterium genus DNA detection and species identification through amplicon sequencing.

Wildlife tuberculosis is a major economic and conservation concern globally. Bovine tuberculosis (bTB), caused by Mycobacterium bovis ( M. bovis ), is the most common form of wildlife tuberculosis. In South Africa, to date, M. bovis infection has been detected in 24 mammalian wildlife species. The identification of M. bovis infection in wildlife species is essential to limit the spread and to control the disease in these populations, sympatric wildlife species and neighboring livestock. The detection of M. bovis -infected individuals is challenging as only severely diseased animals show clinical disease manifestations and diagnostic tools to identify infection are limited. The emergence of novel reagents and technologies to identify M. bovis infection in wildlife species are instrumental in improving the diagnosis and control of bTB. This review provides an update on the diagnostic tools to detect M. bovis infection in South African wildlife but may be a useful guide for other wildlife species.

Diagnosis of bovine tuberculosis (bTB) may be confounded by immunological cross-reactivity to Mycobacterium bovis antigens when animals are sensitised by certain nontuberculous mycobacteria (NTMs). Therefore, this study aimed to investigate NTM species diversity in African buffalo (Syncerus caffer) respiratory secretions and tissue samples, using a combination of novel molecular tools. Oronasal swabs were collected opportunistically from 120 immobilised buffaloes in historically bTB-free herds. In addition, bronchoalveolar lavage fluid (BALF; n = 10) and tissue samples (n = 19) were obtained during post-mortem examination. Mycobacterial species were identified directly from oronasal swab samples using the Xpert MTB/RIF Ultra qPCR (14/120 positive) and GenoType CMdirect (104/120 positive). In addition, all samples underwent mycobacterial culture, and PCRs targeting hsp65 and rpoB were performed. Overall, 55 NTM species were identified in 36 mycobacterial culture-positive swab samples with presence of esat-6 or cfp-10 detected in 20 of 36 isolates. The predominant species were M. avium complex and M. komanii. Nontuberculous mycobacteria were also isolated from 6 of 10 culture-positive BALF and 4 of 19 culture-positive tissue samples. Our findings demonstrate that there is a high diversity of NTMs present in buffaloes, and further investigation should determine their role in confounding bTB diagnosis in this species.

Ante-mortem bovine tuberculosis (bTB) tests for buffaloes include the single comparative intradermal tuberculin test (SCITT), interferon-gamma (IFN-γ) release assay (IGRA) and IFN-γ-inducible protein 10 release assay (IPRA). Although parallel test interpretation increases the detection of Mycobacterium bovis (M. bovis)-infected buffaloes, these algorithms may not be suitable for screening buffaloes in historically bTB-free herds. In this study, the specificities of three assays were determined using M. bovis-unexposed herds, historically negative, and a high-specificity diagnostic algorithm was developed. Serial test interpretation (positive on both) using the IGRA and IPRA showed significantly greater specificity (98.3%) than individual (90.4% and 80.9%, respectively) tests or parallel testing (73%). When the SCITT was added, the algorithm had 100% specificity. Since the cytokine assays had imperfect specificity, potential cross-reactivity with nontuberculous mycobacteria (NTM) was investigated. No association was found between NTM presence (in oronasal swab cultures) and positive cytokine assay results. As a proof-of-principle, serial testing was applied to buffaloes (n = 153) in a historically bTB-free herd. Buffaloes positive on a single test (n = 28) were regarded as test-negative. Four buffaloes were positive on IGRA and IPRA, and M. bovis infection was confirmed by culture. These results demonstrate the value of using IGRA and IPRA in series to screen buffalo herds with no previous history of M. bovis infection.