Anna Olutoyin Okunola

Introduction: Non-sputum tests for people attending primary care with symptoms of tuberculosis (TB) are a global priority. Methods: We performed a cross-sectional two-centre diagnostic accuracy study of a next-generation urine lipoarabinomannan assay (Biopromic TB LAM, BP-LAM) and the lateral flow Determine TB LAM Ag assay (LF-LAM) on urine from 629 adults with presumptive pulmonary TB (315 with TB) attending primary care in South Africa and Uganda. An extended sputum microbiological reference standard (eMRS) was used and, in South Africa, sputum induction done. Results: BP-LAM had higher sensitivity than LF-LAM [95% confidence interval (CI) 63% (58, 69) vs. 22% (18, 27)] and similar specificity [93% (90, 96) vs. 89% (85, 92)]. BP-LAM sensitivity did not differ in HIV-positive vs. -negative people nor by CD4 counts. Specificity was diminished in PLHIV [90% (83, 94) vs. 96% (92, 99)]. The design-locked version of BP-LAM had improved specificity than the prototype [93% (90, 96) vs. 85% (80, 88)]. In people with TB who could not expectorate sputum, 67% (55, 79) were BP-LAM-positive; resulting in similar yield to sputum Xpert MTB/RIF Ultra [64% (49, 78)] in a programmatic scenario where sputum induction is unavailable. BP-LAM false-negatives had, vs. true-positives, less severe disease and were more likely to have previous TB. False-positive results were more likely to be from people who cultured only non-tuberculous mycobacteria [19% (4/21) vs. 4% (12/293) for true-negatives]. Conclusions: BP-LAM has higher sensitivity than LF-LAM, including in HIV-negative people, potentially permitting the expansion of urine LAM testing beyond people with advanced immunosuppression. Funding: The Global Health Technology Fund (GHIT) programs RFP-TRP-2018-001 and RFP-PD-2020-002, EDCTP2 (RIA2020I-3305, CAGE-TB; TMA2020CDF-3209, RADIANT).

Staphylococcus aureus is a bacterium found in the milk of up to 38% of healthy lactating mothers; however, S. aureus is isolated with increased frequency from colostrum and mastitis milk. Both of these milk types have lower lactose concentrations compared to mature milk from healthy lactating mothers, which may indicate that lactose has a role in determining whether S. aureus can survive in human milk. The aim of this study was (1) to investigate whether the presence of S. aureus in human milk is associated with the milk’s lactose concentration, and (2) to determine whether different lactose concentrations can affect the ability of S. aureus isolates to grow in vitro. Human milk samples were collected at 10 weeks postpartum from mothers participating in the Drakenstein Child Health Study (Cape Town, South Africa) and underwent NMR spectroscopy to determine their metabolome. A subset of these samples (n = 117) was cultured to isolate S. aureus. Milk samples with lactose concentrations of less than 166 mM were more likely to have S. aureus present, compared to samples with lactose concentrations of over 166 mM (p < 0.001). In vitro, the growth of S. aureus was negatively correlated with the lactose concentration of axenic culture. Lactose concentrations associated with human milk appear to have an inhibitory effect on the growth of S. aureus human milk isolates. Therefore, low-lactose human milk could potentially be a risk factor for increased S. aureus growth and the development of S. aureus-associated mastitis.

New tuberculosis (TB) diagnostics are at a crossroads: their development, evaluation, and implementation is severely damaged by resource diversion due to COVID-19. Yet several technologies, especially those with potential for non-invasive non-sputum-based testing, hold promise for efficiently triaging and rapidly confirming TB near point-of-care. Such tests are, however, progressing through the pipeline slowly and will take years to reach patients and health workers. Compellingly, such tests will create new opportunities for difficult-to-diagnose populations, including primary care attendees (all-comers in high burden settings irrespective of reason for presentation) and community members (with early stage disease or risk factors like HIV), many of whom cannot easily produce sputum. Critically, all upcoming technologies have limitations that implementers and health workers need to be cognizant of to ensure optimal deployment without undermining confidence in a technology that still offers improvements over the status quo. In this state-of-the-art review, we critically appraise such technologies for active pulmonary TB diagnosis. We highlight strengths, limitations, outstanding research questions, and how current and future tests could be used in the presence of these limitations and uncertainties. Among triage tests, CRP (for which commercial near point-of-care devices exist) and computer-aided detection software with digital chest X-ray hold promise, together with late-stage blood-based assays that detect host and/or microbial biomarkers; however, aside from a handful of prototypes, the latter category has a shortage of promising late-stage alternatives. Furthermore, positive results from new triage tests may have utility in people without TB; however, their utility for informing diagnostic pathways for other diseases is under-researched (most sick people tested for TB do not have TB). For confirmatory tests, few true point-of-care options will be available soon; however, combining novel approaches like tongue swabs with established tests like Ultra have short-term promise but first require optimizations to specimen collection and processing procedures. Concerningly, no technologies yet have compelling evidence of meeting the World Health Organization optimal target product profile performance criteria, especially for important operational criteria crucial for field deployment. This is alarming as the target product profile criteria are themselves almost a decade old and require urgent revision, especially to cater for technologies made prominent by the COVID-19 diagnostic response (e.g., at-home testing and connectivity solutions). Throughout the review, we underscore the importance of how target populations and settings affect test performance and how the criteria by which these tests should be judged vary by use case, including in active case finding. Lastly, we advocate for health workers and researchers to themselves be vocal proponents of the uptake of both new tests and those – already available tests that remain suboptimally utilized.

Culture-independent molecular techniques have advanced the characterization of environmental and human samples including the human milk (HM) bacteriome. However, extraction of high-quality genomic DNA that is representative of the bacterial population in samples is crucial. Lipids removal from HM prior to DNA extraction is common practice, but this may influence the bacterial population detected. The objective of this study was to compare four commercial DNA extraction kits and lipid removal in relation to HM bacterial profiles. Four commercial DNA extraction kits, QIAamp^® DNA Microbiome Kit, ZR Fungal/Bacterial DNA MiniPrep™, QIAsymphony DSP DNA Kit and ZymoBIOMICS™ DNA Miniprep Kit, were assessed using milk collected from ten healthy lactating women. The kits were evaluated based on their ability to extract high quantities of pure DNA from HM and how well they extracted DNA from bacterial communities present in a commercial mock microbial community standard spiked into HM. Finally, the kits were evaluated by assessing their extraction repeatability. Bacterial profiles were assessed using Illumina MiSeq sequencing targeting the V4 region of the 16S rRNA gene. The ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep (Zymo Research Corp., Irvine, CA, USA) kits extracted the highest DNA yields with the best purity. DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ best represented the bacteria in the mock community spiked into HM. In un-spiked HM samples, DNA extracted using the QIAsymphony DSP DNA kit showed statistically significant differences in taxa prevalence from DNA extracted using ZR Fungal/Bacterial DNA MiniPrep™ and ZymoBIOMICS™ DNA Miniprep kits. The only difference between skim and whole milk is observed in bacterial profiles with differing relative abundances of Enhydrobacter and Acinetobacter. DNA extraction, but not lipids removal, substantially influences bacterial profiles detected in HM samples, emphasizing the need for careful selection of a DNA extraction kit to improve DNA recovery from a range of bacterial taxa.

Human milk is needed for optimal growth as it satisfies both the nutritional and biological needs of an infant. The established relationship between breastfeeding and an infant’s health is attributable to the nutritional and non-nutritional, functional components of human milk including metabolites such as the lipids, amino acids, biogenic amines and carbohydrates. These components have diverse roles, including protecting the infant against infections and guiding the development of the infant’s immature immune system. In this review, we provide an in-depth and updated insight into the immune modulatory and anti-infective role of human milk metabolites and their effects on infant health and development. We also review the literature on potential determinants of the human milk metabolome, including maternal infectious diseases such as human immunodeficiency virus and mastitis.

The human breast milk (HBM) bacteriome is an important, continuous source of microbes to the neonate in early life, playing an important role in shaping the infant’s intestinal bacteriome. Study of the composition of the HBM bacteriome is an emerging area of research, with little information available, particularly from low- and middle-income countries. The aim of this study was to characterize the diversity of bacterial communities in HBM samples collected between 6–10 weeks postpartum from lactating South African women and to study potential influencing factors of the bacteriome. Using 16S rRNA gene sequencing of samples from 554 women, we demonstrated that the HBM bacteriome was largely dominated by the phyla Firmicutes (mean relative abundance: 71.1%) and Actinobacteria (mean relative abundance: 16.4%). The most abundant genera identified from the HBM bacteriome were Streptococcus (mean relative abundance: 48.6%), Staphylococcus (mean relative abundance: 17.8%), Rothia (mean relative abundance: 5.8%), and Corynebacterium (mean relative abundance: 4.3%). “Core” bacterial genera including Corynebacterium, Streptococcus, Staphylococcus, Rothia, Veillonella, Gemella, Acinetobacter, Micrococcus and a genus belonging to the Enterobacteriaceae family were present in 80% of samples. HBM samples were classified, according to their bacteriome, into three major clusters, dominated by the genera Staphylococcus (cluster 1), a combination of Staphylococcus and Streptococcus (cluster 2), and Streptococcus (cluster 3). The cluster groups differed significantly for Shannon and chao1 richness indices. Bacterial interactions were studied using co-occurrence networks with positive associations observed between the abundances of Staphylococcus and Corynebacteria (members of the skin microflora) and between Streptococcus, Rothia, Veillonella, and Gemella (members of the oral microflora). HBM from older mothers had a higher Shannon diversity index. The study site was associated with differences in HBM bacteriome composition (permutational multivariate analysis of variance using distance matrices (PERMANOVA), p < 0.05). No other tested socio-demographic or psychosocial factors were associated with HBM bacterial composition.

It is well-known that, beyond nutritional components, human breast milk (HBM) contains a wide variety of non-nutritive bio-factors perfectly suited for the growing infant. In the pre-2000 era, HBM was considered sterile and devoid of micro-organisms. Though HBM was not included as part of the human microbiome project launched in 2007, great strides have been made in studying the bacterial diversity of HBM in both a healthy state and diseased state, and in understanding their role in infant health. HBM provides a vast array of beneficial micro-organisms that play a key role in colonizing the infant’s mucosal system, including that of the gut. They also have a role in priming the infant’s immune system and supporting its maturation. In this review, we provide an in-depth and updated insight into the immunomodulatory, metabolic, and anti-infective role of HBM bacteriome (bacterial community) and its effect on infant health. We also provide key information from the literature by exploring the possible origin of microbial communities in HBM, the bacterial diversity in this niche and the determinants influencing the HBM bacteriome. Lastly, we investigate the role of the HBM bacteriome in maternal infectious disease (human immunodeficiency virus (HIV) and mastitis)), and cancer. Key gaps in HBM bacterial research are also identified.