Human milk: From complex tailored nutrition to bioactive impact on child cognition and behavior.

Human milk is a highly complex liquid food tailor-made to match an infant's needs. Beyond documented positive effects of breastfeeding on infant and maternal health, there is increasing evidence that milk constituents also impact child neurodevelopment. Non-nutrient milk bioactives would contribute to the (long-term) development of child cognition and behavior, a process termed 'Lactocrine Programming'. In this review we discuss the current state of the field on human milk composition and its links with child cognitive and behavioral development. To promote state-of-the-art methodologies and designs that facilitate data pooling and meta-analytic endeavors, we present detailed recommendations and best practices for future studies. Finally, we determine important scientific gaps that need to be filled to advance the field, and discuss innovative directions for future research. Unveiling the mechanisms underlying the links between human milk and child cognition and behavior will deepen our understanding of the broad functions of this complex liquid food, as well as provide necessary information for designing future interventions.

Captive Breeding and Trichomonas gallinae Alter the Oral Microbiome of Bonelli's Eagle Chicks.

Bonelli's eagle (Aquila fasciata) is an endangered raptor species in Europe, and trichomonosis is one of the menaces affecting chicks at nest. In this paper, we attempt to describe the oral microbiome of Bonelli's eagle nestlings and evaluate the influence of several factors, such as captivity breeding, Trichomonas gallinae infection, and the presence of lesions at the oropharynx. The core oral microbiome of Bonelli's eagle is composed of Firmicutes, Bacteroidota, Fusobacteria and Proteobacteria as the most abundant phyla, and Megamonas and Bacteroides as the most abundant genera. None of the factors analysed showed a significant influence on alfa diversity, but beta diversity was affected for some of them. Captivity breeding exerted a high influence on the composition of the oral microbiome, with significant differences in the four most abundant phyla, with a relative increase of Proteobacteria and a decrease of the other three phyla in comparison with chicks bred at nest. Some genera were more abundant in captivity bred chicks, such as Escherichia-Shigella, Enterococcus, Lactobacillus, Corynebacterium, Clostridium and Staphylococcus, while Bacteroides, Oceanivirga, Peptostreptococcus, Gemella, Veillonella, Mycoplasma, Suttonella, Alloscardovia, Varibaculum and Campylobacter were more abundant in nest raised chicks. T. gallinae infection slightly influenced the composition of the microbiome, but chicks displaying trichomonosis lesions had a higher relative abundance of Bacteroides and Gemella, being the last one an opportunistic pathogen of abscess complications in humans. Raptor's microbiomes are scarcely studied. This is the first study on the factors that influence the oral microbiome of Bonelli's eagle.

Garvicins AG1 and AG2: Two Novel Class IId Bacteriocins of Lactococcus garvieae Lg-Granada.

Lactococcus garvieae causes infectious diseases in animals and is considered an emerging zoonotic pathogen involved in human clinical conditions. In silico analysis of plasmid pLG50 of L. garvieae Lg-Granada, an isolate from a patient with endocarditis, revealed the presence of two gene clusters (orf46-47 and orf48-49), each one encoding a novel putative bacteriocin, i.e., garvicin AG1 (GarAG1; orf46) and garvicin AG2 (GarAG2; orf48), and their corresponding immunity proteins (orf47 and orf49). The chemically synthesised bacteriocins GarAG1 and GarAG2 presented inhibitory activity against pathogenic L. garvieae strains, with AG2 also being active against Listeria monocytogenes, Listeria ivanovii and Enterococcus faecalis. Genetic organisation, amino acid sequences and antimicrobial activities of GarAG1 and GarAG2 indicate that they belong to linear non-pediocin-like one-peptide class IId bacteriocins. Gram-positive bacteria that were sensitive to GarAG2 were also able to ferment mannose, suggesting that this bacteriocin could use the mannose phosphotransferase transport system (Man-PTS) involved in mannose uptake as a receptor in sensitive strains. Intriguingly, GarAG1 and GarAG2 were highly active against their own host, L. garvieae Lg-Granada, which could be envisaged as a new strategy to combat pathogens via their own weapons.

Gut microbes and health. / Microbiota intestinal y salud.

The human body is populated by myriads of microorganisms throughout its surface and in the cavities connected to the outside. The microbial colonisers of the intestine (microbiota) are a functional and non-expendable part of the human organism: they provide genes (microbiome) and additional functions to the resources of our species and participate in multiple physiological processes (somatic development, nutrition, immunity, etc.). Some chronic non-communicable diseases of developed society (atopias, metabolic syndrome, inflammatory diseases, cancer and some behaviour disorders) are associated with dysbiosis: loss of species richness in the intestinal microbiota and deviation from the ancestral microbial environment. Changes in the vertical transmission of the microbiome, the use of antiseptics and antibiotics, and dietary habits in industrialised society appear to be at the origin of dysbiosis. Generating and maintaining diversity in the microbiota is a new clinical target for health promotion and disease prevention.

Production of multiple bacteriocins, including the novel bacteriocin gassericin M, by Lactobacillus gasseri LM19, a strain isolated from human milk.

Bacteriocins are antimicrobial peptides produced by bacteria, and their production is regarded as a desirable probiotic trait. We found that Lactobacillus gasseri LM19, a strain isolated from human milk, produces several bacteriocins, including a novel bacteriocin, gassericin M. These bacteriocins were purified from culture and synthesised to investigate their activity and potential synergy. L. gasseri LM19 was tested in a complex environment mimicking human colon conditions; it not only survived, but expressed the seven bacteriocin genes and produced short-chain fatty acids. Metagenomic analysis of these in vitro colon cultures showed that co-inoculation of L. gasseri LM19 with Clostridium perfringens gave 16S ribosomal RNA metagenomic profiles with more similarity to controls than to vessels inoculated with C. perfringens alone. These results indicate that L. gasseri LM19 could be an interesting candidate for maintaining homeostasis in the gut environment.

Early-life intake of major trace elements, bisphenol A, tetrabromobisphenol A and fatty acids: Comparing human milk and commercial infant formulas.

In the present study, the presence of a wide spectrum of major and trace elements (As, Ag, Al, Ba, Cd, Co, Cr, Cu, Hg, Mn, Ni, Sr, Sb, Se, Sn, Pb, V, and Zn), fatty acids, as well as some pollutants like free and total BPA and tetrabromobisphenol A (TBBPA), was analysed in human milk (n = 53) and infant formula (n = 50) samples. In addition, the infant exposure to these chemicals was assessed. The content of free BPA and several elements (Al, Ca, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, Sn, Sr, and Zn) was higher (p < 0.01) in infant formula samples. Furthermore, human milk contained levels of BPA and elements that, in almost all cases, were well below their respective EFSA and/or WHO thresholds, being also independent of the maternal characteristics (e.g., age, BMI or breastfeeding period). The fatty acid profiling also revealed major differences between human milk and infant formulas, which should be taken in account in the development of new formulas as well as in specific recommendations for the diet of breastfeeding mothers. Anyway, the results of this study reinforce that breastfeeding should be always the first feeding option in early life.

Metataxonomic and immunological analysis of milk from ewes with or without a history of mastitis.

Mastitis is a highly prevalent condition that has a great impact on milk production and animal welfare, and often requires substantial management efforts. For this reason, it is generally considered an important threat to the dairy industry. Many microbial, host, and environmental factors can protect against, predispose to, or influence the development of mastitis. The objective of this work was to characterize the milk microbiota of Manchega ewes, and to compare samples from animals with and without a history of mastitis. We analyzed milk samples from 36 ewes belonging to 2 different farms (18 ewes from each farm) using culture-dependent and culture-independent techniques. We also analyzed several immune compounds to investigate associations of mastitis with 3 main variables: farm; history of mastitis or no mastitis; and parity number. Both culture-dependent and culture-independent techniques showed that ewe milk harbored a site-specific complex microbiota and microbiome. Staphylococcus epidermidis was the main species driving the difference between farm A (where it was the dominant species) and B (where it was not). In contrast, samples from farm B were characterized by the presence of a wide spectrum of other coagulase-negative staphylococci. Some of these species have already been associated with subclinical intramammary infections in ruminants. Of the 10 immune compounds assayed in this study, 3 were related to a history of mastitis [IL-8, IFN-γ, and IFN-gamma-induced protein 10 (IP-10)]. Increases in IL-8 concentrations in milk seemed to be a feature of subclinical mastitis in sheep, and in this study, this immune factor was detected only in samples from ewes with some episodes of mastitis and from the group with the highest somatic cell count. We also observed a positive correlation between the samples with the highest somatic cell count and IFN-γ and IP-10 levels. Our results suggest that these 3 compounds could be used as biomarkers for the negative selection of mastitis-prone animals, particularly when somatic cell count is very high.

Prevention of Infectious Mastitis by Oral Administration of Lactobacillus salivarius PS2 During Late Pregnancy.

BACKGROUND: Previous studies have shown that oral administration of lactobacilli can be an efficient approach to treat lactational infectious mastitis. In this trial, we have evaluated the potential of Lactobacillus salivarius PS2 to prevent this condition when orally administered during late pregnancy to women who had experienced infectious mastitis after previous pregnancies. METHODS: In this study, 108 pregnant women were randomly assigned to one of 2 groups. Those in the probiotic group (n = 55) ingested daily 9 log10 colony-forming units of L. salivarius PS2 from approximately week 30 of pregnancy until delivery, whereas those in the placebo group (n = 53) received a placebo. The occurrence of mastitis was evaluated during the first 3 months after delivery. RESULTS: Globally, 44 of 108 women (41%) developed mastitis; however, the percentage of women with mastitis in the probiotic group (25% [n = 14]) was significantly lower than in the control group (57% [n = 30]). When mastitis occurred, the milk bacterial counts in the probiotic group were significantly lower than those obtained in the placebo group. CONCLUSIONS: Oral administration of L. salivarius PS2 during late pregnancy appears to be an efficient method to prevent infectious mastitis in a susceptible population. CLINICAL TRIALS REGISTRATION: NCT01505361.

Purification and genetic characterization of gassericin E, a novel co-culture inducible bacteriocin from Lactobacillus gasseri EV1461 isolated from the vagina of a healthy woman.

BACKGROUND: Lactobacillus gasseri is one of the dominant Lactobacillus species in the vaginal ecosystem. Some strains of this species have a high potential for being used as probiotics in order to maintain vaginal homeostasis, since they may confer colonization resistance against pathogens in the vagina by direct inhibition through production of antimicrobial compounds, as bacteriocins. In this work we have studied bacteriocin production of gassericin E (GasE), a novel bacteriocin produced by L. gasseri EV1461, a strain isolated from the vagina of a healthy woman, and whose production was shown to be promoted by the presence of certain specific bacteria in co-culture. Biochemical and genetic characterization of this novel bacteriocin are addressed. RESULTS: We found that the inhibitory spectrum of L. gasseri EV1461 was broad, being directed to species both related and non-related to the producing strain. Interestingly, L. gasseri EV1461 inhibited the grown of pathogens usually associated with bacterial vaginosis (BV). The antimicrobial activity was due to the production of a novel bacteriocin, gassericin E (GasE). Production of this bacteriocin in broth medium only was achieved at high cell densities. At low cell densities, bacteriocin production ceased and only was restored after the addition of a supernatant from a previous bacteriocin-producing EV1461 culture (autoinduction), or through co-cultivation with several other Gram-positive strains (inducing bacteria). DNA sequence of the GasE locus revealed the presence of two putative operons which could be involved in biosynthesis and immunity of this bacteriocin (gaeAXI), and in regulation, transport and processing (gaePKRTC). The gaePKR encodes a putative three-component regulatory system, involving an autoinducer peptide (GaeP), a histidine protein kinase (GaeK) and a response regulator (GaeR), while the gaeTC encodes for an ABC transporter (GaeT) and their accessory protein (GaeC), involved in transport and processing of the bacteriocin. The gaeAXI, encodes for the bacteriocin gassericin E (GasE), a putative peptide bacteriocin (GaeX), and their immunity protein (GaeI). CONCLUSIONS: The origin of the strain (vagina of healthy woman) and its ability to produce bacteriocins with inhibitory activity against vaginal pathogens may be an advantage for using L. gasseri EV1461 as a probiotic strain to fight and/or prevent bacterial infections as bacterial vaginosis (BV), since it could be better adapted to live and compete into the vaginal environment.

The salivary scavenger and agglutinin in early life: diverse roles in amniotic fluid and in the infant intestine.

The salivary scavenger and agglutinin (SALSA), also known as gp340 and dmbt1, is an antimicrobial and inflammation-regulating molecule located at the mucosal surfaces. The present study revealed that SALSA was present in the amniotic fluid (AF) and exceptionally enriched in both meconium and feces of infants. Based on immunological and mass spectrometric analysis, SALSA was estimated to constitute up to 4-10% of the total protein amount in meconium, making it one of the most abundant proteins. SALSA proteins in the AF and intestinal samples were polymorphic and exhibited varying polypeptide compositions. In particular, a different abundance of peptides corresponding to functionally important structures was found in the AF and intestinal SALSA. The AF form of SALSA had a more intact structure and contained peptides from the zona pellucida domain, which is involved in cell differentiation and oligomerization. In contrast, the intestinal SALSA was more enriched with the scavenger receptor cysteine-rich domains. The AF, but not the meconium SALSA, bound to Streptococcus pyogenes, S. agalactiae, S. gordonii, and Escherichia coli. Furthermore, differential binding was observed also to known endogenous ligands C1q, mannose-binding lectin, and secretory IgA. Our results have thus identified mucosal body compartments, where SALSA is particularly abundant, and suggest that SALSA exhibits varying functions in the different mucosal locations. The high levels of SALSA in AF and the infant intestine suggest a robust and important function for SALSA during the fetal development and in the mucosal innate immune defense of infants.

Evaluation of technological properties of Enterococcus faecium CECT 8849, a strain isolated from human milk, for the dairy industry.

In this work, a variety of biochemical properties of Enterococcus faecium CECT 8849, which had been isolated from breast milk, were analyzed. Its acidifying capacity and proteolytic activity were low but, in contrast, remarkable peptidase and esterase activities were observed. Ethanol and 3-hydroxy-2-butanone were the most abundant volatile compounds found in experimental model cheese manufactured with E. faecium CECT 8849. This strain inhibited the growth of several Listeria monocytogenes and Listeria innocua strains in vitro. Enterocin A and B structural genes were detected in E. faecium CECT 8849. Model fermented milk and cheeses were manufactured from milk inoculated or not with L. innocua CECT 8848 (2.5-3 log10 colony forming units mL(-1)) using E. faecium CECT 8849 or Lactococcus lactis ESI 153 as starter cultures. Although E. faecium CECT 8849 controlled Listeria growth in both dairy models, it led to lower reduction in Listeria counts when compared with L. lactis ESI 153.

Characterization of an outbreak of emerging bovine besnoitiosis in southwestern Spain.

Bovine besnoitiosis is an emerging disease in Europe, presenting quick spread toward central and southern Spain. Characterization of an outbreak in a free-ranging Limousin and Avileña beef cattle herd from southwestern Spain territories is attempted. Serological survey in the herd revealed increase of number of infected animals, from 34.3 % on first diagnoses/exams on December 2013 to 42.5 % in the second on April 2014. Blood analysis and serum biochemistry showed important alterations like leukocytosis (+33.2 % of mean value), with lymphocytosis (+205.3 %) and increase of LDH (+25.1 %), associated with tissue damage. Clinical cases were only observed in Limousin animals. Along with typical lesions of acute and chronic besnoitiosis, inflammatory and degenerative processes and parasitic cysts were present in the corpus cavernosum and the corpus spongiosum of penis. By using polymerase chain reaction (PCR) sequencing of 18S rDNA, Besnoitia besnoiti was confirmed as causative agent; microsatellite sequence analyses showed the homology of isolates with previously studied strains.

The composition of the gut microbiota throughout life, with an emphasis on early life.

The intestinal microbiota has become a relevant aspect of human health. Microbial colonization runs in parallel with immune system maturation and plays a role in intestinal physiology and regulation. Increasing evidence on early microbial contact suggest that human intestinal microbiota is seeded before birth. Maternal microbiota forms the first microbial inoculum, and from birth, the microbial diversity increases and converges toward an adult-like microbiota by the end of the first 3-5 years of life. Perinatal factors such as mode of delivery, diet, genetics, and intestinal mucin glycosylation all contribute to influence microbial colonization. Once established, the composition of the gut microbiota is relatively stable throughout adult life, but can be altered as a result of bacterial infections, antibiotic treatment, lifestyle, surgical, and a long-term change in diet. Shifts in this complex microbial system have been reported to increase the risk of disease. Therefore, an adequate establishment of microbiota and its maintenance throughout life would reduce the risk of disease in early and late life. This review discusses recent studies on the early colonization and factors influencing this process which impact on health.

Urinary metabolomic fingerprinting after consumption of a probiotic strain in women with mastitis.

Infectious mastitis is a common condition among lactating women, with staphylococci and streptococci being the main aetiological agents. In this context, some lactobacilli strains isolated from breast milk appear to be particularly effective for treating mastitis and, therefore, constitute an attractive alternative to antibiotherapy. A (1)H NMR-based metabolomic approach was applied to detect metabolomic differences after consuming a probiotic strain (Lactobacillus salivarius PS2) in women with mastitis. 24h urine of women with lactational mastitis was collected at baseline and after 21 days of probiotic (PB) administration. Multivariate analysis (OSC-PLS-DA and hierarchical clustering) showed metabolome differences after PB treatment. The discriminant metabolites detected at baseline were lactose, and ibuprofen and acetaminophen (two pharmacological drugs commonly used for mastitis pain), while, after PB intake, creatine and the gut microbial co-metabolites hippurate and TMAO were detected. In addition, a voluntary desertion of the pharmacological drugs ibuprofen and acetaminophen was observed after probiotic administration. The application of NMR-based metabolomics enabled the identification of the overall effects of probiotic consumption among women suffering from mastitis and highlighted the potential of this approach in evaluating the outcomes of probiotics consumption. To our knowledge, this is the first time that this approach has been applied in women with mastitis during lactation.

Probiotics in human milk and probiotic supplementation in infant nutrition: a workshop report.

Probiotics in human milk are a very recent field of research, as the existence of the human milk microbiome was discovered only about a decade ago. Current research is focusing on bacterial diversity and the influence of the maternal environment as well as the mode of delivery on human milk microbiota, the pathways of bacterial transfer to milk ducts, possible benefits of specific bacterial strains for the treatment of mastitis in mothers, and disease prevention in children. Recent advances in the assessment of early host-microbe interactions suggest that early colonisation may have an impact on later health. This review article summarises a scientific workshop on probiotics in human milk and their implications for infant health as well as future perspectives for infant feeding.

Investigating the Metabolism of Estrogens in Ligilactobacillus salivarius Strains Isolated from Human Milk and Vaginal Microbiota.

The interplay between enterohepatic circulation and the gut microbiota is the main driver determining systemic levels of estrogens and their metabolites. Nevertheless, the role of potentially probiotic microorganisms in estrogen metabolism has not been investigated so far. In this work, we have explored the ability of six Ligilactobacillus salivarius strains isolated from human milk and vaginal samples to degrade and/or conjugate parental estrogens in vitro and under aerobic conditions. The quantification of estrogens and their derivatives was carried out in cell-free supernatants by LC-QQQ-MS. All the tested L. salivarius strains achieved an average degradation rate of estrone and estriol of 98% and 55%, respectively, whereas 17ß-estradiol was preferentially conjugated (up to 40%). The presence of seven out of ten genes encoding enzymes relevant for estrogen metabolism was further confirmed by PCR, highlighting their genetic potential for degrading, conjugating and/or deconjugating estrogens. The tested L. salivarius strains may be considered potential probiotics affecting the fate of endogenous estrogens. Clinical trials targeting populations with estrogen-dependent conditions will be required to elucidate the true potential of these strains for the restoration and maintenance of a healthy host estrobolome.

Influence of Age of Infection on the Gut Microbiota in Worker Honey Bees (Apis mellifera iberiensis) Experimentally Infected with Nosema ceranae.

The gut microbiota of honey bees has received increasing interest in the past decades due to its crucial role in their health, and can be disrupted by pathogen infection. Nosema ceranae is an intracellular parasite that affects the epithelial cells of the midgut, altering gut homeostasis and representing a major threat to honey bees. Previous studies indicated that younger worker bees are more susceptible to experimental infection by this parasite, although the impact of infection and of age on the gut bacterial communities remains unclear. To address this, honey bees were experimentally infected with a consistent number of N. ceranae spores at various ages post-emergence (p.e.) and the gut bacteria 7 days post-infection (p.i.) were analysed using real-time quantitative PCR, with the results compared to non-infected controls. Infected bees had a significantly higher proportion and load of Gilliamella apicola. In respect to the age of infection, the bees infected just after emergence had elevated loads of G. apicola, Bifidobacterium asteroides, Bombilactobacillus spp., Lactobacillus spp., Bartonella apis, and Bombella apis. Moreover, the G. apicola load was higher in bees infected at nearly all ages, whereas older non-infected bees had higher loads of Bifidobacterium asteroides, Bombilactobacillus spp., Lactobacillus spp., Ba. apis, and Bo apis. These findings suggest that N. ceranae infection and, in particular, the age of bees at infection modulate the gut bacterial community, with G. apicola being the most severely affected species.

Composition and Functional Properties of the Edible Spear and By-Products from Asparagus officinalis L. and Their Potential Prebiotic Effect.

Asparagus is a healthy food appreciated for its organoleptic characteristics, nutritional composition and physiological properties. During its industrial processing, a large amount of by-products are generated, since only the apical part of the vegetable is considered edible and a large amount of by-products are generated that could be of nutritional interest. Therefore, the nutritional composition of the edible part and the two by-products of the plant (root and stem) was evaluated, including dietary fiber, inulin, low-molecular-weight carbohydrates, low-molecular-weight polyphenols and macromolecular polyphenols. The hydration properties, oil retention capacity, glucose retardation index and impact on bacterial growth of both probiotic bacteria and pathogenic strains were determined. All samples were high in fiber (>22 g/100 g dw), fructans (>1.5 g/100 g dw) and polyphenolic compounds (>3 g/100 g dw) and had good water-, oil- and glucose-binding capacity. In addition, they promoted the growth of probiotic strains but not pathogenic ones. The effects were more pronounced in the spear by-product samples and appear to be related to the components of dietary fiber. The results indicate that edible spear has potential beneficial effects on host health and microbiota when ingested as part of a healthy diet, while the by-products could be used as supplements and/or as natural ingredients in fiber-enriched foods that require emulsification and are intended to achieve a prebiotic effect.

Influence of SARS-CoV-2 Status and Aging on the Nasal and Fecal Immunological Profiles of Elderly Individuals Living in Nursing Homes.

In the frame of SARS-CoV-2 infection, studies regarding cytokine profiling of mucosal-related samples are scarce despite being the primary infection sites. The objective of this study was to compare the nasal and fecal inflammatory profiles of elderly individuals living in a nursing home highly affected by COVID-19 (ELD1) with those of elderly individuals living in a nursing home with no cases of SARS-CoV-2 infection (ELD2) and, also, with those of healthy SARS-CoV-2-negative younger adults (YHA). BAFF/TNFSF13B, IL6, IL10 and TNF-α (immunological hallmarks of SARS-CoV-2 infection) were the only immune factors whose concentrations were different in the three groups. Their highest concentrations were achieved in the ELD1 group. Nasal and fecal concentrations of a wide number of pro-inflammatory cytokines were similar in the ELD1 and ELD2 groups but higher than those found in the YHA samples. These results reinforce the hypothesis that immunosenescence and inflammaging rendered the elderly as a highly vulnerable population to a neo-infection, such as COVID-19, which was evidenced during the first pandemic waves.

Comparison of the Fecal Bacteriome of HIV-Positive and HIV-Negative Older Adults.

HIV infection is considered a scenario of accelerated aging. Previous studies have suggested a link between aging, frailty, and gut dysbiosis, but there is a knowledge gap regarding the HIV population. Our objective was to compare the fecal bacteriome of older people with HIV (PWH) and non-HIV controls, and to assess potential links between gut dysbiosis and frailty. A total of 36 fecal samples (24 from PWH and 12 from non-HIV controls) were submitted to a metataxonomic analysis targeting the V3-V4 hypervariable region of the 16S rRNA gene. High-quality reads were assembled and classified into operational taxonomic units. Alpha diversity, assessed using the Shannon index, was higher in the control group than in the HIV group (p < 0.05). The relative abundance of the genus Blautia was higher in the HIV group (p < 0.001). The presence of Blautia was also higher in PWH with depression (p = 0.004), whereas the opposite was observed for the genus Bifidobacterium (p = 0.004). Our study shows shifts in the composition of the PWH bacteriome when compared to that of healthy controls. To our knowledge, this is the first study suggesting a potential link between depression and gut dysbiosis in the HIV population.