Evaluation of the effect of HIV virus on the digestive flora of infected versus non infected infants

Introduction HIV infection is characterized by changes in the composition and functions of gut microbiota. We carried out a study aiming at comparing the compositional changes of the digestive flora of HIV infected infants versus that of non infected infants in Cameroon. Methods A case-control study was carried out during which stool sample was collected from each participant after obtaining the proxy consent. Stools were cultured using aerobic, strict anaerobic, 10% CO2 and micro-aerophilic conditions and specific culture media and bacteria were identified biochemically. Fisher's exact test was used for data analyses. Results From the 80 infants enrolled for the study, 33 (41.3%) were HIV positive. A statistically significant difference was observed between the number of infected versus non infected infants harboring the following bacteria: Clostridium spp. (P=0.009); Enterococcus spp. (p<0.001); Klebsiella (p<0.001); Shigella (<0.001); Staphylococcus aureus (p=0.006) and Streptococcus spp. (P=0.015). Among infected infants, WHO-stage 3 and 4 infants harbored more opportunistic bacteria than stage 1 and stage 2 and Bacteriodes spp. population was depleted as the disease progresses, although not significantly. There was an imbalance in bacteria flora in HIV infected infants harboring qualitatively more bacteria including more opportunistic and pathogenic bacteria than in HIV non-infected infants. Conclusion HIV infected infants presented a qualitatively different flora from HIV non infected infants. They habored more pathogenic bacteria Than non infected infants. Systematic stool culture could benefit for follow-up of HIV infected infants to reduce the risk of gastrointestinal disorders and thus the risk of high morbidity or high mortability.


Introduction
Since the first cases of human immunodeficiency virus (HIV) infection were identified in 1981, the number of infants infected with HIV has increased dramatically in developing countries because the number of HIV-infected women of childbearing age has risen [1]. However, great advances have been made in developed countries to control transmission of the virus from mother to infant. In developing countries some progresses have also been made. Vertically Specifically, bacterial flora has been shown to play a beneficial role when in equilibrium, but becomes harmful when there is imbalance.
While the general dogma is that the placental barrier keeps infants sterile throughout pregnancy, increasing evidence suggests that an infant´s initial bacterial inoculum can be provided by its mother before birth [4,5] and is supplemented by maternal microbes during the delivery [6] and breastfeeding [7,8] processes. Soon after birth, infant flora evolves depending on several factors like mode of delivery, nutrition, age and environment [3,9]. Fooks et al. (2007) [10] reported that the bacterial population in the intestine is quantitatively and qualitatively balanced; each genus has its own growth niche maintaining an "optimal balance" for the physiological performance of the digestive system. However, numerous factors can alter it, either by stimulating beneficial actions through an increase in the number of certain bacteria (Bifidobacteria and Lactobacilli), or by stimulating the proliferation of bacteria considered pathogenic (Clostridia, certain species of Bacteroids, etc.). Knowing that 60% of CD4+ T cells in the human body reside in gut-associated lymphoid tissue, and that the reconstitution of these cell populations and the gut microbial composition is incomplete even under HAART, the human intestine has recently become the focus of attention in HIV research [11]. Moreover, changes in gut microbial composition and function in HIV-positive individuals, aside from being secondary to HIV infection, may also play direct roles in mediating some disease manifestations (recurrent bacterial infections) [11]. The intestinal flora, if well characterized could be balanced before the onset of infection using probiotics. It is for this reason that this research project was initiated with the aim to examine the digestive flora of HIV infected infants born to HIV sero-positive mothers in comparison to that of HIV non-infected infants. produce stool specimen avoiding contamination. Fresh stool sample was collected in a dry sterile stool container.   Table 1. Exposure to HIV: we did not observe any difference in the digestive flora of HIV exposed and non-infected infants versus non-exposed HIV negative infants.

Discussion
Our and up to 60% of the dry mass of feces [12,13]. An estimate of 300 to 1000 different species live in the gut [14,15], with most estimated at about 500 [16][17][18]. However, it is probable that 99% of the bacteria come from about 30 or 40 species [12]. According to the delivery mode, we isolated more bacteria species from infants born by vaginal route than from infants born by caesarean section. The [19][20][21][22][23][24]. According to the feeding mode, our study showed a differential frequency in harboring Staphylococcus spp.
Bifidobacterium and Lactobacillus spp. This is in agreement with other studies that showed a relationship between the digestive flora and the mode of nutrition [9,19,25,26]. We will like to clarify here that formula feeding in Cameroon is based on two main types: plain and enriched. In Cameroon, pediatric milks (0-6 months) are enriched with probiotics (Lactobacillus or Bifidobacteria) and prebiotics (mainly the fructo-oligosaccharides and the galacto-oligosaccharides). These milks represent more than 50% on the market.
The diversity of bacteria identified was analysed according to the HIV status of the infants. Qualitatively we isolated more bacteria in HIV positive children (15 different species of bacteria) than in exposed HIV negative infants and non-exposed negative ( There was an observed shift in gut composition from protective species to pro-inflammatory disease-inducing bacterial species that promote viral replication and chronic immune activation. Bacteroides species also known to benefit their host by excluding potential pathogens from colonizing the gut were least frequently isolated from infected infants. This dysbiosis observed in infants has also been observed in adults [28][29][30]. This is why we had 11.25% on antibiotics, 6.25% with diarrhea and 3.75% with constipation; which were excluded from our analyses. So our study was limited by lack of possibility to cover all the bacteria living in the GIT, and thus was confined to those that could be cultured. Not all the species in the gut have been identified because most cannot be cultured [15], and making identification difficult. An effort to better describe the microflora of the gut and other body fluids using newly developed non-culture based technologies [32] has been achieved. A project termed the "Human Microbiome Project" [33] has been initiated and takes advantage of new, high-throughput technologies to characterize the human microbiome.

Conclusion
We aimed at looking at the effect of HIV infection on the digestive flora of HIV infected infants. Our results showed that HIV infected infants harbor qualitatively more bacteria than HIV non-infected infants. We found that, as HIV disease progresses the digestive flora changes qualitatively and also that as the disease advances (Stage 1-4) the number of opportunistic bacteria also increases. HIV infected infants harbor more opportunistic bacteria than non-infected infants.
The delivery mode affected the flora of HIV infected infants compared to non-infected infants (Staphylococcus spp.  Maternal HIV infection Influences the Microbiome of HIVexposed uninfected infants.

What this study adds
 Differential digestive flora of HIV infected and uninfected infants aged 3-24 months using culture-based methods;  Higher diversity bacteria in HIV infected compared to uninfected infants.

Competing interests
The authors declare no competing interests.

Authors' contributions
Nkenfou did study design, data analyses, manuscript preparation, approval and submission. Abange did samples and data collection, data analyses, approval of the manuscript. Gonsu did data analyses, manuscript review and approval. Kamgaing did data analyses, manuscript review and approval. Lyonga did data analyses, manuscript review and approval. Anoubissi did data analyses, manuscript review and approval. Ndjolo did study supervision, manuscript review and approval. Koki did study supervision, manuscript review and approval. All the authors have read and agreed to the final manuscript.