Poor nutritional status among people living with HIV is a common problem [1
] and has been associated with enhanced HIV progression in numerous observational studies [2
]. Impaired immune function of people living with HIV is further compounded by a deleterious impact of the virus on the gut-associated mucosal immune system [4
], which presumably results in translocation of microbial products from the intestinal tract [6
], an aberrant intestinal microbiota [8
], and an increased incidence of intestinal infections [10
]. Microbial translocation as a result of impaired gut barrier function, in combination with low micronutrient levels, may further enhance the progression of HIV [6
Supplementation of micronutrients, most notably B-complex, vitamin C and E, can reduce the progression and mortality of HIV [13
], but does not impact intestinal barrier impairment [18
]. Supplementation of vitamin A as single supplement does not appear to reduce the progression of HIV or mortality related to the disease in adults [15
Probiotics, defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” [20
], have been used with some success to preserve the immune-function of people living with HIV in the following studies. A small randomized controlled trial (RCT) was performed in Brazil among 77 children infected with HIV. Half of the children received a supplement with probiotic strain Bifidobacterium bifidum
and Streptococcus thermophilus
for two months. After the two month period, CD4 counts among the treatment group increased 118 cells/μL versus
a decrease of 42 cells/μL among the placebo group (p
= 0.05) [21
]. Another small pilot RCT in Nigeria discovered a small, but statistically significant effect, of four weeks of Lactobacillus rhamonus
GR-1 and Lactobacillus reuteri
RC-14 supplementation on the CD4 count of 24 HIV infected women. The treatment group showed an increase in CD4 of 6.7 cells/μL compared to a decrease of 2.2 cells/μL in the control group (p
= 0.05) [22
]. Lastly, an observational study in Tanzania found that yogurt supplemented with Lactobacillus rhamnosus
GR-1 increased the CD4 count with 0.28 cells/μL/day (p
= 0.003) among HIV infected men and women in Tanzania [23
]. The mechanisms are believed to involve enhancing the gut barrier function [24
], alleviating systemic inflammation [26
], or reducing the duration of gastro-intestinal infections [28
] (reviewed in [29
Despite massive efforts, only one out of three people in need of anti-retroviral treatment (ART) currently have access to the treatment [30
]. The application of a food-based intervention, targeting both micronutrient deficiencies and an impaired gut-associated mucosal immune system, could provide an optimal adjunctive intervention to potentially delay the progression of HIV. Since 2005, a small community kitchen in Tanzania has produced yogurt supplemented with probiotics using Lactobacillus rhamnosus
]. We initiated a randomized, double-blind, placebo controlled study to assess whether the addition of probiotic L. rhamnosus
GR-1 to a micronutrient fortified yogurt could positively impact the immune function of HIV patients. We selected L. rhamnosus
GR-1 for its potential to reduce the translocation of Salmonella typhimurium
from the gut to distant organs, as assessed in a mouse model [32
], reduce systemic inflammation in a population with inflammatory bowel disease [33
], and to increase the CD4 count in an HIV infected population [23
2. Experimental Section
2.1. Study Design
The primary outcome was the mean change of CD4 count from baseline to 4 weeks follow-up. Secondary outcomes included hematology indicators (creatinine, albumin, alanine transaminase (ALT), and full blood count), incidence of diarrheal episodes, symptoms, physical energy and their ability to perform activities of daily living. Inclusion criteria were; confirmed HIV infection, naïve to ART and not eligible for ART initiation (i.e.
, CD4 count ≥200 with WHO stage I or II infection or CD4 count ≥350 and CDC stage III infection [34
]), ability to attend the clinic every other day, ≥18 years of age, not pregnant or lactating, and with no known lactose intolerance or milk allergy. We calculated a sample size of 49 per treatment arm to detect a within person difference of 15 cells/μL in CD4 count between the groups with a power of 90%. The calculation assumed an attrition rate of 25% and a standard deviation of 20 cells/μL within-person change in CD4 count. The Medical Research Coordinating Committee of the National Institute for Medical Research, Tanzania, approved the study design and protocol. Participants were informed of the purpose of the trial and gave their signed or thumb printed informed consent before participation. All study procedures were in concordance with the declaration of Helsinki of 1975, as revised in 2008.
2.2. Study Subjects and Data Collection
Recruitment took place at Shaloom health centre and at Sekou-Toure Regional Hospital, Mwanza, Tanzania from June to July 2008. Randomization and follow-up were conducted at Sekou-Toure regional hospital and extended until August 2008. During the baseline visit, a physical examination was performed by a physician, blood was taken to perform biochemical and hematological measurements and a structured interview was performed on demographics, medical history, and physical energy levels. The participants recorded the occurrence of diarrheal episodes using a diary, which was collected at each visit. Diarrhea was defined as three or more loose or watery stools in a 24-h period [35
Participants were randomized to receive a yogurt that was fortified solely with micronutrients (control group) or was fortified with both micronutrients and L. rhamnosus GR-1 (probiotic group) for 4 weeks. Both yogurts were indistinguishable in taste or appearance. Subjects were consecutively assigned to one of the two treatment codes according to a computer generated schedule in blocks of 10. The batches of yogurt were coded by a researcher not involved with data collection to ensure that neither the researchers nor the subjects were aware of their treatment assignments. Participants were asked to return to the clinic every other day to receive a two-day supply of yogurt of which one portion was consumed in the clinic to ensure at least 50% adherence. Subjects returned after 4 weeks for the follow-up visit and were encouraged throughout the duration of the study to report any adverse events or changes in their condition. The procedures of the baseline visit were repeated exactly at the follow-up visit, with additional questions on adverse events. Participants who did not arrive at their follow-up visit were contacted by phone.
The nutrient premix blend was provided by Fortitech Strategic Nutrition (Schenectady, New York, NY, USA), for which the concentrations of nutrients per portion of yogurt (125 mL) are summarized in Table 1
. We based the formulation on that described by Kaiser et al
], but reduced the concentrations of the micronutrients to acceptable physiological levels that were also compatible with inclusion into yogurt. Preparation of the probiotic stock culture took place at National Institute for Medical Research (NIMR), Mwanza, and the organism was added to yogurt produced by a trained group of local women. The viability and abundance of the probiotic strain in the yogurt was assured for every batch using procedures previously described [36
]. Overall, a mean 1.23 × 109
colony forming units/mL of L. rhamnosus
GR-1 was found in the probiotic yogurt resulting in an intended dose of 15.38 × 1010
colony forming units/day.
2.4. Laboratory Measurements
The laboratory measurements were performed at the NIMR, according to good laboratory practices. At baseline and at 4 weeks the CD4 count was measured using the Partec FACS (Partec GmbH, Münster, Germany), hematological parameters were measured using the Beckman Coulter AcT5 Diff Al (Beckman Coulter, Brea, CA, USA) and biochemistry parameters using the Synchron CX®4 PRO (Beckman Coulter, Brea, CA, USA).
All analyses were performed on an intent-to-treat basis. The plan of data analysis was clearly established and stated in the study protocol before start of the study. Normally-distributed continuous (CD4 count, hematology parameters) data were compared using within-subject differences from baseline to follow-up, and tested using an unpaired students t-test. To assess the effect of the CD4 count, HIV symptoms, BMI and anemia at baseline on the treatment response, a linear regression model was fitted. Categorical data (baseline characteristics) were compared using a χ2 test. All tests were performed two-sided at the α = 0.05 level with no adjustments made for multiple comparisons. Compliance with the study treatment was calculated as the number of days the participant consumed the yogurt divided by the total number of days the yogurt should have been consumed. Data was stored in an Access database and analyzed using SPSS 15.0 software.
Probiotic and micronutrient content of the yogurts.
Probiotic and micronutrient content of the yogurts.
|Lactobacillus rhamnosus GR-1 *||109 CFU/mL|
|Vitamin A (as Beta carotene and Palmitate)||1500 IU|
|Vitamin E (as acetate)||5.7 IU|
|Vitamin B1 (thiamin)||0.3 mg|
|Vitamin B12 (cyanocobalamin)||0.6 μg|
|Vitamin B6 (pyroxine)||0.3 mg|
|Vitamin C (ascorbic acid)||21 mg|
|Iron (as ferric pyrophosphate)||3.3 mg|
|Selenium (sodium selenite)||13.8 μg|
|Zinc (zinc sulphate)||2.4 mg|
|DHA (omega-3 from fish oil)||13 mg|
|EPA (omega-3 from fish oil)||19 mg|
In this randomized, controlled study, the addition of a probiotic Lactobacillus
to a micronutrient fortified yogurt did not further substantiate the effect on the CD4 count or clinical symptoms of HIV-infected adults with a starting CD4 count of ≥200 cells/μL. The micronutrient fortified probiotic yogurt was well tolerated among the participants and was not associated with adverse events. This result somewhat contrasts previous studies showing a positive effect of probiotic supplementation on CD4 counts [21
], but these were not performed with micronutrient supplementation which herein and elsewhere [17
] independently can affect on CD4 count. One small RCT in Nigeria had used L. reuteri
RC-14 in combination with the strain studied here (L. rhamnosus
], while the observational study had used L. rhamnosus
GR-1 alone [23
]. The RCT in Brazil had used a combination of different strains for children treated with ART (Bifidobacterium bifidum
and Streptococcus thermophilus
]. The apparent contradiction could have two explanations. Firstly, the study in Nigeria [22
] had found a standard deviation of the change in CD4 count of approximately 10 cells/μL during a 4 week follow-up while in our population we found a standard 330 cells/μL during the same follow-up period. Because we used the previously reported standard deviation for sample size calculations, our study had reduced power to show an effect. The Nigerian study had recruited a homogenous group of women having moderate diarrhea symptoms, which could explain the difference in variability in CD4 counts. Secondly, our treatment period could have been too short to show an effect on CD4 count. Upon reflection, the use of measurements of the intestinal barrier function (e.g., lipo-polysaccharide or lactoferrin: mannitol ratio [6
]), or immune activation and regulation (e.g., CD4+/CD25+ and FoxP3 lymphocytes [30
]) could have served as a screening tool to assess potential benefits of probiotics within an HIV infected population. Therefore, we suggest these markers to be used in future studies to screen or assess the potential effect of specific probiotic strains.
The overall change in CD4 count of a mean −70 cells/μL (CI 95%; −157 to −30) was lower than normally observed among HIV patients [38
]. The decrease among both groups was not expected as the micronutrient formula tested by Kaiser et al.
], on which our micronutrient formula was based, had shown an increase in CD4 count. The nutrient formula used in this study was not intended to provide a high dose of micronutrients at multiple times of the Dietary Reference Intake (DRI) [15
], but rather an excellent dietary source at 25% of the DRI [40
] to an already nutritious food (yogurt). The decrease in CD4 count may be explained by a tendency of parameters to decline when selected above a specific value (i.e
., regression to the mean). Since HIV patients were included only with a CD4 count ≥200 cells/μL, a greater decline in CD4 count could have been observed than within an unselected population. This explanation is supported by the finding that participants with a CD4 count ≥350 cells/μL at baseline experienced decrease (−187 cells/μL) while those with a CD4 count of <350 cells/μL experience an increase in CD4 count (67 cells/μL). The impact of this mechanism on the results was assessed by adjusting for baseline CD4 count levels; this adjustment did not significantly change the inferences of the study, and is therefore likely to be modest. The decrease might further be explained in part by the fact that almost half of the participants had anemia, which has been observed to be factors for enhanced HIV progression [41
]. Again, it is easy in hindsight to consider how better the study could have been set up, but clearly lessons have been learned that even with limited resources, attempts to identify potential responders to the treatment should be made [41
]. In addition, future studies of longer duration should be performed to assess whether benefits take longer to accrue.