Yogurt Containing Probiotic Lactobacillus rhamnosus GR-1 and L. reuteri RC-14 Helps Resolve Moderate Diarrhea and Increases CD4 Count in HIV/AIDS Patients Kingsley C. Anukam, PhD,*w z Emanual O. Osazuwa, PhD,* Humphrey B. Osadolor, MD,y probiotic yogurt on quality of life of women in Nigeria with Abstract: HIV/AIDS is changing the human landscape in sub- HIV/AIDS, and suggests that perhaps a simple fermented food Saharan Africa. Relatively few patients receive antiretroviral can provide some relief in the management of the AIDS therapy, and many suffer from debilitating diarrhea that affects their quality of life. Given the track record of probiotics toalleviate diarrhea, conventional yogurt fermented with Lacto- Key Words: probiotic, yogurt, HIV/AIDS, diarrhea, CD4 bacillus delbruekii var bulgaricus and Streptococcus thermophilus was supplemented with probiotic Lactobacillus rhamnosus GR-1and L. reuteri RC-14. Twenty-four HIV/AIDS adult femalepatients (18 to 44 y) with clinical signs of moderate diarrhea,CD4 counts over 200, and not receiving antiretrovirals or The use of probiotics to alleviate gastrointestinal maladies, in particular diarrhea, has been well dietary supplements, consumed either 100 mL supplemented or documented.1 Fermented milk products, such as yogurt, unsupplemented yogurt per day for 15 days. Hematologic have been shown to play a role in modulating the immune profiles, CD4 cell counts, and quality of life was evaluated at system.2 In sub-Saharan Africa, fermentation of foods baseline, 15 and 30 days postprobiotic-yogurt feeding. There has become less commonplace over the past 50 years, in was no significant alteration in the hematologic parameters of part due to importation of ‘‘Western’’ food practices. This both groups before and after the probiotic-yogurt feeding. The region, already mired in poverty and malnutrition, is now probiotic yogurt group at baseline, 15 and 30 days had a mean engulfed by an HIV/AIDS epidemic that is crippling WBC count of 5.8 ± 0.76 Â 109/L, 6.0 ± 1.02 Â 109/L, and economies and causing relentless suffering. Women are 5.4 ± 0.14 Â 109/L, respectively. However, the mean CD4 cell particularly at risk, and 75% of new infections are count remained the same or increased at 15 and 30 days in 11/12 believed to occur among young women and girls. Less probiotic-treated subjects compared to 3/12 in the control.
than 0.1% of HIV/AIDS patients gain access to the Diarrhea, flatulence, and nausea resolved in 12/12 probiotic- highly active antiretroviral therapy they require, and up treated subjects within 2 days, compared to 2/12 receiving yogurt for 15 days. This is the first study to show the benefits of The CD4 count has become the conventional method of assessing the immune status of HIV-infectedpatients. Decisions regarding the initiation and determi- Received for publication August 17, 2006; accepted October 20, 2006.
nation of antiretroviral efficacy should be guided by From the *Department of Pharmaceutical Microbiology, Faculty of monitoring the plasma HIV-RNA (Viral load) and CD4 Pharmacy, University of Benin; wChidak Medical Diagnostic counts. These parameters give the physician important Laboratories, 200 M. M. Way, Benin City; zDepartment ofMicrobiology, Faculty of Basic and Applied Sciences, Benson information about the virologic and immunologic status Idahosa University, P.M.B. 1100; yDepartment of Chemical pathol- of the patient and the risk of disease progression from ogy, Central Hospital, Benin City, Edo State, Nigeria; JCanadian HIV infection to the development of AIDS. With this Research and Development Centre for Probiotics, Lawson Health CD4 decline comes increased risk of complications, Research Institute, 268 Grosvenor Street; and zDepartment of especially potentially lethal opportunistic infections. In Microbiology and Immunology and Surgery, University of WesternOntario, London, Ontario, Canada.
a study of 974 South African HIV/AIDS patients, those Drs Anukam, Osazuwa and Osadolor have no conflicts.
with a CD4 count between 201 to 350 cells/mL declined Dr Reid declares he holds patents for the use of Lactobacillus GR-1 and 20.5 cells/mL per year (0.8 per 2 wk), those between 351 RC-14 for urogenital health, but not for HIV diarrhea or CD4 and 500 fell 30.6 cells/mL per year (or 1.3 every 2 wk) and This research is funded in part by Urex Biotech Inc and Natural Sciences those greater than 500 cells/mL per year fell 47.1 cells/mL and Engineering Research Council of Canada.
Reprints: Dr Gregor Reid, PhD, MBA, Canadian Research and To date, there have been no studies on the use of Development Centre for Probiotics, Lawson Health Research probiotic yogurt for the management of diarrhea in HIV/ Institute, 268 Grosvenor Street, London, Ontario, N6A 4V2 Canada AIDS patients, nor for halting the drop in CD4 counts.
Copyright r 2008 by Lippincott Williams & Wilkins Lactobacillus reuteri has been shown to be safely J Clin Gastroenterol  Volume 00, Number 00, ’’ 2008 Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Clin Gastroenterol  Volume 00, Number 00, ’’ 2008 administered to HIV/AIDS subjects,5 and to prophylac- biotic L. rhamnosus, GR-1 and L. reuteri RC-14. Ten tically benefit individuals susceptible to cryptosporidio- milliliters of probiotic preparations (2.5 Â 109 CFU/mL) sis.6 The aim of the present study was to determine, in 40 were added to 990 mL of the fermented yogurt. The HIV/AIDS-infected women, whether the addition of yogurt given to the subjects for the 15 days, maintained probiotic strains, L. reuteri (formerly fermentum) RC-14 viable counts of both probiotics at 107/mL, as checked by with L. rhamnosus GR-1 to yogurt, could clear diarrhea culture. Both probiotic strains survive and grow well in and halt the drop in CD4 counts, of subjects with a milk and survive intestinal passage.8 Twelve HIV/AIDS starting CD4 count between 206 and 520 cells/mL.
adult female subjects with clinical signs of moderatediarrhea each consumed 100 mL probiotic yogurt per day for 15 days. For the control group, 12 age-matched HIV/AIDS female subjects consumed 100 mL of unsupple- mented yogurt. Each subject had 4 mL of venous blood Premenopausal women, attending the HIV Volun- collected into ethylenediaminetetraacetic acid containers, tary Counseling Test center in Benin City were recruited.
before study commencement and at 15 and 30 days The ethical review committee of the Faculty of Pharmacy, follow-up. The samples were tested for hematologic University of Benin, gave approval for the study. All parameters and CD4 cell counts using Dynabead testing was voluntary and included precounseling and technique (Dynal A.S., Oslo, Norway) which is the postcounseling by trained HIV counselors. Each of the 24 adopted technique for CD4 estimation under the national subjects consented to the study after thorough explana- ARV programme in Nigeria.9 The CD4 lymphocyte tion by the HIV counselor. All participants who were counts were expressed as cells/mL of blood.
HIV positive were counseled and informed of their HIVstatus. Blood samples (4 mL) were collected into plain bottles for HIV antibodies, detected using Capillus HIV For each test, 155 mL of freshly obtained ethylene- test kits (Cambridge). The reactive samples were further diaminetetraacetic acid anticoagulated blood was added confirmed using the western blot technique (Biorad).
to 350 mL of phosphate-buffered saline; 25 mL of sus-pended magnetic beads coated with anti-CD4 monoclonal antibody was added and the mixture incubated for 10 minutes at room temperature on a dynal mechanical The inclusion criteria was for females aged 18 years rotator, to deplete blood of monocytes. The beads were and above; laboratory evidence of HIV infection; history separated using the magnetic particle concentrator and of no previous antiretroviral therapy; and CD4 cell washed twice with phosphate-buffered saline. After counts above 200 cells/mL. This value was chosen based addition of 50 mL lysing solution, cells were stained with on the recent study7 showing the mean CD4 lymphocyte 50 mL of Turks fluid and layered on a Neubauer counting count of healthy Nigerians to be 685 ± 99, [95% chamber. The nuclei were integrally enumerated under confidence interval (CI) 487-883]. The value was twice light microscopy. Results were expressed as number of that of HIV-infected subjects without AIDS-defining positive cells counted per microliter of whole blood.
illness (314 ± 45, 95% CI 224-404). The CD4 lymphocytecount of HIV-infected with AIDS-defining illness was one-fourth or less (148 ± 30, 95% CI 88-208). The Twelve hematologic parameters were determined at patients who were confirmed positive for HIV were baseline, 15 and 30 days posttreatment, using auto- invited to participate in the probiotic yogurt study.
hematologic analyzer, CELL-DYN 1200 by Abbort, Inc, Lactose intolerance was an exclusion criterion, and none USA [white blood cells, red blood cells, hemoglobin, of the subjects enrolled suffered from this condition.
hematocrit (pack cell volume), mean cell volume, mean Subjects were excluded if they were taking dietary cell hemoglobin, mean cell hemoglobin concentration, red supplements or any anti-retroviral therapies. It was not blood cell distribution width, platelets].
possible to control the dietary intake of the subjects, butinterviews with each did not reveal any significantdifferences in the types of food consumed. The 24 HIV/ Measuring Quality of Life at Baseline, 15 Days, AIDS adult female subjects enrolled were aged 18 to 44 years. The probiotic and placebo products were prepared A structured questionnaire was designed to obtain by an independent laboratory and blinded to the clinical history and quality of life for each patient. The physicians recruiting the patients and the researchers participants were asked to indicate any gastrointestinal analyzing the data. Upon completion of the data analysis discomfort particularly diarrhea which was described as Absent, Moderate (daily episodes of watery stools, mildnausea, and mild flatulence), or Severe (persistent watery stools, urgency and frequency of defecation, nausea and Conventional yogurts fermented with starter cul- flatulence), and the presence of any opportunistic infec- tures of Lactobacillus delbruekii subsp. bulgaricus and tions or skin conditions before and after the probiotic Streptococcus thermophilus were supplemented with pro- Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Clin Gastroenterol  Volume 00, Number 00, ’’ 2008 The subjects treated with the probiotic supplemen- Data are presented as the mean ± standard devia- ted yogurt had rapid (within 2 d) resolution of their tion, and n represents the number of participants from gastrointestinal discomfort, namely diarrhea with flatu- lence and nausea, compared to controls, and this Comparisons were made by use of the Student t test.
remained so for the duration of treatment as well as Differences were regarded significant between the treat- during longer term follow-up (Table 3). Three of the ment groups with a P value less than 0.05.
placebo subjects developed skin rashes at 3 months,whereas none were noted in the probiotic-treated group.
There was 100% compliance at day 15, as deter- mined by return for follow-up and inspection of empty This is the first study to show that probiotic yogurt containers. One subject’s blood sample was not supplemented yogurt, unlike regular yogurt, quickly able to be scored due to technical error. No bacteremia alleviates diarrhea in HIV/AIDS patients, and has a was detected in any subject. Two subjects failed to show positive outcome on CD4 counts. This effect occurred for the 30-day follow-up, despite being contacted. There within days of consumption. The results obtained in this was no significant alteration in the hematologic para- 24-subject study, provide strong evidence for a larger meters (Table 1) of both groups after the treatment.
examination of the use of basic foods for management of The qualitative analysis of the urine parameters (color, the AIDS crisis, particularly in populations without bilirubin, urobilinogen, proteins, ketones, nitrite, glucose, access to antiretrovirals or expensive therapies. In a and blood) did not change significantly in either group.
separate project in a poor area of Tanzania, yogurt However, urine leukocyte esterase, motile bacteria and supplemented with probiotic L. rhamnosus GR-1 is being white blood cells per high power field were significantly made by mothers in a community kitchen. This project, reduced in the probiotic yogurt group compared with the Western Heads East (www.westernheadseast.ca) demon- strates the ability to reach poor people and have them There were significant differences between the engaged in a not-for-profit activity which can have groups with respect to their CD4 cell counts (Table 2).
Eight of 11 subjects at day 15 and 30, consuming Several patients had relatively good increases in unsupplemented yogurt had a drop in CD4 cell count, their CD4 counts, even 15 days after completion of the and the group’s average drop coincided with the probiotic yogurt intake, whereas one subject had a anticipated values predicted by Holmes et al.4 On the significant decrease in her CD4 count in the control contrary, 8/12 subjects at day 15 and 10/11 at day 30, who group. The clinical significance and long term effects consumed the probiotic yogurt, had an increase in CD4 cell count. On average, this resulted in a 3 to 4 fold Diarrhea is not only a problem with HIV/AIDS improvement compared to controls, and this was patients per se,10 but it is one of the adverse effects of statistically significant (P<0.02). No deaths occurred antiretroviral therapy.11 The fact that a food, easily during the study or 3-month follow-up.
produced in developing countries, and indeed part of TABLE 1. Mean Hematologic Profiles of HIV Patients at Baseline, 15 and 30 d Feeding of Yogurt Containing Probiotic Lactobacillusrhamnosus GR-1 and Lactobacillus reuteri RC-14 or Placebo *Statistical analysis = level of significance for change from baseline and probiotic yogurt and placebo to day 30.
MEB indicates monocytes, eosinophils, basophils.
MCH indicates mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; MCV, mean cell volume; PCV, pack cell volume; RDW, red blood cell Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Clin Gastroenterol  Volume 00, Number 00, ’’ 2008 TABLE 2. CD4 Cell Count of HIV Subjects who Consumed Unsupplemented or Probiotic Yogurt Containing Lactobacillus GR-1and RC-14 for 15 Days Subjects were retested at 30 d.
ND indicates not done.
the heritage of many countries, can alleviate diarrhea, retroviral drugs,12 it would be worth investigating the represents a significant potential means of reducing some effects of probiotics with micronutrients supplemented deaths among HIV/AIDS patients. Having stated that, a into yogurt in subjects who do not have access to highly larger study is needed to examine the use of probiotic active antiretroviral therapy therapy.
yogurt in patients with lower CD4 counts and more No side effects were noted, although 3 control severe diarrhea. The effects seemed to carryover for subjects developed skin rashes by the third-month follow- several months after eradication of the diarrhea. Given up. This is not uncommon in patients developing end the apparent success of adding micronutrients to the diet stage AIDS. Again, the extent to which probiotics can of HIV/AIDS patients, albeit in those receiving anti- delay this event, seems worthy of investigation.
TABLE 3. Results of Quality of Life Parameters The 2-d and 3-mo values were ascertained from each subject by personal contact, outside of the 2 follow-up visits on days 15 and 30.
Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
J Clin Gastroenterol  Volume 00, Number 00, ’’ 2008 This study exemplifies the need to bring probiotics 7. Erhabor O, Nwauche CA, Ejele OA, et al. CD4+ lymphocyte from the Northern and Western worlds to Africa, as reference ranges in healthy adults in Port Harcourt, Nigeria. Sahel well as to people in all countries who cannot afford 8. Gardiner G, Heinemann C, Baroja ML, et al. Oral administration pharmaceutical remedies, as recommended by the FAO.13 of the probiotic combination Lactobacillus rhamnosus GR-1 and If such remedies only provide temporary relief from this L. fermentum RC-14 for human intestinal applications. Int Dairy fatal disease, their value will be immense.
9. Odunukwe N, Idigbe O, Kanki P, et al. Haematological and biochemical response to treatment of HIV-1 infection with 1. Walker WA, Goulet O, Morelli L, et al. Progress in the science of a combination of nevirapine+stavudine+lamivudine in Lagos probiotics: from cellular microbiology and applied immunology to Nigeria. Turkish J Haematol. 2005;22:125–131.
clinical nutrition. Eur J Nutr. 2006;45(suppl 9):1–18.
10. Groenewald P, Nannan N, Bourne D, et al. Identifying deaths from 2. Halpem GM, Vruwink KG, Van der Water J, et al. Influence of AIDS in South Africa. AIDS. 2005;19:193–201.
long-term yogurt consumption in young adults. Int J Immunother.
11. Ananworanich J, Gayet-Ageron A, LeBraz M, et al., Staccato Study Group, Swiss HIV Cohort Study. CD4-guided scheduled treatment 3. Harwell JI, Obaro SK. Antiretroviral therapy for children: interruptions compared with continuous therapy for patients substantial benefit but limited access. JAMA. 2006;296:330–331.
infected with HIV-1: results of the Staccato randomised trial.
4. Holmes CB, Wood R, Badri M, et al. CD4 Decline and incidence of opportunistic infections in Cape Town, South Africa: implications 12. Kaiser JD, Campa AM, Ondercin JP, et al. Micronutrient for prophylaxis and treatment. J Acquir Immune Defic Syndr. 2006; supplementation increases CD4 count in HIV-infected individuals on highly active antiretroviral therapy: a prospective, double- 5. Wolf BW, Wheeler KB, Ataya DG, et al. Safety and tolerance of blinded, placebo-controlled trial. J Acquir Immune Defic Syndr.
Lactobacillus reuteri supplementation to a population infected with human immunodeficiency virus. Food Chem Toxicol. 1998;36:1085–1094.
13. FAO/WHO. 2001. Evaluation of health and nutritional properties 6. Alak JIB, Wolf BW, Mdurvwa EG, et al. Effect of Lactobacillus of powder milk and live lactic acid bacteria. Food and Agriculture reuteri on intestinal resistance to Cryptosporidium parvum infection Organization of the United Nations and World Health Organization in a murine model of acquired immunodeficiency syndrome. J Infect Expert Consultation Report. http://www.fao.org/ag/agn/food/food_ Copyright Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

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