The Role of Probiotics L. Plantarum, S. Thermophiles, B. Bifidum on Gut Inflammation, Bacterial Translocation, and CD4+ Cell Count in HIV Patients With Immunological Non-Responder
Phase: PHASE2
Conditions: HIV Infections, Immunological Abnormality
Sponsor: Indonesia University

Research Protocol 
 
The Role of Probiotics L. plantarum, S. thermophiles, B. bifidum on Gut 
inflammation, Bacterial Translocation, and CD4+ Cell Count in HIV Patients 
with Immunological Non-Responder 
(PIONIR) 
 
 
 
 
INVESTIGATORS: 
Principal       : Erni J. Nelwan, MD, Ph.D 
Others 
: Rina Agustina, M.Sc, Ph.D 
 
 
  Prof. Marcellus Simadibrata, MD, Ph.D 
 
 
  
 
 
 
 
FACULTY OF MEDICINE 
UNIVERSITY OF INDONESIA 
Approved May 1st, 2018 

 
I. Background 
Human Immunodeficiency Virus (HIV) infected patients treated with anti retroviral drugs may 
experiencing immunological failure known as immunological non responders (INR). In this 
particular group of patient, the level of CD4 still below normal limit even though the HIV virus 
is suppressed (undetected). Immunological non responders (INR) were associated with high 
morbidity.1 Hypothetically, INR patients have decreased gut integrity and immunity due to  
increased gut inflammation and further causing microbial translocation.2 Probiotics may 
improved gut inflammation condition, however the direct benefits of probiotics supplementation 
for INR HIV patients have not been studied.3 Growing number of INR HIV patients will be 
associated with higher economical burden and decreased quality of life.  
 
Microbial translocation was thought as the underlying mechanism of persistently immune 
activation which may lead to immunological failure condition in virally suppressed HIV 
patients.2 Decreased number of T- helper 17 (Th17) in HIV patients may facilitated microbial 
translocation. HIV Virus destroyed the CD4 T memory cells which mainly found in gut 
associated lymphoid tissue (GALT), thus resulting in lower production of Th17 in gut mucosal 
tissue.2,4 One of the microbial translocation marker is 16S ribosomal DNA (16S rDNA) bacteria 
blood level.5 Gut inflammation may be represented by fecal calprotectin level.6 
 
Probiotics supplementation in HIV patients was proposed may increase gut integrity and 
immunity through several mechanisms: improving tight junction gut mucosal, increasing mucin 
production, regulating Th17; therefore decreasing gut inflammation and microbial translocation. 
Improved gut inflammation and decreased microbial translocation may increased the immune 
recovery in INR HIV patients 7-9  
 
This clinical trial is intended to evaluate the immune recovery effect of Probiotics 
supplementation in INR HIV patients. 
 
 

 
II. 
Objectives  
A. General Objectives 
To determine the effect of Probiotic in immune response for immunological non 
responder HIV patients 
B. Specific Objectives 
a. Primary Objectives 
i. To determine the average CD4 value in immunological non 
responder HIV patients treated with Probiotic compared to 
placebo 
ii. To determine the average T helper 17 value in immunological non 
responder HIV patients treated with Probiotic compared to 
placebo 
iii. To determine the average 16S rDNA value in immunological non 
responder HIV patients treated with Probiotic compared to 
placebo 
iv. To determine the fecal calprotectin value in immunological non 
responder HIV patients treated with Probiotic compared to 
placebo 
b. Secondary Objectives  
To determine the effect of Probiotic supplementation towards quality of 
life improvement in immunological immunological non responder HIV 
patients, using HIV symptom index.  
 
III. 
Methods 
A. Study Design 
This study is double-blind randomized control trial design 
B. Time and Place 
This study will be conducted in Cipto Mangunkusumo National Central General 
Hospital, especially in Human Infectious Disease polyclinic. Enrollment will 
start on May 2018, and recruited until minimum sample size was fulfilled 

C. Study Population 
1. Targeted Population: HIV patients with Immunological Non-responder 
condition in Indonesia 
2. Sample: HIV patients with Immunological Non-responder condition in 
Indonesia 
3. Inclusion Criteria:  
a. HIV positive patients aged 18-55 years old 
b. had already treated with first line Highly Active Anti Retroviral 
Therapy for at least 6 months 
c. had CD4 level of 200-410 cell/L  
d. undetectable Viral Load (<34 copies/ml blood) 
e. agreed to participate in the study and signed the informed consent 
 
4. Exclusion Criteria: 
a. Pregnant patients 
b. Lactating patients 
c. Known Lactobacillus allergy 
d. Body Mass Index (BMI) <16 kg/m2 
e. Under Tuberculosis treatment or other acute illness, acute diarrhea,  
f. routinely taking selenium containing vitamin in last 1 month,  
g. routinely consuming probiotics containing product in last 1 month 
5. Estimation Sample Size: 80 samples. Which will be divided into two groups 
equally. 
6. Study Procedure 
Eligible patients will be approached to be enrolled in the clinical trial. 
Informed consent will be  obtained. Patients will be randomized and 
divided into two groups, intervention and placebo group. Intervention 
group will receive 
Probiotics 
(Chewable tablet) containing 
Lactobacillus plantarum, Stretococcus thermophiles, Bifidobacterium 
bifidum once daily. While, Placebo group will receive placebo which 
had identical shape, flavor, and smell as the interventional drug. 

Intervention will be held for 12 weeks. CD4, Th17, 16S Ribosomal 
RNA, and fecal calprotectin level will be checked at the 0 week and 
repeated after 12 weeks of intervention. Self administered HIV 
Symptoms Index questionnaire will be filled by the respondent on 
0,4,8,12 weeks of intervention. Food frequency questionnaire will be 
filled by the respondents with the help of nutritionist on 0,4,8,12 
weeks of intervention. 
 
7. Side effects 
All side effects that may arise and related to probiotics intake will be assessed 
and reported. Appropriate medical advice will be provided. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
D. Research Flow 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Eligibility of subjects 
Informed Consent 
Randomized 
Probiotic 
group 
Placebo 
group 
Intervention for 12 weeks 
Outcome 
Inclusion and 
Exclusion 
criteria 

 
 
 
 
 
 
E.  Ethics 
This research has been approved by Ethics Committee of the Faculty of Medicine 
Universitas Indonesia (Approval number: 981/UN2.F1/ETIK/2017) 
 
IV. 
Statistical Analysis Plan 
 
All Data will be analyzed using SPSS 24.0 (SPSS) for Windows. Laboratory parameters 
will be compared between two different times and also compared between two groups by using 
paired and independent t test (if normally distributed) or using Wilcoxon and Mann Whitney (if 
not normally distributed). Statistically significance was determined by p value <0.05.  Intention 
to Treat analysis will be used. HIV symptom index and food frequency will be reported 
descriptively. 
 
V. References 
1. Wilson EMP, Sereti I. Immune restoration after antiretroviral therapy: the pitfalls of hasty or 
incomplete repairs. Immunological reviews. 2013;254(1):343-54.  
2. Gaardbo JC, Hartling HJ, Gerstoft J, Nielsen SD. Incomplete Immune Recovery in HIV 
Infection: Mechanisms, Relevance for Clinical Care, and Possible Solutions. Clinical and 
Developmental Immunology. 2012;2012. 3. d'Ettorre G, Ceccarelli G, Giustini N, Serafino S, 
Calantone N, De Girolamo G, et al. Probiotics Reduce Inflammation in Antiretroviral Treated, 
HIV-Infected Individuals: Results of the "Probio-HIV" Clinical Trial. PloS one. 
2015;10(9):e0137200.  
4. Brenchley JM, Douek DC. The mucosal barrier and immune activation in HIV pathogenesis. 
Current opinion in HIV and AIDS. 2008;3(3):356-61.   
5. Harris KA, Hartley JC. Development of broad-range 16S rDNA PCR for use in the routine 
diagnostic clinical microbiology service. Journal of medical microbiology. 2003;52(Pt 8):685-

91.  
6. Xiang JY, Ouyang Q, Li GD, Xiao NP. Clinical value of fecal calprotectin in determining 
disease activity of ulcerative colitis. World journal of gastroenterology. 2008;14(1):53-7.  
7. Karczewski J, Troost FJ, Konings I, Dekker J, Kleerebezem M, Brummer RJ, et al. Regulation 
of human epithelial tight junction proteins by Lactobacillus plantarum in vivo and protective 
effects on the epithelial barrier. American journal of physiology Gastrointestinal and liver 
physiology. 2010;298(6):G851- 9.  
8. Anderson RC, Cookson AL, McNabb WC, Kelly WJ, Roy NC. Lactobacillus plantarum DSM 
2648 is a potential probiotic that enhances intestinal barrier function. FEMS microbiology 
letters. 2010;309(2):184- 92.  
9. Lopez P, Gonzalez-Rodriguez I, Gueimonde M, Margolles A, Suarez A. Immune response to 
Bifidobacterium bifidum strains support Treg/Th17 plasticity. PloS one. 2011;6(9):e24776.