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Study Protocol

Fungal Translocation Marker in People Living with HIV Needing Treatment for Onychomycosis: A Protocol for the Prospective Pilot Study

Chen, Yaling1; Ouyang, Jing1; Isnard, Stéphane2,3; Costiniuk, Cecilia T.2,3; Yan, Jiangyu1; Zhou, Xin1; Routy, Jean-Pierre2,3,4,∗; Chen, Yaokai1,∗

Editor(s): Zhao, Wei

Author Information
Infectious Diseases & Immunity: April 2022 - Volume 2 - Issue 2 - p 122-124
doi: 10.1097/ID9.0000000000000047
  • Open



It is well-known that microbial translocation and chronic activation of the immune system are the hallmarks of progressive HIV infection. Apart from the bacterial content of the gut microbiome, recently, there is increasing evidence that the fungal component is also critical for the regulation of homeostatic and protective immune responses.[1,2] Moreover, fungal diseases are one of the main cause of opportunistic infections in people living with HIV (PLWH); thus antifungal therapeutic agents are, and have been, widely and frequently used amongst this population. However, the influence of antifungal agents on microbial translocation and gut microbiome composition in PLWH are rarely reported.

Onychomycosis is a chronic and mild fungal infection of the finger- and toenails. PLWH are predisposed to onychomycosis, and has been found four times more frequently in PLWH than in the general individuals.[3] Terbinafine, an antifungal agent, has been broadly utilized to treat onychomycosis, and has shown a high mycological and clinical cure rate.[4] It exhibits different antifungal activities against dermatophytes, yeasts, molds, and dimorphic fungi.[5] These differential antifungal properties of terbinafine against different fungi could possibly induce an intestinal mycobiome compositional imbalance and result in significant microbiome diversity change. Moreover, the bioavailability of oral terbinafine is known to be between 70% and 80%,[6] indicating that a large fraction of the drug remains within the gastrointestinal tract, and may thus significantly influence the overall fungal composition of the intestinal microbiome.

In PLWH, we speculate that terbinafine is likely to alter the gut mycobiome, further impacting on intestinal microbiota composition and diversity, and bacterial and fungal microbial translocation. We developed this pilot study protocol to test this hypothesis.


Ethical approval

This study was approved by the Ethics Committee of Chongqing Public Health Medical Center and registered at the Chinese Clinical Trial Registry (ChiCTR2100043617).

Study design

A single-armed prospective study will be conducted atChongqing Public Health Medical Center, which provides care and treatment for 30,000 to 50,000 PLWH in southwestern China. PLWH with comorbid onychomycosis who are prescribed oral terbinafine treatment will be screened to be included in this study. Compared with other opportunistic infections requiring systemic antifungal treatment in PLWH, onychomycosis is a mild, localized chronic fungal infection. We therefore speculate that a relatively stable microbiome and underlying intestinal condition has been maintained in these individuals. A total of four appointments for each participant are planned, that is, one baseline visit (V1) and three follow-up visits (V2 at 1–2weeks, V3 at 12weeks, and V4 at 24weeks). At each visit, plasma and fecal samples will be collected and stored for future reference. During the baseline visit, patients will be screened to verify that all participants fulfill specific inclusion and exclusion criteria, in addition to recording socio-demographic variables, clinical history, and antiretroviral therapy (ART) drug usage. Every study participant will provide signed, written informed consent. During the follow-up visits, evidence of microbial translocation and alterations to microbiome composition will be evaluated using the plasma and fecal samples. Adverse affects will be monitored during the course of treatment.

Sample size calculation

The sample size (n = 22) was chosen based on literature regarding sample sizes and our experience with pilot study design,[7,8] and accounts for a 10% (2 participants) possible drop off or loss to follow-up. This pilot study should provide sufficient data to perform a formal sample size calculation for a proposed more definitive study in the future.

Inclusion criteria

Participants with confirmed HIV-diagnosis needing treatment for onychomycosis, aged ≥18years, and comprising males, females, and transgenders, will be included. It is required that the participant is on ART and did not alter their specific ART regimen for the 6months prior to enrollment, indicating a stable condition. The CD4+ T-cell count of the participants will need to be greater than 200 cells/μL to avoid the use of antibiotics for infection prophylaxis. Participants with onychomycosis should not have been prescribed oral terbinafine treatment and should not have received any antifungal treatment in the previous 3 months before enrollment.

Exclusion criteria

The following groups of patients will be excluded: people participating in other clinical studies (except those in non-interventional studies) in the past 6 months, those who have serious cardiovascular and cerebrovascular diseases, liver and kidney diseases, diabetes, hematological diseases, autoimmune diseases, severe malnutrition, intestinal diseases such as acute and chronic enteritis, inflammatory bowel disease, irritable bowel syndrome, those who are taking drugs that are known to interfere with terbinafine or to have a significant influence on the intestinal barrier, such as metformin, probiotics, and prebiotics in the previous 4 weeks before enrollment, and those undergoing enteroscopy in the previous 4 weeks before enrollment. Participants who have a known allergy to terbinafine or equivalent medications, or who have other factors that may significantly influence the study will not be invited to participate in the study. Patients having any acute infection or medical conditions in the preceding 4 weeks will also not be included.

Diagnostic criteria

In accordance with Chinese Guidelines for Diagnosis and Treatment of Onychomycosis (2015),[9] the diagnosis of onychomycosis will be on clinical evaluation of the nail or based on laboratory examination, including positive results of microscopic mycological nail examination, mycological culture, or histopathological studies. Diagnosis of HIV will be made according to current Chinese Guidelines.[10]


Participants being prescribed a 12-week course of terbinafine treatment for onychomycosis in addition to their ART will be recruited. Terbinafine hydrochloride (AOBANG® Chengdu Open Pharmaceutical Co., Ltd) in the form of 250 mg tablets, once per day, administered orally is recommended. As per standard of care, participants taking drugs with a known interaction with their ART will not be recruited.

Outcomes and measures

Primary outcomes

The primary outcome will be to evaluate the effect of terbinafine on the level of the plasma marker of fungal microbial translocation, 1-3-β-D-glucan (BDG), assessed using the Fungitell® assay, at V2, 3, and 4, compared with V1.

Secondary outcomes

The secondary outcomes will be variations pre- and post-oral terbinafine treatment, and after terbinafine discontinuation, at V2, 3, and 4, compared with V1, and measured as follows:

  • Levels of the gut barrier integrity markers, intestinal fatty acid-binding protein (I-FABP), and regenerating islet-derived protein 3α (REG3α), measured by enzyme-linked-immunosorbent serologic assay (ELISA), in plasma.
  • Levels of the bacterial translocation marker lipopolysaccharide (LPS), assessed using ELISA, in plasma.
  • Microbiota composition and diversity in stools assessed using 16 s and internal transcribed spacer rDNA sequencing.

Exploratory outcomes

The exploratory outcomes are used to assess the effect of terbinafine on the changes of immune cell activation markers Dectin-1 and NKp30, evaluated in peripheral blood mononuclear cells by flow cytometry analyses, at V2, 3, and 4, compared with V1.

Statistical analysis

Comparisons of data from plasma and stool will be made between data from V1, V2, V3, and V4. The Friedman test and the Wilcoxon matched pairs test will be used to analyze the plasma data before and after terbinafine therapy. The number of microbial taxa by type in stool samples will be compared by the Wilcoxon matched pairs test. P-values<0.05 will be deemed to indicate a statistically significant difference.


This study protocol is developed to evaluate the effects of terbinafine on microbial translocation and gut microbiome composition in PLWH requiring therapeutic intervention for onychomycosis, which has been rarely reported. The results of this pilot study may provide a logical and rational basis for future clinical use of antifungal drugs in PLWH. To avoid the effects of inter-individual factors, such as age, gender, and ART, the present study has been designed as a self-controlled study with 22 subjects, comparing changes between pre- and post-oral terbinafine treatment on each participant. However, we acknowledge that further limitations exist as the sample size of the study is small, and not all potential limiting factors are listed in the exclusion criteria. In the future, full-scale studies with formal sample size calculations can be performed, should the outcomes of this study indicate that terbinafine does indeed materially affect microbial translocation and microbiota composition in PLWH.


This work was supported by the Joint Medical Research Project (2020GDRC010) of Chongqing Science & Technology Bureau and Chongqing Health Commission, the Research Project of Chinese Federation of Public Health foundation (GWLM202024), and the Youth Scientific Research and Innovation Fund Project of Chongqing Public Health Medical Center (2019QNKYXM02).

Author Contributions

Jean-Pierre Routy and Yaokai Chen conceived and designed the study, Jing Ouyang and Yaling Chen led the drafting of the manuscript, Stéphane Isnard, Cecilia T. Costinuik, Jiangyu Yan, and Xin Zhou edited the manuscript. All authors have read and approved the final manuscript.

Conflicts of Interest



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HIV; Fungal translocation; Microbial translocation; Onychomycosis; Terbinafine

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