Open Label Phase-II Randomized Trial of Three Liposomal Amphotericin B Regimens as Induction Therapy for Disseminated Histoplasmosis in AIDS Patients
Phase: PHASE2
Conditions: Histoplasmosis, AIDS
Sponsor: Alessandro Pasqualotto

Open Label Phase-II Randomized Trial of 3 Regimens of 
Liposomal Amphotericin B as Induction Therapy for  
Disseminated Histoplasmosis in AIDS patients 
 
 
STUDY PROTOCOL 
 
 
CLINICAL TRIALS REGISTRATION NCT04059770 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
February 24, 2021 
 

STUDY CONCEPT 
 
Background 
Histoplasma capsulatum is the etiologic agent of histoplasmosis, a 
systemic mycosis that is highly endemic in the Americas and also being 
identified in other parts of the globe [1, 2]. Histoplasmosis may disseminate in 
immunosuppressed patients, particularly those infected by the HIV. 
Accordingly, histoplasmosis has been an AIDS-defining infection since 1987 
[3]. Disseminated histoplasmosis (DH) is one of the main AIDS-defining 
infections and a major killer of HIV-infected patients in South and Central 
America [3-5]. 
Most AIDS patients with histoplasmosis require high dose of antifungal 
agents. Even though liposomal amphotericin B (L-AmB) is considered the 
drug of choice for histoplasmosis in AIDS, as agreed in the Infectious 
Diseases Society of America guidelines [6], and supported by a randomized 
clinical trial [7], many patients in Latin America are still treated with high doses 
of deoxycholate amphotericin B (d-AmB) for periods as long as 2-6 weeks. 
These regimens are associated with considerable toxicity, and therefore 
reduced efficacy. Overall mortality in patients using d-AmB in Latin America, 
where Histoplasma antigen detection is rarely available, can be as high as 
50% [5]. A better treatment strategy is ultimately needed. 
L-AmB has several advantages over d-AmB in the treatment o DH: 
lower infusion-related toxicity [8], and reduced hematological and renal toxicity 
[9, 10]. In addition, L-AmB has long half-life and effectively penetrates in 
tissues such as liver, spleen and the central nervous system [11]. Considering 
that the antifungal effect of the polyenes depends on peak concentrations, 
treating patients with a high dose of L-AmB for short periods [instead of 
standard doses for longer periods] is a promising approach. Such strategy 
has successfully been studied in antifungal prophylaxis [12, 13], febrile 
neutropenia [14], leishmaniasis [15], and more recently, cryptococcosis [16]. 
When used for antifungal prophylaxis in hematology patients, doses of up to 
15 mg/kg of L-AmB were given without significant toxicities [17]. Conversely, 
toxicity has been linked to repeated administrations of L-AmB [12]. Despite 

these encouraging data, the strategy of using single high dose of L-AmB has 
never been tried in histoplasmosis. 
 
Research Justification 
 
DH is an endemic disease that frequently occurs in institutions with no 
access to modern diagnostic tests and that have limited availability of L-AmB. 
If a strategy of less frequent, high doses of L-AmB shows activity in DH, this 
will allow for reducing the cost of treatment in developing countries, in addition 
to improving safety and tolerability. Based on what has been published so far, 
safety does not seem to be a concern with high single doses of L-AmB. 
Results of this study may serve as a basis for a future phase III randomized 
comparative trial. 
 
Objectives 
 
To determine and compare the activity and safety of three regimens of 
L-AmB, as induction therapy of DH in AIDS patients, as well as to determine 
the safety of the three regimens. 
 
Methods 
Trial design 
 
This will be a prospective randomized non-comparative multicenter 
open label trial of induction therapy for DH in AIDS, followed by oral therapy 
with itraconazole.  
The total sample size planned is 99 patients (33 patients per study 
arm). This sample size considers 10% of dropout. 
The study will be conducted in compliance with the declaration of 
Helsinki, as well as ICH GCP guidelines. Participation in the study will require 
written consent. The study will be registered at www.clinicaltrials.gov, before 
trial initiation and patient recruitment. 
 
Participants 
We will include adult (>18 years old) patients infected with the HIV 
(AIDS) who are diagnosed with DH, by the means of either (i) positive 
Histoplasma antigen in the urine (IMMY monoclonal antibody test); (ii) 

confirmation by classical methods (microscopy, culture, or histopathology); or 
(iii) positive Histoplasma qualitative PCR (performed in accordance to [18]) in 
BAL samples, as well as bone marrow aspirates and tissue samples. Patients 
who are both antiretroviral treatment (ART) naïve and ART exposed will be 
recruited. 
 
Exclusion criteria 
Patients with previous diagnosis of histoplasmosis will not be 
considered for this study. We will also exclude pregnant or lactating woman, 
patients with renal failure (serum creatinine and urea >1.5x the upper limit of 
normality), abnormal aminotransferases (up to >3x the upper limit of 
normality), and patients with a previous serious reaction to a polyene 
antifungal drug. Patients who received more than one dose of a polyene 
antifungal drug in the last 48 hours will be excluded, as well as patients who 
refuse content to participate in the study. Patients diagnosed with 
histoplasmosis affecting the central nervous system will not be allowed to 
participate in the study. In addition, patients who are expected to die within 48 
hours of enrolment will also be excluded, based on the judgment made by the 
attending physician. Patients diagnosed with tuberculosis will be excluded; as 
well those receiving drugs that cause significant drug-drug interaction (relative 
or absolute) with itraconazole. 
 
Study settings 
 
Six medical centers in Brazil, will recruit patients for this study: Santa 
Casa de Misericórdia de Porto Alegre, Hospital de Clínicas de Porto Alegre, 
Hospital Nossa Senhora da Conceição, Hospital de Doenças Tropicais, 
Hospital São Jose de Doenças Infecciosas, and Hospital Giselda Trigueiro. 
 
Intervention 
AIDS patients with DH will be randomized to one of three study arms: 
(i) single IV dose of L-AmB at 10 mg/kg; (ii) single IV dose of 10 mg/kg of L-
AmB on day 1, followed by 5 mg/kg of L-AmB on day 3; and (iii) L-AmB at 3 
mg/kg IV for 2 weeks. Induction therapy will be followed in all patients by oral 
therapy with itraconazole capsules at 400 mg/daily for a year.  

 
Drug reconstitution and administration 
 
L-AmB (AmBisome; Gilead Sciences, San Dimas, California, USA), a 
lyophilized liposomal preparation of amphotericin B, will be reconstituted 
according to the manufacturer’s instruction and given intravenously at fixed 
doses, according to the randomization arm. L-AmB concentration should be of 
0.2 mg/ml in 5% dextrose, and the drug should be infused over 2 hours, 
preferably in a central line. Patients will be monitored for the occurrence of 
infusion-related toxicity during and after drug infusion. 
 
Primary endpoint 
 
Clinical and mycological successes will be the primary outcomes for 
this study. A successful clinical response to induction therapy will be defined 
according to, as a maximum daily temperature <37.8°C for 72 hours; no 
increase in severity of clinical signs, symptoms, or laboratory abnormalities 
attributable to histoplasmosis; and the resolution of at least one of the signs or 
symptoms of histoplasmosis that qualified the patient for study enrollment [7]. 
 
Primary study outcome will be determined at day 14 of the study. 
 
Secondary endpoints 
 
Overall mortality and investigator-attributable mortality due to 
histoplasmosis will be determined at day 14 of study. Overall Survival (OS) 
will also be determined at 12, 24, and 48 weeks of study. OS will be 
considered as the time from randomization until death from any cause.
 
Secondary endpoint will also include need for an additional antifungal 
course with L-AmB, during 24 weeks of follow up. 
 
Median percentage of change in renal and hepatic function at 14 days 
will be compared with baseline values. 
Frequency of infusion-related toxicity with L-AmB will be recorded. 
Clearance of blood cultures will not be used as a criterion for 
mycological response in this study, due to the limited sensitivity of blood 
culture in histoplasmosis [19]. Fungal clearance will also not be used as a 
criterion for mycological response. Differently from cryptococcosis, in which 
fungal burden is easily obtained and quantified in cerebrospinal fluid samples, 

in histoplasmosis commonly a single positive sample is obtained along patient 
investigation (e.g., microscopy, culture, and/or histopathology). 
Non-culture laboratory markers have proved to be useful adjuncts in 
monitoring the response to systemic histoplasmosis [20, 21]. In this study, we 
will evaluate the effect of a decrease of at least 50% in Histoplasma urinary 
antigen concentrations [19] along the first 2 weeks of therapy as a secondary 
endpoint, and the association of such reductions with clinical response to 
antifungal therapy. 
 
Rescue therapy 
The study protocol will allow patients to receive additional courses of L-
AmB (at 3 mg/kg daily) in case the attendant physician suspect of absence of 
response with the induction regimen. This will apply for failing patients 
receiving a single 10 mg/kg of L-AmB; or 10 mg/kg on day 1, followed by 5 
mg/kg on day 3. In these cases, L-AmB should be given up to a maximum 
period of 2 weeks. The estimated frequency of L-AmB as rescue therapy will 
be based on the frequency of failure with L-AmB at 3 mg/kg in patients with 
DH and AIDS (i.e., 12%) [7]. Considering that 66 patients will be evaluated in 
arms (i) and (ii) of the study (high-doses of L-AmB), in a pessimistic scenario 
we aim to provide 8 additional treatments with L-AmB. 
 
Time for initiating ART 
Physicians will be instructed to initiate ART as soon as possible in 
AIDS patients with DH, since immune reconstitution syndrome is uncommon 
in patients with DH [22]. In Brazil, initial ART consists of dolutegravir, 
lamivudine, and tenofovir. History of previous antifungal use and known 
resistance patters will also be considered in the decisions about ART. 
 
Statistical analyses 
Descriptive statistics will be used to summarize the data. Mean, 
standard deviation, median, interquartile range and minimum and maximum 
will be described for continuous variables. Categorical variables will be 
summarized by absolute and relative frequencies. Kaplan-Meier method will 
be used in order to describe OS. Despite the non-comparative status of the 

trial, exploratory comparisons will be performed across subgroups. 
Continuous variables will be compared using two sample t-test, paired sample 
t-test, Mann-Whitney test, Wilcoxon signed-rank test, one-way ANOVA or 
Kruskal-Wallis test, as appropriate and necessary. Categorical variables will 
be compared with Fisher exact test or chi-square test, as appropriate. All p-
values will be two-sided.  
We expect that the three arms will have the same clinical response. 
For each arm, when the sample size is 29, a two-sided 90% confidence 
interval will extend 10% from the observed proportion for an expected 
proportion of response equal to 88% [as extracted from the Johnson trial]. 
Considering a dropout of 10%, the sample size per arm will be 33. The total 
sample size for the study will be 99 patients. Data analysis will be performed 
using SPSS software. 
 
Ethical aspects 
 
Conduction of this study is conditioned to ethical approval by local 
IRBs. Since this trial will not aim to modify any regulatory aspect related to L-
AmB in Brazil, ANVISA (Brazilian Health Regulatory Agency) will not be 
consulted. The study will also be registered at clinicaltrials.gov. 
 
Study activities 
 
Clinical evaluations will take place at baseline, day 3, day 7, and day 
14 (End of Study Visit). End of follow up will occur at week 48, and mortality 
will be determined at weeks 12, 24, and 48. 
The study will recommend careful attention to hydration status, and 
close monitoring of renal function, hepatic function, and electrolytes. Unless 
clinically advisable, physicians are requested to give one liter of saline 0.9% 
solution intravenously before L-AmB infusion. Patients should also be on 
prophylaxis with daily trimethoprim-sulfamethoxazole. 
 
Table 1 shows the procedures predicted for this study. Clinical and 
laboratory adverse events will be graded using the NIH DAIDS Toxicity Table 
[23]. Itraconazole therapeutic drug monitoring will be offered to sites at day 7 
and day 14 of study. The attending physicians will use this information 
according to their personal judgment. 

 
Table 1. Study activities. 
 
 
Study Plan Schedule Strategy 
 
Table 2 shows the predicted timeline for this study. 
Table 2. Study plan. 
 
 

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