A Randomized Controlled Trial of Bacteriophage Cocktail Therapy for Multidrug-Resistant Gram-Negative Ventilator-Associated Pneumonia
Phase: NA
Conditions: VAP - Ventilator Associated Pneumonia
Sponsor: Chinese PLA General Hospital

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A Randomized Controlled Trial of Bacteriophage
Cocktail Therapy for Multidrug-Resistant
Gram-Negative Ventilator-Associated Pneumonia
Study Protocol
Version: 1.0 | Version Date: May 25, 2025

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Research Purpose and Significance
This study addresses the clinical challenges of high mortality and treatment
difficulties in ventilator-associated pneumonia (VAP) caused by multidrug-resistant
Gram-negative bacteria (Acinetobacter baumannii, Pseudomonas aeruginosa, and
Klebsiella pneumoniae). A Randomized Controlled Trial of Bacteriophage Cocktail
Therapy for Multidrug-Resistant Gram-Negative Ventilator-Associated Pneumonia
aims to meet clinical needs for rapid accessibility and precise bacterial targeting, with
the clinical efficacy for VAP as the primary endpoint, and bacterial clearance rate and
28-day mortality rate as secondary endpoints. The study aims to identify key factors
influencing clinical outcomes, providing robust support for standardizing and
promoting phage therapy.
Research Contents
Clinical Efficacy and Safety Evaluation of Bacteriophage Cocktail Therapy: A
multicenter, double-blind, randomized controlled trial will be conducted to assess the
clinical efficacy, bacterial clearance rate, and 28-day mortality of nebulized
bacteriophage cocktail therapy in patients with multidrug-resistant Gram-negative
bacterial ventilator-associated pneumonia (VAP). This study aims to provide
high-level evidence-based medical support for the application of bacteriophage
cocktail therapy.
Research Design
1.Recruitment
Inclusion Criteria:
(1) Diagnosed with ventilator-associated pneumonia (VAP);
(2) Identified infection with MDR Klebsiella pneumoniae, Acinetobacter baumannii,
or
Pseudomonas
aeruginosa
via
MROSE
evaluation
and
antimicrobial
susceptibility testing (AST) from this or other tertiary hospitals;
(3) Aged 18 to 85 years;
(4) Written informed consent obtained from the patient or legal guardian.
Exclusion Criteria:

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(1) Pregnant or breastfeeding women;
(2) FiO₂ >= 70% or PEEP >= 15 cmH₂O;
(3) Hemodynamic instability (vasopressor dose increased by >=30% in the last 6
hours or norepinephrine > 0.5 μg/kg/min);
(4) Known allergy to phage components.
Withdrawal Criteria:
(1) Participants or their legally authorized representatives voluntarily request
withdrawal of informed consent.
(2) Occurrence of severe adverse reactions attributed to phage therapy, necessitating
discontinuation by the investigator's judgment.
2. Randomization and blinding method
Eligible participants will be randomly assigned to either the Intervention Group
(nebulized phage cocktail plus standard VAP therapy) or the Control Group
(nebulized placebo [normal saline] plus standard VAP therapy). Assignment will
occur via a web-based stratified block randomization system with variable block sizes,
generating a unique random number for each participant. A central computerized
system will perform 1:1 allocation. Random numbers are non-reusable, and
participants failing to complete the study will be classified as withdrawn.
The study employs a double-blind design: participants, investigators, outcome
assessors, and statisticians remain blinded to treatment allocation to minimize bias
and enhance result reliability.
Both groups receive identical nebulization treatments in route, frequency, volume,
appearance, color, and odor. Designated personnel uninvolved in outcome assessment
prepare and administer all treatments to maintain blinding.
An independent third-party data management team manages randomization and
blinding codes through a central online system. Each participant receives a unique
blind code linked to their treatment. Unblinding is prohibited without authorization
unless required for managing serious adverse events, and only with principal
investigator approval. All blinded drug packages are unmarked, and non-assessing

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staff handle drug documentation. Full unblinding will occur after database lock at trial
completion.
3.Treatment Plan
After thorough evaluation of indications and contraindications for bronchoalveolar
lavage,
pre-treatment
respiratory
tract
samples
are
collected
from
subjects.
Gram-negative bacilli infection is identified, and the bacterial species is determined
through M-ROSE detection. The plate count method is employed: the sample is
serially diluted, a specific volume of the diluted solution is spread onto agar plates,
and after incubation (typically 24-48 hours), the number of colonies formed is
counted. Plates with 30-300 colonies are selected for analysis. The formula CFU/mL
= (number of colonies) / (volume plated in mL × dilution factor) is used to calculate
bacterial colony counts in the subject's therapeutic biological samples.
Optimize Phage Therapy:
Phase I (Rapid Intervention):
On the day of enrollment, based on rapid bedside pathogen identification (M-ROSE
combined with rapid PCR testing completed within 2 hours), the intervention group
immediately receives a broad-spectrum phage cocktail targeting the relevant
pathogenic bacterial genus (covering over 90% of clinical isolates in the phage
library). Treatment is administered via nebulized inhalation (twice daily) for 5 days
(the control group receives normal saline nebulization). Concurrently, Phase II
technical preparations are completed: ①Standardized culture and cryopreservation of
the patient's pathogen are initiated immediately based on rapid molecular diagnostic
results; ②Automated microbroth dilution combined with phage plaque formation
assay is employed to complete phage-host specificity validation within 48-72 hours,
establishing an initial personalized phage candidate library.
Phase II (Precision Intervention):
On day 5, lower respiratory tract samples from subjects are collected again, and the
treatment regimen is adjusted according to the phage susceptibility testing results:

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If the original broad-spectrum phage cocktail maintains good lytic activity against the
pathogenic bacteria (manifested as clear or partially turbid plaques), the original
treatment regimen is maintained.
If phages are absent or the titer is significantly reduced (less than 10^5 PFU/mL),
indicating the emergence of a resistant or escape strain, the individualized phage
formulation screened in the previous stage is immediately substituted, and treatment
continues for 5 days.
If neither the broad-spectrum cocktail nor an available individualized phage shows
lytic activity against the pathogen, the original broad-spectrum cocktail regimen is
retained for an additional 5 days of treatment while efficacy is continuously observed.
4.Outcome
Primary Outcome:
Clinical response rate, defined as imaging improvement or lack of progression at the
end of treatment (day 10) OR resolution of pneumonia signs and symptoms.
Secondary Outcomes:
Bacterial clearance rate (Day 10 post-enrollment); 28-day all-cause mortality rate;
Clinical Pulmonary Infection Score (CPIS); Hospitalization duration; ICU stay
duration; Duration of invasive mechanical ventilation; M-ROSE results from lower
respiratory tract specimens; Incidence of antimicrobial resistance progression;
Prevalence of novel drug-resistant bacterial infections; Sequential Organ Failure
Assessment (SOFA) score; Blood cell counts (white blood cells, neutrophils,
lymphocytes);
Inflammatory
markers
(C-reactive
protein,
procalcitonin);
Inflammatory cytokines (IL-6, TNF-α); T lymphocyte subsets.
Safety Outcomes:
Incidence
of
allergic
reactions;
Liver
and
kidney
function;
Emergence
of
phage-resistant bacteria; Phage-neutralizing antibody titers.
5.Sample Size and Calculation Basis
This
study
employs
a
randomized
controlled
trial
design
to
evaluate
the
comprehensive therapeutic effects of phage therapy on VAP patients, with the
primary outcome being the clinical improvement rate. The intervention group receives

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nebulized phage cocktail combined with standard VAP treatment, while the control
group receives nebulized placebo combined with standard VAP treatment. Based on
relevant literature, antibiotic therapy achieves a ~60% clinical improvement rate for
VAP, whereas phage therapy combined with routine anti-infective treatment
demonstrates a ~77.2% improvement rate. Assuming α=0.05 (two-tailed), with 80%
power, PASS software calculated the sample size: 111 cases per group at a 1:1 ratio.
Considering a 10% dropout rate, 124 cases are needed per group, totaling 248 cases.
6.Protocol Deviation
This refers to any of the following scenarios:
(1)During the broad-spectrum or individualized phage susceptibility testing phase, no
lytic phages [Modified: corrected terminology] with detectable activity are identified
through phage plaque assays;
(2)Subjects die within three days after randomization without completing the full
treatment protocol. All such cases shall be considered non-compliant with the study
protocol and classified as protocol deviations.
7. Data Management and Statistical Analysis
Analysis of Intergroup Balance:
Quantitative data will be presented as mean ± standard deviation or median with
interquartile range (for skewed distributions). Intergroup differences will be compared
using the t-test or non-parametric tests (for skewed distributions). Categorical data
will be described using percentages (%). Group comparisons will be conducted using
the chi-square test or Fisher's exact test.
Effectiveness Evaluation Analysis:
This study will adopt the Intention-to-Treat (ITT) principle, including all randomized
patients in the primary outcome analysis, regardless of treatment completion or
dropout. To assess the robustness of the primary analysis results, a sensitivity analysis
(Per-Protocol, PP) will be conducted on participants who complied with the protocol.
For binary outcome measures, Cox regression models will be used to adjust for center
effects, yielding hazard ratios (HR) and 95% confidence intervals to evaluate the
efficacy of phage therapy compared to conventional antimicrobial treatment.

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Continuous outcome measures will be analyzed using Linear Mixed Models suitable
for repeated measurements, comparing differences between the intervention and
control groups.
Subgroup Analysis:
Subgroup analysis will be performed for the primary outcome and important
secondary outcomes to explore potential effect modifiers (e.g., age, sex, invasive
mechanical ventilation status, disease severity indices, underlying comorbidities) that
may influence the efficacy of phage therapy.
8.Management Plan for Common Adverse Reactions / Serious Adverse Reactions
Based on previously published research data, phage therapy demonstrates good
tolerability with rare adverse events and no reported severe complications directly
attributable to phages. Based on existing studies, potential adverse reactions may
include cough, throat discomfort, fever, transient dyspnea, decreased pulse oxygen
saturation, and airway spasms. Any serious adverse events will be documented in
accordance with Good Clinical Practice (GCP) standards, promptly reported, and
properly evaluated and managed.
Management Plan
(1) Cough and Throat Discomfort
For mild cough, reduce the nebulization concentration and/or flow rate and observe
the response.
For severe cough, discontinue nebulization immediately and observe the response.
Administer antitussive medication for symptomatic treatment if necessary, until
symptoms resolve.
(2) Airway Spasm
Discontinue nebulization immediately and observe closely.
If symptoms do not relieve, administer salbutamol via inhaler or nebulizer.
Methylprednisolone 40mg may be administered intravenously if necessary.

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Monitor pulse oximetry (SpO₂). Improve ventilation and hypoxia with nasal catheter
oxygen, high-flow humidified oxygen therapy, or non-invasive ventilation as needed,
until symptoms resolve.
(3) Fever
Discontinue phage therapy immediately.
Monitor body temperature and administer physical cooling or antipyretic drugs as
indicated.
Perform tests including blood routine, IL-6, CRP, procalcitonin, sputum smear,
sputum culture + susceptibility. If temperature >38°C, add blood culture +
susceptibility. Consider chest CT, abdominal ultrasound, or other examinations as
needed to identify the cause of fever.
Detect endotoxin content and cytokine levels in blood.
Retain the phage nebulization solution for bacterial culture, metagenomic detection
analysis, and endotoxin level determination.
(4) Decreased Pulse Oximetry (SpO₂)
Discontinue phage therapy immediately.
Administer oxygen via nasal catheter or mask and observe the change in SpO₂.
Investigate the cause of hypoxia through physical examination and relevant laboratory
tests.
Institute further treatment based on the identified cause of hypoxia.