Sputum bacterial load and bacterial composition correlate with lung function and are altered by long-term azithromycin treatment in children with HIV-associated chronic lung disease

Study information

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study design

randomized controlled trial

Citation

PMID PubMed identifier for scientific articles.

36803868

DOI Digital object identifier for electronic documents.

10.1186/s40168-023-01460-x

URI

Authors

Abotsi RE, Dube FS, Rehman AM, Claassen-Weitz S, Xia Y, Simms V, Mwaikono KS, Gardner-Lubbe S, McHugh G, Ngwira LG, Kwambana-Adams B, Heyderman RS, Odland JØ, Ferrand RA, Nicol MP

Journal

Microbiome

Year

2023

Keywords:

Adolescents, Africa, Bacteriome, FEV1z, HIV, Haemophilus, Microbiome, Moraxella, Obliterative bronchiolitis

BACKGROUND: Long-term azithromycin (AZM) treatment reduces the frequency of acute respiratory exacerbation in children and adolescents with HIV-associated chronic lung disease (HCLD). However, the impact of this treatment on the respiratory bacteriome is unknown. METHOD: African children with HCLD (defined as forced expiratory volume in 1 s z-score (FEV1z) less than - 1.0 with no reversibility) were enrolled in a placebo-controlled trial of once-weekly AZM given for 48-weeks (BREATHE trial). Sputum samples were collected at baseline, 48 weeks (end of treatment) and 72 weeks (6 months post-intervention in participants who reached this timepoint before trial conclusion). Sputum bacterial load and bacteriome profiles were determined using 16S rRNA gene qPCR and V4 region amplicon sequencing, respectively. The primary outcomes were within-participant and within-arm (AZM vs placebo) changes in the sputum bacteriome measured across baseline, 48 weeks and 72 weeks. Associations between clinical or socio-demographic factors and bacteriome profiles were also assessed using linear regression. RESULTS: In total, 347 participants (median age: 15.3 years, interquartile range [12.7-17.7]) were enrolled and randomised to AZM (173) or placebo (174). After 48 weeks, participants in the AZM arm had reduced sputum bacterial load vs placebo arm (16S rRNA copies/µl in log10, mean difference and 95% confidence interval [CI] of AZM vs placebo - 0.54 [- 0.71; - 0.36]). Shannon alpha diversity remained stable in the AZM arm but declined in the placebo arm between baseline and 48 weeks (3.03 vs. 2.80, p = 0.04, Wilcoxon paired test). Bacterial community structure changed in the AZM arm at 48 weeks compared with baseline (PERMANOVA test p = 0.003) but resolved at 72 weeks. The relative abundances of genera previously associated with HCLD decreased in the AZM arm at 48 weeks compared with baseline, including Haemophilus (17.9% vs. 25.8%, p < 0.05, ANCOM ω = 32) and Moraxella (1% vs. 1.9%, p < 0.05, ANCOM ω = 47). This reduction was sustained at 72 weeks relative to baseline. Lung function (FEV1z) was negatively associated with bacterial load (coefficient, [CI]: - 0.09 [- 0.16; - 0.02]) and positively associated with Shannon diversity (0.19 [0.12; 0.27]). The relative abundance of Neisseria (coefficient, [standard error]: (2.85, [0.7], q = 0.01), and Haemophilus (- 6.1, [1.2], q < 0.001) were positively and negatively associated with FEV1z, respectively. An increase in the relative abundance of Streptococcus from baseline to 48 weeks was associated with improvement in FEV1z (3.2 [1.11], q = 0.01) whilst an increase in Moraxella was associated with decline in FEV1z (-2.74 [0.74], q = 0.002). CONCLUSIONS: AZM treatment preserved sputum bacterial diversity and reduced the relative abundances of the HCLD-associated genera Haemophilus and Moraxella. These bacteriological effects were associated with improvement in lung function and may account for reduced respiratory exacerbations associated with AZM treatment of children with HCLD. Video Abstract.

Experiment 1

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Curated date: 2023/10/11

Curator: Chinelsy

Revision editor(s): Chinelsy

Subjects

Location of subjects

Malawi

Zimbabwe

Host species Species from which microbiome was sampled. Contact us to have more species added.

Homo sapiens

Body site Anatomical site where microbial samples were extracted from according to the Uber Anatomy Ontology

Sputum Expectoration,Sputum

Condition The experimental condition / phenotype studied according to the Experimental Factor Ontology

azithromycin (2R,3S,4R,5R,8R,10R,11R,12S,13S,14R)-2-ethyl-3,4,10-trihydroxy-3,5,6,8,10,12,14-heptamethyl-15-oxo-11-,10q24.1-q25.1,aritromicina,Azenil,Azifast,Azigram,Azimakrol,azithromycine,azithromycinum,Azitromin,AZM,Hemomycin,InChIKey=MQTOSJVFKKJCRP-BICOPXKEBK,Zithromax,Zmax,azithromycin

Group 0 name Corresponds to the control (unexposed) group for case-control studies

placebo

Group 1 name Corresponds to the case (exposed) group for case-control studies

azithromycin(AZM)

Group 1 definition Diagnostic criteria applied to define the specific condition / phenotype represented in the case (exposed) group

Participants with HIV-associated Chronic Lung Disease (HCLD) who received azithromycin (AZM)

Group 0 sample size Number of subjects in the control (unexposed) group

174

Group 1 sample size Number of subjects in the case (exposed) group

173

Lab analysis

Sequencing type

16S

16S variable region One or more hypervariable region(s) of the bacterial 16S gene

V4

Sequencing platform Manufacturer and experimental platform used for quantifying microbial abundance

Illumina

Statistical Analysis

Data transformation Data transformation applied to microbial abundance measurements prior to differential abundance testing (if any).

centered log-ratio

Statistical test

ANCOM

Significance threshold p-value or FDR threshold used for differential abundance testing (if any)

0.05

MHT correction Have statistical tests be corrected for multiple hypothesis testing (MHT)?

Yes

Confounders controlled for Confounding factors that have been accounted for by stratification or model adjustment

age, sex

Alpha Diversity

Shannon Estimator of species richness and species evenness: more weight on species richness

increased

Signature 1

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Curated date: 2023/10/11

Curator: Chinelsy

Revision editor(s): Chinelsy, Peace Sandy, ChiomaBlessing

Source: Fig.5

Description: Analysis of composition of microbiomes (ANCOM2) differential abundance testing for samples at each visit and between trial arms Taxa identified as differentially abundant between trial arms at 48 weeks (A, left panel)

Abundance in Group 1: increased abundance in azithromycin(AZM)

NCBI	Quality Control	Links
Actinomyces
Lautropia
Peptococcus
Rothia
Streptobacillus
Treponema
Veillonella
Clostridia
Lachnospiraceae

Revision editor(s): Chinelsy, Peace Sandy, ChiomaBlessing

Signature 2

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Curated date: 2023/10/11

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Revision editor(s): Chinelsy

Source: Fig.5

Description: Analysis of composition of microbiomes (ANCOM2) differential abundance testing for samples at each visit and between trial arms Taxa identified as differentially abundant between trial arms at 48 weeks (A, left panel)

Abundance in Group 1: decreased abundance in azithromycin(AZM)

NCBI	Quality Control	Links
Aggregatibacter
Oribacterium
Moraxella
Haemophilus

Revision editor(s): Chinelsy

Experiment 2

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Differences from previous experiment shown

Subjects

Group 0 name Corresponds to the control (unexposed) group for case-control studies

Participants on azithromycin at baseline (prior to treatment)

Group 1 name Corresponds to the case (exposed) group for case-control studies

Participants on azithromycin at 48 weeks of treatment

Group 0 sample size Number of subjects in the control (unexposed) group

164

Group 1 sample size Number of subjects in the case (exposed) group

154

Lab analysis

Statistical Analysis

Alpha Diversity

Shannon Estimator of species richness and species evenness: more weight on species richness

increased

Signature 1

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Mark reviewed

Your review change was submitted

Needs review

Curated date: 2023/10/12

Curator: Chinelsy

Revision editor(s): Chinelsy, Peace Sandy, Davvve

Source: Fig.5

Description: Analysis of composition of microbiomes (ANCOM2) differential abundance testing for samples at each visit and between trial arms. Taxa identified as differentially abundant between AZM arm at baseline and 48 weeks (A, middle panel).

Abundance in Group 1: increased abundance in Participants on azithromycin at 48 weeks of treatment

NCBI	Quality Control	Links
Lautropia
Peptococcus
Rothia
Streptobacillus
Treponema
Veillonella
Clostridia
Lachnospiraceae
Candidatus Absconditabacteria

Revision editor(s): Chinelsy, Peace Sandy, Davvve

Signature 2

EditHistoryDelete

Mark reviewed

Your review change was submitted

Needs review

Curated date: 2023/10/12

Curator: Chinelsy

Revision editor(s): Chinelsy

Source: Fig.5

Description: Analysis of composition of microbiomes (ANCOM2) differential abundance testing for samples at each visit and between trial arms. Taxa identified as differentially abundant between AZM arm at baseline and 48 weeks (A, middle panel).

Abundance in Group 1: decreased abundance in Participants on azithromycin at 48 weeks of treatment

NCBI	Quality Control	Links
Aggregatibacter
Haemophilus
Moraxella
Oribacterium
F0058 (Paludibacteria)F0058 (Paludibacteria)

Revision editor(s): Chinelsy

Experiment 3

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Differences from previous experiment shown

Subjects

Group 1 name Corresponds to the case (exposed) group for case-control studies

Participants on azithromycin at 72 weeks (24 weeks post treatment)

Group 1 sample size Number of subjects in the case (exposed) group

123

Lab analysis

Statistical Analysis

Alpha Diversity

Shannon Estimator of species richness and species evenness: more weight on species richness

increased

Signature 1

EditHistoryDelete

Mark reviewed

Your review change was submitted

Needs review

Curated date: 2023/10/12

Curator: Chinelsy

Revision editor(s): Chinelsy, Peace Sandy

Source: Fig.5

Description: Analysis of composition of microbiomes (ANCOM2) differential abundance testing for samples at each visit and between trial arms. Taxa identified as differentially abundant between AZM arm at baseline and 72 weeks (A, right panel).

Abundance in Group 1: increased abundance in Participants on azithromycin at 72 weeks (24 weeks post treatment)

NCBI	Quality Control	Links
Lautropia
Oribacterium
Peptococcus
Rothia
Treponema
(F0058 (Paludibacteria)(F0058 (Paludibacteria)
ASV_209 (Clostridia)ASV_209 (Clostridia)
ASV_157 (Absconditabacteria)ASV_157 (Absconditabacteria)

Revision editor(s): Chinelsy, Peace Sandy

Signature 2

EditHistoryDelete

Mark reviewed

Your review change was submitted

Needs review

Curated date: 2023/10/12

Curator: Chinelsy

Revision editor(s): Chinelsy, Davvve

Source: Fig.5

Description: Analysis of composition of microbiomes (ANCOM2) differential abundance testing for samples at each visit and between trial arms. Taxa identified as differentially abundant between AZM arm at baseline and 72 weeks (A, right panel).

Abundance in Group 1: decreased abundance in Participants on azithromycin at 72 weeks (24 weeks post treatment)

NCBI	Quality Control	Links
Aggregatibacter
Haemophilus
Moraxella
Streptobacillus
Veillonella
Clostridia
Lachnospiraceae

Revision editor(s): Chinelsy, Davvve