Project Objectives
• To identify microbial pathogens in HIV-infected Ugandan children and adolescents admitted to Mulago National Referral Hospital, Baylor-Uganda clinic with NMFIs
• To characterize the clinical presentations and determine associations between specific clinical syndromes with both known and novel pathogens in clinical specimens of HIV-infected Ugandan children and adolescents with NMFIs
• To identify and describe the frequency of comorbidities in children and adolescents with perinatally acquired HIV-infection with NMFIs in Uganda in the era of antiretroviral therapy
Project Summary
Rapid diagnostic tests (RDTs) for malaria have underscored the declining proportion of malaria-attributable illnesses in endemic areas. However, once malaria is excluded, accessible diagnostic tools for managing severe febrile illnesses in low-resource settings remain limited. Current RDTs for non-malarial tropical infections typically detect host antibodies against a single infectious agent, but their sensitivities and specificities are inherently constrained. Notably, there is a lack of comprehensive data on the causes of non-malarial febrile illnesses (NMFIs) in HIV-infected children in Uganda, and minimal guidance exists on managing these cases effectively. This highlights the need to better characterize alternative causes of fever in African children to improve diagnostic and therapeutic algorithms.
Given these limitations, there is an urgent need for more sensitive, genomic-based approaches such as shotgun metagenomics sequencing (sMNGS). Incorporating whole-genome-based technologies, including long-read sequencing, into the study of tropical fevers is critical for enhancing the diagnosis and management of severe infections, particularly among vulnerable groups such as HIV-infected children and adolescents with NMFIs. The Pathogen Detection in HIV-Infected Children with Non-Malarial Febrile Illnesses Using Metagenomic Sequencing (PHICAMS) project aims to address these gaps by utilizing sMNGS to identify microbial pathogens in stool samples from HIV-infected Ugandan children and adolescents admitted to Baylor College of Medicine Children's Foundation - Uganda, focusing on NMFIs and associated clinical presentations or comorbidities.
This cross-sectional study enrolled a total of 144 HIV-infected children with NMFI, comprising 73 males and 71 females, with mean ages of 7.1 and 6.9 years, respectively. Initially, 147 stool samples were collected, with one excluded due to poor collection, resulting in 146 samples for DNA extraction. After quality control measures, 144 samples were included in the final analysis. Stool samples were collected and analysed using shotgun metagenomic sequencing at the Chan Zuckerberg Biohub San Francisco to explore the stool microbial landscape and improve diagnostic accuracy for NMFI.
The study identified a diverse microbial composition, including potential pathogens such as Trichomonas vaginalis, Candida albicans, Giardia intestinalis, and Bacteroides spp. Variations in the distribution of these pathogens were observed based on gender and age, with specific microbes associated uniquely with males, females, or certain age groups. The pathogens were transmitted through various routes, including zoonotic, foodborne, and sexually transmitted infections. Although unique pathogen profiles were linked to different antiretroviral regimens, no statistically significant associations were found. Furthermore, the analysis of immune cell responses revealed correlations between pathogen dynamics and variations in white blood cell counts.
The findings of this research, published in BMC Infectious Diseases, demonstrate that shotgun metagenomics has proven to be a powerful tool for identifying pathogens in NMFI cases among HIV-infected children, offering a comprehensive view of microbial interactions. This approach could complement traditional diagnostics to improve early detection and targeted treatment, particularly in resource-limited settings.
Results & Outcomes
• Comprehensive pathogen profiling: We successfully identified a wide range of microbial pathogens, including both well-known and previously unrecognized organisms, contributing to the understanding of NMFI in HIV-infected children.
• Methodological innovation: Demonstrated the efficacy of shotgun metagenomic sequencing in clinical diagnostics within resource-limited settings, paving the way for its broader adoption. However, the metagenomics sequencing platforms are still unavailable in our settings due to high costs.
• Data sharing and collaboration: We shared the genomic sequence data on NCBI under the BioProject ID: PRJNA1137832, a well-established repository and collaborative platform for data and tool sharing. This ensures accessibility and enhances the potential for ongoing research and validation studies.
• Capacity building: For capacity building, the project trained two trainees and me. The trainees completed a two-year MSc in Bioinformatics at Makerere University and have since enrolled in PhD programs in Bioinformatics. They actively apply their expertise professionally, with one securing a full PhD scholarship. This Career Development Fellowship (CDF) also facilitated expanding our research networks, including collaboration with the Chan Zuckerberg Biohub in San Francisco, California.
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