Chondroitin sulfate proteoglycan 4 (CSPG4) increases invasion of recessive dystrophic epidermolysis bullosa-associated cutaneous squamous cell carcinoma by modifying TGFß signaling.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare genetic skin-blistering disorder often progressing to metastatic cutaneous squamous cell carcinoma (cSCC) at chronic wound sites. Chondroitin sulfate proteoglycan 4 (CSPG4) is a cell-surface proteoglycan that is an oncoantigen in multiple malignancies, where it modulates oncogenic signaling, drives epithelial-to-mesenchymal transition (EMT), and enables cell motility. OBJECTIVES: To evaluate CSPG4 expression and function in RDEB-cSCC. METHODS: RDEB-cSCC cell lines were used to assess CSPG4-dependent changes in invasive potential, TGFß1-stimulated signal activation, and clinically relevant cytopathology metrics in an in vitro full-thickness tumor model. CSPG4 expression in RDEB-cSCC and non-RDEB cSCC tumors was analyzed via immunohistochemistry and single-cell RNA sequencing (scRNA-seq), respectively. RESULTS: Inhibiting CSPG4 expression reduced invasive potential in multiple RDEB-cSCC cell lines and altered membrane-proximal TGFß signal activation through changes in SMAD3 phosphorylation. CSPG4 expression was uniformly localized to basal-layer keratinocytes in fibrotic RDEB skin and tumor cells at the tumor/stroma interface at the invasive front in RDEB-cSCC tumors in vivo. Analysis of published scRNA-seq data revealed that CSPG4 expression was correlated with an enhanced EMT transcriptomic signature in cells at the tumor/stroma interface of non-RDEB cSCC tumors. Cytopathological metrics, like nucleus:cell area ratio, were influenced by CSPG4 expression in in vitro tumor models. CONCLUSIONS: We determined that CSPG4 expression in RDEB-cSCC cell lines enhanced invasive potential. Mechanistically, CSPG4 was found to enhance membrane-proximal TGFß-stimulated signaling through SMAD3, which is a key mediator of EMT in RDEB-cSCC. The implication of these studies is that CSPG4 may represent a therapeutic target that can be leveraged for clinical management in patients with RDEB-cSCC.

Human cytomegalovirus in breast milk is associated with milk composition and the infant gut microbiome and growth.

Human cytomegalovirus (CMV) is a highly prevalent herpesvirus that is often transmitted to the neonate via breast milk. Postnatal CMV transmission can have negative health consequences for preterm and immunocompromised infants, but any effects on healthy term infants are thought to be benign. Furthermore, the impact of CMV on the composition of the hundreds of bioactive factors in human milk has not been tested. Here, we utilize a cohort of exclusively breastfeeding full-term mother-infant pairs to test for differences in the milk transcriptome and metabolome associated with CMV, and the impact of CMV in breast milk on the infant gut microbiome and infant growth. We find upregulation of the indoleamine 2,3-dioxygenase (IDO) tryptophan-to-kynurenine metabolic pathway in CMV+ milk samples, and that CMV+ milk is associated with decreased Bifidobacterium in the infant gut. Our data indicate two opposing CMV-associated effects on infant growth; with kynurenine positively correlated, and CMV viral load negatively correlated, with infant weight-for-length at 1 month of age. These results suggest CMV transmission, CMV-related changes in milk composition, or both may be modulators of full-term infant development.

Assembly, stability, and dynamics of the infant gut microbiome are linked to bacterial strains and functions in mother's milk.

The establishment of the gut microbiome in early life is critical for healthy infant development. Although human milk is recommended as the sole source of nutrition for the human infant, little is known about how variation in milk composition, and especially the milk microbiome, shapes the microbial communities in the infant gut. Here, we quantified the similarity between the maternal milk and the infant gut microbiome using 507 metagenomic samples collected from 195 mother-infant pairs at one, three, and six months postpartum. We found that the microbial taxonomic overlap between milk and the infant gut was driven by bifidobacteria, in particular by B. longum. Infant stool samples dominated by B. longum also showed higher temporal stability compared to samples dominated by other species. We identified two instances of strain sharing between maternal milk and the infant gut, one involving a commensal (B. longum) and one a pathobiont (K. pneumoniae). In addition, strain sharing between unrelated infants was higher among infants born at the same hospital compared to infants born in different hospitals, suggesting a potential role of the hospital environment in shaping the infant gut microbiome composition. The infant gut microbiome at one month compared to six months of age was enriched in metabolic pathways associated with de-novo molecule biosynthesis, suggesting that early colonisers might be more versatile and metabolically independent compared to later colonizers. Lastly, we found a significant overlap in antimicrobial resistance genes carriage between the mother's milk and their infant's gut microbiome. Taken together, our results suggest that the human milk microbiome has an important role in the assembly, composition, and stability of the infant gut microbiome.

Trans-eQTL hotspots shape complex traits by modulating cellular states.

Regulatory genetic variation shapes gene expression, providing an important mechanism connecting DNA variation and complex traits. The causal relationships between gene expression and complex traits remain poorly understood. Here, we integrated transcriptomes and 46 genetically complex growth traits in a large cross between two strains of the yeast Saccharomyces cerevisiae. We discovered thousands of genetic correlations between gene expression and growth, suggesting functional connections. Local regulatory variation was a minor source of these genetic correlations. Instead, genetic correlations tended to arise from multiple independent trans-acting regulatory loci. Trans-acting hotspots that affect the expression of numerous genes accounted for particularly large fractions of genetic growth variation and of genetic correlations between gene expression and growth. Genes with genetic correlations were enriched for similar biological processes across traits, but with heterogeneous direction of effect. Our results reveal how trans-acting regulatory hotspots shape complex traits by altering cellular states.

Integration of 168,000 samples reveals global patterns of the human gut microbiome.

Understanding the factors that shape variation in the human microbiome is a major goal of research in biology. While other genomics fields have used large, pre-compiled compendia to extract systematic insights requiring otherwise impractical sample sizes, there has been no comparable resource for the 16S rRNA sequencing data commonly used to quantify microbiome composition. To help close this gap, we have assembled a set of 168,484 publicly available human gut microbiome samples, processed with a single pipeline and combined into the largest unified microbiome dataset to date. We use this resource, which is freely available at microbiomap.org, to shed light on global variation in the human gut microbiome. We find that Firmicutes, particularly Bacilli and Clostridia, are almost universally present in the human gut. At the same time, the relative abundance of the 65 most common microbial genera differ between at least two world regions. We also show that gut microbiomes in undersampled world regions, such as Central and Southern Asia, differ significantly from the more thoroughly characterized microbiomes of Europe and Northern America. Moreover, humans in these overlooked regions likely harbor hundreds of taxa that have not yet been discovered due to this undersampling, highlighting the need for diversity in microbiome studies. We anticipate that this new compendium can serve the community and enable advanced applied and methodological research.

Embracing the Science of Motherhood: Pregnancy's Transformative Effects on the Central Nervous System and the Radiance of Maternal Hormones and Immune Responses.

Pregnancy is often thought of as a time of happiness and anticipation, however, for some women, it can bring about significant emotional distress and feelings of vulnerability. The physiological changes that occur during pregnancy, including hormonal fluctuations and alterations to the immune and physical systems, can affect various parts of the body, including the central nervous system (CNS). As a result, existing conditions may be intensified or new ones, such as neurologic or psychiatric disorders, may arise, exposing women to increased risk of life-threatening conditions or suicide, in the worst-case scenarios. Given the impact of pregnancy on CNS diseases, it is crucial for healthcare providers and patients alike to be aware of these potential effects. By understanding how pregnancy may affect the CNS, clinicians can take appropriate steps to ensure that women receive the care and support they need to minimize any negative outcomes for both the mother and the baby. This paper aims to review the available evidence on the impact of pregnancy on CNS diseases, including mental health conditions, from both the clinical and biomolecular perspectives. By illuminating this crucial subject, this study fosters a delicate understanding within both patients and healthcare providers, thereby paving the way for enhanced outcomes for women throughout their pregnancy journey and beyond.

Human Cytomegalovirus in breast milk is associated with milk composition, the infant gut microbiome, and infant growth.

Human cytomegalovirus (CMV) is a highly prevalent herpesvirus that is often transmitted to the neonate via breast milk. Postnatal CMV transmission can have negative health consequences for preterm and immunocompromised infants, but any effects on healthy term infants are thought to be benign. Furthermore, the impact of CMV on the composition of the hundreds of bioactive factors in human milk has not been tested. Here, we utilize a cohort of exclusively breastfeeding full term mother-infant pairs to test for differences in the milk transcriptome and metabolome associated with CMV, and the impact of CMV in breast milk on the infant gut microbiome and infant growth. We find upregulation of the indoleamine 2,3- dioxygenase (IDO) tryptophan-to-kynurenine metabolic pathway in CMV+ milk samples, and that CMV+ milk is associated with decreased Bifidobacterium in the infant gut. Our data indicate a complex relationship between milk CMV, milk kynurenine, and infant growth; with kynurenine positively correlated, and CMV viral load negatively correlated, with infant weight-for-length at 1 month of age. These results suggest CMV transmission, CMV-related changes in milk composition, or both may be modulators of full term infant development.

Substrate-specific effects of natural genetic variation on proteasome activity.

Protein degradation is an essential biological process that regulates protein abundance and removes misfolded and damaged proteins from cells. In eukaryotes, most protein degradation occurs through the stepwise actions of two functionally distinct entities, the ubiquitin system and the proteasome. Ubiquitin system enzymes attach ubiquitin to cellular proteins, targeting them for degradation. The proteasome then selectively binds and degrades ubiquitinated substrate proteins. Genetic variation in ubiquitin system genes creates heritable differences in the degradation of their substrates. However, the challenges of measuring the degradative activity of the proteasome independently of the ubiquitin system in large samples have limited our understanding of genetic influences on the proteasome. Here, using the yeast Saccharomyces cerevisiae, we built and characterized reporters that provide high-throughput, ubiquitin system-independent measurements of proteasome activity. Using single-cell measurements of proteasome activity from millions of genetically diverse yeast cells, we mapped 15 loci across the genome that influence proteasomal protein degradation. Twelve of these 15 loci exerted specific effects on the degradation of two distinct proteasome substrates, revealing a high degree of substrate-specificity in the genetics of proteasome activity. Using CRISPR-Cas9-based allelic engineering, we resolved a locus to a causal variant in the promoter of RPT6, a gene that encodes a subunit of the proteasome's 19S regulatory particle. The variant increases RPT6 expression, which we show results in increased proteasome activity. Our results reveal the complex genetic architecture of proteasome activity and suggest that genetic influences on the proteasome may be an important source of variation in the many cellular and organismal traits shaped by protein degradation.

Human milk variation is shaped by maternal genetics and impacts the infant gut microbiome.

Human milk is a complex mix of nutritional and bioactive components that provide complete nutrition for the infant. However, we lack a systematic knowledge of the factors shaping milk composition and how milk variation influences infant health. Here, we used multi-omic profiling to characterize interactions between maternal genetics, milk gene expression, milk composition, and the infant fecal microbiome in 242 exclusively breastfeeding mother-infant pairs. We identified 487 genetic loci associated with milk gene expression unique to the lactating mammary gland, including loci that impacted breast cancer risk and human milk oligosaccharide concentration. Integrative analyses uncovered connections between milk gene expression and infant gut microbiome, including an association between the expression of inflammation-related genes with IL-6 concentration in milk and the abundance of Bifidobacteria in the infant gut. Our results show how an improved understanding of the genetics and genomics of human milk connects lactation biology with maternal and infant health.

Targeting the Complement-Sphingolipid System in COVID-19 and Gaucher Diseases: Evidence for a New Treatment Strategy.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2)-induced disease (COVID-19) and Gaucher disease (GD) exhibit upregulation of complement 5a (C5a) and its C5aR1 receptor, and excess synthesis of glycosphingolipids that lead to increased infiltration and activation of innate and adaptive immune cells, resulting in massive generation of pro-inflammatory cytokines, chemokines and growth factors. This C5a-C5aR1-glycosphingolipid pathway- induced pro-inflammatory environment causes the tissue damage in COVID-19 and GD. Strikingly, pharmaceutically targeting the C5a-C5aR1 axis or the glycosphingolipid synthesis pathway led to a reduction in glycosphingolipid synthesis and innate and adaptive immune inflammation, and protection from the tissue destruction in both COVID-19 and GD. These results reveal a common involvement of the complement and glycosphingolipid systems driving immune inflammation and tissue damage in COVID-19 and GD, respectively. It is therefore expected that combined targeting of the complement and sphingolipid pathways could ameliorate the tissue destruction, organ failure, and death in patients at high-risk of developing severe cases of COVID-19.

Variation in ubiquitin system genes creates substrate-specific effects on proteasomal protein degradation.

Precise control of protein degradation is critical for life, yet how natural genetic variation affects this essential process is largely unknown. Here, we developed a statistically powerful mapping approach to characterize how genetic variation affects protein degradation by the ubiquitin-proteasome system (UPS). Using the yeast Saccharomyces cerevisiae, we systematically mapped genetic influences on the N-end rule, a UPS pathway in which protein N-terminal amino acids function as degradation-promoting signals. Across all 20 possible N-terminal amino acids, we identified 149 genomic loci that influence UPS activity, many of which had pathway- or substrate-specific effects. Fine-mapping of four loci identified multiple causal variants in each of four ubiquitin system genes whose products process (NTA1), recognize (UBR1 and DOA10), and ubiquitinate (UBC6) cellular proteins. A cis-acting promoter variant that modulates UPS activity by altering UBR1 expression alters the abundance of 36 proteins without affecting levels of the corresponding mRNA transcripts. Our results reveal a complex genetic basis of variation in UPS activity.

Genes and compounds that increase type VII collagen expression as potential treatments for dystrophic epidermolysis bullosa.

Dystrophic epidermolysis bullosa (DEB) is a skin-blistering disease caused by mutations in COL7A1, which encodes type VII collagen (C7). There is no cure for DEB, but previous work has shown potential therapeutic benefit of increased production of even partially functional C7. Genome-wide screens using CRISPR-Cas9 have enabled the identification of genes involved in cancer development, drug resistance and other genetic diseases, suggesting that they could be used to identify drivers of C7 production. A keratinocyte C7 reporter cell line was created and used in a genome-wide CRISPR activation (CRISPRa) screen to identify genes and pathways that increase C7 expression. The CRISPRa screen results were used to develop a targeted drug screen to identify compounds that upregulate C7 expression. The C7_tdTomato cell line was validated as an effective reporter for detection of C7 upregulation. The CRISPRa screen identified DENND4B and TYROBP as top gene hits plus pathways related to calcium uptake and immune signalling in C7 regulation. The targeted drug screen identified several compounds that increase C7 expression in keratinocytes, of which kaempferol, a plant flavonoid, also significantly increased C7 mRNA and protein in DEB patient cells.

Multiple epistatic DNA variants in a single gene affect gene expression in trans.

DNA variants that alter gene expression in trans are important sources of phenotypic variation. Nevertheless, the identity of trans-acting variants remains poorly understood. Single causal variants in several genes have been reported to affect the expression of numerous distant genes in trans. Whether these simple molecular architectures are representative of trans-acting variation is unknown. Here, we studied the large RAS signaling regulator gene IRA2, which contains variants with extensive trans-acting effects on gene expression in the yeast Saccharomyces cerevisiae. We used systematic CRISPR-based genome engineering and a sensitive phenotyping strategy to dissect causal variants to the nucleotide level. In contrast to the simple molecular architectures known so far, IRA2 contained at least seven causal nonsynonymous variants. The effects of these variants were modulated by nonadditive, epistatic interactions. Two variants at the 5'-end affected gene expression and growth only when combined with a third variant that also had no effect in isolation. Our findings indicate that the molecular basis of trans-acting genetic variation may be considerably more complex than previously appreciated.

C-X-C Motif Chemokine Ligand 9 and Its CXCR3 Receptor Are the Salt and Pepper for T Cells Trafficking in a Mouse Model of Gaucher Disease.

Gaucher disease is a lysosomal storage disease, which happens due to mutations in GBA1/Gba1 that encodes the enzyme termed as lysosomal acid ß-glucosidase. The major function of this enzyme is to catalyze glucosylceramide (GC) into glucose and ceramide. The deficiency of this enzyme and resultant abnormal accumulation of GC cause altered function of several of the innate and adaptive immune cells. For example, augmented infiltration of T cells contributes to the increased production of pro-inflammatory cytokines, (e.g., IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F). This leads to tissue damage in a genetic mouse model (Gba19V/-) of Gaucher disease. The cellular mechanism(s) by which increased tissue infiltration of T cells occurs in this disease is not fully understood. Here, we delineate role of the CXCR3 receptor and its exogenous C-X-C motif chemokine ligand 9 (CXCL9) in induction of increased tissue recruitment of CD4+ T and CD8+ T cells in Gaucher disease. Intracellular FACS staining of macrophages (Mϕs) and dendritic cells (DCs) from Gba19V/- mice showed elevated production of CXCL9. Purified CD4+ T cells and the CD8+ T cells from Gba19V/- mice showed increased expression of CXCR3. Ex vivo and in vivo chemotaxis experiments showed CXCL9 involvement in the recruitment of Gba19V/- T cells. Furthermore, antibody blockade of the CXCL9 receptor (CXCR3) on T cells caused marked reduction in CXCL9- mediated chemotaxis of T cells in Gba19V/- mice. These data implicate abnormalities of the CXCL9-CXCR3 axis leading to enhanced tissue recruitment of T cells in Gaucher disease. Such results provide a rationale for blockade of the CXCL9/CXCR3 axis as potential new therapeutic targets for the treatment of inflammation in Gaucher disease.

Genetic Defects and Pro-inflammatory Cytokines in Parkinson's Disease.

Parkinson's disease (PD) is a movement disorder attributed to the loss of dopaminergic (DA) neurons mainly in the substantia nigra pars compacta. Motor symptoms include resting tremor, rigidity, and bradykinesias, while non-motor symptoms include autonomic dysfunction, anxiety, and sleeping problems. Genetic mutations in a number of genes (e.g., LRRK2, GBA, SNCA, PARK2, PARK6, and PARK7) and the resultant abnormal activation of microglial cells are assumed to be the main reasons for the loss of DA neurons in PD with genetic causes. Additionally, immune cell infiltration and their participation in major histocompatibility complex I (MHCI) and/or MHCII-mediated processing and presentation of cytosolic or mitochondrial antigens activate the microglial cells and cause the massive generation of pro-inflammatory cytokines and chemokines, which are all critical for the propagation of brain inflammation and the neurodegeneration in PD with genetic and idiopathic causes. Despite knowing the involvement of several of such immune devices that trigger neuroinflammation and neurodegeneration in PD, the exact disease mechanism or the innovative biomarker that could detect disease severity in PD linked to LRRK2, GBA, SNCA, PARK2, PARK6, and PARK7 defects is largely unknown. The current review has explored data from genetics, immunology, and in vivo and ex vivo functional studies that demonstrate that certain genetic defects might contribute to microglial cell activation and massive generation of a number of pro-inflammatory cytokines and chemokines, which ultimately drive the brain inflammation and lead to neurodegeneration in PD. Understanding the detailed involvement of a variety of immune mediators, their source, and the target could provide a better understanding of the disease process. This information might be helpful in clinical diagnosis, monitoring of disease progression, and early identification of affected individuals.

Trans-acting genetic variation affects the expression of adjacent genes.

Gene expression differences among individuals are shaped by trans-acting expression quantitative trait loci (eQTLs). Most trans-eQTLs map to hotspot locations that influence many genes. The molecular mechanisms perturbed by hotspots are often assumed to involve "vertical" cascades of effects in pathways that can ultimately affect the expression of thousands of genes. Here, we report that trans-eQTLs can affect the expression of adjacent genes via "horizontal" mechanisms that extend along a chromosome. Genes affected by trans-eQTL hotspots in the yeast Saccharomyces cerevisiae were more likely to be located next to each other than expected by chance. These paired hotspot effects tended to occur at adjacent genes that also show coexpression in response to genetic and environmental perturbations, suggesting shared mechanisms. Physical proximity and shared chromatin state, in addition to regulation of adjacent genes by similar transcription factors, were independently associated with paired hotspot effects among adjacent genes. Paired effects of trans-eQTLs can occur at neighboring genes even when these genes do not share a common function. This phenomenon could result in unexpected connections between regulatory genetic variation and phenotypes.

Simultaneous quantification of mRNA and protein in single cells reveals post-transcriptional effects of genetic variation.

Trans-acting DNA variants may specifically affect mRNA or protein levels of genes located throughout the genome. However, prior work compared trans-acting loci mapped in separate studies, many of which had limited statistical power. Here, we developed a CRISPR-based system for simultaneous quantification of mRNA and protein of a given gene via dual fluorescent reporters in single, live cells of the yeast Saccharomyces cerevisiae. In large populations of recombinant cells from a cross between two genetically divergent strains, we mapped 86 trans-acting loci affecting the expression of ten genes. Less than 20% of these loci had concordant effects on mRNA and protein of the same gene. Most loci influenced protein but not mRNA of a given gene. One locus harbored a premature stop variant in the YAK1 kinase gene that had specific effects on protein or mRNA of dozens of genes. These results demonstrate complex, post-transcriptional genetic effects on gene expression.

Systematic identification of cis-regulatory variants that cause gene expression differences in a yeast cross.

Sequence variation in regulatory DNA alters gene expression and shapes genetically complex traits. However, the identification of individual, causal regulatory variants is challenging. Here, we used a massively parallel reporter assay to measure the cis-regulatory consequences of 5832 natural DNA variants in the promoters of 2503 genes in the yeast Saccharomyces cerevisiae. We identified 451 causal variants, which underlie genetic loci known to affect gene expression. Several promoters harbored multiple causal variants. In five promoters, pairs of variants showed non-additive, epistatic interactions. Causal variants were enriched at conserved nucleotides, tended to have low derived allele frequency, and were depleted from promoters of essential genes, which is consistent with the action of negative selection. Causal variants were also enriched for alterations in transcription factor binding sites. Models integrating these features provided modest, but statistically significant, ability to predict causal variants. This work revealed a complex molecular basis for cis-acting regulatory variation.

Challenges and barriers to HIV service uptake and delivery along the HIV care cascade in Cameroon.

INTRODUCTION: The year 2017 marked a transition period with the end of the implementation of Cameroon´s 2014-2017 HIV/AIDS National Strategic Plan (NSP) and the development of the 2018-2022 NSP. We assessed barriers and challenges to service delivery and uptake along the HIV care cascade in Cameroon to inform decision making within the framework of the new NSP, to achieve the UNAIDS 90-90-90 target. METHODS: We conducted a cross sectional descriptive study nationwide, enrolling HIV infected patients and staff. Data were collected on sociodemographic characteristics, HIV testing, antiretroviral therapy and viral load testing delivery and uptake and factors that limit their access. RESULTS: A total of 137 staff and 642 people living with HIV (PLHIV) were interviewed. Of 642 PLHIV with known status, 339 (53%) repeated their HIV test at least once, with range: 1-10 and median: 2 (IQR: 1-3). Having attained secondary level of education (OR: 2.07, 95% CI: 1.04-4.14; P=0.04) or more (OR: 2.91, 95% CI: 1.16-7.28; P=0.02) were significantly associated with repeat testing. Psychological (refusal of service uptake and existence of HIV), community-level (stigmatization and fear of confidentiality breach) and commodity stock-outs "HIV test kits (21%), antiretrovirals (ARVs) (71.4%), viral load testing reagents (100%)" are the major barriers to service delivery and uptake along the cascade. CONCLUSION: We identified individual, community-level, socio-economic and health care system related barriers which constitute persistent bottlenecks in HIV service delivery and uptake and a high rate of repeat testing by PLHIV with known status. Addressing all these accordingly can help the country achieve the UNAIDS 90-90-90 target.

On estimating the number of people with known HIV positive status.

OBJECTIVE: In 2014, the Joint United Nations Program on HIV and AIDS (UNAIDS) and partners set the '90-90-90 targets'. Many countries are facing the challenge of estimating the first 90. Our objective was to propose an alternative modelling procedure, and to discuss its usefulness for taking into account duplication. RESULTS: For deduplication, we identified two important ingredients: the probability for an HIV+ person of being re-tested during the period and average number of HIV+ tests. Other adjusted factors included: the false positive probability; the death and emigration probabilities. The uncertainty of the adjusted estimate was assessed using the plausibility bounds and sensitivity analysis. The proposed method was applied to Cameroon for the period 1987-2016. Of the 560,000 people living with HIV estimated from UNAIDS in 2016; 504,000 out to know their status. The model estimates that 380,464 [379,257, 381,674] know their status (75.5%); thus 179,536 who do not know their status should be sought through the intensification of testing. These results were subsequently used for constructing the full 2016 Cameroon HIV cascade for identifying programmatic gap, prioritizing the resources, and guiding the strategies of the 2018-2022 National Strategy Plan and funding request.