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PLoS One. 2017 Apr 17;12(4):e0175899. doi: 10.1371/journal.pone.0175899. eCollection 2017.

Sensitive quantification of the HIV-1 reservoir in gut-associated lymphoid tissue.

Author information

AIDS Research Institute IrsiCaixa, Institut d'Investigació en Cièncias de la Salut Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain.
Infectious Diseases Department, Hospital Universitari Vall d'Hebron, Barcelona, Spain.
Department of Gastroenterology, Hospital Universitari Mutua de Terrassa, University of Barcelona, Research Foundation Mutua de Terrassa, Terrassa, Spain.
Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas [CIBERehd], Barcelona, Spain.
CIBEREHD (Centro de Investigación Biomédica en Red, Enfermedades hepáticas y digestivas), IBD Unit, Germans Trias i Pujol research Institute, Badalona, Spain.
Universitat de Vic-Universitat Central de Catalunya, Vic, Spain.
Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain.



The implementation of successful strategies to achieve an HIV cure has become a priority in HIV research. However, the current location and size of HIV reservoirs is still unknown since there are limited tools to evaluate HIV latency in viral sanctuaries such as gut-associated lymphoid tissue (GALT). As reported in the so called "Boston Patients", despite undetectable levels of proviral HIV-1 DNA in blood and GALT, viral rebound happens in just few months after ART interruption. This fact might imply that current methods are not sensitive enough to detect residual reservoirs. Showing that, it is imperative to improve the detection and quantification of HIV-1 reservoir in tissue samples. Herein, we propose a novel non-enzymatic protocol for purification of Lamina Propria Leukocytes (LPL) from gut biopsies combined to viral HIV DNA (vDNA) quantification by droplet digital PCR (ddPCR) to improve the sensitivity and accuracy of viral reservoir measurements (LPL-vDNA assay).


Endoscopic ileum biopsies were sampled from 12 HIV-1-infected cART-suppressed subjects. We performed a DTT/EDTA-based treatment for epithelial layer removal followed by non-enzymatic disruption of the tissue to obtain lamina propria cell suspension (LP). CD45+ cells were subsequently purified by flow sorting and vDNA was determined by ddPCR.


vDNA quantification levels were significantly higher in purified LPLs (CD45+) than in bulk LPs (p<0.01). The levels of vDNA were higher in ileum samples than in concurrent PBMC from the same individuals (p = 0.002). As a result of the increased sensitivity of this purification method, the Poisson 95% confidence intervals of the vDNA quantification data from LPLs were narrower than that from bulk LPs. Of note, vDNA was unambiguously quantified above the detection limit in 100% of LPL samples, while only in 58% of bulk LPs.


We propose an innovative combined protocol for a more sensitive detection of the HIV reservoir in gut-associated viral sanctuaries, which might be used to evaluate any proposed eradication strategy.

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