Detection of Microbial Translocation in HIV and SIV Infection Using the Limulus Amebocyte Lysate Assay is Masked by Serum and Plasma

Abstract

ObjectiveMicrobial translocation (MT) is thought to be a major contributor to the pathogenesis of HIV-related immune activation, and circulating lipopolysaccharide (LPS) from Gram-negative bacteria is the principle measurement of this process. However, related research has been impeded by inconsistent LPS test results.MethodsSpecimens were obtained from HIV-infected adults enrolled in the PEARLS study (ACTG A5175) and HIV-HCV co-infected participants enrolled in a study of liver disease staging using MRI elastography. Pig-tailed macaque specimens were obtained from SIV-infected and –uninfected animals. Samples were tested for LPS using the LAL assay with diazo-coupling modifications to improve sensitive detection.ResultsWhen exogenous LPS was added to macaque plasma, >25% inhibition of LPS detection was found in 10/10 (100%) samples at 20% plasma concentration compared to control; in contrast 5/10 (50%) samples at 2% plasma concentration (p = 0.07) and 0/10 (0%) at 0.1% plasma concentration (p = 0.004) showed >25% inhibition of LPS detection. Similarly, when LPS was added to human serum, >25% inhibition of LPS detection was found in 5/12 (42%) of samples at 2% serum concentration compared to control, while 0/12 (0%) of samples in 0.1% serum showed >25% inhibition of LPS detection (p = 0.07). Likewise, LPS detection in human sera without exogenous LPS was improved by dilution: LPS was detected in 2/12 (17%) human samples in 2% serum, ranging from 3,436–4,736 pg/mL, compared to 9/12 (75%) samples in 0.1% serum, ranging from 123 pg/mL –60,131 pg/mL (p = 0.016). In a separate validation cohort of HIV-HCV co-infected participants sampled at two different times on the same day, LPS measured in 0.2% plasma and with diazo-coupling was closely correlated between the first and second samples (R = 0.66, p<0.05).ConclusionsUndiluted serum and plasma mask LPS detection. The extent of MT may be substantially underestimated.

Quantitative lipopolysaccharide analysis using HPLC/MS/MS and its combination with the limulus amebocyte lysate assay

“…Most importantly, quantitative analysis by HPLC/MS/MS revealed that maximal plasma levels of LPS were actually measured after 6 h, thus indicating that 1 ) the bulk of circulating LPS was still present in the plasma compartment at this time, and 2 ) in accordance with earlier reports ( 8,25 ), the LAL assay can produce false negative results in native plasma. These fi ndings are in agreement with earlier studies, which reported that in vitro incubation of plasma can mask LPS detection in a concentration-dependent manner (26)(27)(28). The combination of the LAL and HPLC/ MS/MS analyses in the present study provided new evidence of the intrinsic capacity of plasma to neutralize the activity of LPS.…”

Soluble T Cell Immunoglobulin Mucin Domain 3 Is Shed from CD8 + T Cells by the Sheddase ADAM10, Is Increased in Plasma during Untreated HIV Infection, and Correlates with HIV Disease Progression

“…However, available assays to measure plasma LPS yielded inconsistent and variable results (data not shown), as previously reported (43). Thus, we measured plasma sCD14, a surrogate marker for plasma LPS (42).…”

Pre-cART Elevation of CRP and CD4+ T-Cell Immune Activation Associated With HIV Clinical Progression in a Multinational Case–Cohort Study

“…LPS, sCD14, and EndoCAb IgM, were measured in one laboratory at Johns Hopkins University. LPS was quantified using an optimized Limulus Amebocyte Lysate (LAL) assay (Lonza, Walkersville, MD) 27 . Commercially available ELISA kits were used for measurement of sCD14 (R&D Systems, Inc., Minneapolis, MN), EndoCAb IgM (Cell Sciences, Canton, MA), and CRP (CRP Quantikine ELISA, R&D Systems, Minneapolis, MN).…”