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

OBJECTIVE Microbial 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. METHODS Specimens 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. RESULTS When 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). CONCLUSIONS Undiluted serum and plasma mask LPS detection. The extent of MT may be substantially underestimated.