Detection of microbial translocation in HIV and SIV infection using the Limulus amebocyte lysate ass Limulus amebocyte lysate assay is masked by serum and plasma
Balagopal A, Gama L, Franco V, Russell JN, Quinn J, Higgins Y, Smeaton LM, Clements JE, Thomas DL, Gupta A; NWCS 319 and ACTG 5175 Study Team
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.
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.
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).
Undiluted serum and plasma mask LPS detection. The extent of MT may be substantially underestimated.
PLoS One. 2012;7(8):e41258. Epub 2012 Aug 1. PubMed PMID: 22870212; PubMed Central PMCID: PMC3409852