title: Detection of microbial translocation in HIV and SIV infection using the Limulus amebocyte lysate assay is masked by serum and plasma.
abstract: 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.
authors.affiliation: Department of Medicine, Johns Hopkins University, Baltimore, Maryland, United States of America. abalago1@jhmi.edu
authors.forename: Ashwin
authors.initials: A
authors.lastname: Balagopal
authors.forename: Lucio
authors.initials: L
authors.lastname: Gama
authors.forename: Veronica
authors.initials: V
authors.lastname: Franco
authors.forename: Julia N
authors.initials: JN
authors.lastname: Russell
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Quinn
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Higgins
authors.forename: Laura M
authors.initials: LM
authors.lastname: Smeaton
authors.forename: Janice E
authors.initials: JE
authors.lastname: Clements
authors.forename: David L
authors.initials: DL
authors.lastname: Thomas
authors.forename: Amita
authors.initials: A
authors.lastname: Gupta
mesh_terms: D000328:Adult; D000818:Animals; D018988:Bacterial Translocation; D005260:Female; D006090:Gram-Negative Bacteria; D016905:Gram-Negative Bacterial Infections / Q000097:blood* / Q000209:etiology; D015658:HIV Infections / Q000097:blood* / Q000150:complications; D006801:Humans; D008033:Limulus Test / Q000379:methods; D008070:Lipopolysaccharides / Q000097:blood*; D008254:Macaca nemestrina; D008297:Male; D016097:Simian Acquired Immunodeficiency Syndrome / Q000097:blood* / Q000150:complications
chemical_list: D008070:Lipopolysaccharides