A Novel Molecular Microbiologic Technique for the Rapid Diagnosis of Microbial Invasion of the Amniotic Cavity and Intra‐Amniotic Infection in Preterm Labor with Intact Membranes

Abstract

Objective The major challenges in using amniotic fluid (AF) cultivation techniques to diagnose microbial invasion of the amniotic cavity (MIAC) are: 1) several days are typically required to obtain results, and 2) many organisms implicated in the pathogenesis of human disease are difficult to culture. Here, we compare the performance of AF culture with a novel technique for the diagnosis of MIAC that can provide results within eight hours by combining broad-range real-time polymerase chain reaction with electrospray ionization mass spectrometry (PCR/ESI-MS) to identify and quantify genomic material from bacteria and viruses in AF. Methods AF samples obtained by transabdominal amniocentesis from 142 women with preterm labor (PTL) and intact membranes were analyzed using cultivation techniques (aerobic, anaerobic and genital mycoplasmas) as well as PCR/ESI-MS. The prevalence and relative magnitude of intra-amniotic inflammation [AF Interleukin 6 (IL-6) concentration ≥ 2.6 ng/mL], acute histologic chorioamnionitis, spontaneous preterm delivery, and perinatal mortality were examined according to the results of these two tests. Results 1) The prevalence of MIAC in patients with preterm labor and intact membranes was 7% using standard cultivation techniques and 12% using PCR/ESI-MS; 2) seven of ten patients with positive AF culture also had positive PCR/ESI-MS [≥17 genome equivalents per PCR reaction well (GE/well)] 3) patients with positive PCR/ESI-MS (≥17 GE/well) and negative AF cultures had significantly higher rates of intra-amniotic inflammation and histologic acute chorioamnionitis, shorter intervals to delivery [median (interquartile range-IQR)], and offspring at higher risk of perinatal mortality, than women with both tests negative [90% (9/10) vs. 32% (39/122); (p<0.001); 70% (7/10) vs. 35% (39/112); (p=0.04); 1 (IQR: <1 – 2) days vs. 25 (IQR: 5 – 51) days; (p=0.002); OR: 5.6; 95% CI: 1.4 – 22, respectively]; 5) there were no significant differences in these factors between patients with positive PCR/ESI-MS (≥17 GE/well) who had negative AF cultures compared to those with positive AF cultures; and 6) PCR/ESI-MS detected genomic material from viruses in two patients (1.4%). Conclusion 1) Rapid diagnosis of intra-amniotic infection is possible using PCR/ESI-MS, which can provide results within 8 hours; 2) the combined use of biomarkers of inflammation and PCR/ESI-MS allows for the rapid identification of specific bacteria and viruses in women with preterm labor and intra-amniotic infection; and 3) this approach may allow for administration of timely and specific interventions to reduce morbidity attributed to infection-induced preterm birth.

Novel biomarkers in amniotic fluid for early assessment of intraamniotic infection

“…On the other hand, some rapid and non-invasive diagnostic tests including maternal serum CRP levels and leukocyte counts, cytokine determination, immunoassays, etc., have showed poor selectivity and sensitivity rendering them inapplicable for decision making in the clinical setting [21][22][23].…”

“…The most widely employed analytical methods that have been used to detect IAI in AF samples are based on enzymatic linked immunosorbent assay (ELISA) [21] or lateral flow-based immunoassay (or point of care test) characterized by rapidity and strong correlation with ELISA results [22,23]. However, these methods lack selectivity and reproducibility.…”

Prevention of preterm birth: Harnessing science to address the global epidemic

“…Infection may infrequently enter the uterus by the hematogenous route, through retrograde seeding via the fallopian tube, or during intrauterine procedures such as amniocentesis (38). The initiating infection may be clinical or subclinical, monomicrobial or polymicrobial, and may require high-throughput, metagenomic tools for identification of common and unusual pathogens (39)(40)(41)(42)(43). The most common bacteria associated with PTB are Ureaplasma urealyticum, Mycoplasma hominis, Streptococcus agalactiae, Escherichia coli, Fusobacterium species, and Gardnerella vaginalis (44).…”

Clinical chorioamnionitis at term: the amniotic fluid fatty acyl lipidome

“…In addition to standard cultivation techniques, the amniotic fluid of patients with clinical chorioamnionitis was analyzed using broad-range real-time PCR/ESI/MS (Ibis®Technology -Athogen), as previously described (34). Briefly, DNA was extracted from 300 µl of amniotic fluid using a method that combines beadbeating cell lysis with a magnetic-bead-based extraction method (38,39).…”

“…The TLB group had yet lower IL-6 concentrations than the TCC-noMIAC group. Intra-amniotic inflammation was diagnosed when the amniotic fluid interleukin (IL)-6 concentrations were 2.6 ng/ml as determined by ELISA (34). Microbial invasion of the amniotic cavity (MIAC) was defined according to the results of amniotic fluid cultures and/or PCR/ESI/MS (Ibis® Technology - Athogen, Carlsbad, CA).…”