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

Problem: The diagnosis of microbial invasion of the amniotic cavity (MIAC) has been traditionally performed using traditional cultivation techniques, which require growth of microorganisms in the laboratory. Shortcomings of culture methods include the time required (days) for identification of microorganisms, and that many microbes involved in the genesis of human diseases are difficult to culture. A novel technique combines broad-range real-time polymerase chain reaction with electrospray ionization time-of-flight mass spectrometry (PCR/ESI-MS) to identify and quantify genomic material from bacteria and viruses.

Method of study: AF samples obtained by transabdominal amniocentesis from 142 women with preterm labor and intact membranes (PTL) 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.

Results: (i) The prevalence of MIAC in patients with PTL was 7% using standard cultivation techniques and 12% using PCR/ESI-MS; (ii) seven of ten patients with positive AF culture also had positive PCR/ESI-MS [≥17 genome equivalents per PCR reaction well (GE/well)]; (iii) patients with positive PCR/ESI-MS (≥17 GE/well) and negative AF cultures had significantly higher rates of intra-amniotic inflammation and acute histologic chorioamnionitis, a shorter interval to delivery [median (interquartile range-IQR)], and offspring at higher risk of perinatal mortality, than women with both tests negative [90% (9/10) versus 32% (39/122) OR: 5.6; 95% CI: 1.4-22; (P < 0.001); 70% (7/10) versus 35% (39/112); (P = 0.04); 1 (IQR: <1-2) days versus 25 (IQR: 5-51) days; (P = 0.002), respectively]; (iv) there were no significant differences in these outcomes between patients with positive PCR/ESI-MS (≥17 GE/well) who had negative AF cultures and those with positive AF cultures; and (v) PCR/ESI-MS detected genomic material from viruses in two patients (1.4%).

Conclusion: (i) Rapid diagnosis of intra-amniotic infection is possible using PCR/ESI-MS; (ii) the combined use of biomarkers of inflammation and PCR/ESI-MS allows for the identification of specific bacteria and viruses in women with preterm labor and intra-amniotic infection; and (iii) this approach may allow for administration of timely and specific interventions to reduce morbidity attributed to infection-induced preterm birth.

Keywords: Broad-range real-time polymerase chain reaction, electrospray ionization mass spectrometry; inflammation; preterm delivery; time-of-flight.

Figure 1

Receiver operator characteristic curve analysis of PCR/ESI-MS genome equivalents per PCR reaction well (GE/well) results for the identification of intra-amniotic inflammation (amniotic fluid IL-6 ≥ 2.6 ng/mL) among patients with detectable DNA of bacteria and/or viruses. Among patients with detectable DNA of bacteria or viruses using PCR/ESI-MS, a GE/well ≥17 had an area under the ROC curve of 0.93 (95% CI 0.83 – 1.0; p<0.001), a positive predictive value of 94.1% (16/17), and a negative predictive value of 84.6% (11/13) for the identification of intra-amniotic inflammation.

Figure 2

Bacteria and viruses detected in amniotic fluid of patients with preterm labor using standard cultivation techniques vs. PCR/ESI-MS. Amniotic fluid culture includes routine cultivation techniques for bacteria (aerobes, anaerobes, and genital mycoplasmas). PCR/ESI-MS refers to broad range polymerase chain reaction (PCR) and Electrospray-Ionization Mass Spectrometry (ESI-MS). Microorganisms detected in the amniotic fluid of patients with preterm labor and intact membranes using standard cultivation techniques versus PCR/ESI-MS. PCR: polymerase chain reaction; ESI-MS: electrospray ionization mass spectrometry; GE/well: genome equivalent per PCR reaction.

Figure 3

Markers of infection in amniotic fluid according to the results of standard AF culture and PCR/ESI-MS. Amniotic fluid concentrations of Interleukin-6 (Panel A) and white blood cells (WBCs) count (Panel B) as a function of PCR/ESI-MS and AF culture results. There was no significant difference in the median AF IL-6 concentration and WBC count between patients with negative AF culture and a positive PCR/ESI-MS (GE/well ≥17) and those patients with positive AF culture and positive PCR/ESI-MS (p= 0.43 and 0.28, respectively). Among patients with negative AF culture, those with a positive PCR/ESI-MS had a significantly higher median AF IL-6 concentration and WBC count than those patients with both tests negative (p<0.001 and p=0.02, respectively). The median AF IL-6 concentration in patients with positive AF culture and negative PCR/ESI-MS (GE/well < 17) was always < 2.6 ng/mL. Amniotic fluid IL-6 concentration and white blood cell count according to the results of standard AF cultures and PCR/ESI-MS.

Figure 4

Prevalence of intra-amniotic inflammation (Panel A) and histologic acute chorioamnionitis (Panel B) according to the results of PCR/ESI-MS and amniotic fluid cultures. Intra-amniotic inflammation was diagnosed in 70% (n=7/10) and 94% (n=16/17) of patients with positive AF culture and positive PCR/ESI-MS (≥17 GE/well) results, respectively. Patients with positive PCR/ESI-MS (GE/well ≥ 17) and negative AF culture have a prevalence of histological acute chorioamnionitis of 70% (7/10). In contrast, the prevalence of histological acute chorioamnionitis in patients with negative AF culture and negative PCR/ESI-MS (GE/well < 17) was 35% (39/112). Prevalence of intra-amniotic inflammation and histologic acute chorioamnionitis by PCR/ESI-MS and amniotic fluid culture results PCR: polymerase chain reaction; ESI-MS: electrospray ionization mass spectrometry.

Figure 5

Kaplan-Meier survival analysis of amniocentesis-to-delivery interval (days) according to amniotic fluid cultures and PCR/ESI-MS results. Patients in whom labor was induced were censored and are represented by crosses. Among patients with negative AF cultures, the amniocentesis-to-delivery interval was significantly shorter in patients with a positive PCR/ESI-MS than in those with a negative PCR/ESI-MS results [1 (IQR: < 1 – 2) days vs. 25 (IQR: 5 – 51) days; p=0.002]. There were no significant differences in the median amniocentesis-to-delivery interval between patients who had a positive PCR/ESI-MS with a negative AF culture [1 (IQR: < 1 – 2) days] and those patients with both tests positive [1 (IQR: 1 – 8) days] (p=0.6). Kaplan-Meier survival analysis of amniocentesis-to-delivery interval (days) according to amniotic fluid cultures and PCR/ESI-MS results.

Figure 6

Forest plot of hazard ratios for spontaneous preterm delivery (<37, <32 weeks) by PCR/ESI-MS and amniotic fluid culture results compared to patients with negative tests. The risks of spontaneous preterm delivery (sPTD) <37 and, separately, <32 weeks, were significantly greater among patients with positive PCR/ESI-MS (GE/well ≥17), both with and without positive AF cultures, when compared to women with negative tests. Forest plot of hazard ratios for spontaneous preterm delivery (<37, <32 weeks) by PCR/ESI-MS and amniotic fluid culture findings compared to patients with negative tests.

Figure 7

Prevalence of perinatal death (fetal and neonatal deaths) by the combined results of amniotic fluid culture and PCR/ESI-MS. Patients with positive PCR/ESI-MS (≥17 GE/well) and negative AF cultures had significantly higher rates of perinatal mortality, than women with both tests negative [40% (4/10) vs. 11% (13/122); (p=0.03)]. There was no difference in the rate of perinatal death for offspring of women with positive PCR/ESI-MS according to the results of AF culture [positive culture 29% (2/7) vs. negative culture 40% (4/10); p=1.0]. Prevalence of perinatal death (fetal and neonatal deaths) by the combined results of amniotic fluid culture and PCR/ESI-MSI.