Preterm labor is frequently caused by IAI

Intraamniotic infection is an acute bacterial infection of the amniotic fluid and intrauterine contents that complicates 4 – 10% of all pregnancies. Intraamniotic infection is also an important cause of preterm birth, responsible for 10 – 20% of all preterm births and more than 50% of preterm births occurring before 30 weeks of gestation.¹ 80% - 90% of cases of intraamniotic infection have no clinical signs or symptoms and often presents as “idiopathic” preterm labor.² There is increasing awareness of the prevalence of IAI in preterm labor and the importance of early detection. In a pooled analysis of 6 studies since 2000, IAI as defined by positive AF culture, including Mycoplasma, was present in 12.7% of all preterm labor cases.³



Although historically microbial culture of AF has been recognized as the “gold standard” for the detection of intraamniotic infection, more recent clinical research has demonstrated that AF culture alone underestimates the true prevalence of IAI.

The use of AF culture alone as a tool for diagnosing intraamniotic infection has a number of disadvantages: culture depends on growth conditions favorable to a select group of known microorganisms. Consequently, difficult to cultivate or uncultivatable bacteria will not be detected by culture alone. In addition, previous antibiotic therapy can cause AF cultures to yield a false negative test result.

When AF culture alone is augmented with AF 16S rDNA PCR assay, the prevalence of IAI increases by 31% to 56% as compared to the prevalence of IAI as measured by culture alone. Recent published clinical research utilizing AF PCR and culture demonstrate that IAI is likely present in 15% to 18% of all PTL cases.⁴

IAI Is Associated With Increased Risk Of Critical Complications

Intraamniotic infection is a critical complication of pregnancy and is associated with significantly risk of morbidity. The table below summarizes published clinical results demonstrating neonates born in the setting of IAI had a 2.7 times -- 3.4 times higher risk of complications such as neonatal sepsis, respiratory distress syndrome, intraventricular hemorrhage, as compared to a similar group of neonates born outside the setting of IAI, but matched for other variables such as gestational age, birth weight, preterm rupture of membranes, preeclampsia, cesarean birth:



In addition:

• IAI is associated with at least one-third of all early-onset neonatal sepsis and pneumonia in United States.⁶

• IAI also accounts for highest proportion of neonatal deaths in the United States and up to 40% of cases of maternal febrile morbidity in the peripartum period.⁷

• A study published in JAMA demonstrated a clear link between IAI and cerebral palsy via meta-analysis of 19 published studies.⁸

• IAI is also responsible for a disproportionate share of aggregate prenatal healthcare costs (estimated in the United States to be $4 to $6 billion annually).⁹

Conventional IAI diagnostic tools have limitations

Despite the increasing awareness of the importance of the diagnosis and treatment of IAI in preterm labor, detection of IAI can be difficult because 80% to 90% of all cases have no clinical signs or symptoms.¹⁰ In addition amniocentesis and commonly available laboratory diagnostic tests with AF have limitations:

• Two of the most common causative pathogens of IAI (Mycoplasma and Ureaplasma species) are present in 30% to 50% of cases. Tests to detect these pathogens are rarely available in clinical laboratories.

• 48 to 72 hours are needed for complete aerobic and anaerobic AF culture results.

• AF Gram stain and glucose concentration tests have relatively poor sensitivity. In a pooled analysis of 6 studies, Gram stain demonstrated average sensitivity of 48%. Glucose concentration demonstrated sensitivity of 63% to 87%.

• Previous antibiotic use can result in the inability of culture to detect pathogenic organisms.

In the absence of clinical symptoms, and given the limitations of current IAI diagnostic tools, routinely assessing PTL patients for IAI can be difficult and cases of IAI can remain undiagnosed and untreated.¹¹

Polymerase Chain Reaction (PCR) Assays in Amniotic Fluid offer advantages versus culture alone for diagnosis of IAI

Historically microbial culture of AF has been recognized as the “gold standard” for the detection of intraamniotic infection. However, the disadvantage of using a culture-based approach for bacterial identification is it depends on growth conditions favorable to a select group of known microorganisms. Consequently, uncultivated or difficult-to-cultivate bacteria will not be detected by culture alone.¹²

In addition, antibiotic use prior to obtaining amniotic fluid for laboratory testing can result in the inability of culture to detect pathogenic organisms.

Molecular microbiology techniques such as Quantitative Polymerase Chain Reaction (qPCR) and Pyrosequencing can detect microbes independent of culture. These techniques amplify highly conserved gene sequences that allow detection and broad taxonomic identification of pathogenic microorganisms in biological fluids.

Ribosomal RNA is the most conserved and least variable gene in all cells including bacteria. For this reason, genes that encode ribosomal RNA have been used extensively to identify medically important microorganisms such as bacteria and fungi. The 16S and 23S rRNA gene sequences are typically used to identify bacteria species while the corresponding 18S rRNA gene sequences are used to identify fungi.

ProteoGenix molecular diagnostics tests are based on the qPCR amplification of ribosomal DNA as well as other unique microbial gene sequences from bacteria and fungi. ProteoGenix has also developed a proprietary specimen collection, transport and preparation system that optimizes the detection and identification of pathogenic bacteria and fungi associated with maternal, fetal and neonatal conditions.

Individual microorganisms are commonly identified by the laboratory by hybridizing an amplified microbial gene sequence to a specific fluorescent molecular probe that can be used to detect and quantify the microorganism present in the clinical sample. In addition to this test approach, ProteoGenix is using breakthrough Pyrosequencing technology to quickly, accurately and specifically identify a host of potential pathogenic microbes down to the species level. Pyrosequencing allows for this simultaneous identification of multiple microbial gene sequences using DNA sequencing technology consisting of an enzyme-cascade system of four enzymes and specific substrates. Also known as “sequencing by synthesis”, Pyrosequencing produces light in the reaction whenever a nucleotide is incorporated as a base pair with the complementary base in a DNA template strand. Pyrosequencing is a rapid process allowing the sequencing of 30 to 40 base pairs in less than an hour.

Numerous investigators have shown that 16S rDNA PCR testing is 30%-50% more sensitive than culture alone in clinical studies using laboratory testing of amniotic fluid to detect intraamniotic infection.¹³

“By using PCR to examine for evidence of microbes, we have confirmed a higher rate of detection of infection associated with spontaneous preterm delivery than detected by standard culture techniques.”

Miralles R, Hodge et al. “Relationship between Antenatal Inflammation and Antenatal Infection Identified by Detection of Microbial Genes by Polymerase Chain Reaction”, Pediatric Research, 2005;57(4):570-577.

“A broad-spectrum bacterial PCR assay may prove to be a useful alternative to culture for the diagnosis of amniotic fluid infection in women in preterm labor.”

Hitti J, et al. “Broad-Spectrum Bacterial Rdna Polymerase Chain Reaction Assay for Detecting Amniotic Fluid Infection Among Women in Premature Labor”, Clinical Infectious Disease, 1997;24(6):1228-1232.

ProteoGenix is the first and only company to offer a commercially available 16S rDNA polymerase chain reaction (PCR) test in amniotic fluid for the detection of bacterial, Mycoplasma and Ureaplasma species DNA to aid in the clinical diagnosis of intraamniotic infection.

ProteoGenix Offers The First Commercially Available AF PCR Test For Detection Of IAI

The ProteoGenix AF Bacteria/Mycoplasma/Ureaplasma End Point PCR Test uses broad-spectrum PCR assay based on 16S ribosomal DNA to provide a qualitative and sensitive tool for detecting the presence of bacterial/Mycoplasma and Ureaplasma species DNA in amniotic fluid. The ProteoGenix AF End Point PCR test offers the following benefits:

• The ProteoGenix PCR analysis is completed on amniotic fluid, which is not commonly available through the majority of hospital and reference laboratories. This provides an additional option to aid physicians in the detection of intraamniotic infection.

• The ability to detect the most common causative pathogens of IAI; including Mycoplasma and Ureaplasma species that have been shown to be present in 30% to 50% of IAI cases. Tests to detect these pathogens are not commonly available in most laboratories.

• Demonstrated sensitivity of 100% and specificity of 96% versus bacterial/Mycoplasma and Ureaplasma species in AF culture.

• ProteoGenix has developed a proprietary specimen collection, transport and preparation system that specifically optimizes the performance of the PCR test in amniotic fluid. This proprietary process improves the accuracy and reproducibility of patient results.
  
  Extraction is a critical step in isolating bacterial DNA. Most extraction techniques utilize “off the shelf” reagents designed for extraction of various types of specimens. ProteoGenix has developed a proprietary extraction process that utilizes specialized reagents to maximize extraction efficiency from amniotic fluid. ProteoGenix has also improved analytical sensitivity and reduce false positive results by optimizing their methods to reduce trace amounts of bacterial DNA found in commercial PCR reagents.

• In a clinical study of the diagnosis of IAI in preterm labor patients with intact fetal membranes; the ProteoGenix AF Endpoint PCR test detected microbial pathogens in ~25% more cases than amniotic fluid bacterial and Mycoplasma culture alone.

• The ProteoGenix Amniotic Fluid 16S rDNA by Endpoint PCR for detection of Bacterial/Mycoplasma and Ureaplasma species is available via the CLIA certified ProteoGenix Clinical Laboratory. Please contact (888) 429-7935 for more information.