Comparison of culture and real-time polymerase chain reaction methods for detection of Mycoplasma hominis in amniotic fluids samples

Abstract

Background: Mycoplasma hominis is often present in the amniotic cavity with microbial invasion associated with spontaneous preterm labor. Conventional culture method is the gold standard for detection of Mycoplasmas, but real‑time polymerase chain reaction (real‑time PCR) has revolutionized the diagnosis of M. hominis.

Objective: The purpose of this study is the comparison of the culture methodology with real‑time PCR for the detection of M. hominis in amniotic fluid samples.

Methods: Amniotic fluid samples were collected from 65 pregnant women (age range: 25–45 years) previously followed at an infertility clinic. They were collected by transabdominal genetic amniocentesis during 16–21 weeks of gestation. Amniotic fluids were inoculated in SP4 broth for 48–72 h, and after becoming alkaline, culture suspension was spread on A7 agar plate for 1 week till the typical colonies seen in “fried‑egg” morphology under stereomicroscope. DNA was extracted using a QIAGEN Mini DNA kit. The real‑time‑PCR was performed using Rotor‑Gene Q Real‑time PCR instrument. A melting‑curve analysis was also performed. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were measured by real‑time PCR by taking culture as gold standard.

Results: Sixty‑five women in 16–21 weeks of gestation, with a mean age of 33 ± 5.06 years, were enrolled into this study. M. hominis detected by culture and real‑time PCR assay was 72% (47/65) and 69% (45/65), respectively. 66% (43/65) specimens were positive by both methods. Real‑time PCR sensitivity was 91.5%, specificity 88.9%, PPV 95.6%, and NPV 80%.

Conclusion: Rapid detection of Mycoplasmas causing maternal complications such as neonatal infections and preterm labor in pregnancy by real‑time PCR may be important and necessary. The high sensitivity and shorter time requirement of real‑time PCR support its further development for diagnosis of Mycoplasma infections.

Keywords: Amniotic fluids, culture, Mycoplasma hominis, real‑time polymerase
chain reaction