A random selection of 20% of the cases of early neonatal deaths that occurred between January 1 and December CH5424802 31, 2003 and fulfilled the inclusion criteria was performed. Cases were separated into two groups according to the judgment or final diagnosis of the institution’s perinatal mortality committee. Group 1 consisted of cases of death due to systemic infection16 without pathogen identification. Group 2 was formed by cases of death due to another cause that was not related with infection. The autopsy organs, specifically lungs, kidneys, brain, liver, and bronchi, which were embedded in paraffin blocks for all participating cases, were located. The study included only cases that

had all these organs available, as well as complete maternal and neonatal clinical records and histopathological studies of the autopsy and ABT-888 cost placenta. The pathologist in chief blindly restudied the histologic samples from the cadavers. Placentas are usually routinely analyzed: hematoxylin and eosin–stained histologic sections are investigated for deciduitis, vasculitis,

endometritis, or chorioamnionitis without special studies. Later, the entire material is discarded. For this reason, only the results of the single study of the placenta were included. The rest of the plates was deparaffinized and processed for DNA extraction. All tests were performed in a blinded manner. Clinical and demographic data were obtained from hospital records. Tissue samples were deparaffinized with xylene at room temperature and washed with ethanol. The obtained tissue was then treated with proteinase K, and the DNA was obtained by the phenol-chloroform-isoamyl technique and ethanol precipitation as previously described.17 End-point polymerase chain reaction and real-time polymerase chain reaction were

performed using PTC-100 Selleck Fludarabine system (MJ Research, Inc. – Waltham, MA, United States) and StepOne (Applied Biosystems – Carlsbad, CA, United States), respectively. The primers used for end-point polymerase chain reaction are those proposed by Dutilh et al.18 and amplify a fragment of the omp1 gene of C. trachomatis (5’-GCCGCTTTGAGT TCTGCTTCCTC-3’; 5’-CCAAGTGGTGCA AGGATCGCA-3’). Each end-point polymerase chain reaction contained 1.75 mM MgCl2, 0.2 mM dNTPs, 25 pM of each proposed primer, 2.5 U Taq polymerase (GoTaq® Flexi DNA polymerase Promega© – Madison, WI, United States), and 5 μL of the DNA sample, for a final volume of 25 μL. The reaction mixture was incubated for 5 min at 95 °C, followed by 35 cycles of 1 min at 95 °C for denaturation, 1 min at 59 °C for alignment, 1 min at 70 °C for extension, and a final elongation step of 5 min at 70 °C. The sample was considered positive when an amplification product of 129 bp was obtained. The real-time polymerase chain reaction was performed using 3 mM of 2.7 mM MgCl2, 25 pM of each of the proposed primers, 2.5 U Taq polymerase (Applied Biosystems), and 5 μL of the DNA sample, for a final volume of 25 μL.