Study Population
Samples included in this study were obtained from patients demanding diagnosis at the university hospital (Hospital de Clínicas "José de San Martín", University of Buenos Aires hospital, HJSM), located in downtown Buenos Aires, and at the national hospital (Hospital Nacional Prof Dr. A. Posadas, HNAP) located in the suburbs of Buenos Aires. Positive samples for C. trachomatis infection collected between January 2001 and December 2006 both from newborn babies (aged under 30 days) with ophthalmia neonatorum and from adults with genital symptoms were studied.
Patients were managed under standard approved hospital procedures. The population attending both hospitals belonged to lower and lower-middle classes and attended general practitioner's office. Neither STD clinic patients nor mother-child pairs were included in this study.
All neonates presented clinical signs of conjunctivitis. The most frequent symptoms described by adult patients from both hospitals included urethral or vaginal discharge, dyspareunia, dysuria, lower abdominal pain, and genital burning or itching sensation.
Sample collection and detection of C. trachomatis
Ocular specimens from neonates and endocervical and urethral specimens from adult patients were collected using sterile Dacron tipped swabs.
All the samples obtained at the university hospital were placed in 2-sucrose phosphate (2SP) solution and were then analysed by omp A PCR and cultured in LLC-MK2 cells.
Specimens collected at the HNAP were analysed with two commercial ELISA tests. Between January 2001 and December 2004 Chlamydiazyme (Abbott Laboratories, Chicago IL) was used, and miniVIDAS (bioMerieux, Marcy l' Etoile, France) was used between January 2005 and December 2006. Antigen detection was performed according to the manufacturer's instructions. Fractions of ELISA-positive samples obtained in the period from 2001 to 2004 and from all samples obtained in 2005 and 2006 were frozen at -20°C and then transported in dry ice from the national hospital to the HJSM for further analysis by omp A PCR.
Cell culture
Confluent cultures of LLC-MK2 cells (kindly provided by Sezione di Microbiologia DMCSS, Universitá degli Studi di Bologna, Bologna, Italy), grown at 37°C and 5% CO2 in culture media (minimum essential medium (MEM, Gibco), supplemented with 10% foetal calf serum (FCS, PAA Lab. GmbH, Austria), 0.1 mM non-essential amino acids (Gibco), 50 mg/L gentamicin, and 2 mM glutamine) were inoculated in duplicate with 500 μl of the 2SP sample suspension, and centrifuged at 700 g for 1 hour at 30°C. After a 72-hour incubation, one of each duplicate was methanol-fixed and stained with a genus-specific fluorescein-conjugated monoclonal antibody (Merifluor Chlamydia, Meridian Diagnostics Inc. Cincinnati, Ohio), following the manufacturer's instructions [20].
OmpA PCR
The omp A gene of C. trachomatis was amplified using the methodology describe by Lan et al. [21]. Briefly, an approximately 1 kb fragment of the omp A gene was amplified using primers SERO1A (5'-ATG AAA AAA CTC TTG AAA TCG G-3') and SERO2A, (5'-TTT CTA GAT CTT CAT TCT TGT T-3'). The reaction was performed in a final volume of 50 μl containing 1.5 mM MgCl2, 0.05 mM of each deoxynucleotide triphosphate, 0.32 μM of each primer, 2 U of Taq DNA polymerase (Invitrogen Corporation, Brazil), and 10 μl of clinical specimen.
Cycling conditions began with an initial 7 min denaturation step at 94°C, followed by 40 cycles of denaturation at 95°C for 1 min, annealing at 45°C for 3 min, and extension at 72°C for 3 min. An additional 7-min extension at 72°C was performed at the end of the 40 cycles.
DNA of C. trachomatis L2/BU/434, (kindly provided by Sezione di Microbiologia DMCSS, Università degli Studi di Bologna, Bologna, Italy) and mock-infected cells were included as positive and negative controls respectively.
Then, 1 μl of the first-round PCR product was used for the semi-nested PCR, which was performed with the same reagents and conditions except for the primers, which were SERO2A, and one nested primer: PCTM3, (PCTM3: 5'-TCC TTG CAA GCT CTG CCT GTG GGG AAT CCT-3'). The PCR products of the second round were checked for correctness on ethidium-bromide stained 1.5% agarose gels. Positive results were routinely subjected to RFLP analysis, to determine the genotype and verify the absence of contamination with positive controls or cross-sample carry-over.
This semi-nested PCR was able to detect 1 to 10 inclusion-forming units in our laboratory conditions.
RFLP genotyping
RFLP analysis of PCR-positive samples was carried out as described by Sayada et al. [22], using the semi-nested PCR product of approximately 1-Kbp. Briefly, 10 μl of the nested PCR product was digested with 2.5 U of Alu I (Promega, Madison, WI). Depending on the Alu I pattern, amplified samples were analyzed, if necessary, with Hinf I, Dde I or Eco RI (Promega, Madison, WI), according to the manufacturer's instructions.
Genotypes were identified by their restriction patterns on ethidium bromide stained 12% polyacrylamide gel electrophoresis.
Genotypes identified by RFLP from samples obtained between January 2005 and December 2006 were sent to omp A sequencing for verification, using the procedure of Jonsdottir et al [23].
Statistical analysis
Epi Info version 2000 software (Center for Disease Control and Prevention, Atlanta, GA) was used to assess differences by application of the chi-square and exact binomial methods. Values of p ≤ 0.05 were considered statistically significant.
