María Angélica Martínez

Bacterial and viral etiology of acute otitis media in Chilean children.

BACKGROUND Acute otitis media (AOM) is a main cause for antimicrobial prescription in Latin America. Pathogen diversity in different geographic regions underscores the need for updated knowledge on AOM microbiology. AIM To prospectively determine the role of bacteria and viruses in Chilean children with AOM. METHODS Between July, 1998, and June, 1999, children >3 months with a presumptive diagnosis of AOM were referred to the study ear, nose and throat physician. Middle ear fluid and nasopharyngeal aspirates were obtained from children with confirmed AOM and processed for common bacteria, Mycoplasma pneumoniae, Chlamydia pneumoniae and viruses. Antimicrobial susceptibility patterns and serotypes of Streptococcus pneumoniae strains were determined. RESULTS An ear, nose and throat physician confirmed diagnoses for 222 (42%) of 529 children referred with diagnosis of AOM, and 170 children met eligibility criteria for the study. One or more pathogens were detected in 140 of 170 (82%) children. Predominant bacteria were S. pneumoniae (37%), Haemophilus influenzae (24%) and Streptococcus pyogenes (13%). M. catarrhalis was detected in 2 children, C. pneumoniae was found in 1 and M. pneumoniae was not detected. Viruses were detected in 22 children (13%) from nasopharyngeal aspirates, and in 6 of them the same virus was detected in middle ear fluid. Penicillin-resistant (intermediate and high) S. pneumoniae represented 40% of isolates and 10% of H. influenzae were beta-lactamase producers. All 10 penicillin-resistant S. pneumoniae strains were resistant to cefuroxime. Eighteen S. pneumoniae serotypes were detected and 19F was associated with high level penicillin resistance. CONCLUSION This study can impact local management of AOM, and it should encourage continuous surveillance of AOM microbiology in Chile and other developing countries.

Detection of Mycoplasma pneumoniae in adult community-acquired pneumonia by PCR and serology

Diagnosis of pneumonia caused by Mycoplasma pneumoniae in adults is hampered by a lack of rapid and standardized tests for detection. This prospective study was conducted to compare the diagnostic values of an indirect immunofluorescence assay and a 16S rRNA gene PCR for the diagnosis of M. pneumoniae pneumonia in adults. From February 2005 to January 2008, 357 patients (53.8 % males, median age 63 years, range 18-94) admitted for community-acquired pneumonia (CAP) to two hospitals in Santiago, Chile, were enrolled in the study. Thirty-two patients (9.0 %) met the criteria of current or recent M. pneumoniae infection, and laboratory diagnosis was definitive in 26 cases (81.2 %) and presumptive in six cases (18.8 %). Among the 32 M. pneumoniae infections, the PCR assay was positive in 23 (71.9 %) and the serology in 27 (84.4 %) of the cases. IgM was positive in acute-phase serum specimens in 13 cases (40.6 %) of M. pneumoniae infections. Using serology as the gold standard, the sensitivity, specificity, and positive and negative predictive values of the PCR were 66.7, 98.5, 78.3 and 97.3 %, respectively, whereas the global agreement of the methods was 343/357 (96.1 %). The frequency of M. pneumoniae CAP cases declined significantly during the second year of study, suggesting the end of an epidemic period. In conclusion, although good global agreement was found between PCR and serology, the lower sensitivity of the PCR leads us to recommend the use of both procedures in parallel to confirm M. pneumoniae in CAP in adults.

Community-acquired pneumonia in Chile: the clinical relevance in the detection of viruses and atypical bacteria

Background Adult community-acquired pneumonia (CAP) is a relevant worldwide cause of morbidity and mortality, however the aetiology often remains uncertain and the therapy is empirical. We applied conventional and molecular diagnostics to identify viruses and atypical bacteria associated with CAP in Chile. Methods We used sputum and blood cultures, IgG/IgM serology and molecular diagnostic techniques (PCR, reverse transcriptase PCR) for detection of classical and atypical bacteria (Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumoniae) and respiratory viruses (adenovirus, respiratory syncytial virus (RSV), human metapneumovirus, influenza virus, parainfluenzavirus, rhinovirus, coronavirus) in adults >18 years old presenting with CAP in Santiago from February 2005 to September 2007. Severity was qualified at admission by Fines pneumonia severity index. Results Overall detection in 356 enrolled adults were 92 (26%) cases of a single bacterial pathogen, 80 (22%) cases of a single viral pathogen, 60 (17%) cases with mixed bacterial and viral infection and 124 (35%) cases with no identified pathogen. Streptococcus pneumoniae and RSV were the most common bacterial and viral pathogens identified. Infectious agent detection by PCR provided greater sensitivity than conventional techniques. To our surprise, no relationship was observed between clinical severity and sole or coinfections. Conclusions The use of molecular diagnostics expanded the detection of viruses and atypical bacteria in adults with CAP, as unique or coinfections. Clinical severity and outcome were independent of the aetiological agents detected.

Role of Neutralizing Antibodies in Adults With Community-Acquired Pneumonia by Respiratory Syncytial Virus

In a study of 356 adults with community-acquired pneumonia, respiratory syncytial virus was a frequent pathogen (13%). Serology and real-time reverse-transcription polymerase chain reaction improved the detection of repiratory syncytial virus. High respiratory syncytial virus serum-neutralizing antibody levels protected against severe pneumonia.

Occurrence and Antimicrobial Susceptibility of Ureaplasma parvum (Ureaplasma urealyticum Biovar 1) and Ureaplasma urealyticum (Ureaplasma urealyticum Biovar 2) from Patients with Adverse Pregnancy Outcomes and Normal Pregnant Women

A recent phylogenetic analysis of Ureaplasma urealyticum resulted in the proposal to divide their 2 biovars into species. We used PCR to compare the distribution of species and the presence of the tet(M) and int-Tn resistance determinants in 63 strains of Ureaplasma spp. isolated from the amniotic fluid of patients with an adverse pregnancy outcome and in 22 strains obtained from the lower genital tract of healthy pregnant women. We also determined the antimicrobial susceptibility of the organisms to erythromycin and tetracycline. U. parvum was the most frequent Ureaplasma species detected in our study. Thus, 50/63 (79.4%) invasive isolates and 17/22 (77.3%) lower genital tract isolates corresponded to U. parvum, whereas 12/63 (19%) invasive isolates and 4/22 (18.2%) non-invasive strains corresponded to U. urealyticum. A mixture of species was found in 2 women. We found no significant differences in the antimicrobial susceptibility of isolates according to species or origin of isolation. Sixty-two strains of Ureaplasma spp. (74.7%) were susceptible to erythromycin, and 21 strains (25.3%) were intermediately susceptible. Sixty-eight isolates (81.9%) were susceptible to tetracycline, 2 strains (2.4%) were intermediate and 13 strains (15.7%) were resistant. DNA sequences related to the tet(M) determinant and the int-Tn gene were found in all tetracycline-resistant isolates.A recent phylogenetic analysis of Ureaplasma urealyticum resulted in the proposal to divide their 2 biovars into species. We used PCR to compare the distribution of species and the presence of the tet(M) and int-Tn resistance determinants in 63 strains of Ureaplasma spp. isolated from the amniotic fluid of patients with an adverse pregnancy outcome and in 22 strains obtained from the lower genital tract of healthy pregnant women. We also determined the antimicrobial susceptibility of the organisms to erythromycin and tetracycline. U. parvum was the most frequent Ureaplasma species detected in our study. Thus, 50/63 (79.4%) invasive isolates and 17/22 (77.3%) lower genital tract isolates corresponded to U. parvum, whereas 12/63 (19%) invasive isolates and 4/22 (18.2%) non-invasive strains corresponded to U. urealyticum. A mixture of species was found in 2 women. We found no significant differences in the antimicrobial susceptibility of isolates according to species or origin of isolation. Sixty-two strains of Ureaplasma spp. (74.7%) were susceptible to erythromycin, and 21 strains (25.3%) were intermediately susceptible. Sixty-eight isolates (81.9%) were susceptible to tetracycline, 2 strains (2.4%) were intermediate and 13 strains (15.7%) were resistant. DNA sequences related to the tet(M) determinant and the int-Tn gene were found in all tetracycline-resistant isolates.

Antibiotic administration in patients with preterm premature rupture of membranes reduces the rate of histological chorioamnionitis: a prospective, randomized, controlled study

Objective: To determine whether antibiotic administration in patients with preterm premature rupture of membranes is associated with a reduction in the rate of histological chorioamnionitis and funisitis. Methods: One hundred consecutive patients with preterm premature rupture of membranes and no labor between 24 and 34 weeks were invited to participate in this study. Eligible patients randomly received either clindamycin-gentamicin for 7 days or placebo, and were managed expectantly until 35 weeks unless fetal or maternal indications developed. Microbial invasion of the amniotic cavity was defined as the presence of a positive amniotic fluid culture obtained by transabdominal amniocentesis. Cervicovaginal infection was diagnosed when bacterial vaginosis or a positive culture for cervicovaginal pathogens or facultative bacteria associated with a significant increase in the white blood cell count were found. Histological chorioamnionitis was based on the observation of polymorphonuclear leukocyte infiltration of the chorionic plate or the extraplacental fetal membranes. Funisitis was diagnosed in the presence of polymorphonuclear leukocyte infiltration into the umbilical vessel walls or Wharton jelly. Statistics were performed using contingency tables. Results: Seventy-one patients with available histological study of the placenta were included. Thirty-five women received antibiotics and 36 were given placebo. Patients who received antibiotics had a significantly lower rate of histological chorioamnionitis than patients who received placebo (46% (16/35) vs. 69% (25/36), respectively; p < 0.05). This effect was more pronounced among women with microbial invasion of the amniotic cavity and/or cervicovaginal infection (58% vs. 89%, respectively; p < 0.01). Antibiotic therapy was associated with an increase in the frequency of placentas without histological abnormalities (29% vs. 6%; p < 0.01). The frequency of funisitis was not different between groups. Conclusion: Administration of antibiotics in patients with preterm premature rupture of membranes is associated with a significant reduction in the incidence of histological chorioamnionitis but it does not modify the frequency of funisitis.

Role of Haemophilus influenzae in Intra-Amniotic Infection in Patients with Preterm Rupture of Membranes

Abstract Haemophilus spp. were isolated from the amniotic fluid of eight of 110 consecutive women with preterm premature rupture of membranes (PROM) between 1992 and 1998. Isolates were nontypeable and classified according to biochemical test results as Haemophilus influenzae biotype I (n=1), biotype II (n=4), biotype III (n=1) or biotype IV (n=2). Primers recognizing specific sequences in the 16S rRNA of the cryptic genospecies of Haemophilus were employed to amplify the DNA of the eight isolates. One isolate classified as Haemophilus influenzae biotype II was confirmed as belonging to the genital cryptic species. Infectious morbidity occurred in five women and two newborns and was associated in most cases with biotype II.

Identification of P1 types and variants of Mycoplasma pneumoniae during an epidemic in Chile.

This study was conducted to determine the types of M. pneumoniae prevalent in adults presenting with community-acquired pneumonia during an epidemic period, and to scrutinize a variable region of the RepMP4 element for the detection of P1 variants. All 23 clinical specimens PCR-positive for M. pneumoniae obtained in two hospitals in Santiago, Chile, from 2005 to 2006 were typed by a multiplex PCR directly and then the RepMP4 fragment of 18 specimens was sequenced. A predominance of M. pneumoniae type 2 was found, 18 (78.3 %) specimens being grouped as type 2 and 5 (21.7 %) as type 1. Co-infection of M. pneumoniae with other respiratory pathogens was found in 10/23 (43.4 %) patients, but their frequency was not related to the M. pneumoniae type. Sequence analysis revealed a single nucleotide polymorphism, a transition mutation, in 50 % of amplicons belonging to type 1 and in 71.4 % of amplicons of type 2. The nucleotide changes were synonymous in each P1 variant. In conclusion, during the 2005-2006 epidemic in Santiago, both types of M. pneumoniae circulated. Although the analysed area in the RepMP4 was small, we detected the existence of P1 variants in the two types of this organism.

Chlamydia trachomatis genotypes associated with pneumonia in Chilean infants

We determined the serovar distribution and genetic variability of the omp1 gene of C. trachomatis in nasopharyngeal aspirates from consecutive infants with pneumonia. C. trachomatis was detected by PCR in 17/94 (18.1%) specimens. Serovar E (47.1%) was the most frequent, followed by serovars F (17.6%), Ja (17.6%), D (11.8%), and G (5.9%). Nucleotide sequence analysis showed polymorphism of Omp1.

Seroprevalence of Chlamydia pneumoniae in Chile

We used microimmunofluorescence to survey the prevalence of antibodies to Chlamydia pneumoniae in 403 serum samples from asymptomatic subjects aged 6 months to 89 y in Santiago, Chile. The results suggest that Chlamydia pneumoniae infection is endemic in Chile, with a seroprevalence of 60% which does not differ by gender.