Vivian Luchsinger

Impaired Immune Response in Severe Human Lower Tract Respiratory Infection by Respiratory Syncytial Virus

Background: Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory infection in infants. The immune response plays a leading role in the severity of the disease. We hypothesized that severe RSV disease is associated with an impaired immune response characterized by low circulating T lymphocytes and plasma cytokine concentrations. Methods: We evaluate the in vivo immune responses of previously healthy infants with their first proven RSV-acute lower respiratory infection that required hospitalization. According to the clinical severity, defined by using a strict scoring system, the in vivo immune response was compared through the analysis of plasma cytokine values and the phenotyping of peripheral blood lymphocyte and natural killer (NK) cells. Results: Absolute blood cell counts of CD4+, CD8+, and CD19+ lymphocytes and NK cells were lower in subjects with RSV than in control infants. Lowest cell counts were observed in more severe RSV-infected infants. Significant low values were obtained in CD8+ lymphocytes (P = 0.03) and nonactive NK cells, that express CD94 antigen (P = 0.046). In contrast, activated NK cells that do not express CD94 molecules were significantly higher in RSV infected infants than in healthy controls (% of cells: P = 0.004). The interferon-&ggr; and tumor necrosis factor-&agr; values in RSV infected patients were lower than in controls subjects. Interleukin-17 cytokine was not detected in healthy infants and the largest concentration was found in moderately ill patients as compared with severe cases (P = 0.033). RSV infection showed significantly higher interleukin-8 chemokine than in control infants (P = 0.024). Conclusion: We propose that severe RSV infection in very young infants is associated with poor blood proinflammatory cytokine production, low counts of CD8+ T cells and with a greater activity of a group of NK cells, that are independent of the major histocompatibility complex class Ib recognition system.

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.

Detection of Pneumocystis carinii f. sp. hominis and Viruses in Presumably Immunocompetent Infants Who Died in the Hospital or in the Community

Fresh-frozen lung and tracheal-aspirate specimens obtained from 112 infants who died in Santiago, Chile, during 1998-2000 were analyzed for the presence of Pneumocystis DNA, by use of nested DNA amplification of the large subunit mitochondrial rRNA, and for the presence of viruses, by use of culture and immunofluorescence. Pneumocystis DNA was detected in specimens from 45 (51.7%) of 87 infants who died in the community and from 5 (20%) of 25 infants who died in the hospital (P=.006). Primary infection with Pneumocystis was highly frequent among infants who die unexpectedly in the community. Infection with viruses was more common in infants who died in the hospital.

Cyclovirus in nasopharyngeal aspirates of Chilean children with respiratory infections

Some respiratory tract infections remain unexplained despite extensive testing for common pathogens. Nasopharyngeal aspirates (NPAs) from 120 Chilean infants from Santiago with acute lower respiratory tract infections were analysed by viral metagenomics, revealing the presence of nucleic acids from anelloviruses, adenovirus-associated virus and 12 known respiratory viral pathogens. A single sequence read showed translated protein similarity to cycloviruses. We used inverse PCR to amplify the complete circular ssDNA genome of a novel cyclovirus we named CyCV-ChileNPA1. Closely related variants were detected using PCR in the NPAs of three other affected children that also contained anelloviruses. This report increases the current knowledge of the genetic diversity of cycloviruses whose detection in multiple NPAs may reflect a tropism for human respiratory tissues.

Comparison of virological profiles of respiratory syncytial virus and rhinovirus in acute lower tract respiratory infections in very young Chilean infants, according to their clinical outcome

Abstract Background Respiratory syncytial virus (RSV) and rhinovirus (HRV) are the main cause of acute lower respiratory tract infections (ALRTIs) in infants. Viral and host-related risk factors for severe disease have also not been clearly established. Objective To assess whether certain viral features of RSV and, or HRV are associated with severe ALRTI. Study design RSV and HRV were studied in nasopharyngeal samples of infants by immunofluorescence, Luminex® and/or real-time RT-PCR assays. Quantitation and genotyping of RSV and HRV by PCR were done. Results Of 124 virus positive specimens, 74 (59.7%) had RSV; 22 (17.7%) HRV and 28 (22.6%) RSV-HRV co-infection. Hospitalization was required in 57/74 RSV infants (77.0%); in 10/22 HRV cases (45.5%) (p =0.006) and in 15/28 co-infected by both viruses (53.6%) (p =0.003). Severe cases were 33/74 (44.6%) RSV infections, 2/22 HRV cases (9.1%), (p <0.002) and 6/28 (21.4%) patients co-infected by RSV–HRV (p <0.026). Three genotypes (NA1, B7, B9) of RSV circulated during the study. In 33 severe infants, NA1 was detected in 19 cases (57.6%); B7 in 13 (39.4%) and B9 in 1 (3.0%) (p <0.01; OR=10.0). RSV loads were similar between outpatients and hospitalized infants (p =0.7) and among different severities (p =0.7). NA1 loads were higher than other strains (p =0.049). Three geno-groups of HRV circulated homogeneously. Conclusion In very young infants, RSV cause more severe disease than HRV. Co-infection does not increase the severity of illness. NA1 RSV genotype was associated with major frequency of hospitalization, severe respiratory disease and higher viral load.

SP-A1, SP-A2 and SP-D gene polymorphisms in severe acute respiratory syncytial infection in Chilean infants.

Respiratory syncytial virus (RSV) is the principal pathogen that causes acute lower respiratory tract infection (ALRI) in infants. Severe RSV-ALRI has been associated with the host genetic susceptibility. To assess whether severe RSV disease in infants is associated with certain single nucleotide polymorphism (SNP) into the gene of SP-A1, SP-A2 and SP-D, a prospective study was performed among blood donors and RSV-infected infants aged <or=6 months, considering their severity, according to a strict scoring system. Allele and genotype frequencies were compared using χ(2)-test. Association studies and haplotype analysis were tested by using Armitagës trend test and Unphased 3.0 program. A total of 118 RSV-infected infants and 104 blood donors were enrolled into the study; 59 infants had a severe respiratory disease, 34 children developed a moderate illness and 25 had a mild disease. There was no difference in the allelic and genotypic frequencies of SP-A1, but intragenic haplotypes showed significant differences among infected infants and blood donors (p=0.0021). 1A(0) variant of SP-A2 was the most frequent allele in all groups. Thr(11) allele of SP-D is significantly higher in RSV infants (p=0.028), as given by its higher frequency in severe disease (p=0.046). Heterozygous Thr(11)/Met(11) was significantly more common in infected infants (p=0.037), because it has higher frequency in critically ill children (p=0.017). Thr(160) allele was significantly higher in severe infants compared with blood donors (p=0.046) and infants with mild disease (p=0.018). Thr(11)-Thr(160)-Ser(270) haplotype was significantly more common in RSV-infants, due to severe (p=0.00000034) and moderate disease (p=0.000009). Differences were also found among severe and mild disease (p=0.026). Differences found with other authors, indicate the need for local studies to identify genetic biomarkers of severity.

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.

Genetic variability of human metapneumovirus isolated from chilean children, 2003–2004

Human metapneumovirus (hMPV) is a significant cause of acute lower respiratory tract infection in all age groups, particularly in children. Two genetic groups and four subgroups of hMPV have been described. They co‐circulate during an epidemic in variable proportions. The aims were to characterize the genotypes of hMPV recovered from children hospitalized for acute lower respiratory tract infection and to establish the molecular epidemiology of strains circulating in Santiago of Chile during a 2‐year period. The detection of the N gene by reverse‐transcription polymerase chain reaction was carried out for screening 545 infants hospitalized for acute lower respiratory tract infection in Santiago during 2003–2004. The genetic typing of hMPV was performed by analyzing the fusion gene sequences. hMPV was detected in 10.2% (56/545 cases). Phylogenetic analysis of F gene sequences from 39 Chilean hMPV strains identified the two groups and four subgroups previously described. Strains clustered into group A were split further into the sub lineages A1, A2, and A3. Most Chilean strains clustered into the proposed novel A3 sub lineage (59%). A3 viruses were present in both years, while A1 and A2 circulated just in 1 year. In conclusion, hMPV is a relevant cause of acute lower respiratory infection in Chilean children and the potential novel cluster of group A emphasize the need for further regional genetic variability studies. J. Med. Virol. 81:340–344, 2009.