Human parechovirus as a minor cause of acute otitis media in children

Human parechoviruses (HPeVs) cause mild upper respiratory infections, gastrointestinal symptoms, central nervous system infections and some studies have linked them with acute otitis media (AOM). The aim of the present study was to study further the role of HPeV infections in AOM by detecting these viruses directly from middle ear fluid (MEF), respiratory and stool samples collected from children during AOM episodes. A total of 91 MEF samples, 98 nasal swab (NS) samples and 92 stool samples were collected during 100 AOM episodes in a total of 87 children aged between five to 42 months. All specimens were analyzed by real time RT-PCR for the presence of HPeV RNA. HPeV infection was diagnosed in 12 (14%) patients. HPeV RNA was detected in altogether 13 samples, including four MEF samples, three NS samples and six stool samples. One patient was positive in both stool and MEF samples. The results suggest that HPeV may play a role in some AOM cases, but it is not a major cause of AOM in children.     

Mycoplasma pneumoniae infections: retrospective study in Basse-Normandie, 1997-2005. Epidemiology--diagnostic utility of serology and PCR for a rapid diagnostic

OBJECTIVES: The objective of this study was to evaluate the epidemiology of Mycoplasma pneumoniae (Mpn) infections in Basse-Normandie by a retrospective analysis of serological and PCR data, and to confirm the diagnostic utility of PCR and serology. METHODS: From 1997 to August 2005, 6156 serum samples and 6123 respiratory tract samples were collected from hospitalised patients and evaluated for the diagnosis of Mpn infection by PCR, serological assays, or by the two tests. During the epidemic period (2004-2005), the results of 1489 patients were analysed. RESULTS: Over the 9-y period, the seroprevalence was 40,4% and we reported on 525 cases with serologically or/and PCR proven Mpn infection, according a cyclic pattern spaced out 7 years. During the epidemic period, the seroprevalence increased to 50,2% and the rate of infections was 8.3%. The analysis of the 124 cases of Mpn infection showed typical epidemiological characteristics: a peak of incidence among the children and young adults, a summer-winter pattern and some coinfections with viral strains. For diagnosis of Mpn infection, the comparison of PCR and serological assays among 36 patients showed a concordance of only 41.7%. CONCLUSION: Mpn infections were endemic and outbreaks were observed according cyclic pattern with a high incidence specially in the children. Sensitive and specific tests were now available for early and reliable diagnosis. In children, the combination of the PCR on nasopharyngeal samples and the IgM EIA serology test were recommended. In adults, the PCR was privilegiated.     

Comparison of PCR assay with bacterial culture for detecting Streptococcus pneumoniae in middle ear fluid of children with acute otitis media.

We have studied etiological diagnosis of acute otitis media (AOM) by comparing a newly developed pneumococcal PCR for Streptococcus pneumoniae to bacterial culture with 180 middle ear fluid (MEF) samples of 125 children with 125 episodes of AOM. For pneumococcal PCR assay, DNA from MEF samples was extracted by phenol-chloroform. The outer primers used amplified a 348-bp region of the pneumolysin gene, and the inner primers amplified a 208-bp region. S. pneumoniae was cultured in 33 (18%) samples, and pneumolysin PCR was positive for 51 (28%) of 180 MEF samples. Only 2 of 21 PCR-positive, S. pneumoniae culture-negative samples were positive for other otitis pathogens. By combining MEF culture and PCR results, 54 (30%) of 180 MEF samples had evidence of pneumococcal etiology. In conclusion, pneumolysin PCR is a sensitive and specific new method to study pneumococcal involvement in MEF samples of children with AOM.     

Comparison of serological and real-time PCR assays to diagnose Bordetella pertussis infection in 2007

Bacterial culture for diagnosing pertussis infection has high specificity but poor sensitivity and is slow. Highly sensitive real-time PCR assays and single-serum pertussis serology have been developed to overcome these limitations, but there are few data available on the relative sensitivities and specificities of such assays for pertussis diagnosis. Using data on 195 participants (>or=7 years old) from an epidemiological study, we assessed the sensitivity, specificity, and performance (Youden index) for pertussis diagnosis of the pertussis toxin enzyme-linked immunosorbent assay (using single and paired serology) and of real-time PCR assays (using the IS481 and ptxA-Pr targets). All available diagnostic information (clinical and laboratory) was pooled to serve as the gold standard. Single serology was the most efficient diagnostic test (Youden index, 0.57 to 0.58), with relatively high sensitivity (>64%) and high specificity (>90%), independent of the cutoff level. IS481 PCR performance was superior to that of ptxA-Pr PCR, and it was the second-most-efficient tool (Youden index, 0.30). Performing both ptxA-Pr and IS481 PCRs did not improve diagnostic performance. The greatest test efficiency (Youden index, 0.69 to 0.74) was achieved when single-serum serology was used in combination with IS481 or ptxA-Pr PCR or paired serology. Combining single serology with one PCR or paired serology increased the sensitivity with an associated limited decrease in specificity. The most specific tests for diagnosis of pertussis were single serology and ptxA-Pr PCR, and the most sensitive diagnostic tool was the combination of IS481 PCR with single serology.     

Higher levels of mucosal antibody to pneumococcal vaccine candidate proteins are associated with reduced acute otitis media caused by Streptococcus pneumoniae in young children

Mucosal immunity has a crucial role in controlling human respiratory tract infections. This study characterizes the naturally acquired mucosal antibody levels to three Streptococcus pneumoniae (Spn) protein antigens, pneumococcal histidine triad protein D (PhtD), pneumococcal choline binding protein A (PcpA), and pneumolysin (Ply), and assesses the association of the mucosal antibody levels with occurrence of acute otitis media (AOM) caused by Spn. Both nasopharyngeal (NP) immunoglobulin G (IgG) and IgA levels to all three proteins slightly decreased in children from 6 to 9 months of age and then gradually increased through 24 months of age. Spn NP colonization was associated with higher mucosal antibody levels to all three proteins. However, children with Spn AOM had 5–8-fold lower IgG and 3–6-fold lower IgA levels to the three proteins than children without AOM but asymptomatically colonized with Spn. Antigen-specific antibody levels in the middle ear fluid (MEF) were correlated with antibody levels in the NP. Children with AOM caused by Spn had lower antibody levels in both the MEF and NP than children with AOM caused by other pathogens. These results indicate that higher naturally acquired mucosal antibody levels to PhtD, PcpA and Ply are associated with reduced AOM caused by Spn.     

Evaluation of an Immunoglobulin G Enzyme-Linked Immunosorbent Assay for Pertussis Toxin and Filamentous Hemagglutinin in Diagnosis of Pertussis in Senegal

The enzyme-linked immunosorbent assay is widely employed for the serological diagnosis of pertussis. It is generally concluded that a significant increase in specific immunoglobulin G (IgG) or IgA against the pertussis toxin (PT) or against filamentous hemagglutinin (FHA) in paired sera correlates with Bordetella pertussis infection. However, this type of diagnosis of pertussis has mainly been applied to unvaccinated children, with timely sampling of acute- and convalescent-phase sera. In current practice and in epidemiological studies, such criteria are not always fulfilled. The aim of this study was to analyze the significance of decreases in IgG antibody titers against PT and FHA between paired sera observed in suspected cases of pertussis infection. Serological results from paired sera were available for 460 children experiencing at least 8 days of cough. An anti-PT IgG decrease was observed in 25% of the children, more frequently than the anti-FHA IgG decrease. Fourteen percent of the serologic decreases were observed in children with culture-confirmed infection, and 59% of the decreases were observed in children with confirmation criteria according to World Health Organization recommendations. Most of the decreases were observed when serum samples were collected according to a standard recommended schedule. Serologic decreases were observed more frequently among vaccinated children than among unvaccinated children. This difference, which was highly significant (P < 0.00001), was explained by the different kinetics of the antibody responses between vaccinated and unvaccinated children. The importance of the antibody response for the evaluation of vaccine efficacy, namely a bias toward higher absolute vaccine efficacy when this response is not taken into account, is discussed. This study supports an earlier recommendation that a significant decrease in PT or FHA should be added to the diagnostic criteria for pertussis.     

Serum intercellular adhesion molecule 1 variations in young children with acute otitis media

Acute otitis media (AOM) is an inflammatory reaction in the middle ear, most often occurring in young children. Streptococcus pneumoniae, nontypeable Haemophilus influenzae, and Moraxella catarrhalis are the most common bacteria isolated. Intercellular adhesion molecule 1 (ICAM-1) is involved in the innate immune response to infection by microorganisms, in effective antigen presentation, and in subsequent T-cell activation. Here we prospectively studied levels of serum soluble ICAM-1 (sICAM-1) before, at the time of, and after antimicrobial treatment of AOM in a group of 138 children ages 6 to 30 months. Middle ear fluids were collected by tympanocentesis to identify otopathogens. We found that (i) serum levels of sICAM-1 were significantly higher in S. pneumoniae-, nontypeable H. influenzae-, and M. catarrhalis-infected children than in well children (P < 0.001), confirming that a systemic inflammatory response occurs during AOM; (ii) sICAM-1 levels varied from no elevation (110 ng/ml) to elevation to high levels (maximum, 1,470 ng/ml) among children with AOM; (iii) in paired samples, sICAM-1 levels increased 4- to 20-fold when children developed AOM compared to their sICAM-1 levels before infection; and (iv) the level of sICAM-1 returned to the pre-AOM level at the convalescent stage of AOM after successful antimicrobial therapy. We conclude that AOM often causes a systemic inflammatory reaction, as measured by elevation of the serum sICAM-1 level, and that a high variability in sICAM-1 responses occurs with the presence of otopathogens during AOM.     

Evaluation of the solid-phase radioimmunoassay for diagnosis of St. Louis encephalitis infection in humans.

Since the use of highly purified viral proteins in the solid-phase radioimmunoassay (SPRIA) procedure is not practical for routine diagnostic serology, the procedure was adapted for use with the crude antigens now commonly used to diagnose St. Louis encephalitis (SLE) virus infections. SLE and various other alpha- and flavivirus antigens were tested by both the SPRIA procedure and conventional serological methods. Standards were established for use of the crude antigens in the SPRIA procedure. Reproducibility of the procedure was similar to that of conventional tests. The SPRIA procedure specifically differentiated SLE from clinically similar alphaviruses, but did not differentiate SLE from related flaviviruses very well. SPRIA diagnosis of SLE infections by the immunoglobulin G response in paired sera was good. Generally, the crude antigen SPRIA procedure was as sensitive as the conventional serological tests, but not as specific.     

Validation of nested Bordetella PCR in pertussis vaccine trial.

A nested PCR, using a 239-bp sequence in the pertussis toxin promoter region, was developed and evaluated. The assay differentiates Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica by restriction enzyme analysis of the amplified fragments. The diagnostic performance of the PCR was evaluated in a Swedish pertussis vaccine efficacy trial which took place from 1992 to 1995, including study children and household members and using culture and serology for laboratory confirmation of suspected cases. In total 2,421 nasopharyngeal aspirates were analyzed. The total diagnostic sensitivity for B. pertussis was 90.2% (194 of 215). During the study period samples were processed with and without the cation-exchange resin Chelex. The PCR diagnostic sensitivity for B. pertussis among the Chelex-treated aspirates was 94.9% (75 of 79), and that for B. pertussis among 124 aspirates in a consecutive non-Chelex-treated material was 89.5% (111 of 124). After Chelex treatment of the 13 PCR-negative samples, an additional six became PCR positive, giving a final sensitivity of 94.3%. In addition, PCR was positive for B. pertussis with 57 of 1,744 samples negative by culture but with available serological data. The specificity of PCR with these samples was supported by a significant increase in antibody levels between acute and convalescent sera in 45 cases and by epidemiological or clinical data in all but two of the remaining cases. PCR was also positive for B. pertussis with 26 of 415 aspirates from episodes lacking serology. The diagnostic sensitivity of PCR for B. parapertussis was 74.0% (37 of 50). There were an additional seven culture-negative B. parapertussis PCR findings, six from cases with significant antibody increases against filamentous hemagglutinin only and one from a case lacking serology. There were no samples positive for B. bronchiseptica. In conclusion, PCR detection of B. pertussis and/or B. parapertussis enabled us to identify 90 positive nasopharyngeal aspirates, in addition to the 262 culture-positive samples (an increase of 34%). Relating these cases to serology and clinical data indicated a PCR specificity approaching 100%.     

Correlation of serum antibody titres with invasive methods for rapid detection of Helicobacter pylori infections in symptomatic children

Helicobacter pylori (H. pylori) is causally associated with peptic ulcer disease and gastric carcinoma. Typically, children get infected during the first decade of life, but diseases associated with H. pylori are seen mainly in adults. Multiple diagnostic methods are available for the detection of H. pylori infection. The aim of this study was to evaluate the correlation and diagnostic accuracy of three invasive methods [rapid urease test (RUT), histology and bacterial culture] and one non-invasive method (IgG serology) for diagnosis of H. pylori infection in a prospective cohort study conducted on 50 symptomatic children between two and eighteen years of age. Endoscopies with gastric biopsies were performed for RUT, culture and histopathological examination, respectively. IgG antibodies were measured in patient sera using a commercially available enzyme-linked immunosorbent assay (ELISA). RUT and positive H. pylori IgG antibodies were concordant in 88% (44/50) of patients. Both tests were negative in 32% (16/50), and both were positive in 56% (28/50). Disagreement occurred in 12% (6/50) of the patients: three of them (6%) had positive RUT and negative H. pylori IgG, and another three (6%) had negative RUT and positive H. pylori IgG. A combination of RUT with non-invasive serology constituted the optimum approach to the diagnosis of H. pylori infection in symptomatic children. The non-invasive serological test (ELISA) could not be used alone as the gold standard because it cannot distinguish between active and recently treated infection; and bacterial culture could not be used alone because of its low sensitivity.     

Comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children.

BACKGROUND: The quality of clinical specimens is a crucial determinant for virological diagnosis. OBJECTIVES: We compared the viral diagnostic yield for influenza A and respiratory syncytial virus (RSV) from the recently developed nasopharyngeal flocked swabs (NPFS) with nasopharyngeal aspirates (NPA) collected in parallel from 196 hospitalized children with acute respiratory infection during the peak period of influenza A and RSV activity in Hong Kong. Specimens were tested by RT-PCR for influenza A and RSV and viral load determined. They were also tested by direct immunofluorescence (DIF) for influenza A and B, RSV, parainfluenza types 1-3 and adenovirus. RESULTS: Both NPA and NPFS had excellent sensitivity (100%) for detecting influenza A by RT-PCR but NPA was slightly more sensitive than NPFS for detecting RSV by both RT-PCR (100% vs. 92.3%) and DIF (87.2% vs. 84.6%) and for detecting influenza A by DIF (90.2% vs. 82.9%). Viral load for influenza A in NPA and NPFS was not significantly different but that for RSV was higher in NPA. CONCLUSION: NPA remains the optimal specimen for diagnosis of respiratory infections by RT-PCR and DIF. However, collection of NPFS is easier to perform in an out-patient setting, was more acceptable to parents and less likely to generate aerosols than NPA engendering potentially less infection control hazard.     

Most human metapneumovirus and human respiratory syncytial virus in infant nasal secretions is cell free.

BACKGROUND: Nasopharyngeal secretions aspirated from infants with bronchiolitis (NPA) are a valuable resource for the study of virus dynamics and local inflammatory responses, however samples are small and difficult to manipulate. OBJECTIVES: To improve yield of NPA from infants. To establish if removal of the cellular component of NPA affects quantification of human metapneumovirus (hMPV) or human respiratory syncytial virus (hRSV) genome. STUDY DESIGN: Weight of NPA collected into traps from 30 infants was compared with that collected in trap plus catheter and washed through with saline from another 30 infants. hMPV (n=33) and hRSV (n=30) genome was measured by reverse-transcribed real-time polymerase chain reaction (RT-RT-PCR) in paired whole and cell-free NPA collected by the improved method. RESULTS: The improved method of NPA collection gave near two-fold greater weight (p = 0.002) of NPA (mean = 0.52 g (S.D. = 0.30 g)) than the traditional method (0.32 g (S.D. 0.19)). There was strong agreement and no significant difference between viral load measured in whole and cell-free fractions of NPA for both viruses (samples (n), correlation coefficient (cc) and significance (p)); hMPV (n=33, cc=0.938, p<0.001) and hRSV (n=30, cc=0.977 and p<0.001). CONCLUSIONS: The majority of hRSV and hMPV in nasal secretions is not associated with cells. Removal of the cellular component of NPA does not interfere with quantification of hRSV and hMPV.     

Dual-label time-resolved fluoroimmunoassay for the simultaneous detection of adenovirus and rotavirus in faeces

Dual-label time-resolved fluoroimmunoassay (TR-FIA) was used for the simultaneous detection of adenovirus and rotavirus in stool specimens. A one-incubation 1 h assay was used. Altogether 169 stool specimens collected from hospitalized children with acute gastroenteritis were tested. The specimens were dispensed with europium and terbium labelled antibodies against adenovirus and rotavirus, respectively, to coated microtitration strip wells. The 11 specimens positive for adenovirus in the EIA were also found positive in the TR-FIA. Fifty-six specimens positive for rotavirus with the reference EIA and one additional specimen were positive with the dual-label TR-FIA. Although the simultaneous assay using terbium and europium chelates gave lower assay sensitivities for both adenovirus and rotavirus when compared to separate assays using only europium chelates, the sensitivities were found to be at least equal to those of conventional standard antispecies EIA. With this one-incubation dual-label TR-FIA the results were obtained in less than 1.5 h for two viruses.     

Determination of human IgG and IgM class antibodies to West Nile virus by enzyme linked immunosorbent assay (ELISA)

An enzyme-linked immunosorbent assay (ELISA) was developed and used for the detection of IgG and IgM antibodies to West Nile virus in human sera. Thirteen paired sera of clinical cases and 24 control sera taken randomly from a blood bank repository were tested. The sera were reacted in microtiter plates coated with PEG-treated WNV antigen. IgG or IgM antibodies were quantitated by the use of alkaline-phosphatase-conjugated anti-human IgG or IgM antibodies. Of the 24 randomly collected serum samples, 7 were positive in the IgG-ELISA test. One positive by the IgM-ELISA was found to contain rheumatoid factor. In 12 of 13 paired sera of clinical cases, IgM as well as IgG antibodies were detected in the second serum sample taken about 3 wk after the onset of clinical signs. The IgM positive sera were screened for rheumatoid factor (RF) on IgG-coated plates. None of them contained RF. Antibody titers obtained by ELISA showed a good correlation with titers obtained by hemagglutination inhibition, complement fixation, and neutralization tests. The ELISA tests for detection of IgM and IgG antibodies to WNV therefore can replace the other serological methods for epidemiological surveillance and diagnostic purposes.     

Diagnosis of respiratory syncytial virus infection in children: Comparison of viral antigen detection and serology

Direct detection of viral antigen in nasopharyngeal secretion by radioimmunoassay was compared with serology by IgG antibody enzyme immunoassay for diagnostic efficacy in 77 children with clinically suspected respiratory syncytial virus (RSV) infections. Antigen detection gave a positive diagnosis in 26 of 33 (79%) children in whom RSV infection was diagnosed by any of the two methods. The diagnostic efficacy of antigen detection was dependent upon the interval after onset at which specimens were collected; 88% of specimens taken during the first 5 days and 50% of specimens taken 6–10 days after onset of illness were positive. It was also dependent on the age of the patients, the diagnostic efficacy being 88 and 76% in children under and over 6 months of age, respectively.     

Comparison of polymerase chain reaction, culture, and western immunoblot serology for diagnosis of Bordetella pertussis infection.

Polymerase chain reaction (PCR) amplification of the pertussis toxin promoter region was used to detect Bordetella pertussis infection in nasopharyngeal aspirates collected from 24 infants and children infected with pertussis and 13 adult contacts during an epidemiological study. The sensitivity of this PCR assay was approximately one bacterium, and the assay was specific for B. pertussis in tests with other Bordetella species and other respiratory pathogens. The pertussis case definition required a cough with a duration of more than 21 days for infants and children and laboratory confirmation by serology as the primary detection method for infants, children, and adults. The sensitivity of PCR and culture on Bordet-Gengou agar medium was assessed with regard to the case definitions. In the group of infants and children (index cases), the sensitivities of the culture and the PCR were 54.1% (13 of 24) and 95.8% (23 of 24), respectively. In the adult group (household contacts), the sensitivities of the two methods were 15.4% (2 of 13) and 61.5% (8 of 13), respectively. PCR combined with pertussis-specific serology appears to be a useful tool for diagnosis of pertussis especially in epidemiological studies.