Primary pneumocystis infection in infants hospitalized with acute respiratory tract infection.

Acquisition of Pneumocystis jirovecii infection early in life has been confirmed by serologic studies. However, no evidence of clinical illness correlated with the primary infection has been found in immunocompetent children. We analyzed 458 nasopharyngeal aspirates from 422 patients hospitalized with 431 episodes of acute respiratory tract infection (RTI) by using a real-time PCR assay. In 68 episodes in 67 infants, P. jirovecii was identified. The odds ratio (95% confidence interval) of a positive signal compared with the first quartile of age (7-49 days) was 47.4 (11.0-203), 8.7 (1.9-39.7), and 0.6 (0.1-6.7) for infants in the second (50-112 days), third (113-265 days), and fourth (268-4,430 days) age quartiles, respectively. Infants with an episode of upper RTI (URTI) were 2.0 (1.05-3.82) times more likely to harbor P. jirovecii than infants with a lower RTI. P. jirovecii may manifest itself as a self-limiting URTI in infants, predominantly those 1.5-4 months of age.

Human metapneumovirus and respiratory syncytial virus in hospitalized danish children with acute respiratory tract infection.

The newly discovered human metapneumovirus (hMPV) has been shown to be associated with respiratory illness. We determined the frequencies and clinical features of hMPV and respiratory syncytial virus (RSV) infections in 374 Danish children with 383 episodes of acute respiratory tract infection (ARTI). Study material comprised routine nasopharyngeal aspirates obtained during 2 winter seasons (November-May) 1999-2000 and 2001-2002 from children hospitalized at the Departments of Paediatrics, Hvidovre Hospital and Amager Hospital, Denmark. hMPV was detected in 11 (2.9%) and RSV in 190 (49.6%) ARTI episodes by real-time reverse transcription-polymerase chain reaction using primers targeting the hMPV N gene and the RSV L gene. Two children were co-infected with hMPV and RSV. They were excluded from statistical analysis. Hospitalization for ARTI caused by hMPV was restricted to very young children 1-6 months of age. Asthmatic bronchitis was diagnosed in 66.7% of hMPV and 10.6% of RSV-infected children (p < 0.001). Overall symptoms and clinical findings were similar among hMPV and RSV positive episodes, but more RSV-infected children required respiratory support. hMPV is present in young Danish children hospitalized with ARTI although less frequent than RSV and with a tendency to a milder clinical course.

A prospective, blinded study of quantitative touch-down polymerase chain reaction using oral-wash samples for diagnosis of Pneumocystis pneumonia in HIV-infected patients.

Oral-wash samples obtained during 113 episodes of suspected Pneumocystis pneumonia (PCP) in human immunodeficiency virus-infected patients were tested by use of a quantitative touch-down PCR (QTD PCR) assay. QTD PCR had a sensitivity of 88% and a specificity of 85%. Treatment for PCP prior to oral wash collection had an impact on the sensitivity, and PCR-positive oral-wash samples obtained within < or =1 day of treatment from patients without PCP had significantly fewer copies per tube than did those from patients with PCP; thus, application of a post hoc cut-off value of 50 copies/tube increased the specificity to 100%. QTD PCR of oral-wash samples can be an accurate and noninvasive method for diagnosis of PCP.

Development of a rapid real-time PCR assay for quantitation of Pneumocystis carinii f. sp. carinii.

A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r = 0.99) over 6 log values for standards containing > or =5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r = 0.99). The assay was applied to a recently reported in vitro axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments.

Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosing Pneumocystis carinii pneumonia.

A rapid (time to completion, <4 h, including DNA extraction) and quantitative touch-down (QTD) real-time diagnostic Pneumocystis carinii PCR assay with an associated internal control was developed, using fluorescence resonance energy transfer (FRET) probes for detection. The touch-down procedure significantly increased the sensitivity of the assay compared to a non-touch-down procedure. Tenfold serial dilutions of a cloned target were used as standards for quantification. P. carinii DNA has been detected in respiratory specimens from patients with P. carinii pneumonia (PCP) and from patients without clinical evidence of PCP. The latter probably represents colonization or subclinical infection. It is logical to hypothesize that quantification might prove helpful in distinguishing between infected and colonized patients: the latter group would have lower copy numbers than PCP patients. A blinded retrospective study of 98 respiratory samples (49 lower respiratory tract specimens and 49 oral washes), from 51 patients with 24 episodes of PCP and 34 episodes of other respiratory disease, was conducted. PCR-positive samples from colonized patients contained a lower concentration of P. carinii DNA than samples from PCP patients: lower respiratory tract samples from PCP and non-PCP patients contained a median of 938 (range, 2.4 to 1,040,000) and 2.6 (range, 0.3 to 248) (P < 0.0004) copies per tube, respectively. Oral washes from PCP and non-PCP patients contained a median of 49 (range, 2.1 to 2,595) and 6.5 (range, 2.2 to 10) (P < 0.03) copies per tube, respectively. These data suggest that this QTD PCR assay can be used to determine if P. carinii is present in respiratory samples and to distinguish between colonization and infection.