Patients
We conducted a retrospective and consecutive study in the Children’s Hospital of Chongqing Medical University, China, from April 2009 to February 2013. For the retrospective study, 6063 male and female children between 1 day and 6 years old were hospitalized for respiratory infection, and 2201 children had pneumonia. There was a total of 509 children conforming to the inclusion criteria (see “Interpretation” section below), and they were included in the retrospective study. From February 2013 to August 2013, blood, urine, and respiratory specimens (sputum or BALF) of 500 children with pneumonia were collected, and 186 of them conformed to the inclusion criteria for the prospective study (see “Interpretation” section below). The study was approved by the Ethics Committee of the Children’s Hospital of Chongqing Medical University and the reference number was chcmu-20090005. Written informed consent was obtained from the parents or legal guardians of the patients.
Data collection
Clinical and laboratory data were collected from the medical records of the patients. The clinical data included sex, age, season, duration of illness, symptoms, signs, and X-ray image information. Laboratory data included peripheral leukocytes, neutrophil proportion, lymphocyte proportion, monocyte proportion, eosinophil proportion, copy number of HCMV DNA, and specific IgM antibody titer for HCMV.
Interpretation
The inclusion criteria for the retrospective study were as follows. (1) Patients were included who were confirmed as having pneumonia with typical symptoms, such as cough, wheezing, sore throat and fever, as confirmed by X-ray [16]; (2) Patients were included who had a normal immune system [including WBC count, absolute lymphocyte count, and proportion of individual subpopulations of lymphocytes, such as T(CD3+), Th(CD4+), and Ts(CD8+)], as detected by flow cytometry (BD Multitest IMK Kit, USA)] [17] (data not shown); (3) Patients were included who were infected by virus alone. Sputum or blood culture was used to show no infection by bacteria, fungus, or Mycoplasma pneumoniae (MP) (Additional file 1: Table S1). A chemiluminescent assay (Abbott Ireland Diagnostics Division, Sligo, Ireland) and respiratory virus antigen detection kit (Diagnostic Hybrids Inc., OH, USA) were used to screen for respiratory virus infection (Additional file 1: Table S2). Chlamydia pneumoniae (CP) was detected by a commercial quantitative diagnostic kit with PCR fluorescence probing (Da’an Gene Co., Ltd., Guangzhou, China); (4) There was no evidence to show that the patients suffered from severe concomitant diseases and nosocomial infections; (5) The patients did not receive any antibiotics or anti-viral drugs 48 h before diagnosis; (6) Patients could be included under the condition that they only had pulmonary HCMV infection, but did not have cytomegalovirus hepatitis or encephalitis. The inclusion criteria for the prospective study were the same as those for the retrospective study.
The guidelines for diagnosis of HCMV pneumonia were based on Paya et al.’s report [18]. Briefly, the patients had typical symptoms of pneumonia. X-rays showed evidence of diffuse lesions. Sputum and blood culture were used to prove that patients were free of bacterial, fungal, and MP infections. The results of PCR, chemiluminescent assay, and detection of virus antigen were used to show that there was no CP or other viral infections. HCMV IgM or HCMV DNA was positive and HCMV IgG was negative. Moreover, antibiotic treatment was invalid, but ganciclovir treatment was valid. Acute HCMV infection in our study was defined as follows: those who were free of bacterial, MP, CP, and fungal infections (shown by sputum and blood culture, and PCR), as well as infection from other viruses (shown by chemiluminescent assay or positive respiratory virus antigen detection); positive HCMV IgM or positive HCMV DNA and negative HCMV IgG; and those receiving invalid antibiotic treatment, but valid ganciclovir treatment. Latent HCMV infection was defined as follows: those who were HCMV IgM-negative, HCMV IgG-positive, and with other viral infections (shown by chemiluminescent assay or positive respiratory virus antigen detection); those who were free of bacterial, MP, CP, and fungal infection (shown by sputum and blood culture, and PCR); and improvement without receiving ganciclovir.
According to the presence of HCMV infection, the patients were classified as the HCMV-infection group, bi/tri-virus-infection group (multiple viral infections), and the non-HCMV-infection group.
Real-time PCR for HCMV DNA
Detection of the copy number of HCMV DNA was performed by using the HCMV DNA quantitative diagnostic kit with PCR fluorescence probing (Da’an Gene Co., Ltd.), as previously reported [19]. This technique is based on TaqMan PCR technology, and the target sequence is in a highly conserved fragment of the HCMV genome (strain AD169).
Briefly, 1 ml of urine specimen was centrifuged at 13,000 × g. Urine supernatant was discarded and the pellet was collected and mixed with 50 μl of DNA extraction solution. The supernatant was then collected. Respiratory specimens were liquefied with 4% NaOH, and then centrifuged at top speed. The pellet was collected and washed. The pellet was then mixed with 50 μl of DNA extraction solution, and the supernatant was collected.
A volume of 2 μl of the supernatant and 43 μl of PCR mix (Da’an Gene Co., Ltd.) were used to perform real-time PCR, using the Applied Biosystems 7500 real-time PCR system (Applied Biosystems, CA, USA) as follows: 93°C for 2 min, 10 cycles of 93°C for 45 s, and 55°C for 60s, followed by 30 cycles of 93°C for 30 s and 55°C for 45 s. For each assay, we used positive viral load standards (104, 105, 106, and 107 copies/mL), plasmid target sequence (500 copies/ml) as a positive control, ddH2O as a blank control, and plasmid without target sequence as a negative quality control. HCMV DNA >500 copies per ml of specimen was regarded as positive.
Chemiluminescent microparticle immunoassay for HCMV IgM antibody
Peripheral blood specimens were used for serological tests. HCMV-specific antibody was detected using a commercial kit (Architect CMV IgM 6C16, Abbott, Sligo, Ireland) according to the manufacturer’s instructions [20]. The Architect CMV IgM assay is a two-step immunoassay for the qualitative detection and semi-quantitative determination of IgM antibodies to HCMV in human serum and plasma. The results were measured as relative light units. If the chemiluminescent signal in the specimen was equal to or greater than 1.00, anti-CMV IgM was considered positive. If the signal in the specimen was lower than 0.85, anti-CMV IgM was considered negative. If the results were uncertain, we examined a second sample within a reasonable period of time (e.g., 2 weeks) to confirm the results. In addition, RSV, adenovirus, and Epstein-Barr virus were tested by chemiluminescent assay kits from Abbott.
Real-time PCR for the pp65 gene in clinical specimens
A total of 186 suspected HCMV-positive cases were included. Urinary and respiratory specimens of these patients were collected and DNA was extracted. The DNA purification process of urinary and respiratory specimens was the same as that mentioned above (“Real-time PCR for HCMV DNA” section). A volume of 2 μl of the resulting supernatant and 18 μl of PCR mix were used to perform real-time PCR. The PCR mixture contained 8 μl 2.5 × Real-MasterMix, 1 μl probe enhancer (Tiangen Biotech, Beijing, China), 1 μl internal TaqMan probe (20 μmol/L), 4 μl nuclease-free water, and 2 μl forward and reverse primers (10 μmol/L) each.
The sequence of the pp65 gene was from Genebank (AY301013). The forward primer was 5′-CCC TCC GGC AAG CTC TTT-3′ and the reverse primer was 5′-CAG GTC CTC TTC CAC GTC AGA-3′. The internal TaqMan probe 5′-TGC ACG TCA CGC TGG-3′ was labeled at the 5′ end with 6-carboxy-fluorescein as the reporter and at the 3′ end with minor groove binding as the quencher [21]. The primers and probe were synthetized by Invitrogen (Life Technologies Corporation, Shanghai, China). Purified genomic DNA from the HCMV-positive specimen was the template and was amplified by PCR. The PCR product was sequenced to confirm whether the sequence was the exact target sequence, and was then ligated to the pBackZero-T vector (TaKaRa, Dalian, China); this was called the pp65-plasmid. The pp65-plasmid concentrations were measured by ultraviolet spectrophotometry to calculate the copy number of the plasmid, and to further obtain viral load standards (104, 105, 106, and 107 copies/mL) and the positive quality control (103 copies/ml) for real-time PCR. Moreover, ddH2O as a blank control, and plasmid without a target sequence as a negative quality control, were prepared and used for each assay. PCR was performed using the 7500 real-time PCR system (Applied Biosystems, CA, USA) under the following conditions: one cycle of 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 55°C for 45 s. All of the standards, controls, and samples were run in triplicate.
Statistical analysis
Quantitative data are expressed as mean ± SD if they followed a normal distribution, or are described as medians and interquartile ranges (25th–75th). Categorical data are expressed as frequencies and percentages. The relationships between acute and chronic pneumonia, pathological classification, sex, age, and HCMV infection were evaluated by logistic regression analysis. Differences in clinical symptoms of categorical variables were determined by the chi-square test. Differences in WBCs, duration of illness, and vital signs of categorical variables were determined using analysis of variance. Paired proportions of discordant serological and PCR results in the different groups were compared with the clinical diagnosis. Paired PCR results of different specimens were tested by McNemar’s test. The receiver operating characteristic curve was used to predict the pp65 gene as a marker for identifying acute HCMV infection or latent HCMV infection in children with pneumonia. Data were analyzed by SAS 9.13 (SAS Institute, Cary, NC, USA).