The community-acquired pneumonia (CAP) is the leading cause of morbidity and mortality worldwide. According to WHO estimates, 450 million cases of pneumonia are recorded each year, with about 4 million deaths from this illness [
Nowadays, the golden standard for CAP diagnosis is still based on the chest radiography; however, a broad range of chest radiographic changes could be induced by various agents, a single and dual bacterium/virus, or mixed pathogens coinfections. These alterations of chest radiography are only helpful in specific cases to confirm a microbial cause of pneumonia [
FilmArray Respiratory Panel (RP) is a multiplexed nucleic acid test for the simultaneous qualitative detection and identification of multiple respiratory virus,
In all specimens of patients from the first and the second affiliated hospitals of Shantou University Medical College, CAP was confirmed by radiological diagnosis. The nasal cavity samples and serum were collected from patients between January and September, 2016.
The specimens collected from nasal mucus were stored in swab storage solution (COPAN, Italy) for multiplex PCR and FilmArray RP assay. The nucleic acid was extracted by using beads viral DNA/RNA extraction kit (TIANLONG, China) and NP968 nucleic acid extraction instrument (TIANLONG, China). We tested 14 viruses, including influenza viruses types A and B (Flu A and Flu B), parainfluenza viruses 1 to 4 (PIV1–4), respiratory syncytial virus (RSV), coronaviruses 229E, OC43, HKU1, and NL63, human metapneumovirus (MPV), human rhinovirus (HRV), and adenovirus (ADV), were detected using a single-tube TaqMan® real-time reverse transcription PCR strategy (AgPath-ID™ One-Step RT-PCR Kit Applied Biosystems, USA). PCR amplification was performed using a 7500 Real-Time PCR System (Applied Biosystems, USA), according to the manufacturer’s instructions.
The nasal mucus specimens were cultured according to China national standard protocols to detect respiratory bacterial (or fungi). Colony identification was undertaken using a VITEK 2 Compact system (Biomérieux, France). Nine bacteria were measured, including
The patient’s serum was collected and placed in −80°C.
We used the BioFire™ Diagnostics Respiratory Panel and FilmArray™ multiplex PCR system (Biomérieux, USA) to detect the same 14 viruses and three other pathogens (
Data were analyzed in SPSS 19.0. The agreement between assays was measured using the kappa statistic. The sensitivity and specificity were also compared for both tests.
Patients were enrolled between January and September, 2016. This study was comprised of both adult (
Patients’ general characteristics.
Cases | |
---|---|
|
74 |
|
|
0–14 | 37 |
≥15 | 37 |
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|
Male | 44 |
Female | 30 |
|
|
Single virus | 26 |
Single bacterial (or fungi) | 10 |
Virus + bacterial (or fungi) | 14 |
Single |
5 |
Virus + |
1 |
Unidentified | 18 |
|
|
Antibiotic | 45 |
Antiviral | 7 |
Antiviral + antibiotic | 11 |
|
|
COPD | 1 |
Tuberculosis | 1 |
Asthma | 2 |
|
|
Diabetes | 4 |
High blood pressure | 14 |
Heart disease | 3 |
Tumor | 1 |
Hepatitis | 6 |
Epilepsy | 4 |
|
|
Mild-moderate | 47 |
Severity | 27 |
|
|
Cough | 56 |
Pharyngeal discomfort | 4 |
Snivel | 10 |
Fever | 42 |
Shortness of breath or dyspnea | 25 |
|
|
Death | 3 |
Alive | 71 |
COPD, chronic obstructive pulmonary disease.
The most prevalent were the RSV (15/74, 20.3%) and the HRV (9/74, 12.2%). Influenza viruses (A + B) were detected in 10 (10/74, 13.5%) samples. Coronaviruses (OC43, HKU1, and NL63) were detected in 9 (9/74, 12.2%) samples, PIV3 in 5 (5/74, 6.8%) samples, ADV in 3 (3/74, 4.1%) samples, and MPV in 2 (2/74, 2.7%) samples (Table
Comparison of positive and negative results in two methods in viral detection.
Virus | Number of specimens (+: positive; −: negative) | Sensitivity (%) | Specificity (%) | PPV (%) | NPV (%) | Kappa | |||
---|---|---|---|---|---|---|---|---|---|
PCR−/FA− | PCR+/FA+ | PCR+/FA− | PCR−/FA+ | ||||||
ADV | 71 | 0 | 0 | 3 | 0 | 95.9 | 0 | 100 | 0 |
229E | 74 | 0 | 0 | 0 | 0 | 100 | 0 | 100 | 1 |
OC43 | 67 | 7 | 0 | 0 | 100 | 100 | 100 | 100 | 1 |
HKU1 | 73 | 1 | 0 | 0 | 100 | 100 | 100 | 100 | 1 |
NL63 | 73 | 1 | 0 | 0 | 100 | 100 | 100 | 100 | 1 |
MPV | 72 | 2 | 0 | 0 | 100 | 100 | 100 | 100 | 1 |
HRV | 65 | 3 | 0 | 6 | 100 | 91.5 | 33.3 | 100 | 0.468 |
FluA | 66 | 8 | 0 | 0 | 100 | 100 | 100 | 100 | 1 |
FluB | 72 | 2 | 0 | 0 | 100 | 100 | 100 | 100 | 1 |
PIV1 | 74 | 0 | 0 | 0 | 0 | 100 | 0 | 100 | 1 |
PIV2 | 74 | 0 | 0 | 0 | 0 | 100 | 0 | 100 | 1 |
PIV3 | 69 | 4 | 1 | 0 | 80 | 100 | 100 | 98.6 | 0.882 |
PIV4 | 74 | 0 | 0 | 0 | 0 | 100 | 0 | 100 | 1 |
RSV | 59 | 10 | 2 | 3 | 83.3 | 95.2 | 76.9 | 96.7 | 0.752 |
FA, FilmArray RP; PPV, positive predictive value; NPV, negative predictive value.
For viruses 229E, OC43, HKU1, NL63, MPV, Flu A, Flu B, PIV1, PIV2, and PIV4, two methods had very good agreement with coincidence rate of 100%. Disagreement was found in 15 specimens, positive results were tested by only one of the two assays (FilmArray RP: 12 cases, multiple PCR: 3 cases). FilmArray RP positive/PCR negative were seen in 3 cases of ADV, 6 cases of HRV, and 3 cases of RSV, while multiple PCR positive/FilmArray RP negative were observed in 1 case of PIV 3 and 2 cases of RSV (Table
In the study, the FilmArray RP detected 50 positive viruses (ADV, 3; OC43, 7; HKU1, 1; NL63, 1; MPV, 2; HRV, 9; Flu A, 8; Flu B, 2; PIV3, 4; and RSV, 13, resp.). A total of 50 (100%) of the 50 pathogens were confirmed by real-time PCR; the comparison of FilmArray RP and multiple PCR for OC43, HKU1, NL63, MPV, Flu A, Flu B, PIV3, and RSV showed satisfactory agreement (kappa
Dual viral infections were detected in 12 (12/74, 16.2%) specimens (Table
The positive virus combination of FilmArray RP and multiplex real-time PCR in detecting dual viral infection.
Multiplex PCR assay | FilmArray RP | |
---|---|---|
Compositions | OC43 + HRV | OC43 + HRV |
Flu A + MPV | Flu A + MPV | |
RSV + HKU1 | RSV + HKU1 | |
RSV + PIV3 | RSV + PIV3 | |
RSV + NL63 | RSV + NL63 | |
RSV | RSV + |
|
RSV | RSV + |
|
RSV | RSV + |
|
RSV | RSV + |
|
Flu A |
| |
OC43 | OC43 + |
|
|
HRV |
Bacteria (or fungi) were detected by culture method in our study. The results showed that bacteria (or fungi) were positive in 25 (25/74, 33.8%) specimens. As FilmArray RP only detects
The positive pathogens of bacterial (or fungi) or
Bacterial (or fungi) or |
FilmArray RP (—: not detected) | Cases |
---|---|---|
Yeast | — | 6 |
Negative |
|
6 |
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— | 4 |
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— | 3 |
|
— | 2 |
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— | 2 |
|
— | 2 |
|
|
1 |
|
— | 1 |
|
— | 1 |
|
|
1 |
|
— | 1 |
Microorganism measurement is an integral part of CAP patient management. Accurate and rapid etiological diagnosis helps prevent secondary infection, prevent the use of unnecessary antibiotics, facilitate more timely use of antiviral drugs, and shorten hospital stays [
The multiplex real-time PCR can detect a broad spectrum of respiratory microorganisms promptly, which makes it possible for clinician to treat the patients with specific antimicrobial agents. However, the main limitations of PCR method include the handling of whole batch samples (at least 8 samples) for cost and labor saving, the different boxes to do extraction, the training of specialized biologist that may be the main reason of DNA/RNA extraction and amplification being easily contaminated, and results interpretation (the frequency of false positive or false negative results hinders the use of PCR in clinical practice). Thus, the PCR is not appropriate for instant point of care especially to some severe patients. Compared with multiple PCR, the measurement time of FilmArray RP needs only about 1.2 hours and there is no need to do the experiment in batch. The single test set makes it meet the requirement of clinician to decide whether the patients should be quarantined or not. For some virus, such as Flu A, rapid diagnosis may help prevent secondary spread, beneficial for preventing virus nosocomial transmission. In children and adults, neuraminidase inhibitors reduce median time to resolution of symptoms by 0.5–2.5 days when administered within 48 h of onset of symptoms [
Due to undeveloped immune system, mixed infections with two or more respiratory viruses are common in children but are not easily detected by conventional methods; hence, the biological significance of dual infections currently is not well understood [
For considering the cost of reagent, the FilmArray RP is more expensive than multiple PCRs; the reagent and consumable costs (in RMB) for the FilmArray RP and the multiple PCR were calculated to be $230 and $40 per patient, respectively. However, the FilmArray RP is essential for the administration of appropriate antiviral therapy, which dramatically shortens hands-on time (62.7 hours versus 1.2 hours); the reduction in average time to discontinuation of oseltamivir resulted in cost savings of approximately US$34.16 per patient [
In the last decades, the emergence of severe acute respiratory syndrome (SARS), avian influenza A (H5N1) virus, and the 2009 pandemic influenza A (H1N1) virus has reemphasized the important role of respiratory viruses as causes of severe pneumonia [
The study was approved by the Ethics Committee of Shantou University Medical College.
Written informed agreements were obtained from patients or their custodians.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Huanzhu Chen, Huilan Weng, and Meirui Lin are equal contributors.
This study was supported by the Department of Education, Guangdong Government, under the Top-Tier University Development Scheme for Research and Control of Infectious Disease.