1. INTRODUCTION
Hospitalized community acquired pneumonia (CAP) is a widespread disease that could affect all ages and genders, which is mostly caused by bacteria and viruses [1,2]. However, defining the pathogenic bacteria and viruses responsible for CAP is challenging because the patients’ sputum (or phlegm) is easily contaminated when passing through oropharynx. Therefore, traditional culture technique has several limitations [3]. Traditional culture technique cannot detect atypical bacteria as well as viruses. In addition, patients often using antibiotics before hospitalization, potentially leading to the destruction of bacteria in the sputum samples while they may still exist in alveolar or bronchial epithelial fluid; a lack of suitable environment to isolate primary bacteria, particularly Streptococcus pneumoniae, Haemophilus influenzae. To overcome those difficulties, we implemented multiplex real-time PCR (MPL-rPCR) technique, known for its high sensitivity and specificity. MPL-rPCR not only enables simultaneous detection of causative bacteria and viruses but also quantifies their quantity to define the primary, thereby delineating and the combined pathogenic agents (co-infection).
Our aims were: (1) to determine the proportion of bacteria and viruses causing CAP in hospitalized adult patients. (2) to determine rate of bacterial and viral combinations.
2. MATERIALS AND METHODS
This study utilized a prospective cross-sectional descriptive design, conducted on adult patients with CAP hospitalized at Respiratory department of Nguyen Tri Phuong Hospital, Nhan Dan Gia Dinh Hospital and University Medical Center from 04/2021 to 03/2023.
Sample selection criteria involved the collecting sputum samples from hospitalized CAP patients diagnosed by clinical doctors according to the Ministry of Health standards specified in Decision No. 4815/QD-BYT. These sputum samples were then transferred to Nam Khoa Company’s Laboratory, where the authors conducted analyses to identify the causative agents. Exclusion criteria included sputum samples from hospitalized CAP patients with lung cancer, advanced tuberculosis, human immunodeficiency virus (HIV) infection, or undergoing treatment with immunosuppressive drugs. Sputum samples collected from the same patient during the treatment period were also excluded.
Deviation control: Strictly comply with diagnostic standards and classification of underlying diseases; select samples based on the Barlett scale (≥2 point); strictly implement exclusion criteria and perform testing procedures according to the standard procedures of Nam Khoa Biotek Company’s Laboratory. For ethical considerations, we only worked with patients’ sputum samples at Nam Khoa Biotek’s Laboratory. The researcher did not get in touch with patients or interfered with the doctors’ treatment process. The Independent Ethics Committee (IEC) of the University of Medicine and Pharmacy HCMC approved our study at Decision No 330/DHYD-HDDD, issue: June 14th, 2019.
This manuscript was prepared and written in accordance with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines [4]. The STROBE check list of the manuscript is described in the supplementary document.
We exclusively examined sputum samples which rated at or above 2 points on the Barlett scale. These samples were transported to Nam Khoa Company’s laboratory for analysis using both traditional culture technique as well as by MPL-rPCR. With the MPL-rPCR method, the nucleic acid was extracted by DNARNAprep-MAGBEAD drug (belongs to Nam Khoa Co., Ltd, Ho Chi Minh City, Vietnam) and the King Fisher FLEX machine (from Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, these DNA extracts were combined with MPL-rPCR mixes specific for bacterial pathogens causing pneumonia (Nam Khoa Co., Ltd) and subject to detection and quantification of the target nucleic acid using CFX 96TM real-time PCR machine (from Biorad, Herculanes, CA, USA). All bacteria with numbers ≥100,000 copies were recognized as pathogens. If atypical bacteria and viruses are detected in sputum samples (regardless of quantity), they are recorded as pathogens. Whichever bacteria were counted with highest number was the main pathogen while the others are combined agents [2]. Bacteria identified by traditional culture technique were all identified as pathogens, regardless of whether they were the main agent or a combination agent.
Study size :
In which : Z = 1.96 (Standard distribution table) p = 0.69 (based on REAL study 2016–2017) [5] d : is the error, with the expectation of a reliability of 95%, choose an error of 5% = 0.05
So,
The number of sputum samples we collected at Nguyen Tri Phương hospital (101 sputum samples), Nhan dân Gia Định hospital (172 sputum samples) and University Medical Center (68 sputum samples). A total of 341 sputum samples (equal 341 patients) were analyzed.
3. RESULTS
There were 341 sputum samples from 341 CAP patients that met the criteria presented above.
The demographic data and the results in bacterial and viral detection by MPL-rPCR technique were shown in Table 1.
Table 1 above indicates that the proportion of causative bacteria in CAP was 67.7%, while the proportion of causative viruses was 57.5%. The difference of these percentages was found to be statistically significant (p<0.05). Furthermore, the relationships between females with males, between age group≤60 years with age group>60 years were also statistically significant (p<0.05).
The causative bacterial pathogens and viruses in CAP detected by MPL-rPCR from sputum samples of 341 hospitalized patients were shown in Table 2.
1) The percentage was over 100% since in many cases, more than one bacteria or virus were found in one sputum of CAP patients. MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-susceptible Staphylococcus aureus; MRSE, methicillin-resistant Staphylococcus epidermidis; CAP, community-acquired pneumonia.
In 341 sputum samples from CAP hospitalized adult patients, there were 231 sputum samples determined as having bacterial patho-gens, reaching a rate of 67.7% and 196 sputum samples detected with viruses, the positive rate was 57.5%. In many cases where bacte-ria pathogens as well as viruses were detected, there were multiple pathogenic agents found in a single sputum sample of CAP patients.
The list of bacterial pathogens showed that, Gram-negative bacilli occurred in higher percentages than Gram-positive cocci (290 vs 108), in which Klebsiella pneumoniae 18.5%, Acinetobacter baumannii 17.3%, H. influenzae 14.1% and Escherichia coli 9.7% while S. pneumoniae was the highest percentage in Gram-positive cocci at 16.4%. Mycoplasma, atypical bacterium was detected at 6.2%.
From the list of viruses, this study showed that Epstein – Barr virus was found at the percentage of 34.9%, followed by Cytomegalo-virus 16.7%, Influenza virus type A 10.3%, Influenza virus type B 4.4%, Rhinovirus and Respiratory syncytial virus were found at the rates of 3.5% and 2.9%.
Based on the quantity of bacterial pathogen discovered by the MPL-rPCR, we categorized the detected bacterial pathogens into main (primary) bacterial pathogens, which exhibited the highest copy numbers, and the co-infected (combined) bacterial pathogens, which had lower copy numbers. The co-infection of bacterial agents was shown in Table 3.
Table 3 illustrates that S. pneumoniae and H. influenzae were frequently detected as a primary bacterium alone, while K. pneu-moniae, A. baumannii, Pseudomonas aeruginosa often acted as primary bacteria in combination with other bacteria. M. morganii and Providencia sp. were exclusively found as combined bacteria.
Analyzed the results showed in Table 4, except Human metapneumovirus, the remain viruses were all in combination with bacterial pathogens causing CAP at the percentages about 60%, in which Epstein-Barr virus, Cytomegalovirus, Influenza virus type A, B had the higher percentages in combination with bacterial pathogens. The primary viruses causing CAP occurred in low percentage 30.6% (60/196). The co-infected bacteria in combination were most common with S. pneumoniae, followed by H. influenzae, K. pneumoniae, A. baumannii.
1) In some cases, virus can combine with 2 or more bacteria. 2) 12 cases (10.1%) infected in combination with fungi or other viruses. 3) 12 cases (21.1%) infected in combination with fungi or other viruses. CAP, community-acquired pneumonia; MRSE, methicillin-resistant Staphylococcus epidermidis; MRSA, methicillin-resistant Staphylococcus aureus.
4. DISCUSSION
There were 341 CAP patients who met the inclusion criteria of our study, and infections by pathogenic bacteria and viruses were predominance in male and in individuals over 60 years of age, consistent with reports by previous authors such as Tao [1], Li [6], Gómez-Junyent [7], Cavallazzii [8], Dang [9], Voiriot [10]. The significant increase in age of CAP patients in previous decades was likely due to the aging population [11]. In this study, pathogenic bacteria were detected at the rate of 67.7%, similar to previous reports by Ly & Pham [12] (69%), Ly & Ly [13] (65.5%), while pathogenic viruses were found at the rate of 57.5%, higher than reports by Tao [1] (23.4%), Voiriot [10] (28%), Self [14] (24.5%), Kim [15] (17.7%), Radovanovic [16] (28.4%), Alimi [17] (22.0%), Ruuskanen [18] (29.0%).
Table 2 showed that, among 231 positive sputum samples from CAP patients, there were 419 bacteria detected by MPL-rPCR, in which Gram-negative bacilli occurred in higher percentages than those in Gram-positive cocci, likely reports by previous authors [8,9,19–21]. Perhaps Gram-negative bacilli, especially A. baumannii and K. pneumoniae has become more prevalent in causing CAP patient in recent days. S. pneumoniae was found at the highest prevalent 16.4% in Gram-positive cocci, which is lower than reported by authors such as Gómez-Junyent [7] (36.5%), Purba [22] (29.2%), Temesgen [23] (35.9%). However, some studies indicated that, although S. pneumoniae occurred less common in recent day but it still plays an significant role in causing CAP in adult patients [8,9,19–21].
In our study, P. aeruginosa causing CAP was counted at the low rate 4.4% (Table 2), but it holds significance in causing CAP, particularly severe CAP, due to its rick factors such as antibiotic resistance and mortality [6,11,21,24–28]. Furthermore, previous studies have reported that P. aeruginosa remained important for patients with severe chronic obstructive pulmonary disease (COPD), especially among the elderly who are receiving regular oral corticosteroid therapy [28–30].
Mycobacteria was the only atypical bacteria detected in low frequency 6.2%, similar to previous research of Liu [31] (6.5%). Some recent authors have commented that atypical bacteria causing CAP were rarely detected and often occurred as co-bacteria with other bacterial pathogens [32–34].
In this study, Epstein-Barr virus was detected at the rate of 34.4%, followed by Cytomegalovirus 16.7% (Table 2). These rates were higher than those of Influenza virus type A 10.3%, Influenza virus type B 4.4%, Rhinovirus 3.5% and Respiratory syncytial virus 2.9%.We were surprised by the high percentages of Epstein-Barr virus and Cytomegalovirus, and we were left wondering if they were co-infections or opportunistic agents, necessitating further careful and thorough examination. Apart from the report by Voiriot [10], where the percentages for Influenza virus type A were at 18.4% , Rhinovirus at 12.6%, almost all reports by previous authors showed that Influenza virus type A, Influenza virus type B, Rhinovirus, Respiratory syncytial virus, H. metapneumovirus were detected at percentages ranging from 2% to 8%, which typically corresponds to the seasons when outbreaks of respiratory viruses are occurring [1,14–18,35] In many cases, more than one pathogenic bacteria and virus were detected in a single sputum sample. Our study, as shown in Table 3, revealed that among 231 sputum samples detected with pathogenic bacteria by MPL-rPCR, there were 96 sputum samples detected with only one % andial pathogens (primary bacteria alone) at the rate of 28.2% (96/341) and 135 sputum samples detected with 2 or more bacterial pathogens at the rate of 39.6% (135/341), similar to previous reports by Ly & Pham [12] 38.3%, Ly & Ly [13] 39.2% and Ta [36] 37.5%. S. pneumoniae and H. influenzae were discovered frequently as a primary bacterium alone while K. pneumoniae, A. baumannii, E. coli, P. aeruginosa were often found as primary bacteria in combination with other bacteria (co-infection). M. morganii and Providencia sp. were found as only combined bacteria (bacterial co-infection).
Analyzing the results in Table 3, almost viruses were in combination with bacterial pathogens causing CAP at the rates from 60% to 80%, in which, Epstein-Barr virus, Cytomegalovirus, Influenza virus type A, B, having the highest percentages. S. pneumoniae, H. influenzae, K. pneumoniae were the most frequent bacteria in viral co-infection [1,10,16,35]. Detections of Influenza virus, Respiratory syncytial virus, H. metapneumovirus in adult patients with CAP likely indicate an etiology role, whereas detections of Epstein-Barr virus and Cytomegalovirus should require further careful and thorough examination [17,18,37].
5. CONCLUSION
Bacterial pathogens and viruses were detected at positive rates of 67.7% and 57.5%, respectively (p<0.05), in which bacterial pathogens extend to Gram-negative bacilli such as K. pneumoniae, A. baumannii, H. influenzae, while predominent viruses occur included Epstein-Barr virus, Cytomegalovirus, Influenza virus type A, B. More than one pathogenic bacteria and viruses are found in one sputum sample. S. pneumoniae, K. pneumoniae, H. influenzae are the most common bacteria in viral co-infections and almost all viruses are co-infected with pathogenic bacteria. Epstein-Barr virus and Cytomegalovirus should require further scrutiny examination.
