Home
|
Table of Content Volume 13 Issue 3- March 2020
Early diagnosis of atypical pneumonia among children by using immunofluorescence biochip method
Bewin Oral J1*, Usha H C2, Madhuri Kulkarni3, Goldy S J4, Rema Devi S5, Jobin S R6
{1Assistant Professor, 5Professor & HOD, 6Assistant Professor, Department of Microbiology} {4Assistant Professor, Department of Obstetrics & Gynaecology} Dr. Somervell Memorial CSI Medical College Hospital, Karakonam, Thiruvananthapuram Dist., Kerala, INDIA. 2Clinical Microbiologist, Cauvery Heart & Multispecialty Hospital, Mysuru, INDIA. 3Retired Professor, Department of Microbiology, JSS Medical College, Mysuru, INDIA. Email: bewin.grace@gmail.com
Abstract Background: Atypical pneumonia, which is caused by Mycoplasma pneumoniae, Legionella spp., Chlamydia pneumoniae and viruses, has been thought to account for 7%‐30% of community‐acquired pneumonia (CAP). The treatment for the pathogens that cause atypical pneumonia is different from that of other bacterial pneumonia. The severity of pneumonia can range from mild to life-threatening, with uncomplicated disease resolving with outpatient antibiotics and complicated cases progressing to septic shock, acute respiratory distress syndrome (ARDS) and death. Therefore this study helps in early detection of the atypical pathogens causing CAP and also in initiation of appropriate treatment which reduces morbidity and mortality. Methods: This is a prospective study conducted in Department of Microbiology, JSS hospital, Mysore for a period of 18 months from January 2014 to August 2015. Results: 70 patients (children) of age less than 15 years with no evidence of typical pathogens causing pneumonia were included in our study. Among the 70 samples tested for atypical pathogens, 44 were positive (63%). Mycoplasma pneumoniae was the predominant atypical pathogen identified in 32.9% of the patients followed by Respiratory syncytial virus (17.1%). Chlamydophila pneumoniae and Influenza B were the next common pathogens detected in 4.3% of the patients. Mixed infections by Legionella pneumophila, Adenovirus and Influenza A and B virus were detected among the atypical pneumonia patients.Conclusion: Atypical pneumonia is in high incidence among CAP, even our study proved their high rate of positivity of 63%. Hence our study facilitates early diagnosis of atypical pneumonia patients by detecting IgM antibodies using Indirect Immunofluorescence and in starting the appropriate treatment in the early phase of infection, which helps in uplifting the public health of community. Key Words: Atypical Pneumonia, Immunofluorescence, IgM antibodies
INTRODUCTION Pneumonia is an inflammation of the lung parenchyma, often due to infection. It is a major cause of morbidity and mortality with an incidence of 20-30% in the developing countries and 3-4% in developed countries 1. Clinically, Pneumonia is often classified as "Typical" or "Atypical pneumonia". In Typical pneumonia, there is sudden onset of fever, chills, pleuritic chest pain and productive cough. Typical pneumonia is usually caused by bacterial pathogens like Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae and Haemophilus influenzae. Atypical pneumonia is a form of pneumonia caused by the atypical pathogens and presents clinically in a pattern which is inconsistent with typical pneumonia 2. “Atypical pneumonia” is caused by atypical organisms. These Atypical organisms may include bacteria, virus, fungi, and protozoa. The Atypical pneumonia patients usually present with scanty to moderate amount of sputum production, limited consolidation, insignificant raise in white cell counts and no alveolar exudates. Even though these infections are called “atypical,” they are not uncommon.3 Atypical pneumonia comprises 20% of all cases of pneumonia and prompt diagnosis is essential for proper clinical management and for better clinical outcome. Childhood Acute Respiratory Infection (ARI) is the largest cause of morbidity among under-five aged children across the world. Pneumonia amounts for almost one-fifth of total mortality in vulnerable age groups4. Mycoplasma pneumoniae and Chlamydophila pneumoniae may exacerbate asthma and are also associated with coronary artery disease and multiple sclerosis (MS). These atypical pathogens are known to cause community acquired pneumonia (CAP) resulting in major public health threat. The importance about the atypical pneumonia patients are the difficulty in early diagnosis and non-responsiveness to β-lactam therapy. Clinical presentation is often indistinguishable from pneumonia due to classical pathogens. Culture identification of these pathogens are technically demanding and time-consuming and even has a low sensitivity, these drawbacks further affect our early and accurate diagnosis of the infected patients, hence affects the management of these patients resulting in high morbidity and mortality. Thus there is a need for advanced diagnostic tools to detect these atypical pathogens causing atypical pneumonia. Hence the main aim of our study was in early detection of Specific IgM antibodies in the early phases of the infection using biochip Indirect Immunofluorescence method in order to initiate appropriate treatment which reduces the morbidity, hospital stay and mortality among the infected individuals.
AIMS AND OBJECTIVES OF THE STUDY
MATERIALS AND METHODS The present study is a prospective study conducted in the Department of Microbiology, JSS hospital, Mysore for a period of 18 months from January 2014 to August 2015. Selection of patients: Inclusion criteria: Patients (children) below 15 years of age with clinical signs and symptoms of pneumonia (productive or non-productive cough, fever and positive lung signs in chest radiography) and children who were not diagnosed of typical pneumonia caused by typical agents were included in this study. Exclusion criteria The patients with culture reports yielding positive result for growth of typical pathogens causing typical pneumonia were excluded from our study. And patients more than 15 years of age were excluded from study. Sample collection Whole blood samples were collected and serum separation was done by centrifugation. The serum samples were then subjected to indirect immunofluorescence method using Biochip (Pneumoslide -M -Vircell slide-Granada, Spain). Manufacturer’s test kit protocol was followed during testing and for interpretation of results. Testing kit It is an indirect immunofluorescence assay (IFA) kit used for the detection of specific IgM antibodies against the main etiological agents causing atypical pneumonia namely, Legionella pneumophila serogroup 1, Mycoplasma pneumoniae, Coxiella burnetii, Chlamydophila pneumoniae, Adenovirus, Respiratory Syncytial Virus, Influenza A, Influenza B and Parainfluenza serotypes 1,2 and 3. Principle of the IFA test The IFA method is based on the reaction between the IgM antibodies present in the patient’s sample and the antigen which is already adsorbed on the slide surface. The specific IgM antibodies present in the sample combines with the antigen in the slide forming an antigen- antibody complex. In the following step, the antigen‐antibody complex combines with the fluorescein‐labelled anti‐human globulin and they can be seen under fluorescent microscope.
VIRCELL PNEUMOSLIDE M SLIDE: 10 slides of 10 wells with the following Antigens:
All the antigens in the slide are obtained in cell culture except L .pneumophila. Each viral well contains infected cells inactivated with formaldehyde and also the non‐infected cells and they are fixed using acetone. Blood should be collected under aseptic precautions by venipuncture. Serum samples are to be refrigerated at 2‐8ºC or even can be stored at ‐20ºC in a deep freezer, if there is an expectation of delay in performing the test for 7 days. Samples should not be repeatedly frozen and thawed, since it might decrease the immunoglobulin titre (especially IgM antibodies). Hyperlipidemic or contaminated sera should be avoided. Serum or plasma samples are the compatible samples for this kit. Procedure:
Quality control: The reaction is considered positive when apple green nuclear, cytoplasmic and/or peripheral fluorescence in 1‐15% of the cells for positive sera to adenovirus, influenza, RSV. Apple green fluorescence noticed all over the organism in samples infected with Legionella, Chlamydophila or Coxiella. In case of mycoplasma infection,Apple green fluorescence is noticed in periphery.The reaction is negative, when no fluorescence noticed in cells of Legionella,Chlamydophila and Coxiella and Red cellular pattern noticed for Mycoplasma, adenovirus, influenza A and B, RSV and parainfluenza.The presence of fluorescence in all the cells or in the well no.10 involves the presence of antinuclear or anticellular antibodies and the result should not be considered as positive, then an alternative technique is required. Be sure to check in all the cases the absence of fluorescence in the cell control well. As cross‐reactive antibody is frequently found in patients with non‐Legionella infection, positive results are evaluated considering the symptomatology and the IgM reports. Due to these reasons that IgM positive Legionella sera at 1/12 were diluted in titres from1/12 to 1/192 and only titres higher than or equal to 1/96 were considered as significant for Legionella infection.
OBSERVATION AND RESULTS The present study was conducted in the Department of Microbiology, JSS hospital, Mysore for a period of 18 months from January 2014 to August 2015. In this study period, patients who presented with features of pneumonia to the hospital were screened for microbiological diagnosis for typical organisms adopting standard laboratory techniques. The patients with positive result for diagnosis of typical pathogens causing typical pneumonia were excluded from the study. 70 patients with no evidence of typical pathogens causing pneumonia were included in the present study. Serum samples from these patients were tested for the presence of IgM antibodies by Indirect Immunofluorescence(IF) to various organisms causing atypical pneumonia, which include Legionella pneumophila serogroup 1 (LP1), Mycoplasma pneumoniae (MP), Coxiella burnetii (COX), Chlamydophila pneumoniae (CP), Adenovirus (ADV), Respiratory syncytial virus (RSV), Influenza A (INFA), Influenza B (INFB) and Parainfluenza 1, 2 and 3 (PIVs). The study included 70 children of age less than15 years. Among the 70 samples tested for atypical pathogens, 44 samples yielded positive report for atypical infections. The rate of positive cases with atypical infection is 63%. Out of these 44 positive samples, 28 were male children and 16 were female children. Chest X-ray of those patients with positive result for atypical infection did not show any positive findings for pneumonia. Around 61.5% of children tested were associated with diarrhoea and vomiting. And around 50% of the children who presented with severe pneumonia were detected with specific IgM antibodies of atypical pathogens, hence diagnosed of atypical pneumonia. Malnutrition was a risk factor found in three children (4.3%) who were diagnosed with atypical pneumonia. Around 59.5% of children had leucocytosis whereas 4.7% had leukopenia. Mycoplasma pneumoniae (32.9%) was the predominant atypical pathogen detected followed by Respiratory syncytial virus (17.1%). Chlamydophila pneumoniae and Influenza B (4.3%) were the next commonly detected atypical pathogens in our study. Mixed infections were noticed among the atypical pneumonia patients. Four patients showed positivity for mixed infection. One child with mixed infection presented with positive result for Legionella pneumophila, Adenovirus and Influenza A virus. Two among the four children with mixed infection had co ˗infection of Mycoplasma pneumoniae and Adenovirus. And the other child had co ˗infection of Mycoplasma pneumonia and Influenza A&B. The common age of Mycoplasma infected patients ranges from 2-4 years and of RSV infected children was less than 1 year, suggesting that RSV is more common among infants. Among 70 children in our study, two of them died. One who died had co- infection of Mycoplasma pneumoniae infection and H1N1 infection. The other child who died had pulmonary hypertension in addition to the current RSV infection.
Table 1: Age wise distribution
Table 2: Gender wise distribution
Table 3: Based on Clinical diagnosis
Table 4: On the basis of Symptoms presented
Table 5: X-Ray findings
Table 6: Pneumoslide M- Organism detected
DISCUSSION The atypical respiratory pathogens namely Mycoplasma pneumoniae,Chlamydophila pneumoniae, Legionella pneumophila and viruses like RSV, Adenovirus ,Influenza virus cause acute respiratory-tract infections and community acquired pneumonia in children especially in developing countries. They can also commonly occur as co pathogens in mixed infectious pneumonia patients resulting in high mortality 5. The prevalence of each pathogen varies from country to country and could be due to the differences in the geographic areas or due to the different diagnostic procedures used in different studies 6. Despite the progress made in the diagnosis of Lower Respiratory Tract Infection, it takes a few days to identify the causative microorganism in blood or sputum samples 7. Cell cultures for viral and atypical bacterial isolation are usually sensitive and detect a broad spectrum of organisms. But the time taken to get results may be as long as 14 days 8. Recently PCR technique has been reported as a rapid method for detection of atypical pathogens. PCR assays need specialized equipments and reagents which are expensive 9. The aim of the present study was to detect various atypical pneumonia causing pathogens by using rapid indirect immunofluorescent assay in facilitating the early initiation of the appropriate treatment for better prognosis. In our study, out of 70 patients (children) selected, 44 samples (63%) yielded positive result proving atypical pneumonia. The positivity rate for atypical pneumonia is 63% in our study. This observation was similar to many other studies conducted throughout the world 10, 11. An Egyptian study by Zaki and Godal has reported a high incidence of atypical pathogens compared to our study and also noted that co-infections of atypical pathogens with agents causing classic pneumonia were high in their study 12. This was not observed in our study, where only atypical pathogens were detected, probably due to the fact that our study population included only those patients who were tested negative for classic pneumonia causing agents by standard microbiological methods. Various other studies which reported the incidence of atypical pneumonia from all parts of the world show considerable variation in the frequency of their occurrence 11, 13. The present study correlates with the study done in USA whose incidence was 54%. Asian countries have lesser incidence rates compared to our study while Nordic countries show similar data 10, 14. Incidence of Bacterial causes of atypical pneumonia also showed comparable results with a study conducted by Agmy et al. in Egypt and was shown to be 34%18,19, whereas a Thailand study by Nuanchan et al. 16 on the contrary showed less incidence of bacterial atypical pneumonia. Studies in various Asian countries including Kuwait have shown similar results for atypical pathogens with incidence ranging from 24-39 % 14, 15. Few INDIAN studies have been done to assess the incidence of atypical pathogens in the community and the causative agents were identified to be of low frequency compared to the present study 7, 17. The lower incidence may be due to the reason that only bacterial pathogens were diagnosed, unlike in our study, where both bacteria and virus were detected. Most studies showed atypical virus as the leading cause of respiratory tract infections in children. However in our study bacterial infections were more common compared to viral among the study population. In our study viral aetiology was detected in 24% of the children, whereas bacterial aetiology was detected in 39 % of children. More male children were affected in our study, concurrent with most studies. Legionella pneumophila is known to cause Legionnaire’s disease which is a systemic infection involving lungs and presents as severe pneumonia requiring hospitalisation and intensive care 18. Mortality with Legionella pneumophila pneumonia is usually high, compared with other atypical pathogens. Kristopher et al. have reported mortality rate of 14 % 19. Our study showed much higher mortality (in 3 out of 4 patients, it was fatal). Co morbid conditions present in our patients’ i.e. pulmonary hypertension, rheumatoid arthritis, and seizure disorder were probably responsible for this trend, as also shown in several other studies 19. Most of our patients presented with cough, expectoration, fever and dyspnoea as the chief complaints, with few of them also had complaints of headache and chest pain. Clinical picture, routine laboratory tests and chest X-ray did not help in predicting the aetiology. Various other studies have also shown that, different etiologic causes for pneumonia could not be distinguished on clinical basis, or radiological or various routine laboratory methods 20, 21, 11. Hence the need for laboratory confirmation in the diagnosis of atypical pneumonia forms a milestone in optimum management. Coxiella burnetti was detected in one patient, who had pleural effusion in addition to bronchopneumonia. She did not have other serious complications like myocarditis, endocarditis or cerebral involvement. Carlos et al. has described that the zoonotic causes of atypical pneumonia may involve pleura, and patients presenting with history of exposure to known risk factors and unexplained pleural effusion, may be suspected to have pneumonia caused by zoonotic atypical pathogens like Coxiella or Francicella. However, there was no history of intimate contact with animals in our patient 22. In the present study, 63% of the hospitalised children aged less than 16 years with acute respiratory tract infection were infected with at least one atypical respiratory pathogen. M. pneumoniae and RSV were the predominant agents causing pneumonia. Multiple pathogens were detected in 5.7% of the patients. Two studies conducted in China by Ji liu et al. and Chen et al. for the detection of atypical pathogens in children, reported causative agents of pneumonia in 25.7% and 58.4% respectively 23,24. In another study Sally et al. had shown prevalence of atypical pathogens in children to be 45 %. 5. however our study showed much higher prevalence compared to these studies. Similar high prevalence was also seen in a Brazilian study, which reported 78% prevalence with viral infections accounting for 60 %. 25. An INDIAN study conducted by Jyostsna et al. in Lucknow showed low prevalence of atypical pathogens with only 10% of the children being diagnosed with atypical pathogens and the only pathogen identified was Mycoplasma pneumoniae 26. Our study also showed high concurrence with the various other studies regarding the most common etiological agent detected, which was found to be M pneumoniae followed by RSV. Chinese studies differ in this matter where the predominant viral pathogens were adenovirus and Influenza B virus. 27, 28, 25, 29.30, 23, 24, 31 This could be due to geographical variation in prevalence of various pathogens 6. Chlamydophila pneumoniae was the etiological agent present in 4.3% of children. The results are in concordance with a study from King George’s Medical University Lucknow, which showed prevalence of Chlamydophila pneumoniae to be 5.5 % 32, 33. Influenza B, Influenza A and Adenovirus were the other viral pathogens detected in 6% of children, which correlated with similar prevalence in other studies 23, 24. Our study had only one child with Legionella pneumophila antibodies. This low prevalence has been reported in other studies also 23,24. Mixed infections (5.7%) found in our study, correlated well with the study conducted by Chen et al. who reported mixed infections in 6.5% of children 24. The main co-pathogen found in mixed infection in our study was Mycoplasma pneumoniae which was also observed in other studies 24, 34, 35, and 36. Sally et al. have hypothesised that Mycoplasma pneumoniae often participated in mixed infections, and it may enhance susceptibility to other infectious agents 21. Patients with features of atypical pneumonia with detectable pathogens, had less severe breathlessness compared to those in whom organisms were not detected (Undiagnosed category), indicating less severe pneumonia and better outcome in them. All the children in our study had similar clinical pictures with cough, fever, and rhinitis. Diarrhoea and vomiting were also common extra-pulmonary symptoms present in significant number of children. Most other studies also recorded similar findings. Esposito et al. and Wubel et al. had made similar observations, and concluded that clinical and routine laboratory parameters did not help in differentiating typical and atypical pneumonia in children 16, 37, 30, 20, and 11. In majority of the studies, M. pneumonia was found in all age groups, with higher incidence in children aged 5-14 years 24. However, in our study, though all age groups were affected, it was most common in children below 5 years. Most of the RSV infection in our study was seen in children below 1 year of age, which was in concordance with other studies 30. Mortality was observed in children (2.9%). RSV pneumonia was reported in this child, however, the associated severe primary pulmonary hypertension in this child contributed to the mortality. Several studies have documented that comorbid conditions worsens the outcome in these children 38. The other child had co-infection with M.pneumoniae and H1N1.
Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12
Figure 1: PneumoslideM Indirect Immunofluoresence Kit for IgM detection of atypical pneumonia; Figure 2: Fluorescent Microscope; Figure 3: Positive for Legionella pneumophila antibodies; Figure 4: Adenovirus positivity showing perinuclear fluorescence; Figure 5: Positive for Chlamydophila pneumoniae antibodies; Figure 6: Positive for Influenza B antibodies showing perinuclear fluorescence; Figure 7: Positive for Coxiella burnetti antibodies; Figure 8: IgM antibodies to Mycoplasma pneumoniae showing perinuclear fluorescence; Figure 9: Perinuclear fluorescence showing presence of antibodies to RSV; Figure 10: Antibodies to Parainfluenza virus showing positive IgM antibodies; Figure 11: Antibodies to Influenza A virus showing perinuclear fluorescence; Figure 12: Cell control
SUMMARY Atypical pneumonia is one of the major causes of community acquired pneumonia in children. The present study titled “Early diagnosis of atypical pneumonia among children by using immunofluorescence biochip method” was taken up with the objective of early diagnosis of different atypical pneumonia causing pathogens and also rate of different rare pathogens. 70 children were included in our study, these children were clinically diagnosed patients of pneumonia yet negative for typical pathogens causing pneumonia. In these 70 serum samples tested for the presence of specific IgM antibodies to various atypical pathogens causing atypical pneumonia, only 44 serum samples yielded positive result. Details of clinical history, examination, associated radiological and routine laboratory findings were noted. The positivity rate in our study was 63%. Predominant atypical pathogens detected in children were Mycoplasma pneumoniae and Respiratory syncytial virus. Clinical history/examination, radiological findings and routine laboratory investigations did not differentiate between atypical pneumonia and typical pneumonia. Legionella pneumophila was associated with severe pneumonia and increased mortality. Mycoplasma pneumoniae was the predominant aetiological atypical agent causing atypical pneumonia in children below five years of age. Respiratory syncytial virus was found to be affecting more infants in our study. Chlamydophila pneumoniae and Adenovirus were causing severe lower respiratory tract infection among children resulting in increasing community acquired pneumonia. Mortality due to atypical pathogens was less in children and the observed mortality rate may be due to associated co morbid conditions.
CONCLUSION The main objective of the study was rapid diagnosis of atypical pneumonia by IgM antibody detection using Indirect Immunofluorescence biochip method. Atypical agents (bacteria and viral) causing atypical pneumonia were detected among 63% of children selected in our study. Clinical features, routine laboratory findings and radiological features could not differentiate the typical and atypical pneumonia in our study thereby emphasising the importance of microbiological diagnosis of atypical pneumonia. Mycoplasma pneumoniae and Respiratory syncytial virus are the major pathogens causing atypical pneumonia in children. Mycoplasma pneumoniae and RSV had good clinical outcome whereas Legionella pneumophila pneumonia had worst outcome. Lower respiratory tract infections are fairly common in children with atypical pathogens being implicated in good number of cases. The clinical presentation can range from mild to severe in children with no significant radiological and laboratory findings. Respiratory syncytial virus is more common in infants whereas Mycoplasma pneumoniae cause diseasein older children. Adenovirus can also cause severe respiratory disease in children and have to be considered while treating children with lower respiratory infections. Most of the children recover from atypical pneumonia, while children with co morbid conditions have very severe manifestations of disease and sometimes even fatal consequences are encountered.
REFERENCES
|
|
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.