Relevance of Serology for Mycoplasma pneumoniae Diagnosis ...

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Abstract. We studied Mycoplasma pneumoniae as the etiologic pathogen in acute exacerbations of asthma and the value of polymerase chain reaction (PCR),.
Microbiology and Infectious Disease / Serology vs PCR and Culture in M pneumoniaee

Relevance of Serology for Mycoplasma pneumoniae Diagnosis Compared With PCR and Culture in Acute Exacerbation of Bronchial Asthma Maysaa El Sayed Zaki, PhD,1 Doaa Raafat, PhD,1 and Amal Abd El Metaal, PhD2 Key Words: Mycoplasma pneumoniae; Asthma; Disease exacerbation; Serology; Polymerase chain reaction; Microbiologic culture DOI: 10.1309/AJCP34YZGEHERWRX

Abstract We studied Mycoplasma pneumoniae as the etiologic pathogen in acute exacerbations of asthma and the value of polymerase chain reaction (PCR), culture, and serologic tests for its accurate diagnosis. For the study, 59 nonsmoking patients with asthma (37 females, 22 males; age, 15-50 years) underwent clinical, radiologic, and laboratory examinations. Bacteria isolated from sputum were Streptococcus pneumoniae (32 [54%]), Staphylococcus aureus (23 [39%]), and M pneumoniae (5 [15%]). All M pneumoniae were associated with S pneumoniae (8/32 [25%]) and S aureus (1/23 [4%]). No M pneumoniae were isolated as single pathogens. Serologic testing for M pneumoniae revealed that all samples were positive for specific IgG; 40 (68%) had a high titer, and 19 (32%) had a moderate titer. Of 59 samples, 29 (49%) were positive by Serodia Myco II gelatin particle testing (Fujirebio, Tokyo, Japan). PCR was positive in 25 samples, all of which had a high IgG titer; all culture-positive cases were PCR+. M pneumoniae is a common bacterial pathogen associated with acute exacerbations of asthma in people 15 years or older. Prompt laboratory diagnosis of M pneumoniae requires direct detection by PCR and culture. A high serologic titer can be a clue for the presence of M pneumoniae.

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The major asthma morbidity, mortality, and health care costs are a result of acute exacerbations. However, exacerbations are only partially responsive to current therapies, and new approaches to treatment are needed.1 The cause of acute exacerbations of asthma is heterogeneous and under discussion. Respiratory virus infection is the most common environmental exposure to cause a severe asthma exacerbation. Airway inflammation is a key part of the lower airway response in asthma exacerbation and occurs together with airflow obstruction and increased airway responsiveness.2 Other findings suggest that viralallergen and viral-bacterial interactions are important for chronicity. Studies are also invoking atypical bacterial infections, Mycoplasma pneumoniae and Chlamydia pneumoniae, as factors in acute exacerbations and chronic asthma.3 In patients with severe chronic obstructive pulmonary disease, acute infective exacerbations are frequent. Streptococcus pneumoniae and Haemophilus influenzae are the most commonly isolated bacteria in sputum cultures from affected patients. Gram-negative bacilli such as Enterobacteriaceae and Pseudomonas spp have also been implicated in such conditions.4 However, few reports discuss the implications of those bacteria and their association with M pneumoniae as the etiologic agents in exacerbation of asthma.5,6 M pneumoniae is a small, cell wall–deficient bacterium that is insensitive to β-lactam antibiotics and cannot be detected by Gram stain. It is a common respiratory pathogen responsible for mild, acute respiratory infections such as sore throat, pharyngitis, and tracheobronchitis in younger children. It is the most common cause of primary atypical pneumonia resistant to β-lactam antibiotics in older children © American Society for Clinical Pathology

Microbiology and Infectious Disease / Original Article

and young adults.7-9 M pneumoniae can also be associated with severe extrapulmonary complications.10-12 The standard laboratory methods for the specific diagnosis of M pneumoniae infection have been isolation in culture and serologic methods. Culture is time-consuming and relatively insensitive. The conventional complement fixation test using a glycolipid antigen gives unspecific reactions and lacks sensitivity.13,14 Alternative tests have been developed to obtain more accurate and prompt diagnosis: indirect enzyme immunoassay (EIA) measuring separately IgG and IgM class antibodies15,16 and polymerase chain reaction (PCR) for rapid and sensitive detection of M pneumoniae in respiratory tract specimens.17,18 The goals of the present study were to investigate the occurrence of M pneumoniae as the etiologic pathogen in acute exacerbations of asthma and to determine the value of culture, PCR, and serologic tests for accurate diagnosis of M pneumoniae.

Materials and Methods The study was performed on 59 nonsmoking patients with asthma (37 females and 22 males; age, 15-50 years). They attended the outpatient clinic of the Thoracic Medicine Department, Mansoura Faculty of Medicine, Mansoura, Egypt, from December 2006 to February 2007. Patients had acute exacerbations of asthma based on the criteria of the New 2002 Global Initiative on Asthma guidelines.19 An exacerbation was defined as episodes of progressive increases in shortness of breath, cough, wheezing, and expectoration. We also studied 20 age- and sex-matched control subjects with asthma. All patients and subjects gave informed written consent. The study was approved by the ethical committee of Mansoura University. Patients were subjected to complete history-taking, plain radiographs to exclude associated abnormalities, and pulmonary function tests using computerized spirometry. Morning sputum samples were obtained from each patient and control subject. Two blood samples were obtained aseptically from each patient and control subject for serologic study, one at admission and the other after 10 days. The serum was separated and stored frozen at –70°C for antibody assay. Sputum Culture Direct Gram staining was performed for each sputum sample, and quantitative culture of the sputum was done on blood agar according to standard operating procedures.20 After aerobic culture of sputum was performed, the remainder of the sputum was frozen at −80°C for further PCR study. Bacterial isolates were identified by biochemical © American Society for Clinical Pathology

reaction using the Sensititre System (TREK Diagnostic Systems, Cleveland, OH). Antibiograms were done, and the zone was measured in millimeters according to World Health Organization recommendations. Mycoplasma pneumoniae Culture Modified Hayflick medium was used. The medium was composed of 7.5 parts of PPLO crystals (Difco, Becton Dickinson, Franklin Lakes, NJ), 1 part heat-inactivated horse serum, 1 part aquos extract of (25%) baker’s yeast, penicillin G (1,000 U), thallium acetate (0.025%), and 1.2% agar. For the sputum culture, 500 µL of sputum was cultured and incubated at 37°C in 5% carbon dioxide up to 10 days. The isolated colonies were identified by their characteristic “fried-egg” morphologic or granular appearance and stained with cresyl–fast violet stain that gives a red-purple color with large and small colonies. Proper identification of the isolated colonies was carried out by nested PCR following DNA extraction with the QIAamp DNA mini kit, QIAGEN, Hilden, Germany; we then amplified the extracted DNA using primer 1 (59-CAA GCC AAA CAC GAG CTC CGG CC-39) and primer 2 (59-CCA GTG TCA GCT GTT TGT CCT TCC CC-39). Samples to be amplified were incubated in a 50-µL reaction volume containing 10 µL of extracted DNA, 40 µL of premixed primers and optimized PCR buffer, and 0.2 µL of Taq DNA polymerase. Samples were amplified for 35 cycles by using a PCR processor (GeneAmp PCR system 2700, Applied Biosystems, Foster City, CA), starting with a 1-minute predenaturing step at 96°C. In the optimized PCR, each cycle consisted of the following steps: denaturation at 94°C for 1 minute; annealing at 60°C for 1 minute; and elongation at 72°C for 1 minute. After 35 cycles, a temperature-delay step of 10 minutes at 72°C was done to complete the elongation. Next, 10 µL of the PCR product was separated by electrophoresis on a 2% agarose gel. A band of 375 base pairs stained with ethidium bromide under UV light was considered a positive result.21,22 Direct PCR for Sputum Samples Sputum was thawed and centrifuged at 2,000 rpm for 15 minutes, and DNA was then extracted using a QIAamp DNA mini kit (QIAGEN) according to the manufacturer’s instructions. M pneumoniae DNA was detected by PCR by the mentioned method. Mycoplasma IgG Antibodies by Enzyme-Linked Immunosorbent Assay Serum IgG antibody against M pneumoniae was measured by enzyme-linked immunosorbent assay (Virotech Diagnostic, Loewenplatz, Germany). IgG antibodies to M pneumoniae were detected by indirect EIA. In this test, the

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antigen solution used to coat the microplate was a solubilized ultrasonicated product of an M pneumoniae culture containing a high proportion of membrane proteins. The specimen arbitrary unit (AU) values were determined from the calibration curve supplied by the manufacturer, and the results were interpreted as follows: A value of less than 10 AU/mL for the rising titer in the second serum specimen was considered insignificant, a value of 10 to 19 AU/mL was considered low, a value of 20 to 39 AU/mL was considered moderate, and a value of more than 40 AU/mL was considered high. The antibodies form an immune complex with the antigen coated on the test strip; then the enzyme conjugate attaches to this complex. Added TMB solution provides a blue color that turns yellow after adding stopping solution, and absorbance is read at 450 nm. Mycoplasma IgM Antibodies by Gelatin Particle Agglutination Test The Serodia Myco II gelatin particle agglutination test (Fujirebio, Tokyo, Japan) is marketed in the United Kingdom by Mast Diagnostics, Bootle, England, and was performed according to the manufacturer’s instructions. It mainly measures IgM for M pneumoniae.21 It is based on the principle that gelatin particles sensitized with M pneumoniae cellmembrane components are agglutinated in the presence of M pneumoniae antibody. Serum samples were inactivated at 56°C for 30 minutes. Rigid U-well microtiter plates (supplied with the kits) were soaked in detergent solution overnight and then rinsed thoroughly under running tap water. They were then washed with distilled water and dried. Using the serum diluents supplied, 25-µL serum samples were double diluted to give dilutions of 1 in 10 to 1 in 10,240. Sensitized and unsensitized lyophilized gelatin particles were suspended in diluents. Next, 25-µL drops of the unsensitized particle suspension were added to the 1 in 10 serum dilutions to give a final dilution of 1 in 20, and 25-µL drops of the sensitized particle suspension were added to the remaining wells giving final dilutions of 1 in 40 to 1 in 20,480. The plates were shaken for 30 seconds and then covered and left undisturbed on a level surface at room temperature for 3 hours (or overnight). Results were considered positive at 1/160 titer or more.

Results The study was performed on 59 nonsmoking patients with asthma (37 females and 22 males; age, 15-50 years) and acute exacerbations of the asthma. Control subjects were 20 subjects with stable asthma. No differences were found among patients in clinical signs, pulmonary function test results, or radiologic findings. 76 76

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Direct Gram stains of sputum from the patients were all positive for neutrophils and epithelial cells. Moreover, 31 samples (53%) had evidence of the causative bacteria, S pneumoniae or S aureus (data not shown). Bacteria Isolated From Patients The bacteria isolated from sputum samples of patients were S pneumoniae (32 [54%]), S aureus (23 [39%]), and M pneumoniae (9 [15%]) zTable 1z. All isolated M pneumoniae were associated with S pneumoniae (8/32 [25%]) and S aureus (1/23 [4%]). No bacterial isolates were found in control subjects (data not shown) zTable 2z. Serologic Study of M pneumoniae By serologic study for M pneumoniae, 29 samples from patients (49%) were positive by the Serodia Myco II gelatin particle test. All patients’ samples were positive for specific IgG; 40 samples (68%) had a high titer and 19 samples (32%) had a moderate titer. Of the control subjects, 10 had positive IgG with a low titer and 2 had positive IgM zTable 3z. Twenty samples with a high IgG titer had a 4-fold increase in titer after 10 days (data not shown). Direct PCR for M pneumoniae In the study, 25 samples were positive with PCR for M pneumoniae. Positive cases by culture were also positive by PCR. Positive PCR samples were associated with a positive high IgG titer, and 17 cases had positive microagglutination particles zTable 4z, zFigure 1z, and zFigure 2z. Serum samples with a 4-fold increase in IgG had positive PCR results (data not shown).

zTable 1z Bacteria Isolated by Culture in 59 Cases of Asthma



Bacteria

No. (%)



Streptococcus pneumoniae Staphylococcus aureus Mycoplasma pneumoniae



32 (54) 23 (39) 9 (15)



zTable 2z Association Between Mycoplasma pneumoniae and Other Bacteria in Cases of Asthma* Mycoplasma pneumoniae Culture



Positive

Total

Streptococcus pneumoniae Staphylococcus aureus Total

8 (25) 1 (4) 9 (16)

32 23 55

*

Data are given as number (percentage).

© American Society for Clinical Pathology

Microbiology and Infectious Disease / Original Article

zTable 3z Comparison of Results for the Serodia Myco II Gelatin Particle and Mycoplasma pneumoniae With Culture for Asthma Patient and Control Samples*

Positive Culture (n = 9)

Negative Culture (n = 70)

Patient samples Serodia Myco II gelatin particle Positive Negative Mycoplasma IgG High titer, >40 AU/mL Moderate titer, 20-39 AU/mL Combined serologic tests Control samples Serodia Myco II gelatin particle Positive Negative Mycoplasma IgG Low titer Combined serologic tests

6 (67) 23 (46) 3 (33) 27 (44) 5 (56) 35 (70) 4 (44) 15 (30) 1 (11) 9 (18) 0 (0) 2 (100) 0 (0) 18 (100) 0 (0) 10 (100) 0 (0) 0 (0)

Total

29 30 40 19 10 2 18 10 0

AU, arbitrary units. * Data are given as number (percentage).

Discussion There is abundant evidence that asthma is frequently exacerbated by infectious agents. Several viruses have been implicated in the inception and exacerbation of asthma. Recent attention has been directed at the role of infections

zTable 4z Positive Culture and/or PCR Serologic Results in 25 PCR+ Cases of Asthma Culture Results/ Case No.



IgG (AU/mL)



PA*



Positive 1 49 0 2 43 0 3 43.7 640 4 43.6 160 5 46.6 0 6 43.2 40 7 43.1 0 8 43.5 80 9 43.6 40 Negative 10 43.7 640 11 47 40 12 43.9 160 13 40 160 14 47.7 40 15 43.3 180 16 44.9 80 17 40 0 18 45 40 19 44 320 20 46.3 0 21 44 80 22 44 0 23 43.5 0 24 43.5 0 25 46.5 0

















AU, arbitrary units; PA, microagglutination particles; PCR, polymerase chain reaction. * Number of patients positive by PA method.

© American Society for Clinical Pathology

with the atypical bacteria M pneumoniae and C pneumoniae as agents capable of triggering asthma exacerbations and potentially as inciting agents for asthma.23 Their ability to elicit a helper T cell (TH) 2 response and promote airway inflammation may be the common pathway in the development of an atopic inflammatory response.24 Symptomatic exacerbations are frequent problems in the management of chronic bronchitis and bronchial asthma. Identification of a bacterial cause of specific exacerbations should be based on changes in clinical symptoms and documentation of significant bronchial bacterial flora and a neutrophilic inflammatory response. In the present study, all sputum samples from patients had high neutrophilic counts that document presence of an infectious agent, but bacteria were present in 53% of patients. Culture is considered more accurate in identifying the causative pathogen.

800 PA IgG

700 600 500 400 300 200 100 0

9

8

7

6

5

4

3

2

1

zFigure 1z Microagglutination particles (PA) and IgG in culturepositive cases of asthma.

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800 PA IgG

700 600 500 400 300 200 100 0

25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9 8 7 6 5 4 3 2 1

zFigure 2z Results in cases of asthma. Microagglutination particles (PA) and IgG for polymerase chain reaction–positive cases.

In our study, the most common bacteria isolated were S pneumoniae (54%), S aureus (39%), and M pneumoniae (15%). Similar results for M pneumoniae isolation rates were reported in previous studies.25-27 However, in those studies, no other pyogenic bacteria were isolated. There is a growing support for the opinion that in infectious exacerbations of asthma, bacteria also can have an important role. The bacteria include typical and atypical bacterial strains.28 Eradication of bacteria in the lower respiratory tract depends on the coordinated expression of proinflammatory cytokines and consequent neutrophilic inflammation.29 The inhalation of lipopolysaccharide components of the bacterial cell wall exacerbates airway inflammation, which is accompanied by mast cell activation and enhanced TH2 responses. These observations provide clues to understanding the mechanisms of bacterial infection– induced exacerbation of the clinical features of asthma.30 The present study emphasizes the importance of laboratory diagnosis of sputum colonization with pathogenic bacteria with its reflection in therapeutic methods in the acute exacerbation of asthma. Moreover, in the present study, there was a strong association between the isolation of pyogenic bacteria and M pneumoniae. This association may participate in augmentation of the immunologic response to foreign organisms. M pneumoniae infection was found to increase production of interleukin (IL)-4 levels and IL-4/IFN-γ. These data suggest a predominant TH2-like cytokine response in M pneumonia, thus representing a favorable condition for IgE production, increasing the tendency for acute exacerbation of asthma.31,32 In M pneumoniae infection, it is difficult to set up criteria for a “gold standard” to detect acute or remote infections. Cultural isolation is 100% specific but lacks sensitivity and is too slow to be of timely diagnostic value. 78 78

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There is no universally agreed-on gold standard serologic assay for the detection of antibodies to M pneumoniae. The present data raise several important issues in the interpretation of M pneumoniae EIAs and Serodia Mycoplasma agglutination particles. In the agglutination particle assay, the concordant positive results were 67%, and the concordant negative results were 33%. This test mainly measures IgM for M pneumoniae and is claimed by the manufacturer to be a substitute for the complement fixation test. The high positive rates (29/59 [49%]) for this test in the patient group underlie the importance of IgM serologic testing to diagnose M pneumoniae at a very early phase of infection. On the other hand, the 2 positive samples in control subjects might be false-positive reactions, probably due to nonspecific binding of macroglobulins or low levels of cross-reacting antibodies binding to nonprotein constituents of the cell membrane antigen preparation.33,34 Because it has been previously demonstrated that the main antigenic components reacting are proteins, better-defined specific protein antigen preparations should give more accurate results and should be more specific than a glycolipid or whole-cell antigen. In the present study, a high titer for IgG (40/59 [68%]) was found among patients with a 56% concordance with positive culture. It is not surprising to find a high endemic IgG seroprevalence; it has been shown previously that healthy persons often have elevated levels of specific M pneumoniae IgG antibodies in their serum, probably because of past M pneumoniae infections.35,36 In our study, 10 control subjects had positive IgG with low titers. However, patients were symptomatic, and we chose a high cutoff value for interpretation. The high sensitivity of serologic testing can be explained by the fact that IgG, once formed, persists for a long time, even though the M pneumoniae organism is cleared from the airways. Another factor for the high sensitivity could be that IgG antibody levels steadily increase with reinfection. The difference in serologic values could be attributed to the difference in the time of sampling and the difference in kinetics of IgG and IgM toward M pneumoniae. It is well known that the serum immunoglobulins produced during an M pneumoniae infection are heterogeneous and that their kinetics are related to the type of antigen.35 Proper laboratory diagnosis of M pneumoniae is a matter of combined test results depending on detection of bacteria and the serologic response. In the present study, PCR was positive in 25 cases associated with a high titer of IgG. Liu et al21 reported positive PCR results for M pneumoniae in 32% of children with lower respiratory tract infections. The difference in rates of positive PCR results can be attributed to age differences with more carriage of M pneumoniae in adults. © American Society for Clinical Pathology

Microbiology and Infectious Disease / Original Article

The association of positive PCR results with high IgG titers and positive microagglutination particle assay results highlights the importance of high titers of immunoglobulins even in single serum samples for predicting the diagnosis of M pneumoniae. From this study, we conclude that M pneumoniae is a common bacterial pathogen associated with acute exacerbation of asthma in people 15 years or older. Prompt laboratory diagnosis of M pneumoniae requires direct detection of the organism by PCR and culture. A high serologic titer can be a clue for presence of M pneumoniae. From 1Clinical Pathology and 2Chest Department, Faculty of Medicine, Mansoura University, Mansoura, Egypt. Address reprint requests to Dr El Sayed Zaki: Clinical Pathology, Faculty of Medicine, Mansoura University, Mansoura City, Egypt.

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© American Society for Clinical Pathology