|Year : 2014 | Volume
| Issue : 1 | Page : 11-14
Research progress in atypical pathogens of community acquired pneumonia
Jiuxin Qu, Bin Cao
Department of Infection and Clinical Microbiology, Beijing Chaoyang Hospital, Affiliated to Capital Medical University, Beijing Institute of Respiratory Diseases, Beijing 100020, China
|Date of Web Publication||25-Sep-2014|
Department of Infection and Clinical Microbiology, Beijing Chaoyang Hospital, Affiliated to Capital Medical University, Beijing Institute of Respiratory Diseases, Beijing 100020
Source of Support: None, Conflict of Interest: None
Atypical pathogen, especially Mycoplasma pneumoniae is a common and important pathogen of community-acquired pneumonia. Physicians should pay more attention on them. Compared with bacteria, the clinical treatment of atypical pathogens is different, as beta-lactams are not effective for atypical pneumonia. Therefore, laboratory diagnostic methods and clinical biology research is particularly important for the diagnosis and treatment of atypical pneumonia. In order to provide more theoretical basis for clinical diagnosis of atypical pathogens infection, we performed a review of the research progress of prevalence, laboratory testing of atypical pathogens related infections.
Keywords: Atypical pneumonia, community-acquired pneumonia, Mycoplasma pneumoniae
|How to cite this article:|
Qu J, Cao B. Research progress in atypical pathogens of community acquired pneumonia. Community Acquir Infect 2014;1:11-4
| Introduction|| |
The atypical respiratory pathogens Mycoplasma pneumoniae, Chlamydia pneumoniae and Legionella pneumophila are now recognised as a significant cause of acute respiratory-tract infections, but they remain colorless after Gram-staining and are difficult to identify by conventional bacterial culture tests. It is reported that patients with atypical pneumonia were more likely to have normal or reduced white blood cell counts.  However some published data showed that between atypical pneumonia and general bacterial pneumonia, there were no significant differences in the symptoms such as fever, cough, productive sputum, and the sign of lung rales. , Also, X-ray findings and the increase of white blood cell counts and percentage of neutrophils were similar between them. Especially, patients with pneumonia caused by L. pneumophila presented with the typical symptoms of Streptococcus pneumonia while patients with S. pneumonia also presented with the symptoms of atypical pneumonia.  However, beta-lactams are not effective for atypical pneumonia. Therefore, laboratory detection methods and clinical biology research on the diagnosis and treatment of atypical pathogens infection is particularly important.
| Atypical Pathogens|| |
Atypical organisms such as M. pneumoniae, C. pneumoniae, and L. pneumophila are implicated in cases of community-acquired pneumonia (CAP). M. pneumoniae in 1962 was successfully isolated.  M. pneumoniae lacks a cell wall, but it can grow in artificial culture medium, and it is the most frequent pathogens found in patients with atypical CAP. C. pneumoniae found in 1986 is intracellular organisms and is a common cause of acute and chronic respiratory-tract infections.  Legionella was found in 1976 because it caused infection outbreak of America veterans. It has been confirmed more than 50 Legionella species, a total of 70 serotypes. L. pneumophila with 16 serotypes is intracellular organisms and closely related to human infection. 
| Atypical Pathogens in Community-acquired Pneumonia|| |
With the widespread use of antibiotics, the change of living environment and constantly updated diagnostic methods, we found atypical pathogen played an important role in CAP. A prospective study was performed on 665 consecutive adult patients with CAP at 12 centers in 7 Chinese cities between 2003 and 2004.  The results showed that atypical pathogens caused 32.4% cases of CAP, of which 20.7% M. pneumoniae, 6.6% C. pneumoniae and 5.1% L. pneumophila. Of 195 patients with a bacterial pathogen, an atypical pathogen was identified in 10.2% cases. Survey of Cao et al. on the etiology and clinical outcomes of CAP treated in an ambulatory setting showed that the most common pathogens were M. pneumoniae (29.4%) and then virus copathogens (2.5%).  The previous research data of "pathogens monitoring network among adults with CAP in Beijing" showed that of 410 patients with CAP, ≥4-fold increase of paired serum M. pneumoniae IgG antibody titer was observed in 18.8% of cases.  Atypical pathogens, rather than S. pneumoniae, become the most important pathogen of adult CAP.
| Laboratory Diagnosis of Atypical Pathogens Infection|| |
Laboratory diagnosis of Mycoplasma pneumoniae infection
At present, several methods are available for the definitive diagnosis of M. pneumoniae infections, including culture, serology, and real-time polymerase chain reaction (PCR) assay. Culture has been reported to be more specific and more sensitive for detection.  However, the culture of M. pneumoniae is time-consuming, requires 2-6 weeks for results, which is less helpful to early diagnosis of acute infection. Culture is essential for investigation of M. pneumoniae clinical isolates and molecular biology and drug sensitivity.  Serologic diagnosis is a common method by measuring serum antibody titers of acute and convalescent-phase serum samples. Sustained high IgM titer (≥l:160) of acute-phase serum samples or a four-fold or greater increase in IgG antibody titer of convalescent-phase serum samples than that of acute-phase serum samples can be regarded as a positive diagnosis.  A recent study showed that a four-fold or greater increase in titer of paired serum samples could be observed within 3-6 days.  Detection of M. pneumoniae by PCR as a rapid, sensitive, and specific method has been reported by many authors. Primary care hospitals rarely perform molecular tests, because it requires specialized equipment and highly trained personnel. With the continuous standardization of quality control, nucleic acid-based tests will become a mainstream technology of laboratory diagnosis of M. pneumoniae infection.
Laboratory diagnosis of Chlamydia pneumoniae infection
The general experience is that the culture of C. pneumoniae is not recommended as a standard of diagnosis, because it is difficult and time-consuming, as well as the sensitivity of isolation is low. Serology has so far been the most commonly used method for diagnosis of C. pneumoniae infections.
Incubation period of C. pneumoniae infection can last up to 1-3 months. IgM appears in the first infection in 3 weeks, IgG in 6-8 weeks. Therefore, serologic diagnosis frequently provides a retrospective diagnosis of C. pneumoniae infection. PCR has been very specific and more sensitive and is a valuable tool in early diagnosis of C. pneumoniae infection.
Laboratory diagnosis of Legionella pneumophila infection
Current diagnostic tests for L. pneumophila infection include culture, serological testing, antigen detection and nucleic acid amplification. Estimated sensitivities of sputum culture range from 15% to 90% and vary according to different comparison standards and by individual laboratories.  Hence culture diagnosis is rarely used in clinical practice, mainly for studies of bacteria biology.  Serological testing for L. pneumophila infection is a valuable epidemiological tool, but has little impact on clinical decision making because of the time delay before a result is available. A four-fold or greater increase in titer of paired serum samples is regarded to indicate current L. pneumophila infection. Detection of soluble Legionella antigen in urine specimens is a rapid method that provides an early diagnosis of L. pneumophila infection. For the detection of L. pneumophila serogroup 1 (accounting for about 70-80% of community-acquired L. pneumophila infection  ), urinary antigen tests have sensitivities in the range of 80-90% and specificities approaching 98-100%.  L. antigenuria can be detected as early as 1 day after onset of symptoms and persists for days to weeks. The inadequacy of urinary antigen testing is a high price. Recently, DNA detection techniques have shown promise for the rapid diagnosis of Legionella infection. PCR has been successfully used to detect Legionella DNA in a range of environmental and clinical samples. The main gene targets used for detection of Legionella nucleic acid are 5S rRNA, 16S rRNA and mip gene. Further work is needed to establish a standard PCR method and procedures.
Comparison of different laboratory diagnosis methods
Mycoplasma pneumoniae is a common cause of CAP. The effect of laboratory diagnostic method has been an issue of great concern to clinicians. First, the standard laboratory diagnosis of M. pneumoniae infections presently relies on conventional serological methods. After infection by M. pneumoniae, IgM antibodies appear in 7 days of the illness, and high antibody titers can be maintained in adolescents. Meanwhile, adults fail to respond with IgM as a result of re-infections. All these result in the low sensitivity of IgM assay, only 31.8% and 33.3% found in foreign studies. , In China, Qu et al. found the sensitivity of the IgM assay was only 7.4%, specificity was 94.9%, which may be relevant with a high rate of M. pneumoniae re-infection.  Second, previous studies reported that the culture was unacceptably insensitive for diagnosing M. pneumoniae infection. But, Qu et al. reported sensitivity and specificity in culture were 55.6% and 94.9%, respectively. In particular, positive likelihood ratio of 10.9% in culture meant that the culture were optimum diagnosis of acute M. pneumoniae infections in adults and adolescents.  It has been shown that PCR as a rapid, sensitive, and specific method may be more useful during the early stages of M. pneumoniae infection.  However, the sensitivity and specificity of PCR commercial kits in diagnosis of acute M. pneumoniae infection were only 40.7% and 88.8%, respectively. This may be relevant with detection ability of kits and the long-term asymptomatic infection caused by M. pneumonia.  Gnarpe et al. demonstrated that during the peak period of M. pneumoniae incidence, About 13.5% of healthy volunteers were found to harbour the bacterium in the throat and during a subsequent period of 11 months, the incidence of M. pneumoniae isolated decreased to 4.6% of volunteers.  Therefore, optimum diagnosis of acute M. pneumoniae infection relies on the use of specialized tests in combination of PCR and serological tests on the basis of clinical symptoms and signs.
| Treatment and Drug Resistance of Atypical Pathogens Infection|| |
Macrolides and fluoroquinolones are generally considered to be the first-choice agents for the treatment of M. pneumoniae infection. Azithromycin and clarithromycin, a new macrolide antibiotics, may offer several advantages over erythromycin, including: Greater antimicrobial activity against certain organisms; longer elimination half-life, thus allowing less frequent administration; lower incidence of adverse gastrointestinal effects. Therefore, the compliance and tolerability of azithromycin and clarithromycin were superior to those of erythromycin.  In the 70s of the last century, resistance to macrolides was firstly reported in M. pneumoniae in a Japanese study.  Recently, macrolide-resistant M. pneumoniae have been spreading worldwide, with prevalences ranging from below 10% in Europe, ,, approximately 40% in America  and 8.2% in Japan.  The resistance rate of M. pneumoniae isolates from adult patients was 69%  in a 2009-2010 Chinese report and 71.7% in a 2013 Chinese report.  This indicates the prevalence of macrolide-resistant M. pneumoniae isolates in Chinese patients has increased sharply.
One report found there was a strong association between macrolide resistance in M. pneumoniae and point mutations in the 23S rRNA.  A2063G and A2064G mutations were responsible for the high-level marcolide resistance in M. pneumonia.  Previous results of 23S rRNA gene sequencing from a Beijing study indicated that all macrolide-resistant isolates harbored an A2063G mutation. , M. pneumoniae can be categorized into 2 genotypes, MP1 and MP2, based on the DNA sequence of the P1 adhesion protein, which is located in the cell membrane and is of vital importance for bacterial adhesion to epithelial cells. Previous studies found M. pneumoniae bacterial load and genotype were not associated with disease severity.  However, multiple-locus variable number tandem repeats analysis (MLVA) is a molecular typing method to be used to genotype several species of bacteria, showing nearly 30 subtypes of M. pneumonia.  "Pneumonia Monitoring Network in Beijing" research group using MLVA typing method to analyze the M. pneumoniae clinical isolates demonstrated that pneumonia severity index scores were significantly higher in patients with M. pneumoniae types U (5-4-5-7-2) and J (3-4-5-7-2) (P < 0.001), and total duration of cough were longer in them (P = 0.011). Moreover, the rate of macrolide nonresistance of isolates harboring Mpn13-14-15-16 as 3-5-6-2 was significantly higher than those with other variable number tandem repeats profiles.  In view of a few relevant studies, only 136 M. pneumoniae strains were covered in those reports. Therefore, it is difficult to accurately determine the influence of different genotypes of M. pneumoniae on clinical features of patients with infection.
In summary, atypical pathogens, especially M. pneumoniae is a common and important pathogen of CAP. Physicians should pay more attention to them and put forward higher requirements for various medical microbiology laboratories in pathogen detection. Meanwhile, with the deepening of relevant studies on pathogens epidemic, drug resistance and the association between genotypes and clinical features, more theoretical basis will be provided for clinical diagnosis of atypical pathogens infection.
| References|| |
|1.||Huang HH, Zhang YY, Huang SG, Lu Q, Zhou X, Xiu QY, et al. Investigation on etiology of community-acquired pneumonia in Shanghai. Chin J Infect Chemother 2003;3:321-4. |
|2.||Porath A, Schlaeffer F, Lieberman D. The epidemiology of community-acquired pneumonia among hospitalized adults. J Infect 1997;34:41-8. |
|3.||Monsieur I, Meysman M, Vincken W, Huyghens L, Lauwers S. Severe community-acquired pneumonia caused by atypical organisms. Acta Clin Belg 1997;52:112-5. |
|4.||Yu RH, Zang GQ, Liu G, Le WL. Etiology and clinical features of 89 patients with leukopenia or normal leucocyte community acquired pneumonia. Chin J Clin Med 2003;3:321-4. |
|5.||Chanock RM, Hayflick L, Barile MF. Growth on artificial medium of an agent associated with atypical pneumonia and its identification as a PPLO. Proc Natl Acad Sci U S A 1962;48:41. |
|6.||Grayston JT, Campbell LA, Kuo CC, Mordhorst CH, Saikku P, Thom DH, et al. A new respiratory tract pathogen: Chlamydia pneumoniae strain TWAR. J Infect Dis 1990;161:618-25. |
|7.||Diederen BM. Legionella spp. and Legionnaires′ disease. J Infect 2008;56:1-12. |
|8.||Liu YN, Chen MJ, Zhao TM, Wang H, Wang R, Liu QF, et al. A multicentre study on the pathogenic agents in 665 adult patients with community-acquired pneumonia in cities of China. Zhonghua Jie He He Hu Xi Za Zhi 2006;29:3-8. |
|9.||Cao B, Ren LL, Zhao F, Gonzalez R, Song SF, Bai L, et al. Viral and Mycoplasma pneumoniae community-acquired pneumonia and novel clinical outcome evaluation in ambulatory adult patients in China. Eur J Clin Microbiol Infect Dis 2010;29:1443-8. |
|10.||Qu JX, Gu L, Wu J, Li XL, Dong JP, Pu ZH, et al. Epidemiological analysis of Mycoplasma pneumoniae acute infection in adults with community-acquired pneumonia. Zhonghua Liu Xing Bing Xue Za Zhi 2012;33:545-6. |
|11.||Hyman CL, Roblin PM, Gaydos CA, Quinn TC, Schachter J, Hammerschlag MR. Prevalence of asymptomatic nasopharyngeal carriage of Chlamydia pneumoniae in subjectively healthy adults: Assessment by polymerase chain reaction-enzyme immunoassay and culture. Clin Infect Dis 1995;20:1174-8. |
|12.||Cao B, Zhao CJ, Yin YD, Zhao F, Song SF, Bai L, et al. High prevalence of macrolide resistance in Mycoplasma pneumoniae isolates from adult and adolescent patients with respiratory tract infection in China. Clin Infect Dis 2010;51:189-94. |
|13.||Kim NH, Lee JA, Eun BW, Shin SH, Chung EH, Park KW, et al. Comparison of polymerase chain reaction and the indirect particle agglutination antibody test for the diagnosis of Mycoplasma pneumoniae pneumonia in children during two outbreaks. Pediatr Infect Dis J 2007;26:897-903. |
|14.||Murdoch DR. Diagnosis of Legionella infection. Clin Infect Dis 2003;36:64-9. |
|15.||Helbig JH, Uldum SA, Bernander S, Lück PC, Wewalka G, Abraham B, et al. Clinical utility of urinary antigen detection for diagnosis of community-acquired, travel-associated, and nosocomial legionnaires′ disease. J Clin Microbiol 2003;41:838-40. |
|16.||Berdal BP, Farshy CE, Feeley JC. Detection of Legionella pneumonophila antigen in urine by enzyme-linked immunospecific assay. J Clin Microbiol 1979;9:575-8. |
|17.||Ozaki T, Nishimura N, Ahn J, Watanabe N, Muto T, Saito A, et al. Utility of a rapid diagnosis kit for Mycoplasma pneumoniae pneumonia in children, and the antimicrobial susceptibility of the isolates. J Infect Chemother 2007;13:204-7. |
|18.||Martínez MA, Ruiz M, Zunino E, Luchsinger V, Avendaño LF. Detection of Mycoplasma pneumoniae in adult community-acquired pneumonia by PCR and serology. J Med Microbiol 2008;57: 1491-5. |
|19.||Qu J, Gu L, Wu J, Dong J, Pu Z, Gao Y, et al. Accuracy of IgM antibody testing, FQ-PCR and culture in laboratory diagnosis of acute infection by Mycoplasma pneumoniae in adults and adolescents with community-acquired pneumonia. BMC Infect Dis 2013;13:172. |
|20.||Morozumi M, Hasegawa K, Chiba N, Iwata S, Kawamura N, Kuroki H, et al. Application of PCR for Mycoplasma pneumoniae detection in children with community-acquired pneumonia. J Infect Chemother 2004;10:274-9. |
|21.||Foy HM. Infections caused by Mycoplasma pneumoniae and possible carrier state in different populations of patients. Clin Infect Dis 1993;17 Suppl 1:S37-46. |
|22.||Gnarpe J, Lundbäck A, Sundelöf B, Gnarpe H. Prevalence of Mycoplasma pneumoniae in subjectively healthy individuals. Scand J Infect Dis 1992;24:161-4. |
|23.||The Respiratory Diseases Branch of Chinese Medical Association Infection. Expert consensus of diagnosis and treatment of Mycoplasma pneumonia in adult. Chin J Tuberc Respir Dis 2010;33:643-5. |
|24.||Atkinson TP, Balish MF, Waites KB. Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections. FEMS Microbiol Rev 2008;32:956-73. |
|25.||Pereyre S, Charron A, Renaudin H, Bébéar C, Bébéar CM. First report of macrolide-resistant strains and description of a novel nucleotide sequence variation in the P1 adhesin gene in Mycoplasma pneumoniae clinical strains isolated in France over 12 years. J Clin Microbiol 2007;45:3534-9. |
|26.||Dumke R, von Baum H, Lück PC, Jacobs E. Occurrence of macrolide-resistant Mycoplasma pneumoniae strains in Germany. Clin Microbiol Infect 2010;16:613-6. |
|27.||Peuchant O, Ménard A, Renaudin H, Morozumi M, Ubukata K, Bébéar CM, et al. Increased macrolide resistance of Mycoplasma pneumoniae in France directly detected in clinical specimens by real-time PCR and melting curve analysis. J Antimicrob Chemother 2009;64:52-8. |
|28.||Morozumi M, Takahashi T, Ubukata K. Macrolide-resistant Mycoplasma pneumoniae: Characteristics of isolates and clinical aspects of community-acquired pneumonia. J Infect Chemother 2010;16:78-86. |
|29.||Yamada M, Buller R, Bledsoe S, Storch GA. Rising rates of macrolide-resistant Mycoplasma pneumoniae in the central United States. Pediatr Infect Dis J 2012;31:409-0. |
|30.||Yin YD, Cao B, Wang H, Wang RT, Liu YM, Gao Y, et al. Survey of macrolide resistance in Mycoplasma pneumoniae in adult patients with community-acquired pneumonia in Beijing, China. Zhonghua Jie He He Hu Xi Za Zhi 2013;36:954-8. |
|31.||Okazaki N, Narita M, Yamada S, Izumikawa K, Umetsu M, Kenri T, et al. Characteristics of macrolide-resistant Mycoplasma pneumoniae strains isolated from patients and induced with erythromycin in vitro. Microbiol Immunol 2001;45:617-20. |
|32.||Qu J, Yu X, Liu Y, Yin Y, Gu L, Cao B, et al. Specific multilocus variable-number tandem-repeat analysis genotypes of Mycoplasma pneumoniae are associated with diseases severity and macrolide susceptibility. PLoS One 2013;8:e82174. |
|33.||Nilsson AC, Björkman P, Welinder-Olsson C, Widell A, Persson K. Clinical severity of Mycoplasma pneumoniae (MP) infection is associated with bacterial load in oropharyngeal secretions but not with MP genotype. BMC Infect Dis 2010;10:39. |
|34.||Dégrange S, Cazanave C, Charron A, Renaudin H, Bébéar C, Bébéar CM. Development of multiple-locus variable-number tandem-repeat analysis for molecular typing of Mycoplasma pneumoniae. J Clin Microbiol 2009;47:914-23. |