Copyright © 2003, American Society for Microbiology. All Rights Reserved.
MINIREVIEW
Molecular Diagnosis of
Mycoplasma pneumoniae
Respiratory
Tract Infections
K. Loens,
1* D. Ursi,
1H. Goossens,
1,2and M. Ieven
1Medical Microbiology, Universitaire Instelling Antwerpen, B2610 Wilrijk, Belgium,1and Department of
Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands2
Mycoplasma pneumoniaeis responsible for 10 to 20% of the
cases of community-acquired pneumonia and has been associ-ated with acute exacerbations of asthma (22).M.pneumoniaeis also implicated in mild acute respiratory infections, such as sore throat, pharyngitis, rhinitis, and tracheobronchitis (2).
Correct diagnosis ofM.pneumoniaeinfections is important to allow the appropriate antibiotic treatment of patients, since it is impossible to identify aM.pneumoniaeinfection solely on the basis of clinical signs and symptoms. It should decrease inappropriate use of antibiotics, influence the patient outcome by reduction of morbidity and mortality, and improve our knowledge of the prevalence of the causes of so-called atypical pneumonia.
Conventional assays for the detection of M. pneumoniae
have their limitations, resulting in the need for more accurate diagnostic methods. Culture is time-consuming and relatively insensitive, because M. pneumoniaegrows slowly in vitro, re-quiring 2 to 5 weeks for colonies to become visible. Serological methods, particularly the complement fixation (CF) test, are most widely used. The sensitivity of these assays depends on whether the first serum sample is collected early or late after the onset of disease and on the availability of paired serum samples collected with an interval of 2 to 3 weeks. Immuno-globulin M (IgM) assays which are more sensitive than the CF test have been developed, but the IgM response may be non-specific (61) or absent, particularly in adults (70). Hybridiza-tion with DNA probes has also been proposed as a rapid and specific procedure to replace culture, but it lacks sensitivity (35).
Nucleic acid amplification techniques (NAATs) have the potential to produce rapid, sensitive, and specific results, al-lowing early appropriate antibiotic therapy.
In the absence of a reference method, the so-called “gold standard” for the diagnosis of an M. pneumoniae infection, either an expanded gold standard or the technique of latent class analysis (LCA) should be applied to calculate the sensi-tivity and specificity of the available diagnostic tests. The nique of LCA can be used if at least three independent tech-niques can be compared. Thus far, only a few PCR tests and a limited number of studies applying culture, serology, and
sev-eral NAATs targeting different genes to detectM.pneumoniae
have been adequately evaluated.
Since NAATs targeting DNA can detect both viable and nonviable organisms, detecting RNA by reverse transcriptase PCR (RT-PCR) or nucleic acid sequence-based amplification (NASBA) may be a useful method to identify productiveM.
pneumoniaeinfections.
The possible long-term carrier state ofM.pneumoniaein the respiratory tract may hinder the evaluation of different diag-nostic tests for the diagnosis of acute infections.
An overview of the peer-reviewed literature on the use of NAATs to detectM.pneumoniaesince 1989 is given. Search combinations wereM.pneumoniaeand PCR,M.pneumoniae
and diagnosis, andM.pneumoniaeand amplification. This mi-nireview describes the molecular biology-based amplification methods to detectM.pneumoniaethat are currently available. Topics discussed include specimen collection and transport, preparation of nucleic acid from clinical specimens, choice of the target sequence, and detection of the amplicons. Methods to recognize and prevent false-positive and false-negative re-sults, the results of NAATs in comparison with results ob-tained by conventional diagnostic tests, and clinical applica-tions are also reviewed.
TECHNICAL ASPECTS
Specimen collection.Specimens suitable for the detection of
M. pneumoniaeinclude sputum (74) and bronchoalveolar
la-vage (BAL) specimens (40, 66), nasopharyngeal and throat swabs (25, 78), nasopharyngeal aspirates (20, 28, 36), tracheal aspirates (1, 26), pleural fluid specimens (52), and transtho-racic needle aspirations (17). More unusual specimens, such as nasal polyps (29), open-lung biopsies, and autopsy specimens (65), have also been tested.
Gnarpe et al. (25) compared nasopharyngeal and throat swabs for the detection of M. pneumoniaeand found throat swabs to be superior to nasopharyngeal swabs. Honda et al. (34) applied capillary PCR to sputum and BAL specimens and throat swabs. Review of the differences in PCR positivity rates as a function of the type of specimens collected showed the highest rate of detection from throat swabs (28.6%). However, there were some problems with proper collection of throat swab specimens due to inadequate scraping of the mucosal surface, resulting in false-negative results due to the collection of an insufficient amount of DNA. The positivity rate was
* Corresponding author. Mailing address: Medical Microbiology, Universitaire Instelling Antwerpen, Universiteitsplein 1 S3, B2610 Wilrijk, Belgium. Phone: 3238202551. Fax: 3238202663. E-mail: katherine.loens@ua.ac.be.
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21.5% for BAL specimens and 14.2% for sputum specimens. The low positivity rate for sputum specimens was attributed to the absence of sputum in many patients with pneumonia due to
M.pneumoniae.
Transport of specimens.Specimens should be transported to the laboratory as soon as possible and stored at 4°C or frozen at⫺70°C. RNA specimens should be processed or fro-zen at⫺70°C as soon as possible to prevent RNA degradation (6, 47).
Processing of specimens.Different extraction methods with and without sample pretreatment have been described: (i) di-lution of the sample with 0.9% sodium chloride, followed by the addition of sodium dodecyl sulfate, extraction with phenol-chloroform, and precipitation with ammonium acetate and ethanol (28); (ii) pretreatment with proteinase K (15), fol-lowed by phenol-chloroform or phenol-chloroform-isoamyl al-cohol extraction and ethanol precipitation (10); (iii) Boom extraction (5) on protease-treated (47) or untreated samples (62); (iv) treatment with Sputazyme (Kobayashi Pharmaceuti-cal Co., Tokyo, Japan), followed by proteinase K digestion (34); (v) incubation with Chelex (Bio-Rad Laboratories, Rich-mond, Calif.) and sodium azide (40); (vi) treatment by sonica-tion or boiling (7); (vii) phenol-chloroform-isoamyl alcohol extraction, followed by ether extraction (79); and (viii) phenol-chloroform extraction and precipitation by sodium acetate or sodium chloride (17).
Extraction methods using commercial kits are available: the Boehringer High Pure viral nucleic acid kit (Boehringer, Mannheim, Germany) (28); the QIAamp blood kit (QIAgen, Hilden, Germany) (69); cell lysis with sodium dodecyl sulfate and proteinase K, followed by purification with QIAamp DNA binding columns (QIAgen) (31); and the Amplicor sputum sample preparation kit (Roche Diagnostic Systems Inc., Branchbury, N.J.) (25), which was found to increase the sen-sitivity of aChlamydia pneumoniaePCR, due to a more com-plete lysis of cells in the specimens (24).
Fahle and Fischer (16) evaluated six commercially available DNA extraction kits for their ability to recover DNA from various dilutions of cytomegalovirus (CMV) added to BAL, cerebrospinal fluid, plasma, or whole-blood specimens. The kits evaluated included the Puregene DNA isolation kit (Gen-tra Systems Inc., Minneapolis, Minn.), the Generation capture column kit (Gentra Systems Inc.), the MasterPure DNA puri-fication kit (Epicentre Technologies, Madison, Wis.), the Iso-Quick nucleic acid extraction kit (MicroProbe Corp., Bothell, Wash.), the QIAamp blood kit (QIAgen), and the NucliSens extraction kit (Organon Teknika Corp., Durham, N.C.). All six kits evaluated effectively removed PCR inhibitors from each of the four specimen types and produced consistently positive results. However, the NucliSens extraction kit and the Pure-gene DNA isolation kit had the most consistently positive results at the lowest concentrations of spiked CMV and were the most sensitive methods for extracting CMV DNA from the four different kinds of spiked specimens.
In contrast, there are very few studies comparing different methods for M. pneumoniae nucleic acid extraction (1, 36). Abele-Horn et al. (1) compared the efficacy of DNA extraction by cell lysis with proteinase K without further nucleic acid purification and DNA extraction after lysis by phenol-chloro-form followed by ethanol precipitation. A 10-fold dilution
se-ries ofM.pneumoniaewas used. The researchers reported that the phenol-chloroform DNA extraction technique was time-consuming and resulted in a 10-fold decrease in sensitivity.
In a study by Ieven et al. (36), 371 nasopharyngeal aspirates from children with acute respiratory infections were examined for the presence of M. pneumoniae by culture and several different PCR protocols in two laboratories. Each laboratory applied one sample preparation method: (i) freeze-boiling (method A) or (ii) isothiocyanate treatment, followed by phe-nol chloroform extraction (method B). Prepared samples were exchanged between the two laboratories. In both laboratories, identical primers were used in a PCR directed at the P1 gene. A specific internal control for P1 amplification was included. After sample preparation method A, laboratory 1 identified 9 positive samples of 13 samples and identified 2 more samples after diluting them 1/10 to eliminate polymerase inhibitors. One additional positive sample was identified after hybridiza-tion. Laboratory 2, using the same material, obtained similar results. After sample preparation method B, 12 positive sam-ples were detected with the primers directed at the P1 adhesin gene in laboratory 1, and 13 positive samples were detected in laboratory 2 after hybridization. It was concluded that, pro-vided a specific internal control is used, sample preparation by freeze-boiling could be recommended.
Target regions and amplification protocols.Several regions in theM.pneumoniaegenome have been used to detect and identifyM. pneumoniaeby PCR or other amplification tech-niques (Table 1).
Van Kuppeveld et al. targeted a fragment of the 16S rRNA for genus and species identification ofM.pneumoniae(73, 74). They compared PCR with RT-PCR, and a 1,000-fold increase in sensitivity was found when rRNA instead of ribosomal DNA (rDNA) was used as a target (73). When the 16S rRNA PCR was compared with culture and serological methods for the diagnosis ofM. pneumoniaeinfections, it was concluded that PCR was the optimal approach (74). Tjhie et al. used the same primers in a comparison of direct PCR and RT-PCR (67), culture, and serology. A positive correlation between the direct PCR and serology, as tested by the microparticle agglutination assay, was found in 88.1% of the cases: PCR and serology gave positive results for 6 of 59 (10.2%) of patients, 3 of 59 (5.1%) patients were positive forM.pneumoniaeonly by PCR, and 4 of 59 (6.8%) patients were positive forM.pneumoniaeonly by serology.
Kessler et al. designed a PCR targeted at the 16S rDNA; the amplicons were identified by probe hybridization in a nonra-dioactive microwell plate format (40). When applied to BAL specimens, 12 specimens were found positive by PCR and serology, of which 7 were subsequently confirmed by culture.
Bernet et al. selected a specific DNA sequence from a genomic library and chose two oligonucleotides in this se-quence to produce an amplicon of 144 bp (3). Analysis of clinical samples showed that PCR was more sensitive than culture for the detection ofM.pneumoniae. These results were confirmed by Skakni et al. (62) but were in contrast with those obtained by Falguera et al. (17) who applied the assay to transthoracic needle aspiration specimens. Vekris et al. (75) developed a microtiter hybridization assay to detect ATPase amplicons, generated by the Bernet primers (3), and found it
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to be cheaper, involving fewer steps and allowing easy handling of a large number of specimens.
Another frequently used target forM.pneumoniaePCR is the P1 adhesin gene. The target sequence for amplification in the assay designed by Buck et al. is a 375-bp segment of this gene (7). The assay proved to be more sensitive than culture and the Gen-Probe assay. De Barbeyrac et al. generated a 466-bp fragment of the P1 gene with primers MP-P11 and MP-P12. Identification was performed by restriction enzyme digestion (11). Leng et al. (46) designed an assay resulting in a 631-bp amplicon and used the primers described by Bernet et al. (3) for comparison. The assays were applied on throat swabs; the latter protocol was the most sensitive.
The PCR protocol described by Ursi et al. (72) was com-pared with culture and serological tests by Dorigo-Zetsma et al. (15). They concluded that PCR results could be added to the criteria for the diagnosis ofM.pneumoniaeinfections and could even replace culture; PCR and the CF test were found to be complementary.
The system developed by Lu¨neberg et al. (48) is based on the gene encoding the elongation factor Tu (tuf) targeted by prim-ers Mpn 38 and Mpn 39 and detected by probe Mpn 46. Compared with culture and serology, the assay had a sensitivity
of 90 and 83%, respectively. Compared with serology, the specificity was 97%.
Studies comparing the performance of PCR methods with different M. pneumoniae target regions and primers are ex-tremely rare. Ieven et al. (36) compared the PCR assay tar-geted at the P1 adhesin gene described by Ursi et al. (72) with the PCR assay described by Van Kuppeveld et al. (74). To confirm the identity of the amplicons, probe hybridization was performed. The P1 adhesin gene primers were found to be more sensitive than the 16S rRNA primers. This most likely results from the P1 cytadhesin gene being present in multiple copies (64).
Three different PCR assays were compared by Abele-Horn et al. (1): (i and ii) the assay originally described by Bernet (3), with and without an additional hybridization step for amplicon detection, and (iii) a newly developed nested PCR format. The PCR was performed directly on the specimens or after over-night incubation in Hayflick broth. All three PCR assays proved to be reliable in detectingM.pneumoniaein respiratory specimens, but the nested format was the most sensitive one. Comparison of sensitivity data from different clinical studies is complicated by differences in sample collection,
transporta-TABLE 1. Nucleic acid amplification assays developed in-house for the detection ofM. pneumoniaeby year of publication
Authors publicationYr of Reference Gene target size (bp)Product Assay typea
Bernet et al. 1989 3 ATPase operon gene 144 S⫹H
Jenssen et al. 1989 38 P1 gene 153 S⫹A
Jenssen et al. 1989 38 16S rRNA gene 584 S⫹A
Buck et al. 1992 7 P1 gene 375 S⫹H
Sasaki et al. 1992 60 16S rRNA gene 809 S⫹H
Ursi et al. 1992 72 P1 gene 209 S⫹A
Van Kuppeveld et al. 1992 73 16S rRNA 277 RT⫹H
Williamson et al. 1992 81 P1 gene 543 S⫹H
Williamson et al. 1992 81 16S rRNA gene 290 S⫹H
Cadieux et al. 1993 9 P1 gene 345 M⫹H
De Barbeyrac et al. 1993 11 P1 gene 466 S⫹H/RE
Kai et al. 1993 39 16S rRNA gene 88 S⫹A
Lu¨neberg et al. 1993 48 tufgene 950 S⫹H
Zingangirova et al. 1993 84 P1 gene 245, 210 N⫹A
Leng et al. 1994 46 P1 gene 631 S⫹A
Tjhie et al. 1994 67 16S rRNA gene 277 S⫹H
Fink et al. 1995 18 P1 gene 466, 183 N⫹A
Ovyn et al. 1996 55 16S rRNA 190 NA⫹H
Ramirez et al. 1996 57 P1 gene 375 S⫹A
Stone et al. 1996 63 16S rRNA NSb Q
Kessler et al. 1997 40 16S rRNA gene 427 S⫹H
Abele-Horn et al. 1998 1 ATPase operon gene 144, 104 N⫹A
Narita et al. 1998 52 ATPase operon gene 144, 108 N⫹A
Corsaro et al. 1999 10 ATPase operon gene 144 M⫹A
Dorigo-Zetsma et al. 1999 14 P1 gene 272, 133 N⫹A
Gro¨ndahl et al. 1999 28 16S rRNA 277 M⫹H
Layani-Milon et al. 1999 45 P1 gene 466 S⫹H
Tong et al. 1999 69 P1 gene 345 M⫹H
Hardegger et al. 2000 31 P1 gene 76 R
Honda et al. 2000 34 ATPase operon gene 250 C⫹A
Kong et al. 2000 43 P1 gene 111–154 N⫹A
Waring et al. 2001 78 P1 gene 309–339 S⫹H
Loens et al. 2002 47 16S rRNA 190 NA⫹H
Welti et al. 2003 80 P1 gene 76 M⫹R
aAbbreviations: A, Agarose gel electrophoresis; C, capillary PCR; H, hybridization; M, multiplex PCR; N, (semi)nested PCR; NA, NASBA; Q, Qreplicase; R,
Real-time PCR; RE, restriction enzyme digestion; RT, RT-PCR; S, single-step PCR.
bNS, not specified.
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[image:3.603.45.539.79.416.2]tion, and extraction procedures, input sample volumes, target genes, primers, cycling parameters, and detection systems.
Furthermore, there is great variation in the units applied to measure the detection limits: CFU, color-changing units (CCU) (1 CCU corresponds to 10 to 100 organisms [3]), num-ber of cells, or quantity of DNA. This makes a comparison of the sensitivity of different assays very difficult. For example, Abele-Horn et al. (1) reported the sensitivity of their assay to be 3,000 genome copies, 30 pg of DNA, 19 CFU, or 1.9⫻103
organisms. Furthermore, different definitions for 1 CFU are used: for example, in one study, 1 CFU corresponds to 160 organisms (32), whereas in another study, 1 CFU corresponds to 10 to 1,000 organisms (58).
More importantly, the lack of a consensus method for ap-propriate evaluation of the different methods precludes com-parisons. In conclusion, standardized highly sensitive and spe-cific NAATs are sorely needed.
Multiplex PCR.Multiplex PCRs to detect two or three dif-ferent respiratory pathogens have been developed by some groups (9, 10, 69). Gro¨ndahl et al. developed a multiplex RT-PCR to detect nine respiratory pathogens in a single tube (28). However, comparisons of monoplex and multiplex PCR assays are rare. Both duplex PCR and the monoplex PCR applied by Corsaro et al. detected 2 CFU of M. pneumoniae in DNA extracts from clinical samples (10). Welti et al. (80) developed a multiplex real-time quantitative PCR assay to detectC.
pneu-moniae,Legionella pneumophila, andM.pneumoniaein
respi-ratory tract specimens. When dilutions of the three pathogen DNAs cloned in plasmids were tested, no significant differ-ences in the sensitivity of each primer set in both the multiplex and monoplex real-time PCR assays were observed. The com-parison of multiplex real-time and conventional PCR assays on 73 respiratory specimens showed an overall agreement of 98.3%, corresponding to 95.8, 100, and 100% agreement forC.
pneumoniae,L.pneumophila, andM.pneumoniae, respectively.
Tong et al. (69) compared the sensitivity and specificity of three PCRs when applied separately and in a triplex format. Sensitivity decreased by about 1 log unit, when the assays were combined. This result was not unexpected, given the complex-ity of variables in a multiplex PCR, including different combi-nations of primer concentrations, magnesium ion concentra-tions, and annealing temperatures. Surprisingly, the sensitivity of the nine-organism multiplex assay of Gro¨ndahl et al. is said to detect one target sequence in nucleic acid extracts from a dilutedM.pneumoniaestock solution (28).
Detection of amplification products.Southern blot hybrid-ization or (semi)nested reamplification are often used to in-crease the sensitivity and to confirm the specificity of the am-plicon. However, such methods are time-consuming and cannot be adapted to process large numbers of specimens. Recently, 5⬘ nuclease assays and retime PCR formats, al-lowing automated PCR amplification and detection of differ-ent pathogens, have been described (31, 54). The advantages of such a real-time system are higher speed, less handling of PCR products, and decreased risk of false-positive results due to carryover contamination. However, the sensitivity of real-time PCR targeting the P1 gene was slightly lower than that of conventional PCR (31). The reasons for this are not clear but may be related to the different assay formats.
Alternative amplification techniques.Besides PCR, alterna-tive amplification techniques, such as NASBA (55), transcrip-tion-mediated amplification, ligase chain reaction, Q repli-case amplification (63), and strand displacement amplification, have been developed. These techniques may be useful alter-natives to PCR, but so far, few studies on using these tech-niques to detectM.pneumoniaein respiratory specimens have been published, except for NASBA. The NASBA assay, tar-geted at 16S rRNA, followed by an enzyme-linked gel assay (ELGA) was used to type M. pneumoniae (55). Later on, NASBA in combination with ELGA and electrochemilumines-cence detection was used to detect M. pneumoniaeRNA in nucleic acid extracts from respiratory specimens (47). The Q
replicase assay was applied to detect syntheticM.pneumoniae
16S rRNA transcripts and seems to be less sensitive than PCR (63).
Quantification of M. pneumoniae nucleic acid. Dorigo-Zetsma et al. (14) investigated the relationship between theM.
pneumoniaeload and disease severity. TheM.pneumoniaeload
in five throat swabs fromM.pneumoniae-positive outpatients and from fiveM.pneumoniae-positive hospitalized patients was assessed semiquantitatively by performing a nested PCR on a series of dilutions of the nucleic acids extracted from these throat swabs. The calculated load varied from 20 to 3,830 CFU/ml. The mean M. pneumoniae load for samples from hospitalized subjects was significantly higher than the load for samples from nonhospitalized subjects.
Quantitative real-time DNA and RNA detection is now also possible by using specialized equipment, such as Perkin-Elmer Taqman (56).
PRACTICAL DIFFICULTIES AND IMPORTANCE OF QUALITY CONTROL
False-positive results.Interlaboratory comparisons illustrate the need for quality control of NAATs. The objective of the study by Ursi et al. (71) was to evaluate the performance of four different assays in detecting M. pneumoniae by PCR in three laboratories through exchange of DNA extracted from respiratory samples. False-positive PCR results were regis-tered in all three participating laboratories, underscoring the importance of including a sufficient number of negative con-trols in the amplification runs for the detection of sample-to-sample carryover, especially when a high proportion of positive samples is expected. In another study by Ieven et al. (36), contamination of entire amplification runs occurred twice, was visualized after hybridization, and was detected by positive results in the negative-control tubes. These contaminations resulted most probably from strong positive samples present in these runs. False-positive results, undetected by the negative controls, occurred in only 0.2% of the samples and were de-tected after hybridization.
Abele-Horn et al. (1) reported that an increase in sensitivity obtained by a nested PCR format was accompanied by an enhanced risk of contamination resulting in false-positive re-sults. In preliminary assays, contamination, detected by posi-tive results after the second amplification step with randomly incorporated negative samples, and probably caused by car-ryover, occurred in 10% of the samples. By strictly following the guidelines for PCR procedures, such as performing
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ent steps in separate rooms, contamination was reduced to 0.5% in their study.
Confirmation of positive findings by repeating the test, in-cluding the extraction procedure on the original specimen, is recommended to detect false-positive results due to sporadic contaminations.
False-negative results.Negative or nonreproducible NAAT results can be due to a low target copy number, inhibition of amplification, primer mismatches due to strain variations at the primer recognition site, or technical and methodological errors. Amplification inhibitors occur frequently and may be difficult to eliminate. These inhibitors include heme com-pounds (33) and polysaccharides in sputum (44), as well as in some reagents (27), and mucolytic agents added to sputum (12). Gibb and Wong found that placing throat swabs in Amies clear medium caused inhibition of PCR, the inhibitory com-ponent being agar that is dissolved by DNAzol and subse-quently precipitated with the DNA by ethanol (21). Others have noted inhibition of PCR by calcium alginate and alumi-num swab shafts (77). Therefore, both sampling devices and transport media should be checked for the presence of inhib-itors.
Different types of internal controls can be used with NAATs to discriminate between a false-negative reaction and a truly nonreactive sample: a homologous extrinsic control, a heter-ologous extrinsic control, and a heterheter-ologous intrinsic control. The former is a wild-type target-derived control, containing a non-target-derived sequence insert. It is added to each sample prior to nucleic acid extraction and coamplified in a single reaction with the same primers as used for amplification of the target sequence. They can be adapted depending on the de-tection format chosen. The advantages of the homologous extrinsic control are that sample-specific effects, the primers and probes, and the number of copies added can be monitored. The disadvantages are that it does not identify false-negative results resulting from degradation of nucleic acids in clinical specimens prior to the addition of the internal control and it
does not identify the absence of cellular material in a clinical specimen.
A heterologous extrinsic control is a non-target-derived con-trol added to each sample prior to nucleic acid extraction. It requires a duplex amplification of target DNA or RNA and control DNA or RNA in a single reaction. Although this kind of control requires primers and probes different from the tar-get, it will still reveal sample-specific effects. This option re-quires optimization to prevent inhibition of the target ampli-fication by the control ampliampli-fication reaction.
A heterologous intrinsic control confirms the presence of human nucleic acid and thus cellular material in the sample. An example is the detection of a low-abundance mRNA de-rived from a human cellular housekeeping gene, encoding the A protein present in the human U1 (U1A) small nuclear ribo-nucleoprotein (snRNP) particle, as a marker of the overall RNA integrity in clinical specimens when RNA is to be ana-lyzed (53). Other examples are the amplification of the -glo-bin or the gamma interferon gene in PCR assays. The disad-vantage of this control is the necessity to perform two separate amplification reactions on the same sample or a duplex ampli-fication, with possible inhibition resulting in the latter (37). This kind of control also allows sample-specific effects to be monitored. It cannot be recommended as a test of inhibition, since the number of copies of the human gene in each sample may be much higher than the number of copies of the M.
pneumoniaetarget gene. Interestingly, the proportion of
respi-ratory specimens revealing inhibition by this type of control is usually much lower than that of the extrinsic homologous con-trol: 0 to 10% versus 15 to 36% (Table 2).
Dilution of samples or nucleic acid extracts is a simple method to improve amplification by the reduction of inhibi-tors; however, the sensitivity may also be reduced as a result of dilution of the target molecules, and consequently, samples with low copy numbers may yield false-negative results.
The study of Ursi et al. (71), revealed also that none of the three participating laboratories was free of false-negative
re-TABLE 2. Application of controls used to monitor inhibition of PCR forM. pneumoniae
Reference Specimen(s)a No. of
specimens Type of controlb Inhibitionc
1 NA, TA 190 -Globin gene 10%, 2%d, 0% after dilution
4 TS 99 -Globin gene 0%
10 BAL, BA, PS 163 IFN-␥gene 0%
14 TS 305 EHC 20%, 0% after dilution
26 RPA, TA 165 G3PDH gene NS
36 NA 371 EHC 24.6%, 0% after dilution
59 TS, NA 54, 45 -Globin gene 0% for TS, 25% for NA
62 NA, BAL 124 -Globin gene 25%
66 BAL 103 EHC 7.8%, 0% after lower sample volume
67 TS, Sp, NSw, BAL, NA 79 -Globin gene 5.1%
68 TS, NSw 462 -Globin gene 11% without extraction, 1.1% after extraction
69 Sp 279 Human mt DNA 3.2%, 0% after dilution
72 NA, BA, NSw, BAL, Sp, ETA, TS, PF 219 EHC 15%, 0% after dilution
78 TS 41 -Globin gene 0%
aAbbreviations: BA, bronchus aspirate; BAL, bronchoalveolar lavage; ETA, endotracheal aspirate; NA, nasopharyngeal aspirate; NSw, nasopharyngeal swab; PF,
pleural fluid; PS, pharyngeal swab; Sp, sputum; TS, throat swab; RPA, rhinopharyngeal aspirate; TA, tracheal aspirate.
bAbbrevations: IFN-␥, gamma interferon; EHC, extrinsic homologous control; G3PDH, glyceraldehyde phosphate dehydrogenase; mt, mitochondrial.
cAbbreviations: NS, not specified; TS, throat swab; NA, nasopharyngeal aspirate.
dWhen PCR is applied on culture-enhanced Hayflick broth (i.e., specimens were received, immediately inoculated into Hayflick broth, and incubated overnight, after
which PCR was applied).
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[image:5.603.40.543.80.237.2]sults: one false-negative result was obtained from 78 samples in two laboratories, and four false-negative results were obtained from 78 samples in the third laboratory. The concordance of the assay results in the three laboratories was 84%.
Validation of amplification techniques. In-house NAATs were validated by assessing their analytical specificity, sensitiv-ity, and reproducibility on both simulated and clinical speci-mens in comparison with the best available conventional meth-ods. To define its analytical specificity, the NAAT developed in-house was applied to cultures of organisms that are taxo-nomically related to the target organism and to organisms commonly present in the clinical specimens to be examined. The analytical sensitivity of the test is defined as the smallest number of organisms that yield a positive result. In practice, this was determined by testing serial dilutions of the target organism, usually under optimal conditions. By repeatedly ap-plying the NAAT to the same series of specimens, the repro-ducibility of the test could be elucidated. Finally, the defined NAAT has to be compared with the best traditional diagnostic procedures in the clinical microbiology laboratory (this may actually require a combination of methods, including culture, serology, or other microscopic methods) on a panel of positive and negative specimens.
Traditionally, NAATs were validated by comparing the NAAT results on a series of clinical specimens with the results of one or two traditional tests.
There are, at present, very few prospective studies compar-ing the performance of two or more amplification protocols, including different specimen preparation methods, on a large number of unselected specimens. Given the multiple amplifi-cation protocols proposed, such studies are clearly needed, but the high cost of multiple amplification protocols limits the number of such studies.
Ideally, a newly proposed NAAT should be validated by comparison with a sensitive culture system and at least one validated PCR or another NAAT that targets a different gene or a different sequence of the same gene.
A comparison of different methods for the diagnosis of re-spiratory tract infections by M. pneumoniae is presented in Table 3. Agreement between different methods is frequently low, and PCR findings by one or more alternative methods have not always been confirmed.
APPLICATION OF NAAT FOR THE DIAGNOSIS OF
M.PNEUMONIAEINFECTIONS IN
RESPIRATORY INFECTIONS
Waring et al. studied a large outbreak ofM.pneumoniaein a closed religious community in New York State (78). Throat swab specimens were collected and processed for culture and PCR. A total of 349 specimens were tested by PCR. Of the 349 specimens, 280 specimens were from the original outbreak and 69 were follow-up specimens. Of the 280 initial specimens, 73 were positive and 207 were negative after PCR and hybridiza-tion and 22 were positive by culture. The first specimens were tested by both culture and PCR. Since no PCR-negative spec-imens were culture positive, the remaining specspec-imens were tested by culture only if they were PCR positive. By this ap-proach, PCR was approximately twice as sensitive as culture. In contrast to these findings, Kai et al. detectedM.pneumoniaeby
PCR in only 22 of the 30 throat swabs that were positive by culture (39). In this study, however, culture was considered positive when the medium changed color without contamina-tion of the growth ofM.pneumoniae. These researchers them-selves stated that their culture method may not have been specific forM.pneumoniae.
Ieven et al. (36) detectedM.pneumoniaein 3.5% of samples from children with acute respiratory tract infections but de-tected it significantly more often (6.9%) in samples from chil-dren above 2 years of age.M.pneumoniaewas the third most common etiologic agent of acute respiratory infections in chil-dren, after respiratory syncytial virus and influenza virus. In lower respiratory tract infections, such as bronchopneumonia and pneumonia, M. pneumoniaewas found as frequently as respiratory syncytial virus (36).
Abele-Horn compared PCR, culture, serology, and the di-rect antigen test for M. pneumoniae (1) in specimens from patients with acute respiratory complaints. A total of 190 pa-tients were divided into three groups: group I (n⫽90) con-sisted of immunocompromised patients with respiratory com-plaints after organ or bone marrow transplantation, group II (n
⫽ 50) were adults with acute respiratory tract disease, and group III (n ⫽ 50) included children with lower respiratory tract infections. Among the 190 patients, 20 (11%) were pos-itive by PCR, 11 (8%) were pospos-itive by the direct antigen test, 8 (4%) were culture positive, and 17 (9%) were positive by serology. In group I, there were 6, 1, 0, and 3 positive results, respectively. In group II, there were 6, 5, 4, and 6 positive results, respectively. In group III, there were 8, 5, 4, and 8 positive results, respectively. In this study, the best correlation between culture, serology, and PCR results was observed among patients with current infections of the lower respiratory tract (groups II and III).
MacFarlane et al. (49) prospectively studied the incidence, etiology, and outcome of lower respiratory tract infections in adult outpatients by serology, culture, and PCR on throat swabs and sputum specimens collected from 316 patients. Twenty-three patients (7.3%) were found to beM.pneumoniae
positive. Surprisingly, all were diagnosed by serology. Kessler et al. prospectively collected BAL specimens for PCR and culture from 116 patients admitted to the hospital with a community-acquired pneumonia (40). Serology forM.
pneumoniaewas done on both acute- and convalescent-phase
sera by the CF test. Twelve samples (10.3%) were PCR posi-tive, and 7 (6.0%) of these were subsequently confirmed by culture. The CF test showed seroconversion for these 12 pa-tients, and the results for all other patients remained negative. In contrast, in a comparison of PCR with serological methods, Menendez et al. (51) found their PCR assay to have a lower sensitivity than serology. Only 3 of 184 community-acquired pneumonia patients were foundM.pneumoniaepositive (51).
M.pneumoniaeinfections seem to be rare in human
immu-nodeficiency virus (HIV)-infected patients (82). Tarp et al., in a retrospective study, applied PCR to BAL fluids obtained from 103 episodes of pneumonia in 83 patients (66).M.
pneu-moniae was found in two patients (2%). In both cases, M.
pneumoniae was present as a coexisting pathogen. The
re-searchers concluded thatM.pneumoniaedoes not seem to play a major role in lower respiratory tract infections in HIV-in-fected adults and children.
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PCR also detectedM. pneumoniaein specimens from 1 to 3% of healthy subjects (48, 67) or in patients after symptomatic infections and even after antibiotic treatment, raising the pos-sibility of a carrier state or persistence of the organism in the respiratory tract (19, 42). Furthermore, Gnarpe et al. found that in endemic and epidemic situations, 4.6 and 13.6%, re-spectively, of healthy blood donors had throat swabs positive
forM.pneumoniae(23).
CONCLUSIONS
Numerous in-house PCR assays to detect M. pneumoniae
have been developed. Proper validation and standardization are often lacking, and quality control studies have revealed frequent deficiencies resulting in both negative and false-positive results.
Consequently, these tests must be submitted to extensive validation before their introduction in the molecular diagnostic laboratory. Validation must be performed at several levels, including sample preparation, amplification, and detection. Since respiratory samples often contain substances inhibiting amplification, special attention should be paid to the efficiency of the reaction with these samples. Once a test is validated, it should be further evaluated in proficiency testing programs.
Whereas quality control is an essential part of quality assur-ance in molecular diagnostics, proficiency panels for the de-tection ofM. pneumoniaeare not readily available yet. They are urgently needed to allow meaningful comparisons between the results obtained in different laboratories.
[image:7.603.48.544.82.442.2]Since it has been reported that application of multiplex NAATs may decrease the sensitivity, these assays have to be
TABLE 3. Comparison of methods for the diagnosis of respiratoryM. pneumoniaeinfection
Reference Specimen(s)a No. of
patients Subject agerangeb
No. (%) of samples positive by detection methodc:
PCR Culture CF DAG Serology
1 NA, TA 190 3–66 20 (10.5) 8 (4.2) ND 11 (5.8) 17 (8.9)
4 TS 99 ⬍1–81 49 (49.5)f ND ND ND 32 (32.3)
8 TS 21 2–18 11 (52.4) ND ND ND 13 (61.9)
10 BAL, BA, Sp 163 1–81 13 (8.0) 9 (5.5) ND ND ND
11 BAL, TS 75 NS 6 (8.0) ND ND ND ND
13 TS, BAL, BA, SP, NSw 144 20–93 15/18 (10.4) ND 10/18 (6.9) ND 4/18 (2.8)
14 TS 305 NS 12 (3.9) ND ND ND ND
15 TS 92 NS 7/9 (7.6) 6/9 (6.5) 7/9 (7.6) ND 7/9 (7.6)
17 TNA 93 NS 8 (8.6) ND ND ND 18 (19.3)
20 NA 132 3m–14y 3 (2.3) ND ND ND ND
25 NSw, TS 66 NS 7 (10.6) ND ND ND ND
28 NA 1,118 NS 91 (8.1) ND ND ND ND
26d RPA, TA 165 2–15 22 (13.3) 13 (7.9) 5 (3.0) ND 12 (7.3)
30 NA 115 6m–29m 30 (26) ND ND ND ND
31 NA, TS 48 ⬍1–16 31 (64.5) ND ND ND 23 (47.9)
34 TS, BAL, Sp 197 NS 25/34 (12.7) ND ND ND 31/34 (15.7)
36 NA 371 ⬍1–16 13 (3.5) 8 (2.2) ND ND ND
39 TS 105 NS 27/31 (25.7) 31 (29.5) ND ND ND
40 BAL 116 1–80 12 (10.3) 7 (6.0) 12 (10.3) ND ND
41 TS 63 21–62 9 (14.3) ND ND ND ND
45 NSw 3,897 NS 283 (7.3) ND ND ND ND
48 TS 102 NS 35 (34.3) 21 (20.5) 40 (39.2) ND ND
49 TS, Sp 316 NS 0 (0) ND ND ND 23 (7.3)
50 NSw, TS 557 0–94 7 (1.3) ND ND ND ND
51 TS, Sp 184 NS 3 (1.6) ND ND ND 2 (1.1)
57 TS 155 7–92 8 (5.2) ND ND ND 9 (5.8)
62 NA, BAL 100 ⬍1–16 20 (20) 1 (1) 5 (5) ND ND
66 BAL 103 NS 2 (1.9)f ND ND ND ND
67 Sp, TS, NSw, BAL, NA 79 ⬍1–85 9/14 (11.4) 2/14 (2.5) 9/14 (11.4)j ND 9/14 (11.4)
68 TS, NSw 462 0–16 11 (2.4) ND ND ND ND
69 Sp 244 5–95 7 (2.9) ND ND ND ND
74 Sp 34 NS 9/10 26.5) 7/10 (20.1) ND ND 9/10 (26.5)
76 TS 473 15–65 4 (0.8)g 2 (0.6)h ND ND 2/4
78 TS 280 NS 73 (26.1) 22 (7.9)i ND ND ND
79e NA 278 ⬍1–16.5 10 (3.6) 8 (2.9) ND ND 31 (11.2)
83 TS, NSw 168 ⬍1–16 5 (3.0) 5 (3.0) ND ND 10 (6.0)
aAbbreviations: BAL, bronchoalveolar lavage; NA, nasopharyngeal aspirate; NSw, nasal swab; PS, pharyngeal swab; RPA, rhinopharyngeal aspirate; Sp, sputum; TA,
tracheal aspirate; TNA, transthoracic needle aspiration; TS, throat swab.
bAge ranges given in years (y) unless specified otherwise. m, months; NS, not specified.
cAbbreviations: CF, complement fixation test; DAG, direct antigen test; ND, not done.
dTwenty PCR-positive specimens were cultured, but complement fixation and IgM serology were not done for all patients.
ePCR and culture were not done for all patients.
fResults confirmed by another PCR targeting another region of the P1 gene.
gTwo of four nasopharyngeal aspirates were PCR positive.
hTwo of 350 throat swab specimens were culture positive.
iCulture was done for a selection of 108 of 280 specimens.
jDone for 59 of 79 patients.
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compared carefully with the corresponding individual assays. In many studies, this comparison is lacking.
Given the high sensitivity and specificity of NAATs, NAATs are the preferred diagnostic procedures for the diagnosis ofM.
pneumoniaeinfections, provided that the quality of the
proce-dures is controlled. Additional studies on large numbers of patients with respiratory signs and symptoms, including hospi-talized and nonhospihospi-talized patients, are necessary to extend our knowledge on the epidemiology ofM.pneumoniae.
As with most applications of NAATs, various amplification targets and the different methods used to detect the targets must be compared to define the most sensitive and specific tests; these studies remain to be undertaken.
ACKNOWLEDGMENT
The financial support from the European Commission QLK2-CT-2000-00294 is gratefully acknowledged.
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