Katherine Loens

Molecular Diagnosis of Mycoplasma pneumoniae Respiratory Tract Infections

Mycoplasma pneumoniae is responsible for 10 to 20% of the cases of community-acquired pneumonia and has been associated with acute exacerbations of asthma ([22][1]). M. pneumoniae is also implicated in mild acute respiratory infections, such as sore throat, pharyngitis, rhinitis, and

Evaluation of NucliSens easyMAG for automated nucleic acid extraction from various clinical specimens.

ABSTRACT The objectives of this study were to evaluate the performance of the NucliSens easyMAG platform for nucleic acid extraction from different clinical specimens compared to NucliSens miniMAG platform and manual QIAGEN extraction. The NucliSens easyMAG and the NucliSens miniMAG showed equal performance on 215 throat swabs since real-time nucleic acid sequence-based amplification scored the same samples positive for Mycoplasma pneumoniae (n = 9) and Chlamydia pneumoniae (n = 5) RNAs, although internal control RNA was slightly better detected with the NucliSens easyMAG (99.3% versus 96.8%). NucliSens easyMAG extracted nucleic acids more efficiently (higher recovery and/or fewer inhibitors) compared to QIAGEN extraction by showing, on average, lower Ct values in real-time LightCycler PCR, although 4 individual specimen out of 45 were found positive only with QIAGEN. For nine M. pneumoniae-positive throat swabs, the mean difference in Ct values between NucliSens easyMAG extraction and QIAGEN extraction was −2.26 (range, −5.77 to +0.60); for the detection of five C. pneumoniae-positive throat swabs, the average difference in Ct values between the two methods was −3.38 (range, −6.62 to −2.02); and for the detection of cytomegalovirus in 24 blood samples, the mean difference in Ct values between the two methods was −0.95 (range, −5.51 to +1.68). The NucliSens easyMAG is considerably easier to perform, efficiently extracts nucleic acids from throat swabs and whole blood, is automated, and has high throughput.

Optimal Sampling Sites and Methods for Detection of Pathogens Possibly Causing Community-Acquired Lower Respiratory Tract Infections

Acute respiratory tract infections (RTIs), both upper (URTIs) and lower respiratory tract infections (LRTIs), are the most common reason for consultation with a general practitioner. RTIs result in about 180 million antibiotic prescriptions per year in the EU-27 member states (ESAC website, 2008; www.esac.ua.ac.be), and 6.4 million antibiotic prescriptions were prescribed for acute bronchitis and cough in 2003 in adults between 16 and 64 years old in the United States (65). The number of pathogens involved in LRTI, with various susceptibilities to antimicrobials, is large constituting an enormous challenge for diagnostic microbiology. In general, in only 50% of cases is an etiologic agent detected. Documented infection is uncommon in community-managed infection and is usually only defined in 25 to 50% of hospital-managed infections. The upper end of the respiratory tract, the rhinopharynx, is widely open to the introduction of airborne microorganisms. It is, however, also a very efficient barrier for invading bacteria. The barrier function of the rhinopharynx results from the local lymphoid tissue producing phagocytic cells and secretory immunoglobulin (Ig) and from the rich commensal flora of aerobic and anaerobic microbes establishing an interfering colonization resistance. The number of these organisms varies from 2.6 × 104 to 4 × 108 CFU of cultivable bacteria per cm2 (95). Aerobes tend to decrease and anaerobes tend to increase with age (98). Colonization starts during the first year of life and is composed not only of commensal organisms but also of potential pathogens: group A β-hemolytic streptococci, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. These are present permanently or intermittently, with rates varying with age (decreasing in adults), exposure to other children, geographic location, socioeconomic status, and vaccination status (39). In most cases, these organisms cause disease in only a small percentage of persons colonized. Viruses do not normally colonize the rhinopharynx. However, most bacterial respiratory infections start with or are accompanied by their proliferation in the nasopharynx. Thus, the URT with its commensal flora acts both as a defense mechanism and as a primary site for LRTI, which creates tremendous diagnostic challenges. Any bacteriological examination of nonsterile respiratory specimens must indeed distinguish between organisms infecting the LRT and organisms colonizing the rhinopharynx. Normally sterile samples therefore are considered the “gold standard.” Different specimens commonly collected to detect pathogens causing LRTI have been compared, but the results were not consistent (12, 43, 49, 58, 103) (Table ​(Table1;1; and see Table S1 in the supplemental material). This minireview presents an overview of the optimal detection of Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Mycoplasma pneumoniae, Chlamydophila pneumoniae, Legionella pneumophila, and respiratory viruses from specimens in patients with community-associated (CA)-LRTI (Table ​(Table22). TABLE 1. Comparison of specimens and sampling methods for the detection of different respiratory pathogensa TABLE 2. Diagnostic approach for the most common specific agents in LRTIsa

Detection of Mycoplasma pneumoniae in respiratory samples by real-time PCR using an inhibition control.

Polymerase chain reaction (PCR) with real-time detection using two adjacent fluorescent probes in a Lightcycler instrument was applied for detection of the Mycoplasma pneumoniae P1 protein gene. To monitor inhibition in each sample an internal control was constructed that can be amplified by the same primers but detected by different probes and dual color detection. The real-time PCR was applied on 115 respiratory samples from 82 patients and compared to a conventional PCR. There was 100% agreement between the assays, but the real-time PCR proved to be highly superior in speed with a much lower risk of false positives by laboratory contamination.

Development of Real-Time Multiplex Nucleic Acid Sequence-Based Amplification for Detection of Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in Respiratory Specimens

ABSTRACT Real-time multiplex isothermal nucleic acid sequence-based amplification (NASBA) was developed to detect Mycoplasma pneumoniae, Chlamydophila pneumoniae, and Legionella spp. in respiratory specimens using the NucliSens Basic Kit (bioMérieux, Boxtel, The Netherlands). Oligonucleotide primers were derived from the M. pneumoniae, C. pneumoniae, and Legionella pneumophila 16S rRNA. For real-time detection, molecular beacons were used. Specificity was established on a panel of bacterial strains. The analytical sensitivity of the assay was determined by testing dilutions of wild-type in vitro-generated RNA in water and dilutions of reference strains in lysis buffer or added to pools of respiratory specimens. Subsequently, a limited number of M. pneumoniae-, C. pneumoniae-, and L. pneumophila-positive and -negative clinical specimens were analyzed. Specific detection of the 16S rRNA of the three organisms was achieved. The analytical sensitivity of the multiplex NASBA on spiked respiratory specimens was slightly diminished compared to the results obtained with the single-target (mono) real-time assays. We conclude that the proposed real-time multiplex NASBA assay, although less sensitive than the real-time mono NASBA assay, is a promising tool for the detection of M. pneumoniae, C. pneumoniae, and Legionella spp. in respiratory specimens, regarding handling, speed, and number of samples that can be analyzed in a single run.

Detection of Mycoplasma pneumoniae by Real-Time Nucleic Acid Sequence-Based Amplification

ABSTRACT Real-time isothermal nucleic acid sequence-based amplification (RT-NASBA) was applied to the detection of Mycoplasma pneumoniae. In vitro-generated M. pneumoniae RNA was used to assess the sensitivity of the assay. The 95% hit rate was 148 molecules of M. pneumoniae RNA in the amplification and 104 molecules of in vitro-generated RNA after nucleic acid extraction. The sensitivity of the RT-NASBA and the conventional NASBA assays corresponded to 5 color-changing units (CCU) of M. pneumoniae. In spiked throat swabs, nasopharyngeal aspirates, bronchoalveolar lavages, and sputum, the sensitivity of both NASBA assays corresponded to 5 to 50 CCU of M. pneumoniae. A total of 17 clinical specimens positive for M. pneumoniae by PCR were also positive by conventional NASBA, but one specimen was negative by RT-NASBA. These results indicate that the sensitivity of detection of M. pneumoniae by RT-NASBA in respiratory samples might be slightly reduced compared to that by conventional NASBA. However, the real-time assay is superior in speed and ease of handling.

Performance of Different Mono- and Multiplex Nucleic Acid Amplification Tests on a Multipathogen External Quality Assessment Panel

ABSTRACT An external quality assessment (EQA) panel consisting of a total of 48 samples in bronchoalveolar lavage (BAL) fluid or transport medium was prepared in collaboration with Quality Control for Molecular Diagnostics (QCMD) (www.qcmd.org). The panel was used to assess the proficiency of the three laboratories that would be responsible for examining the 6,000 samples to be collected in the GRACE Network of Excellence (www.grace-lrti.org). The main objective was to decide on the best-performing testing approach for the detection of influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus (RSV), human metapneumovirus, coronavirus, rhinovirus, adenovirus, Chlamydophila pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila by nucleic acid amplification techniques (NAATs). Two approaches were chosen: (i) laboratories testing samples using their in-house procedures for extraction and amplification and (ii) laboratories using their in-house amplification procedures on centrally extracted samples. Furthermore, three commercially available multiplex NAAT tests—the ResPlex (Qiagen GmbH, Hilden, Germany), RespiFinder plus (PathoFinder, Maastricht, The Netherlands), and RespiFinder Smart 21 (PathoFinder) tests—were evaluated by examination of the same EQA panel by the manufacturer. No large differences among the 3 laboratories were noticed when the performances of the assays developed in-house in combination with the in-house extraction procedures were compared. Also, the extraction procedure (central versus local) had little effect on performance. However, large differences in amplification efficacy were found between the commercially available tests; acceptable results were obtained by using the PathoFinder assays.

Evaluation of different nucleic acid amplification techniques for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from patients with community-acquired pneumonia

The number of pathogens involved in community-acquired pneumonia, with varying susceptibilities to antimicrobials, is numerous constituting an enormous challenge for diagnostic microbiology. Differentiation of infections due to Streptococcus pneumoniae and those due to Mycoplasma pneumoniae, Chlamydophila pneumoniae, or L. pneumophila as well as those due to viruses is essential to allow correct decisions concerning the antibiotics to be administered. The sensitivity and specificity of real-time simplex and multiplex nucleic acid sequence-based amplification (NASBA), and simplex PCR were compared for the detection of M. pneumoniae, C. pneumoniae and Legionella spp. in respiratory specimens from hospitalized and outpatients with community-acquired pneumonia (CAP). Two hundred fifty one respiratory specimens were collected from 147 patients with CAP. NASBA was done using the NucliSens Basic Kit (bioMérieux). PCR for M. pneumoniae and C. pneumoniae was done as described earlier [Ieven, M., Ursi, D., Van Bever, H., Quint, W., Niesters, H. G. M., and Goossens, H. 1996. Detection of Mycoplasma pneumoniae by two polymerase chain reactions and role of M. pneumoniae in acute respiratory tract infections in pediatric patients. J. Infect. Dis. 173, 1445-14452.; Ursi, D., Ieven, M., Van Bever, H. P., and Goossens, H. 1998. Construction of an internal control for the detection of Chlamydia pneumoniae by PCR. Mol. Cellul. Probes. 12, 235-238.]. A real-time PCR was developed to detect L. pneumophila whereas a real-time NASBA was designed to detect Legionella spp. All samples with discordant results were re-analysed. Compared to an expanded gold standard the sensitivities of the different techniques, were 77.8%, 100%, and 100% for detection of M. pneumoniae; and 50%, 100%, and 50% for detection of L. pneumophila by PCR, real-time simplex NASBA, and real-time multiplex NASBA, respectively. C. pneumoniae was detected in two samples only. Simplex real-time NASBA proved to be more sensitive than simplex PCR and was also more sensitive than real-time multiplex NASBA, as previously found with spiked clinical specimens. Its practical attractiveness pleads for further optimalisation of the multiplex approach.

Two Quality Control Exercises Involving Nucleic Acid Amplification Methods for Detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae and Carried Out 2 Years Apart (in 2002 and 2004)

ABSTRACT The quality performance of laboratories for the detection of Mycoplasma pneumoniae and Chlamydophila pneumoniae by two quality control (QC) exercises with a 2-year interval was investigated. For the 2002 QC exercise, specimens were spiked with M. pneumoniae at concentrations of 5,000, 500, 50, and 0 color-changing units (CCU)/100 μl. The limit of detectability was 50 CCU/100 μl. Therefore, this concentration was omitted from the 2004 panel and was excluded from the analysis. In 2002, 2 out of 12 participants obtained 100% correct results, 2 out of 12 produced false-positive results, and 10 out of 12 had between 0 out of 9 and 8 out of 9 correct positive results. In 2004, correct results were obtained in 15 out of 18 tests, and no false-positive results were reported. In 2002, specimens were spiked with C. pneumoniae at concentrations of 490, 49, 4.9, and 0 inclusion-forming units/100 μl (IFU/100 μl). In the 2004 panel, samples spiked with a lower dilution of 0.49 IFU/100 μl were added to the panel. For the C. pneumoniae QC, correct results were produced in 12 out of 16 and 13 out of 18 tests in 2002 and in 2004, respectively. Both multiplex PCR and nucleic acid sequence-based amplification (NASBA) formats scored a smaller number of samples positive than the monoplex reactions.

Development of Conventional and Real-Time Nucleic Acid Sequence-Based Amplification Assays for Detection of Chlamydophila pneumoniae in Respiratory Specimens

ABSTRACT Isothermal nucleic acid sequence-based amplification (NASBA) was applied to the detection of Chlamydophila pneumoniae 16S rRNA by using the NucliSens basic kit (bioMérieux, Boxtel, The Netherlands). The assay was originally developed as a conventional NASBA assay with electrochemiluminescence detection and was subsequently adapted to a real-time NASBA format by using a molecular beacon. C. pneumoniae RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild-type C. pneumoniae RNA and 0.1 inclusion-forming unit (IFU) of C. pneumoniae. In spiked respiratory specimens, the sensitivity of the C. pneumoniae NASBA assay varied between 0.1 and 1 IFU/100 μl sample, depending on the type of specimen. Finally, conventional and real-time NASBA were applied to respiratory specimens previously tested by PCR. A 100% concordance between the test results was obtained.