Detection In Clinical Specimens Research Articles

Third-Generation Sequencing in the Clinical Laboratory: Exploring the Advantages and Challenges of Nanopore Sequencing.

Metagenomic sequencing for infectious disease diagnostics is an important tool that holds promise for use in the clinical laboratory. Challenges for implementation so far include high cost, the length of time to results, and the need for technical and bioinformatics expertise. However, the recent technological innovation of nanopore sequencing from Oxford Nanopore Technologies (ONT) has the potential to address these challenges. ONT sequencing is an attractive platform for clinical laboratories to adopt due to its low cost, rapid turnaround time, and user-friendly bioinformatics pipelines. However, this method still faces the problem of base-calling accuracy compared to other platforms. This review highlights the general challenges of pathogen detection in clinical specimens by metagenomic sequencing, the advantages and disadvantages of the ONT platform, and how research to date supports the potential future use of nanopore sequencing in infectious disease diagnostics.

Detection of bovine pestiviruses in sera of beef calves by a RT-PCR based on a newly designed set of pan-bovine pestivirus primers.

The pestiviruses bovine viral diarrhea virus 1 and 2 (BVDV-1 and -2, respectively) and HoBi-like pestivirus (HoBiPeV) are important pathogens of cattle, and a number of reverse-transcription PCR (RT-PCR)-based assays have been developed for their detection in clinical specimens. We evaluated a newly designed set of pan-bovine pestivirus primers (BP189-389) in a gel-based RT-PCR screening test for pestiviruses in the sera of beef calves destined for export from southern Brazil. Serum samples positive for BVDV antigens by an antigen ELISA ( n = 135) were submitted to RT-PCR assays using different sets of primers, followed by nucleotide sequencing of the amplicons. RT-PCR with pestivirus primers 324-326 detected 110 positive samples: BVDV-1 ( n = 62), BVDV-2 ( n = 38), and HoBiPeV ( n = 10). A PCR using primers HCV90-368 detected 97 positive samples (64 BVDV-1; 33 BVDV-2). An additional RT-PCR round using BVDV-2-specific primers (2F-2R) detected 45 positive samples (including 38 detected by primers 324-326 and 33 by HCV90-368); whereas a RT-PCR using HoBiPeV-specific primers (N2-R5) detected 26 positive samples (including 10 detected by primers 324-326).The assay using the primers BP189-389 detected all 135 ELISA-positive samples, including the 26 HoBiPeV detected by primers N2-R5. Our results demonstrated that primers BP189-389 compare favorably against other primer sets in the detection of bovine pestiviruses, especially HoBiPeV. This conventional PCR may be useful for efficient detection of pestiviruses in bovine sera and other specimens as well, especially in laboratories without real-time PCR equipment.

Abstract 651: Development of an RNA in situ hybridization assay for the detection of prostate cancer cells in urine

Abstract Urine-based detection of prostate cancer represents a promising resource for non-invasive diagnostics. The value of urine as a biospecimen in the management of prostate cancer has already been established, but the utility of urine has yet to be fully explored. The objective of our study was to develop a RNA in situ hybridization (RISH) method for detecting prostate cells in urine and to evaluate the feasibility of using this assay for cancer detection in clinical specimens. We hypothesized that robust and specific labeling of prostate cancer cells could be achieved using this approach, providing benefit over existing urine detection methods, namely the FDA-approved PCA3 test, by allowing for the direct visualization and molecular characterization of individual cells. We optimized the following collection, processing, and preparation procedure for detection of prostate cells in fresh urine specimens collected from patients following digital rectal exam (DRE): the entire urine volume was centrifuged to collect the sediment, which was then formalin fixed, cytocentrifuged onto two slides, and slides were stored at -20°C until proceeding to staining and cytological analysis. Cellularity and sample adequacy was assessed by Papanicolaou staining. For identification of prostate cells in post-DRE urine via RISH, single-probe chromogenic labeling for NKX3.1 or HOXB13 was performed. Pretreatment conditions and staining parameters for RISH were optimized using urine samples spiked with 22Rv1 cells prior to staining patient specimens. We were able to readily identify cells of prostate origin in both spiked urines and patient samples. To further discriminate normal prostate cells from cancer cells, fluorescent multiplex RISH was pursued. We screened a candidate panel of prostate-specific RNA targets that included NKX3.1, HOXB13, KLK3, PRAC1, PRAC2, MALAT1, HOXC6, AMACR, and PCA3. Based on detection specificity, NKX3.1 and PRAC1 were selected as markers to identify cells of prostate origin and PCA3 as a marker of malignancy. Multiplex RISH with these three markers was carried out on 19 patient specimens. Prostate cells, classified as those positive for PRAC1 and/or NKX3.1, were identified in 11 samples, 6 of which were also positive for PCA3. These observations demonstrate that multiplex RISH can be used for the specific detection and visualization of prostate cancer cells in post-DRE urine samples. Although further refinement and validation are necessary, this study provides the first evidence supporting the application of a urinary RISH assay as a potential non-invasive method for prostate cancer detection. Ongoing and future studies will include characterizing performance of additional biomarker panels and validating clinical utility of this technique in a diagnostic setting. Citation Format: Jillian N. Eskra, Daniel Rabizadeh, Christian P. Pavlovich, Leslie Mangold, Elizabeth Fabian, William B. Isaacs, Jun Luo. Development of an RNA in situ hybridization assay for the detection of prostate cancer cells in urine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 651.

Loop-Mediated Isothermal Amplification for Salmonella Detection in Food and Feed: Current Applications and Future Directions.

Loop-mediated isothermal amplification (LAMP) has become a powerful alternative to polymerase chain reaction (PCR) for pathogen detection in clinical specimens and food matrices. Nontyphoidal Salmonella is a zoonotic pathogen of significant food and feed safety concern worldwide. The first study employing LAMP for the rapid detection of Salmonella was reported in 2005, 5 years after the invention of the LAMP technology in Japan. This review provides an overview of international efforts in the past decade on the development and application of Salmonella LAMP assays in a wide array of food and feed matrices. Recent progress in assay design, platform development, commercial application, and method validation is reviewed. Future perspectives toward more practical and wider applications of Salmonella LAMP assays in food and feed testing are discussed.

Experimental platform utilising melting curve technology for detection of mutations in Mycobacterium tuberculosis isolates

Tuberculosis (TB) remains one of the most deadly infections with approximately a quarter of cases not being identified and/or treated mainly due to a lack of resources. Rapid detection of TB or drug-resistant TB enables timely adequate treatment and is a cornerstone of effective TB management. We evaluated the analytical performance of a single-tube assay for multidrug-resistant TB (MDR-TB) on an experimental platform utilising RT-PCR and melting curve analysis that could potentially be operated as a point-of-care (PoC) test in resource-constrained settings with a high burden of TB. Firstly, we developed and evaluated the prototype MDR-TB assay using specimens extracted from well-characterised TB isolates with a variety of distinct rifampicin and isoniazid resistance conferring mutations and nontuberculous Mycobacteria (NTM) strains. Secondly, we validated the experimental platform using 98 clinical sputum samples from pulmonary TB patients collected in high MDR-TB settings. The sensitivity of the platform for TB detection in clinical specimens was 75% for smear-negative and 92.6% for smear-positive sputum samples. The sensitivity of detection for rifampicin and isoniazid resistance was 88.9 and 96.0% and specificity was 87.5 and 100%, respectively. Observed limitations in sensitivity and specificity could be resolved by adjusting the sample preparation methodology and melting curve recognition algorithm. Overall technology could be considered a promising PoC methodology especially in resource-constrained settings based on its combined accuracy, convenience, simplicity, speed, and cost characteristics.

Use of RNALater® Preservation for Virome Sequencing in Outbreak Settings.

Outbreaks of infectious diseases may occur in animal and human populations; this underlines the need for suitable One Health approaches. During outbreak situations, straightforward identification of etiological agents is indispensable for taking countermeasures. A recently published protocol for metagenomic virus detection in clinical specimens (TUViD-VM) was developed for snap-frozen tissues which can be challenging to obtain. Here, we describe the use of RNALater®-treated tissue at ambient temperatures for virome sequencing. This study demonstrates that samples stored in RNALater® buffer yield similar results to those stored snap-frozen.

Evaluation and Comparison of Multiple Test Methods, Including Real-time PCR, for Legionella Detection in Clinical Specimens.

Legionella is a Gram-negative bacterium that can cause Pontiac fever, a mild upper respiratory infection and Legionnaire’s disease, a more severe illness. We aimed to compare the performance of urine antigen, culture, and polymerase chain reaction (PCR) test methods and to determine if sputum is an acceptable alternative to the use of more invasive bronchoalveolar lavage (BAL). Data for this study included specimens tested for Legionella at Public Health Ontario Laboratories from 1st January, 2010 to 30th April, 2014, as part of routine clinical testing. We found sensitivity of urinary antigen test (UAT) compared to culture to be 87%, specificity 94.7%, positive predictive value (PPV) 63.8%, and negative predictive value (NPV) 98.5%. Sensitivity of UAT compared to PCR was 74.7%, specificity 98.3%, PPV 77.7%, and NPV 98.1%. Out of 146 patients who had a Legionella-positive result by PCR, only 66 (45.2%) also had a positive result by culture. Sensitivity for culture was the same using either sputum or BAL (13.6%); sensitivity for PCR was 10.3% for sputum and 12.8% for BAL. Both sputum and BAL yield similar results regardless testing methods (Fisher Exact p-values = 1.0, for each test). In summary, all test methods have inherent weaknesses in identifying Legionella; therefore, more than one testing method should be used. Obtaining a single specimen type from patients with pneumonia limits the ability to diagnose Legionella, particularly when urine is the specimen type submitted. Given ease of collection and similar sensitivity to BAL, clinicians are encouraged to submit sputum in addition to urine when BAL submission is not practical from patients being tested for Legionella.

Colonial morphology of Escherichia coli: impact of detection in clinical specimens

As part of bacteriological identification, the macroscopic observation of the colonies on a culture medium is a critical step as it allows the clinical microbiologist to conduct a primary screening of microorganisms and in some cases even to identify them. This study shows how the macroscopic observation of the colonies, in particular of the colonies of Escherichia coli isolated on MacConkey agar from various clinical specimens, allowed to identify 5 classes of morphological variants of the colonies or <em>colonial morphology</em>, thanks to the knowledge and the experience gained by the clinical microbiologist.

Evaluation of sensitivity of TaqMan RT-PCR for rubella virus detection in clinical specimens

BackgroundAn easy and reliable assay for detection of the rubella virus is required to strengthen rubella surveillance. Although a TaqMan RT-PCR assay for detection of the rubella virus has been established in Japan, its utility for diagnostic purposes has not been tested. ObjectivesTo allow introduction of the TaqMan RT-PCR into the rubella surveillance system in Japan, the sensitivity of the assay was determined using representative strains for all genotypes and clinical specimens. Study designThe detection limits of the method for individual genotypes were examined using viral RNA extracted from 13 representative strains. The assay was also tested at 10 prefectural laboratories in Japan, designated as local reference laboratories for measles and rubella, to allow nationwide application of the assay. ResultsThe detection limits and amplification efficiencies of the assay were similar among all the representative strains of the 13 genotypes. The TaqMan RT-PCR could detect approximately 90% of throat swab and urine samples taken up to 5days of illness. These samples were determined positive by a highly sensitive nested RT-PCR. ConclusionsThe TaqMan RT-PCR could detect at least 10 pfu of rubella virus. Although the sensitivity was somewhat lower than that of the conventional nested RT-PCR, the TaqMan RT-PCR could be more practical to routine tests for rubella laboratory diagnosis and detection in view of the rapid response and reducing risks of contamination.

Protocol for metagenomic virus detection in clinical specimens.

Sixty percent of emerging viruses have a zoonotic origin, making transmission from animals a major threat to public health. Prompt identification and analysis of these pathogens are indispensable to taking action toward prevention and protection of the affected population. We quantifiably compared classical and modern approaches of virus purification and enrichment in theory and experiments. Eventually, we established an unbiased protocol for detection of known and novel emerging viruses from organ tissues (tissue-based universal virus detection for viral metagenomics [TUViD-VM]). The final TUViD-VM protocol was extensively validated by using real-time PCR and next-generation sequencing. We could increase the amount of detectable virus nucleic acids and improved the detection of viruses <75,000-fold compared with other tested approaches. This TUViD-VM protocol can be used in metagenomic and virome studies to increase the likelihood of detecting viruses from any biological source.

Influenza and respiratory syncytial virus detection in clinical specimens without nucleic acid extraction using FOCUS direct disc assay is substantially equivalent to the traditional methods and the FOCUS nucleic acid extraction–dependent RT-PCR assay

In this study, we evaluated FOCUS diagnostic's Flu A/B & RSV direct kit (Direct Disc assay), designed to detect influenza (FLU) and respiratory syncytial viruses (RSV) directly in clinical specimens without nucleic acid extraction. This novel ‘sample-to-answer’, nucleic acid extraction–independent assay uses a unique disc to process, amplify, and detect viral targets in up to 8 specimens at a time. The performance of this assay for detecting FLU and RSV viruses was compared to the traditional methods (culture and/or direct florescent antibody testing) using 945 nasopharyngeal swab specimens. In addition, a total of 150 consecutive clinical specimens positive for FLU (FLU A=50, FLU B=50) or RSV (n=50) were tested in parallel using the novel Direct Disc assay and FOCUS diagnostic's nucleic acid extraction–dependent assay to assess their relative performance. Compared to the traditional methods, the overall (prospective+retrospective) positive/negative percent agreement was determined to be 96.6%/98.1% for FLU A, 98.4%/99.9% for FLU B, and 99.3%/98.8% for RSV. Compared to the nucleic acid extraction–dependent assay, the positive percent agreement was 90% (n=45/50) for FLU A, 92% (n=46/50) for FLU B, and 98% (n=49/50) for RSV. Overall, the Direct Disc assay showed good agreement with both traditional methods and nucleic acid extraction–dependent assay. Although we encountered some failures compared to the nucleic acid extraction–dependent assay, these limitations must be balanced against the substantial advantages of the extraction-free nature of this assay and rapid turnaround time.

Surface plasmon resonance biosensor for detection of feline calicivirus, a surrogate for norovirus

The human noroviruses are the most common non-bacterial cause of gastroenteritis and are responsible for as much as 50% of all gastroenteritis outbreaks worldwide. Norovirus (NoV), a single stranded RNA virus, is highly contagious with an infectious dose of less than 100 viral particles. While techniques exist for the identification of NoV, the lack of a reliable cell culture system, NoV genetic variability, and time-consuming sample preparation steps required to isolate the virus (or its genome) prior to molecular based methods has hindered rapid virus detection. To better protect the public from virus-contaminated food and enable better detection in clinical and environmental samples, sensitive and selective methods with simple sample preparation are needed. Surface plasmon resonance (SPR) biosensors represent an emerging detection platform, and this approach has been applied to the rapid detection of foodborne small molecule toxins, protein toxins, and bacteria. This analytical technique, however, has yet to be fully investigated for rapid virus detection, especially for intact viral particles extracted from food matrices. For this study, the culturable, non-human pathogen feline calicivirus (FCV), which has similar morphology and is genetically related to NoV, was chosen as a surrogate virus for designing and evaluating an SPR assay. An antibody-based assay was performed by first immobilizing anti-FCV to an SPR chip surface and then directly measuring virus binding and subsequent secondary antibody binding. The resulting biosensor directly detected intact FCV particles with limits of detection of approximately 104TCID50FCV/mL from purified cell culture lysates. In addition, intact virus detection in FCV-spiked oyster matrix was possible when using a simple extraction procedure and employing a secondary antibody to FCV for quantitation. The results from these preliminary studies show promise for the development of a rapid assay for detecting intact viruses, such as NoV, using an SPR biosensor. While the current level of sensitivity achieved with this SPR biosensor may be more applicable to virus detection in clinical specimens, broader application and increased sensitivity of this method for foodborne viruses may be achieved when performed in conjunction with efficient virus extraction and concentration methods.

Candida: epidemiología y factores de riesgo para especies no albicans

IntroductionNosocomial fungal infections have increased significantly in the last decade. Candida detection in clinical specimens can mean either colonization or an infection which can be local (muguet) or invasive. Knowledge of the species helps in choosing the best treatment. The aims of this study were to determine the frequency and distribution of Candida species detected in clinical samples, to analyze the clinical characteristics of the involved population and to determine the risk factors for Candida non-albicans species. MethodsRetrospective, observational. Period: 2006-2010. Inclusion criteria: all isolates of Candida in clinical specimens from patients hospitalized —at least 48hours in a neurological center. We analyzed epidemiological characteristics, co morbidities, risk factors, factors associated with Candida non-albicans detection, antifungal treatment, development of adverse events and mortality. ResultsCandida spp. was isolated from 321 clinical specimens: 139 (43.3%) were C. albicans and 182 (56.7%) Candida non-albicans. The distribution of the sample was: urine 122 (Candida non-albicans 67.2%), airway 81, oropharynx 45 (C. albicans) and candidemia 40 (Candida non-albicans 75%). The most frequent co-morbidity was solid tumor (35.5%). The main risk factors were antibiotic therapy (85.5%), steroid therapy (61.7%) and in ICU at diagnosis (61.6%). The analysis of risk factors and the isolation of Candida non-albicans shows that chemotherapy, previous surgery, treatment with aminopenicillins, carbapenems and glycopeptides were statistically significant (P<.05). There is a trend in neutropenic patients (P=.055) and in ICU at diagnosis (P=.076). Overall survival was 71%. ConclusionsCandida species distribution varies with the type of sample analyzed. Non-albicans species make up the majority of the isolates. The identification of the species involved per sample helps to optimize treatment. The high frequency of isolation of Candida in patients on steroids and antibiotics and admitted to ICU, is worth pointing out. Patients with previous surgery, treated with the aforementioned antibiotics or chemotherapy, could receive non-azole antifungals in the initial empirical treatment strategy.

Methods of detection of selected respiratory viruses

Respiratory viruses contribute to significant morbidity and mortality in healthy and immunocompromised individuals and are considered as a significant economic burden in the healthcare system. The similar clinical symptoms in the course of different viral and bacterial respiratory infections make the proper diagnosis difficult. An accurate and prompt diagnostics is crucial for infection control and patient management decisions, especially regarding the use of antibacterial or antiviral therapy and hospitalization. Moreover, the identification of the causative agent eliminates inappropriate use of antibiotics and may reduce the cost of healthcare. A wide variety of diagnostic procedures is applied for the detection of viral agents responsible for respiratory tract infections. For many years, the viral antigen detection and standard isolation technique in cell culture was the main method used in routine diagnostics. However, in recent years the nucleic acid amplification techniques have become widely used and have significantly improved the sensitivity of viral detection in clinical specimens. Molecular diagnostic assays have contributed to revealing high rates of co-infection (multiplex reactions) and allow identification of agents that are difficult to culture. This paper discusses a number of technical aspects of the current most commonly used techniques, their general principles, main benefits and diagnostic value, but also some of their limitations.

Development and clinical verification of a loop-mediated isothermal amplification method for detection of Salmonella species in suspect infected ducks

To detect Salmonella spp. in suspect infected ducks, a loop-mediated isothermal amplification (LAMP) assay was developed. With the help of this assay, we can detect Salmonella enterica serovar Enteriditis and Salmonella enterica serovar Anatis above 6.0 cfu/test and 4.8 cfu/test, respectively, in pure-culture conditions, even in the existence of 0.01 g of duck liver or spleen homogenates; the detection thresholds were still achieved at 6.0 cfu per test tube. Further experiments of the test strains indicated the high specificity of this LAMP assay. In the detection of clinical specimens, a total of 115 Salmonella suspect infected clinical samples was analyzed by traditional culture method, LAMP, and PCR. The results suggested that 11 samples were positive by LAMP and culture methods; however, only 8 specimens were positive by PCR. In consideration of the high sensitivity and fast scanning speed of our LAMP method in clinical specimens, it was more suitable for field application and epidemiological investigation.

Advances in rapid diagnosis of tuberculosis disease and anti-tuberculous drug resistance

Rapid diagnosis of tuberculosis (TB) and multidrug-resistant (resistance to at least rifampin and isoniazid) Mycobacterium tuberculosis (MDR-TB) is one of the cornerstones for global TB control as it allows early epidemiological and therapeutic interventions. The slow growth of the tubercle bacillus is the greatest obstacle to rapid diagnosis of the disease. However, considerable progress has recently been made in developing novel diagnostic tools, especially molecular methods (commercial and 'in-house'), for direct detection in clinical specimens. These methods, based on nucleic acid amplification (NAA) of different targets, aim to identify the M. tuberculosis complex and detect the specific chromosome mutations that are most frequently associated with phenotypic resistance to multiple drugs. In general, commercial methods are recommended since they have a better level of standardization, reproducibility and automation. Although some aspects such as cost-efficiency and the appropriate setting for the implementation of these techniques are not yet well established, organizations such as the WHO are strongly supporting the implementation and universal use of these new molecular methods. This chapter summarizes current knowledge and the available molecular methods for rapid diagnosis of TB and anti-tuberculous drug resistance in clinical microbiology laboratories.

Reply to Schlapbach et al

To the Editor—We thank Schlapbach et al for their comments on our recent prospective study of human metapneumovirus (HMPV) in hospitalized children. Schlapbach and colleagues describe a fatal case of acute respiratory distress syndrome (ARDS) attributed to HMPV and reference several other published case reports of severe HMPV infection. Their letter raises several points worthy of discussion. First, the case described was diagnosed as HMPV infection by direct immunofluorescence assay (DFA). This approach has been shown to have reasonable sensitivity (62%–90%) for HMPV detection in clinical specimens, compared with real-time reverse-transcription polymerase chain reaction (RT-PCR), and excellent specificity [1–3]. HMPV is an established primary cause of lower respiratory infection in children [4, 5]; thus, HMPV was likely to have been the major contributor to the ARDS process. However, DFA also was used to test for other common viruses, which were not detected; DFA and culture are less sensitive than real-time RT-PCR for other respiratory viruses [6, 7]. Therefore, viral coinfection cannot be excluded. Likewise, molecular testing for bacterial copathogens was not performed, making concomitant bacterial sepsis a possibility. Nucleic acid–based testing enhances detection of virtually all pathogens and is rapidly becoming the standard of practice. Second, the report revealed the potential severity of HMPV infection. Schlapbach et al suggest that a lower percentage of children with HMPV infection require intensive care than RSV-infected children; however, the data to support this contention are lacking. Most studies of adequate sample size that compare HMPV and RSV find that the clinical syndromes are virtually indistinguishable (reviewed in Deffrasnes et al [8]). Indeed, in our study, the rates of intensive care unit admission were similar for HMPV (3%) and RSV (2%), and there were no striking clinical differences between illnesses associated with the 2 viruses. As noted by Schlapbach et al, ARDS also has been described only rarely for RSV, with <20 reported cases. We agree that the prevalence of severe infections associated with HMPV, including ARDS, is probably underappreciated because of the lack of widespread use of HMPV diagnostic testing. Although death due to RSV and HMPV is rare in industrialized nations, global mortality associated with RSV in developing countries is substantial [9]. Because HMPV causes clinical disease similar to that associated with RSV, it is likely that HMPV is also associated with a sizable proportion of childhood mortality worldwide. Epidemiologic investigations in developing regions are critical to define these illnesses. One limitation of many hospital-based studies of respiratory viral infection is the inclusion of only 1 or 2 seasons; HMPV shows substantial variation in prevalence from over time [10]. Large, prospective, population-based studies using molecular diagnostics are needed to fully delineate the burden and spectrum of disease due to HMPV.

HER2: Biology, Detection, and Clinical Implications

HER2 is a membrane tyrosine kinase and oncogene that is overexpressed and gene amplified in about 20% of breast cancers. When activated it provides the cell with potent proliferative and antiapoptosis signals and it is the major driver of tumor development and progression for this subset of breast cancer. When shown to be overexpressed or amplified by appropriate methods, HER2 is a valuable treatment target. To review the basic biology of the HER2 signaling network, to discuss various approved methods for its detection in clinical specimens, and to describe the impressive results of therapies targeting HER2. Selected literature searchable on PubMed as well as older studies revealed by the literature review were reviewed. HER2 is an important member of a complex signaling network and when gene amplified, it results in an aggressive subtype of breast cancer. Patients with tumors found to overexpress HER2 protein or to be amplified for the gene are candidates for therapy that significantly reduces mortality.

Is C. Pneumoniae Research in Peril?

Is C. Pneumoniae Research in Peril?

Prevalence of Mycoplasma pneumoniae-associated respiratory tract infections in hospitalized children: results of a 4-year prospective study in Tunis

Specific microbiologic, molecular, and serologic assays are hardly available in Tunis to confirm a suspected infection of Mycoplasma pneumoniae (MP). These diagnosis methods were used for the first time in a Tunisian prospective study to estimate the prevalence of MP infection in children and to evaluate their usefulness for diagnosis. A total of 540 children hospitalized in Tunis for lower respiratory tract infections (LRTIs) between 2005 and 2009 and 580 clinical specimens were investigated for the presence of MP by culture and by end-point polymerase chain reaction (PCR) targeting the P1 and the 16S rRNA genes. Real-time PCR was also used for MP detection on 158 respiratory samples. A total of 525 serum samples were tested for detection of MP-specific IgM and IgG. The P1 adhesin type and the antibiotic susceptibility testing were determined for the 9 clinical strains isolated during the study period. MP was detected in 33 (5.7%) clinical samples. Specific MP seropositivity was confirmed in 54 serum samples (10.3%), among which 19 (3.6%) were indicative of acute MP infection. MP infection was confirmed in 39 (7.2%) patients: 24 positive by PCR and/or culture, 10 serologically positive only, and 5 confirmed positive by both methods. MP infections occurred throughout the year with a slight decrease in autumn. The 9 MP isolates were susceptible to erythromycin, tetracycline, and ciprofloxacin, and all belonged to type I. The prevalence of MP infection in children with LRTI was 7.2% between 2005 and 2009, in Tunisia. Combination of direct detection and serology was required to enhance the clinical sensitivity of MP detection in clinical specimens.