Search

ABSTRACTHaemophilus influenzaeis a significant causative agent of respiratory tract infections (RTI) worldwide. The development of a rapid H. influenzaediagnostic assay that would allow for the implementation of infection control measures and also improve antimicrobial stewardship for patients is required. A number of nucleic acid diagnostics approaches that detect H. influenzaein RTIs have been described in the literature; however, there are reported specificity and sensitivity limitations for these assays. In this study, a novel real-time PCR diagnostic assay targeting the smpBgene was designed to detect all serogroups of H. influenzae. The assay was validated using a panel of well-characterized Haemophilusspp. Subsequently, 44 Haemophilusclinical isolates were collected, and 36 isolates were identified as H. influenzaeusing a gold standard methodology that combined the results of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and a fucKdiagnostic assay. Using the novel smpBdiagnostic assay, 100% concordance was observed with the gold standard, demonstrating a sensitivity of 100% (95% confidence interval [CI], 90.26% to 100.00%) and a specificity of 100% (95% CI, 63.06% to 100.00%) when used on clinical isolates. To demonstrate the clinical utility of the diagnostic assay presented, a panel of lower RTI samples (n= 98) were blindly tested with the gold standard and smpBdiagnostic assays. The results generated were concordant for 94/98 samples tested, demonstrating a sensitivity of 90.91% (95% CI, 78.33% to 97.47%) and a specificity of 100% (95% CI, 93.40% to 100.00%) for the novel smpBassay when used directly on respiratory specimens.

Gastroenteritis is caused by a wide range of viral, bacterial and parasitic pathogens and causes millions of deaths worldwide each year, particularly in infant populations in developing countries. Traditional microbiological culture and immunological based tests are time consuming, laborious and often lack diagnostic specificity and sensitivity. As a result patients can receive suboptimal and/or inappropriate antimicrobial treatment. In recent years, rapid nucleic acid diagnostics (NAD) technologies have become available to complement or even bypass and replace these traditional microbiological culture and immunological based tests.

Respiratory tract infections (RTIs) are caused by a variety of bacterial, viral, fungal, and other pathogens and cause millions of deaths each year. Current standard microbiological culture-based tests are laborious and time consuming. Thus, patients are initially treated empirically, leading to inappropriate use of antibiotics. The purpose of this article is to provide clinicians and scientists with a review of recently available commercial multiparametric molecular diagnostics tests for the detection of RTIs so that they can be considered for use instead of, or in combination with, traditional culture techniques.

ABSTRACTTuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosiscomplex (MTC). The accurate identification of the MTC member causing human infection is important because the treatment of TB caused by some MTC members requires an alteration of the standard drug regimen, it can inform whether transmission is human to human or zoonotic, and it enables accurate epidemiology studies that help improve TB control. In this study, an internally controlled two-stage multiplex real-time PCR-based method, SeekTB, was developed for the accurate identification of all members of the MTC. The method was tested against a panel of well-characterized bacterial strains (n= 180) and determined to be 100% specific for members of the MTC. Additionally, 125 Mycobacteria Growth Indicator Tube (MGIT)-positive cultures were blindly tested by using SeekTB, and the results were compared to those of the GenoType MTBC and TBc ID tests. The SeekTB and GenoType MTBC results were 100% concordant, identifying 84 of these isolates as M. tuberculosisisolates and 41 as non-MTC isolates. Nine discordant results between the molecular methods and the TBc ID culture confirmation test were observed; however, nucleotide sequencing confirmed the results obtained with GenoType MTBC and SeekTB. SeekTB is the first-described internally controlled multiplex real-time PCR diagnostic method for the accurate identification of all eight members of the MTC. This method, designed for use on cultured patient samples, is specific, sensitive, and rapid, with a turnaround time to results of approximately 1.5 to 3.5 h, depending on which, if any, member of the MTC is present.

ABSTRACTTuberculosis (TB) in humans is caused by members of the Mycobacterium tuberculosiscomplex (MTC). Rapid detection of the MTC is necessary for the timely initiation of antibiotic treatment, while differentiation between members of the complex may be important to guide the appropriate antibiotic treatment and provide epidemiological information. In this study, a multiplex real-time PCR diagnostics assay using novel molecular targets was designed to identify the MTC while simultaneously differentiating between M. tuberculosisand M. canettii. The lepAgene was targeted for the detection of members of the MTC, the wbbl1gene was used for the differentiation of M. tuberculosisand M. canettiifrom the remainder of the complex, and a unique region of the M. canettiigenome, a possible novel region of difference (RD), was targeted for the specific identification of M. canettii. The multiplex real-time PCR assay was tested using 125 bacterial strains (64 MTC isolates, 44 nontuberculosis mycobacteria [NTM], and 17 other bacteria). The assay was determined to be 100% specific for the mycobacteria tested. Limits of detection of 2.2, 2.17, and 0.73 cell equivalents were determined for M. tuberculosis/M. canettii, the MTC, and M. canettii, respectively, using probit regression analysis. Further validation of this diagnostics assay, using clinical samples, should demonstrate its potential for the rapid, accurate, and sensitive diagnosis of TB caused by M. tuberculosis, M. canettii, and the other members of the MTC.