ABSTRACT
MALDI-TOF MS identifications of microorganisms in a clinical laboratory were investigated, comparing steel targets with MBT Biotargets. By using MBT Biotargets, the score values of yeast identifications increased, whereas the score values of Gram-negative bacteria decreased. Switching to MBT Biotargets did not negatively impact overall frequencies of high confidence identifications.
Subject(s)
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Steel , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Humans , Steel/chemistry , Bacteria/classification , Bacteria/isolation & purification , Bacteria/genetics , Microbiological Techniques/methods , Yeasts/isolation & purification , Yeasts/classification , Yeasts/geneticsABSTRACT
Patients infected by Mycoplasma genitalium are often treated empirically with the macrolide azithromycin. Macrolide resistance is becoming quite common; empirical treatment is compromised. Sequencing was initially used to detected azithromycin resistance-associated mutations. As this was laborious, qPCRs have been developed for their detection. In the present study, we describe a fast, sensitive, and specific qPCR assay that enables routine testing of M. genitalium and macrolide resistance-associated mutations in a single assay. M. genitalium positive clinical samples were used to compare (i) the commonly used MgPa assay for the detection of M. genitalium infections (MgPa qPCR), (ii) a combined 23S rRNA gene PCR/sequencing assay (Mg23S qPCR/Sequencing) to identify macrolide resistance-associated mutations, and (iii) our newly developed probe-based melt curve qPCR for simultaneous detection of M. genitalium and macrolide resistance-associated mutations (Macrolide-R/MG ELITe MGB Kit, Elitech Bothel USA in short Mg MacrolideR qPCR). Specificity of the qPCR was tested using urogenital samples that were tested positive for a range of other micro-organisms. M. genitalium was detected in 196/236 (83.1%) samples by the MgPa qPCR, versus 172/236 (72.9%) by the combined Mg23S qPCR/Sequencing, and 202/236 (85.6%) by the Mg MacrolideR qPCR. The Mg MacrolideR qPCR showed high concordance to the Mg23S qPCR/Sequencing assay (201 vs 202 could be genotyped, respectively) for the detection of the macrolide resistant mutations. None of the other urogenital pathogens were tested positive in the Mg MacrolideR qPCR, indicating specificity. The Mg MacrolideR qPCR is fast, sensitive, specific, and can easily be implemented in the routine diagnostics.
Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Bacterial , Macrolides/pharmacology , Mycoplasma Infections/diagnosis , Mycoplasma Infections/microbiology , Mycoplasma genitalium/drug effects , Mycoplasma genitalium/genetics , Adolescent , Adult , Antitubercular Agents/therapeutic use , Female , Humans , Macrolides/therapeutic use , Male , Middle Aged , Mutation , Mycoplasma Infections/drug therapy , RNA, Ribosomal, 23S/genetics , Real-Time Polymerase Chain Reaction , Sensitivity and Specificity , Young AdultSubject(s)
Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Macrolides/pharmacology , Mycoplasma Infections/microbiology , Mycoplasma genitalium/drug effects , Mycoplasma genitalium/genetics , RNA, Ribosomal, 23S/genetics , Sexually Transmitted Diseases, Bacterial/microbiology , Adolescent , Adult , Aged , Anti-Bacterial Agents/therapeutic use , Azithromycin/therapeutic use , Female , Humans , Macrolides/therapeutic use , Male , Middle Aged , Mutation , Mycoplasma Infections/drug therapy , Mycoplasma Infections/epidemiology , Mycoplasma genitalium/isolation & purification , Netherlands/epidemiology , Prevalence , Sexually Transmitted Diseases, Bacterial/drug therapy , Sexually Transmitted Diseases, Bacterial/epidemiologyABSTRACT
BACKGROUND: Norovirus P2 domain is commonly used to extrapolate transmission within an outbreak (OB) setting. The current definition is that transmission among cases is considered to be proven when no sequence variation is found. OBJECTIVES: Previous studies have shown a high mutation rate and errors during replication of the norovirus genome, therefore the validity of this criterion must be evaluated. STUDY DESIGN: Sequences of the P2 domain were obtained from patients and health care workers sampled during 4 prospectively GII.4 outbreaks. Fecal samples were tested by RT-PCR for presence of norovirus RNA against a standard control preparation to allow quantification. Estimated time of onset of shedding was derived from shedding kinetics modeled on data from sequential sampling. Thereby P2 sequence variation could be linked to estimated total virus excretion in individual subjects. RESULTS: In all the outbreaks, P2 domain variation was found that resulted in unique codon changes in some patients. Mutations were found in 14% of initial samples and >50% of follow-up samples taken from patients involved in an outbreak. In three patients, aa mutations was observed in or near sites involved in host or antigen binding. CONCLUSIONS: We concluded that P2 domain variation increases with duration of virus shedding, but was unrelated to total amounts of virus shed. Therefore, we propose that cluster identification based on identical sequences should be relaxed to accommodate minor sequence variation. When using sequence data to support outbreak investigations, sequence diversity should be interpreted in relation to timing of sampling since onset of illness.
