Comparison of the Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa ProFlu+™ assay for detecting influenza and respiratory syncytial viruses.

The relative performance of 2 widely used reverse transcription polymerase chain reaction (RT-PCR) assays, the Focus diagnostics Simplexa™ Flu A/B & RSV kit (nucleic acid extraction-dependent assay) and the Prodessa Proflu+™ assay, was evaluated using 735 prospectively and retrospectively collected nasopharyngeal swab specimens. Overall, the assays showed positive and negative agreements of 100% and 99.7% for influenza A, 98.1% and 99.9% for influenza B, and 99.3% and 99.5% for respiratory syncytial virus. The relative analytical sensitivity of the 2 assays was also similar.

Molecular Detection, Quantification, and Toxigenicity Profiling of Aeromonas spp. in Source- and Drinking-Water.

Aeromonas is ubiquitous in aquatic environments and has been associated with a number of extra-gastrointestinal and gastrointestinal illnesses. This warrants monitoring of raw and processed water sources for pathogenic and toxigenic species of this human pathogen. In this study, a total of 17 different water samples [9 raw and 8 treated samples including 4 basin water (partial sand filtration) and 4 finished water samples] were screened for Aeromonas using selective culturing and a genus-specific real-time quantitative PCR assay. The selective culturing yielded Aeromonas counts ranging 0 - 2 x 10(3)CFU/ml and 15 Aeromonas isolates from both raw and treated water samples. The qPCR analysis indicated presence of a considerable nonculturable population (3.4 x 10(1) - 2.4 x 10(4) cells/ml) of Aeromonas in drinking water samples. Virulence potential of the Aeromonas isolates was assessed by multiplex/singleplex PCR-based profiling of the hemolysin and enterotoxin genes viz cytotoxic heat-labile enterotoxin (act), heat-labile cytotonic enterotoxin (alt), heat-stable cytotonic enterotoxin (ast), and aerolysin (aerA) genes. The water isolates yielded five distinct toxigenicity profiles, viz. act, alt, act+alt, aerA+alt, and aerA+alt+act. The alt gene showed the highest frequency of occurrence (40%), followed by the aerA (20%), act (13%), and ast (0%) genes. Taken together, the study demonstrated the occurrence of a considerable population of nonculturable Aeromonads in water and prevalence of toxigenic Aeromonas spp. potentially pathogenic to humans. This emphasizes the importance of routine monitoring of both source and drinking water for this human pathogen and role of the developed molecular approaches in improving the Aeromonas monitoring scheme for water.

Extended tracking of the microbial community structure and dynamics in an industrial synthetic metalworking fluid system.

Understanding of the occupational exposure risk scenario and disease etiology associated with industrial metalworking fluids (MWFs) requires knowledge of the development and composition of their microbial diversity in relation to the underlying fluid management factors. In this study, a managed synthetic MWF operation freshly recharged following the dumping, cleaning, and recharge (DCR) process was tracked in real time for microbial community changes over a period of 1.25 years (65 weeks). The recharged fluid developed very high bacterial counts (viable and nonviable) fairly quickly after the DCR process, indicating its inadequacy. Genus-/group-specific real-time qPCR confirmed the prevalence of six potentially pathogenic/immunogenic microbial genera/groups, viz. pseudomonads, enterics, mycobacteria, legionellae, actinomycetes, and fungi. Selective culturing revealed Acinetobacter and Bacillus as the most frequently isolated Gram-negative and Gram-positive genera, respectively, in addition to the presence of fungi and actinomycetes. Endotoxin perturbations (< 1000 to > 100000 EU mL⁻¹) coincided with temporal increases in Gram-negative bacteria and/or periodic biocide additions. PCR-DGGE-sequencing revealed an expanded estimated bacterial richness (up to 23 bands per sample). Of the 16 dominant bacterial phylotypes identified, the majority were detected for the first time in MWF. Interestingly, the study revealed a crucial role for MWF brand, among other fluid factors, in modulating the community structure and dynamics.

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.

Optimization of a combined human parechovirus-enterovirus real-time reverse transcription-PCR assay and evaluation of a new parechovirus 3-specific assay for cerebrospinal fluid specimen testing.

Human parechoviruses (HPeVs), particularly type 3 (HPeV3), are known central nervous system (CNS) pathogens, causing serious infections in infants similar to those caused by enteroviruses (EVs). The primary aim of this study was to combine and validate HPeV and EV real-time reverse transcription-PCR (RT-PCR) detection assays with the best available RT-PCR reagents and conditions for parallel detection of HPeV and EV on a single platform. The secondary aim was to develop and validate a newly developed HPeV3-specific real-time RT-PCR assay. Five commercially available RT-PCR kits were evaluated with the pan-HPeV and EV assays in one-step and two-step RT-PCRs. Two-step RT-PCR with the AgPath ID RT-PCR (AGP) kit performed best for both pan-HPeV and EV assays. The pan-HPeV-specific assay performed best with the AGP kit in a one-step RT-PCR. Frozen aliquots of 145 (for HPeV, n = 70; for EV, n = 75) previously characterized cerebrospinal fluid (CSF) specimens were tested by EV-, pan-HPeV-, and HPeV3-specific (HPeV specimens only) assays. The pan-HPeV and EV assays demonstrated 100% analytical sensitivity and specificity compared to historic results, while the HPeV3-specific assay demonstrated 97% sensitivity and 100% specificity. We propose a real-time pan-HPeV, EV two-step RT-PCR algorithm for simultaneous detection of HPeV and EV from CSF specimens on a single platform. The HPeV3-specific one-step RT-PCR assay can be used as a rapid and cost-effective assay to detect and identify HPeV3 in pan-HPeV RT-PCR assay-positive CSF specimens.

Characteristics of young infants in whom human parechovirus, enterovirus or neither were detected in cerebrospinal fluid during sepsis evaluations.

BACKGROUND: Human parechovirus (HPeV) causes central nervous system (CNS) infection in infants. To further understand HPeV CNS infection, we describe its clinical, laboratory and epidemiologic characteristics from a Midwestern US tertiary care center. Because HPeV CNS infections have appeared clinically and seasonally similar to enterovirus (EV) infections, we retrospectively compared characteristics of young infants undergoing sepsis evaluations in whom HPeV, EV or neither were detected in cerebrospinal fluid (CSF). METHODS: HPeV real-time reverse-transcription polymerase chain reaction (RT-PCR) assay was performed on frozen nucleic acid extracts of CSF specimens submitted for EV RT-PCR assay from children seen at our hospital in 2009. HPeV genotyping was performed by sequencing of the viral protein 1 region. Clinical data were abstracted from medical records retrospectively for EV-positive, HPeV-positive and age-matched controls in whom neither virus was detected from CSF testing. RESULTS: HPeV was detected in 66 of the 388 (17%) CSF specimens whereas EV was detected in 54 of the 388 (14%) from June through October 2009. Genotyping identified HPeV3 in 51 of the 66 (77%) positive CSF specimens. Males predominated (61%) with the most common presenting symptoms (91%) being fever and irritability. All HPeV-positive patients were <5 months of age. Eight required admission to the pediatric intensive care unit. In multivariate analysis, lower peripheral white blood cell counts with lower absolute lymphocyte count values, higher maximum temperatures, longer fever duration, absence of pleocytosis and longer hospitalization were independently associated with HPeV patients compared with patients with EV or patients negative for both HPeV and EV. CONCLUSIONS: Our data indicate that HPeV3, an emerging CNS pathogen of infants in the United States, should be considered in sepsis-like presentation even without CSF pleocytosis. Addition of HPeV RT-PCR to EV RT-PCR assay for CSF specimens of patients <6 months of age could reduce hospital stay and costs while improving clinical management.

Evaluation of xTAG Respiratory Viral Panel FAST and xTAG Human Parainfluenza Virus Analyte-Specific Reagents for detection of human parainfluenza viruses in respiratory specimens.

The multiplex xTAG(®) Respiratory Viral Panel FAST (RVP FAST) research-use-only assay and xTAG(®) Human Parainfluenza Virus Analyte-Specific Reagent (HPIV-ASR) assay were evaluated with 99 culture-confirmed human parainfluenza virus (HPIV)-positive and -negative specimens and found to have analytical sensitivities of 95.2% and 100% and specificities of 98.3% and 96.6%, respectively. Since the in vitro diagnostic (IVD) version of the RVP FAST assay does not include HPIVs, the HPIV-ASR assay can be tested in parallel with RVP FAST-IVD for optimal detection of HPIVs.

Detection of toxigenic Clostridium difficile in pediatric stool samples: an evaluation of Quik Check Complete Antigen assay, BD GeneOhm Cdiff PCR, and ProGastro Cd PCR assays.

The performance of C. Diff Quik Chek Complete (QCC), BD GeneOhm Cdiff PCR (BD), and ProGastro Cd PCR (PG) assays was evaluated in detecting Clostridium difficile infection (CDI) in children using 200 frozen stool specimens. The results of the tests were compared to the toxigenic culture (TC) as 'gold standard.' The sensitivity, specificity, positive predictive value, and negative predictive value were as follows. QCC antigen (GDH + Toxin-A/B) = 70.8%, 97.4%, 89.5%, and 91.4%; BD PCR = 89.6%, 96.7%, 89.6%, and 96.7%; PG PCR = 100%, 93.4%, 82.8%, and 100%. Polymerase chain reaction (PCR) assays detected an additional 11 positives missed by TC, 7 of which were confirmed positive by an alternate tcdB gene PCR assay. However, retrospective clinical chart review indicated CDI in only 3 of the 11 patients in whom C. difficile was detected by PCR only. A 2-step algorithm utilizing QCC antigen test as a screening test followed by confirmation of GDH-positive and toxin-negative samples with either BD or PG PCR assay will provide rapid and accurate results for majority of the samples and reduce laboratory testing cost.

Molecular epidemiology and clinical presentation of human adenovirus infections in Kansas City children.

BACKGROUND: A significant increase in adenovirus detection among patients at the Children's Mercy Hospital, Kansas City was observed between June 2007 and January 2008. OBJECTIVE: To molecularly characterize the human adenoviruses and describe their association with clinical illness in children. STUDY DESIGN: One hundred adenovirus-positive specimens from 79 children were typed by hexon gene sequence typing method. Restriction enzyme analysis (REA) was performed on isolates of HAdV-3, -7 and -14 to identify genomic variants. Medical records were reviewed to understand the clinical illnesses associated with adenovirus serotypes and genome types. RESULTS: The most prevalent HAdV serotypes were HAdV-3 (37%), HAdV-7 (25%), HAdV-1 (16%), HAdV-2 (8%). HAdV infection was common in children ≤3 years of age (71%) versus children >3 years (29%). Majority of the HAdV-infected children were hospitalized (78%); 22/79 (28%) stayed >3 days and 8/79 (10%) required intensive care unit stay. Hospitalization rates for HAdV-3 (36%) and HAdV-7 (25%) were comparable. REA data indicated that HAdV-3a2 was the predominant HAdV-3 genome type. Two novel genomic variants of HAdV-3 exhibiting unique BglII or BstEII profiles were identified in isolates from patients with bronchiolitis. All HAdV-7 and -14 isolates were identified as corresponding to genome types 7d2 and 14p1, respectively. CONCLUSIONS: In Kansas City, we noticed an increase in the incidence of HAdV-7 (25%; n=24/98) infections compared to the previous two years (6%; n=6/107). Two new genomic variants of HAdV-3 appear to have emerged in our area and seem to be associated with lower respiratory tract infections in children.

Susceptibility of Mycobacterium immunogenum and Pseudomonas fluorescens to formaldehyde and non-formaldehyde biocides in semi-synthetic metalworking fluids.

Mycobacterium immunogenum, a newly identified member of the Mycobacterium chelonae_M. abscessus complex is considered a potential etiological agent for hypersensitivity pneumonitis (HP) in machine workers exposed to contaminated metalworking fluid (MWF). This study investigated the biocidal efficacy of the frequently applied commercial formaldehyde-releasing (HCHO) biocides Grotan and Bioban CS 1135 and non-HCHO type biocides Kathon 886 MW (isothiazolone) and Preventol CMK 40 (phenolic) toward this emerging mycobacterial species (M. immunogenum) in HP-linked MWFs, alone and in presence of a representative of the Gram-negative bacterial contaminants, Pseudomonas fluorescens, using two semi-synthetic MWF matrices (designated Fluid A and Fluid B). Relative biocide susceptibility analysis indicated M immunogenum to be comparatively more resistant (2-1600 fold) than P. fluorescens to the tested biocides under the varied test conditions. In terms of minimum inhibitory concentration, Kathon was the most effective biocide against M. immunogenum. Fluid factors had a major effect on the biocide susceptibility. Fluid A formulation provided greater protective advantage to the test organisms than Fluid B. Fluid dialysis (Fluid A) led to an increased biocidal efficacy of Grotan, Kathon and Preventol against M. immunogenum further implying the role of native fluid components. Used fluid matrix, in general, increased the resistance of the two test organisms against the biocides, with certain exceptions. M. immunogenum resistance increased in presence of the co-contaminant P. fluorescens. Collectively, the results show a multifactorial nature of the biocide susceptibility of MWF-colonizing mycobacteria and highlight the importance of more rigorous efficacy testing and validation of biocides prior to and during their application in metalworking fluid operations.

Human parechovirus 3 causing sepsis-like illness in children from midwestern United States.

BACKGROUND: : Human parechovirus (HPeV) infections of the central nervous system (CNS) in children can be associated with severe outcomes such as neonatal sepsis-like illness, meningitis, or paralysis. We sought to determine the prevalence of HPeV CNS infections and clinical presentation in children from the United States. METHODS: : Frozen nucleic acid extracts from enterovirus-negative cerebrospinal fluid (CSF) obtained at the Children's Mercy Hospitals and Clinics, in Kansas City from 2006 (n = 242), 2007 (n = 324), and 2008 (n = 218) were tested by 2-step HPeV real-time reverse transcription polymerase chain reaction. HPeV genotype was determined by sequencing the VP3/VP1 junction. Demographic and clinical data were abstracted from medical records. RESULTS: : Overall HPeV was detected in 58/780 (7%) of tested CSF samples; 4/218 (2%) in 2006, 54/320 (17%) in 2007, and 0/242 (0%) in 2008. HPeV (17%) and enterovirus (20%) detection were comparable in 2007. HPeV-3 genotype was detected in 52/53 specimens successfully sequenced. Detection was seasonal (June-October). HPeV-3-CNS-infection occurred at a mean age of 6.6 ± 4.4 weeks and predominantly in males (71%). The most common clinical presentation was sepsis-like syndrome (66%). The most common symptoms were irritability (98%), fever (95%), and nonspecific rash (58.6%), while neurologic manifestations were rare (5%). CONCLUSIONS: : To our knowledge, this is the first multiyear prevalence report of HPeV CNS infection in the United States. HPeV CNS infection was detected mostly in male infants with sepsis-like illness during the late summer/autumn season. Routine seasonal CSF testing in infants for HPeV plus enterovirus may improve etiologic detection and clinical management of infantile sepsis-like presentations.

Evaluation of three influenza A and B real-time reverse transcription-PCR assays and a new 2009 H1N1 assay for detection of influenza viruses.

The performance characteristics of three real-time influenza A/B virus reverse transcription-PCR (RT-PCR) assays and two real-time 2009 H1N1 RT-PCR assays were evaluated using previously characterized clinical specimens. A total of 150 respiratory specimens from children (30 influenza A/H1 virus-, 30 influenza A/H3 virus-, 30 2009 H1N1-, and 30 influenza B virus-positive specimens and 30 influenza virus-negative specimens) were tested with the CDC influenza A/B PCR (CDC), ProFlu(+) multiplex real-time RT-PCR assay (ProFlu(+)), and MGB Alert Influenza A/B & RSV RUO (MGB) assays. A second set of 157 respiratory specimens (100 2009 H1N1-, 22 seasonal influenza A/H1-, and 15 seasonal influenza A/H3-positive specimens and 20 influenza-negative specimens) were tested with a new laboratory-developed 2009 H1N1 RT-PCR and the CDC 2009 H1N1 assay. The overall sensitivities of the CDC, ProFlu(+), and MGB assays for detection of influenza A and B viruses were 100%, 98.3%, and 94%, respectively. The ProFlu(+) assay failed to detect one influenza A/H1 virus-positive specimen and yielded one unresolved result with another influenza A/H1 virus-positive specimen. The MGB assay detected 84/87 (96.5%) of influenza A and B viruses and 26/30 (86.6%) of 2009 H1N1 viruses. The new laboratory-developed 2009 H1N1 RT-PCR assay detected 100/100 (100%) 2009 H1N1 virus-positive specimens, while the CDC SW Inf A and SW H1 PCR assays failed to detect one and three low-positive 2009 H1N1-positive specimens, respectively. The CDC influenza A/B virus assay and the newly developed 2009 H1N1 RT-PCR assay with an internal control can be set up in two separate reactions in the same assay for routine clinical testing to detect influenza A and B viruses and to specifically identify the 2009 H1N1 influenza virus.

Evaluation of three analyte-specific reagents for detection and typing of herpes simplex virus in cerebrospinal fluid.

The performance of three analyte-specific reagents (ASRs); Cepheid herpes simplex virus (HSV) Typing Primer Probe set (CD), Eragen MultiCode-Rtx HSV-1/2 kit primer mix (ER), and Roche LightCycler HSV-1/2 Primer/Hybridization Probes (RD), was evaluated for detection and typing of herpes simplex virus (HSV-1 and HSV-2) in cerebrospinal fluid (CSF) specimens. Of 68 CSF specimens, HSV-1 was detected in 8 specimens and HSV-2 was detected in 17 specimens. ER detected all 25 HSV-positive specimens, whereas CD and RD detected 24 and 23 HSV-positive specimens, respectively. The results of HSV typing with the 3 ASRs were in complete agreement. The analytical sensitivity of all ASRs was determined to be about 10(1) copies/reaction. Our results demonstrate that the performances of all 3 ASRs are comparable and reliable for routine clinical testing in detection and typing of HSV DNA in CSF.

Peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) assay for specific detection of Mycobacterium immunogenum and DNA-FISH assay for analysis of pseudomonads in metalworking fluids and sputum.

Specific and rapid detection and quantification of mycobacteria in contaminated metalworking fluid (MWF) are problematic due to complexity of the matrix and heavy background co-occurring microflora. Furthermore, cross-reactivity among neighboring species of Mycobacterium makes species differentiation difficult for this genus. Here, we report for the first time a species-specific peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) method for Mycobacterium immunogenum, a non-tuberculous Mycobacterium species prevalent in MWF and implicated in occupational lung disease hypersensitivity pneumonitis and pseudo-outbreaks. A novel species-specific 14-bp PNA probe was designed for M. immunogenum based on its 16S rRNA gene sequence and was validated for specificity, by testing against a panel of other phylogenetically closely related rapidly growing mycobacteria and representative species of gram-positive, gram-negative, and acid fast organisms. In addition, a DNA-FISH protocol was optimized for co-detection of Pseudomonas, the most predominantly co-occurring genus in contaminated MWF. Reliable quantification for both the test organisms was achieved at or above a cell density of 10(3)cellsml(-1), a recognized minimum limit for microscopic quantification. The mycobacterial PNA-FISH assay was successfully adapted to human sputum demonstrating its potential for clinical diagnostic applications in addition to industrial MWF monitoring, to assess MWF-associated exposures and pseudo-outbreaks.

Differential biocide susceptibility of the multiple genotypes of Mycobacterium immunogenum.

The non-tuberculous mycobacterium Mycobacterium immunogenum colonizes industrial metalworking fluids (MWFs) presumably due to its relative resistance to the currently practiced biocides and has been implicated in occupational respiratory hazards, particularly hypersensitivity pneumonitis. With an aim to understand its inherent biocide susceptibility profile and survival potential in MWF, five different genotypes of this organism, including a reference genotype (700506) and four novel test genotypes (MJY-3, MJY-4, MJY-10 and MJY-12) isolated in our recent study from diverse MWF operations were evaluated. For this, two commercial biocide formulations, Grotan (Hexahydro-1,3,5-tris(2-hydroxyethyl)-s-triazine) and Kathon (5-chloro-2-methyl-4-isothiazolin-3-one) currently practiced for the control of microorganisms, including mycobacteria, in MWF operations were tested. Effect of the fluid matrix on the biocide susceptibility was investigated for the synthetic (S) and semi-synthetic (SS) MWF matrices. In general, the minimum inhibitory concentration values were higher for the HCHO-releasing biocide Grotan than the isothiazolone biocide Kathon. All genotypes (except the reference genotype) showed lower susceptibility in SS as compared to S fluid matrix for Grotan. However, in case of Kathon, a greater susceptibility was observed in SS fluid for majority of the test genotypes (MJY-3, 4 and 10). The test genotypes were more resistant than the reference genotype to either biocide in both fluid types. Furthermore, the individual genotypes showed differential biocidal susceptibility, with MJY-10 being the most resistant. These observations emphasize the importance of using the resistant genotypes of M. immunogenum as the test strains for formulation or development and evaluation of existing and novel biocides, for industrial applications.

Occurrence and characterization of multiple novel genotypes of Mycobacterium immunogenum and Mycobacterium chelonae in metalworking fluids.

Rapidly growing mycobacteria colonize metalworking fluids, leading to contamination of occupational environments and exposure-related respiratory illnesses in machine workers. Lately, it has been emphasized that these fluids are colonizable by a single genotype of a rapidly growing mycobacterium species, Mycobacterium immunogenum. Here, we report on the genotypic diversity of mycobacteria in these fluids, including isolation and characterization of multiple novel genotypes of two distinct species, Mycobacterium chelonae and M. immunogenum. Using agar culturing and Mycobacterium-specific PCR, 13 mycobacterial isolates were recovered from 100 geographically diverse in-use metalworking fluid samples. Based on restriction fragment length polymorphism of PCR products, DNA sequencing (hsp65 gene segment), and phylogenetic analysis of 16S-23S rDNA internal transcribed spacer (ITS) sequences, six isolates were identified as M. immunogenum and seven as M. chelonae; an additional isolate from metalworking fluid diluent water was identified as M. diernhoferi. Genomic DNA macro-restriction fragment pattern analysis, using pulsed-field gel electrophoresis with XbaI and SpeI restriction digestions, showed intraspecies variation among the isolates of M. immunogenum and M. chelonae. Visual and computer-assisted dendrogram analysis of the XbaI macro-restriction patterns revealed three novel genotypes of M. immunogenum and two of M. chelonae, whereas SpeI macro-restriction patterns revealed only two genotypes for each isolate. None of the identified genotypes matched the reportedly dominant one of M. immunogenum from metalworking fluids. Both mycobacterial species are prevalent in metalworking fluids and there is a considerable strain-level genetic diversity within them.

Method for rapid identification and differentiation of the species of the Mycobacterium chelonae complex based on 16S-23S rRNA gene internal transcribed spacer PCR-restriction analysis.

Members of the Mycobacterium chelonae complex (MCC), including M. immunogenum, M. chelonae, and M. abscessus, have been associated with nosocomial infections and occupational hypersensitivity pneumonitis due to metalworking fluid (MWF) exposures. In order to minimize these health hazards, an effective and rapid assay for detection of MCC species and differentiation of MCC species from other species of rapidly growing mycobacteria (RGM) and from one another is warranted. Here we report such a method, based on the variable 16S-23S rRNA gene internal transcribed spacer (ITS) region. Mycobacterium genus-specific primers derived from highly conserved sequences in the ITS region and the flanking 16S rRNA gene were used. Specificity of the primers was verified using the MCC member species, 11 non-MCC RGM species, 3 slow-growing mycobacterial (SGM) species (two strains each), and 19 field isolates, including 18 MCC isolates (from in-use MWF) and one non-MCC isolate (from reverse osmosis water). The ITS amplicon size of M. immunogenum varied from those of M. chelonae and M. abscessus. Sequencing of the approximately 250-bp-long ITS amplicons of the three MCC member species showed differences in 24 to 34 bases, thereby yielding variable deduced restriction maps. ITS PCR-restriction analysis using the in silico-selected restriction enzyme MaeII or HphI differentiated the three MCC members from one another and from other RGM and SGM species without sequencing. The enzyme MaeII discriminated all three member species; however, HphI could only differentiate M. immunogenum from M. chelonae and M. abscessus. Use of an optimized rapid DNA template preparation step based on direct cell lysis in the PCR tube added to the simplicity and adaptability of the developed assay.

A new method for species identification and differentiation of Mycobacterium chelonae complex based on amplified hsp65 restriction analysis (AHSPRA).

Members of the Mycobacterium chelonae complex (MCC), namely M. chelonae, Mycobacterium abscessus and Mycobacterium immunogenum, have been implicated in nosocomial infections and occupational respiratory illnesses like hypersensitivity pneumonitis (HP) associated with contaminated metalworking fluid (MWF) exposures. Close relationship among these member species makes their differentiation cumbersome using the existing methods. Here we report a simple and rapid method for unambiguous identification and differentiation of the three-member species of the MCC group with PCR-restriction analysis targeting a 667-bp segment of a variable region of the 65-kDa-heat shock protein (hsp65) gene. This assay, described as Amplified hsp65 Restriction Analysis (AHSPRA), can discriminate all the three individual species using a one-step restriction digestion using either BbvI or Eco0109I. The enzyme NarI can differentiate M. immunogenum from the other two MCC species (M. chelonae and M. abscessus). The developed method was validated using several non-MCC reference species of other rapidly growing mycobacteria (RGM) and MCC field isolates from MWF samples. Direct cell-lysis was used instead of the conventional DNA template preparation, which improved the rapidity, simplicity and adaptability of the developed method. The results suggest that the developed method can unambiguously differentiate species of the M. chelonae complex from other RGM species and from one another.

Biocidal activity of formaldehyde and nonformaldehyde biocides toward Mycobacterium immunogenum and Pseudomonas fluorescens in pure and mixed suspensions in synthetic metalworking fluid and saline.

The microbicidal activity of four different biocides was studied in synthetic metalworking fluid (MWF) against Mycobacterium immunogenum, a suspected causative agent for hypersensitivity pneumonitis, and Pseudomonas fluorescens, a representative for the predominant gram-negative bacterial contaminants of MWF. The results indicated that M. immunogenum is more resistant than P. fluorescens to the tested formaldehyde-releasing biocides (Grotan and Bioban), isothiazolone (Kathon), and phenolic biocide (Preventol). Kathon was effective against mycobacteria at lower concentrations than the other three test biocides in MWF. In general, there was a marked increase in biocidal resistance of both the test organisms when present in MWF matrix compared to saline. Increased resistance of the two test organisms to biocides was observed when they were in a mixed suspension (1:1 ratio). The results indicate the protective effect of the MWF matrix against the action of commonly used biocides on the MWF-colonizing microbial species of occupational health significance, including mycobacteria.