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Development of a new multiplex real-time PCR assay for rapid screening of hospital-acquired infection agents
Abstract
Aims:
A new multiplex real-time PCR (qPCR) assay was developed to detect antibiotic-resistant hospital-acquired infectious agents in nasal and rectal swab samples in 1.5 h without the need for nucleic acid extraction.
Methods:
Spiked negative clinical specimens were used for the analytical performance evaluation. Double-blind samples were collected from 1788 patients to assess the relative clinical performance of the qPCR assay to the conventional culture-based methods. Bio-Speedy® Fast Lysis Buffer (FLB) and 2× qPCR-Mix for hydrolysis probes (Bioeksen R&D Technologies, Istanbul, Turkey) and LightCycler® 96 Instrument (Roche Inc., Branchburg, NJ, USA) were used for all molecular analyses. The samples were transferred into 400 μl FLB, homogenized and immediately used in qPCRs. The target DNA regions are vanA and vanB genes for vancomycin-resistant Enterococcus (VRE); blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-23, blaOXA-48, blaOXA-58 genes for carbapenem-resistant Enterobacteriaceae (CRE); and mecA, mecC and spa for methicillin-resistant Staphylococcus aureus (MRSA).
Results:
No qPCR tests produced positive results for the samples spiked with the potential cross-reacting organisms. The limit of detection (LOD) of the assay for all targets was 100 colony-forming unit (cfu)/swab-sample. Results of the repeatability studies in two different centers were in 96%-100% (69/72-72/72) agreement. The relative specificity and sensitivity of the qPCR assay were respectively 96.8% and 98.8% for VRE; 94.9% and 95.1% for CRE; 99.9% and 97.1% for MRSA.
Conclusions:
The developed qPCR assay can screen antibiotic-resistant hospital-acquired infectious agents in infected/colonized patients with an equal clinical performance to the culture-based methods.
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Nosocomial infections or healthcare associated infections occur in patients under medical care. These infections occur worldwide both in developed and developing countries. Nosocomial infections accounts for 7% in developed and 10% in developing countries. As these infections occur during hospital stay, they cause prolonged stay, disability, and economic burden. Frequently prevalent infections include central line-associated bloodstream infections, catheter-associated urinary tract infections, surgical site infections and ventilator-associated pneumonia. Nosocomial pathogens include bacteria, viruses and fungal parasites. According to WHO estimates, approximately 15% of all hospitalized patients suffer from these infections. During hospitalization, patient is exposed to pathogens through different sources environment, healthcare staff, and other infected patients. Transmission of these infections should be restricted for prevention. Hospital waste serves as potential source of pathogens and about 20%–25% of hospital waste is termed as hazardous. Nosocomial infections can be controlled by practicing infection control programs, keep check on antimicrobial use and its resistance, adopting antibiotic control policy. Efficient surveillance system can play its part at national and international level. Efforts are required by all stakeholders to prevent and control nosocomial infections.
Background
Group A streptococci (GAS) are the most common bacterial cause of acute pharyngitis and account for 15–30 % of cases of acute pharyngitis in children and 5–10 % of cases in adults. In this study, a real-time quantitative PCR (qPCR) based GAS detection assay in pharyngeal swab specimens was developed. Methods
The qPCR assay was compared with the gold standard bacterial culture and a rapid antigen detection test (RADT) to evaluate its clinical performance in 687 patients. The analytical sensitivity of the assay was 240 cfu/swab. Forty-five different potential cross-reacting organisms did not react with the test. Four different laboratories for the reproducibility studies were in 100 % (60/60) agreement for the contrived GAS positive and negative swab samples. ResultsThe relative sensitivities of the RADT and the qPCR test were 55.9 and 100 %; and the relative specificities were 100 and 96.3 %, respectively. Duration of the total assay for 24 samples including pre-analytical processing and analysis changed between 42 and 55 min depending on the type of qPCR instrument used. A simple DNA extraction method and a low qPCR volume made the developed assay an economical alternative for the GAS detection. Conclusion
We showed that the developed qPCR test is rapid, cheap, sensitive and specific and therefore can be used to replace both antigen detection and culture for diagnosis of acute GAS pharyngitis.
Detecting colonization of patients with carbapenemase-producing bacteria can be difficult. This study compared the sensitivity
and specificity of a PCR-based method (Xpert MDRO) for detecting blaKPC, blaNDM, and blaVIM carbapenem resistance genes using GeneXpert cartridges to the results of culture with and without a broth enrichment step
on 328 rectal, perirectal, and stool samples. The culture method included direct inoculation of a MacConkey agar plate on
which a 10-μg meropenem disk was placed and plating on MacConkey agar after overnight enrichment of the sample in MacConkey
broth containing 1 μg/ml of meropenem. Forty-three (13.1%) samples were positive by PCR for blaKPC and 11 (3.4%) were positive for blaVIM; none were positive for blaNDM. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the PCR assay for
blaKPC were 100%, 99.0%, 93.0%, and 100%, respectively, compared to broth enrichment culture and sequencing of target genes. The
sensitivity, specificity, PPV, and NPV of the assay for blaVIM were 100%, 99.4%, 81.8%, and 100%, respectively. Since none of the clinical samples contained organisms with blaNDM, 66 contrived stool samples were prepared at various dilutions using three Klebsiella pneumoniae isolates containing blaNDM. The PCR assay showed 100% positivity at dilutions from 300 to 1,800 CFU/ml and 93.3% at 150 CFU/ml. The Xpert MDRO PCR assay
required 2 min of hands-on time and 47 min to complete. Rapid identification of patients colonized with carbapenemase-producing
organisms using multiplex PCR may help hospitals to improve infection control activities.
Active screening for vancomycin-resistant enterococci (VRE) using rectal specimens is recommended to limit the spread of antimicrobial resistance within certain high-risk populations. We evaluated the diagnostic performance of Vancomycin Resistance 3 Multiplexed Tandem PCR assay (AusDiagnostics, Australia), a rapid multiplex real-time PCR assay that detects vanA and/or vanB.
Two-hundred-and-eleven rectal swabs from Hematology and Oncology unit were submitted for VRE surveillance via direct detection of vanA and/or vanB by culture and by using Vancomycin Resistance 3 Multiplexed Tandem PCR assay. Enterococci were identified to the species level by using standard biochemical tests and BD Phoenix Automated Microbiology System (BD Diagnostic Systems, USA). Vancomycin susceptibility of enterococci was determined using Etest (BioMerieux, France).
Compared to the culture method, Vancomycin Resistance 3 Multiplexed Tandem PCR assay had a sensitivity of 84.0%, specificity of 98.8%, positive predictive value (PPV) of 91.3%, and negative predictive value (NPV) of 97.6%. The assay failed to detect 18 (8.5%) specimens because of the presence of PCR inhibitors; of the remaining 193 specimens, 25 (12.9%) were positive, 23 for vanA, and 2 for vanB. Although both sensitivity and specificity for vanA VRE was 100% compared to the culture method, all vanB-positive specimens tested negative by VRE culture.
Vancomycin Resistance 3 Multiplexed Tandem PCR assay is a rapid and laborsaving option for VRE surveillance for direct use on rectal swabs. However, the high rate of PCR failure owing to the inhibitors in the specimens and the low specificity for vanB should be considered when interpreting the results.
We assessed the performance of a duplex real-time PCR assay for blaKPC and blaNDM performed directly (D-PCR) on perianal and perirectal swabs and stool. Spiked specimens and 126 clinical surveillance swabs
(comprising a sensitivity panel of 46 perirectal double swabs previously determined to be culture positive for blaKPC-PCR-positive Enterobacteriaceae and a specificity panel of 80 perianal swabs from patients at risk of carbapenemase-producing Enterobacteriaceae [CPE] colonization) were studied. For the surveillance swabs, D-PCR was compared to PCR after broth enrichment (BE-PCR) and
two culture-based methods: the HardyCHROM ESBL agar (HC-A) and the CDC screening (CDC-A) methods. PCR was performed on morphologically
distinct colonies that were isolated by culture. All of the initial PCR testing was done without extraction using a simple
lysis procedure. The analytical sensitivities of D-PCR for blaKPC were 9 CFU/μl (for swabs) and 90 CFU/μl (for stool), and for blaNDM, it was 1.9 CFU/μl (for both swabs and stool). In the clinical sensitivity panel, D-PCR and BE-PCR were initially positive
for blaKPC in 41/46 (89.1%) and 43/46 (93.5%) swabs, respectively. The swabs that were initially negative by D-PCR (n = 5) and BE-PCR (n = 3) were visibly stool soiled; all swabs were blaKPC positive upon repeat testing after lysate extraction. The CDC-A and HC-A yielded blaKPC-positive Enterobacteriaceae from 36/46 (78.3%) and 35/46 (76.1%) swabs, respectively (sensitivities of D-PCR/BE-PCR postextraction of soiled specimens
versus HC-A, P = 0.0009, and versus CDC-A, P = 0.0016). All swabs in the specificity panel were negative for CPE by all four methods. D-PCR allows for the timely detection
of blaKPC and blaNDM carriage with excellent sensitivity when specimens visibly soiled with stool undergo preparatory extraction.
Pertussis continues to be a relevant public-health issue. The high coverage rates achieved have decreased the spread of the pathogen, but the waning of immunity implies a relevant role of adolescents and adults in the infective dynamics as they may represent a significant source of infection for unvaccinated or incompletely immunized newborns. The passive surveillance system is affected by many limitations. The underestimation of pertussis in adolescents, young adults and adults is mainly related to the atypical clinical characteristics of cases and the lack of lab confirmation. The real epidemiological impact of pertussis is not always perceived, anyway, the unavailability of comprehensive data should not hamper the adoption of active prophylactic interventions aimed at preventing the impact of waning immunity on pertussis. To avoid an increase of the mean age of acquisition of the infection, a booster dose of low-antigen content combined vaccine should be adopted in adolescents and adults. A decreased risk of infection in newborns can be achieved with the cocoon strategy, although the debate on this aspect is still open and enhanced surveillance and further studies are needed to fine-tune the pertussis prevention strategy.
Background:
During 9 May 2010-7 May 2011, an outbreak of pertussis-like illness (incidence, 80 cases per 100 000 persons) occurred in Franklin County, Ohio. The majority of cases were identified by IS481-directed polymerase chain reaction (PCR), which does not differentiate among Bordetella species. We sought to determine outbreak etiology and epidemiologic characteristics.
Methods:
We obtained demographic, clinical, and vaccination-related data from the Ohio Disease Reporting System and Impact Statewide Immunization Information System. We tested sera from 14 patients for anti-pertussis toxin (PT) antibodies and used species-specific PCR on 298 nasopharyngeal specimens.
Results:
Reported cases totaled 918. IS481 results were available for 10 serologically tested patients; 5 of 10 had discordant anti-PT antibody and IS481 results, suggestive of Bordetella holmesii, which lacks PT and harbors IS481. We identified specific Bordetella species in 164 of 298 specimens tested with multitarget PCR; B. holmesii and Bordetella pertussis were exclusively detected among 48 (29%) and 112 (68%), respectively; both were detected in 4 (2%). Among 48 patients with B. holmesii infections, 63% were aged 11-18 years, compared with 35% of 112 patients with B. pertussis infections (P = .001). Symptoms were similar among B. holmesii- and B. pertussis-infected patients. Adolescent pertussis ("Tdap") booster vaccinations were more effective against B. pertussis than B. holmesii (effectiveness: 67% and 36%, respectively; 95% confidence intervals, 38%-82% and -33% to 69%, respectively).
Conclusions:
We report the first documented mixed outbreak of B. pertussis and B. holmesii infections. Bordetella holmesii particularly affected adolescents. Although laboratory capacity limitations might inhibit routine use of multitarget PCR for clinical diagnosis, focused testing and enhanced surveillance might improve understanding the burden of B. holmesii infection.
Bacterial load quantification is a critical component of bacterial community analysis, but a culture-independent method capable of detecting and quantifying diverse bacteria is needed. Based on our analysis of a diverse collection of 16 S rRNA gene sequences, we designed a broad-coverage quantitative real-time PCR (qPCR) assay-BactQuant-for quantifying 16 S rRNA gene copy number and estimating bacterial load. We further utilized in silico evaluation to complement laboratory-based qPCR characterization to validate BactQuant.
The aligned core set of 4,938 16 S rRNA gene sequences in the Greengenes database were analyzed for assay design. Cloned plasmid standards were generated and quantified using a qPCR-based approach. Coverage analysis was performed computationally using >670,000 sequences and further evaluated following the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines.
A bacterial TaqMan® qPCR assay targeting a 466 bp region in V3-V4 was designed. Coverage analysis showed that 91% of the phyla, 96% of the genera, and >80% of the 89,537 species analyzed contained at least one perfect sequence match to the BactQuant assay. Of the 106 bacterial species evaluated, amplification efficiencies ranged from 81 to 120%, with r2-value of >0.99, including species with sequence mismatches. Inter- and intra-run coefficient of variance was <3% and <16% for Ct and copy number, respectively.
The BactQuant assay offers significantly broader coverage than a previously reported universal bacterial quantification assay BactQuant in vitro performance was better than the in silico predictions.
We evaluated the performances of 4 commercial real-time PCR kits for Bordetella pertussis IS481 sequence detection in nasopharyngeal aspirates by comparison with an in-house real-time PCR assay. Among them, the Simplexa
Bordetella pertussis/parapertussis assay (Focus Diagnostics), the SmartCycler Bordetella pertussis/parapertussis assay (Cepheid), and Bordetella R-gene (Argene) present sensitivities over 90%. One kit proved unsuitable for routine clinical
use.
The clinical trial conducted in Italy to evaluate the efficacy of acellular pertussis vaccines provided an opportunity to estimate the frequency of clinical infections with Bordetella parapertussis and to compare the clinical characteristics of children suffering from Bordetella pertussis illness with those of children with B. parapertussis illness. This study dealt with 76 B. parapertussis infections diagnosed from a population of 15,601 children participating in the follow-up of suspected cases of pertussis. An overall incidence of 2.1 cases of laboratory-confirmed parapertussis per 1,000 person-years was observed. Children affected by B. parapertussis infections showed a less severe clinical picture both in the duration of symptoms and in the percentage of patients affected, even when compared with vaccinated children with pertussis. To characterize the isolated strains, we performed assays for susceptibility to erythromycin and sulfamethoxazole-trimethoprim, and we examined the genomic DNAs by pulsed-field gel electrophoresis. The results showed a high degree of genetic stability among B. parapertussis strains regardless of time of collection and geographical distribution.
Bordetella holmesii is a human pathogen found mainly in immunocompromised patients. A specific real-time PCR assay was developed and successfully
used to identify specimens from which B. holmesii was misidentified as Bordetella pertussis and to establish the prevalence of B. holmesii in Ontario patients with pertussis-like symptoms.
A PCR-based assay for Bordetella pertussis was inhibited by using a calcium alginate fiber-tipped swab with an aluminum shaft but not by using a Dacron fiber-tipped swab with a plastic shaft. The calcium alginate fiber component inhibited the assay following storage for less than 1 min in a suspension of 10(3) CFU of B. pertussis per ml, whereas the aluminum shaft component required storage for at least 48 h in order to cause inhibition. We recommend the Dacron swab over the calcium alginate swab for collecting specimens for testing in PCR-based assays.
Bordetella pertussis and Bordetella parapertussis are closely related species. Both are responsible for outbreaks of whooping cough in humans and produce similar virulence factors, with the exception of pertussis toxin, specific to B. pertussis. Current pertussis whole-cell vaccine will soon be replaced by acellular vaccines containing major adhesins (filamentous hemagglutinin and pertactin) and major toxin (pertussis toxin). All of these factors are antigens that stimulate a protective immune response in the murine respiratory model and in clinical assays. In the present study, we examined the protective efficacies of these factors, and that of adenylate cyclase-hemolysin, another B. pertussis toxin, against B. parapertussis infection in a murine respiratory model. As expected, pertussis toxin did not protect against B. parapertussis infection, since this bacterium did not express this protein, but the surprising result was that none of the other factors were protective against B. parapertussis infection. Furthermore, B. parapertussis adenylate cyclase-hemolysin, although it protected against B. parapertussis infection, did not protect against B. pertussis infection. Despite a high degree of homology between both B. pertussis and B. parapertussis species, no cross-protection was observed. Our results outline the fact that, as in other gram-negative bacteria, Bordetella surface proteins vary immunologically.
We isolated Bordetella holmesii, generally associated with septicemia in patients with underlying conditions, from nasopharyngeal specimens of otherwise healthy young persons with a cough. The proportion of B. holmesii-positive specimens submitted to the Massachusetts State Laboratory Institute increased from 1995 to 1998. Bordetella holmesii is a recently described gram-negative, asaccharolytic, nonoxidizing, soluble, brown-pigment-producing rod previously known as CDC nonoxidizer group 2 (NO-2) (1). This group consists of 15 closely related, biochemically similar strains of fastidious nonmotile bacteria isolated from human blood cultures. In establishing NO-2 as a species, Weyant et al. (1) performed 16S rRNA sequencing of one NO-2 strain and the type strains of B. pertussis, B. parapertussis, B. bronchiseptica, and B. avium. They found a high degree of homology among them (≥98% over 1,525 bases) and confirmed a close relatedness between NO-2 and Bordetella species by DNA relatedness studies (hydroxyapatite method). Biochemically, the lack of oxidase activity and the production of a brown soluble pigment differentiate B. holmesii from B. pertussis, B. bronchiseptica, and B. avium; the lack of urease activity differentiates it from B. parapertussis (1). Unlike B. pertussis, which causes whooping cough, B. holmesii has been associated most often with septicemia in patients with underlying conditions (1-4). It also has been isolated from sputum from one patient with respiratory symptoms (3). Van den Akker (5) suggested that the difference in lipopolysaccharide expression (important in bacterial pathogenesis) between the closely related B. pertussis and B. holmesii
Standard repetitive extragenic palindromic (REP)-PCR, enterobacterial repetitive intergenic consensus-PCR, and Salmonella enteritidis repetitive element-PCR methods for bacterial strain typing were performed with DNA extracted by boiling members of each of the currently recognized species of human viridans group streptococci. Each of the methods was reproducible. The unique isolates (n = 72) from 15 species of viridans group streptococci were readily distinguishable, with no two isolates showing greater than 90% per cent similarity. The majority of strains exhibited much less than 90% similarity. Isolates identical by REP-PCR were also identical by the other two methods. These PCR-based typing methods, although they do not permit determination of the species of the isolates, are simple to perform and are suitable for clinical and ecological investigations of viridans group streptococci.
The full potential of diagnostic PCR is limited, in part, by the presence of inhibitors in complex biological samples that reduce the amplification efficiency. Therefore, different pre-PCR treatments are being used to reduce the effects of PCR inhibitors. The aim of the present study was to investigate the effects of 16 amplification facilitators to enhance DNA amplification in the presence of blood, feces, or meat. Different concentrations of amplification facilitators and inhibitory samples were added to PCR mixtures containing rTth or Taq DNA polymerase. The addition of 0.6% (wt/vol) bovine serum albumin to reaction mixtures containing Taq DNA polymerase reduced the inhibitory effect of blood and allowed DNA amplification in the presence of 2% instead of 0.2% (vol/vol) blood. Furthermore, the addition of bovine serum albumin (BSA) to reaction mixtures containing feces or meat enhanced the amplification capacities of both polymerases. Taq DNA polymerase was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 4% instead of 0.2% (vol/vol) meat, and rTth was able to amplify DNA in the presence of 4% instead of 0.4% (vol/vol) feces and 20% instead of 2% (vol/vol) meat. The single-stranded DNA binding T4 gene 32 protein (gp32) had a relieving effect similar to that of BSA, except when it was added to PCR mixtures of rTth containing meat and of Taq DNA polymerase containing feces. The relieving effects of betaine and a cocktail of proteinase inhibitors were more sample specific. The addition of 11.7% (wt/vol) betaine allowed Taq DNA polymerase to amplify DNA in the presence of 2% (vol/vol) blood, while the addition of proteinase inhibitors allowed DNA amplification by both polymerases in the presence of 4% (vol/vol) feces. When various combinations of betaine, BSA, gp32, and proteinase inhibitors were tested, no synergistic or additive effects were observed. The effects of facilitators on real-time DNA synthesis instead of conventional PCR were also studied.
A rapid real-time multiplex PCR assay for detecting and differentiating Bordetella pertussis and Bordetella parapertussis in nasopharyngeal swabs was developed. This assay (LC-PCR-IS) targets the insertion sequences IS481 and IS1001 of B. pertussis and B. parapertussis, respectively, and is performed using the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.). The analytical
sensitivity is less than one organism per reaction. Results for Bordetella culture and/or direct fluorescent antibody testing and a second LightCycler PCR assay (target, pertussis toxin gene) were
compared to results of the LC-PCR-IS assay for 111 nasopharyngeal swabs submitted for pertussis testing. Of the specimens,
12 were positive (9 B. pertussis and 3 B. parapertussis) and 68 specimens were negative by all methods. Three other specimens were positive for B. pertussis by at least two of the methods (including the LC-PCR-IS assay), and another 28 specimens were positive for B. pertussis by the LC-PCR-IS assay only. No specimens were negative by the LC-PCR-IS assay and positive by the other methods. A conventional
PCR method (target, IS481) was also compared to the LC-PCR-IS assay for a different group of nasopharyngeal swab specimens (n = 96): 44 specimens were positive and 41 specimens were negative for B. pertussis with both PCR methods. Nine specimens were positive for B. pertussis by the LC-PCR-IS assay and negative by the conventional PCR assay, and two specimens were positive for B. pertussis by the conventional PCR assay and negative by the LC-PCR-IS assay. Positivity of the two assays was not significantly different
(P = 0.0654). The insertion sequence IS481 is also present in Bordetella holmesii; specimens containing B. holmesii may yield false-positive results. The LC-PCR-IS assay takes approximately 45 min to complete post-nucleic acid extraction,
compared to 24 h for the conventional PCR assay previously used in our laboratory. The LC-PCR-IS assay is easier to perform
than the conventional PCR assay, and the closed system decreases the chance of contamination. All of these characteristics
represent a significant improvement in the detection of B. pertussis and B. parapertussis in nasopharyngeal specimens.
Bordetella pertussis, B. bronchiseptica, B. parapertussis(hu) and B. parapertussis(ov) are closely related respiratory pathogens that infect mammalian species. B. pertussis and B. parapertussis(hu) are exclusively human pathogens and cause whooping cough, or pertussis, a disease that has resurged despite vaccination. Although it most often infects animals, infrequently B. bronchiseptica is isolated from humans, and these infections are thought to be zoonotic. B. pertussis and B. parapertussis(hu) are assumed to have evolved from a B. bronchiseptica-like ancestor independently. To determine the phylogenetic relationships among these species, housekeeping and virulence genes were sequenced, comparative genomic hybridizations were performed using DNA microarrays, and the distribution of insertion sequence elements was determined, using a collection of 132 strains. This multifaceted approach distinguished four complexes, representing B. pertussis, B. parapertussis(hur) and two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Of the two B. bronchiseptica complexes, complex IV was more closely related to B. pertussis. Of interest, while only 32% of the complex I strains were isolated from humans, 80% of the complex IV strains were human isolates. Comparative genomic hybridization analysis identified the absence of the pertussis toxin locus and dermonecrotic toxin gene, as well as a polymorphic lipopolysaccharide biosynthesis locus, as associated with adaptation of complex IV strains to the human host. Lipopolysaccharide structural diversity among these strains was confirmed by gel electrophoresis. Thus, complex IV strains may comprise a human-associated lineage of B. bronchiseptica from which B. pertussis evolved. These findings will facilitate the study of pathogen host-adaptation. Our results shed light on the origins of the disease pertussis and suggest that the association of B. pertussis with humans may be more ancient than previously assumed.
In this review we examine the literature related to emerging technologies that will help to reshape the clinical microbiology laboratory. These topics include nucleic acid amplification tests such as isothermal and point-of-care molecular diagnostics, multiplexed panels for syndromic diagnosis, digital PCR, next-generation sequencing, and automation of molecular tests. We also review matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry methods and their role in identification of microorganisms. Lastly, we review the shift to liquid-based microbiology and the integration of partial and full laboratory automation that are beginning to impact the clinical microbiology laboratory.
Background:
Methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus colonization is associated with increased rates of infection. Rapid and reliable detection methods are needed to identify colonization of nares and extra-nare sites, particularly given recent reports of oropharynx-only colonization. Detection methods for MRSA/MSSA colonization include culture, PCR, and novel methods such as PCR/electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS).
Methods:
We evaluated 101 healthy military members for S. aureus colonization in the nares, oropharynx, axilla, and groin, using CHROMagar S. aureus medium and Xpert SA Nasal Complete PCR for MRSA/MSSA detection. The same subjects were screened in the nares, oropharynx, and groin using PCR/ESI-TOF-MS.
Results:
By culture, 3 subjects were MRSA-colonized (all oropharynx) and 34 subjects were MSSA-colonized (all 4 sites). PCR detected oropharyngeal MRSA in 2 subjects, which correlated with culture findings. By PCR, 47 subjects were MSSA-colonized (all 4 sites); however, 43 axillary samples were invalid, 39 of which were associated with deodorant/anti-perspirant use (93%, p < 0.01). By PCR/ESI-TOF-MS, 4 subjects were MRSA-colonized, 2 in the nares and 2 in the oropharynx; however, neither of these correlated with positive MRSA cultures. Twenty-eight subjects had MSSA by PCR/ESI-TOF-MS, and 41 were found to have possible MRSA (S. aureus with mecA and coagulase-negative Staphylococcus (CoNS)).
Conclusion:
The overall 3% MRSA colonization rate is consistent with historical reports, but the oropharynx-only colonization supports more recent findings. In addition, the use of deodorant/anti-perspirant invalidated axillary PCR samples, limiting its utility. Defining MRSA positivity by PCR/ESI-TOF-MS is complicated by co-colonization of S. aureus with CoNS, which can also carry mecA.
Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired (HA-MRSA) infection worldwide. As a result, the rapid and specific detection of MRSA is crucial not only for early prevention of disease spread, but also for the effective treatment of these infections. We report here an integrated modular-based microfluidic system for MRSA identification, which can carry out the multi-step assay used for MRSA identification in a single disposable fluidic cartridge. The multi-step assay included PCR amplification of the mecA gene harboring methicillin resistance loci that can provide information on drug susceptibility, ligase detection reaction (LDR) to generate fluorescent ligation products appended with a zip-code complement that directs the ligation product to a particular address on a universal array containing zip-code probes and a universal DNA array, which consisted of a planar waveguide for evanescent excitation. The fluidic cartridge design was based on a modular format, in which certain steps of the molecular processing pipeline were poised on a module made from a thermoplastic. The cartridge was comprised of a module interconnected to a fluidic motherboard configured in a 3-dimensional network; the motherboard was made from polycarbonate, PC, and was used for PCR and LDR, while the module was made from poly(methylmethacrylate), PMMA, and contained an air-embedded waveguide serving as the support for the universal array. Fluid handling, thermal management and optical readout hardware were situated off-chip and configured into a small footprint instrument. In this work, the cartridge was used to carry out a multiplexed PCR/LDR coupled with the universal array allowed for simultaneous detection of five genes that encode for 16S ribosomal RNA (SG16S), protein A (spa), the femA protein of S. epidermidis (femA), the virulence factor of Panton-Valentine leukocidin (PVL) and the gene that confers methicillin resistance (mecA). Results indicated that this modular system could differentiate community-acquired MRSA (CA-MRSA) from hospital-acquired MRSA (HA-MRSA) based on the presence/absence of the PVL gene as well as S. aureus from other Staphylococcal species using the sequence content in the femA gene. This system can identify strains in <40 min and detect MRSA directly from a mixture of Staphylococci.
In the United States, children receive five doses of diphtheria, tetanus, and acellular pertussis (DTaP) vaccine before 7 years of age. The duration of protection after five doses of DTaP is unknown.
We assessed the risk of pertussis in children in California relative to the time since the fifth dose of DTaP from 2006 to 2011. This period included a large outbreak in 2010. We conducted a case-control study involving members of Kaiser Permanente Northern California who were vaccinated with DTaP at 47 to 84 months of age. We compared children with pertussis confirmed by a positive polymerase-chain-reaction (PCR) assay with two sets of controls: those who were PCR-negative for pertussis and closely matched controls from the general population of health-plan members. We used logistic regression to examine the risk of pertussis in relation to the duration of time since the fifth DTaP dose. Children who received whole-cell pertussis vaccine during infancy or who received any pertussis-containing vaccine after their fifth dose of DTaP were excluded.
We compared 277 children, 4 to 12 years of age, who were PCR-positive for pertussis with 3318 PCR-negative controls and 6086 matched controls. PCR-positive children were more likely to have received the fifth DTaP dose earlier than PCR-negative controls (P<0.001) or matched controls (P=0.005). Comparison with PCR-negative controls yielded an odds ratio of 1.42 (95% confidence interval, 1.21 to 1.66), indicating that after the fifth dose of DTaP, the odds of acquiring pertussis increased by an average of 42% per year.
Protection against pertussis waned during the 5 years after the fifth dose of DTaP. (Funded by Kaiser Permanente).
Clinical specimens from 9 states during 2008-2010 were tested by PCR for Bordetella pertussis and Bordetella parapertussis. Of the positive samples, 13.99% were identified as B. parapertussis. It was concluded that B. parapertussis infections are more common than previously realized and contribute to cases thought to be vaccine failures.
Pertussis is often overlooked as a cause of chronic cough, especially in adolescents and adults. Several symptoms are classically thought to be suggestive of pertussis, but the diagnostic value of each of them is uncertain.
To systematically review the evidence regarding the diagnostic value of 3 classically described symptoms of pertussis: paroxysmal cough, posttussive emesis, and inspiratory whoop.
We searched MEDLINE (January 1966-April 2010), EMBASE (January 1969 to April 2010), and the bibliographies of pertinent articles to identify relevant English-language studies. Articles were selected that included children older than 5 years, adolescents, or adults and confirmed the diagnosis of pertussis among patients with cough illness (of any duration) with an a priori-defined accepted reference standard. Two authors independently extracted data from articles that met selection criteria and resolved any discrepancies by consensus.
Five prospective studies met inclusion criteria; 3 were used in the analysis. Presence of posttussive emesis (summary likelihood ratio [LR], 1.8; 95% confidence interval [CI], 1.4-2.2) or inspiratory whoop (summary LR, 1.9; 95% CI, 1.4-2.6) increases the likelihood of pertussis. Absence of paroxysmal cough (summary LR, 0.52; 95% CI, 0.27-1.0) or posttussive emesis (summary LR, 0.58; 95% CI, 0.44-0.77) reduced the likelihood. Absence of inspiratory whoop was less useful (summary LR, 0.78; 95% CI, 0.66-0.93). No studies evaluated combinations of findings.
In a nonoutbreak setting, data to determine the diagnostic usefulness of symptoms classically associated with pertussis are limited and of relatively weak quality. The presence or absence of posttussive emesis or inspiratory whoop modestly change the likelihood of pertussis; therefore, clinicians must use their overall clinical impression to decide about additional testing or empirical treatment.
Molecular technology has changed the way that clinical laboratories diagnose and manage many infectious diseases. Excellent sensitivity, specificity, and speed have made molecular assays an attractive alternative to culture or enzyme immunoassay methods. Many molecular assays are commercially available and FDA approved. Others, especially those that test for less common analytes, are often laboratory developed. Laboratories also often modify FDA-approved assays to include different extraction systems or additional specimen types. The Clinical Laboratory Improvement Amendments (CLIA) federal regulatory standards require clinical laboratories to establish and document their own performance specifications for laboratory-developed tests to ensure accurate and precise results prior to implementation of the test. The performance characteristics that must be established include accuracy, precision, reportable range, reference interval, analytical sensitivity, and analytical specificity. Clinical laboratories are challenged to understand the requirements and determine the types of experiments and analyses necessary to meet the requirements. A variety of protocols and guidelines are available in various texts and documents. Many of the guidelines are general and more appropriate for assays in chemistry sections of the laboratory but are applied in principle to molecular assays. This review presents information that laboratories may consider in their efforts to meet regulatory requirements.
The polymerase chain reaction (PCR) was recently added to conventional culture and serology for the diagnoses of Bordetella pertussis infection in a large vaccine efficacy trial in Germany. In vaccinees or family members who had illnesses with cough, two nasopharyngeal swabs (calcium alginate for culture and Dacron for PCR) were taken and initial and follow-up clinical data were obtained. PCR was done using oligonucleotide primers PTp1 and PTp2 which amplify a 191-base pair DNA fragment of pertussis toxin operon. From December, 1993, to May, 1994, 555 pairs of swabs were processed; 28 grew B. pertussis and 9 grew B. parapertussis. Twenty-six of the 28 subjects with B. pertussis-positive cultures also had positive PCR results as did one of the 9 B. parapertussis cases and 82 additional samples were positive by PCR. PCR increased the identification of subjects with B. pertussis infections by almost 4-fold. Clinical characteristics were analyzed by laboratory category (Group 1, 28 culture-positive; Group 2, 82 culture-negative, PCR-positive; and Group 3, 436 culture- and PCR-negative). Group 1 subjects were more likely to have a diagnosis of definite or probable pertussis and to have paroxysmal cough, posttussive vomiting, whooping and a cough duration of > or = 4 weeks than Group 2 or 3 subjects. In contrast Group 2 subjects were more likely than Group 1 subjects to have had previous pertussis immunization or prior antibiotics. PCR identified many mild illnesses caused by B. pertussis that were not identified by culture.
While culture for Bordetella species is highly specific, sensitivity is extremely variable due to patient age, immunization status, antibiotic treatment, and specimen transport conditions. We evaluated a real-time multiplex PCR assay as an alternative to culture for the detection and differentiation of Bordetella pertussis and Bordetella parapertussis. The PCR conditions allowed the simultaneous detection of one B. pertussis organism and five B. parapertussis organisms per reaction. An inhibition control was incorporated into the assay. Of 163 total samples evaluated, 37 of 38 samples positive by either culture or direct fluorescent antibody testing (DFA) were also positive by PCR (97% sensitivity). Of 125 culture- or DFA-negative samples, 101 were also negative by PCR (81% specificity). The described multiplex assay is a rapid, sensitive, contamination-limiting, real-time PCR assay that controls for inhibition. The assay performs well using liquid or swab samples and from dried material on slides.
Bordetella parapertussis is one of the bacteria that causes whooping cough. However, little attention has been paid to this bacterium because it causes a milder illness than Bordetella pertussis and the rate of detection is low, even though research suggests that pertussis vaccines have limited efficacy against B. parapertussis infection. However, recent studies have revealed high rates of detection in patients with whooping cough in some field studies. In this review, the relevant studies of B. parapertussis are summarized and it is demonstrated that it is now necessary to pay greater attention to infections by this bacterium.
Bordetella respiratory infections are common in people (B. pertussis) and in animals (B. bronchiseptica). During the last two decades, much has been learned about the virulence determinants, pathogenesis, and immunity of Bordetella. Clinically, the full spectrum of disease due to B. pertussis infection is now understood, and infections in adolescents and adults are recognized as the reservoir for cyclic outbreaks of disease. DTaP vaccines, which are less reactogenic than DTP vaccines, are now in general use in many developed countries, and it is expected that the expansion of their use to adolescents and adults will have a significant impact on reducing pertussis and perhaps decrease the circulation of B. pertussis. Future studies should seek to determine the cause of the unique cough which is associated with Bordetella respiratory infections. It is also hoped that data gathered from molecular Bordetella research will lead to a new generation of DTaP vaccines which provide greater efficacy than is provided by today's vaccines.
Bordetella holmesii is a recently identified gram-negative bacterial species associated with bacteremia, endocarditis, and respiratory illness,
mainly in immunocompromised patients. From isolates submitted to the Centers for Disease Control and Prevention from 1983
through 2000 for further identification, we identified 30 patients with B. holmesii bacteremia. Of the 26 patients for whom data were available, 22 (85%) were anatomically or functionally asplenic. In 25 (96%)
of the 26 patients, B. holmesii was the only organism isolated from blood samples, and 14 patients (54%) had B. holmesii recovered from ⩾2 blood cultures. The clinical course of the infection was generally characterized by a nonspecific febrile
illness. Twenty-one patients (81%) were treated with various antimicrobial agents, and 20 (77%) were admitted to the hospital.
There were no deaths. Our findings support evidence that B. holmesii may be a true pathogen associated with bacteremia among asplenic patients.
Pertussis epidemic-Washington
Centers for Disease Control and Prevention, 2012. Pertussis epidemic-Washington.
MMWR 61, 517-522.