A field-expedient method for direct detection of enterotoxigenic E coli and Shigella from stool.
Efficacious treatment and prevention and control of transmission present significant challenges to clinicians and public health practitioners worldwide. (4) Accurate and rapid diagnosis and epidemiological surveillance focused utilization of public health resources in areas and populations most at risk of developing diarrheal disease can result in significant reductions in morbidity and mortality and economic cost. However, current limitations in diarrheal disease diagnostics drive the need for rapid and sensitive tests. Identification of diarrheagenic Escherichia coli disease, shigellosis, and EIEC causative agent by culture can take days to weeks. Diagnosis, therefore, is often retrospective clinically as well as epidemiologically. Time critical treatment and public health response are unachievable. The time required for routine culture as well as relatively poor sensitivity have driven the development of molecular-based identification technologies that are rapid and highly sensitive and specific. Molecular-based technologies are becoming well established in diarrheal disease diagnostics, surveillance, and food and water safety protocols. This includes restriction enzyme analyses, conventional and RT-PCR, multiplex PCR, loop-mediated isothermal amplification, microarray technologies, and the Taq-Man Array Card. (5-18) Immunochromatographic "dipstick" technologies show promise in diarrheal disease agent detection. (19) However, traditional culture remains the reference methodology.
Disproportionately high diarrheal disease morbidity and mortality occur in developing countries and can increase exponentially during times of natural disaster or political conflict. (4) The risk of diarrhea outbreak is often heightened in these situations due to lack of clean water, poor hygiene, malnutrition, inadequate medical intervention, and limited or absent prevention and control intervention. Medical care providers in regions with underdeveloped medical and public health resources or in situations with failing or totally absent infrastructure operating under austere conditions often have no access to laboratory facilities or fundamental public services such as electricity, water, or an intact transportation system. Under these conditions, culture and microscopy methodologies and molecular-based technologies designed for use within conventional laboratory infrastructure are unsuitable. These obstacles drive the need for mobile, stand-alone analytic capability. (20,21)
In this article we describe a unique analytic capability; highly sensitive and specific thermal-stable hydrolytic enzyme resistant ETEC-STIa, STIb, LT, and Shigella/enteroinvasive E coli (EIEC) TaqMan PCR detection assays with portable, ruggedized, real-time qRT-PCR instrumentation that shows promise as a field-expedient method for direct detection from stool.
Field-evaluation of ETEC-STIa, STIb, LT, and Shigella/EIEC (Shig-ipaH) TaqMan qRT-PCR detection assays was conducted at the Walter Reed Research Unit Nepal (WARUN) central laboratory in Kathmandu. Kathmandu is located in south-central Nepal within the Hill Region (called Pahar in Nepali) where elevations are mostly between 1,000 m and 4,000 m. Pahar is the most heavily populated region of Nepal, however, development of infrastructure and social services have been hindered due to its geographical isolation, limited economic potential, and a history of political instability. Testing was conducted during March 31 to 2 April, 2009. March through April is considered the spring season with daily temperatures ranging from 10[degrees]C to 27[degrees]C and 80% to 100% humidity with occasional short bursts of rain. The ETEC and Shig-ipaH qRT-PCR assays, thermocycler ("Ruggedized" Advanced Pathogen Identification Device (RAPID), BioFire Diagnostics, Inc (BDI), Salt Lake City, UT), and equipment and supplies were packed in 2 hardened cases (Pelican Products, Inc, Torrance, CA). A single individual transported the system by commercial aircraft as checked-baggage from Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand, to WARUN. The field-laboratory was established in a single room of a building without environmental control using 2 tabletops approximately 1 [m.sup.2] in area. The qRT-PCR assays, nucleic acid preparation reagents, and RAPID instrument were transported and stored and sample preparation and analyses conducted under ambient temperature and humidity conditions, vastly simplifying reagent management. The RAPID is powered by 110-220 V source such as an electric generator or, if necessary, from the battery of a vehicle with the engine running using a power inverter. For this study, the field laboratory was equipped with main 220 V power.
Samples and Microbiology
For the purpose of this study, a test panel of 138 well characterized diarrheagenic E coli disease and shigellosis stored stool samples was prepared. Enterotoxigenic Escherichia coli and Shigella infecting agents were previously identified by using a combination of culture with hybridization of DIG-labeled probe (ETEC) and Shigella serotyping. The test panel consisted of STIa (n=21), STIb (n=30), LT (n=40), and Shig-ipaH (n=47) positive samples. Sampling protocol, stool processing, and ETEC and Shigella identification methodologies have been previously described. (22) Under a previous study, clearance for the collection and use of stool samples was obtained from the Ethics Review Board at the Nepal Health Research Council (NHRC) and the Institutional Review Board (IRB), Walter Reed Army Institute of Research (WRAIR), Silver Spring, Maryland (WRAIR IRB #1276 and #1311). Clearance for the use of de-identified samples under this study was obtained from the NHRC and IRB, Privacy Board, Wilford Hall Medical Center, Lackland AFB, San Antonio, Texas.
Stool samples were labeled by code and double-blinded testing was conducted. Routine culture, DIG-labeled probe (ETEC), and serotyping (Shigella), were compared to qRT-PCR and sequences of amplicon products. Prior to field-evaluation, quantification of qRT-PCR assay linearity, limit of detection (LOD), and in vitro sensitivity and specificity were accomplished with well characterized reference strains from culture using the RAPID instrument. Type strains were subcultured on agar plates and several colonies were picked and suspended in normal saline. The measured absorbance at 625 nm was compared to 0.5 McFarland (OD [approximately equal to] 0.088-0.133) spectrophotometrically. Stocks of undiluted cell suspension (viable cell count 1.5 x [10.sup.8] cells/mL) were established; extracted nucleic acid was prepared and serially diluted at 1.5 x [10.sup.8] cells/mL to 1.5 x [10.sup.0] cells/mL. Linearity and LOD of ETEC STIa, STIb, LT, and Shig-ipaH qRT-PCR assays were quantified using ETEC strains ETEC-STIa AF-ETEC727, ETEC-STIb AF-ETEC771, ETEC-LT AF-ETEC966 and Shigella flexneri strain ATCC12022, respectively.
Nucleic Acid Preparation
Extracts were prepared using a commercially available kit; a 10% (wt/vol) stool suspension was prepared with sterile distilled water and clarified by centrifugation at 2,600 x g for 15 minutes. Nucleic acid was extracted with NucliSens Magnetic Extraction Kit (bioMerieux, Inc., Durham, NC) according to the manufacturer's instructions. Briefly, 300 [micro]L of the stool suspension was treated with 1.4 mL lysis-buffer and mixed with paramagnetic silica particles. The bound nucleic acid was washed twice with 400 [micro]L wash buffer I (5 M guanidine thiocyanate, Tris-HCl, Triton X 100, and EDTA), twice with 500 [micro]L wash buffer II (MES), and once with 500 [micro]L wash buffer III (disodium tetraborate). Finally, the nucleic acid was eluted from the silica by incubation in 75 [micro]L elution buffer for 5 minutes at 60[degrees]C. The field site used in this study was equipped with a -70[degrees]C freezer within which purified nucleic acid was preserved until used for qRT-PCR testing.
Polymerase Chain Reaction
Existing wet reagent ETEC-STIa, ETEC-STIb, ETEC-LT and Shig-ipaH TaqMan assays were adapted for use in the preparation of thermal-stabilized, hydrolytic enzyme resistant RApID-based qRT-PCR freeze-dried assays. (5-7,23,24) Separate Shigella and EIEC qRT-PCR assays were not developed because the treatment of shigellosis is the same for infection by either of these agents. Moreover, Shigella and EIEC bacteria differ in endemicity and relative prevalence and as such presumptive identification is often made prior to confirmation testing.
Optimal wet reaction conditions for STIa, STIb, LT, and Shig-ipaH qRT-PCR assays were: 400 nM Forward Primer, 400 nM Reverse Primer, and 150 nM TaqMan Probe, 1:10 dilution of 10X qRT-PCR buffer with BSA and 30 mM Mg[Cl.sub.2] (BDI part number 1770), 1:10 dilution of 10X 2 mM dNTP mixture (BDI part number 1774), and 2 [micro]L of a mixture of 10X Taq polymerase (0.16 [micro]L), antibody (0.16 [micro]L), and enzyme diluent (1.68 [micro]L) (BDI). Reaction volume was 20 [micro]L consisting of 18 [micro]L of master mix and 2 [micro]L of template. A standardized qRT-PCR thermocycling protocol consists of an initial DNA denaturation at 95[degrees]C for 3 minutes, and qRT-PCR for 45 cycles at 95[degrees]C for zero seconds (sinusoidal temperature curve) for template denaturation and 60[degrees]C for 20 seconds of combined annealing and primer extension. Primer and probe sequences were: ETEC STIa Forward Primer (5'-ACCTCG CATATAACATGATGCAA-3'), Reverse Primer (5'-CTAATGTAATTTTCTCTTTTGAAGAGTCA-3'), and Probe (5'-FAM-TTAGCTTTTTCATGTTACCTCCCGTCATGT-TAMRA-3') designed using BLAST database nucleic acid sequence of the E coli toxin I (estAl) gene at GenBank accession number M58746; ETEC STIb Forward Primer (5'-TTCACCTTTC(G/C)CTCAGGATGC-3'), Reverse Primer (5'-ATAGCACCCGGTACAAGCAGG-3'), and Probe (5'-FAM-TCACAGCAGTAATTGCTACTATTCATGCTTTCAGGA-TAMRA-3') target sequence was E coli heat-stable toxin (st) gene at GenBank accession number M29255; ETEC LT Forward Primer (5'-GTTTTATT TACGGCGTTACTATCCT-3'), Reverse Primer (5'-GGGACTTCGACCTGAAATGTT-3'), and Probe (5'-FAM-CTTTTGCCTGCCATCGATTCCGTATAT -TAmRa-3') target sequence was E coli heat-labile enterotoxin B subunit at accession number S60731; Shig-ipaH Forward Primer (5'-CCTTTTCCGCGTTCCTTGA-3'), Reverse Primer (5'-CGGAATCCGGAGGTATTGC-3'), and Probe (5'-FAM-CGCCTTTCCGATACCGTCTCTGCA-TAMRA-3'), target sequence was invasion plasmid antigen H (ipaH) gene at accession number M32063. (24,25) Each of the ETEC-STIa, STIb, LT and Shig-ipaH qRT-PCR assay formulations were optimized by using standardized RAPID master mix reagents (BDI). (26,27)
Thermal-stable qRT-PCR Assays and Validation Testing
Each of the ETEC-STIa, STIb, LT and Shig-ipaH qRT-PCR assays were freeze-dried in a thermal-stable, hydrolytic enzyme resistant formulation by a proprietary process and packaged in a preformatted kit by BDI. The freeze-dried qRT-PCR master mix reagents only required hydration and addition of sample template prior to analysis. Assays were prepared according to manufacturer (BDI) instructions. A standardized qRT-PCR thermal cycling protocol was used (described above). Linear regression analysis was conducted using triplicate dilution series samples spanning 6 orders of magnitude (1.5 x [10.sup.8] cells/mL to 1.5 x [10.sup.3] cells/mL). Correlation coefficient ([R.sup.2]) values were established at unity "best fit" and slope and error calculated by an algorithm provided in the RAPID analytical software (Roche Molecular Biochemicals, Indianapolis, IN). Based on linear regression analysis results, the LOD of each assay was estimated and subjected to replicate testing (n=60) by 2 individuals over 3 days. Validation testing of ETEC-STIa, STIb, LT, and Shig-ipaH qRT-PCR assay in vitro sensitivity and specificity were conducted using panels consisting of well characterized nucleic acid extracts. Stringent cross-reactivity testing of each qRT-PCR assay was conducted using each genetic near neighbor as well as other common diarrheal pathogens. To determine the ability of the assay to detect multiple strains of the same organism (in vitro sensitivity), extract of 1-fold LOD and 10-fold LOD viable cell concentrations were prepared and testing was conducted in triplicate. To determine whether the assay cross-reacts with other closely- and distantly-related organisms (specificity), extract of at least 1000-fold LOD viable cell concentrations were prepared and testing was conducted in triplicate.
Data Acquisition and Analyses
Sample identification and specifications were entered electronically in the RAPID operating system run protocol. Analyses and results were automatically archived. The criterion for a positive result was a significant increase in fluorescence over background levels, ie, critical threshold (Ct), defined by an algorithm provided in the RAPID analytical software (Roche Molecular Biochemicals, Indianapolis, IN, USA). The Ct is defined as the first PCR cycle with significant fluorescence when normalized against background fluorescence. (26,27) The Ct cutoff value was 40 or less.
Nucleic Acid Sequencing
Diarrheal agent identification by methods using qRT-PCR and a combination of culture with DIG-labeled probe (ETEC) and Shigella serotyping were compared to qRT-PCR amplicon product sequencing. The sequencing reaction was one direction. The acceptance standard for identification was at least 90% genetic homology using generated sequence of 150 or more base pairs of an acceptable quality. Several samples tested by qRT-PCR reported fluorescence below the Ct cut-off value, however, these samples produced an insufficient concentration of amplicon for use in sequencing. The associated qRT-PCR results were excluded from sequencing comparison testing. Excluded samples were 2 ETEC STIb (n=30-2 excluded=28) and one Shigella-ipaH sample (n=47-1 excluded=46). Additionally, Shigella positive samples by culture were excluded from sequencing comparison with Shigella-ipaH qRT-PCR (n=46-31 positive culture=15).
Linearity and Limit of Detection of qRT-PCR Assays
Linear regression analyses of all 3 ETEC qRT-PCR assays resulted in an estimated LOD at 30 CFU per 20 [micro]L of reaction volume and 3 CFU per 20 [micro]L reaction volume for Shig-ipaH qRT-PCR assay (Table 1). Replicate testing by 2 different operators, over 3 days, running 20 samples per day at the estimated LOD achieved a replicate test score of 100% (60/60) for all qRT-PCR assays. Results for LOD replicate test results are reported in Table 2.
Positive and Negative Template Control Reactions
Positive template control (PTC) reactions were prepared for each of the 4 assays at 10-fold LOD concentrations. The template was prepared from ETEC and Shigella strains ETEC-STIa AF-ETEC727, ETEC-STIb AF-ETEC771, ETEC-LT AF-ETEC083, and Shigella sonnei ATCC25931, respectively. The PTC reactions consistently reported fluorescence at the expected Ct value ([approximately equal to] 30) under laboratory and field conditions. Negative template control (NTC) reactions consistently reported fluorescence at or below background levels.
Sensitivity and Specificity Testing Using Reference Strains
In laboratory testing, ETEC STIa, STIb, LT and Shig-ipaH qRT-PCR assay sensitivity and specificity test results were concordant with reference strains (Table 3). In sensitivity testing, 3 reference strains representing each pathogen were tested in triplicate. Extracts were tested at LOD and 10-fold LOD concentrations. In specificity testing, 2 reference strains representing each pathogen were tested in triplicate (Table 4). Extracts were tested at 1000-fold LOD concentrations. Cross-reactivity and inhibition of qRT-PCR were not observed. Throughout laboratory-based testing, PTC reactions reported fluorescence at the expected Ct value ([approximately equal to] 30) and NTC reactions did not report fluorescence above background.
Field-evaluation Using Stool Samples
In field-testing, ETEC STIa (n=21), STIb (n=30), LT (n=40) and Shig-ipaH (n=47) qRT-PCR assay sensitivity and specificity test results showed promise when compared to DIG-labeled probe (ETEC) and serotyping (Shigella) results (Table 5). Subsequent comparison of sequences of qRT-PCR amplicon indicated that all 4 assays were 100% sensitive and 100% specific (Table 6). Amplicon product of each of the 4 assays was confirmed as sequences of target pathogen at 90% or greater homology. Sequencing and qRT-PCR concordances were: ETEC-LT 100% (40/40), ETEC-STIa 100% (21/21), ETEC-STIb 100% (28/28), and Shig-ipaH 100% (15/15). Sample preparation, nucleic acid extraction, and analyses were conducted without provision for spatial separation.
Enterotoxigenic Escherichia coli ETEC-STIa, STIb, LT and Shig-ipaH qRT-PCR assay linearity and LOD test results were robust and reproducible. The established LOD of each qRT-PCR assay was validated in stringent replicate sample testing. Using a diverse panel of reference strains representing genotypically similar and clinically significant organisms, all 4 assays proved to be highly sensitive and specific. Under field-deployed conditions, ETEC STIa, STIb, and LT and Shig-ipaH qRT-PCR assays proved to be highly sensitive and specific tests for direct detection of diarrheagenic E coli and shigellosis disease agents from stool samples. Sensitivity test results indicated that the qRT-PCR assays performed at clinically significant detection limits evidenced by DNA sequencing. No false negative or false positive results were observed. Compared to qRT-PCR amplicon sequencing, culture methodology was specific but sensitivity levels and associated negative predictive values were not as robust as qRT-PCR. Furthermore, comparison between DIG-probe (ETEC) and Shigella serotyping to qRT-PCR amplicon sequencing clearly showed that the qRT-PCR assays were more sensitive. Reproducible PTC fluorescence indicated that the assays remained stable at ambient temperatures. Cross-over contamination monitored by NTC consistently reported no fluorescence above background.
In the absence of vaccines against ETEC, Shigella, and EIEC bacteria and current limitations in diagnostics, the risk of an outbreak situation is increased, especially where medical and public health resources are overburdened or absent. In this study, our analytic system was rapidly deployed to an underdeveloped region and integrated into a community health program. In support of ongoing disease surveillance, 138 stool samples were processed and sensitive and specific identification of diarrheagenic E coli disease and bacillary dysentery agents was accomplished in 3 days. Processing and analyses of a batch of 30 samples was completed in less than 3 hours. This has important clinical implications in decision-making of the necessity and appropriate selection of antibiotic therapy and time critical treatment. (22) Diarrheagenic E coli and bacillary dysentery symptoms are easily confused with those of other diarrheal diseases as well as other common infectious diseases and treatment is most effective when started (24) to (48) hours after the onset of diarrhea. (28-30) An accurate diagnosis is needed quickly for efficacious treatment. In contrast to our qRT-PCR system, culture required one week of labor-intensive effort requiring advanced skills, and results showed lower sensitivity and positive predictive values compared to qRT-PCR.
Current limitations in diagnostics drive the need for effective disease prevention and control. The results reported here and in previous studies indicate that diarrheal disease prevalence may be underestimated in epidemiological surveys conducting using culture methodology versus more sensitive technologies. (7,10,13,19) Rapid and accurate identification of causative agents are essential to timely and focused implementation of priority intervention measures directed at preventable conditions. This is especially important in socioeconomic developing regions during times of disaster when local public health resources are often overwhelmed. Human and environmental surveillance data collected in a spatially focused and expedient manner augment the predictive power of transmission risk. Correctly collected and interpreted data on human infection and contaminated food, water, and environment integrated with other key transmission indicators (severity of cases, virulence of the circulating agent, identification of vulnerabilities in critical contamination control points, and climatic and geographical factors) provide for accurate transmission risk assessment. These data assist decision-makers in the appropriate use of antibiotics to mitigate transmission and epidemiological approach, and the dedication of resources for focused application of control measures such as potable water sources, hand washing stations, and monitoring food preparation and hygienic practices. Agile, field-deployable diarrheal agent surveillance capability can provide valuable assistance to local public health practitioners by serving to alert and direct focus of preventive and control measures.
We have described a field-expedient method for direct detection of enterotoxigenic E coli and Shigella from stool unique in its capability to fill a critical role in public health and potentially provide a valuable diagnostic aid.
Sasikorn Silapong, BS
Pimmnapar Neesanant, PhD
Orntipa Sethabutr, MS
Paphavee Lertsethtakarn, PhD
Ladaporn Bodhidatta, MD
James C. McAvin, MS
Carl J. Mason, MD
We thank the bacteriology section and molecular sciences sections in the Department of Enteric Diseases at AFRIMS for serotyping of Shigella and ETEC DIG-hybridization, respectively. We thank the Ministry of Public Health, Nepal, for providing valuable support throughout this study.
This work was funded in part by the Diseases of the Most Impoverished Program, funded by the Bill and Melinda Gates Foundation, the Armed Forces Health Surveillance Center-Global Emerging Infections Surveillance Response System, and the Office of the Air Force Surgeon General, Directorate of Modernization, Falls Church, VA.
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Ms Silapong, Dr Neesanant, Ms Sethabutr, Dr Lertsethtakarn, Dr Bodhitta, and Dr Mason are with the Department of Enteric Diseases, Armed Forces Research Institute of Medical Sciences (AFRIMS), Bangkok, Thailand.
During the conduct of this work, Mr McAvin served as a Molecular Biologist with the 59th Medical Wing, Lackland AFB, Texas. He is now with the Department of Enteric Diseases, AFRIMS, Bangkok, Thailand.
Table 1. Linearity of qRT-PCR assay. Shigella STIa STIb LT flexneri (AF-ETEC727) (AF-ETEC771) (AF-ETEC966) (ATCC12022) Correlation 1.00 1.00 1.00 1.00 coefficient Slope 3.512 3.357 3.438 3.306 Error 0.0428 0.0793 0.0283 0.0787 Table 2. Limit of detection replicate testing of qRT-PCR assays expressed as critical threshold (Ct). ETEC-STIa Ct ETEC-STIb Ct (Mean/STDV) (Mean/STDV) Replicate 1 34.07/0.49 33.66/0.77 Replicate 2 34.63/0.37 34.88/0.29 Replicate 3 35.94/0.35 34.07/0.69 Average 34.88/0.40 34.20/0.58 LT Ct S. flexneri Ct (Mean/STDV) (Mean/STDV) Replicate 1 34.94/0.59 36.03/0.65 Replicate 2 34.56/0.33 36.33/0.65 Replicate 3 34.93/0.43 36.32/0.48 Average 34.81/0.45 36.23/0.59 N=20 for all qRT-PCR assays. Table 3. Results of ETEC and Shigella/EIEC-ipaH qRT-PCR sensitivity testing using reference strains from culture. Strain Pathogen(s) PCR Assay AF-ETEC929 ETEC STIa ETEC-STIa AF-ETEC727 ETEC STIa ETEC-STIa AF-ETEC721 ETEC STIa ETEC-STIa and LT AF-ETEC877 ETEC STIb ETEC-STIb AF-ETEC771 ETEC STIb ETEC-STIb AF-ETEC816 ETEC STIb ETEC-STIb and LT AF-ETEC966 ETEC-LT ETEC-LT AF-ETEC083 ETEC-LT ETEC-LT AF-ETEC816 ETEC-LT ETEC-LT and STIb ATCC12022 Shigella Shigella/EIEC-ipaH ATCC25931 Shigella Shigella/EIEC-ipaH 2457T Shigella Shigella/EIEC-ipaH Ct (1 XLOD) (a) Ct (1XLOD) (a) Strain (Mean/STDV) (b) (Mean/STDV) (b) AF-ETEC929 37.36/0.26 33.91/0.09 AF-ETEC727 35.81/0.07 32.09/0.16 AF-ETEC721 36.43/0.50 32.57/0.47 AF-ETEC877 34.85/0.10 31.09/0.54 AF-ETEC771 34.66/0.66 30.61/0.54 AF-ETEC816 34.09/0.28 30.79/0.09 AF-ETEC966 34.18/0.46 31.05/0.09 AF-ETEC083 33.81/0.50 30.34/0.11 AF-ETEC816 34.23/0.25 31.00/0.08 ATCC12022 36.32/0.61 33.01/0.32 ATCC25931 34.01/0.12 30.59/0.10 2457T 35.57/0.29 32.67/0.21 (a) ETEC-STIa, STIb, and LT assay LOD=1.5 x [10.sup.4] cell/mL; Shigella/EIEC-ipaH assay LOD=1.5 x 103 cell/mL (b) Samples were run in triplicate. Table 4. Results of ETEC and Shigella/EIEC-ipaH qRT-PCR specificity testing using reference strains from culture. ETEC-STIa ETEC-STIb qRT-PCR qRT-PCR Strain Pathogen(s) (1000XLOD) * (1000XLOD) * AF-ETEC929 ETEC STIa Positive Negative AF-ETEC727 ETEC STIa Positive Negative AF-ETEC877 ETEC STIb Negative Positive AF-ETEC771 ETEC STIb Negative Positive AF-ETEC966 ETEC-LT Negative Negative AF-ETEC083 ETEC-LT Negative Negative ATCC25931 Shigella sonnei Negative Negative ATCC25922 Escherichia coli Negative Negative ATCC70819 Campy/obacter Negative Negative jejuni AF-SAL0085 Salmonella gr. E4 Negative Negative AF-SAL445 Salmonella Negative Negative paratyphi A ETEC-LT Shigella-ipaH qRT-PCR qRT-PCR Strain Pathogen(s) (1000XLOD) * (1000XLOD) * AF-ETEC929 ETEC STIa Negative Negative AF-ETEC727 ETEC STIa Negative Negative AF-ETEC877 ETEC STIb Negative Negative AF-ETEC771 ETEC STIb Negative Negative AF-ETEC966 ETEC-LT Positive Negative AF-ETEC083 ETEC-LT Positive Negative ATCC25931 Shigella sonnei Negative Positive ATCC25922 Escherichia coli Negative Negative ATCC70819 Campy/obacter Negative Negative jejuni AF-SAL0085 Salmonella gr. E4 Negative Negative AF-SAL445 Salmonella Negative Negative paratyphi A * ETEC-STIa, STIb, and LT assay LOD = 1.5 x [10.sup.4] cell/mL; Shigella/EIEC-ipaH assay LOD = 1.5 x [10.sup.3] cell/mL. Table 5. Sensitivity and specificity of ETEC and Shigella assays (138 samples) comparing real time qRT-PCR results to hybridization of DIG-labeled probe (ETEC) and serotyping (Shigella) results. qRT-PCR Sensitivity Specificity % % ETEC STIa 100 92.4 ETEC STIb 100 92.6 ETEC LT 100 79.6 Shig-ipaH 100 81.6 Table 6. Analyses of real-time qRT-PCR and standard methods using DNA sequencing as the comparator test. Positive Specimens by Sequence Test Method/ Sensitivity Homology Test Method Sequencing (%) (%) ETEC STIa real-time 21/21 100 98-100 qRT-PCR ETEC STIa 13/21 72.4 hybridization DIG-labeled probe ETEC STIb real- time qRT-PCR 28/28 (a) 100 91-100 ETEC STIb hybridization 23/28 84.8 DIG-labeled probe ETEC LT real- 40/40 100 98-100 time qRT-PCR ETEC LT hybridization 20/40 66.7 DIG-labeled probe Shig-ipaH real- 15/15 (a,b) 100 99-100 time qRT-PCR Shig-ipaH 8/15 (a,b) 68.2 serotyping (a) Positive samples from qRT-PCR with insufficient amplicon concentration for DNA sequencing were excluded from comparison testing (ETEC STIb (n=30-2 excluded=28) and Shigella-ipaH (n = 47-1 excluded = 46). (b) Positive samples from qRT-PCR but negative by culture were selected for sequencing (Shig-ipaH; n = 46-31 positive culture=15).
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|Author:||Silapong, Sasikorn; Neesanant, Pimmnapar; Sethabutr, Orntipa; Lertsethtakarn, Paphavee; Bodhidatta,|
|Publication:||U.S. Army Medical Department Journal|
|Date:||Oct 1, 2015|
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