Printer Friendly

A field-expedient method for direct detection of enterotoxigenic E coli and Shigella from stool.

Diarrhea is a leading cause of disease related morbidity and mortality worldwide, especially for children younger than 5 years. (1) Enterotoxigenic Escherichia coli (ETEC) and Shigella are primary infectious agents of bacterial diarrheal disease. Depending on the ETEC strain, 2 classes of heat-stable (STIa and STIb) and/or heat-labile (LT) plasmid-encoded toxins are produced and manifested by acute diarrhea. (2) The shigellosis or bacillary dysentery form of diarrheal disease is caused by various chromosomal and plasmid produced virulence factors including invasion plasmid antigen H (Shig-ipaH) encoded by a gene conserved across Shigella and enteroinvasive E coli (EIEC) species. (3) In the absence of prompt and appropriate treatment, these pathogens have the ability to cause fatal diarrhea.

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.

METHODS

Study Site

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).

RESULTS

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.

COMMENT

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

ACKNOWLEDGEMENTS

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.

REFERENCES

(1.) Black RE, Cousens S, Johnson HL, et al. Global regional and national causes of child mortality in 2008; a systematic analysis. Lancet. 2010;375(9730):1969-1987.

(2.) Johnson TJ, Nolan LK. Pathogenomics of the virulence plasmids of Escherichia coli. Microbiol Mol Biol Rev. 2009;73:750-774.

(3.) Schroeder GN, Hilbi H. Molecular pathogenesis of Shigella spp: controlling host cell signaling invasion and death by type III secretion. Clin Microbiol Rev. 2008;21(1):134-156.

(4.) Pawlowski SW, Warren CA, Guerrant R. Diagnosis and treatment of acute or persistent diarrhea. Gastroenterology. 2009;136(6):1874-1886.

(5.) Houng HS, Sethabutr O, Echeverria P. A simple polymerase chain reaction technique to detect and differentiate Shigella and enteroinvasive Escherichia coli in human feces. Diagn Microbiol Infect Dis. 1997;28:19-25.

(6.) Sethabutr O, Silapong S, Houng HS, Mason CJ. Evaluation of ipaH-based TaqMan PCR assy for rapid detection and characterization of Shigella and enteroinvasive E coli [abstract]. Presented at :103rd General Meeting of the American Society for Microbiology; May 18-22, 2003; Washington DC. Abstract C-149.

(7.) Vu DT, Sethabutr O, von Seidlein L, et al. Detection of Shigella by a PCR assay targeting the ipaH gene suggests increased prevalence of shigellosis in Nha Trang Vietnam. J Clin Microbiol. 2004;42:2031-2035.

(8.) Song TC, Toma N, Nakasone N, Iwanaga M. Sensitive and rapid detection of Shigella and enteroinvasive Escherichia coli by a loop-mediated isothermal amplification method. FEMS Microbiol Lett. 2005;243(1):259-263.

(9.) Li Y, Liu D, Cao B, et al. Development of a serotype-specific DNA microarray for identification of some Shigella and pathogenic Escherichia coli strains J Clin Microbiol. 2006;44:4376-4383.

(10.) Foley SL, Lynne AM, Nayak RR. Molecular typing methodologies for microbial source tracking and epidemiological investigations of Gram-negative bacterial foodborne pathogens. Infect Genet Evol. 2009;9:430-440.

(11.) O'Leary J, Corcoran D, Lucey B. Comparison of the EntericBio multiplex PCR system with routine culture for detection of bacterial enteric pathogens J Clin Microbiol. 2009;47:3449-3453.

(12.) Wang Q, Wang S, Beutin L, Cao B, Feng L, Wang L. Development of a DNA microaray for detection and serotyping of enterotoxigenic Escherichia coli. J Clin Microbiol. 2010;48:2066-2074.

(13.) Wang SM, Ma JC, Hao ZY, et al. Surveillance of shigellosis by real-time PCR suggests underestimation of shigellosis prevalence by culture-based methods in a population of rural China. J Infect. 2010;61(6):471-475.

(14.) Valadez AM, DebRoy C, Dudley EW, Cutter CN. Multiplex PCR detection of Shiga toxin-producing Escherichia coli strains belonging to serogroups O157 O103 O91 O113 O145 O111 and O26 experimentally inoculated in beef carcass swabs beef trim and ground beef. J Food Prot. 2011;74(2):228-239.

(15.) Velasco JM, Yoon IK, Mason CJ, et al. Applications of PCR (real-time and MassTag) and enzyme-linked immunosorbent assay in diagnosis of respiratory infections and diarrheal illness among deployed US military personnel during exercise Balikatan 2009. Philippines Mil Med. 2011;176(10):1096-1100.

(16.) Sahl JW, Rasko DA. Analysis of global transcriptional profiles of enterotoxigenic Escherichia coli isolate E24377A. Infect Immun. 2012;80(3):1232-1242.

(17.) Liu J, Gratz J, Amour C, et al. A laboratory-developed TaqMan array card for simultaneous detection of 19 enteropathogens. J Clin Microbiol. 2013;51(2):472-480.

(18.) Navidad J, Griswold D, Gradus M, Bhattacharyya S. Evaluation of Luminex xTAG gastrointestinal pathogen analyte-specific reagents for high-throughput simultaneous detection of bacteria viruses and parasites of clinical and public health importance J Clin Microbiol. 2013;51(9):18-24.

(19.) Nato F, Phalipon A, Nguyen LPT, Diep TT, Sansonetti P, Germani Y. Dipstick for rapid diagnosis of Shigella flexneri2a in stool. PLoS One. 2007;2(4):e361.

(20.) Swaby JA, McAvin JC. Support of far forward disease surveillance operations with deployable real-time vector-borne disease agent analytic capability. US Army Med Dep J. April-June 2008:21-24.

(21.) Coleman RE, Hochberg LP, Putnam JL, et al. Use of vector diagnostics during military deployments: recent experience in Iraq and Afghanistan. Mil Med. 2009;174(9):904-920.

(22.) Bodhidatta L, McDaniel P, Sornsakrin S, Srijan A, Serichantlergs O, Mason CJ. Case-contral study of diarrheal disease etiology in a remote rural area in western Thailand. Am J Trop Med Hyg. 2010;83(5):1106-1109.

(23.) Hartman AB, Venkatesan M, Oaks EV, Buysse JM. Sequence and molecular characterization of a multicopy invasion plasmid antigen gene ipaH of Shigella flexneri. J Bacteriol. 1990;172:1905-1915.

(24.) Paniagua M, Espinoza F, Ringman M, Reizenstein E, Svennerholm AM, Hallander H. Analysis of incidence of infection with enterotoxigenic Escherichia coli in a prospective cohort study of infant diarrhea in Nicaragua. J Clin Microbiol. 1997;35(6):1404-1410.

(25.) Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215(3):403-410

(26.) Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP. Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques. 1997;22(1):130-131, 134-138.

(27.) Wittwer CT, Ririe KM, Andrew RV, David DA, Gundry RA, Balis UJ. The LightCycler: a microvolume multismaple fluorimeter with rapid temperature control. Biotechniques. 1997:22(1):176-181.

(28.) Wilson W, Sande M. Current Diagnosis & Treatment in Infectious Diseases. New York: McGraw-Hill; 2001.

(29.) Abubakar I, Irvine C, Aldus F, et al. A systematic review of the clinical public health and cost-effectiveness of rapid diagnostic tests for the detection and identification of bacterial intestinal pathogens in faeces and food. Health Technol Assess. 2007;11(36):1-216.

(30.) Cunningham SA, Sloan LM, Nyre M, Vetter EA, Mandrekar J, Patel R. Three-hour molecular detection of Campylobacter Salmonella Yersinia and Shigella species in feces with accuracy as high as that of culture. J Clin Microbiol. 2010;48(8):2929-2933.

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).
COPYRIGHT 2015 U.S. Army Medical Department Center & School
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2015 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Silapong, Sasikorn; Neesanant, Pimmnapar; Sethabutr, Orntipa; Lertsethtakarn, Paphavee; Bodhidatta,
Publication:U.S. Army Medical Department Journal
Article Type:Report
Geographic Code:7ISRA
Date:Oct 1, 2015
Words:5037
Previous Article:Paralysis as a presenting symptom of hyperthyroidism in an active duty soldier.
Next Article:Data analytics under deployed conditions a case study.
Topics:

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters