Simultaneous Detection Of Multiple Pathogens Research Articles

A Wash-Free Spheres-on-Sphere Strategy for On-Site and Multiplexed Biosensing.

Respiratory infections and food contaminants pose severe challenges to global health and the economy. A rapid on-site platform for the simultaneous detection of multiple pathogens is crucial for accurate diagnosis, appropriate treatment, and a reduced healthcare burden. Herein, we present a spheres-on-sphere (SOS) platform for multiplexed detection using a portable Coulter counter, which employs millimeter- and micron-sized spheres coupled with antibodies as multitarget probes. The assay allows for quantitative detection of multiple analytes within 20 min by simple mixing, enabling on-site detection. The platform shows high accuracy in identifying three respiratory viruses (SARS-CoV-2, influenza A virus, and parainfluenza virus) from throat swab samples, with LOD of 50.7, 32.4, and 49.1 pg/mL. It also demonstrates excellent performance in quantifying three mycotoxins (aflatoxin B1, deoxynivalenol, and ochratoxin A) from food samples. The SOS platform offers a rapid on-site approach with high sensitivity and specificity for applications in resource-limited settings.

Development of rapid nucleic acid testing techniques for common respiratory infectious diseases in the Chinese population.

Background: Most respiratory viruses can cause serious lower respiratory diseases at any age. Therefore, timely and accurate identification of respiratory viruses has become even more important. This study focused on the development of rapid nucleic acid testing techniques for common respiratory infectious diseases in the Chinese population. Methods: Multiplex fluorescent quantitative polymerase chain reaction (PCR) assays were developed and validated for the detection of respiratory pathogens including the novel coronavirus (SARS-CoV-2), influenza A virus (FluA), parainfluenza virus (PIV), and respiratory syncytial virus (RSV). Results: The assays demonstrated high specificity and sensitivity, allowing for the simultaneous detection of multiple pathogens in a single reaction. These techniques offer a rapid and reliable method for screening, diagnosis, and monitoring of respiratory pathogens. Conclusion: The implementation of these techniques might contribute to effective control and prevention measures, leading to improved patient care and public health outcomes in China. Further research and validation are needed to optimize and expand the application of these techniques to a wider range of respiratory pathogens and to enhance their utility in clinical and public health settings.

Multiplex detection of bacterial pathogens by PCR/SERS assay.

Bacterial infections are a leading cause of death globally. The detection of DNA sequences correlated to the causative pathogen has become a vital tool in medical diagnostics. In practice, PCR-based assays for the simultaneous detection of multiple pathogens currently rely on probe-based quantitative strategies that require expensive equipment but have limited sensitivity or multiplexing capabilities. Hence, novel approaches to address the limitations of the current gold standard methods are still in high demand. In this study, we propose a simple multiplex PCR/SERS assay for the simultaneous detection of four bacterial pathogens, namely P. aeruginosa, S. aureus, S. epidermidis, and M. smegmatis. Wherein, specific primers for amplifying each target gDNA were applied, followed by applying SERS nanotags functionalized with complementary DNA probes and Raman reporters for specific identification of the target bacterial pathogens. The PCR/SERS assay showed high specificity and sensitivity for genotyping bacterial pathogen gDNA, whereby as few as 100 copies of the target gDNA could be detected. With high sensitivity and the convenience of standard PCR amplification, the proposed assay shows great potential for the sensitive detection of multiple pathogen infections to aid clinical decision-making.

Comparing single versusmultiple virus detection in pediatric acute gastroenteritis postimplementation of routine multiplex RT-PCR diagnostic testing.

Utilizing multiplex real time polymerase chain reaction (RT-PCR) for rapid diagnosis of gastroenteritis, enables simultaneous detection of multiple pathogens. A comparative analysis of disease characteristics was conducted between cases with single and multiple viruses. Rotavirus vaccine was introduced in 2010, reaching a 70% coverage in 2 years. All rectal swabs collected from diarrheic children (<5 years) between December 2017 and March 2022 were included. Detection of the same viruses within 2 months was considered a single episode. Episodes with positive stool bacterial PCR were excluded. A total of 5879 samples were collected, revealing 86.9% (1509) with single virus detection and 13.1% (227) with multiple viruses. The most frequent combination was rotavirus and norovirus (27.8%), these infections followed a winter-spring seasonality akin to rotavirus. Children with multivirus infections exhibited higher immunodeficiency (OR 2.06) rates, but lower food allergy (OR 0.45) and prematurity rates (OR 0.55) compared to single infections. Greater disease severity, evaluated by the Vesikari score, was observed in multivirus episodes (p < 0.001, OR 1.12). Multivirus infections accounted for 13.1% of symptomatic cases in hospitalized young children. Despite vaccination efforts, rotavirus remained prominent, frequently in co-infections with norovirus. Overall, multivirus infections were linked to more severe diseases than single virus cases.

An immobilization-free electrochemical biosensor based on CRISPR/Cas13a and FAM-RNA-MB for simultaneous detection of multiple pathogens

To better respond to biosecurity issues, we need to build good technology and material reserves for pathogenic microorganism screening. Here, we designed an electrochemical/optical signal probe with a common fluorophore and an electrochemically active group, breaking the previous perception that the signal probe is composed of a fluorophore and a quenching group and realizing the response of three signals: electrochemistry, fluorescence, and direct observation. Then, we proposed a homogeneous electrochemical nucleic acid detection system based on CRISPR/Cas named “HELEN-CR” by integrating free electrochemical/optical signal probes and Cas13a cleavage, achieving a limit of detection of 1 pM within 25 min. To improve the detection sensitivity, we applied recombinase polymerase amplification to amplify the target nucleic acid, achieving a limit of detection of 30 zM within 45 min. Complemented by our self-developed multi-chamber microfluidic chip and portable electrochemical instrument, simultaneous detection of multiple pathogens can be achieved within 50 min, facilitating minimally trained personnel to obtain detection results quickly in a difficult environment. This study proposes a simple, scalable, and general idea and solution for the rapid detection of pathogenic microorganisms and biosecurity monitoring.

Advances in Multiplexed Microfluidics for Infectious Disease Detection

Abstract Microfluidics enables miniaturization, functionality, high throughput and reproducibility of multipathogen detection. Multiplexed microfluidic devices are electrochemical sensor–based, optical sensor–based, immunosensor-based and paper-based multiplexed microfluidics. However, the simultaneous detection of multiple pathogens is limited because of the complexity and diversity of infectious disease sources and mutual interference among analytes. This review provides an overview of recent advances in developing multiplex diagnostic microfluidic devices for detecting infectious diseases and discusses practical issues and perspectives. This review also covers microfluidic nucleic acid amplification strategies to improve detection sensitivity. Finally, we discuss the limitations and challenges in the design of multiplexed microfluidics.

Rapid and simultaneous detection of multiple pathogens in the lower reproductive tract during pregnancy based on loop-mediated isothermal amplification-microfluidic chip

BackgroundFemale reproductive tract infection (RTI) is the common source of varied diseases, especially as an important risk factor for pregnancy outcomes, therefore the rapid, accurate and simultaneous detection of multiple pathogens is in urgent need for assisting the diagnosis and treatment of RTI in pregnant women. Streptococcus agalactiae (S. agalactiae), Enterococcus faecalis (E. faecalis), Gardnerella vaginalis (G. vaginalis), Candida albicans (C. albicans) and Chlamydia trachomatis (C. trachomatis) are five main pathogens in lower genital tract with high risk, serious consequences and clinical demands. The combination of loop-mediated isothermal amplification (LAMP) and microfluidic technology was used to develop the LAMP-microfluidic chip for rapid, simple, sensitive and simultaneous detection of the five target pathogens above.ResultsStandard strains and clinical isolates were used for the establishment of the novel LAMP method in tube and LAMP-microfluidic chip, followed by the chip detection on 103 clinical samples and PCR verification partially. The sensitivities of LAMP of S. agalactiae, E. faecalis, G. vaginalis, and C. albicans in tube were 22.0, 76.0, 13.2, 1.11 CFU/μL, respectively, and C. trachomatis was 41.3 copies/μL; on LAMP-microfluidic chip they were 260, 154, 3.9 and 7.53 CFU/μL, respectively, and C. trachomatis was 120 copies/μL. The positive coincidence rates of clinical stains in tube and on chip experiments were 100%. Compared with the classic culture method performed in hospitals, the positive coincidence rate of the 103 clinical samples detected by LAMP-microfluidic chip were 100%. For the six inconsistent ones, including four G. vaginalis and two C. albicans positive samples tested by LAMP-microfluidic chip and verified by PCR were negative by culturing method in hospitals, indicating the lack of efficient detection by the classic culturing method.ConclusionOur study suggested that the LAMP-microfluidic chips could simultaneously, efficiently, and accurately detect multiple main pathogens, including S. agalactiae, E. faecalis, G. vaginalis, C. albicans and C. trachomatis, in clinical samples of female RTI to give a great clinical value. Accordingly, this novel method has the potential to provide a valuable reference for female RTI screening and early diagnosis during pregnancy.

Advances in the differential molecular diagnosis of vesicular disease pathogens in swine.

Foot-and-mouth disease virus (FMDV), Senecavirus A (SVA) and swine vesicular disease virus (SVDV) are members of the family Picornaviridae, which can cause similar symptoms - vesicular lesions in the tissues of the mouth, nose, feet, skin and mucous membrane of animals. Rapid and accurate diagnosis of these viruses allows for control measures to prevent the spread of these diseases. Reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR are traditional and reliable methods for pathogen detection, while their amplification reaction requires a thermocycler. Isothermal amplification methods including loop-mediated isothermal amplification and recombinase polymerase amplification developed in recent years are simple, rapid and do not require specialized equipment, allowing for point of care diagnostics. Luminex technology allows for simultaneous detection of multiple pathogens. CRISPR-Cas diagnostic systems also emerging nucleic acid detection technologies which are very sensitivity and specificity. In this paper, various nucleic acid detection methods aimed at vesicular disease pathogens in swine (including FMDV, SVA and SVDV) are summarized.

Fast screening of enteropathogens in marine water samples.

This study aimed to fast screen the microbiological contamination of recreational waters using a TaqMan Array Card (TAC), a multiplexed platform designed for the simultaneous detection of 35 enteropathogens. Surface and deep marine water samples were concentrated by skimmed milk flocculation and processed for nucleic acid extraction protocol using QIAamp Fast DNA Stool Mini Kit. Twelve microorganisms and parasites, including bacteria (n = 6), protozoa (4), and viruses (2), were detected in 85.7% (24/28) of samples. Campylobacter (82.1%), Cryptosporidium (39.3%), and adenovirus (14.3%) were the most detected pathogens. Neither fungi nor helminths were detected. A spatial pollution profile of microbiological contamination was observed in the area. Methodologies for simultaneous detection of multiple pathogens, such as TAC, can assist decision-makers by providing a quick assessment of the microbiological water quality in areas used for recreational purposes, which in many cases are in accordance with the bacteriological indicators.

Simultaneous detection of multiple pathogens with the TaqMan Array Card

Quantitative polymerase chain reaction (qPCR) is a gold standard method for the detection and quantification of pathogenic organisms. Standard qPCR is inexpensive, sensitive and highly specific to the pathogen of interest. While qPCR assays can be multiplexed to allow the detection of multiple organisms in one reaction, it is prohibitively labour intensive to screen large numbers of samples for several pathogens at the same time. The TaqMan Array Card (TAC) is a cost-effective and accurate technique that expands the number of assays that can be simultaneously performed on a sample, with no increase in set-up time and only small reductions in sensitivity. This approach is highly beneficial in settings where there is a need to monitor a large panel of pathogens. We illustrate the application of TAC to the monitoring of gastrointestinal pathogens, which span viral, bacterial, protist and helminth taxa. This protocol outlines the laboratory set-up of a TaqMan Array Card, and some recommended data processing steps to aid in accurate interpretation of the results. A video protocol is additionally provided to assist in the use of the technique.•The TAC is designed primarily for gene expression assays, but has recently been utilised in several studies for pathogen detection in human clinical samples.•We expand the use of TAC for pathogen detection across human, animal and environmental sample types, and have developed a protocol and guidelines for the processing and interpretation of results that circumvents issues with the automated outputs.•This technique is applicable to pathogen or organism detection in any context, if quality nucleic acid extracts can be obtained from the sample type of interest.

A magnet-actuated microfluidic array chip for high-throughput pretreatment and amplification and detection of multiple pathogens.

The outbreak of global infectious diseases has posed a significant threat to public health, requiring the rapid and accurate diagnosis of pathogens promptly for the society to implement immediate control measures to prevent widespread pandemics. In this work, a magnet-actuated microfluidic array chip (MMAC) is developed with integrated sample processing and nucleic acid amplification for the rapid detection of multiple pathogens by loop-mediated isothermal amplification. In comparison to previous works, where fluid control was dependent on external equipment or finger-based manual pressing, the fluid control of the MMAC is realized by magnetically actuating a ferric oxide (Fe3O4) doped polydimethylsiloxane (PDMS) layer that separates the sample from the LAMP reagent in a high-throughput manner, which not only reduces the complexity of fluid control but also enhances the repeatability of detection by eliminating variations in operation by different users. Examination with a testing sample containing Salmonella typhimurium and Escherichia coli showed high specificity for pathogen detection without cross-contamination. The lowest detection concentration was 5.2 copies per μL for Salmonella typhimurium with a detection time of 60 min. The proposed method demonstrated the simultaneous detection of multiple pathogens, which is potentially helpful in applications of immediate diagnosis.

Reagentless and Ultrasensitive Electrochemical Dnazyme Based Sensing Platform for Clinical Detection of Uropathogenic Escherichia coli

Urinary tract infections (UTIs) are one of the most ubiquitous bacterial infection diseases affecting half of the global population at least once in their lifetime. Given the occurrence of the false positive result in dipstics and the time-consuming identification of bacteria using culturing method to detect UroPathogenic Escherichia coli (UPEC), there is the need for a point-of-care diagnostic platform that is reliable, sensitive, specific, cost-effective, fast and easy to use. With the advent electrochemical methods coupled with the specific biorecognition elements, the detection of any biomarker has become possible with one touch. Among many biorecognition molecules, DNAzymes are gaining attention due to their robustness, stability, specificity, and the ease of selection without prior knowledge of the biomarker. Their structure switching and functionality (RNA cleaving) can be utilized with redox active species (like methylene blue) to design highly sensitive and selective electrochemical assays for pathogen detection. In this work, we have developed a dual signaling electrochemical DNAzyme based platform for specific and sensitive detection of Escherichia coli (E.coli) in UTI patient urine samples. The two on-chip integrated working electrodes named as release channel and capture channel are integrated with DNAzyme-redox barcode and DNA probe respectively. In the presence of the bacterial target the DNAzyme catalyzes the cleavage of the redox DNA barcode in the release channel, which is subsequently translated into a redox signal upon hybridization with the probe on the capture channel. The differential signal generated by the two channels in the sensor demonstrated the sensitive detection of 10 CFU for E.coli in buffer and urine. Additionally, the sensor specifically detected E. coli in the presence of a panel of other gram positive and negative bacteria. Clinical validation of the assay was performed using 45 E.coli+ and E.coli- patient urines delineating a clinical sensitivity of 100% and specificity of 78% in a 30 minute testing time. This cost effective, reagent-free, time-efficient, and amplification-free electrochemical assay is suitable for detecting a wide range of pathogens, also alleviates the additional knowledge requirement of the biomarker as compared to other protein and aptamer-based assay. With the advancement in the chip fabrication and microfluidics, this assay can also be multiplexed with other pathogen for simultaneous detection of multiple pathogens in a single urine sample.

Microfluidic devices for multiplexed detection of foodborne pathogens

The global burden of foodborne diseases is substantial and foodborne pathogens are the major cause for human illnesses. In order to prevent the spread of foodborne pathogens, detection methods are constantly being updated towards rapid, portable, inexpensive, and multiplexed on-site detection. Due to the nature of the small size and low volume, microfluidics has been applied to rapid, time-saving, sensitive, and portable devices to meet the requirements of on-site detection. Simultaneous detection of multiple pathogens is another key parameter to ensure food safety. Multiplexed detection technology, including microfluidic chip design, offers a new opportunity to achieve this goal. In this review, we introduced several sample preparation and corresponding detection methods on microfluidic devices for multiplexed detection of foodborne pathogens. In the sample preparation section, methods of cell capture and enrichment, as well as nucleic acid sample preparation, were described in detail, and in the section of detection methods, amplification, immunoassay, surface plasmon resonance and impedance spectroscopy were exhaustively illustrated. The limitations and advantages of all available experimental options were also summarized and discussed in order to form a comprehensive understanding of cutting-edge technologies and provide a comparative assessment for future investigation and in-field application.

Ultrasensitive Electrochemical Dnazyme Based Sensing Platform for Clinical Detection of Uropathogenic Escherichia coli

Urinary tract infections (UTIs) are one of the most ubiquitous bacterial infection diseases affecting half of the global population at least once in their lifetime. Given the occurrence of the false positive result in Dipstics and the time-consuming identification of bacteria using culturing method to detect UroPathogenic Escherichia coli (UPEC), there is the need for a point-of-care diagnostic platform that is reliable, sensitive, specific, cost-effective, fast and easy to use. With the advent electrochemical methods coupled with the specific biorecognition elements, the detection of any biomarker has become possible with one touch. Among many biorecognition molecules, DNAzymes are gaining attention due to their robustness, stability, specificity, and the ease of selection without prior knowledge of the biomarker. Their structure switching and functionality (RNA cleaving) can be utilized with redox active species (like methylene blue) to design highly sensitive and selective electrochemical assays for pathogen detection.In this work, we have developed a dual signaling electrochemical DNAzyme based platform for specific and sensitive detection of Escherichia coli (E.coli) in UTI patient urine samples. The two on-chip working electrodes named as release channel and capture channel are integrated with DNAzyme-redox barcode and DNA probe respectively. In the presence of the bacterial target the DNAzyme catalyzes the cleavage of the redox DNA barcode in the release channel, which is subsequently translated into a redox signal upon hybridization with the probe on the capture channel. The differential signal generated by the two channels in the sensor demonstrated the sensitive detection of 10 CFU for E.coli in buffer and urine. Additionally, the sensor specifically detected E. coli in the presence of a panel of other gram positive and negative bacteria. Clinical validation of the assay was performed using 40 E.coli+ and E.coli- patient urine delineating a clinical sensitivity of 100% and specificity of 78% in a 30 minute testing time. This cost effective, reagent-free, time-efficient, and amplification-free electrochemical assay is suitable for detecting a wide range of pathogens, also alleviates the additional knowledge requirement of the biomarker as compared to other protein and aptamer-based assay. With the advancement in the chip fabrication and microfluidics, this assay can also be multiplexed with other pathogen for simultaneous detection of multiple pathogens in a single urine sample.

Assessment of STI screening in Romania using a multiplex PCR technique.

Most sexually transmitted infections (STIs) are curable, but inappropriate treatment can lead to serious complications. The importance of setting up STI screening programs has been highlighted in various studies, the absence of such national programs accounting for the lack of STI statistics in Romania. The purpose of our study was to evaluate multiplex PCR as a screening method for the most common 6 STIs and establish their frequency in a group of symptomatic and asymptomatic patients. We aimed to highlight STI associations and correlations between STI pathogens and symptomatology, demographic status, antecedents or sexual partners. A total of 249 patients, both symptomatic and asymptomatic, were included in this study. Chlamydia trachomatis (CT), Neisseia gonorrhoeae (NG), Trichomonas vaginalis (TV), Ureaplasma urealyticum (UU), Mycoplasma hominis (MH) and Mycoplasma genitalium (MG) were all identified in urine samples via multiplex Polymerase Chain Reaction (PCR). The SPSS IBM program was employed for statistical analysis. 32.12% of the patients were found positive, some presenting multiple infections. The results are representative for the Romanian male population. 107 STI pathogens were identified, most frequent being CT, UU and NG. Several statistical correlations between patient characteristics and the presence of STIs have been demonstrated. The results suggest that multiplex PCR meets all the prerequisites for a screening method, allowing the use of multiple specimens and enabling simultaneous detection of multiple pathogens in a short period of time. STI identification via multiplex PCR proved to be an effective method for quantifying their frequency in Romania.

Syndrome Evaluation System for Simultaneous Detection of Pathogens Causing Acute Encephalitic Syndrome in India, Part-2: Validation Using Well Characterized Clinical Samples.

Diagnosis of the aetiological agent in case of acute encephalitic syndrome (AES) continues to pose a challenge in clinical practice as a variety of pathogens are known to cause AES. Here, we report the validation of a Syndrome Evaluation System (SES) developed for simultaneous detection of multiple AES pathogens using a well characterized set of Cerebrospinal fluid (CSF) samples. The validation of the SES was carried out in two phases. In the first phase, the SES was validated using 51 CSF samples obtained from autopsy proven cases and 50 samples obtained from apparently healthy individuals undergoing spinal anesthesia for minor surgeries served as “controls.” The SES detected etilogical agent in 48/51 (94.11 %) samples obtained from autopsy proven AES cases while all the 50 CSF samples obtained from “controls” were negative. In the second phase, the SES was validated using well characterized CSF samples obtained from AES patients fulfilling the WHO case definition of AES (Group I; n = 207) and samples that were collected from patients with non-infectious neurological disorder (Group II; n = 90). All the samples were tested using multiple conventional/serological assays and categorized into various groups. Amongst the AES cases fulfilling WHO case definition, the SES detected AES pathogens in 160/207 (77.29%) cases while conventional serological/molecular assays were able to detect AES pathogens only in 77/207 (37.1%) of cases. Further, in 12/83 CSF samples that were positive by SES and negative by conventional serological/molecular tests, the results were additionally confirmed by sequencing the PCR products to rule out non-specific amplification in the SES. In patients with non-infectious neurological disorders the SES detected latent viruses 12/90 CSF samples. These results indicate that the SES, apart being a rapid, sensitive, specific, and cost-effective method provides the major advantage of simultaneous detection of multiple pathogens using as single specimen of CSF.

Smartphone-based pathogen diagnosis in urinary sepsis patients.

BackgroundThere is an urgent need for rapid, sensitive, and affordable diagnostics for microbial infections at the point-of-care. Although a number of innovative systems have been reported that transform mobile phones into potential diagnostic tools, the translational challenge to clinical diagnostics remains a significant hurdle to overcome.MethodsA smartphone-based real-time loop-mediated isothermal amplification (smaRT-LAMP) system was developed for pathogen ID in urinary sepsis patients. The free, custom-built mobile phone app allows the phone to serve as a stand-alone device for quantitative diagnostics, allowing the determination of genome copy-number of bacterial pathogens in real time.FindingsA head-to-head comparative bacterial analysis of urine from sepsis patients revealed that the performance of smaRT-LAMP matched that of clinical diagnostics at the admitting hospital in a fraction of the time (~1 h vs. 18–28 h). Among patients with bacteremic complications of their urinary sepsis, pathogen ID from the urine matched that from the blood – potentially allowing pathogen diagnosis shortly after hospital admission. Additionally, smaRT-LAMP did not exhibit false positives in sepsis patients with clinically negative urine cultures.InterpretationThe smaRT-LAMP system is effective against diverse Gram-negative and -positive pathogens and biological specimens, costs less than $100 US to fabricate (in addition to the smartphone), and is configurable for the simultaneous detection of multiple pathogens. SmaRT-LAMP thus offers the potential to deliver rapid diagnosis and treatment of urinary tract infections and urinary sepsis with a simple test that can be performed at low cost at the point-of-care.FundNational Institutes of Health, Chan-Zuckerberg Biohub, Bill and Melinda Gates Foundation.

Sensitive detection of live Escherichia coli by bacteriophage amplification-coupled immunoassay on the Luminex® MAGPIX instrument

Phages are natural predators of bacteria and have been exploited in bacterial detection because of their exquisite specificity to their cognate bacterial hosts. In this study, we present a “proof of concept” bacteriophage amplification-coupled assay as a surrogate for detecting a bacterium present in a sample. The assay entails detection of progeny phage resulting from infection and subsequent growth inside the bacterium present in suspected samples. This approach reduces testing time and enhances sensitivity to identify pathogens compared to traditional overnight plaque assay. Further, the assay has the ability to discriminate between live and dead cells since phages require live host cells to infect and replicate. To demonstrate its utility, phage MS2 amplification-coupled, bead-based sandwich type immunoassay on the Luminex® MAGPIX instrument for Escherichia coli detection was performed. The assay not only showed live cell discrimination ability but also a limit of E. coli detection of 1 × 102 cells/mL of live cells after a 3-h incubation. In addition, the sensitivity of the assay was not impaired in the presence of dead cells. These results demonstrate that bacteriophage amplification-coupled assay can be a rapid live cell detection assay compared to traditional culture methods and a promising tool for quick validation of bacterial inactivation. Combined with the unique multiplex bead chemistry of the Luminex® MAGPIX platform, the phage assay can be expanded to be an ultra-deep multiplex assay for the simultaneous detection of multiple pathogens using specific phages directed against the target pathogens.

Travelers' Diarrhea in Thailand: A Quantitative Analysis Using TaqMan® Array Card.

Travelers' diarrhea (TD) is a common illness experienced by travelers from developed countries who visit developing countries. Recent questionnaire-based surveillance studies showed that approximately 6%-16% of travelers experienced TD while visiting Thailand; however, a majority of TD information was limited mainly to US military populations. A TD surveillance study was conducted at Bumrungrad International Hospital in 2012-2014 in Bangkok, Thailand. Enteropathogens were identified using conventional methods and the TaqMan® array card (TAC), which uses real-time polymerase chain reaction for the simultaneous detection of multiple pathogens. Analyses to determine pathogen-disease and symptoms association were performed to elucidate the clinical relevance of each enteropathogen. TAC identified more pathogens per sample than conventional methods. Campylobacter spp. were the most prevalent, followed by the diarrheagenic Escherichia coli and norovirus GII. These agents had significant pathogen-disease associations as well as high attributable fractions among diarrheal cases. A wide range of pathogen loads for Campylobacter spp. was associated with TD, while heat-labile toxin enterotoxigenic Escherichia coli was associated with an increased pathogen load. Most cases were associated with inflammatory diarrhea, while Campylobacter spp. and Shigella spp. were associated with dysentery. A pan-molecular diagnostic method such as TAC produces quantifiable and comparable results of all tested pathogens, thereby reducing the variability associated with multiple conventional methods. This allows better determination of the clinical relevance of each diarrhea etiologic agent, as well as their geographical relevance in Thailand.

Laboratory diagnosis of cryptosporidiosis.

Cryptosporidiosis is a major etiology of persistent and chronic diarrhea in children and immunocompromised patients. In addition, it is also one of the important pathogens causing zoonotic or waterborne outbreaks. A number of conventional and molecular tests are available, but each test is riddled with few limitations. Further, there is a need to develop point of care tests and multiplexed tests for simultaneous detection of multiple pathogens.