Jean Gratz

A Laboratory-Developed TaqMan Array Card for Simultaneous Detection of 19 Enteropathogens

ABSTRACT The TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here we developed an enteric TaqMan Array Card to detect 19 enteropathogens, including viruses (adenovirus, astrovirus, norovirus GII, rotavirus, and sapovirus), bacteria (Campylobacter jejuni/C. coli, Clostridium difficile, Salmonella, Vibrio cholerae, diarrheagenic Escherichia coli strains including enteroaggregative E. coli [EAEC], enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], and Shiga-toxigenic E. coli [STEC]), Shigella/enteroinvasive E. coli (EIEC), protozoa (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica), and helminths (Ascaris lumbricoides and Trichuris trichiura), as well as two extrinsic controls to monitor extraction and amplification efficiency (the bacteriophage MS2 and phocine herpesvirus). Primers and probes were newly designed or adapted from published sources and spotted onto microfluidic cards. Fecal samples were spiked with extrinsic controls, and DNA and RNA were extracted using the QiaAmp Stool DNA minikit and the QuickGene RNA Tissue kit, respectively, and then mixed with Ag-Path-ID One Step real-time reverse transcription-PCR (RT-PCR) reagents and loaded into cards. PCR efficiencies were between 90% and 105%, with linearities of 0.988 to 1. The limit of detection of the assays in the TAC was within a 10-fold difference from the cognate assays performed on plates. Precision testing demonstrated a coefficient of variation of below 5% within a run and 14% between runs. Accuracy was evaluated for 109 selected clinical specimens and revealed an average sensitivity and specificity of 85% and 77%, respectively, compared with conventional methods (including microscopy, culture, and immunoassay) and 98% and 96%, respectively, compared with our laboratory-developed PCR-Luminex assays. This TAC allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes.

Simultaneous Detection of Six Diarrhea-Causing Bacterial Pathogens with an In-House PCR-Luminex Assay

ABSTRACT Diarrhea can be caused by a range of pathogens, including several bacteria. Conventional diagnostic methods, such as culture, biochemical tests, and enzyme-linked immunosorbent assay (ELISA), are laborious. We developed a 7-plex PCR-Luminex assay to simultaneously screen for several of the major diarrhea-causing bacteria directly in fecal specimens, including pathogenic Aeromonas, Campylobacter jejuni, Campylobacter coli, Salmonella, Shigella, enteroinvasive Escherichia coli (EIEC), Vibrio, and Yersinia. We included an extrinsic control to verify extraction and amplification. The assay was first validated with reference strains or isolates and exhibited a limit of detection of 103 to 105 CFU/g of stool for each pathogen as well as quantitative detection up to 109 CFU/g. A total of 205 clinical fecal specimens from individuals with diarrhea, previously cultured for enteric pathogens and tested for Campylobacter by ELISA, were evaluated. Using these predicate methods as standards, sensitivities and specificities of the PCR-Luminex assay were 89% and 94% for Aeromonas, 89% and 93% for Campylobacter, 96% and 95% for Salmonella, 94% and 94% for Shigella, 92% and 97% for Vibrio, and 100% and 100% for Yersinia, respectively. All discrepant results were further examined by singleplex real-time PCR assays targeting different gene regions, which revealed 89% (55/62 results) concordance with the PCR-Luminex assay. The fluorescent signals obtained with this approach exhibited a statistically significant correlation with the cycle threshold (CT ) values from the cognate real-time PCR assays (P < 0.05). This multiplex PCR-Luminex assay enables sensitive, specific, and quantitative detection of the major bacterial causes of gastroenteritis.

Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples

Cyclospora, Cystoisospora, and Microsporidia are eukaryotic enteropathogens that are difficult to detect in stool samples because they require special stains and microscopy. We developed a multiplex polymerase chain reaction (PCR) reaction with 4 primer sets to amplify Cyclospora cayetanensis, Cystoisospora belli, Enterocytozoon bieneusi, and Encephalitozoon intestinalis. Detection of the amplicon is through specific probes coupled to Luminex beads. Sensitivity of the assay was evaluated using Encephalitozoon intestinalis spores and revealed detection of 10(1) spores spiked into stool. No cross-reactivity was observed. We evaluated the assay on diarrheal specimens from Thailand, Tanzania, Indonesia, and the Netherlands that had been previously tested by microscopy, and the assay yielded 87-100% sensitivity and 88-100% specificity. Microscopy-negative/PCR-positive samples had lower Luminex values, suggesting they were true but with lower burden infections. In summary, this is a convenient single PCR reaction that can detect Cyclospora, Cystoisospora, and Microsporidia without the need for cumbersome microscopic analysis.

Association Between Stool Enteropathogen Quantity and Disease in Tanzanian Children Using TaqMan Array Cards: A Nested Case-Control Study

Etiologic studies of diarrhea are limited by uneven diagnostic methods and frequent asymptomatic detection of enteropathogens. Polymerase chain reaction-based stool pathogen quantification may help distinguish clinically significant infections. We performed a nested case-control study of diarrhea in infants from a community-based birth cohort in Tanzania. We tested 71 diarrheal samples and pre-diarrheal matched controls with a laboratory-developed TaqMan Array Card for 19 enteropathogens. With qualitative detection, no pathogens were significantly associated with diarrhea. When pathogen quantity was considered, rotavirus (odds ratio [OR] = 2.70 per log10 increase, P < 0.001), astrovirus (OR = 1.49, P = 0.01), and Shigella/enteroinvasive Escherichia coli (OR = 1.47, P = 0.04) were associated with diarrhea. Enterotoxigenic E. coli (0.15 SD decline in length-for-age z score after 3 months per log10 increase, P < 0.001) and Campylobacter jejuni/C. coli (0.11 SD decline, P = 0.003) in pre-diarrheal stools were associated with poor linear growth. Quantitative analysis can help refine the association between enteropathogens and disease in endemic settings.

Establishment of haematological and immunological reference values for healthy Tanzanian children in Kilimanjaro Region

Objective  To determine the normal haematological and immunological reference intervals for healthy Tanzanian children.

Correction: Prevalence and extent of heteroresistance by next generation sequencing of multidrug-resistant tuberculosis

[This corrects the article DOI: 10.1371/journal.pone.0176522.].

Multiplex real time PCR panels to identify fourteen colonization factors of enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) is a leading cause of childhood diarrhea in low income countries and in travelers to those areas. Inactivated enterotoxins and colonization factors (CFs) are leading vaccine candidates, therefore it is important to determine the prevailing CF types in different geographic locations and populations. Here we developed real time PCR (qPCR) assays for 14 colonization factors, including the common vaccine targets. These assays, along with three enterotoxin targets (STh, STp, and LT) were formulated into three 5-plex qPCR panels, and validated on 120 ETEC isolates and 74 E. coli colony pools. The overall sensitivity and specificity was 99% (199/202) and 99% (2497/2514), respectively, compared to the CF results obtained with conventional PCR. Amplicon sequencing of discrepant samples revealed that the qPCR was 100% accurate. qPCR panels were also performed on nucleic acid extracted from stool and compared to the results of the ETEC isolates or E. coli colony pools cultured from them. 95% (105/110) of the CF detections in the cultures were confirmed in the stool. Additionally, direct testing of stool yielded 30 more CF detections. Among 74 randomly selected E. coli colony pools with paired stool, at least one CF was detected in 63% (32/51) of the colony pools while at least one CF was detected in 78% (47/60) of the stool samples (P = NS). We conclude that these ETEC CF assays can be used on both cultures and stool samples to facilitate better understanding of CF distribution for ETEC epidemiology and vaccine development.

Etiology of Diarrhea, Nutritional Outcomes, and Novel Intestinal Biomarkers in Tanzanian Infants

Objective: Diarrheal diseases are a leading cause of morbidity and mortality worldwide, but the etiology of diarrhea and its relation to nutritional outcomes in resource-limited settings is poorly defined. We sought to determine the etiology of community-acquired diarrhea in Tanzanian infants and to assess the association with anthropometrics and novel intestinal biomarkers. Methods: A convenience sample of infants in a trial of zinc and/or multivitamin supplementation in Tanzania was selected. Subjects were enrolled at age 6 weeks and studied for 18 months. Stool samples were obtained from children with acute diarrhea. A novel, polymerase chain reaction-based TaqMan array was used to screen stool for 15 enteropathogens. A subset of subjects had serum gastrointestinal biomarkers measured. Results: One hundred twenty-three subjects with diarrhea were enrolled. The mean ± SD age at stool sample collection was 12.4 ± 3.9 months. Thirty-five enteropathogens were identified in 34 (27.6%) subjects: 11 rotavirus, 9 Cryptosporidium spp, 7 Shigella spp, 3 Campylobacter jejuni/coli, 3 heat stable-enterotoxigenic Escherichia coli, and 2 enteropathogenic E coli. Subjects with any identified enteropathogen had significantly lower weight-for-length z scores (−0.55 ± 1.10 vs 0.03 ± 1.30, P = 0.03) at the final clinic visit than those without an identified pathogen. Fifty of the 123 subjects (40.7%) had serum analyzed for antibodies to lipopolysaccharide (LPS) and flagellin. Subjects with any identified enteropathogen had lower immunoglobulin (IgA) antibodies to LPS (0.75 ± 0.27 vs 1.13 ± 0.77, P = 0.01) and flagellin (0.52 ± 0.16 vs 0.73 ± 0.47, P = 0.02) than those without an identified pathogen. Conclusions: This quantitative polymerase chain reaction method may allow identification of enteropathogens that place children at higher risk for suboptimal growth. IgA anti-LPS and flagellin antibodies hold promise as emerging intestinal biomarkers.