Diagnostic microbiologic methods in the GEMS-1 case/control study

Abstract

To understand the etiology of moderate-to-severe diarrhea among children in high mortality areas of sub-Saharan Africa and South Asia, we performed a comprehensive case/control study of children aged <5 years at 7 sites. Each site employed an identical case/control study design and each utilized a uniform comprehensive set of microbiological assays to identify the likely bacterial, viral and protozoal etiologies. The selected assays effected a balanced consideration of cost, robustness and performance, and all assays were performed at the study sites. Identification of bacterial pathogens employed streamlined conventional bacteriologic biochemical and serological algorithms. Diarrheagenic Escherichia coli were identified by application of a multiplex polymerase chain reaction assay for enterotoxigenic, enteroaggregative, and enteropathogenic E. coli. Rotavirus, adenovirus, Entamoeba histolytica, Giardia enterica, and Cryptosporidium species were detected by commercially available enzyme immunoassays on stool samples. Samples positive for adenovirus were further evaluated for adenovirus serotypes 40 and 41. We developed a novel multiplex assay to detect norovirus (types 1 and 2), astrovirus, and sapovirus. The portfolio of diagnostic assays used in the GEMS study can be broadly applied in developing countries seeking robust cost-effective methods for enteric pathogen detection.

Figure 1.

Appearance of diarrheagenic Escherichia coli amplicons separated by agarose gel electrophoresis. Lane 1, enteropathogenic E. coli; lane 2, enteroaggregative E. coli; 3, enterotoxigenic E. coli; lanes 4 and 5, negative control strains; lane 6, 100 bp DNA ladder (New England Biolabs).

Figure 2.

An example of the graphical results of real-time polymerase chain reaction performed on 4 eae-positive specimens (red), 4 unknown specimens (green), and negative controls (yellow and blue). A threshold for detection of DNA-based fluorescence is set slightly above background fluorescence levels.

Figure 3.

Gels showing the results of a multiplex polymerase chain reaction (PCR) assay for enteropathogenic Escherichia coli (EPEC), Shiga toxin–producing E. coli, and enterohemorrhagic E. coli (EHEC). Individual isolates from 34 specimens were subjected to a multiplex PCR as described in the text. Each specimen, separated by yellow vertical lines, consists of 3 individual isolates. The yellow values indicate the cycle threshold obtained for each specimen in the real-time PCR used in the initial screening for eae. The amplicons produced by the positive controls, EPEC E2348/69 (eae and bfpA) and EHEC EH48 (stx1, stx2, and ehxA) are also shown. 100 bp DNA ladder was used as a molecular size marker. Abbreviation: NTC, no template control.

Figure 4.

Appearance of enteric viral amplicons separated by agarose gel electrophoresis. Lane M, 100 bp DNA ladder (New England Biolabs); lane 1, Norovirus GI (330 bp); lane 2, Norovirus GII positive (387 bp); lane 3, sapovirus (434 bp); lane 4, astrovirus (719 bp).