title: Quantitative PCR for detection of Shigella improves ascertainment of Shigella burden in children with moderate-to-severe diarrhea in low-income countries.
abstract: Estimates of the prevalence of Shigella spp. are limited by the suboptimal sensitivity of current diagnostic and surveillance methods. We used a quantitative PCR (qPCR) assay to detect Shigella in the stool samples of 3,533 children aged <59 months from the Gambia, Mali, Kenya, and Bangladesh, with or without moderate-to-severe diarrhea (MSD). We compared the results from conventional culture to those from qPCR for the Shigella ipaH gene. Using MSD as the reference standard, we determined the optimal cutpoint to be 2.9 × 10(4) ipaH copies per 100 ng of stool DNA for set 1 (n = 877). One hundred fifty-eight (18%) specimens yielded >2.9 × 10(4) ipaH copies. Ninety (10%) specimens were positive by traditional culture for Shigella. Individuals with ≥ 2.9 × 10(4) ipaH copies have 5.6-times-higher odds of having diarrhea than those with <2.9 × 10(4) ipaH copies (95% confidence interval, 3.7 to 8.5; P < 0.0001). Nearly identical results were found using an independent set of samples. qPCR detected 155 additional MSD cases with high copy numbers of ipaH, a 90% increase from the 172 cases detected by culture in both samples. Among a subset (n = 2,874) comprising MSD cases and their age-, gender-, and location-matched controls, the fraction of MSD cases that were attributable to Shigella infection increased from 9.6% (n = 129) for culture to 17.6% (n = 262) for qPCR when employing our cutpoint. We suggest that qPCR with a cutpoint of approximately 1.4 × 10(4) ipaH copies be the new reference standard for the detection and diagnosis of shigellosis in children in low-income countries. The acceptance of this new standard would substantially increase the fraction of MSD cases that are attributable to Shigella.
authors.affiliation: University of Maryland, School of Medicine, Baltimore, Maryland, USA. blind002@umaryland.edu
authors.forename: Brianna
authors.initials: B
authors.lastname: Lindsay
authors.forename: John B
authors.initials: JB
authors.lastname: Ochieng
authors.forename: Usman N
authors.initials: UN
authors.lastname: Ikumapayi
authors.forename: Aliou
authors.initials: A
authors.lastname: Toure
authors.forename: Dilruba
authors.initials: D
authors.lastname: Ahmed
authors.forename: Shan
authors.initials: S
authors.lastname: Li
authors.forename: Sandra
authors.initials: S
authors.lastname: Panchalingam
authors.forename: Myron M
authors.initials: MM
authors.lastname: Levine
authors.forename: Karen
authors.initials: K
authors.lastname: Kotloff
authors.forename: David A
authors.initials: DA
authors.lastname: Rasko
authors.forename: Carolyn R
authors.initials: CR
authors.lastname: Morris
authors.forename: Jane
authors.initials: J
authors.lastname: Juma
authors.forename: Barry S
authors.initials: BS
authors.lastname: Fields
authors.forename: Michel
authors.initials: M
authors.lastname: Dione
authors.forename: Dramane
authors.initials: D
authors.lastname: Malle
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Becker
authors.forename: Eric R
authors.initials: ER
authors.lastname: Houpt
authors.forename: James P
authors.initials: JP
authors.lastname: Nataro
authors.forename: Halvor
authors.initials: H
authors.lastname: Sommerfelt
authors.forename: Mihai
authors.initials: M
authors.lastname: Pop
authors.forename: Joe
authors.initials: J
authors.lastname: Oundo
authors.forename: Martin
authors.initials: M
authors.lastname: Antonio
authors.forename: Anowar
authors.initials: A
authors.lastname: Hossain
authors.forename: Boubou
authors.initials: B
authors.lastname: Tamboura
authors.forename: O Colin
authors.initials: OC
authors.lastname: Stine
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D016022:Case-Control Studies; D002675:Child, Preschool; D003906:Developing Countries; D003967:Diarrhea / Q000175:diagnosis* / Q000453:epidemiology* / Q000382:microbiology; D004405:Dysentery, Bacillary / Q000175:diagnosis* / Q000453:epidemiology* / Q000382:microbiology; D005243:Feces / Q000382:microbiology; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008297:Male; D015995:Prevalence; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012760:Shigella / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C063500:ipaH protein, Shigella flexneri
references.citation: Diagn Microbiol Infect Dis. 2012 Jun;73(2):121-8
references.pmid: 22541788
references.citation: Clin Infect Dis. 2011 Dec;53(12):1223-9
references.pmid: 22028433
references.citation: Am J Epidemiol. 1985 Nov;122(5):904-14
references.pmid: 4050778
references.citation: Clin Infect Dis. 2012 Dec;55 Suppl 4:S232-45
references.pmid: 23169936
references.citation: Acta Paediatr. 2007 May;96(5):644-7
references.pmid: 17376185
references.citation: J Clin Microbiol. 2004 May;42(5):2031-5
references.pmid: 15131166
references.citation: PLoS Med. 2006 Sep;3(9):e353
references.pmid: 16968124
references.citation: Clin Infect Dis. 2012 Dec;55 Suppl 4:S294-302
references.pmid: 23169941
references.citation: BMC Infect Dis. 2009 May 14;9:63
references.pmid: 19442278
references.citation: Epidemiology. 2005 Sep;16(5):604-12
references.pmid: 16135935
references.citation: Jpn J Infect Dis. 2005 Aug;58(4):232-4
references.pmid: 16116257
references.citation: Vaccine. 1995 Nov;13(16):1488-94
references.pmid: 8578831
references.citation: J Infect Dis. 1997 Oct;176(4):1013-8
references.pmid: 9333160
references.citation: N Engl J Med. 2011 Aug 25;365(8):709-17
references.pmid: 21793740
references.citation: Clin Infect Dis. 2012 Dec;55 Suppl 4:S303-11
references.pmid: 23169942
references.citation: Infect Immun. 1997 Sep;65(9):3852-6
references.pmid: 9284163
references.citation: Ann Ist Super Sanita. 2009;45(2):168-72
references.pmid: 19636168
references.citation: Am J Trop Med Hyg. 1997 May;56(5):533-7
references.pmid: 9180604
references.citation: Infect Immun. 2011 Feb;79(2):950-60
references.pmid: 21078854