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Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen’s extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.

Acute febrile illness (AFI) is associated with substantial morbidity and mortality worldwide, yet an etiologic agent is often not identified. Convalescent-phase serology is impractical, blood culture is slow, and many pathogens are fastidious or impossible to cultivate. We developed a real-time PCR-based TaqMan array card (TAC) that can test six to eight samples within 2. Fever is a symptom common to a wide variety of infectious diseases, including some of the leading causes of death in subSaharan Africa (SSA). Many etiologic studies have been performed for respiratory infections, diarrheal illness, and meningitis (1, 2). However, the incidence and etiology of undifferentiated fever are less clear (3). Most research has examined individual agents such as Plasmodium, Salmonella, and specific zoonotic or arboviral pathogens (4-6) by utilizing blood culture (7) or a complex mixture of rapid, serologic, culture, and molecular assays and algorithms to determine an etiologic agent (8).We describe our initial development and validation of a TaqMan array card (TAC) that uses quantitative reverse transcription-PCR (qRT-PCR) for the simultaneous detection of 15 viruses, 8 bacteria, and 3 protozoa of particular relevance to SSA (5, 9-13), with the intended use for outbreak investigation and acute febrile illness (AFI) surveillance. Previous TAC assays have been developed for respiratory diseases, enteric diseases, and etiologies of neonatal sepsis (14-16), and we have shown their robust and comparable performance across several countries (17). Once developed, TaqMan array cards are stable at 4°C for 2 years, can be shipped at ambient temperature, and minimize several cumbersome steps in the field, such that they are as easy to perform as individual quantitative PCR (qPCR) assays.This work was primarily a development exercise since clinical

Background-Several viruses can cause diarrheal disease, a leading cause of morbidity and mortality worldwide. Existing diagnostic methods include ELISA and nucleic acid amplification, usually performed individually.

Abstract. 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 communitybased birth cohort in Tanzania. We tested 71 diarrheal samples and pre-diarrheal matched controls with a laboratorydeveloped 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 log 10 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 log 10 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.