Publications (10)31.93 Total impact
-
Article: Development of a multiplex polymerase chain reaction assay for diarrheagenic Escherichia coli and Shigella spp. and its evaluation on colonies, culture broths, and stool.
[show abstract] [hide abstract]
ABSTRACT: Detection of diarrheagenic Escherichia coli (DEC) typically depends on identification of virulence genes from stool cultures, not on stool itself. We developed a multiplex polymerase chain reaction (PCR) assay that detects key DEC virulence genes (stx1, stx2, eae, bfpA, ipaH, LT, STh, aaiC, aatA). The assay involved a multiplex PCR reaction followed by detection of amplicon(s) using Luminex beads. The assay was evaluated on over 100 colony and broth specimens. We then evaluated the assay using DNA extracted from stool, colony pools, and Gram-negative broths, using stool spiked with known quantities of DEC. Performance of the assay on stool DNA was most quantitative, while stool broth DNA offered the lowest limit of detection. The assay was prospectively evaluated on clinical specimens in Tanzania. Stool DNA yielded higher sensitivity than colony pools compared with broth DNA as the standard. We propose using this assay to screen for DEC directly in stool or stool broths.Diagnostic microbiology and infectious disease 04/2012; 73(2):121-8. · 2.45 Impact Factor -
Article: Multiplex polymerase chain reaction method to detect Cyclospora, Cystoisospora, and Microsporidia in stool samples.
[show abstract] [hide abstract]
ABSTRACT: 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.Diagnostic microbiology and infectious disease 12/2011; 71(4):386-90. · 2.45 Impact Factor -
Article: Molecular techniques in ecohealth research toolkit: facilitating estimation of aggregate gastroenteritis burden in an irrigated periurban landscape.
[show abstract] [hide abstract]
ABSTRACT: Assessment of microbial hazards associated with certain environmental matrices, livelihood strategies, and food handling practices are constrained by time-consuming conventional microbiological techniques that lead to health risk assessments of narrow geographic or time scope, often targeting very few pathogens. Health risk assessment based on one or few indicator organisms underestimates true disease burden due a number of coexisting causative pathogens. Here, we employed molecular techniques in a survey of Cryptosporidium parvum, Giardia lamblia, Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., Shigella spp., Vibrio cholera, and Rotavirus A densities in canal water with respect to seasonality and spatial distribution of point-nonpoint pollution sources. Three irrigational canals stretching across nearly a 150-km(2) periurban landscape, traditionally used for agricultural irrigation but function as vital part of municipal wastewater stabilization in recent years, were investigated. Compiled stochastic data (pathogen concentration, susceptible populations) and literature-obtained deterministic data (pathogen dose-response model parameter values) were used in estimating waterborne gastroenteritis burden. Exposure scenarios include swimming or fishing, consuming canal water-irrigated vegetables, and ingesting or inhaling water aerosols while working in canal water-irrigated fields. Estimated annual gastroenteritis burden due individual pathogens among the sampling points was -10.6log(10) to -2.2log(10) DALYs. Aggregated annual gastroenteritis burden due all the target pathogens per sampling point was -3.1log(10) to -1.9log(10) DALYs, far exceeding WHO acceptable limit of -6.0log(10) DALYs. The present approach will facilitate the comprehensive collection of surface water microbiological baseline data and setting of benchmarks for interventions aimed at reducing microbial hazards in similar landscapes worldwide.EcoHealth 09/2011; 8(3):349-64. · 1.70 Impact Factor -
Article: Human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections.
[show abstract] [hide abstract]
ABSTRACT: A new species of parvovirus, tentatively named human bocavirus 4 (HBoV4), was genetically characterized. Among 641 feces samples obtained from children and adults, the most commonly detected bocavirus species were, in descending order, HBoV2, HBoV3, HBoV4, and HBoV1, with an HBoV2 prevalence of 21% and 26% in Nigerian and Tunisian children, respectively. HBoV3 or HBoV4 species were found in 12 of 192 patients with non-polio acute flaccid paralysis in Tunisia and Nigeria and 0 of 96 healthy Tunisian contacts (P = .01). Evidence of extensive recombination at the NP1 and VP1 gene boundary between and within bocavirus species was found. The high degree of genetic diversity seen among the human bocaviruses found in feces specimens, relative to the highly homogeneous HBoV1, suggest that this worldwide-distributed respiratory pathogen may have recently evolved from an enteric bocavirus after acquiring an expanded tropism favoring the respiratory tract. Elucidating the possible role of the newly identified enteric bocaviruses in human diseases, including acute flaccid paralysis and diarrhea, will require further epidemiological studies.The Journal of Infectious Diseases 06/2010; 201(11):1633-43. · 6.41 Impact Factor -
Article: Human Bocaviruses Are Highly Diverse, Dispersed, Recombination Prone, and Prevalent in Enteric Infections
[show abstract] [hide abstract]
ABSTRACT: A new species of parvovirus, tentatively named human bocavirus 4 (HBoV4), was genetically characterized. Among 641 feces samples obtained from children and adults, the most commonly detected bocavirus species were, in descending order, HBoV2, HBoV3, HBoV4, and HBoV1, with an HBoV2 prevalence of 21% and 26% in Nigerian and Tunisian children, respectively. HBoV3 or HBoV4 species were found in 12 of 192 patients with non-polio acute flaccid paralysis in Tunisia and Nigeria and 0 of 96 healthy Tunisian contacts (P p). Evidence of extensive recombination at the NP1 and VP1 gene boundary between and within bocavirus .01 species was found. The high degree of genetic diversity seen among the human bocaviruses found in feces specimens, relative to the highly homogeneous HBoV1, suggest that this worldwide-distributed respiratory pathogen may have recently evolved from an enteric bocavirus after acquiring an expanded tropism favoring the respiratory tract. Elucidating the possible role of the newly identified enteric bocaviruses in human diseases, including acute flaccid paralysis and diarrhea, will require further epidemiological studies.The Journal of Infectious Diseases 06/2010; 201(11):1633-1643. · 6.41 Impact Factor -
Article: Antimicrobial resistance patterns and prevalence of class 1 and 2 integrons in Shigella flexneri and Shigella sonnei isolated in Uzbekistan.
[show abstract] [hide abstract]
ABSTRACT: Shigella is a frequent cause of bacterial dysentery in the developing world. Treatment with effective antibiotics is recommended for shigellosis, but options become limited due to globally emerging resistance. One of the mechanisms for the development of resistance utilizes integrons. This study described the antibiotic susceptibility and the presence of class 1 and 2 integrons in S. flexneri and S. sonnei isolated in Uzbekistan. We studied 31 isolates of S. flexneri and 21 isolates of S. sonnei isolated in Uzbekistan between 1992 and 2007 for the susceptibility or resistance to ampicillin (Am), chloramphenicol (Cl), tetracycline (Te), co-trimoxazole (Sxt), kanamycin (Km), streptomycin (Str), gentamicin (Gm), cefazolin (Czn), cefoperazone (Cpr), cefuroxime (Cur), ceftazidime (Ctz), nalidixic acid (NA) and ciprofloxacin (Cip). Am/Str/Cl/Te and Am/Str/Cl/Te/Sxt resistance patterns were found most frequently in S. flexneri. Single isolates were resistant to aminoglycoside, quinolones and cephalosporins. The resistance patterns were different in the two species. Integrons were detected in 93.5% of S. flexneri (29/31) and 81.0% of S. sonnei (17/21) isolates. In addition, 61.3% of S. flexneri (19/31) isolates and 19.0% of S. sonnei (4/21) isolates carried both classes of integrons. In 29.0% of S. flexneri (9/31) isolates, only class 1 integrons were identified. In S. flexneri isolates, the presence of class 1 integrons was associated with resistance to ampicillin and chloramphenicol. Only Class 2 integrons were present in 61.9% of S. sonnei (13/21) isolates. Our study documents antibiotic resistance among Shigella spp. in Uzbekistan. Ninety percent of Shigella strains were resistant to previously used antibiotics. Differences among S. flexneri and S. sonnei isolates in patterns of antimicrobial resistance to routinely used shigellosis antibiotics were observed. The majority of S. flexneri were resistant to ampicillin, chloramphenicol, tetracycline and streptomycin. Class 1 and 2 integrons were widely present in these Shigella strains. Resistance to ampicillin/chloramphenicol was associated with the presence of class 1 integrons. Though several mechanisms are possible, the resistance of Shigella isolates to ampicillin/chloramphenicol may be associated with the expression of genes within class 1 integrons.Gut Pathogens 01/2010; 2(1):18. · 2.11 Impact Factor -
Article: Diagnostic approach to acute diarrheal illness in a military population on training exercises in Thailand, a region of campylobacter hyperendemicity.
[show abstract] [hide abstract]
ABSTRACT: High rates of Campylobacter fluoroquinolone resistance highlight the need to evaluate diagnostic strategies that can be used to assist with clinical management. Diagnostic tests were evaluated with U.S. soldiers presenting with acute diarrhea during deployment in Thailand. The results of bedside and field laboratory diagnostic tests were compared to stool microbiology findings for 182 enrolled patients. Campylobacter jejuni was isolated from 62% of the cases. Clinical and laboratory findings at the time of presentation were evaluated to determine their impact on the posttest probability, defined as the likelihood of a diagnosis of Campylobacter infection. Clinical findings, the results of tests for inflammation (stool occult blood testing [Hemoccult], fecal leukocytes, fecal lactoferrin, plasma C-reactive protein), and the numbers of Campylobacter-specific antibody-secreting cells in peripheral blood failed to increase the posttest probability above 90% in this setting of Campylobacter hyperendemicity when these findings were present. Positive results by a Campylobacter-specific commercial enzyme immunoassay (EIA) and, less so, a research PCR were strong positive predictors. The negative predictive value for ruling out Campylobacter infection, defined as a posttest probability of less than 10%, was similarly observed with these Campylobacter-specific stool-based tests as well the fecal leukocyte test. Compared to the other tests evaluated, the Campylobacter EIA is a sensitive and specific rapid diagnostic test that may assist with diagnostic evaluation, with consideration of the epidemiological setting, logistics, and cost.Journal of clinical microbiology 05/2008; 46(4):1418-25. · 4.16 Impact Factor -
Article: An AFLP-based database of Shigella flexneri and Shigella sonnei isolates and its use for the identification of untypable Shigella strains.
[show abstract] [hide abstract]
ABSTRACT: Amplified fragment length polymorphism (AFLP) can be used to assess the genetic diversity of closely related microbial genomes. In this study, the first of its kind for identification of Shigella, the high discriminatory power of AFLP has been used to determine the genetic relatedness of 230 isolates of Shigella flexneri and Shigella sonnei strains. An AFLP database was generated to demonstrate its utility in the discrimination of closely related strains. Based on AFLP, S. flexneri strains could be grouped into separate clusters according to their serotypes. Within each serotype, strains demonstrated 80-100% similarity indicating that identical strains and closely related strains could be distinguished by this technique. S. flexneri 6 formed a distinct cluster with 55% similarity to the rest of the S. flexneri strains showing significant divergence from the rest of the S. flexneri strains. Significantly, S. sonnei isolates formed a distinct group and showed approximately the same level of genetic linkage to S. flexneri as Escherichia coli strains. Untypable isolates that showed conflicting agglutination reactions with conventional typing sera were identifiable by AFLP. Thus AFLP can be used for genetic fingerprinting of Shigella strains and aid in the identification of variant untypable isolates.Journal of Microbiological Methods 01/2007; 67(3):487-95. · 2.09 Impact Factor -
Article: Detection of Shigella by a PCR assay targeting the ipaH gene suggests increased prevalence of shigellosis in Nha Trang, Vietnam.
[show abstract] [hide abstract]
ABSTRACT: Shigella spp. are exquisitely fastidious gram-negative organisms which frequently escape detection by traditional culture methods. To get a more complete understanding of the disease burden caused by Shigella in Nha Trang, Vietnam, real-time PCR was used to detect Shigella DNA. Randomly selected rectal swab specimens from 60 Shigella culture-positive patients and 500 Shigella culture-negative patients detected by population-based surveillance of patients seeking care for diarrhea were processed by real-time PCR. The target of the primer pair is the invasion plasmid antigen H gene sequence (ipaH), carried by all four Shigella species and enteroinvasive Escherichia coli. Shigella spp. could be isolated from the rectal swabs of 547 of 19,206 (3%) patients with diarrhea. IpaH was detected in 55 of 60 (93%) Shigella culture-positive specimens, whereas it was detected in 87 of 245 (36%) culture-negative patients free of dysentery (P < 0.001). The number of PCR cycles required to detect a PCR product was highest for culture-negative, nonbloody diarrheal specimens (mean number of cycles to detection, 36.6) and was lowest for children with culture-positive, bloody diarrheal specimens (mean number of cycles, 25.3) (P < 0.001). The data from real-time PCR amplification indicate that the culture-proven prevalence of Shigella among patients with diarrhea may underestimate the prevalence of Shigella infections. The clinical presentation of shigellosis may be directly related to the bacterial load.Journal of Clinical Microbiology 06/2004; 42(5):2031-5. · 4.15 Impact Factor -
Article: A simple polymerase chain reaction technique to detect and differentiate Shigella and enteroinvasive escherichia coli in human feces
[show abstract] [hide abstract]
ABSTRACT: A simple polymerase chain reaction (PCR) procedure using IS630-specific primers was developed as a general diagnostic probe to detect Shigella and enteroinvasive Escherichia coli (EIEC). However, IS630 and the other two previously reported molecular probes, ipaH and ial, cannot be used to differentiate among Shigella serotypes and EIEC strains that cause dysentery. The sensitivity of PCR protocol was determined to be 100–200 shigellae for each PCR reaction. An enrichment incubation would allow the detection of shigellae in stool samples with low bacterial concentration; i.e., <104 CFU/gram. Serotype-specific primers derived from the rfc genes of different Shigella strains were used in PCR reactions to differentiate among Shigella serotypes in the laboratory, such as S. sonnei, S. flexneri, and S. dysenteriae 1. It was demonstrated further that the multiplex PCR system containing rfc-specific primers can efficiently identify the most prominent Shigella serotypes in raw stool samples of acute diarrheal patients.Diagnostic Microbiology and Infectious Disease.
