Suzanne Stroup

Giardia Assemblage A Infection and Diarrhea in Bangladesh

Giardia lamblia is the most prevalent human intestinal protozoan worldwide, but only a minority of infections result in diarrhea. We tested here whether the 2 major G. lamblia genotypes, assemblages A and B, differ in their propensity to cause disease. To determine whether an association exists between infection with assemblage A or B and diarrhea, 2534 Bangladeshi patients were enrolled in a case-control study. A total of 322 Giardia infections were identified and assayed for genotype by real-time polymerase chain reaction. Higher odds ratios for diarrhea were observed for assemblage A and A2 infections, whereas higher parasite DNA loads and a higher overall prevalence were observed for assemblage B infections. Our findings indicate that genotypic differences in virulence and fecundity may help to explain why not every Giardia infection results in disease, but they need to be confirmed in other urban populations of the developing world.

Use of quantitative molecular diagnostic methods to identify causes of diarrhoea in children: a reanalysis of the GEMS case-control study

BACKGROUND Diarrhoea is the second leading cause of mortality in children worldwide, but establishing the cause can be complicated by diverse diagnostic approaches and varying test characteristics. We used quantitative molecular diagnostic methods to reassess causes of diarrhoea in the Global Enteric Multicenter Study (GEMS). METHODS GEMS was a study of moderate to severe diarrhoea in children younger than 5 years in Africa and Asia. We used quantitative real-time PCR (qPCR) to test for 32 enteropathogens in stool samples from cases and matched asymptomatic controls from GEMS, and compared pathogen-specific attributable incidences with those found with the original GEMS microbiological methods, including culture, EIA, and reverse-transcriptase PCR. We calculated revised pathogen-specific burdens of disease and assessed causes in individual children. FINDINGS We analysed 5304 sample pairs. For most pathogens, incidence was greater with qPCR than with the original methods, particularly for adenovirus 40/41 (around five times), Shigella spp or enteroinvasive Escherichia coli (EIEC) and Campylobactor jejuni o C coli (around two times), and heat-stable enterotoxin-producing E coli ([ST-ETEC] around 1·5 times). The six most attributable pathogens became, in descending order, Shigella spp, rotavirus, adenovirus 40/41, ST-ETEC, Cryptosporidium spp, and Campylobacter spp. Pathogen-attributable diarrhoeal burden was 89·3% (95% CI 83·2-96·0) at the population level, compared with 51·5% (48·0-55·0) in the original GEMS analysis. The top six pathogens accounted for 77·8% (74·6-80·9) of all attributable diarrhoea. With use of model-derived quantitative cutoffs to assess individual diarrhoeal cases, 2254 (42·5%) of 5304 cases had one diarrhoea-associated pathogen detected and 2063 (38·9%) had two or more, with Shigella spp and rotavirus being the pathogens most strongly associated with diarrhoea in children with mixed infections. INTERPRETATION A quantitative molecular diagnostic approach improved population-level and case-level characterisation of the causes of diarrhoea and indicated a high burden of disease associated with six pathogens, for which targeted treatment should be prioritised. FUNDING Bill & Melinda Gates Foundation.

Resistance of C57BL/6 Mice to Amoebiasis Is Mediated by Nonhemopoietic Cells but Requires Hemopoietic IL-10 Production

Resistance to intestinal amoebiasis is mouse strain dependent. C57BL/6 (B6) mice clear Entamoeba histolytica within hours of challenge, whereas C3H and CBA strains are susceptible to infection and disease. In this study, we show using bone marrow (BM) chimeric mice that mouse strain-dependent resistance is mediated by nonhemopoietic cells; specifically, B6 BM → CBA recipients remained susceptible as measured by amoeba score and culture, whereas CBA BM → B6 recipients remained resistant. Interestingly, hemopoietic IL-10 was required for maintaining the resistance of B6 mice, in that B6 IL-10-deficient mice and IL-10−/− BM → wild-type recipients, but not IL-10+/+ BM → IL-10−/− recipients, exhibited higher amoeba scores than their wild-type controls. Additionally, C57BL/10 IL-10−/−Rag2−/− mice exhibited diminished amoeba scores and culture rates vs IL-10−/− mice, indicating that lymphocytes potentiated the susceptibility of IL-10-deficient mice. We conclude that nonhemopoietic cells mediate the natural resistance to intestinal amoebiasis of B6 mice, yet this resistance depends on hemopoietic IL-10 activity.

Comparative Genomic Hybridizations of Entamoeba Strains Reveal Unique Genetic Fingerprints That Correlate with Virulence

ABSTRACT Variable phenotypes have been identified for Entamoeba species. Entamoeba histolytica is invasive and causes colitis and liver abscesses but only in ∼10% of infected individuals; 90% remain asymptomatically colonized. Entamoeba dispar, a closely related species, is avirulent. To determine the extent of genetic diversity among Entamoeba isolates and potential genotype-phenotype correlations, we have developed an E. histolytica genomic DNA microarray and used it to genotype strains of E. histolytica and E. dispar. On the basis of the identification of divergent genetic loci, all strains had unique genetic fingerprints. Comparison of divergent genetic regions allowed us to distinguish between E. histolytica and E. dispar, identify novel genetic regions usable for strain and species typing, and identify a number of genes restricted to virulent strains. Among the four E. histolytica strains, a strain with attenuated virulence was the most divergent and phylogenetically distinct strain, raising the intriguing possibility that genetic subtypes of E. histolytica may be partially responsible for the observed variability in clinical outcomes. This microarray-based genotyping assay can readily be applied to the study of E. histolytica clinical isolates to determine genetic diversity and potential genotypic-phenotypic associations.

Microbiologic Methods Utilized in the MAL-ED Cohort Study

A central hypothesis of The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) study is that enteropathogens contribute to growth faltering. To examine this question, the MAL-ED network of investigators set out to achieve 3 goals: (1) develop harmonized protocols to test for a diverse range of enteropathogens, (2) provide quality-assured and comparable results from 8 global sites, and (3) achieve maximum laboratory throughput and minimum cost. This paper describes the rationale for the microbiologic assays chosen and methodologies used to accomplish the 3 goals.

Discordance across Several Methods for Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates in a Single Laboratory

ABSTRACT Given the increases in drug-resistant tuberculosis, laboratory capacities for drug susceptibility testing are being scaled up worldwide. A laboratory must decide among several endorsed methodologies. We evaluated 87 Mycobacterium tuberculosis isolates for concordance of susceptibility results across six methods: the L-J proportion method, MGIT 960 SIRE AST, Gene/Xpert MTB/RIF, GenoType MTBDRplus line probe assay, MycoTB MIC plate, and a laboratory-developed mycobacteriophage quantitative PCR (qPCR)-based method. Most (80%) isolates were multidrug resistant. Of the culture-based methods, the mycobacteriophage qPCR method was fastest, the L-J proportion method was the slowest, and the MGIT method required the most repeat testing (P < 0.05). For isoniazid (INH), 82% of isolates were susceptible by all methods or resistant by all methods, whereas for rifampin (RIF), ethambutol (EMB), and streptomycin (STR), such complete concordance was observed in 77%, 50%, and 51% of isolates, respectively (P < 0.05 for INH or RIF versus EMB or STR). The discrepancies of EMB and STR stemmed largely from diminished concordance of the MGIT EMB results (kappa coefficient range, 0.26 to 0.30) and the L-J STR result (kappa range, 0.35 to 0.45) versus other methods. Phage qPCR and the MycoTB MIC plate were the only methods that yielded second-line susceptibilities and revealed significant quantitative correlations for all drugs except cycloserine, as well as moderate to excellent kappa coefficients for all drugs except for para-aminosalicylic acid. In summary, the performance of M. tuberculosis susceptibility testing differs by platform and by drug. Laboratories should carefully consider these factors before choosing one methodology, particularly in settings where EMB and STR results are clinically important.

Digital PCR to Detect and Quantify Heteroresistance in Drug Resistant Mycobacterium tuberculosis

Drug resistance in Mycobacterium tuberculosis presents an enormous public health threat. It is typically defined as >1% of drug resistant colonies using the agar proportion method. Detecting small numbers of drug resistant Tb in a population, also known as heteroresistance, is challenging with current methodologies. Here we have utilized digital PCR to detect heteroresistance within M. tuberculosis populations with excellent accuracy versus the agar proportion method. We designed dual TaqMan-MGB probes to detect wild-type and mutant sequences of katG (315), rpoB (531), gyrA (94,95) and rrs (1401), genes that associate with resistance to isoniazid, rifampin, fluoroquinolone, and aminoglycoside respectively. We generated heteroresistant mixtures of susceptible and extensively drug resistant Tb, followed by DNA extraction and digital PCR. Digital PCR yielded a close approximation to agar proportions percentages of resistant colonies, and yielded 100% concordance with agar proportions susceptible/resistant results. Indeed, the digital PCR method was able to identify mutant sequence in mixtures containing as little as 1000∶1 susceptible:resistant Tb. By contrast, real-time PCR or PCR followed by Sanger sequencing were less sensitive and had little resolution to detect heteroresistance, requiring fully 1∶1 or 10∶1 susceptible:resistant ratios in order to detect resistance. Our assay can also work in sputum so long as sufficient quantities of Tb are present (>1000 cfu/ml). This work demonstrates the utility of digital PCR to detect and quantify heteroresistance in drug resistant Tb, which may be useful to inform treatment decisions faster than agar proportion.

Ehrlichia and Spotted Fever Group Rickettsiae Surveillance in Amblyomma americanum in Virginia Through Use of a Novel Six-Plex Real-Time PCR Assay

The population of the lone star tick Amblyomma americanum has expanded in North America over the last several decades. It is known to be an aggressive and nondiscriminatory biter and is by far the most common human-biting tick encountered in Virginia. Few studies of human pathogen prevalence in ticks have been conducted in our state since the mid-twentieth century. We developed a six-plex real-time PCR assay to detect three Ehrlichia species (E. chaffeensis, E. ewingii, and Panola Mountain Ehrlichia) and three spotted fever group Rickettsiae (SFGR; R. amblyommii, R. parkeri, and R. rickettsii) and used it to test A. americanum from around the state. Our studies revealed a presence of all three Ehrlichia species (0-24.5%) and a high prevalence (50-80%) of R. amblyommii, a presumptively nonpathogenic SFGR, in all regions surveyed. R. parkeri, previously only detected in Virginias Amblyomma maculatum ticks, was found in A. americanum in several surveyed areas within two regions having established A. maculatum populations. R. rickettsii was not found in any sample tested. Our study provides the first state-wide screening of A. americanum ticks in recent history and indicates that human exposure to R. amblyommii and to Ehrlichiae may be common. The high prevalence of R. amblyommii, serological cross-reactivity of all SFGR members, and the apparent rarity of R. rickettsii in human biting ticks across the eastern United States suggest that clinical cases of tick-borne disease, including ehrlichiosis, may be commonly misdiagnosed as Rocky Mountain spotted fever, and that suspicion of other SFGR as well as Ehrlichia should be increased. These data may be of relevance to other regions where A. americanum is prevalent.

Integrated Microfluidic Card with TaqMan Probes and High-Resolution Melt Analysis To Detect Tuberculosis Drug Resistance Mutations across 10 Genes

ABSTRACT Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes. IMPORTANCE Multidrug-resistant tuberculosis threatens global tuberculosis control efforts. Optimal therapy utilizes susceptibility test results to guide individualized treatment regimens; however, the susceptibility testing methods in use are technically difficult and slow. We developed an integrated TaqMan array card method with high-resolution melt analysis that interrogates 10 genes to yield a fast, comprehensive, and accurate drug susceptibility result for the 9 major antituberculosis antibiotics. Multidrug-resistant tuberculosis threatens global tuberculosis control efforts. Optimal therapy utilizes susceptibility test results to guide individualized treatment regimens; however, the susceptibility testing methods in use are technically difficult and slow. We developed an integrated TaqMan array card method with high-resolution melt analysis that interrogates 10 genes to yield a fast, comprehensive, and accurate drug susceptibility result for the 9 major antituberculosis antibiotics.

Rapid First- and Second-Line Drug Susceptibility Assay for Mycobacterium tuberculosis Isolates by Use of Quantitative PCR

ABSTRACT The slow turnaround time for Mycobacterium tuberculosis drug susceptibility results is a barrier to care. We developed a rapid quantitative PCR (qPCR)-based phenotypic antimicrobial susceptibility test that utilizes amplification of the M. tuberculosis 16S rRNA gene after 3 days of incubation with antituberculosis drugs. To decrease background from killed organisms, we used propidium monoazide (PMA), a DNA-binding dye that penetrates damaged bacterial cells and renders DNA unamplifiable. M. tuberculosis was cultured in broth media containing PMA with or without drugs for 3 days prior to DNA extraction and real-time PCR amplification. 16S rRNA qPCR exhibited a significant decrease in threshold cycle (CT ) time values (CT control − CT drug treated) with drug-susceptible strains compared with resistant strains. Susceptibility data were reported as ΔCT or as 2Δ CT and with appropriate cutoffs yielded an accuracy of 89 to 100% on 38 susceptible, multidrug-resistant, and extensively drug-resistant strains compared with conventional agar proportion susceptibility results for isoniazid, rifampin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide, para-aminosalicylic acid, linezolid, and cycloserine and compared with Bactec MGIT results for pyrazinamide. This PMA-qPCR assay is useful as a rapid 3-day first- and second-line drug susceptibility test for M. tuberculosis.