First description and phylogenetic analysis of coxsackie virus A non-polio enteroviruses and parechoviruses A in South Sudanese children.

Enteroviruses (EV) and parechoviruses A (PeV-A) are commonly circulating viruses able to cause severe disease. Surveillance studies from sub-Saharan Africa are limited and show high but variable infection rates and a high variation in genotypes. This is the first study to describe EV and PeV-A circulation in children in South Sudan. Of the fecal samples collected, 35% and 10% were positive for EV and PeV-A, respectively. A wide range of genotypes were found, including several rarely described EV and PeV-A types. Coxsackie virus A (CVA) EV-C types, particularly CVA13, were the most dominant EV types. The CVA13 types had a high diversity with the majority belonging to four different previously described clusters. PeV-A1 and -A14 were the most common PeV-A genotypes. A lack of representative data from our and other studies from sub-Saharan Africa demonstrates the need for more systematic surveillance of non-polio EV and PeV-A types in this region.

Surveillance of leishmaniasis cases from 15 European centres, 2014 to 2019: a retrospective analysis.

BackgroundSurveillance of human leishmaniasis in Europe is mostly limited to country-specific information from autochthonous infections in the southern part. As at the end of 2021, no integrated analysis has been performed for cases seen across centres in different European countries.AimTo provide a broad perspective on autochthonous and imported leishmaniasis cases in endemic and non-endemic countries in Europe.MethodsWe retrospectively collected records from cutaneous, mucosal and visceral leishmaniasis cases diagnosed in 15 centres between 2014 and 2019. Centres were located in 11 countries: Belgium, France, Germany, Italy, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Data on country of infection, reason for travelling, infecting species, age and sex were analysed.ResultsWe obtained diagnostic files from 1,142 cases, of which 76%, 21% and 3% had cutaneous, visceral, and mucosal disease, respectively. Of these, 68% were men, and 32% women, with the median age of 37 years (range: 0-90) at diagnosis. Visceral leishmaniasis was mainly acquired in Europe (88%; 167/190), while cutaneous leishmaniasis was primarily imported from outside Europe (77%; 575/749). Sixty-two percent of cutaneous leishmaniasis cases from outside Europe were from the Old World, and 38% from the New World. Geographic species distribution largely confirmed known epidemiology, with notable exceptions.ConclusionsOur study confirms previous reports regarding geographic origin, species, and traveller subgroups importing leishmaniasis into Europe. We demonstrate the importance of pooling species typing data from many centres, even from areas where the aetiology is presumably known, to monitor changing epidemiology.

The Impact of Intensive Versus Standard Anthelminthic Treatment on Allergy-related Outcomes, Helminth Infection Intensity, and Helminth-related Morbidity in Lake Victoria Fishing Communities, Uganda: Results From the LaVIISWA Cluster-randomized Trial.

BACKGROUND: The prevalence of allergy-related diseases is increasing in low-income countries. Parasitic helminths, common in these settings, may be protective. We hypothesized that intensive, community-wide, anthelminthic mass drug administration (MDA) would increase allergy-related diseases, while reducing helminth-related morbidity. METHODS: In an open, cluster-randomized trial (ISRCTN47196031), we randomized 26 high-schistosomiasis-transmission fishing villages in Lake Victoria, Uganda, in a 1:1 ratio to receive community-wide intensive (quarterly single-dose praziquantel plus albendazole daily for 3 days) or standard (annual praziquantel plus 6 monthly single-dose albendazole) MDA. Primary outcomes were recent wheezing, skin prick test positivity (SPT), and allergen-specific immunoglobulin E (asIgE) after 3 years of intervention. Secondary outcomes included helminths, haemoglobin, and hepatosplenomegaly. RESULTS: The outcome survey comprised 3350 individuals. Intensive MDA had no effect on wheezing (risk ratio [RR] 1.11, 95% confidence interval [CI] 0.64-1.93), SPT (RR 1.10, 95% CI 0.85-1.42), or asIgE (RR 0.96, 95% CI 0.82-1.12). Intensive MDA reduced Schistosoma mansoni infection intensity: the prevalence from Kato Katz examinations of single stool samples from each patient was 23% versus 39% (RR 0.70, 95% CI 0.55-0.88), but the urine circulating cathodic antigen test remained positive in 85% participants in both trial arms. Hookworm prevalence was 8% versus 11% (RR 0.55, 95% CI 0.31-1.00). There were no differences in anemia or hepatospenomegaly between trial arms. CONCLUSIONS: Despite reductions in S. mansoni intensity and hookworm prevalence, intensive MDA had no effect on atopy, allergy-related diseases, or helminth-related pathology. This could be due to sustained low-intensity infections; thus, a causal link between helminths and allergy outcomes cannot be discounted. Intensive community-based MDA has a limited impact in high-schistosomiasis-transmission fishing communities, in the absence of other interventions. CLINICAL TRIALS REGISTRATION: ISRCTN47196031.

Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC ß-lactamase production in Escherichia coli by elaborate phenotypic and genotypic characterization.

OBJECTIVES: AmpC-ß-lactamase production is an under-recognized antibiotic resistance mechanism that renders Gram-negative bacteria resistant to common ß-lactam antibiotics, similar to the well-known ESBLs. For infection control purposes, it is important to be able to discriminate between plasmid-mediated AmpC (pAmpC) production and chromosomal-mediated AmpC (cAmpC) hyperproduction in Gram-negative bacteria as pAmpC requires isolation precautions to minimize the risk of horizontal gene transmission. Detecting pAmpC in Escherichia coli is challenging, as both pAmpC production and cAmpC hyperproduction may lead to third-generation cephalosporin resistance. METHODS: We tested a collection of E. coli strains suspected to produce AmpC. Elaborate susceptibility testing for third-generation cephalosporins, WGS and machine learning were used to develop an algorithm to determine ampC genotypes in E. coli. WGS was applied to detect pampC genes, cAmpC hyperproducers and STs. RESULTS: In total, 172 E. coli strains (n=75 ST) were divided into a training set and two validation sets. Ninety strains were pampC positive, the predominant gene being blaCMY-2 (86.7%), followed by blaDHA-1 (7.8%), and 59 strains were cAmpC hyperproducers. The algorithm used a cefotaxime MIC value above 6 mg/L to identify pampC-positive E. coli and an MIC value of 0.5 mg/L to discriminate between cAmpC-hyperproducing and non-cAmpC-hyperproducing E. coli strains. Accuracy was 0.88 (95% CI=0.79-0.94) on the training set, 0.79 (95% CI=0.64-0.89) on validation set 1 and 0.85 (95% CI=0.71-0.94) on validation set 2. CONCLUSIONS: This approach resulted in a pragmatic algorithm for differentiating ampC genotypes in E. coli based on phenotypic susceptibility testing.

Community deworming alleviates geohelminth-induced immune hyporesponsiveness.

In cross-sectional studies, chronic helminth infections have been associated with immunological hyporesponsiveness that can affect responses to unrelated antigens. To study the immunological effects of deworming, we conducted a cluster-randomized, double-blind, placebo-controlled trial in Indonesia and assigned 954 households to receive albendazole or placebo once every 3 mo for 2 y. Helminth-specific and nonspecific whole-blood cytokine responses were assessed in 1,059 subjects of all ages, whereas phenotyping of regulatory molecules was undertaken in 121 school-aged children. All measurements were performed before and at 9 and 21 mo after initiation of treatment. Anthelmintic treatment resulted in significant increases in proinflammatory cytokine responses to Plasmodium falciparum-infected red blood cells (PfRBCs) and mitogen, with the largest effect on TNF responses to PfRBCs at 9 mo-estimate [95% confidence interval], 0.37 [0.21-0.53], P value over time (Ptime) < 0.0001. Although the frequency of regulatory T cells did not change after treatment, there was a significant decline in the expression of the inhibitory molecule cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) on CD4+ T cells of albendazole-treated individuals, -0.060 [-0.107 to -0.013] and -0.057 [-0.105 to -0.008] at 9 and 21 mo, respectively; Ptime = 0.017. This trial shows the capacity of helminths to up-regulate inhibitory molecules and to suppress proinflammatory immune responses in humans. This could help to explain the inferior immunological responses to vaccines and lower prevalence of inflammatory diseases in low- compared with high-income countries.

Developing a real-time PCR assay based on multiplex high-resolution melt-curve analysis: a pilot study in detection and discrimination of soil-transmitted helminth and schistosome species.

With the push towards control and elimination of soil-transmitted helminthiasis and schistosomiasis in low- and middle-income countries, there is a need to develop alternative diagnostic assays that complement the current in-country resources, preferably at a lower cost. Here, we describe a novel high-resolution melt (HRM) curve assay with six PCR primer pairs, designed to sub-regions of the nuclear ribosomal locus. Used within a single reaction and dye detection channel, they are able to discriminate Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Ascaris lumbricoides, Trichuris trichiuria and Schistosoma spp. by HRM curve analysis. Here we describe the primers and the results of a pilot assessment whereby the HRM assay was tested against a selection of archived fecal samples from Ghanaian children as characterized by Kato-Katz and real-time PCR analysis with species-specific TaqMan hydrolysis probes. The resulting sensitivity and specificity of the HRM was 80 and 98.6% respectively. We judge the assay to be appropriate in modestly equipped and resourced laboratories. This method provides a potentially cheaper alternative to the TaqMan method for laboratories in lower resource settings. However, the assay requires a more extensive assessment as the samples used were not representative of all target organisms.

Improved diagnosis of Trichuris trichiura by using a bead-beating procedure on ethanol preserved stool samples prior to DNA isolation and the performance of multiplex real-time PCR for intestinal parasites.

For the majority of intestinal parasites, real-time PCR-based diagnosis outperforms microscopy. However, the data for Trichuris trichiura have been less convincing and most comparative studies have been performed in populations with low prevalence. This study aims to improve detection of T. trichuria DNA in human stool by evaluating four sample preparation methods. Faecal samples (n = 60) were collected at Flores island, Indonesia and examined by microscopy. Aliquots were taken and a bead-beating procedure was used both on directly frozen stool and on material preserved with 96% ethanol. PCR on frozen samples showed 40% to be positive for T. trichiura, compared with 45% positive by microscopy. The percentage positive increased when using ethanol preservation (45·0%), bead-beating (51·7%) and a combination (55·0%) and all three methods showed significantly higher DNA loads. The various procedures had a less pronounced effect on the PCR results of nine other parasite targets tested. Most prevalent were Ascaris lumbricoides (≈60%), Necator americanus (≈60%), Dientamoeba fragilis (≈50%) and Giardia lamblia (≈12%). To validate the practicality of the procedure, bead-beating was applied in a population-based survey testing 910 stool samples. Findings confirmed bead-beating before DNA extraction to be a highly efficient procedure for the detection of T. trichiura DNA in stool.

Protection against malaria after immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity.

Volunteers immunized under chloroquine chemoprophylaxis with Plasmodium falciparum sporozoites (CPS) develop complete, long-lasting protection against homologous sporozoite challenge. Chloroquine affects neither sporozoites nor liver-stages, but kills only asexual forms in erythrocytes once released from the liver into the circulation. Consequently, CPS immunization exposes the host to antigens from both preerythrocytic and blood stages, and induced immunity might target either of these stages. We therefore explored the life cycle stage specificity of CPS-induced protection. Twenty-five malaria-naïve volunteers were enrolled in a clinical trial, 15 of whom received CPS immunization. Five immunized subjects and five controls received a sporozoite challenge by mosquito bites, whereas nine immunized and five control subjects received an i.v. challenge with P. falciparum-infected erythrocytes. The latter approach completely bypasses preerythrocytic stages, enabling a direct comparison of protection against either life cycle stage. CPS-immunized subjects (13 of 14) developed anticircumsporozoite antibodies, whereas only one volunteer generated minimal titers against typical blood-stage antigens. IgG from CPS-immunized volunteers did not inhibit asexual blood-stage growth in vitro. All CPS-immunized subjects (5 of 5) were protected against sporozoite challenge. In contrast, nine of nine CPS-immunized subjects developed parasitemia after blood-stage challenge, with identical prepatent periods and blood-stage multiplication rates compared with controls. Intravenously challenged CPS-immunized subjects showed earlier fever and increased plasma concentrations of inflammatory markers D-dimer, IFN-γ, and monokine induced by IFN-γ than i.v. challenged controls. The complete lack of protection against blood-stage challenge indicates that CPS-induced protection is mediated by immunity against preerythrocytic stages. However, evidence is presented for immune recognition of P. falciparum-infected erythrocytes, suggesting memory responses unable to generate functional immunity.

Increase in ECHOvirus 6 infections associated with neurological symptoms in the Netherlands, June to August 2016.

The Dutch virus-typing network VIRO-TypeNed reported an increase in ECHOvirus 6 (E-6) infections with neurological symptoms in the Netherlands between June and August 2016. Of the 31 cases detected from January through August 2016, 15 presented with neurological symptoms. Ten of 15 neurological cases were detected in the same province and the identified viruses were genetically related. This report is to alert medical and public health professionals of the circulation of E-6 associated with neurological symptoms.

VIRO-TypeNed, systematic molecular surveillance of enteroviruses in the Netherlands between 2010 and 2014.

VIRO-TypeNed is a collaborative molecular surveillance platform facilitated through a web-based database. Genetic data in combination with epidemiological, clinical and patient data are shared between clinical and public health laboratories, as part of the surveillance underpinning poliovirus eradication. We analysed the combination of data submitted from 2010 to 2014 to understand circulation patterns of non-polio enteroviruses (NPEV) of public health relevance. Two epidemiological patterns were observed based on VIRO-TypeNed data and classical surveillance data dating back to 1996: (i) endemic cyclic, characterised by predictable upsurges/outbreaks every two to four years, and (ii) epidemic, where rare virus types caused upsurges/outbreaks. Genetic analysis suggests continuous temporal displacement of virus lineages due to the accumulation of (silent) genetic changes. Non-synonymous changes in the antigenic B/C loop suggest antigenic diversification, which may affect population susceptibility. Infections were frequently detected at an age under three months and at an older, parenting age (25-49 years) pointing to a distinct role of immunity in the circulation patterns. Upsurges were detected in the summer and winter which can promote increased transmissibility underlying new (cyclic) upsurges and requires close monitoring. The combination of data provide a better understanding of NPEV circulation required to control and curtail upsurges and outbreaks.

Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014.

Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally.

Molecular testing for clinical diagnosis and epidemiological investigations of intestinal parasitic infections.

Over the past few decades, nucleic acid-based methods have been developed for the diagnosis of intestinal parasitic infections. Advantages of nucleic acid-based methods are numerous; typically, these include increased sensitivity and specificity and simpler standardization of diagnostic procedures. DNA samples can also be stored and used for genetic characterization and molecular typing, providing a valuable tool for surveys and surveillance studies. A variety of technologies have been applied, and some specific and general pitfalls and limitations have been identified. This review provides an overview of the multitude of methods that have been reported for the detection of intestinal parasites and offers some guidance in applying these methods in the clinical laboratory and in epidemiological studies.

Effect of Daily Antenatal Iron Supplementation on Plasmodium Infection in Kenyan Women: A Randomized Clinical Trial.

IMPORTANCE: Anemia affects most pregnant African women and is predominantly due to iron deficiency, but antenatal iron supplementation has uncertain health benefits and can increase the malaria burden. OBJECTIVE: To measure the effect of antenatal iron supplementation on maternal Plasmodium infection risk, maternal iron status, and neonatal outcomes. DESIGN, SETTING, AND PARTICIPANTS: Randomized placebo-controlled trial conducted October 2011 through April 2013 in a malaria endemic area among 470 rural Kenyan women aged 15 to 45 years with singleton pregnancies, gestational age of 13 to 23 weeks, and hemoglobin concentration of 9 g/dL or greater. All women received 5.7 mg iron/day through flour fortification during intervention, and usual intermittent preventive treatment against malaria was given. INTERVENTIONS: Supervised daily supplementation with 60 mg of elemental iron (as ferrous fumarate, n = 237 women) or placebo (n = 233) from randomization until 1 month postpartum. MAIN OUTCOMES AND MEASURES: Primary outcome was maternal Plasmodium infection at birth. Predefined secondary outcomes were birth weight and gestational age at delivery, intrauterine growth, and maternal and infant iron status at 1 month after birth. RESULTS: Among the 470 participating women, 40 women (22 iron, 18 placebo) were lost to follow-up or excluded at birth; 12 mothers were lost to follow-up postpartum (5 iron, 7 placebo). At baseline, 190 of 318 women (59.7%) were iron-deficient. In intention-to-treat analysis, comparison of women who received iron vs placebo, respectively, yielded the following results at birth: Plasmodium infection risk: 50.9% vs 52.1% (crude difference, -1.2%, 95% CI, -11.8% to 9.5%; P = .83); birth weight: 3202 g vs 3053 g (crude difference, 150 g, 95% CI, 56 to 244; P = .002); birth-weight-for-gestational-age z score: 0.52 vs 0.31 (crude difference, 0.21, 95% CI, -0.11 to 0.52; P = .20); and at 1 month after birth: maternal hemoglobin concentration: 12.89 g/dL vs 11.99 g/dL (crude difference, 0.90 g/dL, 95% CI, 0.61 to 1.19; P < .001); geometric mean maternal plasma ferritin concentration: 32.1 µg/L vs 14.4 µg/L (crude difference, 123.4%, 95% CI, 85.5% to 169.1%; P < .001); geometric mean neonatal plasma ferritin concentration: 163.0 µg/L vs 138.7 µg/L (crude difference, 17.5%, 95% CI, 2.4% to 34.8%; P = .02). Serious adverse events were reported for 9 and 12 women who received iron and placebo, respectively. There was no evidence that intervention effects on Plasmodium infection risk were modified by intermittent preventive treatment use. CONCLUSIONS AND RELEVANCE: Among rural Kenyan women with singleton pregnancies, administration of daily iron supplementation, compared with administration of placebo, resulted in no significant differences in overall maternal Plasmodium infection risk. Iron supplementation led to increased birth weight. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01308112.

Evaluation of four single-locus markers for Leishmania species discrimination by sequencing.

Several genetic markers have been described for discriminating Leishmania species. In most reported cases, one or a few polymorphisms are the basis of species identification, and the methods were validated on a limited number of strains from a particular geographical region. Therefore, most techniques may underestimate the global intraspecies variability and are applicable only in certain areas. In addition, interlaboratory standardization is mostly absent, complicating comparisons among different studies. Here, we compared species typing results from all sequence polymorphisms found in four popular markers that can be applied directly on clinical samples: the miniexon or spliced leader, the internal transcribed spacer of the ribosomal DNA array, the 7SL RNA gene, and the heat shock protein 70 gene. Clustering was evaluated among 74 Leishmania strains, selected to represent a wide geographic distribution and genetic variability of the medically relevant species of the genus. Results were compared with a multilocus sequence typing (MLST) approach using 7 single-copy household genes and with multilocus enzyme electrophoresis (MLEE), still considered the gold standard by some. We show that strain groupings are highly congruent across the four different single-locus markers, MLST, and MLEE. Overall, the heat shock protein 70 gene and the miniexon presented the best resolutions for separating medically relevant species. As gene sequence analysis is validated here on a global scale, it is advocated as the method of choice for use in genetic, clinical, and epidemiological studies and for managing patients with unknown origins of infection, especially in Western infectious disease clinics dealing with imported leishmaniasis.

A quality control program within a clinical trial Consortium for PCR protocols to detect Plasmodium species.

Malaria parasite infections that are only detectable by molecular methods are highly prevalent and represent a potential transmission reservoir. The methods used to detect these infections are not standardized, and their operating characteristics are often unknown. We designed a proficiency panel of Plasmodium spp. in order to compare the accuracy of parasite detection of molecular protocols used by labs in a clinical trial consortium. Ten dried blood spots (DBSs) were assembled that contained P. falciparum, P. vivax, P. malariae, and P. ovale; DBSs contained either a single species or a species mixed with P. falciparum. DBS panels were tested in 9 participating laboratories in a masked fashion. Of 90 tests, 68 (75.6%) were correct; there were 20 false-negative results and 2 false positives. The detection rate was 77.8% (49/63) for P. falciparum, 91.7% (11/12) for P. vivax, 83.3% (10/12) for P. malariae, and 70% (7/10) for P. ovale. Most false-negative P. falciparum results were from samples with an estimated ≤ 5 parasites per µl of blood. Between labs, accuracy ranged from 100% to 50%. In one lab, the inability to detect species in mixed-species infections prompted a redesign and improvement of the assay. Most PCR-based protocols were able to detect P. falciparum and P. vivax at higher densities, but these assays may not reliably detect parasites in samples with low P. falciparum densities. Accordingly, formal quality assurance for PCR should be employed whenever this method is used for diagnosis or surveillance. Such efforts will be important if PCR is to be widely employed to assist malaria elimination efforts.

Application of PCR-based methods for diagnosis of intestinal parasitic infections in the clinical laboratory.

For many years PCR- and other DNA-based methods of pathogen detection have been available in most clinical microbiology laboratories; however, until recently these tools were not routinely exploited for the diagnosis of parasitic infections. Laboratories were initially reluctant to implement PCR as incorporation of such assays within the algorithm of tools available for the most accurate diagnosis of a large variety of parasites was unclear. With regard to diagnosis of intestinal parasitic infections, the diversity of parasites that one can expect in most settings is far less than the parasitological textbooks would have you believe, hence developing a simplified diagnostic triage is feasible. Therefore the classical algorithm based on population, patient groups, use of immuno-suppressive drugs, travel history etc. is also applicable to decide when to perform and which additional techniques are to be used, if a multiplex PCR panel is used as a first-line screening diagnostic.

Asymptomatic plasmodial infection is associated with increased tumor necrosis factor receptor II-expressing regulatory T cells and suppressed type 2 immune responses.

BACKGROUND: In malaria-endemic areas, a proportion of individuals becomes chronic carriers of parasites with few or no clinical signs. There is little information on cellular immune responses in asymptomatic parasite carriers. METHODS: In 80 schoolchildren residing in a malaria-endemic area of Flores Island, Indonesia, T-helper subsets, regulatory T-cell (Treg) frequencies, tumor necrosis factor receptor type II (TNFRII) expression on Tregs, and cytokine responses induced by Plasmodium falciparum-infected red blood cells (RBCs) were measured, and values for asymptomatic infected subjects were compared to those for uninfected controls. To ascertain that alterations found were due to the presence of malaria parasites, the immune responses were analyzed in 16 children before and 1 month after antimalarial treatment. RESULTS: TNFRII expression, a marker of activation on Tregs, was higher during infection but decreased upon treatment. GATA3-positive cells and the level of interleukin 13 secretion in response to P. falciparum-infected RBCs appeared to be suppressed by plasmodial infection, as both increased after antimalarial treatment. TNFRII expression on Tregs correlated positively with TNF in response to P. falciparum-infected RBCs, but this association disappeared following treatment. CONCLUSIONS: Malaria parasites associated with asymptomatic infections seem to result in increased TNFRII expression on Tregs, as well as suppressed Th2 cytokine responses, features that might be important for survival of the parasites in asymptomatic carriers.

Genomic distribution of SINEs in Entamoeba histolytica strains: implication for genotyping.

BACKGROUND: The major clinical manifestations of Entamoeba histolytica infection include amebic colitis and liver abscess. However the majority of infections remain asymptomatic. Earlier reports have shown that some E. histolytica isolates are more virulent than others, suggesting that virulence may be linked to genotype. Here we have looked at the genomic distribution of the retrotransposable short interspersed nuclear elements EhSINE1 and EhSINE2. Due to their mobile nature, some EhSINE copies may occupy different genomic locations among isolates of E. histolytica possibly affecting adjacent gene expression; this variability in location can be exploited to differentiate strains. RESULTS: We have looked for EhSINE1- and EhSINE2-occupied loci in the genome sequence of Entamoeba histolytica HM-1:IMSS and searched for homologous loci in other strains to determine the insertion status of these elements. A total of 393 EhSINE1 and 119 EhSINE2 loci were analyzed in the available sequenced strains (Rahman, DS4-868, HM1:CA, KU48, KU50, KU27 and MS96-3382. Seventeen loci (13 EhSINE1 and 4 EhSINE2) were identified where a EhSINE1/EhSINE2 sequence was missing from the corresponding locus of other strains. Most of these loci were unoccupied in more than one strain. Some of the loci were analyzed experimentally for SINE occupancy using DNA from strain Rahman. These data helped to correctly assemble the nucleotide sequence at three loci in Rahman. SINE occupancy was also checked at these three loci in 7 other axenically cultivated E. histolytica strains and 16 clinical isolates. Each locus gave a single, specific amplicon with the primer sets used, making this a suitable method for strain typing. Based on presence/absence of SINE and amplification with locus-specific primers, the 23 strains could be divided into eleven genotypes. The results obtained by our method correlated with the data from other typing methods. We also report a bioinformatic analysis of EhSINE2 copies. CONCLUSIONS: Our results reveal several loci with extensive polymorphism of SINE occupancy among different strains of E. histolytica and prove the principle that the genomic distribution of SINEs is a valid method for typing of E. histolytica strains.

A laboratory-developed TaqMan Array Card for simultaneous detection of 19 enteropathogens.

The TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here we developed an enteric TaqMan Array Card to detect 19 enteropathogens, including viruses (adenovirus, astrovirus, norovirus GII, rotavirus, and sapovirus), bacteria (Campylobacter jejuni/C. coli, Clostridium difficile, Salmonella, Vibrio cholerae, diarrheagenic Escherichia coli strains including enteroaggregative E. coli [EAEC], enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], and Shiga-toxigenic E. coli [STEC]), Shigella/enteroinvasive E. coli (EIEC), protozoa (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica), and helminths (Ascaris lumbricoides and Trichuris trichiura), as well as two extrinsic controls to monitor extraction and amplification efficiency (the bacteriophage MS2 and phocine herpesvirus). Primers and probes were newly designed or adapted from published sources and spotted onto microfluidic cards. Fecal samples were spiked with extrinsic controls, and DNA and RNA were extracted using the QiaAmp Stool DNA minikit and the QuickGene RNA Tissue kit, respectively, and then mixed with Ag-Path-ID One Step real-time reverse transcription-PCR (RT-PCR) reagents and loaded into cards. PCR efficiencies were between 90% and 105%, with linearities of 0.988 to 1. The limit of detection of the assays in the TAC was within a 10-fold difference from the cognate assays performed on plates. Precision testing demonstrated a coefficient of variation of below 5% within a run and 14% between runs. Accuracy was evaluated for 109 selected clinical specimens and revealed an average sensitivity and specificity of 85% and 77%, respectively, compared with conventional methods (including microscopy, culture, and immunoassay) and 98% and 96%, respectively, compared with our laboratory-developed PCR-Luminex assays. This TAC allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes.

Bayesian modelling to estimate the test characteristics of coprology, coproantigen ELISA and a novel real-time PCR for the diagnosis of taeniasis.

OBJECTIVE: To estimate and compare the performances of coprology, copro-Ag ELISA and real-time polymerase chain reaction assay (copro-PCR) for detection of Taenia solium tapeworm carriers. METHODS: The three diagnostic tests were applied on 817 stool samples collected in two Zambian communities where taeniasis is endemic. A Bayesian approach was used to allow estimation of the test characteristics. Two (0.2%; 95% Confidence Interval (CI): 0-0.8), 67 (8.2%; 95% CI: 6.4-10.3) and 10 (1.2%; 95% CI: 0.5-2.2) samples were positive using coprology, copro-Ag ELISA and copro-PCR, respectively. RESULTS: Specificities of 99.9%, 92.0% and 99.0% were determined for coprology, copro-Ag ELISA and copro-PCR, respectively. Sensitivities of 52.5%, 84.5% and 82.7% were determined for coprology, copro-Ag ELISA and copro-PCR, respectively. CONCLUSIONS: We urge for additional studies exploring possible cross-reactions of the copro-Ag ELISA and for the use of more sensitive tests, such as copro-PCR, for the detection of tapeworm carriers, which is a key factor in controlling the parasite in endemic areas.