Multicenter evaluation of the BD max enteric bacterial panel PCR assay for rapid detection of Salmonella spp., Shigella spp., Campylobacter spp. (C. jejuni and C. coli), and Shiga toxin 1 and 2 genes.

Diarrhea due to enteric bacterial pathogens causes significant morbidity and mortality in the United States and worldwide. However, bacterial pathogens may be infrequently identified. Currently, culture and enzyme immunoassays (EIAs) are the primary methods used by clinical laboratories to detect enteric bacterial pathogens. We conducted a multicenter evaluation of the BD Max enteric bacterial panel (EBP) PCR assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2. A total of 4,242 preserved or unpreserved stool specimens, including 3,457 specimens collected prospectively and 785 frozen, retrospective samples, were evaluated. Compared to culture or EIA, the positive percent agreement (PPA) and negative percent agreement (NPA) values for the BD Max EBP assay for all specimens combined were as follows: 97.1% and 99.2% for Salmonella spp., 99.1% and 99.7% for Shigella spp., 97.2% and 98.4% for C. jejuni and C. coli, and 97.4% and 99.3% for Shiga toxins, respectively. Discrepant results for prospective samples were resolved with alternate PCR assays and bidirectional sequencing of amplicons. Following discrepant analysis, PPA and NPA values were as follows: 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C. jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. No differences in detection were observed for samples preserved in Cary-Blair medium and unpreserved samples. In this large, multicenter study, the BD Max EBP assay showed superior sensitivity compared to conventional methods and excellent specificity for the detection of enteric bacterial pathogens in stool specimens.

Impact of isolation precautions on quality of life: a meta-analysis.

Impact of isolation precautions on psychological wellbeing of patients has yet to be fully quantified. To assess the impact of isolation precautions on patients' health-related quality of life and depression or anxiety scales and estimate per day cost of anxiety and depression. Literature pertaining to impact of isolation precautions was searched on EMBASE and PubMed databases and Google Scholar. A two-step independent screening of the articles was performed. Articles that compared isolated and non-isolated patients using different quality of life and psychological burden scales were included. A meta-analysis was conducted using the Hospital Anxiety and Depression Scales (HADS-A and HADS-D). Psychological burden measures from selected literature were presented in a graph as effect sizes. Per day cost of anxiety and depression was estimated using pooled mean difference from meta-analysis. Out of 106 articles, 94 were excluded due to inclusion criteria, leaving 12 for full text review. After review of full text of the articles, seven articles were shortlisted for empirical analysis and four out of these seven for meta-analysis. The pooled mean difference estimates for HADS-A was -1.4 (P=0.15) and that for HADS-D was -1.85 (P=0.09). In the empirical analysis of psychological burden scales, the effect in all studies except one was negative. Results from meta-analysis and empirical analysis of psychological burden implied that isolated patients are worse off in general. The implied estimated per day cost of anxiety and depression in terms of quality-adjusted life years (QALYs) is approximately US$10.

Recent developments in amoebiasis:the Gal/GalNAc lectins of Entamoeba histolytica and Entamoeba dispar.

Amoebiasis is responsible for 50000-100000 deaths annually. Invasive amoebic disease begins with the attachment of Entamoeba histolytica trophozoites to colonic mucin, a process mediated by the amoebic Gal/GalNAc lectin. The non-pathogenic counterpart, E. dispar, is morphologically identical but genetically distinct. Investigations comparing the Gal/GalNac lectin from these two organisms are under way.

Immunochromatographic strip-based detection of Entamoeba histolytica-E. dispar and Giardia lamblia coproantigen.

BIOSITE Triage was 68.3% sensitive and 100% specific for the detection of Entamoeba histolytica-E. dispar (n = 71) compared to Alexon-Trend's ProSpecT test (reference standard) using fresh-frozen stool. Neither test is able to distinguish E. histolytica from E. dispar. Triage was 83.3% sensitive and 100% specific compared to microscopy (formalin-ether concentrates and permanent stains) for the detection of Giardia lamblia.

Entamoeba dispar: molecular characterization of the galactose/N-acetyl-d-galactosamine lectin.

Amebiasis contributes to approximately 50 million cases of life-threatening dysentery worldwide. Comparison of the lectins from Entamoeba histolytica (pathogenic) and Entamoeba dispar (nonpathogenic) was undertaken to elucidate the differential roles of this molecule in invasion versus colonization. Surface lectin was less abundant on axenic E. dispar than on axenic E. histolytica, commensurate with differences in lectin (heavy and light subunits) RNA when assessed by semiquantitative RT-PCR. The 1G7 epitope, which falls within the immunodominant and immunoprotective cysteine-rich region (480-900), was absent on axenic E. dispar. Indirect immunofluorescence, transient transection of COS7, and immunoprecipitation demonstrated that the 1G7 epitope was conserved in the nonpathogenic lectin homologue but not exposed on live E. dispar trophozoites. Hgl2 (E. histolytica) and Dhgl2 (E. dispar) lectin homologues demonstrated comparable high-affinity binding to multivalent GalNAc(19) BSA. These data provide evidence for relative gene and conformational regulation of the E.dispar lectin.

The cysteine-rich region of the Entamoeba histolytica adherence lectin (170-kilodalton subunit) is sufficient for high-affinity Gal/GalNAc-specific binding in vitro.

Adherence of Entamoeba histolytica trophozoites to colonic mucin, epithelium, and other target cells is mediated by the amebic Gal/GalNAc lectin. We constructed in vitro expression vectors containing full-length (residues 1 to 1280), cysteine-poor (1 to 353 and 1 to 480), and cysteine-rich (356 to 1143 and 480 to 900) fragments of the gene encoding the heavy subunit of the adherence lectin, hgl2. In vitro transcription followed by translation using a nuclease-treated rabbit reticulocyte lysate system was carried out. Immunoreactivity of in vitro-translated Hgl2 was confirmed by immunoprecipitation with lectin-specific monoclonal antibodies (MAbs) 1G7 and 8A3, which recognize linear epitopes. Protein disulfide isomerase (PDI) refolding of Hgl2 enhanced immunoreactivity (P < 0.05) with the conformationally dependent MAb 3F4. Binding of PDI-refolded full-length (P < 0.001) and cysteine-rich (P = 0.005) Hgl2 to CHO cells was galactose dependent and competitively inhibited by native hololectin (50% inhibitory concentration of 39.6 ng/ml). The cysteine-poor region (1 to 353) did not bind CHO cells. Both full-length (1 to 1280) and cysteine-rich (356 to 1143) Hgl2 bound the glyconeoconjugate GalNAc(19)BSA in a GalNAc-specific manner. The smaller cysteine-rich fragment (480 to 900) also exhibited GalNAc-specific binding but to a lesser extent (P < 0.05) than residues 1 to 1280 and 356 to 1143. Neither the cysteine-poor fragment (1 to 480), luciferase (protein control), nor control translation reactions (without hgl2 lectin mRNA) bound GalNAc(19)BSA. Binding to GalNAc(19)BSA was shown to be dependent on the concentration of GalNAc(19)BSA coated in each well or (35)S-lectin added (K(D) = 0.85 +/- 0.37 pM). Binding was competitively inhibited by the terminal GalNAc-containing glycoprotein asialofetuin (P < 0.005). Taken together, these data provide direct evidence that the cysteine-rich region of the Gal/GalNAc lectin heavy subunit contains one or more carbohydrate-binding domains.

Entamoeba histolytica and Entamoeba dispar: epidemiology and comparison of diagnostic methods in a setting of nonendemicity.

Recent studies suggest that stool antigen assays are more sensitive and specific than microscopy for the diagnosis of Entamoeba histolytica infection. One hundred twelve patients presenting at 3 centers with symptoms or risk factors of E. histolytica infection were prospectively enrolled in this study to evaluate new diagnostic tests for infections with E. histolytica and Entamoeba dispar. Four ELISA-based stool antigen kits for detecting E. histolytica or E. dispar were blindly compared with stool microscopy. Amebic serology was assessed by indirect hemagglutination. When antigen assays were used as the reference standard, microscopy performed at referral centers was more specific (68.4% vs. 9.5%) but less sensitive (70.4% vs. 92.1%) than microscopy performed in community laboratories. Diagnosis with the E. histolytica test and Merlin Optimun S ELISA indicated that only 3 (4.2%) of 72 coproantigen-positive stools were positive for E. histolytica. Indirect hemagglutination was a good predictor of E. histolytica infection when titers of antibody to ameba were >/=1:512.

The performance and utility of rapid diagnostic assays for Plasmodium falciparum malaria in a field setting in the Lao People's Democratic Republic.

Rapid diagnostic assays for malaria have the potential to improve the management and control of the disease in developing countries. The objectives of the present study were to evaluate, in a field setting, the performance of several such assays for Plasmodium falciparum infection and to examine the usefulness of these assays in identifying subjects for treatment trials in rural field sites. Residents of 12 villages in Laos who presented with fever were eligible for inclusion. Blood was collected by fingerprick for a dipstick assay, developed by the Program for Appropriate Technology in Health (PATH), performed and interpreted in the field by local healthcare workers. Compared with 'blinded' reference microscopy (N =196), the sensitivity and specificity of the PATH assay were 96.2% and 93.0%, respectively. Two rapid diagnostic assays (PATH and OptiMAL) were also performed on the subset of subjects eligible to participate in an in-vivo treatment trial (N = 97), and the results again compared with those of 'blinded' reference microscopy. In this subset, a subject was considered a 'true positive' if found positive by microscopy or the alternate rapid assay. Using this modified reference standard, the sensitivity and specificity of the PATH assay were 96.7% and 94.4%, and those of the OptiMAL assay were 91.8% and 100%, respectively. Both of the rapid assays tested therefore appear suitable for use in rural field settings by local healthcare providers and can accurately identify participants for treatment trials.

Molecular markers for chloroquine-resistant Plasmodium falciparum malaria in Thailand and Laos.

Chloroquine-resistant Plasmodium falciparum is well documented in Thailand. Laos, however, continues to use chloroquine (CQ) as the first-line therapy for the treatment of P. falciparum malaria. The objective of the present study was to determine the prevalence, in these two areas, of the cg2, pfmdr1 and pfcrt allelic types that have previously been associated with CQ resistance. Isolates of P. falciparum were collected from participants in ongoing treatment studies conducted in Thailand (near the Thai-Cambodian border) and in Laos (Vang Vieng district). The pfmdr1 and pfcrt alleles were characterized by PCR-RFLP and mutations in cg2 were characterized by PCR and single-stranded-conformation-polymorphism (SSCP) electrophoresis. Eight (32%) of the 25 Laotian isolates but only one (4%) of the 25 Thai isolates were found to contain the pfmdr1 mutation N86Y (P = 0.02). In contrast, the cg2 polymorphisms previously associated with CQ resistance were present in only 10 of the isolates from Laos but 24 of those from Thailand (40% v. 96%; P < 0.001). All the samples from both countries contained the pfcrt K76T mutant allele reported to confer resistance to CQ. The results may indicate that drug pressure for the maintenance of the pfmdr1 and cg2 alleles varies in intensity in the Thai and Laotian study areas, probably reflecting differences in the national malaria-treatment policies of Thailand and Laos.

Plasmodium falciparum malaria in Laos: chloroquine treatment outcome and predictive value of molecular markers.

A 28-day treatment trial was undertaken, to determine the efficacy of chloroquine in Laos and to assess the predictive value of molecular markers (cg2, pfmdr1, and pfcrt) that were previously linked to chloroquine resistance. In total, 522 febrile patients were screened for falciparum malaria by rapid diagnostic assays. Of 81 patients (15.5% prevalence) who were positive by the assays and microscopy, 48 were eligible to participate in the 28-day trial. Nine patients defaulted. Chloroquine cured 54% (95% confidence interval, 45.8-61.8) of falciparum-infected patients. Of 18 (46%) patients with treatment failure, 13 (72%) experienced high-grade resistance. Polymorphisms in cg2 and the N86Y mutation in PfMDR1 were not predictive of treatment outcome. A mutation in PfCRT (K76T) was perfectly associated with in vivo chloroquine resistance. However, K76T was also present in in vivo-sensitive isolates, which suggests that the presence of this mutation was necessary, but not sufficient, to predict in vivo outcome in this cohort.