Rapid Detection of Trypanosoma cruzi in Human Serum by Use of an Immunochromatographic Dipstick Test

We evaluated a commercially available immunochromatographic dipstick test to detect Trypanosoma cruzi infection in 366 human serum samples with known serological results from Argentina, Ecuador, Mexico, and Venezuela. One hundred forty-nine of 366 (40.7%) and 171/366 (46.7%) samples tested positive by dipstick and serology, respectively. Dipstick sensitivity was calculated to be 84.8% (range between countries, 77.5 to 95%), and specificity was 97.9% (95.9 to 100%).Chagas disease is caused by Trypanosoma cruzi and is found in wildlife, domestic animals, and humans in rural as well as peri-urban areas of Mexico, Central America, and South America; in the United States, T. cruzi is found in wildlife, but human cases are rare (29). Although transmission of T. cruzi can occur orally, congenitally, or transfusionally, most transmission to mammalian hosts is through the feces of blood-feeding triatomine bugs when T. cruzi trypomastigotes in the feces contaminate the bite wound or enter the host through mucosal surfaces (22). By causing the loss of an estimated 670,000 disability-adjusted life years (i.e., a measure that sums years of potential life lost due to premature mortality and years of productive life lost due to disability), Chagas disease is the most important parasitic disease in the Americas; 8 to 10 million people are currently infected with T. cruzi, with up to 100 million at risk of contracting the disease (32).There are several methods to diagnose T. cruzi infection (11), but none are ideal when mass screening of samples is required (e.g., epidemiological surveys, blood unit screening). While comparatively easy to use and sensitive, serological tests (i.e., enzyme-linked immunosorbent assay [ELISA], immunofluorescence antibody test [IFAT], indirect hemagglutination test [IHAT], or radioimmunosorbent assay [RIA]) are of varied specificities (i.e., 60 to 100%) (12, 16, 26). Molecular tests, including PCR-based approaches, are very specific but lack sensitivity (i.e., 30 to 95%) and require technological expertise and specialized, expensive laboratory equipment (11, 21, 23). Hemoculture and xenodiagnosis are the current gold standard for T. cruzi parasitological diagnosis (6, 11, 21). Though these techniques are specific, their sensitivity in the chronic phase of infection is quite variable (e.g., 0 to 50% [6]); they also are labor-intensive and time-consuming (e.g., because of the necessity of mass-rearing bugs for xenodiagnosis and examination of them). Thus, a rapid, sensitive, and specific diagnostic test to detect T. cruzi infection would be extremely valuable for mass-screening surveys and intervention campaigns as well as during the onset of outbreaks; results could be read immediately, and control measures could be implemented in situ.Immunochromatographic dipstick tests have been developed for a range of tropical diseases, including malaria (31), leishmaniasis (7), and schistosomiasis (3); until recently (4, 5, 8, 14, 17, 20, 25, 28, 30), none was available for Chagas disease.Recently, the World Health Organization announced renewed efforts to eliminate Chagas disease (27). For such efforts to succeed, an easy-to-use, sensitive, and specific diagnostic test will be crucial for both detecting and treating cases early as well as monitoring the implementation of elimination efforts and evaluating their impact (18, 24).We evaluated the sensitivity and specificity of a commercially available immunochromatographic dipstick test to detect antibodies to T. cruzi infection in human serum samples with known serological results collected in areas of both Chagas disease endemicity and nonendemicity in four different Latin American countries.     

Identification of Novel Diagnostic Serum Biomarkers for Chagas' Disease in Asymptomatic Subjects by Mass Spectrometric Profiling

More than 10 million people are thought to be infected with Trypanosoma cruzi, primarily in the Americas. The clinical manifestations of Chagas'' disease (CD) are variable, but most subjects remain asymptomatic for decades. Only 15 to 30% eventually develop terminal complications. All current diagnostic tests have limitations. New approaches are needed for blood bank screening as well as for improved diagnosis and prognosis. Sera from subjects with asymptomatic CD (n = 131) were compared to those from uninfected controls (n = 164) and subjects with other parasitic diseases (n = 140), using protein array mass spectrometry. To identify biomarkers associated with CD, sera were fractionated by anion-exchange chromatography and bound to two commercial ProteinChip array chemistries: WCX2 and IMAC3. Multiple candidate biomarkers were found in CD sera (3 to 75.4 kDa). Algorithms employing 3 to 5 of these biomarkers achieved up to 100% sensitivity and 98% specificity for CD. The biomarkers most useful for diagnosis were identified and validated. These included MIP1 alpha, C3a anaphylatoxin, and unusually truncated forms of fibronectin, apolipoprotein A1 (ApoA1), and C3. An antipeptide antiserum against the 28.9-kDa C terminus of the fibronectin fragment achieved good specificity (90%) for CD in a Western blot format. We identified full-length ApoA1 (28.1 kDa), the major structural and functional protein component of high-density lipoprotein (HDL), as an important negative biomarker for CD, and relatively little full-length ApoA1 was detected in CD sera. This work provides proof of principle that both platform-dependent (i.e., mass spectrometry-based) and platform-independent (i.e., Western blot) tests can be generated using high-throughput mass profiling.Chagas'' disease (CD), caused by the protozoan parasite Trypanosoma cruzi, is transmitted by triatomid bugs in resource-poor settings. Over 10 million people are thought to be infected worldwide (51), and vector-mediated transmission can occur from regions in the southernmost United States to temperate South America (4, 7). While some CD patients become acutely ill (e.g., local swelling, fever), most are unaware that they are infected and can remain asymptomatic for decades (24). Only 15 to 30% of patients will eventually develop cardiac or gastrointestinal complications (28). CD can also enter into or be maintained in populations through migration, vertical transmission, organ transplantation (11), and blood transfusion (25).Transfusion-associated transmission of CD occurs both in countries where CD is endemic and in those where it is not (26, 49). Although serologic screening tests have greatly reduced the risk of transfusion-acquired CD in Latin America (25), serologic methods are imperfect, and >10,000 cases may occur annually in Sao Paulo alone (1). False-positive results can also be a serious concern, resulting in wasted units and permanent losses from the blood donor pool (42). A review of serological procedures in 16 Latin American blood banks from 1997 to 2000 reported false-negative rates of 0.7% to 3.7% and false-positive rates of 0.3% to 3.2%. We recently demonstrated that up to 50% of true CD cases are missed by routine screening in some Venezuelan blood banks (6). Transmission of CD by transfusion/transplantation is also a concern in countries where it is not endemic but where there are large Latin American populations. As many as 100,000 of the 7 to 8 million North American residents of Latin American origin may be silent carriers of T. cruzi (24, 44, 48). Geographic exclusion policies can mitigate risk, but donors do not always provide complete information (32). This situation is further confounded because CD can pass unsuspected from mother to child for several generations (33). In 2007, the CDC reported that 1/4,655 blood donations in the United States was positive for T. cruzi (12), and a small serosurvey of 102 Latin American immigrants in Ontario demonstrated that 1% had CD antibodies (45). The relatively small number of transfusion-transmitted T. cruzi cases reported in North America in the last 2 decades may therefore represent only the tip of the iceberg (22, 25, 35). Several recent reports of CD following organ transplantation further underscore the vulnerability of transfusion and transplantation services in North America (11). A new enzyme-linked immunosorbent assay (ELISA), the Ortho T. cruzi ELISA test system, was approved by the FDA for screening of blood donors and was licensed in December 2006 (21). However, the long history of similar tests in the Americas suggests that new diagnostic strategies should also be pursued.Surface-enhanced laser desorption ionization-time-of-flight mass spectrometry (SELDI-TOF MS) can provide protein profiles of body fluids. This platform has been used primarily as a discovery tool for biomarkers under neoplastic and inflammatory conditions (53). However, we and others have applied SELDI-TOF MS to studies of human infections, such as African sleeping sickness (37), hepatitis C (43), severe acute respiratory syndrome (SARS) (54), and bacterial endocarditis (19), as well as to studies of fasciolosis in sheep (38) and cysticercosis in pigs (16). There has been some justified criticism of protein profiling efforts, including SELDI-TOF data, when authors have failed to proceed to identification and validation of putative biomarkers (50). In this work, we used SELDI-TOF MS as the first step to compare serum protein profiles of asymptomatic CD-infected subjects, healthy controls, and subjects with other parasitic diseases. Promising biomarkers were independently validated, and second-generation reagents were produced (e.g., antipeptide antibodies). We report on both SELDI-TOF MS platform-based tests and alternative assays derived from this biomarker discovery program (i.e., MS platform independent) that can achieve high sensitivity and specificity for latent CD.(These data were presented in part at the American Society for Tropical Medicine and Hygiene annual meetings in Miami [7 to 11 November 2004; Proteomic Symposium] and Washington [11 to 15 December 2005; Symposium 17].)     

Vancomycin MIC plus Heteroresistance and Outcome of Methicillin-Resistant Staphylococcus aureus Bacteremia: Trends over 11 Years

Vancomycin MICs (V-MIC) and the frequency of heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) isolates are increasing among methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates, but their relevance remains uncertain. We compared the V-MIC (Etest) and the frequency of hVISA (Etest macromethod) for all MRSA blood isolates saved over an 11-year span and correlated the results with the clinical outcome. We tested 489 isolates: 61, 55, 187, and 186 isolates recovered in 1996-1997, 2000, 2002-2003, and 2005-2006, respectively. The V-MICs were ≤1, 1.5, 2, and 3 μg/ml for 74 (15.1%), 355 (72.6%), 50 (10.2%), and 10 (2.1%) isolates, respectively. We detected hVISA in 0/74, 48/355 (13.5%), 15/50 (30.0%), and 8/10 (80.0%) isolates with V-MICs of ≤1, 1.5, 2, and 3 μg/ml, respectively (P < 0.001). The V-MIC distribution and the hVISA frequency were stable over the 11-year period. Most patients (89.0%) received vancomycin. The mortality rate (evaluated with 285 patients for whose isolates the trough V-MIC was ≥10 μg/ml) was comparable for patients whose isolates had V-MICs of ≤1 and 1.5 μg/ml (19.4% and 27.0%, respectively; P = 0.2) but higher for patients whose isolates had V-MICs of ≥2 μg/ml (47.6%; P = 0.03). However, the impact of V-MIC and hVISA status on mortality or persistent (≥7 days) bacteremia was not substantiated by multivariate analysis. Staphylococcal chromosome cassette mec (SCCmec) typing of 261 isolates (including all hVISA isolates) revealed that 93.0% of the hVISA isolates were SCCmec type II. These findings demonstrate that the V-MIC distribution and hVISA frequencies were stable over an 11-year span. A V-MIC of ≥2 μg/ml was associated with a higher rate of mortality by univariate analysis, but the relevance of the V-MIC and the presence of hVISA remain uncertain. A multicenter prospective randomized study by the use of standardized methods is needed to evaluate the relevance of hVISA and determine the optimal treatment of patients whose isolates have V-MICs of ≥2.0 μg/ml.The treatment of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) bacteremia with vancomycin is often associated with a poor clinical outcome (6, 15, 28, 40). Treatment failure was reported among patients infected with isolates whose vancomycin MICs were ≥4 μg/ml (6, 9, 12, 25, 28, 42). This prompted the Clinical and Laboratory Standards Institute to lower the cutoffs for S. aureus susceptibility to ≤2 μg/ml for susceptible, 4 to 8 μg/ml for intermediate (vancomycin-intermediate S. aureus [VISA]), and 16 μg/ml for resistance (39). Within the susceptibility range, the MIC is reported to increase over time (14, 25, 35-40). This is often referred to as MIC creep (38). Additionally, isolates with heteroresistance (heteroresistant vancomycin-intermediate S. aureus [hVISA]) are emerging, and this has uncertain implications for laboratory detection and clinical management (2, 5, 15, 24, 40-42). The first isolate of hVISA to be identified was reported from Japan in 1997 (11). Since then, it has been reported worldwide at frequencies of 0 to 50% (2, 4, 6, 9, 12, 19, 20, 21, 24, 26, 27, 31, 40, 42, 44). This disparity in frequency is probably a result of its variable incidence and the different testing methodologies used. Likewise, the frequency of isolates with MICs of 1.5 to <4 μg/ml varies according to the testing method used (3, 32). The relevance of an MIC on the higher side of the susceptibility range and the presence of hVISA isolates remains uncertain (8, 19, 21). Therapeutic failure was reported in patients infected with isolates with vancomycin MICs of 2 μg/ml (6, 12, 28) and 1.5 or 1 μg/ml (25, 34, 37). Most clinical microbiology laboratories use automated testing methods that are known to underestimate the vancomycin MIC (13, 24). Additionally, most previous studies addressing the relevance of such isolates were observational and usually involved only a few patients and poorly selected controls (1, 4, 7, 9, 12, 14, 25, 35, 38, 42). At our institution, we found the frequency of hVISA isolates among isolates from patients with persistent MRSA bacteremia to be 14%; however, heteroresistance did not correlate with the mortality rate (19). In the current study, we tested all blood MRSA isolates collected over 11 years to determine whether the vancomycin MIC and the prevalence of hVISA have changed over time and to evaluate the effects of increasing vancomycin MICs and the hVISA frequency on patient outcomes.     

Evaluation of a Rapid Serological Test for the Determination of Mycobacterium bovis Infection in Badgers (Meles meles) Found Dead

Between October 2005 and May 2006, a total of 727 badgers found dead in Wales were reported, and 550 were delivered to the Regional Laboratories of the Veterinary Laboratories Agency (VLA). Of the 459 carcasses suitable for examination, 55 were deemed to be infected with Mycobacterium bovis on the basis of culture, spoligotyping, and variable-number tandem repeat typing. Acid-fast bacteria were observed histologically in a further six badgers, but these bacteria were not confirmed as M. bovis by culture. A rapid serological test (BrockTB Stat-Pak) performed on thoracic blood showed a sensitivity of 35% and a specificity of 99%. Presence of M. bovis infection was 45 times more likely to be confirmed postmortem by culture in BrockTB Stat-Pak-reactive animals than in seronegative ones. Using visible carcass lesions as a marker of bovine tuberculosis (bTB) infection had a similar sensitivity (38%) but was significantly less specific (84%) than serology. The overall accuracy of the antibody detection was 93% (346 correct results from 374 tests), whereas the accuracy of regarding visible lesions as a marker for bTB infection was 78% (354 correct from 453 carcasses examined). Culture remains the gold standard method for detecting M. bovis infection in badgers. However, where resources are limited and/or an instant result is preferred, the BrockTB Stat-Pak could be used in field surveillance efforts to identify animals which should be examined further by only submitting test-negative animals to more detailed postmortem examination and culture.Mycobacterium bovis infection is the cause of bovine tuberculosis (bTB) in a wide range of mammal species, including domestic livestock and captive and free-ranging wildlife. Bovine TB remains an important zoonotic disease with significant impacts on the economy in many countries (6, 22, 23). Eurasian badgers (Meles meles) are a wildlife maintenance host of bTB in Great Britain and Ireland (5, 15) and are implicated in the maintenance and onward transmission of M. bovis infection to cattle (10, 19).Surveillance of wildlife vectors of disease for prevalence estimates of infection may be valuable in disease control strategies and for the assessment of risk of transmission to livestock. Diagnosis of bTB in live badgers has been demonstrated using assays of both serological (4, 20) and cell-mediated (8, 9) immunity. While isolation of M. bovis from clinical samples is definitive, it is too insensitive for badgers, as infected animals yield positive samples infrequently and intermittently (3). A rapid serological test (BrockTB Stat-Pak; Chembio Diagnostic Systems, Inc.) has recently been developed for the diagnosis of bTB in multiple wildlife species (20). The test has modest sensitivity (46 to 55%) for antibody detection in live, infected badgers, but it has the advantages of being simple, rapid, inexpensive, and suitable for field application. Its utility as an animal-side test for badgers, however, is limited by the difficulties associated with obtaining a blood sample from a nonanesthetized animal.Where carcasses are recovered and submitted for mycobacterial culture, the sensitivity of diagnosis depends on the effort taken for careful examination and on the number of tissue samples submitted for culture testing and histopathology (7), as well as on the condition of the carcass. In many cases, the cost involved may prove prohibitive. Reliance on the presence of visible lesions as indicative of bTB is fraught with difficulties, as infected animals may present with no visible lesions or lesions may be the result of other infections while having the appearance of bTB (reviewed in reference 13). The purpose of this study was to determine whether the BrockTB Stat-Pak test could detect M. bovis antibody in blood collected from the carcasses of dead badgers as an alternative means of diagnosis and decision making. Animals were obtained as part of a separate government-funded study to determine the prevalence of bTB in badgers found dead in Wales (http://new.wales.gov.uk/depc/publications/environmentandcountryside/animalhealthandwelfare/diseasesurveillancecontrol/bovinetb/2567889/publicationindex/2326585/badgerfounddeadreport?lang=en). Our results reveal that the BrockTB Stat-Pak test used on thoracic blood samples was very specific (99%) but less sensitive (35%) than found previously for live badgers (2, 14). However, bTB was 45 times more likely to be confirmed in BrockTB Stat-Pak-positive animals than in BrockTB Stat-Pak-negative ones, whereas using visible carcass lesions as a marker of infection was less reliable.     

Evaluation of a New Selective Medium,BD BBL CHROMagar MRSA II,for Detection of Methicillin-Resistant Staphylococcus aureus in Different Specimens

The sensitivity of screening for methicillin-resistant Staphylococcus aureus (MRSA) can be improved by adding other specimen sites to nares. We describe an evaluation of a new selective medium, BBL CHROMagar MRSA II (CMRSAII), for its ability to detect MRSA from different specimen types. CMRSAII is a chromogenic medium which incorporates cefoxitin for the detection of MRSA. A study was performed at four clinical laboratories with the following specimens: 1,446 respiratory, 694 stool, 1,275 skin, and 948 wound specimens and 688 blood culture bottles containing Gram-positive cocci. The recovery of MRSA on traditional culture media was compared to results with CMRSAII. S. aureus was tested by cefoxitin disk diffusion. CMRSAII was interpreted as positive for MRSA at 24 h (range, 18 to 28 h) based solely on the visualization of mauve-colored colonies and at 48 h (range, 36 to 52 h) based on detection of mauve colonies with subsequent confirmation as S. aureus (by coagulase or latex agglutination testing). MRSA was recovered more frequently on CMRSAII (89.8% at 24 h and 95.6% at 48 h) than on traditional culture plates (83.1% at 24 h and 79.8% at 48 h) for all specimen types combined (P < 0.001). The percent sensitivities of CMRSAII at 24- and 48-h reads, respectively, were 85.5 and 92.4% for respiratory specimens, 87.9% and 98.3% for stool specimens, 88.4% and 96.1% for skin specimens, 92.1% and 94.6% for wound specimens, and 100% and 100% for positive blood cultures. The specificity was 99.8% for respiratory specimens and 100% for all others. In conclusion, CMRSAII is a reliable screening medium for multiple specimen types.Controlling the spread of multidrug-resistant microorganisms and especially methicillin-resistant Staphylococcus aureus (MRSA) has become a major infection control objective in the United States (4) and many European countries (3, 4, 21). A part of most programs to control the spread of MRSA is screening of patients (4, 8, 14), and screening has even become mandatory in some countries (11, 31).Traditionally, MRSA screening included mainly the culturing of naris swabs. However, it has been demonstrated that up to 35% of MRSA carriers may be colonized only from sites other than the nares, for example, the throat or the rectum (1, 2, 16).Usage of chromogenic media can improve the sensitivity and pace of MRSA detection (5, 6, 9, 10, 12, 13, 15, 17,19, 20, 22-24, 26-30); however, currently available media that have been marketed at this time are recommended only for nasal specimens.This study was designed to compare the performance of BBL CHROMagar MRSAII (CMRSAII), a chromogenic medium which incorporates cefoxitin, with traditional culture media in the recovery and identification of MRSA isolates from clinical specimens, including respiratory, lower gastrointestinal, and skin specimens as well as wound cultures and blood culture bottles with Gram-positive cocci. In addition, it was designed to determine whether CMRSAII results may be reported as presumptive or definitive with no (or one) confirmatory test at 24 and 48 h of incubation.(These data were presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008.)     

Probing Population Dynamics of Trypanosoma cruzi during Progression of the Chronic Phase in Chagasic Patients

Our research aimed to characterize the genetic profiles of 102 Trypanosoma cruzi isolates recently obtained from 44 chronic chagasic patients from different regions of the states of Minas Gerais and Goiás in Brazil. At least two isolates were obtained from each patient at different times in order to study the parasite population dynamics during disease progression in the chronic phase. The isolates were characterized molecularly by genotyping the 3′ region of the 24Sα rRNA, the mitochondrial cytochrome oxidase subunit 2 (COII) gene, and the intergenic region of the spliced leader intergenic region (SL-IR) gene. Seventy-seven isolates were analyzed for nine microsatellite loci. The data presented here show a strong correlation between the T. cruzi lineage II (T. cruzi II) and human infection in these regions of Brazil. Interestingly, isolates from two patients were initially characterized (by rRNA genotyping) as T. cruzi I and hybrid strains, but subsequent analyses of the COII and SL-IR genes confirmed that those isolates belonged to T. cruzi III and a hybrid group, respectively. Our results confirm the risk of misclassifying T. cruzi isolates on the basis of analysis of a single molecular marker. The microsatellite profiles showed that different isolates obtained from the same patient were genetically identical and monoclonal. Exceptions were observed for T. cruzi isolates from two patients who presented differences for the SCLE11 locus and also from two other patients who showed amplification of three peaks for a microsatellite locus (TcAAAT6), implying that they were multiclonal. On the basis of the findings of the studies described here, we were not able to establish a correlation between the clinical forms of Chagas'' disease and the genetic profiles of the T. cruzi isolates.The protozoan Trypanosoma cruzi is both phenotypically and genotypically heterogeneous. Strains of T. cruzi exhibit large differences in growth rate, histotropism, antigenicity, pathogenicity, their infectivity of potential insect vectors, drug susceptibility, and DNA content. T. cruzi causes Chagas'' disease, which is characterized by a variable clinical course that ranges from the absence of symptoms to severe disease with cardiac involvement and/or digestive tract damage. This variability has been attributed both to differences in the host response and to the genomic heterogeneity of the parasite (2, 7, 12, 23, 25, 27, 31, 33, 34, 42, 44).T. cruzi is a diploid organism. Its genome has recently been sequenced; however, most aspects of its population structure and evolution remain to be elucidated (17). Despite their high degrees of genetic variability, T. cruzi isolates can be classified into two major phylogenetic lineages: T. cruzi lineage I (T. cruzi I) and T. cruzi II (4). These lineages are very divergent, as revealed by several biological and molecular markers, including isoenzymes, 24Sα rRNA, the miniexon gene, and microsatellite polymorphisms, among others (4, 10, 35, 36, 39, 42, 45, 50).There are two different transmission cycles for T. cruzi. The sylvatic cycle involves wild triatomine species and nonhuman vertebrate hosts. The domestic cycle involves home-dwelling triatomines and primarily humans and household animals. In the Southern Cone, T. cruzi I and T. cruzi II strains predominantly belong to distinct ecological environments: the sylvatic and domestic transmission cycles of Chagas'' disease, respectively (8, 49). In Brazil, T. cruzi II strains appear to be responsible for tissue lesions in Chagas'' disease, while human cases of infection caused by T. cruzi I strains are rare and are usually asymptomatic (8, 20, 50). However, growing evidence has revealed that T. cruzi I is more associated with clinical cases of Chagas'' disease than was initially realized, especially in the northern part of South America (3, 15, 41). Recently, the existence of a third ancestral lineage, T. cruzi III, was proposed, but the epidemiological importance of these parasites in humans remains to be determined (21).Many research groups have tried to correlate the genetic variability of the parasite with the clinical manifestation of the disease, but they were unsuccessful (6, 16, 22, 23, 26, 49). In light of the histotropic-clonal model proposed for Chagas'' disease in the beginning of the 1980s, at least two explanations can be devised. (i) With regard to the hypothesis of T. cruzi subpopulation selection, vertebrate hosts (particularly humans) may act as selective biological filters as the host immune response selects a subset of the parasitic clones present in the original infecting parasite population. In addition, tissue tropism can differ between parasitic clones. Therefore, parasites isolated from patients'' blood and maintained under laboratory conditions for analyses can differ from those parasites directly involved in the specific tissue lesions (1, 2, 22, 27, 30, 33, 36, 43). (ii) Different T. cruzi life-cycle stages are represented in an infected host. At the time of parasite isolation from host blood, one particular clone could predominate over others, resulting in the isolation of an underrepresentative parasite population (13).In order to address the last possibility, we analyzed the genetic profiles of 102 T. cruzi populations isolated on different occasions from 44 chronic chagasic patients from two different regions of endemicity of the states of Minas Gerais (MG) and Goiás (GO) in Brazil.     

Evaluation of Two Immunoblot Assays and a Western Blot Assay for the Detection of Antisyphilis Immunoglobulin G Antibodies

In the present study, two immunoglobulin G (IgG) immunoblot assays and one IgG Western blot assay were compared to the rapid plasma reagin test (RPR), the fluorescent treponemal antibody absorption test (FTA-ABS), and the Treponema pallidum particle agglutination assay (TP-PA). The agreement levels of the Viramed, Virotech, and MarDx assays were 97.0%, 96.4%, and 99.4%, and the agreements of samples inconclusive by FTA-ABS and resolved by TP-PA were 91.7%, 83.3%, and 69.4%, respectively.Syphilis, a disease caused by Treponema pallidum, is transmitted congenitally or through sexual intercourse (8-9). Non-treponema-based tests such as the rapid plasma reagin test (RPR) are used to detect syphilis infection (6, 9-10). These tests may produce false-positive results in pregnant women and patients with infections (3, 5-6, 9, 11). An algorithm has been developed for the serological diagnosis of syphilis which includes a non-treponema-based screening test and a treponema-based confirmatory assay (1-2, 7, 11). Traditional confirmatory assays include the fluorescent treponemal antibody absorption test (FTA-ABS) and the T. pallidum particle agglutination assay (TP-PA) (9).Western blot-based assays to detect immunoglobulin G (IgG) antibodies may prove useful, especially in cases where the FTA-ABS is inconclusive. In the present study, results of two immunoblot assays and one Western blot assay were compared to FTA-ABS/TP-PA and RPR results, as well as to each other.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Three Doses of an Experimental Detoxified L3-Derived Lipooligosaccharide Meningococcal Vaccine Offer Good Safety but Low Immunogenicity in Healthy Young Adults

This open, randomized phase I study evaluated the safety and reactogenicity of an experimental meningococcal serogroup B (MenB) vaccine obtained from outer membrane vesicle detoxified L3-derived lipooligosaccharide. Healthy young adults (n = 150) were randomized to receive either experimental vaccine (provided in five formulations, n = 25 in each group) or VA-Mengoc-BC (control, n = 25) administered on a 0- to 6-week/6-month schedule. Serum bactericidal assays performed against three MenB wild-type strains assessed the immune response, defined as a 4-fold increase from pre- to postvaccination. No serious adverse events related to vaccination were reported. Pain at the injection site, fatigue, and headache were the most commonly reported adverse events. Solicited adverse events graded level 3 (i.e., preventing daily activity) were pain (up to 17% of the test subjects versus 32% of the controls), fatigue (up to 12% of the test subjects versus 8% of the controls), and headache (up to 4% of any group). Swelling graded level 3 (greater than 50 mm) occurred in up to 4% of the test subjects versus 8% of the controls. The immune responses ranged from 5% to 36% across experimental vaccines for the L3 H44-76 strain (versus 27% for the control), from 0% to 11% for the L3 NZ98/124 strain (versus 23% for the control), and from 0% to 13% for the L2 760676 strain (versus 59% for the control). All geometric mean titers were below those measured with the control vaccine. The five experimental formulations were safe and well tolerated but tended to be less immunogenic than the control vaccine.Meningococcal diseases caused by Neisseria meningitidis are a significant health burden throughout the world, leading to death and permanent sequelae (15). Whereas polysaccharide or polysaccharide conjugate vaccines are effective against serogroups A, C, Y, and W135, N. meningitidis serogroup B (MenB) remains a major cause of death and morbidity throughout the world, infants less than 1 year of age being affected the most (5, 8). Serogroup B outbreaks were reported in Europe, Latin America, Australia, New Zealand, and the United States (3, 7, 22, 33). Immunization against MenB presents a challenge, as the capsular polysaccharide is poorly immunogenic in humans (4) and shares molecular mimicry with human antigens (11), which guided the search for outer membrane vesicle (OMV) vaccines (16).Three MenB OMV vaccines with PorA protein as the dominant antigen have been brought to the market (VA-Mengoc-BC [Finlay Institute], MeNZB [Chiron], and MenBvac [Norwegian Institute of Public Health]), but although they have shown protection against PorA-heterologous strains in older children and adults, protection of the youngest is mostly against PorA-homologous MenB strains and their accessibility is geographically limited (7, 9, 18, 21, 25, 26, 31, 34, 36, 37). To be immunogenic in the pediatric and adult populations, a more comprehensive MenB vaccine should include antigens inducing cross-reactive serum bactericidal antibodies (SBA) against a broad spectrum of circulating strains (16, 17, 20, 21, 35). That could best be achieved with non-PorA vaccines (20).Natural immunity against MenB is also induced by protein and lipooligosaccharide (LOS) antigens (28), but proteins and LOS may vary substantially across meningococcal strains. However, at least 70% of invasive MenB isolates express LOS of immunotype L3,7 (19, 27, 29, 30). Hence, GlaxoSmithKline (GSK) Biologicals has developed an experimental vaccine based on the LOS L3 immunotype that was shown to induce bactericidal antibodies in preclinical studies (39). Two detoxified LOS type 3 MenB experimental vaccines differing by the length of the LOS were developed. Such formulations have shown good safety and immunogenicity during preclinical and toxicological studies (39).The primary objective of this study was to evaluate the safety and reactogenicity of several formulations of the experimental vaccines given to healthy young adults. The secondary objective was to assess the immunogenicity of the different formulations.     

Nasal Colonization of and Clonal Transmission of Methicillin-Susceptible Staphylococcus aureus among Chinese Military Volunteers

Military facilities provide unique opportunities for studying Staphylococcus aureus nasal colonization and transmission patterns. In this cross-sectional observational study, we assessed the prevalence of S. aureus nasal colonization among Chinese military volunteers in two camps in the Beijing area. Antimicrobial resistance patterns, risk factors for colonization, and transmission patterns using pulsed-field gel electrophoresis were also evaluated. From May to July 2007, 1,044 nasal swabs were collected from military volunteers from suburban (560) and urban (484) camps. A total of 209 S. aureus isolates were recovered, of which all were methicillin susceptible. Independent factors associated with methicillin-susceptible S. aureus (MSSA) nasal colonization included younger age (odds ratio [OR] = 1.51, 95% confidence interval [95% CI] = 1.03 to 2.21, P = 0.0347), higher education (OR = 1.38, 95% CI = 1.10 to 1.73, P = 0.0056), shorter length of service (OR = 1.74, 95% CI = 1.28 to 2.36, P = 0.0004), nonsmoking (OR = 1.61, 95% CI = 1.14 to 2.28, P = 0.0069), and inactive participation in social events (OR = 2.40, 95% CI = 1.25 to 5.49, P = 0.0082). Among 209 MSSA isolates, 126 (60.3%) were determined to be epidemic and a total of 12 genotypes were identified, of which four (90 isolates [71.4%]) represented the majority of strains. Length of service and camp location were statistically related to the four major MSSA genotype clonal transmissions. Our data indicated that MSSA, not methicillin-resistant S. aureus (MRSA), nasal colonization and clonal transmission occur in healthy military volunteers in Beijing. Younger, female, nonsmoking volunteers with higher education, little or no participation in social events, and less time in service are at higher risk for nasal MSSA carriage.Staphylococcus aureus is an important cause of skin and soft tissue infections, as well as invasive infections in humans (25). Since methicillin-resistant S. aureus (MRSA) was first reported, it has become endemic in hospitals and communities around the world (10). The recent emergence of a highly virulent community-associated MRSA (CA-MRSA) and vancomycin-resistant, intermediate-resistant, or heteroresistant S. aureus further heightens public health concerns (14, 17, 37, 46). Prevention of S. aureus infection and reduction of the spread of virulent and resistant strains are therefore of great importance.On the other hand, S. aureus is a member of the commensal microflora. The anterior nares of the nose are the primary reservoirs of S. aureus colonization in humans, and many S. aureus infections occur in persons with prior nasal bacterial carriage (47). Nasal colonization is an important step in the pathogenesis of S. aureus infection and is a risk factor for acquiring nosocomial infection (22). It has been shown that 80% of nosocomial S. aureus bacteremia episodes in carriers of this bacteria were attributed to an endogenous source (44). Nosocomial S. aureus bacteremia was three times more frequent in S. aureus carriers than in noncarriers (48). Numerous studies of S. aureus nasal carriage have been carried out in various geographic regions in the United States and the Netherlands (2, 5, 7, 21, 23, 27, 28, 41). Cross-section surveys of nasal carriage prevalence and transmission mechanisms in special healthy populations are beneficial in assessing risk factors associated with S. aureus infections (2, 8, 13, 26, 32-35). Military facilities provide unique opportunities for studying S. aureus nasal colonization and transmission (11, 19, 52).In China, MRSA was shown in 63% of S. aureus isolates, among which 77% nosocomial and 43% community isolates were MRSA (49). According to a study conducted in 2005, the mean incidence of MRSA across China was over 50%, and in Shanghai, the prevalence was over 80%, contributed to by two major epidemic MRSA clones with unique geographic distribution (24, 45, 50, 51). Therefore, understanding and controlling the spread of MRSA in both hospital and community settings in China are now of paramount importance. The majority of S. aureus isolates studied in China have been limited to clinical patients, and S. aureus isolates recovered from healthy populations or those from healthy military volunteers have not been previously reported.In this study, we reported a cross-sectional observational study conducted in two military camps in the Beijing area, People''s Republic of China. The prevalence of S. aureus nasal colonization and risk factors associated with colonization were assessed. Nasal carriage S. aureus isolates were genotyped to determine potential clonal transmission in military facilities and related transmission factors.(This study was presented in part at the 109th General Meeting of the American Society for Microbiology, Philadelphia, PA, 17 to 21 May 2009.)     

Evaluation of Adult Chronic Chagas' Heart Disease Diagnosis by Molecular and Serological Methods

Chagas'' disease caused by Trypanosoma cruzi is endemic in Latin America. T. cruzi presents heterogeneous populations and comprises two main genetic lineages, named T. cruzi I and T. cruzi II. Diagnosis in the chronic phase is based on conventional serological tests, including indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA), and diagnosis in the acute phase based on parasitological methods, including hemoculture. The objective of this study was to evaluate the diagnostic procedures of Chagas'' disease in adult patients in the chronic phase by using a PCR assay and conventional serological tests, including TESA-blot as the gold standard. Samples were obtained from 240 clinical chronic chagasic patients. The sensitivities, compared to that of TESA-blot, were 70% for PCR using the kinetoplast region, 75% for PCR using the nuclear repetitive region, 99% for IIF, and 95% for ELISA. According to the serological tests results, we recommend that researchers assess the reliability and sensitivity of the commercial kit Chagatest ELISA recombinant, version 3.0 (Chagatest Rec v3.0; Wiener Lab, Rosario, Argentina), due to the lack of sensitivity. Based on our analysis, we concluded that PCR cannot be validated as a conventional diagnostic technique for Chagas'' disease. These data have been corroborated by low levels of concordance with serology test results. It is recommended that PCR be used only for alternative diagnostic support. Using the nuclear repetitive region of T. cruzi, PCR could also be applicable for monitoring patients receiving etiologic treatment.Chagas'' disease is a complex zoonosis caused by the parasite Trypanosoma cruzi. This parasite can be genetically classified into two major lineages, namely, T. cruzi I, which is found in northern South American countries, and T. cruzi II, which is found in southern South American countries (46). Chagas'' disease is a chronic systemic disease endemic to both South and Central America. T. cruzi is transmitted through the infected dejections of triatomine insects by blood transfusion, congenital infection, laboratory accidents, or oral infection (45). Chagas'' disease constitutes a serious public health problem in terms of both social and economic impact. The disease currently affects 15 million people, and about 28 million are at risk of acquiring the infection. In America, nearly 41,200 new cases occur each year, along with an average of 12,500 deaths per year (16).Chagas'' disease presents two distinct clinical stages. The acute phase begins about 1 week after initial infection, and nearly 30% of patients recall having had relevant symptoms and signs during this period. During the chronic disease stage, the parasites are no longer easily detectable in the bloodstream but serological tests remain positive. Diagnosis of Chagas'' disease is based on parasitological and serological methods. Infection can usually be detected by microscopic examination or by parasitological tests such as hemoculture or PCR (6, 28). There are several targets for the detection of T. cruzi by PCR. The variable region of the minicircle kinetoplast DNA (kDNA) and a repeat tandem sequence of nuclear DNA (stDNA) of the parasite have been the regions most widely used as target sequences for diagnosis via PCR (2, 6, 13, 32, 44). Serological diagnosis of T. cruzi infection is typically performed by using two of three individual tests, according to availability (45). Enzyme-linked immunosorbent assay (ELISA), indirect immunofluorescence (IIF), and indirect hemagglutination are often used. These three tests, also referred to as the conventional tests, usually employ recombinant and/or crude antigenic T. cruzi preparations (21). The major innovation in Chagas'' disease diagnosis with the detection of antibodies against T. cruzi is the TESA-blot (TESA stands for trypomastigote excreted-secreted antigen). This is an immunoblot assay that has been widely used because of its high sensitivity and specificity compared to those of conventional serological methods (38). The isolation and gene cloning of this immunodominant peptide have been intended, and ELISAs based on TESAs have been performed with high-quality results (25). In addition, this test has shown the presence of false negatives when this technique was compared with conventional serology in a cohort from Bolivia (47). These reasons make TESA-blot one of the most feasible and available tests for the diagnosis of Chagas'' disease (38, 39, 40). Recently, TESA-blot has shown great usefulness in resolving doubtful serology and cross-antigenicity issues with related protozoan parasites in regions where the disease is endemic (41). Because of these previous reports, TESA-blot has been selected as the gold standard in several different studies due to the high sensitivity and specificity of the test.Among the conventional techniques used for the serological diagnosis of Chagas'' disease are ELISA because of its high sensitivity and IIF due to its specificity. However, it has been observed that these tests can produce a certain number of false positives and false negatives. This makes it necessary to search for diagnostic tests that provide more reliable results (5). As a routine test for the diagnosis of Chagas'' disease, the World Health Organization recommends immunological techniques according to the type of diagnosis, and a minimum of two positive serological tests are required for considering a patient to be infected with T. cruzi. Nevertheless, in some cases, there is a need to implement other techniques for the diagnosis of T. cruzi. Because of the number of copies and organization of the kinetoplast and nuclear repetitive DNA of T. cruzi, a PCR assay has been developed (29, 42). Comparative studies of PCR, hemoculture, and serology showed that individuals with positive hemoculture and serology results had a detection rate of 36.5%. When PCR was used, the detection of infection was 83.5%. These results demonstrate the higher sensitivity of PCR compared to hemoculture (13). There is great variability within the results obtained by PCR, xenodiagnosis, and hemoculture that makes PCR a controversial tool of choice for the accurate diagnosis of Chagas'' disease.The specificity of serological techniques has been questioned because of the cross-antigenicity between T. cruzi and parasites of related protozoan diseases, particularly leishmaniasis and infection with T. rangeli (5, 39). This questioning arises because these techniques use crude or partially purified parasite extracts, which can cause false-positive results. In order to avoid false-positive results, recombinant antigens and/or synthetic peptides have been used with success (18, 27, 30, 39, 40). These problems may be overcome by using recombinant antigens containing specific T. cruzi epitopes that elicit an immune response in the majority of chagasic patients (8, 14, 21, 39). Therefore, parasitological tests are still extremely necessary to detect T. cruzi, especially in patients with doubtful serology results and to determine the treatment response.The present study is a substudy of the BENEFIT (BENznidazol Evaluation For Interrupting Trypanosomiasis) population recruited in Colombia (23), and the objective was to compare serological diagnosis by ELISA, IIF, and TESA-blot to PCR amplification of the variable region of the kDNA and the stDNA of T. cruzi in clinical and serologically ascertained chagasic patients from Colombia. We also aimed to optimize the procedure of DNA parasite amplification by selecting the most suitable DNA extraction method. Similarly, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and the kappa index were calculated. TESA-blot was used as the gold standard due to the above-mentioned characteristics of this test.     

Comparison of Flocked and Rayon Swabs for Detection of Nasal Carriage of Staphylococcus aureus among Pathology Staff Members

Comparison of flocked swabs (E-swabs; Copan) to the standard rayon swabs (Copan) was undertaken for detection of Staphylococcus aureus nasal carriage among staff at Dorevitch Pathology in Heidelberg, Melbourne, Australia. Among 100 volunteers, 36 were found to be colonized with S. aureus by one or both swab results. The prevalence detected by E-swabs was 35%, and the prevalence through rayon swabs was 34% (95% confidence interval [CI] for the difference in proportions, −12 to 14). Thirty-three volunteers tested positive with both types of swabs, while 2 were detected on E-swabs alone and another on rayon swab testing alone. There was no evidence of a significant difference in carriage detected by E-swabs or rayon swabs.Staphylococcus aureus is a common cause of infections in the community and a major cause of hospital-associated morbidity (18). Colonization is well described, with up to 30% of the population thought to be carriers (7, 16, 18), and is associated with a higher risk of infection in the hospital setting (2, 4, 9, 11, 12, 15, 17, 18). The anterior nares have been shown to be the most frequent site of carriage and therefore a single site for detection (9, 10, 18). Nasal carriage is defined as “persistent” or “intermittent or noncarriage,” with persistent carriers showing an increased risk of infection, compared with intermittent carriers who share the same low risk as noncarriers (13). Given the clinical relevance, it is imperative to use the best swab system which would provide the highest yield in detecting nasal carriage. Flocked swabs have been described as improving uptake of epithelial cells and, therefore, microorganisms and viruses (1, 5, 6, 14), but are more expensive than standard rayon swabs, so it is therefore worth investigating whether there is evidence that E-swabs perform better in detecting nasal carriage.     

Development and Application of a Broadly Sensitive Dried-Blood-Spot-Based Genotyping Assay for Global Surveillance of HIV-1 Drug Resistance

As antiretroviral therapy (ART) is scaled up in resource-limited countries, surveillance for HIV drug resistance (DR) is vital to ensure sustained effectiveness of first-line ART. We have developed and applied a broadly sensitive dried-blood-spot (DBS)-based genotyping assay for surveillance of HIV-1 DR in international settings. In 2005 and 2006, 171 DBS samples were collected under field conditions from newly diagnosed HIV-1-infected individuals from Malawi (n = 58), Tanzania (n = 60), and China (n =53). In addition, 30 DBS and 40 plasma specimens collected from ART patients in China and Cameroon, respectively, were also tested. Of the 171 DBS analyzed at the protease and RT regions, 149 (87.1%) could be genotyped, including 49 (81.7%) from Tanzania, 47 (88.7%) from China, and 53 (91.4%) from Malawi. Among the 70 ART patient samples analyzed, 100% (30/30) of the Chinese DBS and 90% (36/40) of the Cameroonian plasma specimens were genotyped, including 8 samples with a viral load of <400 copies/ml. The results of phylogenetic analyses indicated that the subtype, circulating recombinant form (CRF), and unique recombinant form (URF) distribution was as follows: 73 strains were subtype C (34%), 37 were subtype B (17.2%), 24 each were CRF01_AE or CRF02_AG (11.2% each), 22 were subtype A1 (10.2%), and 9 were unclassifiable (UC) (4.2%). The remaining samples were minor strains comprised of 6 that were CRF07_BC (2.8%), 5 that were CRF10_CD (2.3%), 3 each that were URF_A1C and CRF08_BC (1.4%), 2 each that were G, URF_BC, and URF_D/UC (0.9%), and 1 each that were subtype F1, subtype F2, and URF_A1D (0.5%). Our results indicate that this broadly sensitive genotyping assay can be used to genotype DBS collected from areas with diverse HIV-1 group M subtypes and CRFs. Thus, the assay is likely to become a useful screening tool in the global resistance surveillance and monitoring of HIV-1 where multiple subtypes and CRFs are found.Filter papers have been useful for the collection, storage, and testing of blood specimens in the diagnostic screening of metabolic and inherited disorders in newborn babies in the United States for many years (13, 14). More recently, many resource-limited countries have been using them for HIV-related molecular assays, including early infant diagnosis using Roche Amplicor HIV-1 DNA PCR testing (supported by the U.S. President''s Emergency Plan for AIDS Relief [PEPFAR]) (31), real-time PCR-based HIV-1 diagnosis (22, 27) and viral load measurement (6, 17, 18, 20, 26), and HIV-1 drug resistance (DR) genotyping (5, 7, 8, 12, 16, 19, 23-25, 28, 30, 35-37).Dried blood spots (DBS) offer several advantages over conventional plasma or serum for sample collection and storage. First, DBS circumvent the need for phlebotomy and reduce the risk of needle-stick-related HIV exposure when they are collected using finger or heel stick. Second, they do not require cold-chain transportation of specimens from collection sites to the testing laboratories; thus, they can be transported using standard postal services, resulting in the reduction of cost for storage and transportation when dried and processed appropriately. Overall, DBS provide resource-limited countries with opportunities for sustainable laboratory services and surveillance programs for HIV diagnosis and DR genotyping. Very few studies have been conducted using DBS collected under real field conditions. Furthermore, the DR genotyping assays used were not evaluated for multiple HIV-1 subtypes and circulating recombinant forms (CRFs) (5, 16, 19, 23, 25, 37).The aim of the current study was to determine the performance of a broadly sensitive genotyping assay for detecting multiple HIV-1 group M subtypes and CRFs that cocirculate in different countries, using DBS collected under field conditions.     

Development of a Bead Immunoassay To Measure Vi Polysaccharide-Specific Serum IgG after Vaccination with the Salmonella enterica Serovar Typhi Vi Polysaccharide

Vi polysaccharide from Salmonella enterica serotype Typhi is used as one of the available vaccines to prevent typhoid fever. Measurement of Vi-specific serum antibodies after vaccination with Vi polysaccharide by enzyme-linked immunosorbent assay (ELISA) may be complicated due to poor binding of the Vi polysaccharide to ELISA plates resulting in poor reproducibility of measured antibody responses. We chemically conjugated Vi polysaccharide to fluorescent beads and performed studies to determine if a bead-based immunoassay provided a reproducible method to measure vaccine-induced anti-Vi serum IgG antibodies. Compared to ELISA, the Vi bead immunoassay had a lower background and therefore a greater signal-to-noise ratio. The Vi bead immunoassay was used to evaluate serum anti-Vi IgG in 996 subjects from the city of Kolkata, India, before and after vaccination. Due to the location being one where Salmonella serotype Typhi is endemic, approximately 45% of the subjects had protective levels of anti-Vi serum IgG (i.e., 1 μg/ml anti-Vi IgG) before vaccination, and nearly 98% of the subjects had protective levels of anti-Vi serum IgG after vaccination. Our results demonstrate that a bead-based immunoassay provides an effective, reproducible method to measure serum anti-Vi IgG responses before and after vaccination with the Vi polysaccharide vaccine.Typhoid fever is caused by Salmonella enterica serotype Typhi (32). Humans are the only natural host and reservoir of S. enterica serotype Typhi (32, 41). Typhoid fever represents a spectrum of diseases ranging from an acute uncomplicated disease—including fever, headache, malaise, and disturbances of bowel function (constipation in adults and diarrhea in children)—to a more severe, complicated form of disease in 10 to 20% of infected patients that includes bleeding in the gastrointestinal tract, intestinal perforation (in 1 to 3% of hospital typhoid fever cases) and an altered mental state (32, 41). The case fatality rate is highly variable, depending on the medical treatment available and geographic location. For example, the average fatality rate is less than 1% overall but may range between 2% fatality in hospitalized patients in Pakistan and Vietnam and 50% fatality in hospitalized patients in some parts of Indonesia and Papua New Guinea (32, 41). Worldwide, typhoid fever remains a significant public health problem, with an estimated 17,000,000 cases of typhoid fever each year and up to 600,000 deaths (2, 10, 32, 41).Typhoid vaccines currently available are composed of purified Vi polysaccharide or live attenuated S. enterica serotype Typhi (Ty21a) organisms (10, 39). The Vi polysaccharide vaccine induces protective serum antibody responses that reach a maximum at 28 days after a single intramuscular vaccination with 25 μg purified Vi polysaccharide (39), a capsular polysaccharide (Vi for virulence) that increases the virulence of S. enterica serotype Typhi (32). Protective antibody levels have been estimated to be 1 μg/ml anti-Vi IgG antibody in the serum (20). Protective efficacy of the Vi polysaccharide vaccine as determined by protection against disease is modest, with only 55 to 72% of subjects protected against disease through 3 years postvaccination (1, 20, 39). The live attenuated Ty21a vaccine is administered orally as three or four doses of enteric capsules (39). Due to its use as an oral, mucosally administered vaccine, the Ty21a vaccine induces protection against typhoid fever by induction of mucosal IgA and serum IgG antibodies specific for lipopolysaccharide antigens (39). The protective efficacy of the Ty21a vaccine at 3 years postvaccination was reported to range from 42 to 67% when using three doses of Ty21a enteric capsules (11, 39). Next-generation vaccines that utilize Vi conjugated to protein carriers that provide superior induction of anti-Vi antibodies are currently in development (14, 21, 25, 36).Despite its ability to induce protective immune responses when used alone or conjugated to protein carriers, the use of Vi polysaccharide as a coating antigen in enzyme-linked immunosorbent assay (ELISA) to measure vaccine-induced anti-Vi antibody responses has been reported to be problematic. The use of polysaccharides (lipopolysaccharide [LPS], Haemophilus influenzae type b capsular polysaccharide, Vi polysaccharide) as coating antigens for immunoassays is plagued by problems such as a poor binding of polysaccharides to ELISA plates and inconsistent results (3, 15, 16, 26, 33). To increase binding of Vi antigen to ELISA plates and produce more-robust assays, others have biotinylated Vi and then added it to streptavidin-coated plates (12) or conjugated Vi to tyramine (22, 26). However, some reports indicate that Vi was used without any additional treatment as an ELISA coating antigen (7, 19, 21) although a Vi ELISA performed on plates was less sensitive than a radioimmunoassay procedure (19).Immunoassays based on the use of fluorescent beads as the solid surface have recently been developed and compared to ELISA for the measurement of antigen-specific antibodies for polysaccharides from Streptococcus pneumoniae, Neisseria meningitidis, or Haemophilus influenzae type b (HiB) (5, 8, 23, 27, 34, 35). The fluorescent bead assays were comparable to ELISA and sometimes were noted as having enhanced dynamic ranges or increased sensitivity (5, 8, 27, 35). An additional benefit of fluorescent bead immunoassays is their ability to be multiplexed to permit the simultaneous measurement of antibodies specific for different antigens (8, 23, 27, 34, 35). This study was performed to evaluate a fluorescent bead immunoassay for its ability to measure vaccine-induced antibodies specific for Salmonella serotype Typhi Vi polysaccharide. The performance of the fluorescent bead assay was compared to that of ELISA.     

High-Throughput Identification of Bacteria and Yeast by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Conventional Medical Microbiology Laboratories

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.Identification of bacteria and yeasts is generally based on conventional phenotypic methods, encompassing culture and growth patterns on specific media, Gram staining, and morphological and biochemical characteristics. Although results of Gram staining can be achieved within minutes, complete identification usually takes 1 or more days. In addition, tests may be difficult to interpret or inconclusive and require specialized staff. Recent molecular methods for microbial identification, such as real-time PCR, sequence analysis, or microarray analysis, have found some application in bacteriology. However, these methods do not provide the complete solution in routine bacterial identifications. To optimize care of patients with infectious diseases, there still is an urgent need for rapid and simple techniques for microbial identification.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been used to analyze many different biological molecules. The application of microbial identification based on species-specific spectra of peptides and protein masses by mass spectrometry was first reported about 30 years ago (1). By further improvement of the technique, a rapid, accurate, easy-to-use, and inexpensive method has become available for identification of microorganisms (4, 14, 27). MALDI-TOF MS can be used for accurate and rapid identification of various microorganisms, such as Gram-positive bacteria (2, 3, 9, 10, 22, 26), Enterobacteriaceae (5), nonfermenting bacteria (6, 19-21), mycobacteria (12, 16, 24), anaerobes (10, 23), and yeasts (18, 25). Most studies have reported on MALDI-TOF MS identification of a single strain or family of microorganisms in a research setting. Only one study applied MALDI-TOF MS for identification of bacteria—but not yeasts—in conventional microbiology settings but did not evaluate the results for individual bacteria at the species level (27). In the present study, identification of bacteria by MALDI-TOF MS was extensively evaluated for both bacterial and yeast species identification in an academic medical microbiologic laboratory.     

Clonal Analysis of Colonizing Group B Streptococcus,Serotype IV,an Emerging Pathogen in the United States

Colonizing group B Streptococcus (GBS) capsular polysaccharide (CPS) type IV isolates were recovered from vaginal and rectal samples obtained from 97 (8.4%) nonpregnant women of 1,160 women enrolled in a U.S. multicenter GBS vaccine study from 2004 to 2008. Since this rate was much higher than the rate of prevalence of 0.4 to 0.6% that we found in previous studies, the isolates were analyzed by using surface protein profile identification, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) to characterize them and identify trends in DNA clonality and divergence. Of the 101 type IV isolates studied, 53 expressed α and group B protective surface (BPS) proteins, 27 expressed BPS only, 20 expressed α only, and 1 had no detectable surface proteins. The isolates spanned three PFGE macrorestriction profile groups, groups 37, 38, and 39, of which group 37 was predominant. The isolates in group 37 expressed the α and BPS proteins, while those in groups 38 and 39 expressed the α protein only, with two exceptions. MLST studies of selective isolates from the four protein profile groups showed that isolates expressing α,BPS or BPS only were of a new sequence type, sequence type 452, while those expressing α only or no proteins were mainly of a new sequence type, sequence type 459. Overall, our study revealed a limited diversity in surface proteins, MLST types, and DNA macrorestriction profiles for type IV GBS. There appeared to be an association between the MLST types and protein expression profiles. The increased prevalence of type IV GBS colonization suggested the possibility that this serotype may emerge as a GBS pathogen.Group B Streptococcus (GBS) (Streptococcus agalactiae) is a leading cause of neonatal infection in the United States, with maternal vaginal or rectal colonization often resulting in the transmission of GBS to the infant during the perinatal period (8, 23). GBS isolates are classified according to nine capsular polysaccharide (CPS) types: types Ia, Ib, and II to VIII and the recently proposed type IX (9, 15, 21, 23, 46, 52). Isolates that do not express any of the known CPS types are designated nontypeable (NT) (2, 6, 21, 40). In addition to CPS, GBS may express one or more surface-localized proteins, including the α and β components of the c protein (24); the alpha-like R proteins, specifically R1, R4(Rib), and R1,R4 (also known as Alp3) (14, 17, 19, 30, 40); and the group B protective surface (BPS) protein (12). Certain protein profiles are associated with each capsular polysaccharide CPS type (2), for example, the c(α only) protein with types Ia and II, c(α + β) with type Ib, and R4(Rib) with type III (2, 14). BPS, expressed by fewer than 3% of colonizing isolates, can be found alone or with another protein in type Ia, II, and V isolates (12, 14).In the United States, the predominant serotypes over the past 2 decades, constituting 70 to 75% of all GBS isolates, have been type Ia, type III, and the more recently emerged type V (14, 15, 20, 52). The remaining isolates consisted primarily of types Ib and II, with types IV, VI, VII, and VIII making up a small fraction of the isolates. We found type IV to represent between 0.4 and 0.6% of colonizing GBS isolates (14, 15), but only rare type IV isolates were found in invasive GBS disease during that same time period (14, 43, 52).In contrast to the previously low percentage of type IV isolates reported for the United States, recent studies in the United Arab Emirates, Turkey, and Zimbabwe showed large proportions of type IV isolates among their GBS isolates. In the United Arab Emirates, type IV was the predominant serotype among colonized pregnant women, representing 26.3% of the GBS isolates (1). In eastern Turkey, it was the second most common serotype, at 8.3%, among colonizing isolates (10), and in Zimbabwe, it was the fourth most common serotype, comprising 5.1% of GBS isolates from colonized pregnant women and 4.0% of all GBS isolates from various sites, including blood and cerebrospinal fluid (CSF), from hospitalized patients (36).Immunization studies of humans (3, 28) and protection studies with mice (37) have shown the potential of vaccines against the common GBS serotypes to prevent invasive neonatal GBS disease through the vaccination of pregnant women (3, 28). The GBS strains described here are from a phase II randomized, double-blinded clinical trial of a GBS serotype III-tetanus toxoid (CPS III-TT) vaccine to prevent the vaginal acquisition of GBS type III in nonpregnant women in three areas of the United States: Pittsburgh (PA), Georgia, and Texas (S. Hillier, unpublished data). Because we found type IV isolates for almost 10% of these patients, we examined the type IV isolates for surface proteins and clonality.Pulsed-field gel electrophoresis (PFGE) was used in this analysis because it is a widely used method that can further characterize GBS isolates within particular CPS type and/or protein profile groups (2, 4, 6, 48). Multilocus sequence typing (MLST) was performed in order to assess the general relatedness of strains within and across laboratories (25, 50). Together, the discriminatory power of PFGE and the objectivity of MLST gave insight into the GBS type IV population genetic structure and the identification of emerging clones (2, 5, 13, 18, 19).     

Prevalence of and Risk Factors for Colonization by Methicillin-Resistant Staphylococcus aureus among Adults in Community Settings in Taiwan

In order to determine the prevalence of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) colonization among adults in community settings in Taiwan and identify its risk factors, we conducted the present study. For a 3-month period, we enrolled all adults who attended mandatory health examinations at three medical centers and signed the informed consent. Nasal swabs were taken for the isolation of S. aureus. For each MRSA isolate, we performed multilocus sequence typing, identification of the staphylococcal cassette chromosome mec, tests for the presence of the Panton-Valentine leukocidin gene, and tests for drug susceptibilities. Risk factors for MRSA colonization were determined. The results indicated that the MRSA colonization rate among adults in the community settings in Taiwan was 3.8% (119/3,098). Most MRSA isolates belonged to sequence type 59 (84.0%). Independent risk factors for MRSA colonization included the presence of household members less than 7 years old (P < 0.0001) and the use of antibiotics within the past year (P = 0.0031). Smoking appeared to be protective against MRSA colonization (P < 0.0001).Before the late 1990s, nearly all methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) infections occurred in patients with specific risk factors who were in health care facilities (31). However, the emergence of MRSA infections among previously healthy persons in community settings (without exposure to health care facilities) was noted thereafter (6, 31). Therefore, MRSA infections are now classified as health care-associated MRSA (HA-MRSA) infections and community-associated MRSA (CA-MRSA) infections (38).Strains responsible for CA-MRSA infections differ from those for HA-MRSA infections in several phenotypic and genetic features (1, 28). CA-MRSA strains carry type IV or V staphylococcal cassette chromosome mec (SCCmec) elements, are usually Panton-Valentine leukocidin (PVL) producing, and are not multidrug resistant; HA-MRSA strains carry type I, II, or III SCCmec elements, are usually not PVL producing, and are multidrug resistant (15, 22, 28).Initially, CA-MRSA infections were mostly reported in young children (36). However, as CA-MRSA infections became more common, infections were reported among people of all ages and contributed to the increase of community-associated S. aureus infections with significance (25, 29, 36). MRSA colonization is an important risk factor for subsequent MRSA infection (30), so several studies in the United States have characterized the MRSA colonization rate in a community setting (13, 16). These studies demonstrated that the nasal colonization rates among healthy children increased from 0.8% in 2001 to 9.2% in 2004 (13). The colonization rate was 0.84% among people participating in the 2001 to 2002 National Health and Nutrition Examination Survey (NHANES) (16).In Taiwan, MRSA strains of sequence type 59 (ST59), determined by multilocus sequence typing (MLST) and carrying type IV or V SCCmec elements, were recently found to be the major strains of CA-MRSA (5, 7, 27). Other studies demonstrated that these CA-MRSA strains were responsible for the rapid increase in the number of CA-MRSA infections among children and adults in Taiwan (7, 37). The MRSA colonization rates among Taiwanese children increased from 1.5% from 2001 to 2002 to 7.2% from 2005 to 2006 (18, 19). However, the MRSA colonization rate among adults in community settings in Taiwan is unclear. This study was conducted to determine the prevalence and risk factors for the colonization of MRSA among adults in community settings in Taiwan.     

Trends in Methicillin-Resistant Staphylococcus aureus Anovaginal Colonization in Pregnant Women in 2005 versus 2009

In 2005, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) anovaginal colonization in pregnant women at our center (Columbia University Medical Center) was 0.5%, and MRSA-colonized women were less likely to carry group B streptococcus (GBS). In this study, our objectives were to identify changing trends in the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) anovaginal colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to characterize the MRSA strains. From February to July 2009, Lim broths from GBS surveillance samples were cultured for S. aureus. MRSA strains were identified by resistance to cefoxitin and characterized by MicroScan, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa typing, and Panton-Valentine leukocidin PCR. A total of 2,921 specimens from different patients were analyzed. The prevalences of MSSA, MRSA, and GBS colonization were 11.8%, 0.6% and 23.3%, respectively. GBS colonization was associated with S. aureus colonization (odds ratio [OR], 1.9; 95% confidence interval [95% CI], 1.5 to 2.4). The frequencies of GBS colonization were similar in MRSA-positive (34.2%) versus MRSA-negative patients (21.8%) (P = 0.4). All MRSA isolates from 2009 and 13/14 isolates from 2005 were SCCmec type IV or V, consistent with community-associated MRSA; 12/18 (2009) and 0/14 (2005) isolates were the USA300 clone. Levofloxacin resistance increased from 14.3% (2005) to 55.6% (2009) (P = 0.028). In conclusion, the prevalence of MRSA anovaginal colonization in pregnant women in New York City, NY, remained stable from 2005 to 2009, and USA300 emerged as the predominant clone with a significant increase in levofloxacin-resistant isolates.The rapid spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) across the United States has been associated primarily with the dissemination of a specific clone that has the pulsed-field gel electrophoresis (PFGE) pattern termed USA300 (20). CA-MRSA can cause infections in pregnant and postpartum women and outbreaks in newborn nurseries and neonatal intensive care units (NICUs) (5, 22, 26, 32, 35, 36, 38). CA-MRSA strains, including USA300, have replaced health care-associated (HA)-MRSA as the predominant strains isolated from infants in some NICUs (6, 35). CA-MRSA infections appear to be increasing in otherwise healthy neonates in the nursery (18, 38) who may acquire S. aureus from health care workers or from their mothers and other family members (19, 23, 25, 28).Vertical transmission from mothers to infants at delivery has also been proposed as a possible mechanism of acquisition of CA-MRSA (1, 2, 7, 28). S. aureus has been reported to colonize the vagina in 4 to 22% of pregnant women (2, 4, 9, 13, 14). In 2005, following an outbreak of USA300 in postpartum women at our medical center (Columbia University Medical Center), we determined that the prevalence of methicillin-susceptible S. aureus (MSSA) anovaginal colonization was 16.6% and the prevalence of MRSA colonization was 0.5% (9). Overall, 93% of MRSA strains were staphylococcal cassette chromosome mec (SCCmec) type IV or V, which is consistent with CA-MRSA (9). More recent studies conducted in other locales have suggested that the prevalence of anovaginal colonization with MRSA is increasing, with reported rates ranging from 3.5 to 10.4% (2, 13). The association of MRSA colonization with group B streptococcus (GBS) colonization is less clear; some reports have shown an increased rate of MRSA colonization in GBS-positive women (2), while others have shown a decreased rate (8, 32).Reports suggesting an increasing prevalence of MRSA among pregnant women, coupled with the recent emergence of USA300 in our NICU (6), led us to question whether the epidemiology of MRSA colonization was also changing in pregnant women in our population in New York City, NY. The objectives of this study were to determine the current prevalence of MRSA and MSSA colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to use molecular methods to characterize MRSA strains from the current study and compare those with strains from our 2005 study.     

Loss of Ability To Self-Heal Malaria upon Taurine Transporter Deletion

Deletion of the taurine transporter gene (taut) results in lowered levels of taurine, the most abundant amino acid in mammals. Here, we show that taut^−/− mice have lost their ability to self-heal blood-stage infections with Plasmodium chabaudi malaria. All taut^−/− mice succumb to infections during crisis, while about 90% of the control taut^+/+ mice survive. The latter retain unchanged taurine levels even at peak parasitemia. Deletion of taut, however, results in the lowering of circulating taurine levels from 540 to 264 μmol/liter, and infections cause additional lowering to 192 μmol/liter. Peak parasitemia levels in taut^−/− mice are approximately 60% higher than those in taut^+/+ mice, an elevation that is associated with increased systemic tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) levels, as well as with liver injuries. The latter manifest as increased systemic ammonia levels, a perturbed capacity to entrap injected particles, and increased expression of genes encoding TNF-α, IL-1β, IL-6, inducible nitric oxide synthase (iNOS), NF-κB, and vitamin D receptor (VDR). Autopsy reveals multiorgan failure as the cause of death for malaria-infected taut^−/− mice. Our data indicate that taut-controlled taurine homeostasis is essential for resistance to P. chabaudi malaria. Taurine deficiency due to taut deletion, however, impairs the eryptosis of P. chabaudi-parasitized erythrocytes and expedites increases in systemic TNF-α, IL-1β, and ammonia levels, presumably contributing to multiorgan failure in P. chabaudi-infected taut^−/− mice.Taurine, a nonprotein sulfur-containing amino acid, is the most abundant amino acid in mammals, occurring both in cells and in blood plasma (7, 22). It plays an important role in diverse biological processes, such as cell volume regulation, neuromodulation, antioxidant defense, protein stabilization, and stress responses (11, 16, 46, 48, 54). Taurine may protect cells against various types of injury (11, 16, 29, 38, 45, 48, 54, 56). In particular, taurine is considered a basic regulator of cell homeostasis, presumably as an osmolyte and chaperone (19). According to the current view, taurine is synthesized from cysteine primarily—if not exclusively—in hepatocytes, and it is then exported to the plasma and imported into other cells via the taurine transporter (22).The disruption of the taurine transporter gene (taut) by homologous recombination causes reductions of 80 to 98% in taurine levels in plasma and blood cells, as well as in tissues such as the brain, the kidney, and skeletal and heart muscles (20, 50, 51). Moreover, taut^−/− mice exhibit decreased resistance to osmotic shock and oxidation stress (31). Taurine levels in the liver are decreased by about 70% in adult taut^−/− mice; in particular, they are decreased by more than 80% in Kupffer and sinusoidal endothelial cells and by only approximately 30% in liver parenchymal cells (50). Taurine availability is an important modulator of Kupffer cell functions, such as phagocytosis and eicosanoid synthesis (52). taut-deficient mice develop moderate unspecific hepatitis and liver fibrosis at older than 1 year (50, 51). Moreover, though our knowledge is still poor, some information is available on how the immune system is affected by taut depletion (17). For instance, taurine modulates basic functions of leukocytes, such as phagocytosis, prostanoid formation, and cytokine formation (22, 39, 43, 45, 52, 53). However, the consequences of lowered taurine levels for the outcomes of infectious diseases have not been investigated to date.Malaria is one of the major infectious diseases worldwide, with about 1 million to 3 millions deaths per year (13). The liver plays a central role in malaria: it is the site where the preerythrocytic stages of the malaria parasites have to develop and multiply (in hepatocytes), but it is also the site where the intraerythrocytic stages of the parasites, which are responsible for disease and death, can be trapped and even destroyed (2, 3, 28). Predominantly the Kupffer cells, which constitute approximately 80 to 90% of total macrophages, contribute to the trapping capacity of the liver. Recent evidence obtained in experimental Plasmodium chabaudi malaria indicates that during the crisis phase of infection, when parasitemia drops from about 50% to below 1% within 3 to 4 days, the liver improves its trapping capacity, whereas the spleen is largely closed (27).In the present study, we show that the lowering of taurine levels due to taut deletion results in a lethal outcome of otherwise self-healing blood-stage malaria caused by P. chabaudi in mice.     

Algorithm Combining Toxin Immunoassay and Stool Culture for Diagnosis of Clostridium difficile Infection

Enzyme immunoassays (EIA) to detect glutamate dehydrogenase or toxins A (TcdA) and B (TcdB), a cytotoxicity assay, and bacteriologic culture have disadvantages when applied individually to diagnosis of Clostridium difficile infections. Stool specimens (n = 1,596) were subjected to toxin detection via an enzyme-linked fluorescent immunoassay (ELFA; Vidas CDAB assay) and bacteriologic culture for toxigenic C. difficile in a three-step algorithm with additional toxigenic culture. Isolates (n = 163) from ELFA-negative stool specimens were examined via ELFA for toxin production. We amplified tcdA and tcdB from C. difficile isolates and tcdB from stool specimens that were ELFA positive or equivocal and culture negative, and we compared the results to those obtained with the three-step algorithm. More than 26% of stool specimens (419/1,596) were culture positive, yielding 248 isolates (59.2%) with both toxin genes (tcdA- and tcdB-positive isolates), 88 isolates (21.0%) with either tcdA or tcdB, and 83 (19.8%) that had no toxin genes (tcdA- and tcdB-negative isolates). Among 49 (culture-negative/ELFA-positive or -equivocal) stool specimens, 53.1% (26/49) represented tcdB-positive isolates. Therefore, the total number of PCR-positive cases was 362, and 27.1% (98/362) of these were detected through toxigenic culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 63.3%, 96.7%, 90.5%, and 92.4% (ELFA alone); 92.8%, 93.3%, 80.2%, and 97.8% (culture); and 70.7%, 91.4%, 95.5%, and 100% (three-step algorithm ELFA and bacterial culture with toxigenic culture), respectively, with culture and PCR for tcdA and tcdB as the standards. Thus, sensitivity and specificity were highest using culture and ELFA, respectively, but we recommend the three-step algorithm comprising EIA to detect both toxins and toxigenic culture for C. difficile as a practical method for achieving better PPV and NPV.Clostridium difficile is an important nosocomial pathogen, causing antimicrobial-associated diarrhea and pseudomembranous colitis. Toxins A (TcdA) and B (TcdB) mediate the pathogenesis of C. difficile infection (CDI), and toxin detection is an important part of diagnosis. A cytotoxicity neutralization assay (CNA) is the reference method for toxin detection, but it is expensive and time-consuming and requires tissue culture facilities (34, 35). Most laboratories now use a commercial enzyme immunoassay (EIA) to detect TcdA and/or TcdB, with the benefits of rapid turnaround time and ease of use (3, 21, 22, 23, 26, 27, 33, 35). The putative >90% sensitivity of toxin EIAs is not often realized in practice, but EIA is the only toxin detection method available to many routine medical laboratories. The demand for EIA kits detecting both TcdA and TcdB has increased due to increased worldwide prevalence of TcdA-negative, TcdB-positive (TcdA− TcdB+) strains (1, 12, 24, 29, 32).A two-step algorithm, based upon EIA-based detection of species-specific antigen glutamate dehydrogenase (GDH-Ag) and toxin detection via CNA, was suggested to have improved sensitivity and specificity in the detection of toxigenic C. difficile (34). However, the GDH-Ag assay detects both nontoxigenic and toxigenic strains, and the aforementioned shortcomings of the CNA assay make it unavailable to many routine laboratories.Bacteriologic culture can be time-consuming, but it is more straightforward and sensitive than CNA for the detection of toxigenic C. difficile. Furthermore, it provides isolates for characterization, yielding information about CDI epidemiology and antimicrobial susceptibility (11, 28, 36). We evaluated the combination of bacteriologic culture and EIA-based detection of TcdA and TcdB as a new strategy for diagnosis of CDI, especially in areas where TcdA− TcdB+ strains are prevalent.