Distribution of the Escherichia coli Common Pilus among Diverse Strains of Human Enterotoxigenic E. coli

The Escherichia coli common pilus (ECP) is produced by commensal and pathogenic E. coli strains. This pilus is unrelated to any of the known colonization factors (CFs) of enterotoxigenic E. coli (ETEC). In this study, we investigated the distribution and production of ECP among a collection of 136 human CF-positive and CF-negative ETEC strains of different geographic origins. The major pilus subunit gene, ecpA, was found in 109 (80%) of these strains, suggesting that it is widely distributed among ETEC strains. Phenotypic analysis of a subset of 43 strains chosen randomly showed that 58% of them produced ECP independently of the presence or absence of CFs, a percentage even higher than that of the most prevalent CFs. These data suggest an important role for ECP in the biology of ETEC, particularly in CF-negative strains, and in human infection.Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and mortality for children living in developing countries (11). The presence of ETEC in these areas is associated with a lack of sanitation or poor sanitation and the consumption of contaminated water or food. The main virulence factors of ETEC are a heat-labile (LT) and/or a heat-stable (ST) enterotoxin and multiple adhesive pili called colonization factors (CFs) (1, 7), which are produced in the small intestine and can cause life-threatening, cholera-like watery diarrhea (7). Since the early 1970s, more than 25 different CFs have been reported in ETEC strains of diverse geographic origins, and the prevalence of these pili differs by geographic region (7, 11). Studies of the prevalence and distribution of CFs among ETEC strains worldwide have shown that the most common CFs are CFA/I and combinations of E. coli surface antigens CS1, CS2, and CS3 or of antigens CS4, CS5, and CS6. Approximately 50% of ETEC strains contain at least one of these CFs (7), leaving 50% of strains that do not produce any of the CFs known or characterized so far. The presence of type IV pili, which are associated with host colonization and virulence in many gram-negative bacteria, has also been demonstrated in a significant number (30 to 50%, depending on the geographic source) of ETEC strains, including strains that do not harbor any of the known CFs. These pili provide a mechanism for the organisms to colonize the human gut and establish gastrointestinal disease. Epidemiological studies have shown that protective immunity, attributed to the antigenic variety of the CFs produced, can be achieved through multiple infections. Thus, it is believed that vaccines aimed at preventing ETEC infections, particularly in the young population and travelers, should contain the immunogenic B subunit of the LT and a combination of the most common CFs (7, 9, 10).Previously, it was reported that meningitis-associated E. coli strains, and not other E. coli pathogroups, were able to assemble a “meningitis-associated temperature-dependent pilus” (Mat) after growth at 20°C in Luria-Bertani (LB) medium. The major pilus subunit of the Mat pilus is encoded by the yagZ gene, commonly found in all E. coli strains. Recently, our laboratory reported that most (75%) strains of human and animal E. coli pathogroups (including ETEC), as well as commensal E. coli strains, produce at 37°C a pilus adhesive structure composed of a major 21-kDa protein pilin subunit corresponding to the product of the yagZ gene (8). Because this gene was demonstrated to be widely distributed and highly conserved among E. coli strains, and because production of the pili was shown in the major E. coli pathovars, it was proposed that the pilus be renamed “E. coli common pilus,” or ECP, and that the gene encoding the pilin subunit be designated ecpA. A role for ECP in adherence to cultured human epithelial cells was demonstrated in enterohemorrhagic E. coli (EHEC) O157:H7 and commensal E. coli strains (8).ECP is not related to any of the known ETEC CFs. The present study was carried out to further investigate the presence of ecpA and to determine the production of ECP in a collection of human ETEC strains that had previously been characterized as CF positive or CF negative. We found ECP production in both groups of strains at rates comparable to those found for the most common CFs. Our data suggest that the production of ECP in ETEC strains may contribute to the adhesive properties of this organism and may represent a target for vaccine development and the prevention of ETEC infections.     

Children with the Le(a+b?) Blood Group Have Increased Susceptibility to Diarrhea Caused by Enterotoxigenic Escherichia coli Expressing Colonization Factor I Group Fimbriae

Recent studies have shown that children with blood group A have increased susceptibility to enterotoxigenic Escherichia coli (ETEC) diarrhea and that Lewis blood group “a” antigen (Le^a) may be a candidate receptor for ETEC colonization factor (CF) antigen I (CFA/I) fimbriae. Based on these findings, we have attempted to determine if children with the Le(a+b−) phenotype may be more susceptible to diarrhea caused by ETEC, in particular ETEC expressing CFA/I and related fimbriae of the CFA/I group, than Le(a−b+) children. To test this hypothesis, we have determined the Lewis antigen expression in 179 Bangladeshi children from a prospective birth cohort study in urban Dhaka in which ETEC expressing major CFs such as CFA/I, CS3, CS5, and CS6 was the most commonly isolated diarrhea pathogen during the first 2 years of life. The Lewis blood group phenotypes were determined by a dot blot immunoassay using saliva samples and by a tube agglutination test using fresh red blood cells. The results indicate that Le(a+b−) children more often had symptomatic than asymptomatic ETEC infections (P < 0.001), whereas symptomatic and asymptomatic ETEC infections were equally frequent in Le(a−b+) children. We also show that children with the Le(a+b−) blood type had significantly higher incidences of diarrhea caused by ETEC expressing fimbriae of the CFA/I group than Le(a−b+) children (P < 0.001). In contrast, we did not find any association between the Lewis blood group phenotype and diarrhea caused by ETEC expressing CS6 or rotavirus.Expression of Lewis or ABO histo-blood group types has been shown to be associated with different risks of enteric infections (4, 5, 12, 15, 24, 27), presumably through differential expression of cell surface glycoconjugates that are used as receptors for pathogens of the intestinal mucosa. The Lewis blood group antigens on the intestinal mucosa are synthesized through a group of glycosyltransferases, which insert fucose residues in type 1 and type 2 oligosaccharide precursors (21, 29, 30). The synthesis of Lewis antigens is dependent on the FUT2 and FUT3 genes. If both genes are functional, the phenotype will be Le(a−b+), i.e., the secretor type, whereas individuals in whom the FUT2 gene is not expressed will have the Le(a+b−) phenotype, i.e., the nonsecretor type. Failure to express both FUT2 and FUT3 will result in Le(a−b−) (9).A predisposition for obtaining dehydrating cholera has been seen in blood group O individuals (8, 12, 14, 19, 28). In contrast, our recent study showed that enterotoxigenic Escherichia coli (ETEC) diarrheal episodes were more common in children with blood group AB or A than in individuals with blood group O (24). We have also shown that colonization factor (CF) antigen I (CFA/I) expressed by ETEC binds to glycosphingolipids that are associated with blood group antigens, e.g., Le^a, that may be expressed on epithelial cells in the small intestine in humans (16). The glycosphingolipid binding capacity of CFA/I fimbriae resides in the major CfaB subunit protein (3, 8, 16). CFA/I was the first identified human-specific CF of ETEC bacteria (11). Subsequently, seven other genetically related fimbriae, CS1, CS2, CS4, CS14, CS17, CS19, and putative CF O71, denoted as the CFA/I group (1), have been shown to be related to CFA/I both in the structural subunits (26) and tip-localized minor adhesive subunits (1). A glycosphingolipid binding pattern similar to that of CFA/I has been demonstrated for CS1 and CS4 that might be due to related N-terminal sequences (3, 8, 16). In addition, in another study (25) we have also shown that the conserved regions of the CF subunit proteins (shared by the CFA/I group fimbriae) are likely to be responsible for the receptor binding, since monoclonal antibodies against this region prevented enterocyte binding and protected against challenge with ETEC expressing CFA/I and CS4.In a recent longitudinal birth cohort (BC) study in Dhaka, we showed that ETEC was a major pathogen in children up to 2 years old and that a high proportion of symptomatic infections were caused by ETEC expressing the CFA/I group fimbriae (24). In this study, we present additional data to determine whether children with specific Lewis blood group antigen phenotypes, e.g., Le(a+b−) or Le(a−b+), have different susceptibilities to diarrhea caused by ETEC, in particular ETEC expressing the CFA/I group fimbriae, as well as susceptibilities to diarrhea caused by rotavirus.     

Ancestral Lineages of Human Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea among children living in and among travelers visiting developing countries. Human ETEC strains represent an epidemiologically and phenotypically diverse group of pathogens, and there is a need to identify natural groupings of these organisms that may help to explain this diversity. Here, we sought to identify most of the important human ETEC lineages that exist in the E. coli population, because strains that originate from the same lineage may also have inherited many of the same epidemiological and phenotypic traits. We performed multilocus sequence typing (MLST) on 1,019 ETEC isolates obtained from humans in different countries and analyzed the data against a backdrop of MLST data from 1,250 non-ETEC E. coli and eight ETEC isolates from pigs. A total of 42 different lineages were identified, 15 of which, representing 792 (78%) of the strains, were estimated to have emerged >900 years ago. Twenty of the lineages were represented in more than one country. There was evidence of extensive exchange of enterotoxin and colonization factor genes between different lineages. Human and porcine ETEC have probably emerged from the same ancestral ETEC lineage on at least three occasions. Our findings suggest that most ETEC strains circulating in the human population today originate from well-established, globally widespread ETEC lineages. Some of the more important lineages identified here may represent a smaller and more manageable target for the ongoing efforts to develop effective ETEC vaccines.Enterotoxigenic Escherichia coli (ETEC) infections are an important cause of childhood diarrhea and diarrheal deaths among young children in developing countries (59) and of diarrhea among travelers to these countries (6, 54). Human ETEC strains are E. coli that produce one or more of three plasmid-encoded protein enterotoxins called human heat-stable toxin (STh or STaII), porcine heat-stable toxin (STp or STaI), and heat-labile toxin (LT or LT-I). The enterotoxins induce secretion of salts and water into the intestinal lumen (29). Many ETEC strains also produce surface appendages, called colonization factors (CFs), which help anchor the bacteria to the small intestinal wall (20). The toxins and all but one known CF are plasmid encoded (20, 34).Human ETEC strains are phenotypically and epidemiologically diverse: more than 20 different CFs have thus far been described (20), the characterization of ETEC strains collected from different parts of the world has yielded 117 different serotypes (57), and some ETEC strains appear to be more pathogenic than others (9, 36, 46). This diversity poses a challenge for the ongoing efforts to develop effective ETEC vaccines (7). Many studies have shown that ETEC have emerged from E. coli on several occasions, probably through horizontal transfer of the enterotoxin-encoding virulence plasmids, and that some of these ETEC lineages appear to be widespread (4, 11, 31-33, 37, 38, 43, 47, 51). Because strains that originate from the same ETEC lineage may also have inherited many of the same epidemiological and phenotypic traits, identifying and defining these lineages may improve our understanding of the ETEC diversity and may lead to the identification of lineage-specific protective antigens that can be used in vaccines. To identify these lineages, we performed multilocus sequence typing (MLST) and phylogenetic analyses on a collection of ETEC strains that had been isolated from humans in different countries. We also estimated each lineage''s age as a measure of how stable and well established these lineages are in the E. coli population. If the ancestral origin of the human ETEC population changes frequently, it would complicate efforts to identify new, chromosomally encoded antigens capable of inducing protective immune responses against ETEC. In that case, today''s main vaccine development strategy of targeting plasmid-encoded virulence factors, such as the toxins and CFs, would probably continue to be the best approach for developing effective ETEC vaccines.     

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 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.)     

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.     

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.     

Characterization and Studies of the Cellular Interaction of Native Colonization Factor CS6 Purified from a Clinical Isolate of Enterotoxigenic Escherichia coli

CS6 is a widely expressed colonization factor of enterotoxigenic Escherichia coli (ETEC). To date, CS6 has not been well characterized in its native state. Here, we purified CS6 for the first time from an ETEC clinical isolate. Purified CS6 was composed of two structural subunits, CssA and CssB, which were present in equal amounts and tightly linked through noncovalent, detergent-stable association. The CssA subunit was poorly immunogenic, whereas CssB was highly immunogenic. Although the predicted molecular mass of CssA is 15 kDa, the purified CssA has an effective molecular mass of 18.5 kDa due to fatty acid modification. When purified CS6 was screened for its ability to bind with different extracellular matrix proteins, fibronectin (Fn) was found to interact with CS6 as well as CssA in a dose-dependent and saturable manner. This interaction was inhibited both by a synthetic peptide corresponding to the C-terminal hydrophilic, surface-exposed region of CssA (positions 112 to 126) and by the antibody derived against this region. Enzyme-linked immunosorbent assay results showed that CssA interacted with the 70-kDa N-terminal domain of Fn. The modifications on CssA probably do not play a role in Fn binding. Preincubation of INT 407 cells with CssA, but not CssB, inhibited ETEC binding to these cells. The results suggested that CS6-expressing ETEC binds to Fn of INT 407 cells through the C-terminal region of CssA. Purified CS6 was found to colocalize with Fn along the junctions of INT 407 cells. Based on the results obtained, we propose that CS6-expressing ETEC binds to the intestinal cells through Fn for colonization.Enterotoxigenic Escherichia coli (ETEC) infection is the leading cause of infantile diarrhea in developing countries and an important etiologic agent for traveler''s diarrhea. ETEC accounts for approximately 210 million diarrhea episodes and 380,000 deaths annually (35). Community-based studies conducted in developing countries with children younger than 5 years have shown that ETEC was the most frequently isolated enteropathogen (34, 35). As a cause of traveler''s diarrhea, ETEC was found to be associated with 40 to 70% of the cases, with drastic outcome in terms of morbidity and economic consequences (34).In order to initiate pathogenesis, ETEC strains must adhere to the small intestine (14). This event is mediated by several proteinaceous surface antigens, collectively known as colonization factor antigens (CFAs) (6). To date, more than 25 distinct colonization factors have been identified, of which CS6 is the most prevalent in many countries (7, 20, 22). Many of the colonization factors have morphology of fimbriae or pili (14). However, the morphology of CS6 has not so far been defined. CS6 was assumed to be either a nonfimbrial or a short oligomeric assembly that does not protrude enough to be visualized under an electron microscope (17). Functional CS6 is expressed and transported to the bacterial surface in a chaperone-usher pathway. CssC and CssD are the chaperone and usher proteins, respectively, that help surface expression of the CS6 structural subunits, CssA and CssB (33).The role of CS6 in intestinal adherence has been demonstrated using CS6-expressing whole bacteria, but the receptor specificity is still unknown (11). A recent report has shown that when CssB is mutated, binding of bacteria to a colonic cell line (CaCo-2) is reduced slightly compared to that of the bacteria expressing whole CS6 (30).Here, we have purified CS6 to homogeneity from a clinical isolate of ETEC and separated its subunits (CssA and CssB) for the first time. We have characterized CS6 in its native form and demonstrated that fibronectin (Fn) is the interacting matrix for adherence. The carboxy-terminal (C-terminal) region of CssA plays a key role in this interaction with the amino-terminal (N-terminal) region of Fn.     

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.)     

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.     

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.     

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).     

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.     

Development of a DNA Microarray for Detection and Serotyping of Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a common pathogen worldwide causing infectious diarrhea, especially traveler''s diarrhea. Traditional physiological assays, immunoassays, and PCR-based methods for the detection of ETEC target the heat-labile enterotoxin and/or the heat-stable enterotoxin. Separate serotyping methods using antisera are required to determine the ETEC serogroup. In this study, we developed a DNA microarray that can simultaneously detect enterotoxin genes and the 19 most common O serogroup genes in ETEC strains. The specificity and reproducibility of this approach were verified by hybridization to 223 strains: 50 target reference or clinical strains and 173 other strains, including those belonging to other E. coli O serogroups and closely related species. The sensitivity of detection was determined to be 50 ng of genomic DNA or 10⁸ CFU per ml of organisms in pure culture. The random PCR strategy used in this study with minimal bias provides an effective alternative to multiplex PCR for the detection of pathogens using DNA microarrays. The assay holds promise for applications in the clinical diagnosis and epidemiological surveillance of pathogenic microorganisms.Enterotoxigenic Escherichia coli (ETEC) is the leading bacterial cause of infectious diarrhea in the developing world, causing infantile or cholera-like disease in all age groups (2). It is among the major etiologic agents, leading to an estimated 1.5 million deaths per year worldwide (13, 14). ETEC is also a major cause of traveler''s diarrhea (3, 8, 11) and the most common pathogen among the six recognized diarrheagenic categories of E. coli, especially in the developing world (18). ETEC strains produce one or both of the following two enterotoxins: heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). Two classes of STs—STa and STb—and two variants of STa—STp (initially discovered in isolates from pigs) and STh (initially discovered in isolates from humans)—have been described. The elt, estA, and estB genes encode the enterotoxins LT, STa, and STb, respectively (6, 23, 26).The O antigen comprises the outermost domain of the lipopolysaccharide molecule and is attached to the core oligosaccharide on the surfaces of Gram-negative bacteria (20). O antigens are among the most variable cellular constituents, imparting antigenic specificity. The composition of the O chain differs from strain to strain; more than 180 O-antigen structures are produced by different E. coli strains (25). The most common O serogroups reported in ETEC are O6, O8, O11, O15, O25, O27, O78, O85, O114, O115, O126, O128, O139, O148, O149, O159, O166, O167, and O173 (5, 18, 19, 31).Detection of ETEC has long relied on detection of the enterotoxins LT and/or ST by physiological assays and immunoassays, and serotyping has depended on assays using O-serogroup-specific antisera. These traditional approaches are slow and labor-intensive, and assays using antisera can be impeded by cross-reactivity. PCR assays, which are more rapid, sensitive, and specific, have also been widely used for ETEC diagnosis (15, 24). However, molecular methods for the serotyping of ETEC have not been developed.Molecular detection and typing by PCR and microarray techniques have many advantages over traditional methods. DNA microarrays provide an efficient approach for the parallel detection and analysis of a large number of pathogenic microorganisms. This technique has been applied to the detection of pathogens from all kinds of biological samples, including water, food, and soil (4, 7, 12, 17, 21).In this study, we developed a DNA microarray for the detection and typing of ETEC. The genes encoding the enterotoxins LT and ST were used for the detection of ETEC, and the serogroup-specific genes wzx and/or wzy were used for the typing of the 19 most common ETEC O serogroups. The microarray was examined for its specificity and sensitivity, and the findings of this study indicate that it is highly sensitive and reproducible.     

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.     

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.     

Safety and Immunogenicity of the Merck Adenovirus Serotype 5 (MRKAd5) and MRKAd6 Human Immunodeficiency Virus Type 1 Trigene Vaccines Alone and in Combination in Healthy Adults

Preexisting immunity to adenovirus serotype 5 (Ad5) diminishes immune responses to vaccines using Ad5 as a vector. Alternate Ad serotypes as vaccine vectors might overcome Ad5-specific neutralizing antibodies and enhance immune responses in populations with a high prevalence of Ad5 immunity. To test this hypothesis, healthy human immunodeficiency virus (HIV)-seronegative adults were enrolled in a blinded, randomized, dose-escalating, placebo-controlled study. In part A, subjects with baseline Ad6 titers of ≤18 received the Merck Ad6 (MRKAd6) HIV type 1 (HIV-1) trigene vaccine at weeks 0, 4, and 26. In part B, subjects stratified by Ad5 titers (≤200 or >200) and Ad6 titers (≤18 or >18) received the MRKAd5-plus-MRKAd6 (MRKAd5+6) HIV-1 trigene vaccine at weeks 0, 4, and 26. Immunogenicity was assessed by an enzyme-linked immunospot (ELISPOT) assay at week 30. No serious adverse events occurred. MRKAd6 trigene vaccine recipients responded more often to Nef than to Gag or Pol. In part A, ELISPOT response rates to ≥2 vaccine antigens were 14%, 63%, and 71% at 10⁹, 10¹⁰, and 10¹¹ viral genomes (vg)/dose, respectively. All responders had positive Nef-specific ELISPOT results. In part B, Nef-ELISPOT response rates at 10¹⁰ vg/dose of the MRKAd5+6 trigene vaccine were 50% in the low-Ad5/low-Ad6 stratum (n = 8), 78% in the low-Ad5/high-Ad6 stratum (n = 9), 75% in the high-Ad5/low-Ad6 stratum (n = 8), and 44% in the high-Ad5/high-Ad6 stratum (n = 9). The MRKAd6 and MRKAd5+6 trigene vaccines elicited dose-dependent responses predominantly to Nef and were generally well tolerated, indicating that Ad6 should be considered a candidate vector for future vaccines. Although small sample sizes limit the conclusions that can be drawn from this exploratory study, combining two Ad vectors may be a useful vaccine strategy for circumventing isolated immunity to a single Ad serotype.Adenovirus (Ad) vectors have been investigated as a vaccination strategy for inducing cell-mediated immunity (CMI) to several viral and bacterial pathogens (11, 13, 22, 24, 26). In preclinical and phase I studies, vaccination with attenuated Ad serotype 5 (Ad5) vectors expressing human immunodeficiency virus type 1 (HIV-1) gag elicited strong CMI responses in both macaques and humans (4, 5, 14, 20, 23). Although a similar Ad5-vectored trivalent HIV-1 vaccine did not prevent or modulate infection in the proof-of-concept STEP trial (2), adenoviruses remain attractive candidates as vectors for inducing CMI against a variety of common infections.Diminished immune responses to transgenes carried by Ad5 vectors as a result of preexisting Ad5-specific immunity have been a concern from the advent of Ad5-based vaccine trials in humans (2, 5, 13, 16, 18, 25). High preexisting titers of neutralizing antibodies against Ad5 substantially diminished CMI responses to HIV-1 vaccines using Ad5 vectors (2, 5, 16, 18). Most North American adults have demonstrable neutralizing antibody against Ad5, and nearly one-third have relatively high titers (21, 25, 26). The frequency and magnitude of Ad5 titers are even higher in other parts of the world (8, 21). Neutralizing antibody against Ad6 is present less frequently and in lower titers (8, 21). Relatively few individuals would be expected to have high titers of antibodies against both Ad5 and Ad6.Strategies for overcoming preexisting Ad5 immunity include increasing the dose of Ad5-based vaccines, employing heterologous prime-boost regimens, or using different vectors, such as alternative adenovirus serotypes (3, 15, 26). The current trial was designed to explore the use of Ad6 with or without Ad5 as a vaccine vector for delivering HIV-1 gag, nef, and pol transgenes.(These data have been presented in part at the AIDS Vaccine 2007 Conference, Seattle, WA, August 2007 [12a, 12b].)     

Association of Single Nucleotide Polymorphisms in Cytotoxic T-Lymphocyte Antigen 4 and Susceptibility to Autoimmune Type 1 Diabetes in Tunisians

In addition to HLA and insulin genes, the costimulatory molecule CTLA-4 gene is a confirmed type 1 diabetes (T1D) susceptibility gene. Previous studies investigated the association of CTLA-4 genetic variants with the risk of T1D, but with inconclusive findings. Here, we tested the contributions of common CTLA-4 gene variants to T1D susceptibility in Tunisian patients and control subjects. The study subjects comprised 228 T1D patients (47.8% females) and 193 unrelated healthy controls (45.6% females). Genotyping for CTLA-4 CT60A/G (rs3087243), +49A/G (rs231775), and −318C/T (rs5742909) was performed by PCR-restriction fragment length polymorphism (RFLP) analysis. The minor-allele frequencies (MAF) for the three CTLA-4 variants were significantly higher in T1D patients, and significantly higher frequencies of homozygous +49G/G and homozygous CT60G/G genotypes were seen in patients, which was confirmed by univariate regression analysis (taking the homozygous wild type as a reference). Of the eight possible three-locus CTLA-4 haplotypes (+49A/G, −318C/T, and CT60A/G) identified, multivariate regression analysis confirmed the positive association of ACG (odds ratio [OR], 1.93; 95% confidence interval [CI], 1.26 to 2.94), GCG (OR, 2.40; 95% CI, 1.11 to 5.21), and GTA (OR, 4.67; 95% CI, 1.52 to 14.39) haplotypes with T1D, after confounding variables were adjusted for. Our results indicate that CTLA-4 gene variants are associated with increased T1D susceptibility in Tunisian patients, further supporting a central role for altered T-cell costimulation in T1D pathogenesis.Type 1 (insulin-dependent) diabetes (T1D) is the most prevalent form of diabetes in children and young adults and results from autoimmune CD4⁺ and CD8⁺ T-cell-directed destruction of insulin-producing pancreatic β islet cells in genetically susceptible individuals (3, 12), leading to irreversible hyperglycemia and related complications (13). There is a strong genetic component to T1D pathogenesis, evidenced by its clustering in families and by the contributions of a number of susceptibility gene variants to its pathogenesis (10, 12, 29). They include the human leukocyte antigen (HLA) locus, in particular the class II region (DR and DQ), which accounts for 40 to 50% of T1D familial clustering (1, 12, 18), and non-HLA susceptibility loci, several of which were mapped by genome-scanning (11, 29) and/or candidate gene (7, 18, 31) approaches. They include insulin promoter gene variants, which reportedly may modulate immunological tolerance by controlling the expansion of the autoreactive cell pool (26), and the T-cell costimulator cytotoxic T-lymphocyte antigen 4 (CTLA-4) transmembrane glycoprotein, which plays a key role in the fine tuning of T-cell immunity (9, 32, 33).CTLA-4 is a 40-kDa transmembrane glycoprotein expressed on resting and activated T cells and nonlymphoid cells (33), and along with the related CD28 costimulatory molecule, it regulates T-cell activation (and is itself primarily mediated by engagement of the T-cell receptor [TCR]) but does recognize major histocompatibility complex (MHC)-bound antigenic peptides (9, 33). CTLA-4 negatively regulates T-cell activation and effector function, in part by inhibiting Th1 (interleukin 2 [IL-2] and gamma interferon [IFN-γ]) cytokine production and IL-2 receptor α-chain (p55; Tac) expression by engaging antigen-presenting cell (APC)-bound B7.1 (CD80) and B7.2 (CD86) ligands (9, 33). Functionally, CTLA-4 attenuates T-cell signaling by interference with intracellular signal transduction events, including TCR signaling, and reduced CTLA-4 expression and/or activity results in uncontrolled T-cell-associated autoimmunity and lymphoproliferative disease (9, 21). In this regard, it was shown that CTLA-4 polymorphisms significantly influence the risk of autoimmune diseases, including Graves'' disease, systemic lupus erythematosus, autoimmune hypothyroidism, celiac disease, and type 1 diabetes (15, 21, 32).First observed in Italian subjects (25), and confirmed subsequently by case control and family studies, CTLA-4 polymorphic variants were linked with T1D pathogenesis (14, 20, 31, 32). While this association was detected in different ethnic groups (14, 23, 30), it appears more likely to be Caucasian selective (10, 29, 33) and absent from non-Caucasians (5, 6, 8, 19, 22). A recent report from the Type I Diabetes Genetics Consortium bearing on 2,300 affected sib pair families demonstrated that among the 24 single nucleotide polymorphisms (SNPs) genotyped in the CTLA-4 region, only the +49A/G and CT60 SNPs were replicated in the nine combined collections (27). In the present study, we investigated the association of three common CTLA-4 SNPs (−318C/T; +49A/G, and CT60A/G) and the corresponding haplotypes with T1D in Tunisian Arab patients.     

Evaluation of the C.Diff Quik Chek Complete Assay,a New Glutamate Dehydrogenase and A/B Toxin Combination Lateral Flow Assay for Use in Rapid,Simple Diagnosis of Clostridium difficile Disease

The diagnosis of Clostridium difficile infection continues to be a challenge for many clinical microbiology laboratories. A new lateral flow assay, the C.Diff Quik Chek Complete assay, which tests for the presence of both glutamate dehydrogenase (GDH) and C. difficile toxins A and B, was evaluated for its ability to diagnose C. difficile disease. The results of this assay were compared to those of both PCR and toxigenic culture. The results showed that this assay allows 88% of specimens to be accurately screened as either positive (both tests positive) or negative (both tests negative) for the presence of toxigenic C. difficile in less than 30 min and with minimal hands-on time. Use of a random-access PCR for the analysis of specimens with discrepant results (one test positive and the other negative) allows the easy, rapid, and highly sensitive (100%; 95% confidence interval [CI], 89.6 to 100%) and specific (99.6%; 95% CI, 97.3 to 99.9%) diagnosis of C. difficile disease. The use of this algorithm would save institutional costs, curtail unnecessary isolation days, reduce the nosocomial transmission of disease, and increase the quality of care for patients.The laboratory diagnosis of Clostridium difficile disease has evolved significantly over the last several years, and many tests that may be used to assist with the detection of C. difficile infection are now available. These assays include enzyme immunoassays (EIAs), lateral flow tests, PCR assays, tissue culture cytotoxicity neutralization tests, and toxigenic culture. Many recent papers have reported on the use of different algorithms that use the tests mentioned above to allow the better diagnosis of C. difficile disease (4, 5, 8, 18, 19, 20, 23, 26). Many of these approaches incorporate cytotoxicity neutralization (CTN) assays or anaerobic agar culture with identification of the organism, followed by toxin testing. However, many laboratories do not have the technical expertise, facilities, or training to perform CTN assays (which are labor-intensive and somewhat subjective), and an anaerobic agar culture with toxin detection may take several days; both of these methods delay the reporting of results (14, 17, 19, 20, 21, 29). The use of PCR for the diagnosis of this disease has been shown to be very specific and sensitive but often does not allow for random-access (i.e., real-time) results and can be quite costly to perform as a stand-alone testing method (2, 6, 10, 12, 15, 17, 27, 28). There is a real need for a rapid and simple testing strategy for the accurate diagnosis of C. difficile disease. With this in mind, we examined a simple, rapid two-step algorithm for the laboratory diagnosis of Clostridium difficile disease utilizing the new C.Diff Quik Chek Complete assay, a lateral flow kit that uses a combination of glutamate dehydrogenase (GDH) antigen detection plus toxin A and B detection for initial screening and a random-access, highly sensitive Xpert C. difficile PCR assay which detects the toxin B gene for samples with discrepant results.     

Performance of Purified Antigens for Serodiagnosis of Pulmonary Tuberculosis: a Meta-Analysis

Serological antibody detection tests for tuberculosis may offer the potential to improve diagnosis. Recent meta-analyses have shown that commercially available tests have variable accuracies and a limited clinical role. We reviewed the immunodiagnostic potential of antigens evaluated in research laboratories (in-house) for the serodiagnosis of pulmonary tuberculosis and conducted a meta-analysis to evaluate the performance of comparable antigens. Selection criteria included the participation of at least 25 pulmonary tuberculosis patients and the use of purified antigens. Studies evaluating 38 kDa, MPT51, malate synthase, culture filtrate protein 10, TbF6, antigen 85B, α-crystallin, 2,3-diacyltrehalose, 2,3,6-triacyltrehalose, 2,3,6,6′-tetraacyltrehalose 2′-sulfate, cord factor, and TbF6 plus DPEP (multiple antigen) were included in the meta-analysis. The results demonstrated that (i) in sputum smear-positive patients, sensitivities significantly ≥50% were provided for recombinant malate synthase (73%; 95% confidence interval [CI], 58 to 85) and TbF6 plus DPEP (75%; 95% CI, 50 to 91); (ii) protein antigens achieved high specificities; (iii) among the lipid antigens, cord factor had the best overall performance (sensitivity, 69% [95% CI, 28 to 94]; specificity, 91% [95% CI, 78 to 97]); (iv) compared with the sensitivities achieved with single antigens (median sensitivity, 53%; range, 2% to 100%), multiple antigens yielded higher sensitivities (median sensitivity, 76%; range, 16% to 96%); (v) in human immunodeficiency virus (HIV)-infected patients who are sputum smear positive, antibodies to several single and multiple antigens were detected; and (vi) data on seroreactivity to antigens in sputum smear-negative or pediatric patients were insufficient. Potential candidate antigens for an antibody detection test for pulmonary tuberculosis in HIV-infected and -uninfected patients have been identified, although no antigen achieves sufficient sensitivity to replace sputum smear microscopy. Combinations of select antigens provide higher sensitivities than single antigens. The use of a case-control design with healthy controls for the majority of studies was a limitation of the review. Efforts are needed to improve the methodological quality of tuberculosis diagnostic studies.The failure to diagnose tuberculosis (TB) accurately and rapidly is a key challenge in curbing the epidemic (45, 88, 116). Sputum microscopy, currently the sole diagnostic test in most areas where TB is endemic, has several limitations; in particular, the sensitivity compared with that of culture is variable (80, 97, 104, 116), multiple patient visits are required (56, 93, 114), considerable technical training is necessary, and the procedure is labor-intensive (45, 65). Antibody detection tests (serological tests) are used for the diagnosis of many infectious diseases and could potentially improve the means of diagnosis of TB. These tests measure the presence of specific antibodies (most often immunoglobulin G [IgG]) directed against immunodominant antigens of the pathogen in question. Compared with microscopy, antibody detection methods may enable the rapid diagnosis of TB, as these tests have the advantages of speed (results can be available within hours), technological simplicity, and minimal training requirements. In addition, these tests can be adapted to point-of-care formats that can be implemented at lower levels of health services in low- and middle-income countries (21, 22, 57, 65).Efforts to develop antibody detection tests for the diagnosis of TB have been under way for decades, and the performance of these tests has been well described (13, 17, 22, 32, 40, 47, 48, 52, 60, 64, 100, 107). Several systematic reviews of these tests have been published (discussed below) (28, 94, 95).First-generation antibody detection tests were based on crude mixtures of constituents and products of Mycobacterium tuberculosis, for example, culture filtrate proteins and purified protein derivative, the preparation used in the tuberculin skin test. Several of these early tests had low specificities, as the tests contained antigens shared among different bacterial species (1, 22, 48, 57). During the past two decades, an increased understanding of humoral immune responses to M. tuberculosis and the new tools of genomics and proteomics have led to the discovery of new antigens reported to provide improved sensitivities and specificities for the diagnosis of TB compared with those achieved with the antigens in the first-generation tests (48).We reviewed the immunodiagnostic potential of different antigens evaluated in research laboratories (in-house) for the serodiagnosis of pulmonary TB and carried out a meta-analysis to evaluate the performance of various antigens singly and in combination. Previous meta-analyses have shown that commercially available serological tests for both pulmonary TB (94) and extrapulmonary TB (95) have variable accuracies and, consequently, a limited clinical role. Another systematic review (searches through 2003) limited studies to the cohort or case series type of design and included only nine studies relating to in-house anti-TB antibody serological tests (28). A recently published expert review (1) did not include a meta-analysis. We are unaware of other systematic reviews on this topic.The current review addresses the following questions. (i) What is the performance of different antigens in the serodiagnosis of pulmonary TB in sputum smear-positive and smear-negative patients? (ii) What is the performance of these antigens in the serodiagnosis of pulmonary TB in patients with human immunodeficiency virus (HIV) infection?