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.     

Expression of Homing Receptors on IgA1 and IgA2 Plasmablasts in Blood Reflects Differential Distribution of IgA1 and IgA2 in Various Body Fluids

Although secretory IgA is the most abundantly produced Ig isotype, the mechanisms underlying the differential distribution of IgA subclasses in various body fluids remain unclear. To explore these mechanisms, we examined the distribution of IgA subclasses, the influence of the nature and sites of encounters with antigens, and the correlation between IgA subclass distribution and homing potentials of circulating IgA plasmablasts. IgA1 predominated in serum, tears, nasal wash fluid, and saliva; the levels of IgA1 and IgA2 were comparable in vaginal wash fluid; and IgA2 predominated in intestinal lavage fluids. Seventy-one percent of circulating IgA plasmablasts secreted IgA1. The intestinal homing receptor (HR), α4β7, was expressed more frequently on IgA2 than on IgA1 plasmablasts, with no differences in the expression of other HRs. IgA subclass distribution among circulating antigen-specific antibody-secreting cells (ASC) was dependent on the nature of the antigen: following vaccination with Salmonella enterica serovar Typhi, unconjugated pneumococcal polysaccharide, or Haemophilus influenzae polysaccharide-diphtheria toxoid conjugate, the proportions of specific IgA1 ASC were 74%, 47%, 56%, and 80%, respectively. HR expression depended on the route of administration: expression of HRs was different after oral than after parenteral vaccination, while no difference was seen between HR expression of antigen-specific IgA1 and IgA2 ASC induced via the same route. The key factors determining IgA subclass distribution in a given secretion are the nature of the antigens encountered at a particular site and the site-specific homing instructions given to lymphocytes at that site. These two factors are reflected as differences in the homing profiles of the total populations of circulating IgA1 and IgA2 plasmablasts.Because humoral immunity is most frequently evaluated by titers of serum antibodies, which are dominated by IgG, the role of IgA as the major Ig isotype produced in humans has not been adequately appreciated: the daily production of IgA (66 mg/kg of body weight/day) exceeds that of all other immunoglobulin classes combined (10, 14, 40). Even if IgA levels in serum are lower than those of IgG, due to a shorter half-life in the circulation, IgA is the predominant immunoglobulin in most external secretions (3, 14, 40). IgA, as the mucosal Ig isotype, plays a dominant role as the first immunological defense barrier in the body, since the mucosal sites act as a portal of entry to the majority of human pathogens (3, 40, 53).In humans, IgA comprises two subclasses, IgA1 and IgA2, which are unequally distributed in the body fluids (4, 13, 34, 36, 41). In serum, IgA1 is dominant, while in secretions, there is a significant contribution of IgA2. In external secretions, both IgA1 and IgA2 are present as secretory IgA (S-IgA), a polymeric form which is more resistant to proteolytic enzymes than any of the other isotypes (40). S-IgA is known to provide protection of mucosal membranes in several complementary ways: it can effectively neutralize viruses (46, 47, 49) or toxins (47) and displays antibacterial activity (47, 51). To evade the protective effect of S-IgA at the mucosal sites, some pathogenic bacteria (e.g., Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Neisseria gonorrhoeae) produce proteases which cleave the IgA1 molecule (31), leaving the IgA2 intact. The unequal distribution of IgA subclasses in different body fluids (5, 13, 28) and among IgA-secreting cells in the circulation (25) and mucosal and systemic lymphoid tissues (11, 30) has been reported in several independent studies. The differential distribution of the two IgA subclasses in secretions has been shown to be accompanied by a similar distribution of IgA1- and IgA2-producing cells at those sites (11, 30). IgA1 cells predominate in the bone marrow, and they are also present in higher proportions in upper parts of the orogastric and the respiratory tracts, while IgA2 cells predominate in the lower part of the gastrointestinal tract (11, 30). The basis for this differential distribution of IgA subclasses in various mucosal sites is not understood.More than two-thirds of all Ig-producing cells in the body reside in the various mucosal and exocrine sites (40), particularly along the gastrointestinal tract, and most of them produce IgA specific for antigens encountered at mucosal surfaces. The major source of the precursors of the intestinal IgA plasma cells is the naïve B cells in the organized lymphoepithelial tissues, such as the Peyer''s patches (12). B cells in the germinal centers of Peyer''s patches switch from IgM⁺ to IgA⁺ under the influence of resident cells and various cytokines (8, 14, 38). Subsequently, the cells migrate to the draining mesenteric lymph node, where they continue to divide and differentiate into plasmablasts (12, 15, 16). Finally, they exit the lymph nodes and migrate via thoracic duct lymph into the blood, which carries them to the target tissues, such as the lamina propria of the gut, to secrete IgA. The homing of these activated lymphocytes from the circulation into mucosal tissues involves multiple steps (33, 43, 48) in which the two central events are (i) the interactions between the chemokine receptors (CCR) on the lymphocyte surface and the chemokines produced in the tissue and (ii) the recognition by the lymphocyte surface homing receptors (HR) of their tissue-specific ligands, called addressins, on the surface of the endothelial cells in the mucosa. The key homing receptor-addressin interaction in lymphocyte homing to the intestinal tissue is believed to be the binding of the gut HR, α4β7, on the lymphocyte surface to its ligand, MAdCAM-1, on the high endothelial venules of the intestinal lamina propria (2). A fraction of IgA⁺ plasmablasts in the circulation expresses α4β7 (21, 27), consistent with their ability to home to intestinal lamina propria. Some IgA plasmablasts express other HR (20-22, 26), such as the peripheral lymph node HR, l-selectin (6, 32), and the skin HR, cutaneous lymphocyte-associated antigen (CLA) (44), consistent with the ability of a portion of IgA plasmablasts to home to extraintestinal lymphoid tissues at systemic and mucosal sites (48). The HR guiding the cells to the different extraintestinal mucosal sites have not been identified, but it has been suggested that there would be several different HR and CCR contributing to the selective homing to extraintestinal mucosal sites (4, 54). In humans, it is possible to determine the expression of HR on circulating plasmablasts and interpret these data as homing potentials or homing profiles of these cells (22, 26, 27, 45). Furthermore, it might be assumed that the differential distribution of IgA1 and IgA2 plasma cells in the various sites in the body would depend on a differential homing potential of circulating IgA1 and IgA2 plasmablasts and, thus, could be predicted by the differential expression of HR.Although there are data concerning the differential distribution of IgA subclasses in various body fluids (5, 9, 13, 34, 36, 41, 42), studies involving parallel distribution of IgA subclasses in various body fluids and circulating plasmablasts expressing mucosal and/or systemic HR are not available. In the present study, we (i) determined the differential distribution of IgA subclasses in various body fluids using identical assays and reagents, (ii) evaluated the factors influencing IgA subclass distribution of a specific immune response, and (iii) explored whether the differential distribution of IgA subclasses in the various body fluids is associated with differential homing profiles of IgA1 and IgA2 plasmablasts in the circulation.     

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.     

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.     

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.     

Comparative Evaluation of a Commercially Available Automated System for Extraction of Viral DNA from Whole Blood: Application to Monitoring of Epstein-Barr Virus and Cytomegalovirus Load

The NucliSENS easyMAG automated system was compared to the column-based Qiagen method for Epstein-Barr virus (EBV) or cytomegalovirus (CMV) DNA extraction from whole blood before viral load determination using the corresponding R-gene amplification kits. Both extraction techniques exhibited a total agreement of 81.3% for EBV and 87.2% for CMV.Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections represent a significant clinical threat for immunocompromised patients. The frequent determination of EBV and CMV viral load permits the early diagnosis of infection, start of preemptive or curative therapy, and monitoring of treatment efficiency (5, 17, 20). By comparison to serum, plasma, or white-blood-cell fractions, whole-blood samples are now recognized as the most suitable sample for the determination of viral loads for EBV and CMV (3, 4, 7, 9, 10, 12, 13, 18, 19).Although the methods relying on silica columns are time-consuming and need trained experimenters, these methods are considered the gold standard for the extraction of nucleic acids from whole-blood samples. Due to the large amount of genetic material in such samples, new extraction methods must be carefully evaluated, including those relying on automated devices (1, 7, 10, 14, 15). The fully automated NucliSENS easyMAG instrument (bioMérieux) using magnetic silica particles (2) allows the simultaneous process of up to 24 extractions. The use of magnetic particles eliminates the several centrifugation steps that could be a source of cross-contamination, and manual steps are limited to the loading of samples, reagents, and disposables. The performance of this method in the extraction of DNA from whole-blood samples prior to viral quantification has not been yet evaluated. The present study was undertaken to answer this question in the clinical context of EBV or CMV infection.The whole-blood specimens selected for this study included 80 samples for EBV analysis and 94 samples for CMV analysis, taken from patients hospitalized at the University Hospital of Saint-Etienne, Saint-Etienne, France, from December 2007 to September 2008. The samples were kept frozen at −20°C. After the samples were thawed, whole-blood aliquots were tested and kept at 4°C for up to 24 h for potential retest. Two hundred microliters of each selected sample was extracted by two different technicians either by the reference manual method, i.e., QIAamp column DNA blood extraction kit according to the manufacturer''s recommendations (Qiagen), or by the new specific B protocol on the NucliSENS easyMAG instrument. The latter protocol consists of the treatment of 200 μl of whole blood in 2 ml of lysis buffer and the capture of nucleic acids by 140 μl of magnetic silica. After incubation and washing procedures, nucleic acids were recovered in 50 μl of elution buffer. EBV and CMV loads were quantified by using the respective R-gene amplification kit (Argene Biosoft) according to the manufacturer''s recommendations. Both amplification kits have been previously validated for quantification of EBV and CMV load in whole blood (8, 11). DNA extracts from both methods were amplified in the same run using an ABI 7500 instrument (Applied Biosystems).     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Diversity of Group B Streptococcus Serotypes Causing Urinary Tract Infection in Adults

Serotypes of group B streptococcus (GBS) that cause urinary tract infection (UTI) are poorly characterized. We conducted a prospective study of GBS UTI in adults to define the clinical and microbiological characteristics of these infections, including which serotypes cause disease. Patients who had GBS cultured from urine over a 1-year period were grouped according to symptoms, bacteriuria, and urinalysis. Demographic data were obtained by reviewing medical records. Isolates were serotyped by latex agglutination and multiplex PCR-reverse line blotting (mPCR/RLB). Antibiotic susceptibilities were determined by disc diffusion. GBS was cultured from 387/34,367 consecutive urine samples (1.1%): 62 patients had bacteriuria of >10⁷ CFU/liter and at least one UTI symptom; of these patients, 31 had urinary leukocyte esterase and pyuria (others not tested), 50 (81%) had symptoms consistent with cystitis, and 12 (19%) had symptoms of pyelonephritis. Compared with controls (who had GBS isolated without symptoms), a prior history of UTI was an independent risk factor for disease. Increased age was also significantly associated with acute infection. Serotyping results were consistent between latex agglutination and mPCR/RLB for 331/387 (85.5%) isolates; 22 (5.7%) and 7 (1.8%) isolates were nontypeable with antisera and by mPCR/RLB, respectively; and 45/56 (80.4%) isolates with discrepant results were typed by mPCR/RLB as belonging to serotype V. Serotypes V, Ia, and III caused the most UTIs; serotypes II, Ib, and IV were less common. Nontypeable GBS was not associated with UTI. Erythromycin (39.5%) and clindamycin (26.4%) resistance was common. We conclude that a more diverse spectrum of GBS serotypes causes UTI than previously recognized, with the exception of nontypeable GBS.Group B streptococcus (GBS) is a leading cause of infection in newborns, pregnant women, and older persons with chronic medical illness (3, 8). In addition to maternal cervicovaginal colonization and neonatal infection that results from the vertical transmission of bacteria from mothers to their infants, GBS can also cause urinary tract infection (UTI). The spectrum of GBS UTI includes asymptomatic bacteriuria (ABU), cystitis, pyelonephritis, urethritis, and urosepsis (6, 8, 10, 20, 23, 26). GBS ABU is particularly common among pregnant women, although those most at risk for cystitis due to GBS appear to be the elderly and immunocompromised individuals (8, 9, 25). Despite the uropathogenic nature of GBS, the clinical and microbiological features of GBS UTI, including risk factors for disease and whether there is a tendency for particular GBS serotypes to cause UTI, are poorly understood.Clinically, UTI due to GBS may be indistinguishable from UTI caused by other uropathogens (25). However, a recent study of multiple uropathogens and host characteristics highlighted unique frequencies of host characteristics in UTI groups defined by the causal organism (37). This suggests that the clinical and microbiological features of UTI may differ depending on the infecting uropathogen. GBS colonization of the urinary tract in women most likely occurs by an ascending route from the vagina, where GBS can persist asymptomatically. While the overall prevalence of GBS UTI in the adult population remains unclear, GBS bacteriuria during pregnancy occurs at rates of between 1 and 3.5% (4, 23, 41). Many of these episodes represent ABU (2, 18); however, GBS ABU is considered to be a surrogate for heavy maternal colonization (29, 42) and is currently recommended for intrapartum antibiotic chemoprophylaxis (23, 34). In addition, up to 7% of pregnancies may be complicated by GBS UTI, and GBS reportedly accounts for approximately 10% of all cases of pyelonephritis during pregnancy (25, 28). GBS UTI may also contribute to chorioamnionitis (1), premature onset of labor (24), and an increased risk of vertical transmission of GBS (29, 42).Several studies have also reported high rates of GBS UTI in nonpregnant adults (8, 9, 25, 39). In one study, GBS was cultured from 39% of all cases of symptomatic UTI among nursing home residents >70 years of age (40). Other studies reported that GBS UTI may account for up to one-third of all invasive infections due to GBS in adults (9, 12, 19, 26). Several independent surveys have reported the recovery of GBS from between 1 and 2% of all UTI cases (7, 26, 30). GBS UTI may also account for up to 7% of late-onset disease in neonates (43). Thus, while there is an increasing amount of data regarding the prevalence of GBS UTI in adults, little is known regarding the clinical and microbiological features associated with these infections or the GBS serotypes that cause UTI. In this study, we carried out a single-center analysis of adult patients at the University of Alabama at Birmingham Hospital between August 2007 and August 2008 who had GBS cultured from urine during routine assessments for UTI to define the clinical and microbiological characteristics of GBS UTI including which serotypes cause disease.     

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.)     

Simultaneous Genotyping and Quantification of Hepatitis B Virus for Genotypes B and C by Real-Time PCR Assay

Hepatitis B virus (HBV) is an important cause of human chronic liver diseases and is a major public health problem. Viral load and HBV genotype play critical roles in determining clinical outcomes and response to antiviral treatment in hepatitis B patients. Viral genotype detection and quantification assays are currently in use with different levels of effectiveness. In this study, the performance of a real-time genotyping and quantitative PCR (GQ-PCR)-based assay was evaluated. Through the use of genotype-specific primers and probes, this assay provides simultaneous identification and quantification of genotypes B and C in a single reaction. Our GQ-PCR correctly identified all predefined genotypes B and C, and no cross-reaction between genotypes B and C were observed. The GQ-PCR identified more cases of HBV infections with mixed genotypes B and C than direct sequencing did. Samples from 127 HBV-infected Chinese patients were genotyped with GQ-PCR, revealing 56.7% HBV as genotype B, 13.4% as genotype C, and 29.8% as mixed genotypes B and C. This assay provides a reliable, efficient, and cost-effective means for quantification of the B and C genotypes of HBV in single or mixed infections. This assay is suitable for sequential monitoring of viral load levels and for determining the relationship between the genotype viral load and stage of disease in Asians.Hepatitis B virus (HBV) is one of the most serious and prevalent health problems, affecting more than 2 billion people worldwide. Chronic HBV infection greatly increases the risk for liver cirrhosis and hepatocellular carcinoma (HCC). HBV infection is associated with up to 80 to 90% of HCC patients in China, India, North and South Korea, Singapore, and Vietnam (17). Although highly effective vaccines against hepatitis B virus have been available since 1982, there are still more than 400 million chronic carriers, 75% of whom reside in the Asia-Pacific region (18).HBV has been classified into eight genotypes (A to H) based on divergence over the entire HBV genomic sequence of greater than 8% (16). The clinical picture, the response to treatment, and the long-term prognosis, as well as the serocoversion profile, are influenced by the HBV genotypes (8). China is a country seriously affected by the burden of chronic HBV infection. The prevalence of chronic HBV infection in China is 5 to 20% of the general population (16, 29). The most prevalent genotypes in China are genotypes B and C (36). In mainland China, an area of HBV endemicity with one-third of the HBV carriers in the world and chronic carriers of hepatitis B surface antigen (HBsAg), patients are commonly infected during early childhood (31). Chronic HBV infection greatly increases the risk for liver cirrhosis and HCC. HBV infection is associated with up to 80 to 90% of HCC patients in China (17).Previous studies indicated that HBV genotype C takes a more aggressive disease course than genotype B (2, 3, 4, 7, 9, 11, 14, 22, 34). Genotype C, in comparison to genotype B, is also associated with a lower response rate to antiviral therapy (10, 13, 15, 19, 27, 28, 29, 35) and precore mutations (30). Therefore, the role of HBV genotypes in predicting outcome should be evaluated further. Evidence has suggested that coinfection with different HBV genotypes is associated with higher viral replication and a more severe course of the disease (5, 6, 12, 26, 33). Several methods have been developed for HBV quantification and genotyping in a single reaction by real-time PCR (20, 23, 32). However, those assays include additional melting-curve analyses for HBV genotypes.Reliable and easy methods to concurrently genotype and quantitate HBV genotypes B and C are prerequisites for molecular epidemiological tests, clinical studies, antiviral therapy, and detection of coinfection. Owing to its high sensitivity, specificity, and broad dynamic range, quantitative real-time PCR (QRT-PCR) has become increasingly important in the diagnostic laboratory and has been used for HBV detection, genotyping, and quantification (1). In this study, a novel real-time PCR-based assay was developed for simultaneous genotyping and quantification of HBV for individual B and C genotypes and for mixed genotypes.     

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).     

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?     

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.     

Evidence of Prior Exposure to Human Bocavirus as Determined by a Retrospective Serological Study of 404 Serum Samples from Adults in the United States

Recently, molecular screening for pathogenic agents has identified a partial genome of a novel parvovirus, called human bocavirus (HBoV). The presence of this newly described parvovirus correlated with upper and lower respiratory tract infections in children. Lower respiratory tract infections are a leading cause of hospital admission in children, and the etiological agent has not been identified in up to 39% of these cases. Using baculovirus expression vectors (BEVs) and an insect cell system, we produced virus-like particles (VLPs) of HBoV. The engineered BEVs express the HBoV capsid proteins stoichiometrically from a single open reading frame. Three capsid proteins assemble into the VLP rather than two proteins predicted from the HBoV genome sequence. The denatured capsid proteins VP1, VP2, and VP3 resolve on silver-stained sodium dodecyl sulfate-polyacrylamide gels as three bands with apparent molecular masses of 72 kDa, 68 kDa, and 62 kDa, respectively. VP2 apparently initiates at a GCT codon (alanine) 273 nucleotides downstream from the VP1 start site and 114 nucleotides upstream from the VP3 initiation site. We characterized the stable capsids using physical, biochemical, and serological techniques. We found that the density of the VLP is 1.32 g/cm³ and is consistent with an icosahedral symmetry with approximately a 25-nm diameter. Rabbit antiserum against the capsid of HBoV, which did not cross-react with adeno-associated virus type 2, was used to develop enzyme-linked immunosorbent assays (ELISAs) for anti-HBoV antibodies in human serum. Using ELISA, we tested 404 human serum samples and established a range of antibody titers in a large U.S. adult population sample.Among the family Parvoviridae, the genus Parvovirinae has many pathogenic species such as feline panleukopenia virus (38, 46), canine parvovirus (39), and Aleutian disease virus of mink (7). However, the only human-pathogenic parvovirus is the sole member of the Erythrovirinae, strain B-19 (3, 26, 52). Recently, a second potentially pathogenic human parvovirus was isolated and assigned the species named human bocavirus (HBoV) (2). Although HBoV DNA was detected in clinical isolates of children with lower respiratory tract infections (4, 5, 12, 30), it is unclear whether HBoV was the etiological agent or contributed to the pathogenicity of the respiratory infection (34, 45). Until recently, the presence of HBoV in respiratory secretions relied on PCR (13, 16, 32, 35, 37, 40). Using PCR, HBoV DNA has been detected worldwide with 5 to 10% prevalence among children with upper or lower respiratory tract infections (6, 22, 24, 27, 33, 36, 41, 51). However, 80% of the HBoV DNA-positive patients were coinfected with common human respiratory viruses (8, 10, 17). Thus, whatever role HBoV plays in lower respiratory tract infection remains unclear.Using the baculovirus expression vector (BEV) system, we produced virus-like particles (VLPs) of HBoV. Two recent reports developed enzyme-linked immunosorbent assays (ELISAs) using HBoV antigen that associated into a homomeric particle with apparent parvovirus-like size and symmetry. As reported previously by Kahn et al. (23), 270 serum specimens, mostly from infants, were tested using a VP2 VLP-based ELISA. Using a cutoff of 0.150 absorption units at a dilution of 1:80, 90% of infants ≤2 months old were considered to be HBoV seropositive. The percentage dropped to 25% in the 4-month-old age group but increased to 85% for individuals in the >48-month- to 20-year-old age group. In a previously reported study (29) also using VP2 VLP-HBoV, 394 sera of children under 15 years old were tested. Using a cutoff of 0.3 absorption units and a 1:200 serum dilution, those authors reported an age-related increase in seroprevalence in healthy children and an even higher seroprevalence in children (birth to 3 years old) with lower respiratory tract infection. The incomplete VLP described in recent reports (23, 29, 31) might have resulted in the relatively low titers perhaps due to a lack of native epitopes derived from tertiary protein interactions. Based on precedence with adeno-associated virus (AAV), proper capsid formation requires the major coat protein and VP2 for AAV VLP assembly using baculovirus and Sf9 cells (44).Based on the reported HBoV sequence, we designed a BEV to express the HBoV capsid proteins from a single open reading frame. Using a non-AUG initiation codon for VP1, the ratio of the smaller, major capsid protein VP2 to the minor, larger capsid protein VP1 was approximately 5:1. However, in both the BEV-infected insect cell lysates and purified VLPs, three capsid proteins were observed, not two as predicted from the HBoV genomic sequence. Rabbit antiserum produced in response to immunizations with purified VLP developed high-titer immunoglobulins (Igs) specific for HBoV. Thus, we developed an ELISA and tested 404 serum samples from adults. The data obtained from these sera produced a broad range of titers, suggesting that the prevalence of prior exposure to HBoV in the United States adult population is 59 to 67%.     

Lipoprotein Lipase and Hydrofluoric Acid Deactivate Both Bacterial Lipoproteins and Lipoteichoic Acids,but Platelet-Activating Factor-Acetylhydrolase Degrades Only Lipoteichoic Acids

To identify the Toll-like receptor 2 ligand critically involved in infections with gram-positive bacteria, lipoprotein lipase (LPL) or hydrogen peroxide (H₂O₂) is often used to selectively inactivate lipoproteins, and hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) is used to selectively inactivate lipoteichoic acid (LTA). However, the specificities of these chemical reactions are unknown. We investigated the reaction specificities by using two synthetic lipoproteins (Pam₃CSK₄ and FSL-1) and LTAs from pneumococci and staphylococci. Changes in the structures of the two synthetic proteins and the LTAs were monitored by mass spectrometry, and biological activity changes were evaluated by measuring tumor necrosis factor alpha production by mouse macrophage cells (RAW 264.7) following stimulation. PAF-AH inactivated LTA without reducing the biological activities of Pam₃CSK₄ and FSL-1. Mass spectroscopy confirmed that PAF-AH monodeacylated pneumococcal LTA but did not alter the structure of either Pam₃CSK₄ or FSL-1. As expected, HF treatment reduced the biological activity of LTA by more than 80% and degraded LTA. HF treatment not only deacylated Pam₃CSK₄ and FSL-1 but also reduced the activities of the lipoproteins by more than 60%. Treatment with LPL decreased the biological activities by more than 80%. LPL also removed an acyl chain from the LTA and reduced its activity. Our results indicate that treatment with 1% H₂O₂ for 6 h at 37°C inactivates Pam₃CSK₄, FSL-1, and LTA by more than 80%. Although HF, LPL, and H₂O₂ treatments degrade and inactivate both lipopeptides and LTA, PAF-AH selectively inactivated LTA with no effect on the biological and structural properties of the two lipopeptides. Also, the ability of PAF-AH to reduce the inflammatory activities of cell wall extracts from gram-positive bacteria suggests LTA to be essential in inflammatory responses to gram-positive bacteria.Bacterial sepsis is a leading cause of death within intensive care units (43). Although bacterial sepsis was traditionally associated with gram-negative (Gr−) bacteria, recently, the prevalence of sepsis caused by gram-positive (Gr+) bacteria has rapidly increased (2, 3, 38). In fact, in 2000, Gr+ bacteria accounted for 52% of sepsis cases whereas Gr− bacteria accounted for only 37.6% (7, 31, 38). In bacterial sepsis, the innate immune system provides both the initial immune responses and the early inflammatory responses (1, 8, 12). Early responses to infections with Gr+ and Gr− bacteria have been shown in previous studies to involve different cytokine profiles (9, 16, 25, 51, 54). Other studies have found that infections with Gr− bacteria activate Toll-like receptor 4 (TLR4) primarily with lipopolysaccharide (LPS), a membrane component of Gr− bacteria (26, 27, 44, 53). In contrast, infections with Gr+ bacteria involve TLR2, but the nature of the key TLR2 ligand is still controversial (34, 52, 56).Two components of the cell walls of Gr+ bacteria have been proposed to be TLR2 ligands. One group of studies suggests that lipoteichoic acid (LTA) is the key ligand (10, 46, 49, 57). LTA is a polyphosphate attached to the cell membrane via a diacyl glycolipid and is an abundant component of the envelopes of Gr+ bacteria (47). Highly purified LTA, as well as its synthetic analogs, has been shown to trigger TLR2-mediated inflammatory responses (10, 15, 20, 35). However, the biological role of the LTA is unclear because it is difficult to purify natural LTA without introducing contaminants or damaging the structure of the LTA (41). Another group proposes bacterial lipoproteins as the critical ligand (22). Lipoproteins are a functionally diverse class of bacterial membrane proteins characterized by an N-terminal lipid moiety (4) and are TLR2 ligands (22-24). Although synthetic analogs of lipoproteins were found to be potent TLR2 ligands (5, 6, 42), natural lipoproteins are difficult to purify, and their properties are poorly understood.To avoid the technical difficulties involved in purification, a different investigational approach was developed. This approach uses methods to selectively inactivate either LTA or lipoproteins in bacterial culture supernatants or crude bacterial cell wall extracts (22-24, 49). LTA inactivation is usually performed with hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) (23, 48, 49), which, respectively, hydrolyzes the phosphodiester bonds in the LTA or deacylates one of its acyl chains (17, 28, 36, 55). Lipoprotein inactivation is commonly achieved by deacylation with a lipoprotein lipase (LPL) or by oxidation with hydrogen peroxide (H₂O₂) (22, 24, 62). Despite their wide use, the reaction selectivities of these methods have not been evaluated. Thus, we investigated the reaction specificities of these methods by studying the impacts of these four reactions on the biological properties as well as the chemical structures of LTA and lipoprotein analogs.     

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.     

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.