Development of a Human-Murine Chimeric Immunoglobulin M Antibody for Use in the Serological Detection of Human Flavivirus Antibodies

Current diagnosis of human flaviviral infections relies heavily on serological techniques such as the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Broad application of this assay is hindered by a lack of standardized human positive-control sera that react with the wide variety of flaviviruses that can cause human disease, e.g., dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and St. Louis encephalitis virus (SLEV). We have created a human-murine chimeric antibody combining the variable regions of the broadly flavivirus cross-reactive murine monoclonal antibody (MAb) 6B6C-1 and the constant region of human IgM to produce a standardized reagent capable of replacing human positive-control sera in a MAC-ELISA for the diagnosis of all human flaviviral infections. The human-murine chimeric IgM antibody secreted from plasmid-transformed Sp2/0-Ag14 cells had a level of serological activity identical to that of 6B6C-1 as measured by ELISA, immunoblotting, and MAC-ELISA for multiple members of the flavivirus genus, including WNV, SLEV, YFV, DENV, and JEV.Flaviviruses are positive-stranded RNA viruses, members of the family Flaviviridae, and are responsible for a number of medically important human diseases. Flaviviruses are arthropod-borne viruses (arboviruses) that are most commonly transmitted seasonally and in specific geographic locations. In the United States, flaviviruses are transmitted mainly by mosquitoes (17, 21). More than 70 different flaviviruses are known to exist; however, the majority are not currently associated with human disease (21). The tenuous nature of the status quo was proven by the emergence of West Nile virus (WNV) in the western hemisphere. Prior to the 1999 outbreak of WNV encephalitis in New York City, St. Louis encephalitis virus (SLEV) was the most important agent of epidemic viral encephalitis in North America, last causing a major epidemic in the mid-1970s (22, 24, 28). Since 1999, the distribution of WNV has rapidly expanded from New York to the rest of the United States and into Canada and Central and South America. As of August 2008, 27,841 human WNV cases in the United States have been reported to the Centers for Disease Control and Prevention (CDC) (http://www.cdc.gov/ncidod/dvbid/westnile/surv&controlCaseCount08_detailed.htm). Given the globalization of commerce and travel, virus-infected people, animals, and arthropod vectors are able to move easily between distant locations with great speed (13). Thus, it is likely that other arboviruses will follow the example of WNV, resulting in new or novel disease outbreaks in regions of the world outside their normal geographic ranges. Because of this, a rapid and standardized approach to the identification of arboviral infections is needed, worldwide, for the diagnosis and tracking of current and reemerging arboviral diseases.The most commonly employed serological technique for the diagnosis of human flaviviral infections is the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA), which detects virus-reactive IgM, an effective marker of acute viral infection (17, 18, 21), in serum or cerebrospinal fluid from a person with a clinically compatible illness. In the diagnostic setting, the MAC-ELISA and a similar ELISA measuring virus-reactive human IgG are often used in tandem to provide clear diagnostic profiles (9, 13, 18).Application of the MAC-ELISA in the serodiagnosis of flaviviral infection is hampered by the limited availability of human infection-immune sera for use as virus-reactive, antibody-positive control specimens. For the most part, antibody-positive control sera are derived from small volumes of diagnostic serum specimens. The specimens are typically collected only from the most prevalent flaviviral agents (17, 18). The lot-to-lot variability of these specimens can be high, and constant recalibration of antibody-positive and negative-control sera is necessary to ensure that test parameters remain valid (8, 18). Of even greater concern is the lack of broadly cross-reactive antibody-positive control sera that can be used in the MAC-ELISA for the identification of atypical flaviviral infections (17).The availability of a flavivirus group-reactive human IgM antibody would be a tremendous asset in the serological diagnosis of flaviviral infections. Although a number of murine monoclonal antibodies (MAbs) demonstrating flavivirus group reactivity exist, they are unsuitable for use in the human MAC-ELISA. Fortunately, advances in the humanization of murine MAbs have made it possible to overcome these limitations (25). One such method, described by Hackett et al., involves the incorporation of the heavy (H)- and light (L)-chain variable (V) regions of a given murine MAb into an expression plasmid (pJH2-24-95B1; referred to below as pJH2) that contains the constant (Cμ) region of human IgM (8). Upon transfection of cells, the resulting plasmid construct expresses a human-murine hybrid (chimeric) IgM molecule that retains the specificity of the “parent” murine MAb but reacts like human IgM in the MAC-ELISA (8, 10).In this report we describe the development and characterization of such a human-murine chimeric IgM antibody prepared by using the IgM expression plasmid described by Hackett et al. (8). This chimeric IgM was created by incorporating the V regions of the broadly flavivirus cross-reactive murine MAb 6B6C-1 into a plasmid construct containing the human IgM μ chain. The murine MAb 6B6C-1 was originally raised against SLEV and is specific for the flaviviral envelope (E) protein (19, 23). The flavivirus group reactivity of chimeric 6B6C-1 IgM was confirmed, and the chimeric 6B6C-1 IgM was evaluated in the standard MAC-ELISA and shown to be a satisfactory replacement for antibody-positive human control sera against all flaviviruses tested.     

Evaluation of Extracellular Subviral Particles of Dengue Virus Type 2 and Japanese Encephalitis Virus Produced by Spodoptera frugiperda Cells for Use as Vaccine and Diagnostic Antigens

New or improved vaccines against dengue virus types 1 to 4 (DENV1 to DENV4) and Japanese encephalitis virus (JEV), the causative agents of dengue fever and Japanese encephalitis (JE), respectively, are urgently required. The use of noninfectious subviral extracellular particles (EPs) is an inexpensive and safe strategy for the production of protein-based flavivirus vaccines. Although coexpression of premembrane (prM) and envelope (E) proteins has been demonstrated to produce EPs in mammalian cells, low yields have hindered their commercial application. Therefore, we used an insect cell expression system with Spodoptera frugiperda-derived Sf9 cells to investigate high-level production of DENV2 and JEV EPs. Sf9 cells transfected with the prM and E genes of DENV2 or JEV secreted corresponding viral antigens in a particulate form that were biochemically and biophysically equivalent to the authentic antigens obtained from infected C6/36 mosquito cells. Additionally, equivalent neutralizing antibody titers were induced in mice immunized either with EPs produced by transfected Sf9 cells or with EPs produced by transfected mammalian cells, in the context of coimmunization with a DNA vaccine that expresses EPs. Furthermore, the results of an enzyme-linked immunosorbent assay (ELISA) using an EP antigen derived from Sf9 cells correlated significantly with the results obtained by a neutralization test and an ELISA using an EP antigen derived from mammalian cells. Finally, Sf9 cells could produce 10- to 100-fold larger amounts of E antigen than mammalian cells. These results indicate the potential of Sf9 cells for high-level production of flavivirus protein vaccines and diagnostic antigens.Dengue virus types 1 to 4 (DENV1-4) and Japanese encephalitis virus (JEV), the causative agents of dengue fever and Japanese encephalitis (JE), respectively, are globally important human pathogens (10) for which new or improved vaccines are urgently required. DENV1-4 cause dengue fever and dengue hemorrhagic fever in tropical areas and many subtropical areas. An estimated 50 million to 100 million dengue cases occur annually, with 2.5 billion people at risk of infection (11). However, there is no approved vaccine for dengue diseases, and the development of such a vaccine is urgently needed (12). JEV is the single largest cause of childhood viral encephalitis in the world, with an estimated 50,000 cases annually. Mortality rates can reach 30% among confirmed cases, and as many as one-third of survivors suffer from permanent and severe psychoneurological sequelae (13, 39). Although inactivated vaccines are used internationally for JE, they are too expensive for widespread use in most developing countries (3), and therefore, more cost-effective alternatives are needed.Neutralizing antibodies are important in host protection against dengue diseases and JE (10, 34). For JE, previously used mouse brain-derived (16, 44) and more recently used Vero cell-derived (20, 25, 35) inactivated vaccines can efficiently induce neutralizing antibody responses. However, these protein-based vaccines are produced from infectious agents, and their production therefore requires biosafety level 2 or 3 containment facilities and complex purification protocols, thus increasing the cost of the vaccine. Vaccine production without infective procedures can be achieved using genetic engineering techniques (29, 55).DENV1-4 and JEV are members of the genus Flavivirus in the family Flaviviridae (37). The envelope (E) protein is the major component of the envelopes of flavivirus virion particles and possesses most of the neutralizing epitopes (46). The other protein on the envelopes of mature virions is the membrane (M) protein, which is synthesized as the precursor membrane (prM) protein in infected cells. Cells expressing flavivirus prM and E proteins are known to secrete nucleocapsid-free subviral extracellular particles (EPs), which are similar to slowly sedimenting hemagglutinin (SHA) particles secreted from flavivirus-infected cells (47). EPs of JEV synthesized in mammalian expression systems have been evaluated for their immunogenicity and/or protective efficacy in mice (15, 24, 43). Two of these studies (24, 43) demonstrated that the EPs induced neutralizing antibodies at levels comparable to those induced by an inactivated JE vaccine. In our laboratory, mammalian cell lines continuously expressing EPs of dengue type 2 virus (DENV2) (26) or JEV (27) have been generated and designated D cells and F cells, respectively. The EPs contained an E protein that was antigenically and biochemically equivalent to the authentic E protein, and the EPs were immunogenic and protective in mice. However, the yields of viral antigens produced from D and F cells were low and would not meet the requirements for commercial vaccine production. Increasing the levels of viral antigen production from transfected cells would reduce the cost of vaccine preparation.Recently, insect cell expression systems have been increasingly used in various fields of medical sciences (1, 6, 17, 53). In general, insect cells are easier to cultivate than mammalian cells, because they often do not require serum supplementation in the culture medium or incubation under CO₂. In addition, insect cells can be adapted to suspension culture, allowing cultures to be simply scaled up. Furthermore, various techniques have been developed for high-density culture of insect cells; for example, in one study, the immobilization of insect cells within biomass support particles achieved a density of approximately 3 × 10⁷ cells/cm³ (50). Thus, the insect cell expression system can be a simple and inexpensive strategy for vaccine antigen production.In addition to their use as vaccine antigens, EPs derived from mammalian cells could be used as serodiagnostic antigens (27, 32). The production of serodiagnostic antigens may also encounter problems when the antigens are sourced directly from infectious agents. Currently, numerous commercial assays utilizing several different formats, such as the immunochromatography test and the IgM capture enzyme-linked immunosorbent assay (ELISA), are available for the diagnosis of DENV and JEV infections (4, 49). These commercial tests use viral antigens derived from transfected or infected cultured cells. Thus, the application of insect cell-derived EPs as diagnostic antigens would be an attractive alternative.In this study, we produced EPs of DENV2 and JEV in a transient expression system using the Sf9 cell line, which was derived from the pupal ovarian tissue of the fall armyworm, Spodoptera frugiperda. These proteins were evaluated for vaccine and diagnostic antigens, mainly by direct comparison with mammalian-cell-derived EPs. The EPs produced from Sf9 cells were immunogenic in mice and useful as antigens for ELISA. In addition, Sf9 cells produced larger amounts of antigen than CHO cells, suggesting the potential applicability of insect cells for the production of DENV2 and JEV antigens for vaccines and serodiagnostic tests.     

Immunogenicity and Protective Efficacy of a Recombinant Subunit West Nile Virus Vaccine in Rhesus Monkeys

The immunogenicity and protective efficacy of a recombinant subunit West Nile virus (WNV) vaccine was evaluated in rhesus macaques (Macaca mulatta). The vaccine consisted of a recombinant envelope (E) protein truncated at the C-terminal end, resulting in a polypeptide containing 80% of the N-terminal amino acids of the native WNV protein (WN-80E), mixed with an adjuvant (GPI-0100). WN-80E was produced in a Drosophila melanogaster expression system with high yield and purified by immunoaffinity chromatography using a monoclonal antibody specific for flavivirus E proteins. Groups of monkeys were vaccinated with formulations containing 1 or 25 μg of WN-80E antigen, and both humoral and cellular immunity were assessed after vaccination. The results demonstrated potent antibody responses to vaccination, as determined by both enzyme-linked immunosorbent assay and virus-neutralizing antibody assays. All vaccinated animals responded favorably, and there was little difference in response between animals immunized with 1 or 25 μg of WN-80E. Cellular immunity was determined by lymphocyte proliferation and cytokine production assays using peripheral blood mononuclear cells from vaccinated animals stimulated in vitro with WN-80E. Cell-mediated immune responses varied from animal to animal within each group. About half of the animals responded with lymphoproliferation, cytokine production, or both. Again, there was little difference in response between animals immunized with a 1- or 25-μg dose of WN-80E in the vaccine formulations. In a separate experiment, groups of monkeys were immunized with the WN-80E/GPI-0100 vaccine or an adjuvant-only control formulation. Animals were then challenged by inoculation of wild-type WNV, and the level of viremia in each animal was monitored daily for 10 days. The results showed that whereas all animals in the control group had detectable viremia for at least 3 days after challenge, all of the vaccinated animals were negative on all days after challenge. Thus, the WN-80E vaccine was 100% efficacious in protecting monkeys against infection with WNV.West Nile virus (WNV) was first detected in North America in 1999 and spread rapidly across the continental United States (3, 32), as well as into Canada (8), Mexico (9), and Central and South America (17). The virus is transmitted via mosquitoes, primarily through the bite of Culex species but also by many other genera of mosquitoes (14). Birds are the natural hosts and serve as the zoonotic reservoir, while mammals and reptiles are considered to be incidental hosts from which, it is believed, further transmission generally does not occur (12). This is thought to be due to the relatively low levels of viremia that develop in these latter hosts, which may be insufficient to allow for secondary mosquito transmission (11). However, more recent studies (2, 38) have suggested that, in some mammals and reptiles, sufficient viremia may develop to yield at least a low competence for transmission.Based on retrospective seroepidemiological surveys conducted after the initial discovery of this virus in North America, it was determined that about 20% of those individuals infected developed clinical disease (3). The large majority of clinical cases resulted in a self-limiting, influenza-like syndrome (3); however, about 1 in 150 infected patients developed neurological complications (28). These complications included cases of meningitis; encephalitis; meningoencephalitis; and an acute, flaccid paralytic, poliomyelitis-like syndrome (13). The cases with WNV-associated neurological complications tend to be severe, often resulting in permanent disabilities, with reported case fatality rates of 5 to 15% (3). However, in a recent study (4), it was reported that even in those cases of mild, nonneuropathological disease, after resolution of the infection had apparently occurred, residual defects in neuromotor and cognitive function could be measured for at least 1 year after the original diagnosis.Moreover, the disease course tends to be much more severe in elderly individuals, with significantly higher case fatality rates of about 30% in neuroinvasive cases (5, 30, 33, 42). This may be due to declining immunocompetence concomitant with aging (“immunosenescence”). In addition to the elderly population, individuals whose immune systems have been compromised through primary immune deficiencies, acquired deficiencies, or immunosuppressive therapies are also at increased risk of severe disease caused by WNV infection (10, 18). Certain other chronic diseases, such as diabetes mellitus and hypertension, may also render individuals infected with WNV more susceptible to developing severe disease (16).WNV is a member of the Flaviviridae family, genus Flavivirus. It is an enveloped, positive-strand RNA virus. The RNA genome comprises 10 genes, coding for three structural and seven nonstructural proteins (31). The structural proteins are the core or capsid protein (C); a premembrane protein (prM), which is cleaved to yield the membrane protein in the mature virion; and the envelope protein (E). The latter two are glycosylated. The E protein shares significant homology with the E proteins of other flaviviruses, particularly those of the other members of the Japanese encephalitis virus (JEV) serocomplex, JEV itself, St. Louis encephalitis virus (SLEV), and Murray Valley encephalitis virus. Antibodies directed against particular epitopes contained within the E protein are capable of virus neutralization. These epitopes have recently been mapped to at least two of three domains of the E protein, domains II and III, using sets of monoclonal antibodies for dengue virus (DENV) (6), as well as JEV (20) and WNV (1, 41). Neutralizing antibodies reacting with domain III are generally specific for each virus and do not cross-neutralize other viruses (or other serotypes of the same virus if multiple serotypes exist), while those targeting domain II are often cross-reactive. A high titer of virus-neutralizing antibodies is generally accepted as the best in vitro correlate of in vivo protection against virus infection or immunity to subsequent infection (23, 45). For this reason, the E protein was selected as the appropriate immunogen for use in the development of a WNV vaccine candidate.In previous studies at Hawaii Biotech, Inc. (HBI), a proprietary method of expression was used successfully to produce recombinant E proteins from flaviviruses, such as DENV serotypes 1 to 4, JEV, hepatitis C virus, and WNV (7, 19, 25, 26, 35). These proteins are truncated at the C terminus, leaving 80% of the native E protein (80E). The truncation deletes the membrane anchor portion of the protein, thus allowing it to be secreted into the extracellular medium, facilitating recovery. Furthermore, the expressed DENV and WNV proteins have been shown to be properly glycosylated and to maintain native conformation as determined by reactivity with conformationally sensitive monoclonal antibodies 4G2 and 9D12 (B. -A. Coller, D. E. Clements, and G. S. Bignami, unpublished data) and X-ray crystallography structure determination (25, 26). The immunogenicity of the vaccine formulations using the truncated WNV E protein (WN-80E [amino acids 1 to 401]) was demonstrated in mice (19), and its protective efficacy documented in hamsters (39, 43) and geese (15). The present report for the first time documents the immunogenicity and protective efficacy of a WN-80E vaccine formulation in a nonhuman primate animal model.     

Enterococcal Biofilm Formation and Virulence in an Optimized Murine Model of Foreign Body-Associated Urinary Tract Infections

Catheter-associated urinary tract infections (CAUTIs) constitute the majority of nosocomial UTIs and pose significant clinical challenges. Enterococcal species are among the predominant causative agents of CAUTIs. However, very little is known about the pathophysiology of Enterococcus-mediated UTIs. We optimized a murine model of foreign body-associated UTI in order to mimic conditions of indwelling catheters in patients. In this model, the presence of a foreign body elicits major histological changes and induces the expression of several proinflammatory cytokines in the bladder. In addition, in contrast to naïve mice, infection of catheter-implanted mice with Enterococcus faecalis induced the specific expression of interleukin 1β (IL-1β) and macrophage inflammatory protein 1α (MIP-1α) in the bladder. These responses resulted in a favorable niche for the development of persistent E. faecalis infections in the murine bladders and kidneys. Furthermore, biofilm formation on the catheter implant in vivo correlated with persistent infections. However, the enterococcal autolytic factors GelE and Atn (also known as AtlA), which are important in biofilm formation in vitro, are dispensable in vivo. In contrast, the housekeeping sortase A (SrtA) is critical for biofilm formation and virulence in CAUTIs. Overall, this murine model represents a significant advance in the understanding of CAUTIs and underscores the importance of urinary catheterization during E. faecalis uropathogenesis. This model is also a valuable tool for the identification of virulence determinants that can serve as potential antimicrobial targets for the treatment of enterococcal infections.Catheter-associated urinary tract infections (CAUTIs) are the most common complications resulting from the use of indwelling urinary catheters (25, 70). CAUTIs account for 40% of all nosocomial infections (48) with more than one million cases diagnosed annually in hospitals and nursing homes in the United States, generating approximately $600 million in medical expenditures every year (26, 69, 70). The pathophysiology of CAUTIs results from the disruption of the normal mechanical and antimicrobial defenses of the bladder and the injuries ensuing from urinary catheterization, which render the bladder environment vulnerable to microbial adhesion, multiplication, and dissemination within the urinary tract (15, 47, 49, 51, 78). Furthermore, indwelling urinary catheters provide an additional surface for microbial attachment and biofilm formation, which is a major component of the pathophysiology of CAUTIs and other chronic device-associated persistent infections (4, 6, 12, 14, 37, 46, 68). Once formed, biofilms provide a favorable milieu for microbial survival within the host as the organisms are shielded from the host immune response, as well as antibiotics and antimicrobial agents (36, 67, 81), leading to chronic or recurrent infections that are difficult to treat. If untreated, CAUTIs can lead to more severe diseases such as acute pyelonephritis, bacteremia, urosepsis, and in some cases, death (38, 77). The high incidence of CAUTIs and their medical and economic challenges underscore the need for a better understanding of CAUTI pathogenesis.While community-acquired UTIs are most commonly due to uropathogenic Escherichia coli (UPEC), UPEC represent only 50% of bacterial isolates derived from patients with CAUTIs (34). The Enterococcus species, especially Enterococcus faecalis and Enterococcus faecium, account for 15% to 30% of CAUTIs (38) and are now considered the third leading cause of hospital-acquired UTIs (20, 68). The ability of many enterococcal isolates to produce biofilms (41, 60, 73) and the increasing microbial resistance to antibiotics, including vancomycin, pose significant challenges for the treatment of enterococcal infections (11, 80).Several rodent models have been developed to study E. faecalis pathogenesis in the urinary tract in single and mixed infections with other Gram-negative pathogens (21, 29, 43, 61, 75). These models involve the transurethral inoculation of bacterial suspensions into the bladders of healthy animals or streptozocin-induced diabetic animals (56) and have been shown to be primarily useful for the study of E. faecalis-mediated pyelonephritis. The experimental conditions defined by these models are inadequate for the investigation of persistent enterococcal CAUTI, since the bacteria are readily cleared from the bladder and fail to establish chronic cystitis (29, 56, 61). Nonetheless, data obtained from these models have implicated some enterococcal factors in UTI pathogenesis, including the enterococcal surface protein Esp (61), the pilus-associated sortase C (SrtC) (30), and the endocarditis and biofilm-associated pilus (Ebp) (65). However, well-characterized adhesins and biofilm determinants often associated with enterococcal UTI isolates, like aggregation substance (AS) and the housekeeping sortase A (SrtA) (17, 27, 33), were reported to be dispensable for virulence in the urinary tract (27, 30). Since these conclusions are drawn from models where persistent infections cannot be established, it is imperative to reexamine the existing paradigm in an animal model that better mimics the transition of E. faecalis from a commensal organism to a virulent pathogen in the urinary tract.In this study, we optimized a rodent model of foreign body-associated UTI, developed in rats by Kurosaka et al. (35) and adapted for mice by Kadurugamuwa et al. (28), to investigate the pathophysiology of E. faecalis-mediated CAUTIs. We report that the presence of silicone catheter implants causes major physiological changes in the bladder, which becomes predisposing to E. faecalis biofilm formation, and is associated with persistent enterococcal cystitis and pyelonephritis. We further demonstrate that biofilm formation on the silicone implants and the development of successful enterococcal UTI are independent of autolytic factors GelE and Atn, also known as AtlA (13), but requires the presence of the housekeeping sortase A (SrtA). Overall, this optimized murine model is well suited to identify host and enterococcal factors critical for pathogenesis in the urinary tract which will provide a better understanding of the mechanisms underlying the pathophysiology of CAUTIs.     

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.     

Mobilization of CD34+ Progenitor Cells in Association with Decreased Proliferation in the Bone Marrow of Macaques after Administration of the Fms-Like Tyrosine Kinase 3 Ligand

Fms-like tyrosine kinase 3 ligand (FLT3-L) is critical for the differentiation and self-renewal of CD34⁺ progenitor cells in primates and has been used therapeutically to mobilize progenitor and dendritic cells in vivo. However, little is known regarding the expansion of progenitor cells outside of peripheral blood, particularly in bone marrow (BM), where progenitor cells primarily reside. Evaluation of FLT3-L-mediated cell mobilization during lentivirus infections, where the numbers of CD34⁺ progenitor cells are reduced, is limited. We enumerated frequencies and absolute numbers of CD34⁺ progenitor cells in blood and BM of naive and SIV- or SHIV-infected macaques during and after the administration of FLT3-L. Flow cytometric analyses revealed that, while CD34⁺ cells increased in the circulation, no expansion was observed in BM. Furthermore, in the BM intracellular Ki67, a marker of cell proliferation, was downregulated in CD34⁺ progenitor cells but was upregulated significantly in the bulk cell population. Although the exact mechanism(s) remains unclear, these data suggest that CD34⁺ cell mobilization in blood was the result of cellular emigration from BM and not the proliferation of CD34⁺ cells already in the periphery. It is possible that the decreased progenitor cell proliferation observed in BM is evidence of a negative regulatory mechanism preventing hyperproliferation and development of neoplastic cells.The cytokine receptor Fms-like tyrosine kinase 3 (FLT3) is expressed at high levels on both primitive and early lymphoid/myeloid CD34⁺ progenitor cells (3, 21). Interaction with its cognate ligand (FLT3-L), found in both soluble and membrane-bound isoforms, contributes to the regulation of self-renewal and differentiation potential of these cells (43, 44). However, dysregulation of FLT3/FLT3-L signaling can result in the development of various leukemias (1, 6, 22, 29), and increased serum levels of FLT3-L are often indicative of other hematologic and autoimmune abnormalities (17, 25, 39). Nonetheless, after both murine and human FLT3-L were cloned in the early 1990s (24), this hematopoietic cytokine was used effectively in vitro to expand and maintain CD34⁺ progenitor cells (26, 32, 33) and, in combination with other growth factors, was used to induce differentiation of myeloid lineage cells (4), dendritic cells (2, 15), natural killer (NK) cells (42), erythroid precursors (12), and even endothelial cells (41). In addition, FLT3-L was shown to specifically suppress apoptosis of CD34⁺ progenitor cells (27).Early in vivo studies in mice demonstrated that FLT3-L administration not only mobilized and expanded murine CD34⁺ progenitor cells but also promoted expansion of human CD34⁺ cells transferred into SCID mice (8, 9, 24). In nonhuman primates FLT3-L was used to expand dendritic cell subsets (7, 30, 35), to treat radiation-induced myelosuppression (13, 14, 19), and as an adjuvant for various vaccines (23, 40). Although CD34⁺ cells primarily reside in the bone marrow (BM), examination of mobilization of these cells in vivo in nonhuman primates has been limited and typically restricted to analyses of blood (5, 18, 28). CD34⁺ cell mobilization and hematopoiesis is of particular interest in macaque models of lentivirus infections because, during both HIV and SIV infections, BM damage and reduced hematopoiesis is evident early after infection and is associated with decreased numbers and clonogenic potential of CD34⁺ progenitors, despite low levels of infection and virus replication in these cells (10, 16, 20, 34, 36, 37). Therefore, in the present study we quantified and characterized mobilization of CD34⁺ progenitor cells in BM in relation to that observed in peripheral blood by examining BM aspirates taken at various times during and after FLT3-L administration to naive and SIV- or SHIV-infected macaques.     

Propionibacterium acnes and Staphylococcus epidermidis Isolated from Refractory Endodontic Lesions Are Opportunistic Pathogens

The predominant cultivable microbiota from 20 refractory endodontic lesions (9 with abscesses and 11 without abscesses) were determined, and Propionibacterium acnes and Staphylococcus epidermidis were among the most predominant organisms. The number of species identified from lesions with abscesses (14.1 ± 2.6) was significantly greater (P < 0.001) than the number from lesions without abscesses (7.4 ± 5.9). Comparison of perioral isolates using repetitive extragenic palindromic PCR of the same species from the same subjects demonstrated that the endodontic and skin populations were significantly different. The P. acnes isolates were typed on the basis of recA gene sequence comparison, and only three types (types I, II, and III) were identified among 125 isolates examined. However, we found that type I (type IA and IB) isolates were primarily isolated from the skin, while types II and III were significantly more likely to be isolated from the endodontic lesions (P < 10⁻¹⁰). We found that the robustness of the recA phylotypes was not strong by comparing the partial gene sequences of six putative virulence determinants, PAmce, PAp60, PA-25957, PA-5541, PA-21293, and PA-4687. The resulting neighbor-joining trees were incongruent, and significant (phi test; P = 2.2 × 10⁻⁷) evidence of recombination was demonstrated, with significant phylogenetic heterogeneity being apparent within the clusters. P. acnes and S. epidermidis isolated from refractory endodontic infections, with or without periapical abscesses, are likely to be nosocomial infections.Propionibacterium acnes and coagulase-negative staphylococci, including Staphylococcus epidermidis, have been identified among the microflora of endodontic infections (8, 12, 40, 50, 52, 55, 56, 63, 64), but their importance as endodontic pathogens has largely been ignored due to their nearly universal presence on the skin and the consequent likelihood of sample contamination. However, there is now considerable evidence that these organisms are increasingly isolated from human infections, and so their association with endodontic infections requires clarification. P. acnes, a non-spore-forming, Gram-positive anaerobic or aerotolerant rod, is a member of the resident microflora of the large intestine, conjunctiva, and external ear canal (10, 15) and accounts for approximately half of the total skin microbiota (59), predominating over other pilosebaceous flora (16, 39). Although traditionally considered to be relatively nonpathogenic, an increasing number of studies have implicated P. acnes as an opportunistic pathogen responsible for a wide range of infections and inflammatory conditions. In addition to its well-established role in the pathogenesis of acnes vulgaris (16, 34), it has also been linked to synovitis-acnes-pustulosis-hyperostosis-osteitis syndrome (44, 54), sarcoidosis (17), and prostate cancer (14). Recent studies have revealed trauma and surgery as the predisposing factors associated with numerous P. acnes infections, which include brain abscesses (36), osteomyelitis after lumbar puncture (1), discitis after surgery (23), spodylodiscitis following epidural catheterization (22, 25), postoperative mediastinitis (19, 58), endophthalmitis (7), and endocarditis (21). Furthermore, it is also emerging as an important pathogen in infections related to medical foreign-body implants, such as intraocular lenses (65), ventroperitoneal shunts, orthopedic implants (9, 28, 51), silicone implants (2), prosthetic heart valves (33, 42), and prosthetic joints (38, 61).S. epidermidis is part of the human skin microflora, where, as a commensal organism, it usually exists in a benign relationship with the host. Although it is ranked first in nosocomial and implant-based infections, perhaps due to its ubiquity on the skin, it is more likely to contaminate devices at the time of insertion. As a recognized opportunistic pathogen, it is responsible for nosocomial infections of indwelling medical devices (43), such as peripheral or central intravenous catheters (CVCs), prosthetic joints, vascular grafts, and central nervous system shunts (46), and cardiac device infections, such as prosthetic valve endocarditis (PVE) (13), as well as ventricular assist device driveline-related infections (4). Clearly, a characteristic shared by both of these organisms is an association with the infection of prosthetic devices.The treatment of endodontic infections involves the insertion of gutta-percha into the debrided and disinfected root canal and the restoration of the tooth. This treatment may fail, with the root canal becoming infected. A range of bacteria have been isolated from such infected sites, including P. acnes and S. epidermidis, but these have generally been considered contaminants. The objective of the present study was to confirm the endodontic origin of P. acnes and S. epidermidis isolates recovered from refractory endodontic infections. We have used DNA fingerprinting of the P. acnes and S. epidermidis isolates from endodontic lesions and samples from the perioral skin of the same individual using repetitive extragenic palindromic PCRs (REP-PCRs) (3). The recA phylotypes of the P. acnes isolates were determined, as we expected to find that the predominant phylotypes from the endodontic lesions would be types II and III, which are almost exclusively associated with infections of implanted prostheses, while types IA and IB are usually isolated from skin (26, 30, 31, 62). We also investigated the phylogenetic status and robustness of the recA phylotypes by comparing the intrastrain relationships of partial gene sequences of six putative virulence determinants, PAmce (mammalian cell entry gene), PAp60, PA-25957, PA-5541, PA-21293, and PA-4687 (20, 35). Here we have set out to determine if P. acnes and S. epidermidis isolated from refractory endodontic lesions are contaminants arising during the sample collection process or if they represent nosocomial infections.     

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.     

Development of Real-Time PCR Assays for Detection of the Streptococcus milleri Group from Cystic Fibrosis Clinical Specimens by Targeting the cpn60 and 16S rRNA Genes

Cystic fibrosis (CF) is a multiorgan disease, with the majority of mortalities resulting from pulmonary failure due to repeated pulmonary exacerbations. Recently, members of the Streptococcus anginosus group (S. anginosus, S. constellatus, and S. intermedius), herein referred to as the “Streptococcus milleri group” (SMG) have been implicated as important etiological pathogens contributing to pulmonary exacerbations in CF patients. This is partly due to better microbiological detection of the SMG species through the development of a novel specific medium termed “McKay agar.” McKay agar demonstrated that SMG has been an underreported respiratory pathogen contributing to lung exacerbations. Our aim was to develop a real-time PCR assay to expedite the detection of SMG within diagnostic samples. The cpn60 gene was chosen as a target, with all three members amplified using a single hybridization probe set. SMG strain analysis showed that speciation based on melting curve analysis allowed for the majority of the S. constellatus (96%), S. intermedius (94%), and S. anginosus (60%) strains to be correctly identified. To increase specificity for S. anginosus, two 16S rRNA real-time PCR assays were developed targeting the 16S rRNA gene. The 16s_SA assay is specific for S. anginosus (100%), while the 16s_SCI assay is specific for S. constellatus and S. intermedius (100%). These assays can detect <10 genome equivalents in pure culture and >10⁴ genome equivalents in sputum samples, making this a great tool for assessment of the presence of SMG in complex polymicrobial samples. Novel molecular methods were developed providing detection ability for SMG, an emerging opportunistic pathogen.Cystic fibrosis (CF) is the most common fatal genetic disease affecting young Caucasians (13). It is a multiorgan disease that primarily affects the lungs and digestive system. Within the CF lungs, there is a buildup of thick mucus that is difficult to clear, leading to chronic bacterial colonization with high bacterial loads (34, 41, 49). However, it is not solely the presence of high bacterial loads in the lungs of CF patients but periods of pulmonary exacerbation, an overt immune response that leads to the majority of irreversible lung damage, that ultimately lead to pulmonary failure in 90% of afflicted individuals (34-36). Classically there are relatively few bacterial pathogens described in CF lung disease (15, 17, 18); however, CF should be considered a polymicrobial infectious disease, as the CF lungs are colonized by a diverse and dynamic consortium of bacteria, fungi, and viruses (1, 21-23, 44-46, 53).Recently the Streptococcus anginosus group, herein referred to as the “Streptococcus milleri group” (SMG), which includes the three species S. anginosus, S. constellatus and S. intermedius, has been implicated in pulmonary exacerbations of CF patients (4, 37, 45). The SMG species have been identified as part of the microbiota of the respiratory tract, gastrointestinal tract, and genitourinary tract in 15 to 30% of healthy individuals (20, 38, 39, 43). However, each species within the SMG has the capacity to cause severe invasive infections throughout the body. SMG infection is the most common cause of brain and liver abscesses (8, 20, 32, 54) and is a major cause of empyema (30). Members of the SMG have been implicated in infection at all body sites, associated with skin and soft tissue (3, 56), abdomen (48), head and neck (16, 33, 48), pleuropulmonary (28, 48), cardiovascular and blood (25, 40), and genitourinary and musculoskeletal (7, 48) infections. There does appear to be species bias to some infections, as S. anginosus has been found to cause the majority of abdominal infections, while S. intermedius has been more often linked to liver and central nervous system (CNS) infections (10, 56). Members of the SMG have also been implicated as a common etiology of intra-abdominal abscesses developed by individuals who have received solid organ transplants and may have been underestimated as a cause of disease within this population (50).SMG strains are phenotypically diverse, even within each species. However, most strains share some common characteristics such as slow growth rate, a distinctive “caramel smell,” their ability to hydrolyze arginine, acetoin production from glucose, and an inability to ferment sorbitol (9, 20, 38, 43). Microbiological differentiation of the three species within the SMG can be problematic. A few methods have been designed that allow for the differentiation of these three species; unfortunately, they are time-consuming, and results are variable (14, 31, 58). Recently a new medium that has been developed, McKay agar, that allows for the isolation of SMG from complex clinical samples; however, other organisms, including additional Streptococcus strains, can also be cultured on this medium (46a). Numerous molecular assays have been developed to differentiate SMG using cpn60 (53), rnpB (27, 52, 55), 16S rRNA genes (7, 10, 31), 16S-to-23S rRNA gene intergenic spacer (ITS) region (5, 11, 52, 57), and the penicillin-binding protein (51). These assays are limited by their need for nucleic acid sequence analysis or further PCR analysis required to differentiate SMG species.The increased importance of SMG in human infections and the difficulty in microbial detection suggest a need for a rapid and reliable test to detect SMG from pure culture as well as complex polymicrobial diagnostic samples such as CF sputum samples (4, 37, 45). The development of a real-time PCR assay in combination with McKay agar isolation would reduce microbial identification time, thereby decreasing the period before the initiation of appropriate antibiotics, which in turn would resolve clinical symptoms more efficiently for all types of infections. This would also afford the opportunity for clinical intervention before the onset of pulmonary exacerbation preventing increased lung damage.We have developed three real-time PCR assays. The first assay is based on cpn60, which detects S. constellatus and S. intermedius and many S. anginosus strains and allows for melting curve-based speciation. The second assay specifically detects S. anginosus, based on a conserved region of the 16S rRNA gene. Finally, the third assay specifically detects S. constellatus and S. intermedius based on a conserved region of the 16S rRNA gene. These assays provide a novel culture-independent strategy for the detection of this important group of emerging pathogens.     

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.     

Molecular Identification and Susceptibility of Trichosporon Species Isolated from Clinical Specimens in Qatar: Isolation of Trichosporon dohaense Taj-Aldeen,Meis & Boekhout sp. nov.

Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid leukemia. Sequence analysis revealed the isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp. nov., with CBS 10761^T as the holotype strain, belonging to the Ovoides clade. In the D1-D2 large-subunit rRNA gene analysis, T. dohaense is a sister species to T. coremiiforme, and in the internal transcribed spacer analysis, the species is basal to the other species of this clade. Molecular identification of the strains yielded 17 T. asahii, 3 T. inkin, 2 T. japonicum, 2 T. faecale, and 3 T. dohaense isolates. The former four species exhibited low MICs for five antifungal azoles but showed high MICs for amphotericin B. T. dohaense demonstrated the lowest amphotericin B MIC (1 mg/liter). For the majority of T. asahii isolates, amphotericin B MICs were high (MIC at which 90% of isolates were inhibited [MIC₉₀], ≥16 mg/liter), and except for fluconazole (MIC₉₀, 8 mg/liter), the azole MICs were low: MIC₉₀s were 0.5 mg/liter for itraconazole, 0.25 mg/liter for voriconazole, 0.25 mg/liter for posaconazole, and 0.125 mg/liter for isavuconazole. The echinocandins, caspofungin and anidulafungin, demonstrated no activity against Trichosporon species.Trichosporon species are yeast-like fungi, widely distributed in nature and commonly isolated from soil and other environmental sources, which have been involved in a variety of opportunistic infections and have been recognized as emerging fungal pathogens in immunocompromised hosts (19, 79, 80). Disseminated Trichosporon infections are potentially life-threatening and are often fatal in neutropenic patients (7, 22). Although uncommon, pathogenic species of this genus have been reported increasingly, mostly in patients with malignant diseases (3, 6, 9, 10, 11, 20, 32, 44, 47, 48, 63, 77), neonates (18, 56, 84), a bone marrow transplant recipient (22), a solid organ transplant recipient (50), and patients with human immunodeficiency virus (34, 35, 46). Trichosporon has also been reported to cause fungemia (5, 9, 25, 29, 30, 33, 53, 62). Members of the genus Trichosporon have occasionally been implicated as nail pathogens (16, 28, 74) and in subcutaneous infections (66). Trichosporon is considered an opportunistic agent, and therefore, recovery of Trichosporon species capable of growing at 37°C, especially from immunocompromised patients, should be regarded as potentially significant. Several reports have addressed the difficulty of identifying Trichosporon to the species level by physiological and biochemical characteristics (2, 64); therefore, molecular methods based on the sequencing of the internal transcribed spacer (ITS) have been developed (15, 69, 71, 72).In the present paper, we report the isolation of Trichosporon species from clinical specimens over a 4-year period in Qatar, the poor performance of biochemical identification methods, the significance of molecular identification, and the antifungal susceptibility data for the isolates. While investigating the molecular identification of Trichosporon species, we found three strains that do not match any of the published strains in the literature. We describe this organism as Trichosporon dohaense Taj-Aldeen, Meis & Boekhout, sp. nov., the name proposed for this species.     

Identification of a Variety of Staphylococcus Species by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Whole-cell fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) in combination with a dedicated bioinformatic software tool (MALDI Biotyper 2.0) was used to identify 152 staphylococcal strains corresponding to 22 staphylococcal species. Spectra of the 152 isolates, previously identified at the species level using a sodA gene-based oligonucleotide array, were analyzed against the main spectra of 3,030 microorganisms. A total of 151 strains out of 152 (99.3%) were correctly identified at the species level; only one strain was identified at the genus level. The MALDI-TOF MS method revealed different clonal lineages of Staphylococcus epidermidis that were of either human or environmental origin, which suggests that the MALDI-TOF MS method could be useful in the profiling of staphylococcal strains. The topology of the dendrogram generated by the MALDI Biotyper 2.0 software from the spectra of 120 Staphylococcus reference strains (representing 36 species) was in general agreement with that inferred from the 16S rRNA gene-based analysis. Our findings indicate that the MALDI-TOF MS technology, associated with a broad-spectrum reference database, is an effective tool for the swift and reliable identification of Staphylococci.Most staphylococci are harmless and reside normally on the skin and mucous membranes of humans and other organisms (16, 22, 34). Staphylococcal strains are isolated from various food products in which they are involved in fermentation (18, 29). Staphylococcus species can cause a wide variety of diseases in humans and other animals (2, 22, 30-32, 35). S. aureus is a major pathogen in human infections (31). Several other Staphylococcus species have also been implicated in human infections, notably S. saprophyticus, S. epidermidis, S. lugdunensis, and S. schleiferi (4, 16, 31, 34). Coagulase-negative staphylococci (CoNS) have emerged as predominant pathogens in hospital-acquired infections (4, 16, 31, 34). One of the major challenges of daily diagnostic work is therefore to identify Staphylococcus species.Several manual and automated methods based on phenotypic characteristics have been developed for the identification of Staphylococci (12, 24). Unfortunately, these systems have their limitations, mostly due to phenotypic differences between strains from the same species (6, 10, 19, 21). Over the last 10 years, many genotypic methods based on the analysis of selected DNA targets have been designed for species-level identification of most common isolated CoNS (20, 26, 33). The sequence polymorphism of the sodA gene has significant discriminatory power (20) and allows the development of assays based on DNA chip technologies (“Staph array”) (8). Recently, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using protein “fingerprints” was used for the identification of microorganisms (1, 3, 5, 9, 11, 14, 25, 36). In the present study, we assessed the ability of the MALDI Biotyper system (Bruker Daltonique, Wissembourg, France) to identify Staphylococcus species of clinical and environmental origins previously identified by sodA gene-based oligonucleotide array (8).     

Tracking the Emerging Human Pathogen Pseudallescheria boydii by Using Highly Specific Monoclonal Antibodies

Pseudallescheria boydii has long been known to cause white grain mycetoma in immunocompetent humans, but it has recently emerged as an opportunistic pathogen of humans, causing potentially fatal invasive infections in immunocompromised individuals and evacuees of natural disasters, such as tsunamis and hurricanes. The diagnosis of P. boydii is problematic since it exhibits morphological characteristics similar to those of other hyaline fungi that cause infectious diseases, such as Aspergillus fumigatus and Scedosporium prolificans. This paper describes the development of immunoglobulin M (IgM) and IgG1 κ-light chain monoclonal antibodies (MAbs) specific to P. boydii and certain closely related fungi. The MAbs bind to an immunodominant carbohydrate epitope on an extracellular 120-kDa antigen present in the spore and hyphal cell walls of P. boydii and Scedosporium apiospermum. The MAbs do not react with S. prolificans, Scedosporium dehoogii, or a large number of clinically relevant fungi, including A. fumigatus, Candida albicans, Cryptococcus neoformans, Fusarium solani, and Rhizopus oryzae. The MAbs were used in immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to accurately differentiate P. boydii from other infectious fungi and to track the pathogen in environmental samples. Specificity of the DAS-ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of environmental isolates.Pseudallescheria boydii is an infectious fungal pathogen of humans (7, 16, 40, 58, 59). It is the etiologic agent of white grain mycetoma in immunocompetent humans (7) and has emerged over recent years as the cause of fatal disseminated infections in individuals with neutropenia, AIDS, diabetes, renal failure, bone marrow or solid organ transplants, systemic lupus erythematous, and Crohn''s disease; in those undergoing corticosteroid treatment; and in leukemia and lymphoma patients (1, 2, 3, 18, 27, 31, 32, 34, 36, 37, 38, 47, 49, 52). The fungus is the most prevalent species after Aspergillus fumigatus in the lungs of cystic fibrosis patients (8), where it causes allergic bronchopulmonary disease (5) and chronic lung lesions simulating aspergillosis (24). Near-drowning incidents and recent natural disasters, such as the Indonesian tsunami in 2004, have shown P. boydii and the related species Scedosporium apiospermum and Scedosporium aurantiacum to be the causes of fatal central nervous system infections and pneumonia in immunocompetent victims who have aspirated polluted water (4, 11, 12, 21, 22, 25, 30, 33, 57). Its significance as a potential pathogen of disaster evacuees has led to its recent inclusion in the Centers for Disease Control and Prevention list of infectious etiologies in persons with altered mental statuses, central nervous system syndromes, or respiratory illness.P. boydii is thought to be an underdiagnosed fungus (60), and misidentification is one of the reasons that the mortality rate due to invasive pseudallescheriasis is high. Detection of invasive P. boydii infections, based on cytopathology and histopathology, is problematic since it can occur in tissue and bronchoalveolar and bronchial washing specimens with other hyaline septated fungi, such as Aspergillus and Fusarium spp. (7, 23, 53, 60), which exhibit similar morphological characteristics upon microscopic examination (2, 23, 24, 28, 37, 44, 53, 60). Early diagnosis of infection by P. boydii and differentiation from other agents of hyalohyphomycosis is imperative, since it is refractory to antifungal compounds, such as amphotericin B, that are commonly administered for the control of fungal infections (10, 39, 58).The immunological diagnosis of Pseudallescheria infections has focused on the detection of antigens by counterimmunoelectrophoresis, and by immunohistological techniques using polyclonal fluorescent antibodies, but cross-reactions with antigens from other fungi, such as Aspergillus species, occurs (7, 19, 23). Pinto and coworkers (41, 42) isolated a peptidorhamnomannan from hyphae of P. boydii and proposed the antigen as a diagnostic marker for the pathogen. Cross-reactivity with Sporothrix schenckii and with Aspergillus have, however, been noted (23, 41). Furthermore, it is uncertain whether a similar antigen is present in the related pathogenic species S. prolificans, an important consideration in patient groups susceptible to mixed Scedosporium infections (6, 18).Hybridoma technology allows the production of highly specific MAbs that are able to differentiate between closely related species of fungi (54, 55, 56). The purpose of this paper is to report the development of MAbs specific to P. boydii and certain closely related species and their use to accurately discriminate among P. boydii, A. fumigatus, and other human pathogenic fungi by using immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs).Currently, the natural environmental habitat of P. boydii is unknown, but nutrient-rich, brackish waters, such as estuaries, have been suggested (9, 17). In combination with a semiselective isolation procedure, I show how the DAS-ELISA can be used to rapidly and accurately track the pathogen in naturally infested estuarine muds, and in doing so illustrate the potential of the DAS-ELISA as a diagnostic platform for detection of P. boydii and related species within the Pseudallescheria complex.     

Gelatinase Contributes to the Pathogenesis of Endocarditis Caused by Enterococcus faecalis

The Gram-positive pathogen Enterococcus faecalis is a leading agent of nosocomial infections, including urinary tract infections, surgical site infections, and bacteremia. Among the infections caused by E. faecalis, endocarditis remains a serious clinical manifestation and unique in that it is commonly acquired in a community setting. Infective endocarditis is a complex disease, with many host and microbial components contributing to the formation of bacterial biofilm-like vegetations on the aortic valve and adjacent areas within the heart. In the current study, we compared the pathogenic potential of the vancomycin-resistant E. faecalis V583 and three isogenic protease mutants (ΔgelE, ΔsprE, and ΔgelE ΔsprE mutants) in a rabbit model of enterococcal endocarditis. The bacterial burdens displayed by GelE⁻ mutants (ΔgelE and ΔgelE ΔsprE mutants) in the heart were significantly lower than those of V583 or the SprE⁻ mutant. Vegetations on the aortic valve infected with GelE⁻ mutants (ΔgelE and ΔgelE ΔsprE mutants) also showed a significant increase in deposition of fibrinous matrix layer and increased chemotaxis of inflammatory cells. In support of a role for proteolytic modulation of the immune response to E. faecalis, we also demonstrate that GelE can cleave the anaphylatoxin complement C5a and that this proteolysis leads to decreased neutrophil migration in vitro. In vivo, a decreased heterophil (neutrophil-like cell) migration was observed at tissue sites infected with GelE-producing strains but not at those infected with SprE-producing strains. Taken together, these observations suggest that of the two enterococcal proteases, gelatinase is the principal mediator of pathogenesis in endocarditis.Enterococci are leading causes of hospital-acquired infections, including bacteremia, surgical site infections, and urinary tract infections (31). However, one of the most serious clinical manifestations of enterococcal infection is endocarditis, with mortality rates ranging from 15 to 20% (23). Enterococci, most commonly E. faecalis, are the third leading cause of infective endocarditis (21). Enteroccoci cause subacute-chronic endocarditis and are the causative agents of up to 20% of native valve endocarditis and 15% of prosthetic valve endocarditis (21, 23). Unlike other enterococcal infections, endocarditis is most often community acquired, although recent studies indicate that there is a significant risk of acquiring enterococcal endocarditis in a clinical environment (7, 8).The pathological progression of infective endocarditis initially involves the development of vegetations on heart valves, followed by embolization and dissemination to other body sites (15). In experimental endocarditis in rabbits, mortality is often associated with embolization to secondary infectious sites, including blood vessels of the heart, brain, and kidneys (10). Occasionally the emboli occlude blood vessels in the secondary infection sites, leading to tissue damage. Previous studies indicated that the presence of extracellular proteases (GelE and SprE) significantly increased mortality in animal infection models, but the relative contribution of each protease in experimental endocarditis has not been examined to date (10, 35).Multiple bacterial species produce extracellular proteases that contribute to pathogenesis through manipulation of the host immune response (28). These proteases target several components of the host innate immune system, including complement, antimicrobial peptides (AMPs), cytokines, and cytokine receptors (28). Complement C3a is an anaphylatoxin involved in activation and recruitment of eosinophils but is limited in its ability to activate and recruit neutrophils (3, 4, 6, 19). Compared to C3a, the complement C5a is at least 100-fold more potent in activation and recruitment of neutrophils (6). Determination of the effects of E. faecalis proteases on C5a is of particular importance because of the relevance of neutrophil recruitment for bacterial clearance. In addition, thrombin activation that is commonly observed as a consequence of microbial infection on the heart valve results in direct C5 cleavage generating functional C5a in the absence of C3 (16).The E. faecalis proteases GelE and SprE are cotranscribed through regulation by the fsr regulatory system (29, 30). SprE has been shown to contribute to disease in animal models (5, 30, 34, 36), but mechanistically how it contributes is not known at this time. Gelatinase is a zinc-metalloprotease (18) that is related to aureolysin from Staphylococcus aureus and elastase from Pseudomonas aeruginosa (28). Gelatinase is known for its contribution to biofilm formation (12, 38) and is also thought to contribute to virulence through degradation of a broad range of host substrates, including collagen, fibrinogen, fibrin, endothelin-1, bradykinin, LL-37, and complement components C3 and C3a (18, 19, 26, 27, 33, 39). The broad substrate specificity of GelE probably contributes significantly to the complexity of endocarditis pathology, but specific mechanistic contributions to endocarditis have not been elucidated. We sought to elucidate the specific contributions of each protease to endocarditis as well as assess mechanisms that are likely associated with increased pathogenesis.     

Genotype-Specific Clearance of Genital Human Papillomavirus (HPV) Infections among Mothers in the Finnish Family HPV Study

The majority of cervical human papillomavirus (HPV) infections in young women are transient, but whether the clearance differs among different HPV genotypes and the different factors predicting genotype-specific clearance are partly unknown. In the Finnish Family HPV Study, 131 of 252 women (mean age, 25.5 years) cleared their infection during the prospective follow-up of 6 years (median, 62.4 months; range, 1.6 to 94.5 months). Cervical scrapings collected at each visit were tested for 24 low-risk and high-risk (HR) HPV types with multiplex HPV genotyping. Poison regression (panel data) was used to estimate predictors for the clearance of species 7 and 9 HPV genotypes. Of all HPV genotypes detected in these women, multiple-type and HPV type 16 (HPV16) infections showed clearance least frequently (46.1% and 50.5%, respectively). The actuarial and crude mean times to first clearance were variable among different genotypes. The actuarial clearance rate (events/person-time at risk) was highest for HPV16 and multiple-type infections, while HPV66 and -82 had the highest crude clearance rate. Independent predictors increasing type-specific clearance of species 7/9 HPV genotypes were older age (incidence rate ratio [IRR] = 1.1; 95% confidence interval [95% CI], 1.03 to 1.18; P = 0.002) and baseline oral HR HPV DNA-negative status (IRR = 2.94; 95% CI, 1.03 to 8.36; P = 0.042), while a higher number of sexual partners during the follow-up decreased the probability of clearance (IIR = 0.35; 95% CI, 0.15 to 0.83; P = 0.018). To conclude, HPV16 and multiple-type infections showed the lowest clearance among young mothers. Increasing age and negative oral HR HPV DNA status at baseline were associated with increased clearance, whereas a higher number of current sexual partners decreased the probability of species 7/9 HPV genotype clearance.Genital human papillomavirus (HPV) infections are transient in most cases (3, 7). Most studies on HPV clearance have addressed high-risk (HR) HPV types collectively and/or have compared clearance between HR and low-risk (LR) HPV types (3, 4, 12, 13, 17, 18, 21, 23, 24). Earlier data suggest that HR HPV infections usually clear more slowly than LR HPV infections (4, 25) and that the likelihood of an infection not clearing increases in parallel with its duration (7, 13).It was not until recently that data on HPV clearance at the genotype level were available (5, 9, 19, 25). The results indicate that infection with HPV type 16 (HPV16) has the lowest tendency for clearance. Accurate data on actual and crude clearance times and clearance rates (CRs) for individual genotypes are needed to understand the natural history of HPV infections.The present study is one of the first to assess the frequency of HPV type-specific clearance as well as the actuarial and crude clearance times and clearance rates for the 24 most common LR and HR HPV genotypes. The study was performed among newly delivered mothers who were followed up for 6 years in the Finnish Family HPV Study. In addition, predictors of species 7/9 HPV genotype clearance were analyzed in a panel Poisson regression model.     

Screening of Urine Samples by Flow Cytometry Reduces the Need for Culture

Urine samples constitute a large proportion of samples tested in clinical microbiology laboratories. Culturing of the samples is fairly time- and labor-consuming, and most of the samples will yield no growth or insignificant growth. We analyzed the feasibility of the flow cytometry-based UF-500i instrument (Sysmex, Japan) to screen out urine samples with no growth or insignificant growth and reduce the number of samples to be cultured. A total of 1,094 urine specimens sent to our laboratory for culture during 4 months in the spring of 2009 in Lahti, Finland, were included in the study. After culture, all samples were analyzed with the Sysmex UF-500i for bacterial and leukocyte (white blood cell [WBC]) counts. Youden index and closest (0,1) methods were used to determine the cutoff values for bacterial and WBC counts in culture-positive and -negative groups. By flow cytometry, samples considered positive for UTI in culture had bacterial and WBC values that were significantly higher than those for samples considered negative. The flow cytometric screening worked best when both bacterial counts and WBC counts were used with age- and gender-specific cutoff values for all patient groups, excluding patients with urological disease or anomaly. By use of these cutoff values, 5/167 (3.0%) of culture-positive samples were missed by UF-500i and the percentage of samples that did not need to be cultured was 64.5%. Use of the UF-500i instrument is a reliable method for screening out a major part of the UTI-negative samples, significantly diminishing the amount of work required in the microbiology laboratory.Urinary tract infections (UTIs) are among the most common infections treated by community health care centers and hospitals (5, 6, 13, 19, 24). In Finland, urinary tract infections account for approximately 6% of all infectious disease diagnoses in primary care (20) and urine samples constitute a large proportion of the samples tested in clinical microbiology laboratories (13, 18, 24). The gold standard for UTI diagnosis is bacterial culture, which is based on bacterial counts and identification. Culturing of the samples is fairly time- and labor-consuming, and most of the samples yield no growth or insignificant growth (10, 15, 22, 24). In order to improve the efficiency of handling of the urine samples, methods for screening out the culture-negative samples from the culture-positive samples have been developed. Chemical screening with strips for nitrite, pH, leukocytes, erythrocytes, albumin, and glucose is widely used (17, 18, 22, 23), but a meta-analysis of the literature (4) has shown that the method is insensitive and is suitable as a rule-out test only when both nitrite and leukocyte-esterase are negative. Cells, particles, and microorganisms in urine can be examined by microscopic-urine-sediment analysis, but this method is time-consuming, labor-intensive, and sensitive to interobserver variability (2, 7, 8, 10, 12, 21).Pyuria with bacteria predicts bladder infection better than the presence of bacteria alone, and therefore, a screening method that detects both leukocytes and bacteria is favorable for the identification of patients with urinary tract infections (18). During the last 10 years, the use of flow cytometry-based analyzers that measure quantitatively both leukocytes and bacteria has been evaluated (2, 6, 8, 10-16, 21, 25). The studies done with the first-generation Sysmex instruments, UF-50 and UF-100, showed variable results concerning the suitability of this technology for screening purposes (3, 6, 15, 25). The second-generation Sysmex analyzers, UF-500i and UF-1000i, have a separate measurement channel for bacteria which improves the specificity for counting of bacterial organisms.The aim of this study was to evaluate the feasibility of flow cytometry using a UF-500i instrument (Sysmex Corporation, Japan) in routine diagnosis of UTI. We sought to develop a screening strategy in which as few samples as possible needed to be cultured, while maintaining a low level of false negatives and a high negative predictive value.     

Virulence and Cellular Interactions of Burkholderia multivorans in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens, but not with Escherichia coli. In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1β. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.Chronic granulomatous disease (CGD) is a rare primary immunodeficiency resulting from genetic defects in the phagocyte NAPDH oxidase. It is characterized by life-threatening infections caused by specific bacteria and fungi, leading to pneumonias, tissue abscesses, and exuberant granuloma formation (38). The Burkholderia cepacia complex (Bcc) includes at least 10 distinct species and is a leading cause of bacterial infections in CGD (44). Patients with cystic fibrosis (CF) also develop Bcc infections with various outcomes, ranging from no change in clinical course to a more rapid deterioration of lung function to the dreadful cepacia syndrome, which is characterized by necrotizing pneumonia and sepsis (25, 45). Interestingly, Bcc rarely causes infection in healthy individuals, but it can infect patients undergoing bronchoscopies and other procedures (4).Within the Bcc, Burkholderia cenocepacia and Burkholderia multivorans are commonly isolated from CF and non-CF patients (4, 32); the rate of B. multivorans infection now exceeds that of B. cenocepacia at several CF centers (15). In contrast to the high transmissibility of some CF B. cenocepacia strains (i.e., the epidemic lineage ET12) (24, 25), CF B. multivorans infections likely reflect independent acquisitions from unrelated sources (24). Curiously, unlike B. cenocepacia, B. multivorans has been recovered from environmental samples only rarely (1, 24), and it is the most frequently found species among CGD patients (16, 17).The mechanisms by which the Bcc causes disease specifically in CF are not known. Bcc isolates can survive within macrophages (28, 33) and respiratory epithelial cells (5, 21) and can invade epithelial cells in vivo (8, 10) and persist in the lung (9, 10). Cell infection assays using monocytes, macrophages, and epithelial cells (10, 11, 29, 46) show great variability among individual Bcc strains, with no clear correlation between those isolated from CF patients and those isolated from the environment (22). For the most part, these studies have been carried out using tissue culture models (28, 29, 43) and, in some cases, CF human or CF mouse cell systems (34, 35).Much less is known about the interaction between the Bcc and CGD despite the availability of animal models for the disease (20, 31). B. cenocepacia induced the necrosis of human CGD neutrophils but not normal controls (6). Similarly to healthy people, normal mice are resistant to the Bcc and usually show only transient infections upon inoculation (8, 37). On the other hand, CGD mice are highly susceptible to Bcc infection and show clinical signs that are similar to those of the human disease (20, 31, 37).To address why B. multivorans is a pathogen in CGD, we initiated studies with strains isolated from CGD patients and CGD cells. Strains of B. multivorans differed in cell association and cell entry. We found a preferential association of bacteria with CGD instead of normal leukocytes as shown by microscopy and culture techniques. This preferential association is shared by another CGD pathogen, Serratia marcescens, but not by Escherichia coli. Finally, we demonstrate dramatic differences in virulence in B. multivorans strains in a mouse model of CGD.     

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.