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.     

Diagnostic Value of PCR Analysis of Bacteria and Fungi from Blood in Empiric-Therapy-Resistant Febrile Neutropenia

This study aimed to assess the clinical utility of PCR for the analysis of bacteria and fungi from blood for the management of febrile neutropenic patients with hematologic malignancies. Using a PCR system able to detect a broad range of bacteria and fungi, we conducted a prospective pilot study of periodic analyses of blood from patients following intensive chemotherapy. When fever occurred, it was treated with empirical antibiotic therapy, basically without knowledge of the PCR results. In 23 febrile episodes during the neutropenic period, bacteria were detected by PCR in 11 cases, while the same species were identified by blood culture in 3 cases. In 10 out of 11 PCR-positive cases, fever could be managed by empirical therapy. In the empirical-therapy-resistant case, the identification of Stenotrophomonas maltophilia by PCR led to improvement of fever. No fungi were detected by PCR in febrile cases, while Aspergillus fumigatus was detected in one afebrile patient, several days before a clinical diagnosis was made. In subsequent sporadic PCR analyses in 15 cases of febrile neutropenia, bacteria were detected by both PCR and blood culture in 7 cases and by PCR alone in 6. Fungi were not detected. While fever was improved by empirical therapy in 12 out of the 13 PCR-positive cases, the identification of Pseudomonas aeruginosa by PCR in one therapy-resistant case contributed to the successful treatment of persistent fever. Our results indicate that PCR analysis of bacteria from blood provides essential information for managing empirical-therapy-resistant febrile neutropenia.Management of febrile neutropenia in hematological patients undergoing intensive chemotherapy is important, because bacterial or fungal infections during prolonged neutropenia are major causes of morbidity and mortality in these patients (3, 14, 17, 26, 28, 32). These infections can rapidly become life-threatening if not treated appropriately and promptly (5, 23, 32). Therapeutic decisions should ideally be made based on microbial isolation. However, the sensitivity of microbial culture tests remains low. Despite clinicians'' best efforts, it has been shown that specific pathogens were identified in only about 20 to 30% of febrile neutropenia cases (19, 20, 28, 33, 39). PCR-based molecular detection of fungal DNA from blood may be a promising tool for the early diagnosis of invasive fungal diseases; however, standardization and clinical validation are tasks that remain (1, 2, 9, 13, 22, 29). PCR analysis of bacteria from blood is reported to be more sensitive than blood culture (16, 19, 37-40). Nevertheless, prompt empirical therapy with broad-spectrum antimicrobial agents, sometimes modified by the results of blood cultures, has led to a dramatic reduction in the mortality rate in febrile neutropenia (14, 17, 23, 26, 28, 30, 32, 34). Thus, even though bacterial PCR analysis is a sensitive tool for diagnosing bacterial infections, it is more costly than blood culture, and its general utility during empirical antibiotic therapy in febrile neutropenia patients remains unclear.To address this issue, we initially performed a pilot study to prospectively detect bacterial or fungal infections in peripheral blood after chemotherapy, using a PCR analysis system able to detect a broad range of species of bacteria (24, 36) and fungi. In this pilot study, the PCR results were not disclosed to the physicians unless more than two physicians agreed that disclosure of the PCR results was required for treatment. Based on the results of this study, we performed bacterial PCR analysis in sporadic febrile neutropenic cases but disclosed the PCR results as soon as possible. We observed that, although bacteria were detected by PCR analysis more often than by blood cultures, most febrile neutropenia cases could be managed with empirical antibacterial therapy, without access to PCR data. However, PCR analysis was useful in the diagnosis and treatment of empirical-therapy-resistant febrile neutropenia.     

The O Antigen Is a Critical Antigen for the Development of a Protective Immune Response to Bordetella parapertussis

Despite excellent vaccine coverage in developed countries, whooping cough is a reemerging disease that can be caused by two closely related pathogens, Bordetella pertussis and B. parapertussis. The two are antigenically distinct, and current vaccines, containing only B. pertussis-derived antigens, confer efficient protection against B. pertussis but not against B. parapertussis. B. pertussis does not express the O antigen, while B. parapertussis retains it as a dominant surface antigen. Since the O antigen is a protective antigen for many pathogenic bacteria, we examined whether this factor is a potential protective antigen for B. parapertussis. In a mouse model of infection, immunization with wild-type B. parapertussis elicited a strong antibody response to the O antigen and conferred efficient protection against a subsequent B. parapertussis challenge. However, immunization with an isogenic mutant lacking the O antigen, B. parapertussis Δwbm, induced antibodies that recognized other antigens but did not efficiently mediate opsonophagocytosis of B. parapertussis. The passive transfer of sera raised against B. parapertussis, but not B. parapertussis Δwbm, reduced B. parapertussis loads in the lower respiratory tracts of mice. The addition of 10 μg of purified B. parapertussis lipopolysaccharide (LPS), which contains the O antigen, but not B. parapertussis Δwbm LPS drastically improved the efficacy of the acellular vaccine Adacel against B. parapertussis. These data suggest that the O antigen is a critical protective antigen of B. parapertussis and its inclusion can substantially improve whooping cough vaccine efficacy against this pathogen.Bordetella pertussis and B. parapertussis are the causative agents of whooping cough, resulting in approximately 50 million cases and 300,000 deaths annually worldwide (28). While whooping cough is considered by the CDC to be a reemerging disease (5), the relative incidences of B. pertussis and B. parapertussis are not clear (50). It is known, however, that the resurgence of whooping cough roughly correlates with the introduction of acellular pertussis vaccines (5). These vaccines contain only B. pertussis-derived antigens and confer little to no protection against B. parapertussis (9, 14, 15, 23, 27, 28). Current acellular pertussis vaccines contain some combination of filamentous hemagglutinin, pertactin, and fimbriae 2 and 3, all of which are expressed by both B. pertussis and B. parapertussis, and pertussis toxin, which is B. pertussis specific (33, 34). Based on genome sequences, the levels of amino acid sequence identity between B. pertussis and B. parapertussis filamentous hemagglutinin, pertactin, and fimbria 2 and 3 proteins are about 98, 91, 71, and 92% (35), and antibodies raised against these antigens from B. pertussis cross-react with B. parapertussis (17, 31). However, immunization with purified B. pertussis filamentous hemagglutinin or pertactin does not confer protection against B. parapertussis (17). B. pertussis fimbriae confer some protection against B. parapertussis, but at much lower levels than they protect against B. pertussis (52). Based on these observations and the fact that B. parapertussis infection induces protective immunity to itself (56, 58), we hypothesized that the lack of protective antigens from B. parapertussis may be part of the reason why current whooping cough vaccines are ineffective against this bacterium.Although B. pertussis and B. parapertussis are very closely related (8, 35, 48), they differ in the structure of their lipopolysaccharides (LPS) (1, 2, 39, 40, 47). B. pertussis produces a lipooligosaccharide containing lipid A and a branched-chain core oligosaccharide with a complex trisaccharide modification but lacks the O antigen due to a natural deletion of the wbm locus responsible for its synthesis (39, 47). B. parapertussis LPS is similar to B. pertussis LPS but lacks the trisaccharide modification and includes an O antigen (39, 40). In addition to conferring serum resistance by inhibiting C3 deposition onto the surfaces of bacteria (11), the O antigen enables B. parapertussis to avoid B. pertussis-induced immunity by preventing antibody binding to cross-reactive antigens on the surfaces of B. parapertussis cells (56, 59). Since the O antigen is one dominant surface antigen recognized by B. parapertussis immune sera (56) and has been shown previously to be a protective antigen of various pathogenic bacteria (22, 36), we hypothesized that the O antigen is a protective antigen of B. parapertussis.To assess the role of the O antigen in the generation of an adaptive immune response to B. parapertussis, the immunity and protection generated by B. parapertussis infection or vaccination were compared to those generated by an isogenic mutant of B. parapertussis lacking the O antigen (Δwbm) (39). Animals immunized with B. parapertussis, but not B. parapertussis Δwbm, were protected against subsequent challenge with B. parapertussis. Mice immunized with B. parapertussis Δwbm were also deficient in the production of B. parapertussis-specific antibodies, and sera collected from these mice were less effective at reducing B. parapertussis colonization upon passive transfer than sera raised against B. parapertussis. The inclusion of LPS from B. parapertussis, but not from B. parapertussis Δwbm, rendered the acellular B. pertussis vaccine Adacel efficacious against B. parapertussis challenge. Together, these data indicate that the O antigen is an important protective antigen of B. parapertussis.     

Diagnosis of Bacteremia in Whole-Blood Samples by Use of a Commercial Universal 16S rRNA Gene-Based PCR and Sequence Analysis

In a prospective, multicenter study of 342 blood samples from 187 patients with systemic inflammatory response syndrome, sepsis, or neutropenic fever, a new commercial PCR test (SepsiTest; Molzym) was evaluated for rapid diagnosis of bacteremia. The test comprises a universal PCR from the 16S rRNA gene, with subsequent identification of bacteria from positive samples by sequence analysis of amplicons. Compared to blood culture (BC), the diagnostic sensitivity and specificity of the PCR were 87.0 and 85.8%, respectively. Considering the 34 BC-positive patients, 28 were also PCR positive in at least one of the samples, resulting in a patient-related sensitivity of 82.4%. The concordance of PCR and BC for both positive and negative samples was (47 + 247)/342, i.e., 86.0%. In total, 31 patients were PCR/sequencing positive and BC negative, in whom the PCR result was judged as possible or probable to true bacteremia in 25. In conclusion, the PCR approach facilitates the detection of bacteremia in blood samples within a few hours. Despite the indispensability of BC diagnostics, the rapid detection of bacteria by SepsiTest appears to be a valuable tool, allowing earlier pathogen-adapted antimicrobial therapy in critically ill patients.Bloodstream infection is a life-threatening condition with a high mortality rate, especially in intensive care and neutropenic patients (5, 19, 35, 38). Pathogenic bacteria are the most frequent causes of bloodstream infection, although fungi can also be isolated in a minority of patients (7, 17, 21, 32, 34). Currently, inoculation of blood cultures (BC) is the standard method for microbiological diagnosis of bloodstream infections. However, the limitations of BC include relatively low sensitivities and a long time-to-result for detection and identification of the pathogen, generally over 2 days, and even longer for fastidious organisms (13, 20, 27).In contrast, DNA-based procedures may offer faster and more reliable diagnoses (3, 30). PCR amplification of microbial genes, followed by detection of amplified products by gel electrophoresis or real-time PCR monitoring using fluorescent dyes or target-directed fluorescent probes, is a quick process allowing pathogen detection within a few hours (18). Identification of microorganisms can be performed by PCR algorithms, taxon-specific oligonucleotide microarrays, or sequencing amplicons (30).PCR amplification of conserved regions of the bacterial genome, in particular the 16S rRNA gene, combined with sequence analysis is a well-established technique for the identification of bacterial pathogens (18). The main advantages of targeting the 16S rRNA gene are the broad range of pathogens detectable and the independence of this method from the in vitro viability of strains (6). The high sensitivity of detection by PCR of bacterial DNA (15) suggests its use in the diagnosis of bacteremia (16). Initial disadvantages of PCR, notably the incidence of false-positive results from bacterial DNA contaminating PCR reagents (4, 39), have been counteracted by the development of purification methods (12, 28) and the availability of commercial products (22).We present here a prospective, multicenter study investigating blood samples from patients with suspected bloodstream infections by a new, commercial, universal 16S rRNA gene-based PCR assay (SepsiTest; Molzym, Bremen, Germany) combined with sequence analysis of the amplicon for the identification of pathogens. The results obtained were compared to standard BC diagnostics.     

Multiplexed Luminex xMAP Assay for Detection and Identification of Five Adenovirus Serotypes Associated with Epidemics of Respiratory Disease in Adults

Several serotypes of human adenovirus (HAdV) cause acute respiratory disease (ARD) among healthy adults, sometimes generating broad outbreaks with high attack rates and occasional fatalities. Timely serotype identification provides valuable epidemiological information and significantly contributes to prevention (vaccination) strategies. The prevalence of specific serotypes causing ARD varies geographically. HAdV-3, HAdV-4, HAdV-7, HAdV-14, and HAdV-21 are the serotypes most commonly found in adult populations in the Western Hemisphere. Unfortunately, conventional serotype identification is a tedious process which can take a week or longer. For this reason, new molecular methods for serotype identification are needed. Commercially available rapid antigen and PCR assays for the detection of HAdV are universal but do not distinguish between the different serotypes. We describe the development of a sensitive and specific multiplex assay capable of identifying serotypes 3, 4, 7, 14, and 21. Two sets of primers were used for nonspecific (universal) PCR amplification, and serotype-specific probes coupled to Luminex tags were used for target-specific extension (TSE). PCR and TSE primers were designed using known hexon gene sequences of HAdV. The TSE products of HAdV-3, HAdV-4, HAdV-7, HAdV-14, and HAdV-21 were correctly identified using the Luminex xMAP fluid microsphere-based array system. No cross-reactivity with other respiratory pathogens or other HAdV serotypes was observed. This multiplexed assay can be expanded to include more serotypes and will allow broad and rapid detection and identification of adenoviral serotypes in a high-throughput environment.Human adenoviruses (HAdVs) cause a wide range of diseases in humans, including upper and lower respiratory illness, urinary tract infections, conjunctivitis, and gastroenteritis. There are 51 different serotypes based on type-specific serum neutralization, and these are classified into six species (A, B, C, D, E, and F) on the basis of hemagglutination, oncogenic, and phylogenetic properties (13, 24, 28, 31).The most common serotypes known to cause respiratory illness in the adult population are 3, 4, 7, 11, 14, and 21 (21, 24, 25, 37). All of these can cause locally severe outbreaks with high attack rates. These types of outbreaks are rarely reported in civilian populations but are essentially continuous at military training camps, particularly with serotype 4 (HAdV-4) and, to a lesser extent, HAdV-7 (10). Some recent studies have suggested that specific serotypes cause more severe disease, especially in immunocompromised patients (8, 13, 14, 24, 28, 32). The military previously established universal vaccination of new recruits for HAdV-4 and -7, which reduced adenovirus-induced acute respiratory disease (ARD) by 95 to 99% (20). Production of these vaccines ceased in 1996, but similar replacement vaccines are currently in the final stages of licensure. In 2006 and thereafter, HAdV-14 and HAdV-21 produced extensive outbreaks of ARD in military recruits (20).It is vital to be able to identify the serotype and changes in the serotype over time in order to evaluate viral virulence, vaccine efficacy, and the potential impact of antiviral use. Identification of serotypes was traditionally done by virus isolation in cell culture, followed by neutralization tests, antibody studies, and/or antigen detection by immunofluorescence (3, 16, 19, 23). These techniques are time-consuming and labor-intensive (11, 34). Another technique that has been used is whole-genome restriction endonuclease analysis, which relies on large-scale viral culture to generate the full genomic substrate (2, 4, 5).More recently, PCR-based detection and discrimination methods have been developed (1, 21, 35, 36). These techniques are faster and can also detect coinfections when used in a multiplex assay (20, 21, 33, 35, 36), thus reducing cost, labor, and sample volume needed for analysis. Current PCR assays identify the six subgenera (A to F) or up to three serotypes per reaction mixture (1, 21, 33, 35, 36). Real-time PCR also exists for generic detection (but not discrimination) of all 51 serotypes (6, 7, 12, 21, 33), and sequence analysis of the genomic region coding for the seven hypervariable loops of the hexon (the primary antigenic determinant) can identify and discriminate all 51 serotypes with a single assay (27), albeit a relatively time-consuming and complex one.Luminex has designed an xMAP system that in theory can detect up to 100 pathogens in a single sample by coupling bioassays with digital signal processing in real time. The platform is a suspension array where specific capture moieties are covalently coupled to the surfaces of internally dyed microspheres (22). The diversity of these microspheres increases the number of targets that can be identified in a single sample approximately 20-fold over traditional real-time PCR.In this study we designed and tested a Luminex-based assay capable of detecting and identifying HAdV-3, HAdV-4, HAdV-7, HAdV-14, and HAdV-21 in a single reaction. The assay was tested, and the results indicate its potential as a diagnostic tool.     

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.     

Validation and Clinical Application of a Molecular Method for Identification of Histoplasma capsulatum in Human Specimens in Colombia,South America

The conventional means of diagnosis of histoplasmosis presents difficulties because of the delay to the time that the diagnosis is made, indicating the need for the implementation of molecular assays. We evaluated 146 clinical samples from 135 patients suspected of having histoplasmosis using a previously reported nested PCR assay for the Histoplasma capsulatum-specific 100-kDa protein (the Hc100 PCR). In order to determine the specificity of this molecular test, we also used samples from healthy individuals (n = 20), patients suspected of having respiratory disease with negative fungal cultures (n = 29), and patients with other proven infections (n = 60). Additionally, a sizable collection of DNA from cultures of H. capsulatum and other medically relevant pathogens was studied. A panfungal PCR assay that amplified the internal transcribed spacer 2 region was also used to identify all fungal DNAs. All PCR-amplified products were sequenced. Of the 146 clinical samples, 67 (45.9%) were positive by culture and PCR, while 9 samples negative by culture were positive by PCR. All the sequences corresponding to the 76 amplified products presented ≥98% identity with H. capsulatum. The Hc100 PCR exhibited a sensitivity of 100% and specificities of 92.4% and 95.2% when the results were compared to those for the negative controls and samples from other proven clinical entities, respectively; the positive predictive value was 83% and the negative predictive value was 100%; the positive and negative likelihood rates were 25 and 0, respectively. These results suggest that the Hc100 nested PCR assay for the detection of H. capsulatum DNA is a useful test in areas where mycosis caused by this organism is endemic.Histoplasmosis is the most important mycosis endemic in the Americas and occurs by inhalation of the infectious propagules (microconidia) produced by the dimorphic fungus Histoplasma capsulatum (19, 32). It is amply distributed in most countries, being more prevalent in specific regions of United States, such as the Mississippi and Ohio River Valleys (14, 19). A high prevalence of histoplasmosis has also been observed in Central America (Mexico, Panama, Honduras, Guatemala, and Nicaragua), the Caribbean (Jamaica, Puerto Rico, Cuba, and Martinique), and South America (Venezuela, French Guyana, Colombia, Peru, Brazil, and Argentina) (16, 25).The severity of histoplasmosis varies greatly depending on the intensity of the exposure to the fungus and on the immune status of the infected individual (18, 29). In patients with immunodeficiency disorders, and especially in those infected with HIV, histoplasmosis is considered an opportunistic infection (17, 20, 27); in addition, in a high proportion of cases, this fungal infection is manifested as a severe disseminated process which often leads to death if it is not treated promptly (17, 20, 27).The diagnosis of histoplasmosis is usually accomplished by culture and microscopic examination of respiratory tract, biopsy, and body fluid specimens; nevertheless, these techniques yield positive results in only approximately 50% of the cases (9, 16, 18, 32). In addition, culturing of the fungus usually takes from 2 to 6 weeks, thus delaying the times to diagnosis and the initiation of therapy. Immunological tests that detect antibodies and/or antigens are also of value and may give results faster than culture. However, they show variable values of sensitivity and specificity and may often be negative for immunodeficient patients (18). The detection of antigen in serum and urine samples appears to be a sensitive and specific diagnostic tool, especially in HIV-infected patients (81 to 95% sensitivity with urine) (8, 12, 13, 26), although antigen detection shows cross-reactivity with the causative agents of other mycoses (12, 13, 16, 18, 30, 31).In the last decade, several molecular approaches have been developed for the detection of H. capsulatum DNA in human clinical samples. Various studies have obtained high sensitivity and specificity values for PCR-based molecular tests, including a PCR (the Hc100 PCR) that detects a gene that codes for an H. capsulatum 100-kDa protein (Hc100), which is essential for the survival of H. capsulatum in human cells (3); a PCR that detects 18S rRNA (2); a PCR that detects the internal transcribed spacer (ITS) region of the rRNA gene complex (21); and a PCR that detects the M and H antigens (4, 15). Some of these PCR assays have been tested with paraffin-embedded biopsy samples (3), blood specimens (22), infected mouse tissues (2), and samples from in vitro cultures; however, the DNA-based diagnosis of this fungal infection has not yet been established as a regular diagnostic tool, nor is a PCR assay commercially available (19).In the present study, we evaluated over a 2-year period a cohort of patients with suspected or clinically diagnosed histoplasmosis, using a nested PCR targeting the gene coding for the 100-kDa protein previously described by Bialek et al. (3) and using fungal isolation in culture as the “gold standard” technique.(The results presented here are part of Cesar Muñoz′s master''s thesis for the Corporation of Biomedical Basic Sciences Master''s Program, Universidad de Antioquia, Medellín, Colombia.)     

Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination

The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 10⁵ to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r² = 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient.Since the discovery of human T-lymphotropic virus type 1 (HTLV-1) in 1980 (16, 40), three other genotypes and 10 subtypes have been recognized. The precise geographical distribution and the clinical consequences of these infections are still a matter of debate. This can be attributed at least in part to the fact that there are insufficient accurate tools for HTLV diagnosis, genotyping, and measurement of viral burden.HTLV-1 is endemic in several geographical areas, including sub-Saharan Africa, South America, the Caribbean Islands, Japan, and Melanesia. It has been estimated that worldwide 10 to 25 million people are infected with this retrovirus (41, 53). Most HTLV-1-infected individuals remain asymptomatic throughout their lifetimes. However, 5 to 10% of infected people develop clinical complications, among which adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are the most severe. Other manifestations of HTLV-1 infection include infective dermatitis (25), uveitis (34), arthritis (38), and Strongyloides stercoralis infection (53). Some of these manifestations could accelerate disease development and/or progression (12, 16). For HTLV-1, a distinction is made between seven subtypes: the worldwide, cosmopolitan subtype HTLV-1a; the Central African subtypes HTLV-1b, -d, -e, -f, and -g; and the Australo-Melanesic subtype HTLV-1c (8, 23, 41, 52).HTLV-2 was discovered in 1982. This retrovirus is endemic in Amerindian and pygmy populations and epidemic in intravenous drug users (16, 49). In contrast to the case for HTLV-1, convincing epidemiological demonstrations of a definitive etiological role of HTLV-2 in human disease are limited. Nevertheless, HTLV-2 has been linked with the development of neurological disorders similar to HAM/TSP, with arthritis, with pulmonary disorders, and with increased mortality (2, 16, 42). HTLV-2 is divided into three subtypes, namely, HTLV-2a and HTLV-2b, mostly found on the American continent, and HTLV-2d, mostly found in Africa (10, 41, 44, 52).In 2005, two more genotypes, HTLV-3 and -4, were discovered in asymptomatic individuals from Cameroon (6, 7, 47, 56). To date, no diseases have been reported in association with HTLV-3 or -4. Further research is needed to determine the distribution and prevalence as well as the pathogenicity of these two new genotypes.The routine diagnosis of HTLV infections is based on conventional serological techniques such as enzyme-linked immunosorbent assay and Western blotting. However, among samples infected with HTLV-1 or HTLV-2, the proportion of seroindeterminate results is high (20, 21, 28, 57). Moreover, in the cases of HTLV-3 and HTLV-4, an indeterminate Western blot pattern appears to be the rule rather than the exception (6, 29). To confirm and/or support serological assays, diagnostic HTLV PCR techniques were created (51, 54). In the next phase, real-time or quantitative PCR (qPCR) assays were developed that confirm the diagnosis and at the same time quantify the HTLV proviral load (PVL). The majority of the published HTLV qPCR assays are singleplex assays, which detect one HTLV genotype per amplification reaction and hereby were developed for the most prevalent variant of HTLV-1, the cosmopolitan HTLV-1a, or for HTLV-2 infection (11, 22, 26, 32, 55). Multiplex qPCR allows the simultaneous detection and amplification of two or more target DNA sequences in only one amplification reaction. To our knowledge, one specific and one generic biplex qPCR for HTLV-1 and -2 (13, 26) and, just recently, one triplex qPCR for HTLV-1, -2, and -3 have been described (3).To address the current problems with HTLV diagnosis and quantitation, taking into account the diversity in HTLV genotypes and subtypes, we developed a novel triplex qPCR assay for simultaneous detection, genotyping, and quantification of PVL of HTLV-1, -2, and -3 infections. In the future, HTLV-4 can be incorporated into our qPCR technique, provided that viral cell culture is possible. Furthermore, considering the increasing number of HTLV qPCR techniques available at present, together with the lack of validation, we performed the first comparative analysis between two qPCR assays developed at different institutions.     

Development and Application of Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation of Mycobacterium Species from Isolates and Sputum Specimens

We developed a multiprobe real-time PCR assay targeting hsp65 (HMPRT-PCR) to detect and identify mycobacterial isolates and isolates directly from sputum specimens. Primers and probes for HMPRT-PCR were designed on the basis of the hsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, including Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, M. abscessus, M. massiliense, and M. fortuitum. This technique was applied to 24 reference and 133 clinical isolates and differentiated between all strains with 100% sensitivity and specificity. Furthermore, this method was applied to sputum specimens from 117 consecutive smear-positive patients with smear results of from a trace to 3+. These results were then compared to those obtained using the rpoB PCR-restriction analysis method with samples from cultures of the same sputum specimens. The HMPRT-PCR method correctly identified the mycobacteria in 89 samples (76.0%, 89/117), and moreover, the sensitivity level was increased to 94.3% (50/53) for sputa with an acid-fast bacillus score equal to or greater than 2+. Our data suggest that this novel HMPRT-PCR method could be a promising approach for detecting pathogenic mycobacterial species from sputum samples and culture isolates routinely in a clinical setting.Of the known species in the genus Mycobacterium, Mycobacterium tuberculosis is the most common and most important pathogen, causing 2 million deaths and over 8 million cases of tuberculosis worldwide annually (2, 3, 4, 7). In addition to M. tuberculosis, infections with nontuberculosis mycobacteria (NTM) can also cause clinical problems. Because of the different pathogenic potentials and susceptibilities of different mycobacterial species, the treatments of mycobacterial infections are different (13, 30, 33, 34). Thus, it is very important to differentiate between mycobacteria at the species level during early-stage diagnostics.Instead of a culture-based identification scheme, which may take 4 to 6 weeks or longer to identify slowly growing mycobacteria, PCR-based protocols (sequencing or PCR-restriction analysis [PRA]) targeting chronometer molecules, such as 16S rRNA (5, 6, 28), hsp65 (17, 19, 25), and rpoB (1, 16, 21), have been widely used to identify mycobacteria. However, in spite of the successful application of these conventional PCR-based methods to culture isolates, there are some drawbacks in their direct application to clinical specimens. This is especially true for sputum samples, which also contain numbers of commensal bacteria from the respiratory tract, producing confusing results by the simultaneous amplification of both commensals and mycobacterial strains. We have recently developed several methods for mycobacterial species identification based on amplification of hsp65 gene sequences directly from sputum samples (15, 27). Limitations due to the intrinsic features of conventional PCR prevented feasible identification of mycobacterial species from sputum samples using this method.The use of the real-time PCR assay in the diagnosis of many infectious diseases has been increasing, as it represents an appealing alternative to conventional PCR. It is an improvement over conventional methods because of its increased sensitivity and specificity, low contamination risk, and ease of performance and speed (8). In particular, fluorescence resonance energy transfer (FRET)-based real-time PCR permits not only the simultaneous identification of multiple target species but also the direct identification of target species from primary specimens such as sputum specimens through melting curve analysis of the amplification product (8). These characteristics of FRET-based real-time PCR provide a useful advantage for the identification of mycobacteria from sputum samples. Recently, several real-time PCR-based methods for mycobacterial detection and identification have been developed and evaluated (9, 22, 23, 26, 29). However, direct application of the real-time PCR-based method to primary specimens was generally limited to M. tuberculosis alone (11, 26). So far, a method which can simultaneously identify several pathogenic NTM as well as M. tuberculosis from primary sputum samples in a single reaction has not been developed.In the present study, we sought to develop a multiprobe real-time PCR targeting the hsp65 gene (HMPRT-PCR) based on melting curve analysis (HybProbes). This enabled the simultaneous identification of several pathogenic mycobacteria, including M. tuberculosis, in a single PCR performed on cultures and sputum samples. The usefulness of these methods was evaluated by blindly applying them to cultured and sputum samples.     

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.     

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.     

Rapid Diagnosis of Azole-Resistant Aspergillosis by Direct PCR Using Tissue Specimens

We report the use of PCR techniques on a formalin-fixed and paraffin-embedded tissue specimen for direct detection of one dominant azole resistance mechanism in a case of disseminated invasive aspergillosis. Rapid detection of mutations associated with azole resistance directly in tissue significantly reduces diagnostic delay.Invasive infections due to Aspergillus fumigatus are associated with significant morbidity and mortality, although the prognosis of patients with invasive aspergillosis has improved with the clinical use of mold-active antifungal azoles, most notably voriconazole (9, 11). However, the survival of patients may be threatened by the emergence of azole resistance of aspergilli (1, 7, 13). Resistance is commonly due to point mutations in the cyp51A gene, which is the target for antifungal azoles (1, 4, 8, 13, 14). The isolates commonly exhibit a cross-resistant phenotype (4), and patients with azole-resistant disease may fail azole therapy (1, 7, 10, 12). One problem in the management of azole-resistant aspergillosis is the early detection of resistance as cultures are negative in up to 50% of patients with focal pulmonary lesions (2), and in vitro susceptibility testing takes at least 5 to 7 days to complete. In this report, molecular tools were utilized to rapidly confirm the diagnosis of disseminated azole-resistant aspergillosis.     

Evaluation of Multiplex Type-Specific Real-Time PCR Assays Using the LightCycler and Joint Biological Agent Identification and Diagnostic System Platforms for Detection and Quantitation of Adult Human Respiratory Adenoviruses

Every year, thousands of basic military trainees in each service of the U.S. Armed Forces experience acute respiratory disease. The majority of this disease burden results from infection with human adenoviruses. We designed single- and multiplex assays that detect and discriminate adenovirus types B3, E4, B7, B11, B14, and B21. A total of 116 oropharyngeal swab specimens obtained from patients at the Naval Health Research Center were used to validate the new assays. Type-specific singleplex assays were designed and used independently to successfully identify 94 representative patient specimens. The lower limits of detection for our singleplex real-time PCR assays were calculated to be 50, 500, 500, 50, 50, and 50 genomic copies per reaction for human adenovirus type B3 (HAdV-B3), HAdV-E4, HAdV-B7, HAdV-B11, HAdV-B14, and HAdV-B21, respectively. These were then multiplexed to increase efficiency and tested against singleplex assays using titrated controls. The HAdV-B3/B11 and HAdV-E4/B7 multiplex assays were as sensitive and specific as they were individually. The HAdV-B14/B21 multiplex assay was not as efficient at detecting HAdV-B14 as the singleplex assay. Interestingly, a statistically significant difference was found between the viral loads of HAdV-B14 and those of HAdV-B3, -E4, -B7, and -B21 (P < 0.001). The assays did not cross-react with other adenoviruses, influenza virus, respiratory syncytial virus, or respiratory disease-causing bacteria. These assays have the potential to be useful as clinical diagnostic tools for the detection of HAdV infection in adult populations.Human adenoviruses (HAdVs) were first associated with clinical illness among military trainees with respiratory disease in the early 1950s (6, 17). HAdVs were the first respiratory viruses to be isolated and characterized. Epidemiological studies showed that adenoviruses are a primary cause of acute respiratory disease (ARD) among military recruits (3, 5) and are a common cause of epidemic respiratory illness in crowded adult civilian populations (18, 21). The diverse human pathogens in the genus Mastadenovirus are categorized into 54 types of adenoviruses. The 54 known types have been grouped into seven species (A to G), based on their immunochemical responses, nucleic acid characteristics, hexon and fiber protein characteristics, biological properties, and phylogenetic analysis (7, 24). They are associated with a broad range of symptoms, including those associated with ARD, conjunctivitis, genitourinary infections, and gastroenteritis, and specific types of adenovirus are associated with specific types of disease (18, 21).Adenoviruses of species B, C, and E are associated with ARD. Species C types, including HAdV type C1 (HAdV-C1), -C2, -C5, and -C6, are common causes of endemic respiratory illness in pediatric populations, and the majority of adults have acquired immunity to these types (8, 16). These may generate asymptomatic carrier-state infections that last into young adulthood, resulting in viral shedding detectable by PCR. Diverse species B types, including HAdV-B3, -B7, -B11, -B14, and -B21, and the sole species E type, HAdV-E4, cause epidemic outbreaks of ARD and conjunctivitis among adults and children (14, 22). These are also associated with essentially continuous outbreaks among unvaccinated military recruits (14, 23). Symptoms range from those associated with mild ARD to severe pneumonia, occasionally resulting in death, even among these otherwise healthy young adults (1, 4, 11, 19). Among U.S. military trainees alone, HAdVs are estimated to cause 22,000 cases of ARD that is severe enough to require medical attention, delaying training schedules, decreasing the quality of life of trainees, and costing the U.S. government $40 million annually (19).The impact and distribution of different types varies over time and can be drastically altered through use of type-specific vaccines (14, 22, 23). An effective vaccine for HAdV-B7 and HAdV-E4 was used in the United States between 1971 and 1999, when production ceased and it became unavailable. Currently, a replacement vaccine is in clinical trials and may soon be available (19, 22), but this vaccine will be limited to use for types 4 and 7. The recent emergence in the United States of a new type, HAdV-B14a (which had not previously been seen in the Western Hemisphere), caused severe outbreaks among both military trainees and civilians and was the subject of several specific outbreak investigations (2, 12, 22). Similarly, in other regions and other circumstances, the severity, attack rates, specific symptoms, and relative risk of fatality may vary greatly between types. It is clearly of significant public health interest to track adult respiratory adenoviruses in a type-specific manner.Historically, adenoviruses were detected by tissue culture methods and discriminated by type-specific serum neutralization methods (13). However, traditional (probeless) PCR assays have since replaced these methods, owing to their greater speed, their significantly lower cost, and the decline in availability of type-specific antisera (15). A variety of reliable PCR assays have been developed and used, including species-specific (26) and type-specific (25) tests. Universal PCR assays paired with sequence analysis have been used to provide a truly comprehensive detection and discrimination method for all HAdV types (20). Real-time (probe-based) PCR platforms now offer even greater efficiency, improved sensitivity and specificity, and the added information value resulting from quantitative analysis of viral titers (9).In this study, we developed a series of single- and multiplexed real-time PCR assays for both the LightCycler and the military Joint Biological Agent Identification and Diagnostic System (JBAIDS) platforms which can detect and discriminate all HAdVs implicated in adult epidemic ARD in the United States, including HAdV-B3, -E4, -B7, -B11, -B14, and -B21. Combined, these tools offer a rapid, high-throughput method for universal detection, discrimination, and quantitation of HAdVs in uncultured throat swab specimens. These assays will allow for much more rapid outbreak assessment and, if validated as in-house diagnostic assays, more rapid individual and public health responses.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Physical and Chemical Characterization and Immunologic Properties of Salmonella enterica Serovar Typhi Capsular Polysaccharide-Diphtheria Toxoid Conjugates

Typhoid fever remains a serious public health problem in developing countries, especially among young children. Recent studies showed more than 50% of typhoid cases are in children under 5 years old. Licensed vaccines, such as Salmonella enterica serovar Typhi capsular Vi, did not confer protection against typhoid fever for this age group. Vi conjugate, prepared by binding Vi to Pseudomonas aeruginosa recombinant exoprotein A (rEPA), induces protective levels of antibody at as young as 2 years old. Because of the lack of regulatory precedent for rEPA in licensing vaccines, we employed diphtheria toxoid (DT) as the carrier protein to accommodate accessibility in developing countries. Five lots of Vi-DT conjugates were prepared using adipic acid dihydrazide (ADH) as the linker. All 5 lots showed consistency in their physical and chemical characteristics and final yields. These Vi-DT conjugates elicited levels of IgG anti-Vi in young mice significantly higher than those in mice injected with Vi alone and induced a booster response upon reinjection. This booster effect was absent if the Vi replaced one of the two conjugate injections. Vi-DT was stable under repeated freeze-thaw (20 cycles). We plan to perform clinical evaluation of the safety and immunogenicity of Vi-DT when added to the infant combination vaccines.Typhoid fever, a serious systemic infection caused by Salmonella enterica serovar Typhi, remains a major public health problem in Central Asia, Southeast Asia, Africa, and Latin America (11, 52, 53). It was estimated that more than 21 million cases of typhoid fever and >200,000 deaths occurred in 2000 (10). The treatment of patients and management of asymptomatic carriers are becoming more difficult due to the worldwide emergence of multidrug-resistant (MDR) strains (2, 15, 29, 42, 43). Vaccination is considered the most promising strategy for the control of typhoid fever in developing countries (11, 19, 52, 53).Typhoid fever in children younger than 5 years old has often been unrecognized due to atypical clinical symptoms, difficulties in the number and volume of blood drawings, and use of less than optimal culture media (35, 46). Several studies have shown that the incidence of typhoid fever among children less than 5 years old is similar to that in school age children and young adults (14, 27, 34, 50, 51).The 3 licensed typhoid vaccines have limited efficacy, and none are suitable for young children under 5 years old. The use of heat-inactivated whole-cell vaccine was suspended in many countries because of its reactogenicity. The parenteral Vi polysaccharide and the live attenuated oral Ty21a vaccine were introduced in the late 1980s; both vaccines are well accepted and confer moderate protection (50 to 70%) in older children and adults. However, neither vaccine is licensed for routine immunization of infants (52).The Vi capsular polysaccharide is both an essential virulence factor and a protective antigen for S. Typhi (36, 38, 39). The concentration of serum IgG anti-Vi is correlated with immunity to the pathogen (22, 25, 26, 28, 36, 38, 49). However, Vi is not suitable for routine immunization of infants and young children because of its age-related immunogenicity and T-cell independence. As was shown for other capsular polysaccharides, such as Haemophilus influenzae type b (8, 37); meningococcus groups A, C, and W135; and Streptococcus pneumoniae (12, 20), Vi covalently bound with protein conferred T-cell dependence and increased immunogenicity (48-50). To date, diphtheria toxoid (DT), tetanus toxoid (TT), cholera toxins (CT), the B subunit of the heat-labile toxin (LT-B) of Escherichia coli, recombinant outer membrane protein of Klebsiella pneumoniae (rP40), and iron-regulated outer-membrane proteins (IROMPs) of S. Typhi have served as carriers for Vi polysaccharide in laboratory studies (16, 17, 32, 48-50; personal communications). An improved method was developed (24), utilizing adipic acid dihydrazide (ADH) as the linker and Pseudomonas aeruginosa recombinant exoprotein A (rEPA) as the carrier. Clinical trials of Vi-rEPA conjugates conferred 89% protection in Vietnamese children 2 to 5 years old for 46 months (23, 26, 28). The level of serum IgG anti-Vi induced by Vi-rEPA conjugates was correlated with prevention of typhoid fever in these studies (7, 21-23, 26, 28).One limitation of using rEPA as the carrier protein is the lack of regulatory precedent in licensing vaccines. In this report, five lots of Vi conjugates using DT manufactured by pharmaceutical companies in China and India were prepared (24, 48, 49). Modifications of conjugation procedures were made for the purposes of easy adoption and scale up by manufacturers. The stability of Vi-DT was studied for the feasibility of stockpiling in disaster relief.Another important aspect of conjugate vaccine implementation is the optimum immunization formulation and schedule using alternating injections of polysaccharide and conjugate. Priming or boosting effects of polysaccharide on its conjugate vaccine have been observed in infants injected with pneumococcal and meningococcal vaccines (3, 4, 31, 40). There was no consistent conclusion about various types of polysaccharides studied (6, 9, 31, 40, 41). Here, we compared the immune response of Vi polysaccharide injected before or after the administration of Vi-DT with the responses of those receiving 2 injections of Vi-DT. We also investigated the dosage effect for the purpose of better formulation.     

Protection of Sheep against Rift Valley Fever Virus and Sheep Poxvirus with a Recombinant Capripoxvirus Vaccine

Rift Valley fever (RVF) is an epizootic viral disease of sheep that can be transmitted from sheep to humans, particularly by contact with aborted fetuses. A capripoxvirus (CPV) recombinant virus (rKS1/RVFV) was developed, which expressed the Rift Valley fever virus (RVFV) Gn and Gc glycoproteins. These expressed glycoproteins had the correct size and reacted with monoclonal antibodies (MAb) to native glycoproteins. Mice vaccinated with rKS1/RVFV were protected against RVFV challenge. Sheep vaccinated with rKS1/RVFV twice developed neutralizing antibodies and were significantly protected against RVFV and sheep poxvirus challenge. These findings further document the value of CPV recombinants as ruminant vaccine vectors and support the inclusion of RVFV genes encoding glycoproteins in multivalent recombinant vaccines to be used where RVF occurs.Rift Valley fever (RFV) virus (RVFV) is a mosquito-borne member of the genus Phlebovirus, family Bunyaviridae. It is widely distributed in Africa, causing endemic and epidemic disease in both humans and livestock, including sheep, cattle, and goats. RVF was first described in Kenya and was shown to be caused by a filterable virus transmissible via blood (9). Acute RVF in lambs is characterized by fever and death within 24 to 48 h of being detected (43). Signs in adult sheep include fever, mucopurulent nasal discharge, hemorrhagic diarrhea, and abortion in pregnant ewes (43). RVFV can be transmitted from infected sheep to humans, particularly when humans are exposed to aborted sheep fetuses and blood.Attenuated live RVFV vaccines are available for use in livestock. A mutagen-attenuated RVFV vaccine induces protective immune responses in lambs and appears to be safe (25); however, other studies documented teratogenic effects on lambs from vaccinated pregnant ewes similar to those caused by the attenuated RVFV strain Smithburn (18). An inactivated RVFV vaccine induces neutralizing antibody responses in humans (33), and its use in sheep would not induce teratogenic effects or abortions. However, the inactivated vaccine requires 3 doses (33) and is expensive to produce. Efforts to make RVFV vaccines without these disadvantages include an attenuated RVFV developed by reverse genetics and lacking the NSs and NSm genes (4) and other new-generation RVFV vaccines (reviewed in reference 19) that protect mice against virus challenge (7, 16, 24, 27).The middle (M) RNA segment of the RVFV genome encodes the viral glycoproteins Gn and Gc (8, 20), and recombinant vaccinia virus expressing these glycoproteins induces neutralizing antibody and protective immunity to RVFV in mice (7). Vaccinia virus is safe for animals, but there is some risk to humans, as it was reported previously to spread from human vaccinees to contacts (28, 55) and to cause serious clinical disease in human immunodeficiency virus-infected patients (36). Although modified vaccinia virus Ankara is a safer alternative for humans (6, 57), there are animal poxviruses with naturally restricted host ranges for vaccine vectors in animals (1, 13, 30, 31, 40, 46, 47, 52, 53).For ruminants, the genus Capripoxvirus (CPV) of the family Poxviridae has been an effective recombinant vector to induce protective immunity against several other viruses (3, 17, 29, 32, 40, 41, 51). This genus has three closely related species causing sheep pox, goat pox, and lumpy skin disease (LSD) of cattle. A recombinant LSD vaccine expressing the Gn and Gc glycoproteins of RVFV induced protection against RVFV challenge in mice (52, 53) and sheep (52). The three species of CPV have 96 to 97% nucleotide identity (49) and are restricted to ruminants, with no evidence of human infections (10, 11). Furthermore, attenuated CPV vaccines are in use in Africa and the Middle East to control ruminant poxvirus disease (11, 21). The use of a CPV vector to deliver virus vaccines to ruminants also induces immunity to the CPV vector, thus increasing the valence of the vaccine (3, 17, 39, 40). We report here the construction of a recombinant CPV that expresses the RVFV Gn and Gc glycoproteins and induces protective immunity against RVFV and sheep poxvirus (SPV) challenge in sheep.