Evaluation of Sensitivity of Multiplex PCR for Detection of Mycobacterium tuberculosis and Pneumocystis jirovecii in Clinical Samples

A multiplex PCR assay for the simultaneous detection of Mycobacterium tuberculosis and Pneumocystis jirovecii was developed using IS6110-based detection for M. tuberculosis and mitochondrial large-subunit (mtLSU) rRNA gene detection for P. jirovecii. Ninety-five pulmonary blinded samples were examined using the developed multiplex PCR assay, and the results were compared with those obtained by the single nested PCRs targeting IS6110 for M. tuberculosis and mtLSU rRNA for P. jirovecii. Of the 95 pulmonary samples tested, the multiplex nested PCR developed here could detect 36 cases of M. tuberculosis infection, 35 cases of P. jirovecii infection, and 17 cases of M. tuberculosis and P. jirovecii coinfections. The sensitivities of the multiplex nested PCR in detecting M. tuberculosis and P. jirovecii were 92.1% and 81.4%, respectively, whereas the specificities in detecting M. tuberculosis and P. jirovecii were 98.2% and 100%, respectively.Pulmonary tuberculosis (TB) and Pneumocystis jirovecii pneumonia are two of the most common opportunistic infections found in association with AIDS worldwide (4), including Thailand (2, 3, 6, 10). About one-third of the world''s population and one-third of people infected with HIV are infected with Mycobacterium tuberculosis. The World Health Organization (WHO) reported that globally 9.2 million new cases of TB and 1.7 million deaths from TB occurred in 2006, and of these, 0.7 million cases and 0.2 million deaths, respectively, were in HIV-positive people (21). At present, Pneumocystis pneumonia, caused by Pneumocystis jirovecii (previously known as P. carinii f. sp. hominis), remains one of the most common AIDS-defining illnesses and is a frequent cause of morbidity and mortality in HIV-infected patients (7). Geographically, TB is the most common respiratory opportunistic infection in people infected with HIV worldwide, especially in the developing world (1, 4, 6, 8, 9, 12), whereas P. jirovecii pneumonia is more prevalent in industrialized countries (4, 5, 13, 16). In Thailand, TB has been the most common opportunistic infection in people with AIDS, whereas P. jirovecii pneumonia has been the second most common (12, 18). The total number of the two infections represents one-half of opportunistic infections in AIDS cases.TB and P. jirovecii pneumonia can clinically and radiologically mimic each other, including having similar presentations in patients, and they cannot always be diagnosed by clinical presentation or sputum examination. In addition, coinfection in individuals may also occur. Therefore, accurate and rapid diagnosis is required. The molecular means of diagnosis is considered to be a reliable technique, and it is essential that it be developed or improved to simultaneously diagnose TB and P. jirovecii pneumonia. Having a technique for differential diagnosis of the two infections would contribute to the ability to provide immediate treatment, controlling the diseases and decreasing the rates of transmission. The aim of the present study was to develop a multiplex PCR technique for the detection of M. tuberculosis and P. jirovecii simultaneously in clinical samples. In the present study, the development of a multiplex PCR involved selection of the appropriate genes, as well as the optimum PCR mixture and PCR thermal profile. The multiplex PCR was applied to test its sensitivity and specificity with clinical specimens.     

Mycobacterium tuberculosis Transmission Is Not Related to Household Genotype in a Setting of High Endemicity

Among the different strains of Mycobacterium tuberculosis, Beijing has been identified as an emerging genotype. Enhanced transmissibility provides a potential mechanism for genotype selection. This study evaluated whether the Beijing genotype is more readily transmitted than other prevalent genotypes to children in contact with an adult tuberculosis (TB) index case in the child''s household. We conducted a prospective, community-based study at two primary health care clinics in Cape Town, South Africa, from January 2003 through December 2004. Bacteriologically confirmed new adult pulmonary TB cases were genotyped by IS6110 DNA fingerprinting; household contacts less than 5 years were traced and screened for M. tuberculosis infection and/or disease. A total of 187 adult index cases were identified from 174 households with children aged less than 5 years. Of 261 child contacts aged 0 to 5 years, 219 (83.9%) were completely evaluated and the isolate from the index case was successfully genotyped. M. tuberculosis infection (induration of ≥10 mm by Mantoux tuberculin skin test) was documented in 118/219 (53.9%) children; 34 (15.5%) had radiographic signs suggestive of active TB. There was no significant difference in the ratio of infected children among those exposed to the Beijing genotype (51/89; 57.3%) and those exposed to non-Beijing genotypes (55/115; 47.8%) (odds ratio, 1.5; 95% confidence interval, 0.8 to 2.7). Genotyping was successful for six children diagnosed with active TB; the isolates from only two children had IS6110 fingerprints that were identical to the IS6110 fingerprint of the isolate from the presumed index case. We found no significant association between the M. tuberculosis genotype and transmissibility within the household. However, undocumented M. tuberculosis exposure may have been a major confounding factor in this setting with a high burden of TB.From an evolutionary perspective, the global tuberculosis (TB) epidemic presents a dynamic picture. Mycobacterium tuberculosis generates significant genetic diversity through deletion, duplication, and recombination events; but unlike most other bacterial pathogens, gene exchange is rare (31, 33). The absence of horizontal gene transfer results in strict clonality with distinct genetic lineages that permit accurate phylogenetic reconstruction. Selection of the most successful genotypes is mediated by genotype-specific differences in host-pathogen interactions (15, 18), some of which have been well characterized in animal models (11, 24, 25). Pathogen-related factors that may contribute to M. tuberculosis genotype selection include variability in transmissibility (the ability to spread from person to person), pathogenicity (the ability to cause clinical disease), the level of protection afforded by Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccination, and the acquisition of drug resistance.The Beijing genotype predominates in parts of East Asia (17, 23, 38, 41), northern Eurasia (12, 31), and southern Africa (8, 39). Beijing has been regarded as an emerging genotype on the basis of its global distribution, its association with young age (4), and its proportional increase in prevalence over time (8, 39). An increased ability to circumvent the protection afforded by BCG vaccination is suggested by the positive association (of the Beijing genotype) with the presence of a BCG scar in human populations (4) and has been observed in mice (24), although more recent findings challenge this observation (20). Multiple mechanisms have been explored to explain the potential link between the emergence of the Beijing genotype and low-level BCG protection (1), which may provide the Beijing genotype with a selective advantage in populations in which universal BCG vaccination is practiced.The association between the Beijing genotype and drug-resistant TB is well documented in multiple settings (2, 9, 31, 32, 37). Although it has been demonstrated that the acquisition of drug resistance is usually associated with a fitness cost, this finding seems variable and strain dependent (14) and may be insufficient to prevent transmission (16). Some Beijing genotypes retain their fitness in vitro, despite the acquisition of drug resistance (36), while compensatory evolution may account for significantly higher levels of fitness in clinical strains than in their progenitors (16). The geographic clustering of drug-resistant cases with evidence of clonal expansion suggests the successful transmission of drug-resistant Beijing genotypes (37). This is supported by the frequency with which isolates of the Beijing genotype are identified among children with drug-resistant TB (30), which indicates successful transmission within the community (34).Variable transmissibility, irrespective of drug resistance, represents a relatively unexplored potential mechanism for the emergence of an M. tuberculosis genotype. Conventional molecular tools are limited by an inability to distinguish factors related to transmissibility from those related to pathogenicity, since only patients with active disease can be evaluated. The value of experimental animal models is equally limited, since artificially induced infection does not allow simulation of the natural airborne transmission of M. tuberculosis. The household provides an appropriate setting in which variables related to recent M. tuberculosis transmission in the human host may be studied and allows the evaluation of young children likely to have been infected through contact with others at the household level.The study described here aimed to determine whether the Beijing genotype is more readily transmitted than other prevalent genotypes to children in household contact with an adult TB index case.     

Influence of Advanced Age on Mycobacterium bovis BCG Vaccination in Guinea Pigs Aerogenically Infected with Mycobacterium tuberculosis

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only tuberculosis (TB) vaccine currently available, but its efficacy against adult pulmonary TB remains controversial. BCG induces specific immune responses to mycobacterial antigens and may elicit protective immunity against TB. TB remains a major public health problem, especially among the elderly, yet the efficacy of BCG in the elderly is unknown. We investigated the ability of BCG vaccination to prevent TB in young (6-week-old), middle-aged (18-month-old), and old (60-month-old) guinea pigs. BCG-Tokyo vaccination reduced the growth of Mycobacterium tuberculosis H37Rv in all three groups. By use of an enzyme-linked immunospot (ELISPOT) assay, antigen-specific gamma interferon (IFN-γ)-producing cells were detected in the 60-month-old guinea pigs after a booster vaccination with BCG-Tokyo. Our findings suggest that BCG-Tokyo has a protective effect against tuberculosis infection regardless of age.Tuberculosis (TB) remains a major public health problem, especially among elderly people. Patients ≥60 years of age account for ≥50% of new cases in Japan (29). The increasing susceptibility of the elderly to Mycobacterium tuberculosis is generally thought to be associated with age-related changes in immune system function, especially losses or delays in antigen-specific CD4⁺ T-cell function (14). Compromised antigen-specific CD4⁺ T-cell responses may contribute to increased susceptibility to M. tuberculosis infection in mice (27).Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only TB vaccine currently available. BCG has been used for more than 80 years (41), and vaccination with BCG is the standard for TB prevention in most countries. BCG induces specific immune responses to mycobacterial antigens and may elicit protective immunity against tuberculosis. BCG provides efficient protection against severe and disseminated TB, such as tuberculosis meningitis and miliary tuberculosis, in children (33, 34, 40). Although the long-term efficacy of BCG has been documented (3, 6), with several reports indicating efficient protection against disseminated TB in newborns and children, it appears to have less efficacy against adult pulmonary TB (2). In fact, its efficacy against pulmonary TB in both adults and the elderly is controversial, as is the efficacy of revaccination (5).In the present study, we examined the efficacy of BCG against TB at different ages in a common guinea pig model (15, 25, 30). We used three age-segregated groups—young (6 weeks old), middle-aged (18 months old), and old (60 months old)—and we measured the number of antigen-specific gamma interferon (IFN-γ)-producing cells as an indicator of the efficacy of the vaccine against TB.     

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.     

Mycobacterium tuberculosis Culture Filtrate Proteins plus CpG Oligodeoxynucleotides Confer Protection to Mycobacterium bovis BCG-Primed Mice by Inhibiting Interleukin-4 Secretion

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-γ) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-γ-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-γ and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-γ and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.The attenuated Mycobacterium bovis strain bacillus Calmette-Guérin (BCG) is the currently used vaccine against tuberculosis (TB). In spite of its wide use, the BCG vaccine only protects against severe forms of childhood TB and generally does not prevent adult pulmonary TB (11, 30, 47).Considering that one-third of the world population is thought to be infected with Mycobacterium tuberculosis and that only a small proportion of these individuals will develop active disease, new vaccine candidates to prevent the establishment of infection could also boost and improve the cellular immunity of already latently infected individuals. Vaccine candidates currently in clinical trials include improved recombinant BCG vaccines, virus-based recombinant vaccines, and subunit vaccines comprised of dominant secreted antigens (1, 32). Secreted proteins, regularly described as culture filtrate proteins (CFP), are the main targets of the T-cell response in mice, both at the height of infection and in a state of memory immunity, as well as in humans with active TB (1, 4, 5, 7, 23). Immunization with these antigens in the presence of different adjuvants provided protection in mice challenged with M. tuberculosis, and protection was mediated by gamma interferon (IFN-γ)-producing CD4⁺ cells (29, 38).We previously showed that a homologous immunization schedule based on three doses of CFP antigens plus CpG oligodeoxynucleotide adjuvant stimulated significant IFN-γ production by spleen cells and in the lungs of challenged mice. In spite of high IFN-γ concentrations, immunized and challenged mice were not protected and indeed had extensive lung damage (16).Since IFN-γ is the best indicator of protective immunity defined thus far, we changed the schedule of homologous immunization to heterologous immunization, also known as a prime-boost regimen, to induce protection.Several studies have demonstrated the efficacy of prime-boost vaccination strategies in generating cellular immunity to a variety of pathogens (3, 10, 14, 17, 34, 36, 44, 45, 49). Recently, our group also showed that a single dose of a DNA-HSP65 vaccine booster significantly enhanced the protection conferred against TB by a single subcutaneous dose of BCG (18). In addition, secreted antigens such as the 6-kDa early-secretion antigen target (ESAT-6), 85A or 85B antigens, and Mtb72F have proven to be promising candidates for BCG-boosting vaccines in mice, guinea pigs, and nonhuman primates (6, 9, 12, 19, 33, 37, 46, 48). Because a single dominant antigen may not confer the same level of protection to all vaccinated individuals, and based on high CFP antigen-mediated IFN-γ production in the presence of CpG adjuvant, in this study we used CFP plus CpG oligodeoxynucleotides to boost BCG vaccination in order to improve protection and lung preservation following M. tuberculosis challenge.     

Quantitative Drug Susceptibility Testing of Mycobacterium tuberculosis by Use of MGIT 960 and EpiCenter Instrumentation

Since numbers of drug-resistant Mycobacterium tuberculosis strains are on the rise, the simple classification into “susceptible” and “resistant” strains based on susceptibility testing at “critical concentrations” has to be reconsidered. While future studies have to address the correlation of phenotypic resistance levels and treatment outcomes, a prerequisite for corresponding investigations is the ability to exactly determine levels of quantitative drug resistance in clinical M. tuberculosis isolates. Here we have established the conditions for quantitative drug susceptibility testing for first- and second-line agents using MGIT 960 instrumentation and EpiCenter software equipped with the TB eXiST module. In-depth comparative analysis of a range of well-characterized susceptible and resistant clinical isolates has allowed us to propose conditions for testing and to develop criteria for interpretation.Tuberculosis (TB) is a leading cause of morbidity and mortality worldwide. Developing countries are the most vulnerable, with more than 95% of the cases (32, 33). The present trend is characterized by an alarming emergence of drug resistance (7, 8, 30). Much attention has focused on the burden of multidrug-resistant (MDR) TB, i.e., resistance to the first-line drugs isoniazid and rifampin (rifampicin) (32, 33), and the emergence of extensively drug resistant TB (6, 26). The rise of drug-resistant TB and the increased susceptibility of the human population to TB due to coinfection with human immunodeficiency virus are driving the worldwide TB pandemic and will worsen the situation in the years ahead, with devastating effects in poor countries, whose economies suffer most from this development (19, 20).In the diagnostic laboratory, testing of mycobacteria for drug susceptibility is substantially different from the general testing procedures used in bacteriology. Rather than determining MICs, a single drug concentration, termed the critical concentration, is usually used to categorize a clinical isolate as susceptible or resistant. This “critical concentration” is more an epidemiological parameter (to distinguish “wild-type” strains from “non-wild-type” strains that are able to grow in the presence of higher drug concentrations [5]) than a clinical cutoff value established to guide treatment decisions (14). With growing knowledge about the mechanisms that underlie drug resistance, it has become evident that drug resistance is multifaceted and that different mutations may lead to different levels of resistance. The acquisition of a resistance mutation leading to a decrease in drug susceptibility should not inevitably exclude an anti-TB drug from a treatment regimen, since low-level resistance does not necessarily imply clinical resistance (3). However, until now, different levels of phenotypic resistance have only rarely been taken into account in the procedures used for in vitro drug susceptibility testing (DST) of mycobacteria (4).“Critical concentration”-based DST of primary and secondary drugs has been established for the radiometric Bactec 460 instrumentation (Becton Dickinson Microbiology Systems, Sparks, MD) and is considered the “gold standard” in the testing of second-line drugs (22, 25). However, the Bactec 460 system has several drawbacks: (i) it involves the use of sharps and radioisotopes with the need for disposal; (ii) it is only semiautomated; and (iii) it needs considerable hands-on time. The nonradiometric MGIT (mycobacterial growth indicator tube) 960 platform (Becton Dickinsion) has been evaluated extensively for DST of first-line drugs (1, 2, 15, 29) and has recently also been evaluated for second-line DST (16, 25). In contrast to the Bactec 460 instrumentation, the MGIT 960 platform is a fully automated system that uses a fluorescence-quenching-based oxygen sensor for growth detection.We have previously characterized quantitative drug resistance levels in clinical strains of drug-susceptible and drug-resistant Mycobacterium tuberculosis using radiometric Bactec 460 measurements (28). However, for widespread implementation of quantitative DST in diagnostic mycobacteriology, the technique chosen should be fully automated, compatible with a computerized expert system for interpretation (so as to avoid individual errors and subjectivity), and safe and reliable (e.g., if possible, the system should not use radioactive material nor needles, such as syringes, for inoculation nor needles inside the instrument). To this end, we have subjected a carefully chosen subset of a previously described and well-characterized collection of clinical M. tuberculosis isolates (28) to quantitative measurements of drug susceptibility using the MGIT 960 platform in conjunction with EpiCenter software equipped with the TB eXiST module.     

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.     

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

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

Humoral Immune Responses against the Mycobacterium tuberculosis 38-Kilodalton,MTB48, and CFP-10/ESAT-6 Antigens in Tuberculosis

The diagnosis of smear-negative and culture-negative patients with active tuberculosis (TB) is challenging. The detection of Mycobacterium tuberculosis-specific antibodies in human sera has been an important diagnostic aid. However, detection of antibody responses to a single antigen usually has a low sensitivity for diagnosis of TB. In this study, humoral immune responses against recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 (culture filtrate protein 10/6-kDa early secreted antigen target of M. tuberculosis) antigens in 250 Chinese TB patients and 260 healthy subjects were evaluated by an enzyme-linked immunosorbent assay (ELISA). The levels of antibodies against those antigens in TB patients, even in bacterium-negative ones, were significantly higher than those in healthy subjects (P < 0.001). The serodiagnostic sensitivities to detect antibodies against individual antigens, i.e., recombinant M. tuberculosis 38-kDa, MTB48, and CFP-10/ESAT-6 antigens, in TB patients were 73.6%, 73.2%, and 60.4%, respectively, with specificities of 85.4%, 77.7%, and 73.8%, respectively. Importantly, the sensitivity to positively detect humoral responses to one of the antigens increased further. Our data suggest that the humoral immune responses to M. tuberculosis antigens in TB patients are heterogeneous. The 38-kDa, MTB48, and CFP-10/ESAT-6 antigens can be used as the cocktail antigens in the serodiagnosis of active TB, especially for smear- or culture-negative TB cases.The control of tuberculosis (TB) remains challenging in China (18). Currently, the diagnosis of active TB mainly relies on clinical symptoms, radiologic findings, and the detection of Mycobacterium tuberculosis in clinical samples using smear staining and mycobacterial culture. However, the diagnosis of TB in smear- and culture-negative TB patients is difficult. The detection of M. tuberculosis-specific antibodies in human sera has been an important aid in diagnosis of TB. Notably, several antigens have been demonstrated to have merit in TB diagnosis, including the 38-kDa protein, which is commonly used in serodiagnostic tests (4, 5, 8, 13, 19, 22, 23). Previous studies suggest that the antibody responses to M. tuberculosis antigens are heterogeneous among individuals (17) so that the detection of antibodies against a single antigen usually has a low sensitivity for diagnosis of TB, especially for bacterium-negative cases. Therefore, it may be valuable to evaluate antibodies against the 38-kDa antigen and other major antigens for the diagnosis of active TB (14, 15).Notably, the MTB48, CFP-10 (culture filtrate protein 10), and ESAT-6 (6-kDa early secreted antigen target of M. tuberculosis) genes are conserved in M. tuberculosis and Mycobacterium bovis isolates but partially deleted or absent in M. bovis BCG as well as in most nontuberculous mycobacteria (NTM) (1-3, 10, 16). Importantly, the proteins encoded by these genes are immunogenic (7, 9, 12, 16). In this study, we cloned the 38-kDa, MTB48, CFP-10, and ESAT-6 genes and generated recombinant 38-kDa, MTB48, and CFP-10/ESAT-6 fusion proteins in Escherichia coli. Subsequently, we developed an enzyme-linked immunosorbent assay (ELISA) for the characterization of serum antibodies against 38-kDa, MTB48, and CFP-10/ESAT-6 antigens in a population of 250 active TB patients and 260 healthy subjects. We found that characterization of antibodies against multiple M. tuberculosis antigens were valuable for the diagnosis of active TB.     

Rapid Detection of Rifampicin- and Isoniazid-Resistant Mycobacterium tuberculosis by High-Resolution Melting Analysis

We have developed a high-resolution melting (HRM) assay to scan for mutations in the rpoB, inhA, ahpC, and katG genes and/or promoter regions for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis. For assay development, 23 drug-resistant isolates of M. tuberculosis having 29 different mutations, together with 40 drug-susceptible isolates, were utilized. All 29 mutations were accurately detected by our assay. We further validated the assay with a series of 59 samples tested in a blind manner. All sequence alterations that were within the regions targeted by the HRM assay were correctly identified. Compared against results of DNA sequencing, the sensitivity and specificity of our HRM assay were 100%. For the blinded samples, the specificities and sensitivities were 89.3% and 100%, respectively, for detecting rifampin resistance and 98.1% and 83.3%, respectively, for detecting isoniazid resistance, as isolates with mutations in regions not encompassed by our assay were not detected. A C-to-T sequence alteration at position −15 of the ahpC regulatory region, which was previously reported to be associated with isoniazid resistance, may possibly be a polymorphism, as it was detected in an isoniazid-susceptible M. tuberculosis isolate. HRM is a rapid, accurate, simple, closed-tube, and low-cost method. It is thus an ideal assay to be used in countries with a high prevalence of drug-resistant M. tuberculosis and where cost-effectiveness is essential. As a mutation-scanning assay for detecting drug-resistant M. tuberculosis, it can potentially lead to better treatment outcomes resulting from earlier treatment with the appropriate antibiotics.The emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant TB (XDR-TB) has hampered the control and treatment of TB (45). MDR-TB is defined as TB that is resistant to at least isoniazid (INH) and rifampin (RIF), two main first-line antitubercular drugs, while XDR-TB is MDR-TB that is additionally resistant to three or more second-line drugs. MDR-TB accounts for an estimated 5% of all TB cases (44); however, patients are often not expeditiously diagnosed, resulting in the delay of appropriate treatment as well as poorer treatment outcomes for patients and the propagation and spread of MDR-TB. Conventional methods for drug susceptibility testing of MDR-TB require an additional culture period, typically between 2 and 5 weeks. An easy-to-implement, cost-effective, and rapid method for drug susceptibility testing is thus of paramount importance to limit the spread of drug-resistant tuberculosis.Drug resistance in Mycobacterium tuberculosis is due to mutations in genes or promoters of genes activating the drug or encoding the drug targets, which are detectable in the majority of drug-resistant isolates (41). Mutations associated with RIF resistance occur mainly in an 81-bp RIF resistance-determining region (RRDR) of the rpoB gene (codons 507 to 533; numbering according to the Escherichia coli rpoB sequence), with >95% of RIF-resistant isolates containing at least one mutation in this region (12, 13, 22, 28, 31, 36, 43). Mutations associated with INH resistance occur mainly in the katG gene (codon 315), the inhA gene and regulatory region, and the ahpC regulatory region (11, 20, 27, 29, 34, 40, 42).While several molecular methods have been previously described for drug susceptibility testing of M. tuberculosis (2, 6, 7, 9, 26, 38), the cost and technical demands of the assays restrict their usage, especially in countries where funds are scarce. Another problem with the majority of PCR-based molecular methods is the requirement of downstream processing of PCR products, which exposes the PCR products to the environment, increasing the risk of cross-contamination of subsequent assays.The high-resolution melting (HRM) analysis is a simple, cost-effective, closed-tube method with sensitivity and specificity reported to be higher than those of denaturing high-performance liquid chromatography (dHPLC) (3). HRM does not require the use of costly fluorescent probes and requires no post-PCR handling, making it an attractive alternative method for genotypic drug susceptibility testing of M. tuberculosis. The method involves performing a PCR with a saturating double-stranded DNA-binding dye such as Syto9, followed by a high-resolution melt analysis, whereby the amplicons are slowly heated to denaturation with real-time monitoring of the decrease in fluorescence during denaturation. By comparing the melting profile of the sample with a reference, any sequence variance can be detected. Homoduplexes are usually detected by a change in melting temperature (T_m), while heteroduplexes are usually detected by a change in the melt curve shape (24). As it is easier to identify a change in melt curve shape (10), the sample and reference DNA can be mixed together and amplified together by PCR to produce heteroduplexes, as in the method we have developed.However, as HRM analysis detects all mutations within the PCR amplicon, known polymorphisms that lie within the amplicon can be excluded by the use of an unlabeled oligonucleotide probe as described by Zhou et al. (48). Briefly, a 3′-blocked unlabeled probe, designed to encompass the polymorphism, is included in the PCR.In this study, we evaluated the efficacy of our assay in comparison with that of standard drug susceptibility testing for the detection of RIF- and/or INH-resistant M. tuberculosis strains from clinical specimens.     

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.     

Identification of Mycobacterium tuberculosis Antigens of High Serodiagnostic Value

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis, with several million new cases detected each year. Current methods of diagnosis are time-consuming and/or expensive or have a low level of accuracy. Therefore, new diagnostics are urgently needed to address the global tuberculosis burden and to improve control programs. Serological assays remain attractive for use in resource-limited settings because they are simple, rapid, and inexpensive and offer the possibility of detecting cases often missed by routine sputum smear microscopy. The aim of this study was to identify M. tuberculosis seroreactive antigens from a panel of 103 recombinant proteins selected as diagnostic candidates. Initial library screening by protein array analysis and enzyme-linked immunosorbent assay (ELISA) identified 42 antigens with serodiagnostic potential. Among these, 25 were novel proteins. The reactive antigens demonstrated various individual sensitivities, ranging from 12% to 78% (specificities, 76 to 100%). When the antigens were analyzed in combinations, up to 93% of antibody responders could be identified among the TB patients. Selected seroreactive proteins were used to design 3 new polyepitope fusion proteins. Characterization of these antigens by multiantigen print immunoassay (MAPIA) revealed that the vast majority of the TB patients (90%) produced antibody responses. The results confirmed that due to the remarkable variation in immune recognition patterns, an optimal multiantigen cocktail should be designed to cover the heterogeneity of antibody responses and thus achieve the highest possible test sensitivity.Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis and is one of the leading causes of mortality due to infectious disease worldwide (9). Nearly one-third of the world''s population is believed to be infected, with approximately 8.8 million new cases detected each year (30, 45). The World Health Organization (WHO) cites TB as the single most important fatal infection, with over 1.6 million deaths per year, the majority (95%) of which are in developing countries (45).Because of logistical and technical shortcomings, human TB testing in most countries is limited to clinical evaluation of symptomatic individuals and screening of high-risk populations. Compounding the severity of TB is the realization that a leading cause of death among HIV-positive people is concomitant TB, accounting for about one-third of AIDS-related deaths. It is estimated that a rapid and widely available diagnostic with 85% sensitivity and 95% specificity would result in 400,000 fewer deaths each year and would greatly reduce the global health cost of TB (18).Existing TB diagnostic methods are either too time-consuming, too complex and labor-intensive, too inaccurate, or too expensive for routine use in resource-limited settings (2, 36). For active pulmonary disease, sputum smear microscopy, culture, and/or PCR-based probes can be used to support X-ray findings and/or clinical observations suggestive of TB. Of these, microscopic examination of sputum is the only rapid, relatively simple, and inexpensive test for TB. The reported sensitivity of Ziehl-Neelsen staining of unprocessed sputum smears from immunocompetent adults is only 40 to 70% (19, 21), and it may be significantly lower for children and/or HIV-infected patients (12). A delayed or missed TB diagnosis certainly contributes to M. tuberculosis transmission and increased TB mortality (22, 27).Mycobacterial culture is the gold standard method of TB diagnosis. However, it requires up to 8 weeks for the isolation of M. tuberculosis from a clinical specimen, and importantly, in 10 to 20% of positive cases, the bacillus is not successfully cultured (3). Culture is more expensive than microscopy and requires a high standard of technical expertise. Therefore, a sensitive and specific point-of-care test for the rapid diagnosis of patients with active TB would facilitate early treatment and reduce M. tuberculosis transmission.An antibody test for TB has long been sought. Serologic assays remain attractive for use in resource-limited settings because they generally are simple, rapid, and relatively inexpensive compared to other methods. For TB, serological tests may also offer the possibility of detecting cases that are usually missed by routine sputum smear microscopy, such as extrapulmonary disease and pediatric TB. Numerous in-house serological assays for TB, using a variety of antigens to detect circulating antibodies, have been developed over the years, including complement fixation tests, hemagglutination tests, radioimmunoassays, and enzyme-linked immunosorbent assays (ELISAs) (11, 38-40). Both lateral-flow and enzyme immunoassay formats have been developed and are currently available commercially, but so far none has demonstrated adequate sensitivity and specificity (7, 13, 31, 38).In this study, we assessed a large panel of recombinant TB antigens for their serodiagnostic potential. From an initial screen of 103 recombinant proteins by protein microarray analysis and ELISA, 42 previously known and novel TB antigens were found to elicit specific antibody responses in TB patients. Several fusion proteins comprised of tandem arrangements of the selected antigens were made and serologically characterized by ELISA and multiple-antigen print immunoassays (MAPIA). The antigens identified hold promise for the development of a rapid and highly sensitive serodiagnostic test for TB.     

Kinetics of a Tuberculosis-Specific Gamma Interferon Release Assay in Military Personnel with a Positive Tuberculin Skin Test

Treatment of latent Mycobacterium tuberculosis infection on the basis of the tuberculin skin test (TST) result is inaccurate due to the false-positive TST results that occur after Mycobacterium bovis BCG vaccination or exposure to nontuberculous mycobacteria (NTM). Gamma interferon release assays (IGRAs) are based on M. tuberculosis-specific antigens. In a previous study among BCG-naïve military employees, a positive TST result after deployment was mostly associated with a negative IGRA result, suggesting exposure to NTM. Data regarding the kinetics of IGRAs are limited and controversial. The present study aimed to reassess the rate of false-positive TST results and to evaluate the kinetics of the Quantiferon TB Gold In-Tube assay (QFT-Git) in military personnel with a positive TST result. QFT-Git was performed at the time of inclusion in the study and was repeated after 2, 6, 12, and 18 or 24 months. Of 192 participants, 17 were recruits and 175 were screened after deployment (n = 169) or because of travel or health care work. Baseline positive QFT-Git results were observed in 7/17 (41.2%) and 12/174 (6.9%) participants, respectively. During follow-up, a negative QFT-Git result remained negative in 163/165 (98.8%) participants. Of 18 subjects with an initial positive QFT-Git result, reversion to a negative result occurred in 1/6 (16%) recruits, whereas it occurred in 8/12 (66%) subjects after deployment or with other risk factors (P = 0.046). The quantitative result was significantly lower in subjects with reversion than in those with consistent positive results (P = 0.017). This study confirmed a low rate of positive QFT-Git results among military personnel with a positive TST result after deployment, supporting the hypothesis of exposure to NTM. Reversion of the majority of initially low-positive QFT-Git results indicates that QFT-Git may be useful for the diagnosis of later reinfections.Each year, about 3,000 Dutch army personnel are deployed to regions where tuberculosis (TB) is highly endemic. Screening of military personnel for latent Mycobacterium tuberculosis infection (LTBI) has thus far been based on the tuberculin skin test (TST). The Netherlands is a country with a low prevalence of TB, with a yearly incidence of 5.9 cases/100,000 population in 2007, only one-third of which occurred among native Dutch persons (Tuberculosis in The Netherlands 2007 [www.kncvtbc.nl]). Personnel are screened by the TST upon initial recruitment into the army, after deployment, or in the presence of other risk factors for TB exposure. Military personnel with TST conversion are prescribed isoniazid for 6 months to prevent TB disease. The risk of progression from untreated LTBI to active TB is generally believed to be about 10%, with half of the cases occurring within 2 years after infection. However, the risks observed in different studies comparing subjects treated with isoniazid or placebo varied widely, depending on the setting and the characteristics of the study population (38). A major disadvantage of the current policy is that a substantial proportion of TST conversions in this setting are thought to be caused by exposure to nontuberculous mycobacteria (NTM), skewing the risk-benefit ratio of preventive treatment (8). In addition, increasing proportions of the Dutch population and Dutch military recruits originate from countries where M. bovis BCG vaccination is routinely used. In BCG-vaccinated Dutch military personnel or those with a previous positive TST result, TST is not performed, as a rule, and chest radiography is used as an alternative, but radiography lacks sensitivity for the detection of LTBI. Finally, a positive TST result often remains positive, thus precluding the detection of reinfection.In order to overcome the disadvantages of the TST, gamma interferon (IFN-γ) release assays (IGRAs) that use M. tuberculosis-specific antigens and that are not affected by BCG and most NTM were developed (2-4, 32, 34). In contact investigations, the results of IGRAs had a better correlation with measures of exposure (5, 19, 21, 43). Since 2005, IGRAs have increasingly been used for the detection of LTBIs either as a replacement of or as adjunct to the TST (18, 26, 28). Of the two presently commercially available IGRAs, the Quantiferon TB Gold In-Tube assay (QFT-Git) is a robust whole-blood-based test suitable for use for large-scale testing (5, 7, 22). In a previous study, QFT-Git was positive for only a minority of military personnel with a positive TST result after deployment (13). Those results were considered to be related to NTM exposure, in accordance with the high proportion of false-positive TST responses assessed by dual skin testing of army recruits by the use of tuberculin and atypical sensitin (8). The destinations of deployment at the time of the earlier study were mainly Iraq and Bosnia (13), but the destination has changed to Afghanistan in the past few years. As the incidence of TB is higher in Afghanistan than in most of the countries where the military personnel were deployed, in order to justify a change in treatment policy, the previously observed low rate of positive IGRA results in association with a positive TST result needed to be studied in the current setting. In the previous study (13), QFT-Git was performed only once, and the subjects were not assessed for eventual later conversion or reversion. Previous studies of the kinetics of IGRAs gave variable and partly conflicting results (9, 12, 14, 15, 17, 20, 29, 30, 35-37, 40), although the main trend was for high-positive results to usually remain positive, and reversion can occur when the results are low or moderately positive (12, 15, 20, 29, 30, 33, 35-37, 42). In a setting of LTBIs, the relevance of follow-up testing by IGRAs may lie in the possibility of detecting later reinfection if reversion to a negative result has been documented.The aims of this study were to study the kinetics of QFT-Git during at least 6 months of follow-up in order to evaluate the possibility of detection of later reinfection and to confirm the previously observed very low rate of positive QFT-Git results in military personnel with a positive TST result after deployment.     

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.     

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.     

Prospective Universal Application of Mycobacterial Interspersed Repetitive-Unit-Variable-Number Tandem-Repeat Genotyping To Characterize Mycobacterium tuberculosis Isolates for Fast Identification of Clustered and Orphan Cases

The use of molecular tools for genotyping Mycobacterium tuberculosis isolates in epidemiological surveys in order to identify clustered and orphan strains requires faster response times than those offered by the reference method, IS6110 restriction fragment length polymorphism (RFLP) genotyping. A method based on PCR, the mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping technique, is an option for fast fingerprinting of M. tuberculosis, although precise evaluations of correlation between MIRU-VNTR and RFLP findings in population-based studies in different contexts are required before the methods are switched. In this study, we evaluated MIRU-VNTR genotyping (with a set of 15 loci [MIRU-15]) in parallel to RFLP genotyping in a 39-month universal population-based study in a challenging setting with a high proportion of immigrants. For 81.9% (281/343) of the M. tuberculosis isolates, both RFLP and MIRU-VNTR types were obtained. The percentages of clustered cases were 39.9% (112/281) and 43.1% (121/281) for RFLP and MIRU-15 analyses, and the numbers of clusters identified were 42 and 45, respectively. For 85.4% of the cases, the RFLP and MIRU-15 results were concordant, identifying the same cases as clustered and orphan (kappa, 0.7). However, for the remaining 14.6% of the cases, discrepancies were observed: 16 of the cases clustered by RFLP analysis were identified as orphan by MIRU-15 analysis, and 25 cases identified as orphan by RFLP analysis were clustered by MIRU-15 analysis. When discrepant cases showing subtle genotypic differences were tolerated, the discrepancies fell from 14.6% to 8.6%. Epidemiological links were found for 83.8% of the cases clustered by both RFLP and MIRU-15 analyses, whereas for the cases clustered by RFLP or MIRU-VNTR analysis alone, links were identified for only 30.8% or 38.9% of the cases, respectively. The latter group of cases mainly comprised isolates that could also have been clustered, if subtle genotypic differences had been tolerated. MIRU-15 genotyping seems to be a good alternative to RFLP genotyping for real-time interventional schemes. The correlation between MIRU-15 and IS6110 RFLP findings was reasonable, although some uncertainties as to the assignation of clusters by MIRU-15 analysis were identified.Molecular tools have been widely used to characterize Mycobacterium tuberculosis isolates, with the aim of better understanding the epidemiology of tuberculosis (TB) (1, 6, 8, 18, 23). This has enabled us to document suspected outbreaks (4, 28, 34), identify risk factors associated with TB transmission (13, 20, 36), and evaluate the efficiency of control programs by observing the dynamics of clustered cases (9, 12, 17, 22, 24).Restriction fragment length polymorphism (RFLP) analysis based on the IS6110 sequence is the reference genotyping method for M. tuberculosis (35). However, its limitations (mainly response times) make its adaptation unsuitable for real-time intervention epidemiological schemes. New genotyping techniques based on PCR have recently been developed and are more suitable for these purposes.One of the most promising PCR-based methods is mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) genotyping (21, 30-32). A novel format based on 15 loci has improved upon the initial 12-loci version. Its discriminatory power has been found to be equivalent to that of the standard approach on the basis of reference method, and its response time could be competitive. However, very few long-term analyses apply this technique universally in parallel to the reference method to identify advantages and pitfalls (1, 25, 27, 33).In order to compare both techniques, we selected the province of Almería, in southeast Spain, because of the complexity of its socioepidemiological population profile, which challenges us to develop new and more-efficient methods of surveying TB transmission. In Almería, around 60% of the cases involve immigrants who are dispersed throughout the province and who are highly mobile and difficult to access. Our group had already developed a new advanced system for studying clustered cases by active compilation of data through a newly developed computer application, GenContacTB, and standardized interviews of the patients on the basis of nominal and photographic identification (26). These aspects make Almería a suitable context for exploring novel, rapid M. tuberculosis-genotyping tools. Our study aimed to evaluate MIRU-VNTR genotyping with a set of 15 loci (MIRU-15) over a 39-month period by using a prospective design for most of the period (data for 2005 were retrospectively studied to increase analytical power), and the coverage of the population was universal (all M. tuberculosis isolates were included).     

Variation in Gamma Interferon Responses to Different Infecting Strains of Mycobacterium tuberculosis in Acid-Fast Bacillus Smear-Positive Patients and Household Contacts in Antananarivo,Madagascar

The majority of healthy individuals exposed to Mycobacterium tuberculosis will not develop tuberculosis (TB), though many may become latently infected. More precise measurement of the human immune response to M. tuberculosis infection may help us understand this difference and potentially identify those subjects most at risk of developing active disease. Gamma interferon (IFN-γ) production has been widely used as a proxy marker to study infection and to examine the human immune response to specific M. tuberculosis antigens. It has been suggested that genetically distinct M. tuberculosis strains may invoke different immune responses, although how these differences influence the immune responses and clinical outcome in human tuberculosis is still poorly understood. We therefore evaluated the antigen-specific IFN-γ production responses in peripheral blood mononuclear cells from two cohorts of subjects recruited in Antananarivo, Madagascar, from 2004 to 2006 and examined the influence of the infecting M. tuberculosis strains on this response. The cohorts were sputum-positive index cases and their household contacts. Clinical strains isolated from the TB patients were typed by spoligotyping. Comparison of the IFN-γ responses with the spoligotype of the infecting clinical strains showed that “modern” M. tuberculosis strains, like Beijing and Central Asian (CAS) strains, tended to induce lower IFN-γ responses than “ancient” strains, like East African-Indian (EAI) strains, in index cases and their household contacts. These results suggest that new strains may have evolved to induce a host response different from that of ancient strains. These findings could have important implications in the development of therapeutic and diagnostic strategies.Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a major cause of global morbidity and mortality throughout the world. It is estimated that there are in excess of new 8 million cases of TB each year, and this represents just the tip of the iceberg. Infection with M. tuberculosis leads to clinically active TB in about 5 to 10% of exposed individuals. A much higher proportion of exposed individuals apparently become latently infected, and these individuals may remain noninfectious and symptom free for years. Approximately one-third of the world population is thought to be latently infected with M. tuberculosis. However, under some circumstances (in about 5% of the latently infected people), the host immune response is perturbed and latent M. tuberculosis infection may develop into clinically active TB (52). This process is most prominent in individuals coinfected with human immunodeficiency virus (HIV), but it can also occur with impairment of the immune system associated with old age, malnutrition, anti-inflammatory drug treatment, etc. Reactivation of latent disease is thought to contribute roughly half of all TB cases, and thus, understanding the factors controlling the development of acute primary TB or latent infection is crucial to TB control (64).Gamma interferon (IFN-γ) production has been widely used to study infection and to examine the human immune response to specific M. tuberculosis antigens. The 6-kDa early secreted antigenic target (ESAT-6) antigen, encoded by genes located within region of difference 1 (RD1) of the M. tuberculosis genome, is much more specific for M. tuberculosis than purified protein derivative (PPD), as these genes were deleted from M. bovis in the development of BCG substrains or are not found in most environmental mycobacteria (29, 53). Some studies showed that the level of IFN-γ release in response to ESAT-6 could identify TB contacts at risk of developing active disease after recent infection (3, 18, 30). CFP7 or TB10.4 is an immunodominant antigen recognized by TB patients and M. bovis BCG-vaccinated subjects, while ESAT-6 is specific to TB patients and induces a strong IFN-γ response (51). Moreover, since CFP7 induces strong protection against infection by M. tuberculosis, it was proposed to be a TB vaccine candidate (1, 19).There is a growing number of observations indicating that TB cases resulting from infection with epidemic strains, such as the W-Beijing strains (22, 35, 39, 44), may display a more severe pathology or more severe symptoms. Beijing strains were also found to induce higher fevers in pulmonary TB patients than other strains (62). In addition, the Beijing genotype, which is responsible for more than 80% of TB cases in China, was associated with virulence and high transmissibility (7, 28). The same has been found more recently with the RD(Rio) strains belonging to the Latin America-Mediterranean (LAM) family (38). Despite the fact that other epidemiological and clinical studies have failed to confirm any association between the mycobacterial genotype and the clinical presentation (8, 41, 43), the immunological aspects of infection with these strains is still of interest and poorly described.Epidemiological studies carried out in Madagascar showed no association between IS6110 patterns and clinical tuberculosis presentation (47), but they did reveal a heterologous population of M. tuberculosis strains, including the existence of a high frequency of unusual genotypes, such as the shared type 109 from the EAI8-MDG family (SpolDB4) (10) or strains with a single copy of IS6110 (24, 46). Since there are limited data on the correlation of the strain genotype with clinical features or the host immune response in patients and their contacts (57, 59), we investigated the IFN-γ response to the ESAT-6, CFP7, and PPD antigens in pulmonary TB patients and their household contacts (as this is commonly used as a biomarker to identify M. tuberculosis infection) and examined the influence of the M. tuberculosis genotype on this response.