Evaluation of the BD GeneOhm StaphSR Assay for Detection of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Spiked Positive Blood Culture Bottles

To improve the clinical outcome of Staphylococcus aureus septicemia, the early selection of appropriate antibiotic treatment is crucial. Molecular diagnostics represents an attractive approach for the rapid identification of S. aureus and the determination of its methicillin (meticillin) resistance. In direct comparison to other molecular assays (sa442 and mecA real-time PCRs) and standard laboratory procedures, we evaluated the BD GeneOhm StaphSR assay for its use in the detection of S. aureus and methicillin-resistant S. aureus (MRSA) from spiked blood culture bottles (n = 134). In the case of detecting S. aureus (n = 90; for methicillin-susceptible S. aureus, n = 45; for MRSA, n = 45), the BD GeneOhm StaphSR assay had a sensitivity and a specificity of 100% each (95% confidence intervals [CIs], 96.0 to 100% and 82.4 to 100%, respectively). For MRSA (n = 45), the test was 95.6% (95% CI, 84.9 to 99.5%) sensitive and 95.3% (95% CI, 86.9 to 99.0%) specific. Overall, five discrepant results arose with this assay due to the presence of methicillin-susceptible, revertant MRSA strains (3/45) and MRSA strains that were not detected by the BD GeneOhm StaphSR assay (2/45). Compared to other real-time PCR-based molecular approaches and to conventional standard laboratory methods, the BD GeneOhm StaphSR turned out to be an appropriate diagnostic tool for a rapid (∼1.5 h), preliminary identification of S. aureus and MRSA from blood cultures.Staphylococcus aureus septicemia is associated with high mortality rates, prolonged hospitalization, and increased costs (3, 5). The prevalence of S. aureus septicemia is increasing, primarily due to infections caused by methicillin (meticillin)-resistant S. aureus (MRSA) (20). Several studies have shown that mortality rates among patients suffering from MRSA septicemia is significantly higher than those of patients suffering from infections caused by methicillin-susceptible S. aureus (MSSA) (5, 18, 19).The early selection of an appropriate antibiotic regime for the treatment of MSSA or MRSA is crucial for the patient''s outcome (4, 14, 15). However, bacterial identification and preliminary antibiotic susceptibility testing by standard microbiological procedures still requires 24 to 48 h after growth detection by automated incubation systems. In contrast, new real-time PCR-based methods that use samples directly from positive blood culture bottles allows differentiation of MSSA, MRSA, and coagulase-negative staphylococci (CoNS) within 1.5 to 3 h (7, 12, 13, 16). Such tests promote an early appropriate antibiotic selection, thus avoiding the unnecessary use of vancomycin, and they reduce mortality, the length of hospitalization, and costs associated with bloodstream infections caused by these bacteria (3).We described recently a real-time PCR method for the detection of MSSA, MRSA, and CoNS directly from positive blood cultures; it turned out to have 100% sensitivity and 100% specificity for the detection of MSSA and MRSA (7). In this study, the differentiation between MSSA and MRSA directly from signal-positive blood cultures was achieved by the separate detection of the S. aureus-specific chromosomal fragment sa442 and the mecA gene (encoding methicillin resistance). However, since this test is not a commercialized system and does not run on a common platform like, e.g., the SmartCycler (Cepheid, Sunnyvale, CA), its widespread application is limited. Moreover, in blood cultures containing a mixture of MSSA (sa442⁺ but mecA negative) and methicillin-resistant CoNS (MR-CoNS; sa442 negative but mecA⁺), the test is prone to lead to the incorrect identification of MRSA (sa442⁺ mecA⁺).The BD GeneOhm StaphSR assay (BD Diagnostics GeneOhm, Québec, Canada) provides a rapid, simple, commercially available diagnostic test that runs on the SmartCycler for the detection of S. aureus and MRSA from nasal swabs, wounds, and blood cultures. This multiplex real-time PCR amplifies an S. aureus-specific target sequence and a specific target near the staphylococcal cassette chromosome mec (SCCmec) insertion site and the orfX junction in MRSA, thereby differentiating between MSSA and MRSA (9, 17).Using the herein-described setting, we evaluated the BD GeneOhm StaphSR assay and the PCR that detects sa442 and mecA (designated sa442-mecA-PCR) for the detection of MSSA and MRSA directly from spiked blood cultures.     

Blood Cultures Positive for Coagulase-Negative Staphylococci: Antisepsis,Pseudobacteremia, and Therapy of Patients

A blood culture cohort study investigating issues related to isolation of coagulase-negative staphylococci (CoNS) and other skin microflora is reported. Data were collected over 12 weeks to determine the incidence of significant CoNS bacteremia versus that of pseudobacteremia (contaminants) and to evaluate drug therapy in patients with cultures positive for CoNS. In addition, the effectiveness of 0.2% chlorine peroxide as a bactericidal disinfectant was compared to that of 10% providone iodine. A total of 3,276 cultures of blood from 1,433 patients were evaluated in the study. Eighty-nine cultures were positive for skin flora, with 81 of 89 (91%) involving CoNS. The incidence of significant CoNS bacteremia was 20 of 81 (24.7%), that of indeterminate bacteremia was 10 of 81 (12.3%), and that of contamination was 59 of 81 (72.8%). The incidence of significant bacteremia involving CoNS was double the 10 to 12% rate based on previous estimations at our institutions. In tests with the two bactericidal disinfectants, 22 of 1,639 cultures (1.3%) in the chlorine peroxide group versus 37 of 1,637 (2.3%) in the providone iodine group were considered contaminated (P = 0.065). Rates of contamination for venipuncture versus catheter collection were not significantly different (P = 0.46). The overall contamination rate was 59 of 3,276 (1.8%), which is consistent with the lower end of published quality assurance benchmark standards. The low rate was believed to be due to the professional phlebotomy staff in our institutions. There was excellent agreement between retrospective analysis by reviewers, when formal criteria were used, and the attending physicians’ intuitive clinical impressions in the classification of significant bloodstream infections (100% agreement) or contamination (95% agreement). However, physicians still used antimicrobial agents to treat nearly one-half of the patients with contaminated blood cultures, with vancomycin being misused in 34% of patients. In addition, 10% of patients with significant bacteremia were treated with inappropriate agents. There were no significant adverse events or prolonged hospital stays due to the unnecessary use of vancomycin; however, the additional costs of treating patients whose cultures contained CoNS contaminants was estimated to be $1,000 per patient. Measures to limit the unnecessary use of vancomycin (and other agents) are important.Coagulase-negative staphylococci (CoNS), the most frequent blood culture isolates, are predominantly blood culture contaminants, but they are also a significant cause of bacteremia (2–5, 7, 9, 13). Institution-specific contamination rates vary from 2 to more than 6% (3, 5, 23, 26, 27). In the past 5 years, estimated contamination rates at our hospitals ranged from 2.5 to 3.5%. During this period, CoNS accounted for 45 to 60% of total blood isolates, and we estimated, using laboratory criteria, that 10 to 12% of CoNS isolates from blood were implicated in significant bloodstream infections. A relatively large proportion of the patient population with presumed false-positive blood cultures due to contaminants (pseudobacteremia) were treated with antimicrobial agents, in particular, vancomycin.Clinical and microbiologic guidelines for the differentiation of true bacteremia from pseudobacteremia or contamination have been published (5, 13, 15). Suggested laboratory criteria for true bacteremia include growth within 48 h and multiple blood cultures positive for the same organism. In contrast, increased duration of time before positivity, polymicrobial growth of skin organisms, or growth during antibiotic treatment suggest contamination. Others recommended that the addition of clinical guidelines is essential for the appropriate classification of bacteremia (4, 8, 9, 15, 18).We conducted a cohort study to evaluate clinical and laboratory data for adult patients with blood cultures positive for CoNS. The study was done at two tertiary-care teaching centers, Deaconess Medical Center (DMC) and Sacred Heart Medical Center (SHMC), with a combined capacity of 900 beds. We examined problems associated with false-positive bacteremia and determined the incidence of significant bacteremia. Our goal was to make recommendations to improve clinicians’ ability to recognize the significance of potentially contaminating organisms and to evaluate treatment given to patients with CoNS-positive blood cultures. To attempt to minimize contamination, we evaluated the nontoxic, antiseptic and disinfectant chlorine peroxide in comparison to a standard disinfectant.(This work was previously presented in abstract form at the 96th General Meeting of the American Society for Microbiology, New Orleans, La., 19 to 23 May 1996 [24a].)     

Algorithm Combining Toxin Immunoassay and Stool Culture for Diagnosis of Clostridium difficile Infection

Enzyme immunoassays (EIA) to detect glutamate dehydrogenase or toxins A (TcdA) and B (TcdB), a cytotoxicity assay, and bacteriologic culture have disadvantages when applied individually to diagnosis of Clostridium difficile infections. Stool specimens (n = 1,596) were subjected to toxin detection via an enzyme-linked fluorescent immunoassay (ELFA; Vidas CDAB assay) and bacteriologic culture for toxigenic C. difficile in a three-step algorithm with additional toxigenic culture. Isolates (n = 163) from ELFA-negative stool specimens were examined via ELFA for toxin production. We amplified tcdA and tcdB from C. difficile isolates and tcdB from stool specimens that were ELFA positive or equivocal and culture negative, and we compared the results to those obtained with the three-step algorithm. More than 26% of stool specimens (419/1,596) were culture positive, yielding 248 isolates (59.2%) with both toxin genes (tcdA- and tcdB-positive isolates), 88 isolates (21.0%) with either tcdA or tcdB, and 83 (19.8%) that had no toxin genes (tcdA- and tcdB-negative isolates). Among 49 (culture-negative/ELFA-positive or -equivocal) stool specimens, 53.1% (26/49) represented tcdB-positive isolates. Therefore, the total number of PCR-positive cases was 362, and 27.1% (98/362) of these were detected through toxigenic culture. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were 63.3%, 96.7%, 90.5%, and 92.4% (ELFA alone); 92.8%, 93.3%, 80.2%, and 97.8% (culture); and 70.7%, 91.4%, 95.5%, and 100% (three-step algorithm ELFA and bacterial culture with toxigenic culture), respectively, with culture and PCR for tcdA and tcdB as the standards. Thus, sensitivity and specificity were highest using culture and ELFA, respectively, but we recommend the three-step algorithm comprising EIA to detect both toxins and toxigenic culture for C. difficile as a practical method for achieving better PPV and NPV.Clostridium difficile is an important nosocomial pathogen, causing antimicrobial-associated diarrhea and pseudomembranous colitis. Toxins A (TcdA) and B (TcdB) mediate the pathogenesis of C. difficile infection (CDI), and toxin detection is an important part of diagnosis. A cytotoxicity neutralization assay (CNA) is the reference method for toxin detection, but it is expensive and time-consuming and requires tissue culture facilities (34, 35). Most laboratories now use a commercial enzyme immunoassay (EIA) to detect TcdA and/or TcdB, with the benefits of rapid turnaround time and ease of use (3, 21, 22, 23, 26, 27, 33, 35). The putative >90% sensitivity of toxin EIAs is not often realized in practice, but EIA is the only toxin detection method available to many routine medical laboratories. The demand for EIA kits detecting both TcdA and TcdB has increased due to increased worldwide prevalence of TcdA-negative, TcdB-positive (TcdA− TcdB+) strains (1, 12, 24, 29, 32).A two-step algorithm, based upon EIA-based detection of species-specific antigen glutamate dehydrogenase (GDH-Ag) and toxin detection via CNA, was suggested to have improved sensitivity and specificity in the detection of toxigenic C. difficile (34). However, the GDH-Ag assay detects both nontoxigenic and toxigenic strains, and the aforementioned shortcomings of the CNA assay make it unavailable to many routine laboratories.Bacteriologic culture can be time-consuming, but it is more straightforward and sensitive than CNA for the detection of toxigenic C. difficile. Furthermore, it provides isolates for characterization, yielding information about CDI epidemiology and antimicrobial susceptibility (11, 28, 36). We evaluated the combination of bacteriologic culture and EIA-based detection of TcdA and TcdB as a new strategy for diagnosis of CDI, especially in areas where TcdA− TcdB+ strains are prevalent.     

Nasal Colonization of and Clonal Transmission of Methicillin-Susceptible Staphylococcus aureus among Chinese Military Volunteers

Military facilities provide unique opportunities for studying Staphylococcus aureus nasal colonization and transmission patterns. In this cross-sectional observational study, we assessed the prevalence of S. aureus nasal colonization among Chinese military volunteers in two camps in the Beijing area. Antimicrobial resistance patterns, risk factors for colonization, and transmission patterns using pulsed-field gel electrophoresis were also evaluated. From May to July 2007, 1,044 nasal swabs were collected from military volunteers from suburban (560) and urban (484) camps. A total of 209 S. aureus isolates were recovered, of which all were methicillin susceptible. Independent factors associated with methicillin-susceptible S. aureus (MSSA) nasal colonization included younger age (odds ratio [OR] = 1.51, 95% confidence interval [95% CI] = 1.03 to 2.21, P = 0.0347), higher education (OR = 1.38, 95% CI = 1.10 to 1.73, P = 0.0056), shorter length of service (OR = 1.74, 95% CI = 1.28 to 2.36, P = 0.0004), nonsmoking (OR = 1.61, 95% CI = 1.14 to 2.28, P = 0.0069), and inactive participation in social events (OR = 2.40, 95% CI = 1.25 to 5.49, P = 0.0082). Among 209 MSSA isolates, 126 (60.3%) were determined to be epidemic and a total of 12 genotypes were identified, of which four (90 isolates [71.4%]) represented the majority of strains. Length of service and camp location were statistically related to the four major MSSA genotype clonal transmissions. Our data indicated that MSSA, not methicillin-resistant S. aureus (MRSA), nasal colonization and clonal transmission occur in healthy military volunteers in Beijing. Younger, female, nonsmoking volunteers with higher education, little or no participation in social events, and less time in service are at higher risk for nasal MSSA carriage.Staphylococcus aureus is an important cause of skin and soft tissue infections, as well as invasive infections in humans (25). Since methicillin-resistant S. aureus (MRSA) was first reported, it has become endemic in hospitals and communities around the world (10). The recent emergence of a highly virulent community-associated MRSA (CA-MRSA) and vancomycin-resistant, intermediate-resistant, or heteroresistant S. aureus further heightens public health concerns (14, 17, 37, 46). Prevention of S. aureus infection and reduction of the spread of virulent and resistant strains are therefore of great importance.On the other hand, S. aureus is a member of the commensal microflora. The anterior nares of the nose are the primary reservoirs of S. aureus colonization in humans, and many S. aureus infections occur in persons with prior nasal bacterial carriage (47). Nasal colonization is an important step in the pathogenesis of S. aureus infection and is a risk factor for acquiring nosocomial infection (22). It has been shown that 80% of nosocomial S. aureus bacteremia episodes in carriers of this bacteria were attributed to an endogenous source (44). Nosocomial S. aureus bacteremia was three times more frequent in S. aureus carriers than in noncarriers (48). Numerous studies of S. aureus nasal carriage have been carried out in various geographic regions in the United States and the Netherlands (2, 5, 7, 21, 23, 27, 28, 41). Cross-section surveys of nasal carriage prevalence and transmission mechanisms in special healthy populations are beneficial in assessing risk factors associated with S. aureus infections (2, 8, 13, 26, 32-35). Military facilities provide unique opportunities for studying S. aureus nasal colonization and transmission (11, 19, 52).In China, MRSA was shown in 63% of S. aureus isolates, among which 77% nosocomial and 43% community isolates were MRSA (49). According to a study conducted in 2005, the mean incidence of MRSA across China was over 50%, and in Shanghai, the prevalence was over 80%, contributed to by two major epidemic MRSA clones with unique geographic distribution (24, 45, 50, 51). Therefore, understanding and controlling the spread of MRSA in both hospital and community settings in China are now of paramount importance. The majority of S. aureus isolates studied in China have been limited to clinical patients, and S. aureus isolates recovered from healthy populations or those from healthy military volunteers have not been previously reported.In this study, we reported a cross-sectional observational study conducted in two military camps in the Beijing area, People''s Republic of China. The prevalence of S. aureus nasal colonization and risk factors associated with colonization were assessed. Nasal carriage S. aureus isolates were genotyped to determine potential clonal transmission in military facilities and related transmission factors.(This study was presented in part at the 109th General Meeting of the American Society for Microbiology, Philadelphia, PA, 17 to 21 May 2009.)     

Evaluation of a New Selective Medium,BD BBL CHROMagar MRSA II,for Detection of Methicillin-Resistant Staphylococcus aureus in Different Specimens

The sensitivity of screening for methicillin-resistant Staphylococcus aureus (MRSA) can be improved by adding other specimen sites to nares. We describe an evaluation of a new selective medium, BBL CHROMagar MRSA II (CMRSAII), for its ability to detect MRSA from different specimen types. CMRSAII is a chromogenic medium which incorporates cefoxitin for the detection of MRSA. A study was performed at four clinical laboratories with the following specimens: 1,446 respiratory, 694 stool, 1,275 skin, and 948 wound specimens and 688 blood culture bottles containing Gram-positive cocci. The recovery of MRSA on traditional culture media was compared to results with CMRSAII. S. aureus was tested by cefoxitin disk diffusion. CMRSAII was interpreted as positive for MRSA at 24 h (range, 18 to 28 h) based solely on the visualization of mauve-colored colonies and at 48 h (range, 36 to 52 h) based on detection of mauve colonies with subsequent confirmation as S. aureus (by coagulase or latex agglutination testing). MRSA was recovered more frequently on CMRSAII (89.8% at 24 h and 95.6% at 48 h) than on traditional culture plates (83.1% at 24 h and 79.8% at 48 h) for all specimen types combined (P < 0.001). The percent sensitivities of CMRSAII at 24- and 48-h reads, respectively, were 85.5 and 92.4% for respiratory specimens, 87.9% and 98.3% for stool specimens, 88.4% and 96.1% for skin specimens, 92.1% and 94.6% for wound specimens, and 100% and 100% for positive blood cultures. The specificity was 99.8% for respiratory specimens and 100% for all others. In conclusion, CMRSAII is a reliable screening medium for multiple specimen types.Controlling the spread of multidrug-resistant microorganisms and especially methicillin-resistant Staphylococcus aureus (MRSA) has become a major infection control objective in the United States (4) and many European countries (3, 4, 21). A part of most programs to control the spread of MRSA is screening of patients (4, 8, 14), and screening has even become mandatory in some countries (11, 31).Traditionally, MRSA screening included mainly the culturing of naris swabs. However, it has been demonstrated that up to 35% of MRSA carriers may be colonized only from sites other than the nares, for example, the throat or the rectum (1, 2, 16).Usage of chromogenic media can improve the sensitivity and pace of MRSA detection (5, 6, 9, 10, 12, 13, 15, 17,19, 20, 22-24, 26-30); however, currently available media that have been marketed at this time are recommended only for nasal specimens.This study was designed to compare the performance of BBL CHROMagar MRSAII (CMRSAII), a chromogenic medium which incorporates cefoxitin, with traditional culture media in the recovery and identification of MRSA isolates from clinical specimens, including respiratory, lower gastrointestinal, and skin specimens as well as wound cultures and blood culture bottles with Gram-positive cocci. In addition, it was designed to determine whether CMRSAII results may be reported as presumptive or definitive with no (or one) confirmatory test at 24 and 48 h of incubation.(These data were presented in part at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 25 to 28 October 2008.)     

Vancomycin MIC plus Heteroresistance and Outcome of Methicillin-Resistant Staphylococcus aureus Bacteremia: Trends over 11 Years

Vancomycin MICs (V-MIC) and the frequency of heteroresistant vancomycin-intermediate Staphylococcus aureus (hVISA) isolates are increasing among methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates, but their relevance remains uncertain. We compared the V-MIC (Etest) and the frequency of hVISA (Etest macromethod) for all MRSA blood isolates saved over an 11-year span and correlated the results with the clinical outcome. We tested 489 isolates: 61, 55, 187, and 186 isolates recovered in 1996-1997, 2000, 2002-2003, and 2005-2006, respectively. The V-MICs were ≤1, 1.5, 2, and 3 μg/ml for 74 (15.1%), 355 (72.6%), 50 (10.2%), and 10 (2.1%) isolates, respectively. We detected hVISA in 0/74, 48/355 (13.5%), 15/50 (30.0%), and 8/10 (80.0%) isolates with V-MICs of ≤1, 1.5, 2, and 3 μg/ml, respectively (P < 0.001). The V-MIC distribution and the hVISA frequency were stable over the 11-year period. Most patients (89.0%) received vancomycin. The mortality rate (evaluated with 285 patients for whose isolates the trough V-MIC was ≥10 μg/ml) was comparable for patients whose isolates had V-MICs of ≤1 and 1.5 μg/ml (19.4% and 27.0%, respectively; P = 0.2) but higher for patients whose isolates had V-MICs of ≥2 μg/ml (47.6%; P = 0.03). However, the impact of V-MIC and hVISA status on mortality or persistent (≥7 days) bacteremia was not substantiated by multivariate analysis. Staphylococcal chromosome cassette mec (SCCmec) typing of 261 isolates (including all hVISA isolates) revealed that 93.0% of the hVISA isolates were SCCmec type II. These findings demonstrate that the V-MIC distribution and hVISA frequencies were stable over an 11-year span. A V-MIC of ≥2 μg/ml was associated with a higher rate of mortality by univariate analysis, but the relevance of the V-MIC and the presence of hVISA remain uncertain. A multicenter prospective randomized study by the use of standardized methods is needed to evaluate the relevance of hVISA and determine the optimal treatment of patients whose isolates have V-MICs of ≥2.0 μg/ml.The treatment of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) bacteremia with vancomycin is often associated with a poor clinical outcome (6, 15, 28, 40). Treatment failure was reported among patients infected with isolates whose vancomycin MICs were ≥4 μg/ml (6, 9, 12, 25, 28, 42). This prompted the Clinical and Laboratory Standards Institute to lower the cutoffs for S. aureus susceptibility to ≤2 μg/ml for susceptible, 4 to 8 μg/ml for intermediate (vancomycin-intermediate S. aureus [VISA]), and 16 μg/ml for resistance (39). Within the susceptibility range, the MIC is reported to increase over time (14, 25, 35-40). This is often referred to as MIC creep (38). Additionally, isolates with heteroresistance (heteroresistant vancomycin-intermediate S. aureus [hVISA]) are emerging, and this has uncertain implications for laboratory detection and clinical management (2, 5, 15, 24, 40-42). The first isolate of hVISA to be identified was reported from Japan in 1997 (11). Since then, it has been reported worldwide at frequencies of 0 to 50% (2, 4, 6, 9, 12, 19, 20, 21, 24, 26, 27, 31, 40, 42, 44). This disparity in frequency is probably a result of its variable incidence and the different testing methodologies used. Likewise, the frequency of isolates with MICs of 1.5 to <4 μg/ml varies according to the testing method used (3, 32). The relevance of an MIC on the higher side of the susceptibility range and the presence of hVISA isolates remains uncertain (8, 19, 21). Therapeutic failure was reported in patients infected with isolates with vancomycin MICs of 2 μg/ml (6, 12, 28) and 1.5 or 1 μg/ml (25, 34, 37). Most clinical microbiology laboratories use automated testing methods that are known to underestimate the vancomycin MIC (13, 24). Additionally, most previous studies addressing the relevance of such isolates were observational and usually involved only a few patients and poorly selected controls (1, 4, 7, 9, 12, 14, 25, 35, 38, 42). At our institution, we found the frequency of hVISA isolates among isolates from patients with persistent MRSA bacteremia to be 14%; however, heteroresistance did not correlate with the mortality rate (19). In the current study, we tested all blood MRSA isolates collected over 11 years to determine whether the vancomycin MIC and the prevalence of hVISA have changed over time and to evaluate the effects of increasing vancomycin MICs and the hVISA frequency on patient outcomes.     

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.)     

Hemagglutination Test for Rapid Serodiagnosis of Human Pythiosis

Human pythiosis is an emerging, life-threatening infectious disease, caused by the oomycete Pythium insidiosum. Thailand is an area where human pythiosis is endemic and the genetic blood disorder thalassemia is a predisposing factor. Patients with pythiosis present with arterial occlusions of the lower extremities, corneal ulcers, or chronic cutaneous infections. Diagnosis relies on time-consuming, relatively insensitive tests such as culture identification and immunodiffusion assay. Most patients undergo surgical removal of infected organs, and many die from the infection. Delayed diagnosis results in a poor prognosis. Here, we describe a hemagglutination (HA) test for rapid diagnosis of human pythiosis. Sheep red blood cells were coated with P. insidiosum protein extract and used in duplicated detection assays using serum samples from 33 patients with vascular (n = 27), cutaneous (n = 2), or ocular (n = 4) pythiosis and serum samples from 289 control patients with other infectious diseases (n = 77), with highly positive antinuclear antibody (n = 5), with thalassemia (n = 21), or with no known disorder (i.e., healthy blood donors) (n = 186). Based on receiver-operating characteristic analysis, a serum titer of 1:160 was selected as the cutoff point for the HA test. Serum samples that generated HA at the cutoff titer were read as positive, while samples that did not were read as negative. Positive results were obtained with the serum samples of all patients with vascular and cutaneous pythiosis and with two serum samples from the control group. Negative results were obtained with serum samples from all ocular pythiosis patients and the 287 remaining serum samples from the control group. Sensitivity and specificity of the HA were 88% and 99%, respectively. In conclusion, the HA test for detection of anti-Pythium antibodies is a simple, rapid, and reliable test for serodiagnosis of vascular and cutaneous pythiosis.Pythiosis is can be a fatal infectious disease of humans and animals living in tropical and subtropical countries (2, 9, 15, 16, 18, 27, 30). The causative agent is the fungus-like organism Pythium insidiosum, which is a member of the family Pythiaceae, order Pythiales, class Oomycetes, and the phylum Pseudofungi in the kingdom Chromista (Stramenopila) (5, 9, 18, 30). Naturally, P. insidiosum inhabits swampy areas, where it is present in the form of mycelium or biflagellate zoospores (5, 19). The zoospore is an infective stage where it can swim, attach to, and penetrate host tissue, possibly leading to pathology (18, 19).Although pythiosis in animals has been increasingly reported worldwide, most human pythiosis cases have been reported in Thailand, where it is considered to be endemic (8, 14, 16, 17, 26, 28, 30, 33). Thalassemia and agriculture-related careers are predisposing factors for human pythiosis (16, 17, 28). Clinical features of human pythiosis can be categorized into four forms as follows. (i) Vascular pythiosis (59% of reported cases) is an infection of the arteries leading to arterial occlusion and aneurysm. In advanced cases, many patients die, and since the main treatment is limb amputation, many patients become handicapped. (ii) Ocular pythiosis (33%) is an infection of the eyes, in which patients usually present with corneal ulcers or keratitis. Most of these patients undergo enucleation therapy to control the infection. (iii) Patients with cutaneous pythiosis (5%) present with granulomatous and ulcerative lesions confined to cutaneous and subcutaneous tissues. (iv) Disseminated pythiosis (3%) is an infection of other internal organs, such as the brain, sinuses, or gastrointestinal tract. The use of conventional antifungal drugs is ineffective in treatment of pythiosis because Pythium is only distantly related phylogenetically to fungi, and radical surgery is the main treatment option (16, 17, 29).Delayed diagnosis leads to delayed treatment and a poorer prognosis in patients with pythiosis. Diagnosis by culture identification of P. insidiosum is time-consuming and laborious (3, 23). Serodiagnosis of pythiosis commonly relies on an immunodiffusion (ID) test. Although the ID test is highly specific, it has very poor sensitivity (11, 12, 21, 25). Subsequently, other diagnostic methods, such as an in-house enzyme-linked immunosorbent assay (ELISA), an immunochromatographic test (ICT), a Western blot assay, and a PCR assay, were developed and have good specificity and sensitivity (11-13, 20, 22, 32). However, the lack of diagnostic materials and special equipment needed for these tests limits their use, especially in rural areas where the disease is prevalent. Here, we describe a hemagglutination (HA) test to assist a rapid diagnosis of human pythiosis. The test is easy to perform, requires only routine laboratory equipment and could easily be adapted to a simple kit format.     

Effects of Volume and Site of Blood Draw on Blood Culture Results

Blood culture contamination greatly affects clinical decisions. Hence, it is of interest to assess the influence of factors such as the volume of blood drawn and the site of blood draw on the rates of blood culture contamination. In a retrospective study, blood cultures from infants and children up to 18 years of age who had at least one positive blood culture during the year 2006 were analyzed for their volume of blood drawn, patient''s weight, site of blood draw used, and blood culture results. Blood cultures were deemed adequate collections if they contained an appropriate weight-related volume of blood. Moreover, blood culture results were categorized as true pathogens, contaminants, and negative cultures; these were then compared and analyzed with respect to their volume and site of blood draw. A total of 5,023 blood cultures were collected during 2006, of which 843 were analyzed. There were 306 (36%) positive cultures among the 843 cultures analyzed. Of the 306 positive cultures, 98 (32%) were contaminants and 208 (68%) cultures grew significant pathogens. Thirty-five percent of the contaminant cultures had adequate volume compared to 60% in the true bacteremia group (P < 0.001). Also, of the 843 cultures, the rates of contamination among the different sites of blood draw were as follows: peripheral venipuncture, 36%; arterial, 10%; and central venous access, 7% (P = 0.155). The rate of contamination was higher with lower blood volumes, and there was no significant difference in the rates of contamination among the different sites of blood draw.Blood cultures are vital for identifying pathogens causing serious infections and in directing appropriate antibiotic therapy. Moreover, they remain the standard method for detecting bacteremia in the evaluation of sick patients (14). Unfortunately, blood culture contamination is a common occurrence and may lead to confusion regarding the significance of a positive blood culture. The most common contaminants are coagulase-negative staphylococcus species which are also becoming more prevalent as a primary pathogen in immunocompromised patients and in patients with indwelling intravascular devices (9, 15). The uncertain clinical significance of potential contaminants leads to longer hospital stays, unnecessary antibiotic therapy, and additional laboratory testing; as a result, the cost incurred by a hospital is many times that incurred by the laboratory (2).Many factors influence the yield of blood cultures, but the single most important factor is blood volume. Several studies have shown that the rate of isolation of pathogens from blood cultures increases with the quantity of blood submitted (12). Hence, a blood culture may be falsely negative from an inadequate-volume blood culture (6). Furthermore, the blood culture contamination rate inversely correlates with the volume of blood (3). The site and method of blood collection have also been known to influence the rate of contamination of blood cultures (8). Vascular-access devices, such as arterial and central venous catheters, pass through the skin and are susceptible to bacterial colonization. Hence, it is easy for these bacteria to colonize and multiply in and around these ports, and they can be pulled into blood specimens drawn from those sites. Hence, the primary aims of this study were to determine the volume of blood obtained for culture in routine clinical practice and to evaluate if inadequate blood volumes lead to an increased incidence of contaminants. Finally, this study also assessed whether the site of blood draw was related to an increased frequency of contaminated cultures.     

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.     

The Pan Genera Detection Immunoassay: a Novel Point-of-Issue Method for Detection of Bacterial Contamination in Platelet Concentrates

Bacterial contamination of platelet concentrates (PCs) still represents an ongoing risk in transfusion-transmitted sepsis. Recently the Pan Genera Detection (PGD) system was developed and FDA licensed for screening of bacterial contamination of PCs directly prior to transfusion. The test principle is based on the immunological detection of lipopolysaccharide (for Gram-negative bacteria) or lipoteichoic acid (for Gram-positive bacteria). In the present study we analyzed the applicability of this method with regard to detection limit, practicability, implementation, and performance. PCs were spiked with Staphylococcus aureus, Bacillus subtilis, and five different Klebsiella pneumoniae strains, as well as eight different Escherichia coli strains. The presence of bacteria was assessed by the PGD immunoassay, and bacteria were enumerated by plating cultures. Application of the PGD immunoassay showed that it is a rapid test with a short hands-on time for sample processing and no demand for special technical equipment and instrument operation. The lower detection limits of the assay for Gram-positive bacteria showed a good agreement with the manufacturer''s specifications (8.2 × 10³ to 5.5 × 10⁴ CFU/ml). For some strains of K. pneumoniae and E. coli, the PGD test showed analytical sensitivities (>10⁶ CFU/ml) that were divergent from the designated values (K. pneumoniae, 2.0 × 10⁴ CFU/ml; E. coli, 2.8 × 10⁴ CFU/ml). Result interpretation is sometimes difficult due to very faint bands. In conclusion, our study demonstrates that the PGD immunoassay is an easy-to-perform bedside test for the detection of bacterial contamination in PCs. However, to date there are some shortcomings in the interpretation of results and in the detection limits for some strains of Gram-negative bacteria.Bacterial contamination of platelet concentrates (PCs) still represents an ongoing risk in transfusion-transmitted sepsis. In 2004, sterility testing of PCs was recommended by the American Association of Blood Banks, and the detection of bacterial contamination in PCs has been implemented in several blood centers and transfusion services as routine quality control testing (11). However, transfusion-transmitted bacterial sepsis has still not been completely eliminated, with septic complications observed particularly with older PCs (6, 8, 11, 12, 20, 26, 32, 34). At present, the detection of microbiological contamination in PCs can be divided into two major methodological concepts (19): (i) incubation or cultivation methods and (ii) rapid detection methods, such as nucleic acid amplification techniques (NAT) (9, 16, 28), fluorescence-activated cell sorting (FACS) (10, 13, 17, 30, 31), or immunological detection methods (Pan Genera Detection [PGD] system) (24, 25).Incubation or cultivation methods are currently the most sensitive detection methods and are utilized predominantly for sterility testing of PCs (7). Nevertheless, culture-based methods require 24 h prior to sampling and at least 18 h to 24 h of incubation to obtain a positive result (5, 15, 23, 24). Therefore, culture-based methods have to be combined with an early sampling strategy, carrying a high risk of sampling errors. The initial levels of most skin-based bacteria in PC units are usually remarkably low, and it has been demonstrated that 57% of cultures are false negative at low contamination levels of 10 to 100 CFU per PC unit (22). Application of rapid detection methods combined with an early sampling strategy will also invariably miss bacterial contamination as a result of the sampling error. Therefore, a prolonged time frame between sampling and testing will increase the probability that most contaminated PCs will be identified. With cultural approaches, results are not available in time to avoid transfusion of the contaminated PC.Hence, substantial interest focuses on rapid detection methods for bacterial screening combined with a late sampling strategy. In this context, a sensitive, specific, cost-effective, rapid, and easy-to-perform point-of-issue bacterial detection test immediately before transfusion (24, 25), requiring only a small test sample volume, is considered optimal. Recently, the Pan Genera Detection (PGD) system was developed and FDA licensed [501(k) clearance] for the screening of bacterial contamination directly prior to transfusion. Experience regarding the implementation and performance of this technology has seldom been reported (24, 25). A first study using the PGD test for screening of 7,733 whole-blood derived PCs has been published (38). The test principle is based on the immunological detection of the conserved bacterial antigens lipopolysaccharide (LPS) (for Gram-negative bacteria) or lipoteichoic acid (LTA) (for Gram-positive bacteria) by lateral-flow immunoprecipitation. In a previous study, we developed a novel rapid screening method based on flow cytometric detection (10), which was compared with the PGD test, among others. Preliminary results revealed that the PGD test detected Gram-positive bacteria in the given range, but Gram-negative bacterial species such as Klebsiella pneumoniae were detected with considerably divergent detection limits, as specified by the manufacturer. Based on these data, we have evaluated this effect systematically and in detail in the present study.     

Development and Application of a Broadly Sensitive Dried-Blood-Spot-Based Genotyping Assay for Global Surveillance of HIV-1 Drug Resistance

As antiretroviral therapy (ART) is scaled up in resource-limited countries, surveillance for HIV drug resistance (DR) is vital to ensure sustained effectiveness of first-line ART. We have developed and applied a broadly sensitive dried-blood-spot (DBS)-based genotyping assay for surveillance of HIV-1 DR in international settings. In 2005 and 2006, 171 DBS samples were collected under field conditions from newly diagnosed HIV-1-infected individuals from Malawi (n = 58), Tanzania (n = 60), and China (n =53). In addition, 30 DBS and 40 plasma specimens collected from ART patients in China and Cameroon, respectively, were also tested. Of the 171 DBS analyzed at the protease and RT regions, 149 (87.1%) could be genotyped, including 49 (81.7%) from Tanzania, 47 (88.7%) from China, and 53 (91.4%) from Malawi. Among the 70 ART patient samples analyzed, 100% (30/30) of the Chinese DBS and 90% (36/40) of the Cameroonian plasma specimens were genotyped, including 8 samples with a viral load of <400 copies/ml. The results of phylogenetic analyses indicated that the subtype, circulating recombinant form (CRF), and unique recombinant form (URF) distribution was as follows: 73 strains were subtype C (34%), 37 were subtype B (17.2%), 24 each were CRF01_AE or CRF02_AG (11.2% each), 22 were subtype A1 (10.2%), and 9 were unclassifiable (UC) (4.2%). The remaining samples were minor strains comprised of 6 that were CRF07_BC (2.8%), 5 that were CRF10_CD (2.3%), 3 each that were URF_A1C and CRF08_BC (1.4%), 2 each that were G, URF_BC, and URF_D/UC (0.9%), and 1 each that were subtype F1, subtype F2, and URF_A1D (0.5%). Our results indicate that this broadly sensitive genotyping assay can be used to genotype DBS collected from areas with diverse HIV-1 group M subtypes and CRFs. Thus, the assay is likely to become a useful screening tool in the global resistance surveillance and monitoring of HIV-1 where multiple subtypes and CRFs are found.Filter papers have been useful for the collection, storage, and testing of blood specimens in the diagnostic screening of metabolic and inherited disorders in newborn babies in the United States for many years (13, 14). More recently, many resource-limited countries have been using them for HIV-related molecular assays, including early infant diagnosis using Roche Amplicor HIV-1 DNA PCR testing (supported by the U.S. President''s Emergency Plan for AIDS Relief [PEPFAR]) (31), real-time PCR-based HIV-1 diagnosis (22, 27) and viral load measurement (6, 17, 18, 20, 26), and HIV-1 drug resistance (DR) genotyping (5, 7, 8, 12, 16, 19, 23-25, 28, 30, 35-37).Dried blood spots (DBS) offer several advantages over conventional plasma or serum for sample collection and storage. First, DBS circumvent the need for phlebotomy and reduce the risk of needle-stick-related HIV exposure when they are collected using finger or heel stick. Second, they do not require cold-chain transportation of specimens from collection sites to the testing laboratories; thus, they can be transported using standard postal services, resulting in the reduction of cost for storage and transportation when dried and processed appropriately. Overall, DBS provide resource-limited countries with opportunities for sustainable laboratory services and surveillance programs for HIV diagnosis and DR genotyping. Very few studies have been conducted using DBS collected under real field conditions. Furthermore, the DR genotyping assays used were not evaluated for multiple HIV-1 subtypes and circulating recombinant forms (CRFs) (5, 16, 19, 23, 25, 37).The aim of the current study was to determine the performance of a broadly sensitive genotyping assay for detecting multiple HIV-1 group M subtypes and CRFs that cocirculate in different countries, using DBS collected under field conditions.     

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

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

Prospective Comparison of Eubacterial PCR and Measurement of Procalcitonin Levels with Blood Culture for Diagnosing Septicemia in Intensive Care Unit Patients

Rapid identification of infection has a major impact on the clinical course, management, and outcome of critically ill intensive care unit (ICU) patients. We compared the results of PCR and procalcitonin with blood culture for ICU patients suspected of having septicemia. Ninety patients (60 patients meeting the criteria for sepsis and 30 patients not meeting the criteria for sepsis) were evaluated. Compared with blood culture as the gold standard, the sensitivity, specificity, and positive and negative predictive values for PCR were 100%, 43.33%, 46.87%, and 100%, respectively, and for procalcitonin were 100%, 61.66%, 56.6%, and 100%, respectively. The average times required to produce a final result were as follows: PCR, 10 h; blood culture, 33 h; procalcitonin, 45 min. Both PCR and procalcitonin may be useful as rapid tests for detecting septicemia but compared with blood cultures lacked specificity.A rapid and reliable system to detect bacteria in the bloodstream would be clinically useful as it could guide early appropriate antibiotic treatment and result in improved patient survival (14). The gold standard for the diagnosis of infection is the isolation and identification of organisms by culture (27). This process usually requires 24 h or more. A large proportion of patients suspected of having septicemia have negative blood cultures (3) due to either previous antibiotic treatment, samples of small volume, transient bacteremias, or sepsis of nonbacterial origin (8, 30). Given the slowness and low sensitivity of blood culture, there is a need for more-rapid and more-sensitive techniques. PCR, which amplifies characteristic genes of microorganisms, is one such technique. In clinical conditions with diverse etiological agents in sterile sites, e.g., blood in sepsis, a broad-range bacterial PCR which uses a primer pair aimed at highly conserved DNA coding regions on bacterial rRNA can be used (8, 10, 11, 20). This is described as eubacterial PCR as well as broad-range bacterial PCR as it detects an rRNA gene component present in all bacteria. PCR cannot differentiate DNA sequences from viable and nonviable bacteria. The value of this test may be enhanced if it is coupled with a host response biomarker indicative of infection and systemic inflammation. Procalcitonin is one such marker and is gaining increasing importance in identification of sepsis (1, 15, 16). Procalcitonin levels are undetectable in healthy individuals but increase in patients with bacterial sepsis and correlate well with the severity of the illness (5, 19, 29).The aim of this study was to compare the results for eubacterial PCR and procalcitonin with blood culture in intensive care unit (ICU) patients suspected of having septicemia.     

Comparison of the Bactec 9240 and BacT/Alert Blood Culture Systems for Evaluation of Placental Cord Blood for Transfusion in Neonates

The Bactec 9240 and the BacT/Alert blood culture systems were compared as a means for detection of bacterial contaminants in whole blood, concentrated red cells, and plasma preparations prepared from umbilical cord blood (UCB) samples. Ninety-two UCB units seeded with low levels of various bacteria were evaluated. In more than 50% of cases, growth was not detected in plasma using either system (P < 0.001). When concentrated red cells and whole blood were compared, the Bactec system detected bacterial growth consistently sooner than the BacT/Alert system in all seeded bacteria except Staphylococcus species in whole blood. The median lengths of time to detection (LTD) for whole blood and concentrated cells in BacT/Alert were 18.7 h and 18.5 h, respectively. The median LTD for the same blood fractions using the Bactec system were 16.05 h and 15.64 h. These differences in LTD by blood culture system and sample type were statistically significant (whole blood, P = 0.0449; concentrated cells, P = 0.0037). Based on the results of our study, we recommend the use of either concentrated red cells or whole blood for sterility testing in UCB samples. In our laboratory, the Bactec system compared to the BacT/Alert system was the superior method for rapid detection of bacterial contaminants in cord blood.While all neonates experience a decline in their circulating red blood cells immediately after birth, anemia is a more common complication for premature neonates (27, 28). Annually in the United States, an estimated 130,000 anemic, critically ill infants receive approximately one million red blood cell transfusions (31). Autologous blood transfusions have been shown to be safe in both adult and pediatric patients (17, 21, 25). Umbilical/placental cord blood is autologous blood from a neonate (20), and the use of autologous umbilical cord blood (UCB) has long been discussed among neonatologists (5, 6, 9, 10, 29). Owing to the increasing utilization of UCB for the transplantation of hematopoietic stem cells, significant progress has been made in developing safer and more efficient collection techniques for UCB (12, 14). In neonates, bacterial contamination has been described as the third most common cause of transfusion-related fatality, with most fatalities occurring in gram-negative sepsis (13). Unfortunately, many cases of transfusion-transmitted bacterial infection remain unrecognized and underreported (4, 18, 30). While much experience exists now regarding the efficacy, recovery, and safety of UCB, only few studies investigated the prevalence of bacterial contamination of cord blood. These studies report variable bacterial contamination rates of between 1.85 and 12% (3, 7, 8, 10, 14). Bacterial contamination consists predominantly of organisms known as typical skin contaminants similar to those described in adult blood culture collections. Organisms of the vaginal flora have been described as an additional and important component of contaminants in UCB. The American Association of Blood Banks (AABB) standards require that a small volume of collected UCB be used for sterility testing. While general regulations exist for the evaluation of safety, including bacterial and viral pathogens, in adult blood and platelet collections as well as human cell therapy products, to our knowledge no specific method requirements for the evaluation of bacterial contamination of UCB for autologous transfusions have been published to date (1, 11). The two most frequently used FDA-approved automated, continuous-monitoring blood culture systems in the United States are the BacT/Alert system (bioMérieux, Durham, NC) and the Bactec system (BD Microbiology, Sparks, MD). In the current study, we investigated the performance of the Bactec 9240 and BacT/Alert continuous-monitoring blood culture systems for the detection of “seeded” bacterial contaminants in UCB samples compared to adult blood collections, with an additional focus on detection of “seeded” bacteria in various fractions of cord blood components.     

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.     

Three Doses of an Experimental Detoxified L3-Derived Lipooligosaccharide Meningococcal Vaccine Offer Good Safety but Low Immunogenicity in Healthy Young Adults

This open, randomized phase I study evaluated the safety and reactogenicity of an experimental meningococcal serogroup B (MenB) vaccine obtained from outer membrane vesicle detoxified L3-derived lipooligosaccharide. Healthy young adults (n = 150) were randomized to receive either experimental vaccine (provided in five formulations, n = 25 in each group) or VA-Mengoc-BC (control, n = 25) administered on a 0- to 6-week/6-month schedule. Serum bactericidal assays performed against three MenB wild-type strains assessed the immune response, defined as a 4-fold increase from pre- to postvaccination. No serious adverse events related to vaccination were reported. Pain at the injection site, fatigue, and headache were the most commonly reported adverse events. Solicited adverse events graded level 3 (i.e., preventing daily activity) were pain (up to 17% of the test subjects versus 32% of the controls), fatigue (up to 12% of the test subjects versus 8% of the controls), and headache (up to 4% of any group). Swelling graded level 3 (greater than 50 mm) occurred in up to 4% of the test subjects versus 8% of the controls. The immune responses ranged from 5% to 36% across experimental vaccines for the L3 H44-76 strain (versus 27% for the control), from 0% to 11% for the L3 NZ98/124 strain (versus 23% for the control), and from 0% to 13% for the L2 760676 strain (versus 59% for the control). All geometric mean titers were below those measured with the control vaccine. The five experimental formulations were safe and well tolerated but tended to be less immunogenic than the control vaccine.Meningococcal diseases caused by Neisseria meningitidis are a significant health burden throughout the world, leading to death and permanent sequelae (15). Whereas polysaccharide or polysaccharide conjugate vaccines are effective against serogroups A, C, Y, and W135, N. meningitidis serogroup B (MenB) remains a major cause of death and morbidity throughout the world, infants less than 1 year of age being affected the most (5, 8). Serogroup B outbreaks were reported in Europe, Latin America, Australia, New Zealand, and the United States (3, 7, 22, 33). Immunization against MenB presents a challenge, as the capsular polysaccharide is poorly immunogenic in humans (4) and shares molecular mimicry with human antigens (11), which guided the search for outer membrane vesicle (OMV) vaccines (16).Three MenB OMV vaccines with PorA protein as the dominant antigen have been brought to the market (VA-Mengoc-BC [Finlay Institute], MeNZB [Chiron], and MenBvac [Norwegian Institute of Public Health]), but although they have shown protection against PorA-heterologous strains in older children and adults, protection of the youngest is mostly against PorA-homologous MenB strains and their accessibility is geographically limited (7, 9, 18, 21, 25, 26, 31, 34, 36, 37). To be immunogenic in the pediatric and adult populations, a more comprehensive MenB vaccine should include antigens inducing cross-reactive serum bactericidal antibodies (SBA) against a broad spectrum of circulating strains (16, 17, 20, 21, 35). That could best be achieved with non-PorA vaccines (20).Natural immunity against MenB is also induced by protein and lipooligosaccharide (LOS) antigens (28), but proteins and LOS may vary substantially across meningococcal strains. However, at least 70% of invasive MenB isolates express LOS of immunotype L3,7 (19, 27, 29, 30). Hence, GlaxoSmithKline (GSK) Biologicals has developed an experimental vaccine based on the LOS L3 immunotype that was shown to induce bactericidal antibodies in preclinical studies (39). Two detoxified LOS type 3 MenB experimental vaccines differing by the length of the LOS were developed. Such formulations have shown good safety and immunogenicity during preclinical and toxicological studies (39).The primary objective of this study was to evaluate the safety and reactogenicity of several formulations of the experimental vaccines given to healthy young adults. The secondary objective was to assess the immunogenicity of the different formulations.     

Rapid Mycobacterial Liquid Culture-Screening Method for Mycobacterium avium Complex Based on Secreted Antigen-Capture Enzyme-Linked Immunosorbent Assay

Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer''s instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1.4%) were MAC-ELISA false negative, and only 7 of 219 MGIT signal-negative cultures (3.2%) were false positive. The MAC-ELISA is a low-cost, rapid, sensitive, and specific test for MAC in liquid cultures. It could be used in conjunction with or independent of automated culture reading instrumentation. For maximal accuracy and subspecies-specific identification, use of a confirmatory multiplex MAC PCR is recommended.Members of the Mycobacterium avium complex (MAC) are a family of intracellular bacterial pathogens causing significant disease in both animals and humans. The complex contains four subspecies of M. avium: M. avium subsp. avium, M. avium subsp. paratuberculosis, M. avium subsp. hominissuis, and M. avium subsp. silvaticum (24, 35). Mycobacterium intracellulare is also a member of the complex (20, 35).The clinical importance of MAC infection has increased in recent decades because of the greater population of immunocompromised individuals with longer life expectancies, immunosuppressive chemotherapy, and the spread of human immunodeficiency virus infection (8, 20, 25, 27). With AIDS patients, the incidence of disseminated mycobacterial infection caused by MAC strains can reach up to 50% (19). Although these mycobacterial infections are not often characterized to subspecies, it appears that M. avium subsp. hominissuis is most often involved with AIDS patients (3, 4, 18, 24, 35). In addition, M. avium subsp. hominissuis causes infection in a subset of patients without an obvious immune defect (13) or underlying pulmonary disease and in children with lymphadenitis or cystic fibrosis (31). In virtually all cases, these organisms are believed to be of environmental origin: surface water, tap water, soil, dust, or food (22, 24, 29, 38). M. avium subsp. avium, ubiquitous in the environment and more virulent than M. avium subsp. hominissuis, is distinguished by the insertion element IS901 (24). While capable of infecting multiple animal species, M. avium subsp. avium is commonly isolated from birds as one of the causes of avian tuberculosis (26, 32). M. avium subsp. silvaticum, also called the “wood pigeon bacillus,” is uncommonly isolated but reported to cause enteritis in ruminants as well as disseminated infection in other hosts (33).M. avium subsp. paratuberculosis infection causes paratuberculosis (Johne''s disease) characterized by chronic granulomatous enteritis in animals, most often ruminants (9, 21). This organism grows very slowly in vitro (slower than most “slow-growing” mycobacteria), is dependent on mycobactin for growth in vitro, and is alone in containing IS900 in its genome (15, 16, 23). M. avium subsp. paratuberculosis has a broad host range and is implicated by some in the pathogenesis of Crohn''s disease in humans (1, 12). The inability of M. avium subsp. paratuberculosis to produce the siderophore mycobactin renders it incapable of replication in the environment, with the possible exception of inside free-living amoeba, and so it is considered an obligate parasite of animals and possibly humans (6). Paratuberculosis has emerged as a common and costly disease for the dairy industry (16). Surveys indicate that at least 68% of U.S. dairy herds are M. avium subsp. paratuberculosis infected (36).Microbiological culture remains a mainstay for diagnosis of mycobacterial infections, since it has greater sensitivity than PCR-based methods and yields the living isolates necessary for antibiotic susceptibility testing and molecular epidemiology. Because culture on conventional solid bacteriological media is laborious and slow, liquid culture-based mycobacterial detection systems, such the Bactec, MGIT, Trek ESP, and BacT/Alert 3D systems, have become commonplace in clinical laboratories, offering the advantages of automation and shorter detection times from clinical samples (5, 7, 17, 37). However, a positive signal during culture with any of these systems is simply a nonspecific indication of any sort of microbial growth (37). Thus, specimen processing and decontamination protocols to selectively kill nonmycobacterial microflora in the clinical or environmental samples are key components for an effective assay (7, 34). Although a number of different protocols have been described (7, 11, 28, 34), a standard protocol specifically designed for optimal recovery of MAC has not yet been established.Numerous PCRs are performed in our laboratory in response to these signal-positive cultures; in the last year, approximately 45% did not contain the pathogen of interest, MAC (unpublished data). This sample management approach is inefficient and labor-intensive.To better focus PCR resources on those cultures most likely to contain MAC, a novel enzyme-linked immunosorbent assay (ELISA) was designed to detect secreted MAC antigens in culture medium fluid. This assay, called the MAC-ELISA, was then evaluated for analytical and diagnostic specificity and sensitivity, first using pure cultures and then cultures derived from clinical samples.