Pilot Study of Association of Bacteria on Breast Implants with Capsular Contracture

Capsular contracture is the most common and frustrating complication in women who have undergone breast implantation. Its cause and, accordingly, treatment and prevention remain to be elucidated fully. The aim of this prospective observational pilot study was to test the hypothesis that the presence of bacteria on breast implants is associated with capsular contracture. We prospectively studied consecutive patients who underwent breast implant removal for reasons other than overt infection at the Mayo Clinic from February through September 2008. Removed breast implants were processed using a vortexing/sonication procedure and then subjected to semiquantitative culture. Twenty-seven of the 45 implants collected were removed due to significant capsular contracture, among which 9 (33%) had ≥20 CFU bacteria/10 ml sonicate fluid; 18 were removed for reasons other than significant capsular contracture, among which 1 (5%) had ≥20 CFU/10 ml sonicate fluid (P = 0.034). Propionibacterium species, coagulase-negative staphylococci, and Corynebacterium species were the microorganisms isolated. The results of this study demonstrate that there is a significant association between capsular contracture and the presence of bacteria on the implant. The role of these bacteria in the pathogenesis of capsular contracture deserves further study.Breast implants are used for reconstruction after mastectomy and for breast augmentation. According to the American Society of Plastic Surgery, in 2007 breast augmentation became the leading cosmetic surgical procedure in the United States, with 347,500 procedures performed annually (http://www.plasticsurgery.org/media/statistics/index.cfm). Women who have undergone breast implantation may experience local complications during the ensuing years. Capsular contracture is the most common and frustrating complication (1, 3), with a reported incidence as high as 50 to 74% according to some studies (4, 9). Capsular contracture is classified according to the Baker classification system (14), as follows: grade I, breast absolutely natural; grade II, minimum contracture; grade III, moderate contracture; and grade IV, severe contracture. The cause of capsular contracture and, accordingly, its treatment and prevention remain to be elucidated fully. A number of factors, including foreign body reaction, hematoma, and peri-implant infection, have been suggested (17). Several lines of evidence suggest, in a preliminary way, a role of subclinical infection in capsular contracture pathogenesis (7, 16, 21). Local skin flora (e.g., coagulase-negative staphylococci, Propionibacterium acnes, and Corynebacterium species) may gain access to breast implants during or following placement. Some have suggested that biofilms form on the implant, stimulating fibrosis around the implant and, ultimately, capsular contracture (6, 15, 17, 21). However, studies examining this issue have not used techniques to specifically sample implant-associated biofilms in a quantitative fashion; accordingly, findings of some prior studies may represent contamination.We have developed a new technique which uses a combination of vortexing and sonication to sample biofilm bacteria on the surfaces of implants (20). We have shown that this technique is more sensitive than periprosthetic tissue culture for the diagnosis of prosthetic joint infection (20). Moreover, the combination of vortexing and sonication to disrupt bacterial biofilms, followed by culture, has been demonstrated to be a sensitive method for detecting bacteria adherent to bone cement and other surfaces (11). We hypothesized that bacterial biofilms are present in some patients with breast implant capsular contracture. We performed a prospective observational pilot study to test our hypothesis.     

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

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

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.)     

Evaluation of the C.Diff Quik Chek Complete Assay,a New Glutamate Dehydrogenase and A/B Toxin Combination Lateral Flow Assay for Use in Rapid,Simple Diagnosis of Clostridium difficile Disease

The diagnosis of Clostridium difficile infection continues to be a challenge for many clinical microbiology laboratories. A new lateral flow assay, the C.Diff Quik Chek Complete assay, which tests for the presence of both glutamate dehydrogenase (GDH) and C. difficile toxins A and B, was evaluated for its ability to diagnose C. difficile disease. The results of this assay were compared to those of both PCR and toxigenic culture. The results showed that this assay allows 88% of specimens to be accurately screened as either positive (both tests positive) or negative (both tests negative) for the presence of toxigenic C. difficile in less than 30 min and with minimal hands-on time. Use of a random-access PCR for the analysis of specimens with discrepant results (one test positive and the other negative) allows the easy, rapid, and highly sensitive (100%; 95% confidence interval [CI], 89.6 to 100%) and specific (99.6%; 95% CI, 97.3 to 99.9%) diagnosis of C. difficile disease. The use of this algorithm would save institutional costs, curtail unnecessary isolation days, reduce the nosocomial transmission of disease, and increase the quality of care for patients.The laboratory diagnosis of Clostridium difficile disease has evolved significantly over the last several years, and many tests that may be used to assist with the detection of C. difficile infection are now available. These assays include enzyme immunoassays (EIAs), lateral flow tests, PCR assays, tissue culture cytotoxicity neutralization tests, and toxigenic culture. Many recent papers have reported on the use of different algorithms that use the tests mentioned above to allow the better diagnosis of C. difficile disease (4, 5, 8, 18, 19, 20, 23, 26). Many of these approaches incorporate cytotoxicity neutralization (CTN) assays or anaerobic agar culture with identification of the organism, followed by toxin testing. However, many laboratories do not have the technical expertise, facilities, or training to perform CTN assays (which are labor-intensive and somewhat subjective), and an anaerobic agar culture with toxin detection may take several days; both of these methods delay the reporting of results (14, 17, 19, 20, 21, 29). The use of PCR for the diagnosis of this disease has been shown to be very specific and sensitive but often does not allow for random-access (i.e., real-time) results and can be quite costly to perform as a stand-alone testing method (2, 6, 10, 12, 15, 17, 27, 28). There is a real need for a rapid and simple testing strategy for the accurate diagnosis of C. difficile disease. With this in mind, we examined a simple, rapid two-step algorithm for the laboratory diagnosis of Clostridium difficile disease utilizing the new C.Diff Quik Chek Complete assay, a lateral flow kit that uses a combination of glutamate dehydrogenase (GDH) antigen detection plus toxin A and B detection for initial screening and a random-access, highly sensitive Xpert C. difficile PCR assay which detects the toxin B gene for samples with discrepant results.     

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.     

Efficient Diagnosis of Vulvovaginal Candidiasis by Use of a New Rapid Immunochromatography Test

The clinical symptoms of vulvovaginal candidiasis (VVC) are nonspecific, and misdiagnosis is common, leading to a delay in the initiation of antifungal treatment. We evaluated a new immunochromatography test (ICT), the CandiVagi assay (SR2B, Avrille, France), for the rapid diagnosis of VVC. This test, which employs an immunoglobulin M antibody directed against the β-1,2-mannopyranosyl epitopes found in the yeast cell wall, was compared with direct microscopic examination and culture of vaginal swabs. Two-hundred five women were investigated, including 130 women with symptomatic vaginitis and 75 asymptomatic controls. Two vaginal swabs were obtained from each woman: one was used to prepare a wet mount and Gram-stained preparations for direct microscopic examination and was also cultured on Sabouraud dextrose agar for the isolation of Candida spp., and the second swab was used for ICT. The sensitivities of microscopic examination, culture, and ICT for the diagnosis of VVC were 61%, 100%, and 96.6%, respectively, while the specificities of the three methods were 100%, 82%, and 98.6%, respectively. ICT had a negative predictive value of 98.6%, a positive predictive value of 96.6%, and an efficiency of 98%. ICT provided a rapid result and a better compromise between sensitivity and specificity than conventional microscopy and culture for the diagnosis of VVC. This easy-to-perform diagnostic test will be useful to practitioners treating women with symptoms of vaginitis.Vaginitis is the commonest reason for gynecological consultation in women of childbearing age. Anaerobic bacteria are the most prevalent cause of vaginal infection in the United States and Europe, followed closely by Candida spp. (34, 37). It is estimated that at least 75% of healthy adult women will suffer one episode of Candida vulvovaginitis during their reproductive lives and that 5% will have recurrent infectious episodes (20, 30). Candida albicans is responsible for infection in 80 to 90% of cases, although the incidence of vulvovaginal candidiasis (VVC) due to non-C. albicans species such as C. glabrata has increased steadily over the past few decades (21, 36).The main symptoms of VVC have been widely described and include vulvar and vaginal pruritus, pain or a burning sensation, and external dysuria (8). Physical examination may reveal perineal edema, vulvar and vaginal erythema, fissures, and a thick curdy discharge (8). However, these symptoms are nonspecific and do not enable clinicians to distinguish confidently between VVC, bacterial vaginosis, and Trichomonas vaginalis infection (2, 22, 23, 31), leading to subsequent suboptimal care. The accurate diagnosis of VVC currently depends on the demonstration of a Candida sp. in vaginal swabs by direct microscopic examination and/or culture. A positive Gram stain, the absence of a watery discharge, and patient self-diagnosis of “another yeast infection” have been identified as the best predictors of a positive culture for patients with VVC (1).Several rapid diagnostic tests have been developed over the past 25 years in an attempt to speed up the diagnosis of VVC (17, 29). Latex particle agglutination (LPA) was found to be more sensitive than KOH microscopy (19, 28, 29) and was more specific than other diagnostic criteria (10). However, a few studies reported false-positive reactions with this test (39) or a sensitivity that was lower than that of KOH microscopic examination carried out by experienced clinical practitioners (38).We have developed a sensitive immunochromatography test (ICT), the CandiVagi assay (SR2B, Avrille, France), for the rapid diagnosis of VVC using an immunoglobulin M (IgM) monoclonal antibody (MAb) directed against the Candida mannan (18, 40). Here, we present the results of a preliminary evaluation of this test and a comparison of the results obtained by ICT with those obtained by conventional microscopy and culture. Specific attention was focused on the ability of ICT to discriminate between Candida carriage and Candida infection and its specificity for women with bacterial/trichomonal vaginitis.     

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.     

Streptococcus pneumoniae DNA Load in Blood as a Marker of Infection in Patients with Community-Acquired Pneumonia

Direct detection of Streptococcus pneumoniae DNA in blood adds to culture results in the etiological diagnosis of patients with community-acquired pneumonia (CAP). Quantification of the amount of DNA, the bacterial DNA load (BDL), provides a measurement of DNAemia that may increase the understanding of the clinical relevance of S. pneumoniae DNA in blood. We evaluated the S. pneumoniae BDL as a diagnostic tool in adult patients with CAP. The BDL was determined in whole-blood samples collected simultaneously with blood for culture from 45 adult patients with CAP. After DNA extraction, S. pneumoniae DNA was detected with specific real-time PCR amplification, and the BDL was calculated with a standard curve. PCR and microbiological results were compared, and the BDL was related to clinical and laboratory parameters. S. pneumoniae DNA was detected in 10/13 patients with positive blood cultures and in 67% of patients with microbiologically confirmed pneumococcal pneumonia. The positive predictive values of the receiver operating characteristic curves for the BDLs for pneumococcal infection (100%) and pneumococcal bacteremia (69%) were higher than those for the level of C-reactive protein (CRP; 43% and 23%, respectively) and the white blood cell count (WBC; 42% and 35%, respectively); the negative predictive values of these three parameters were in the same range (±90 and ±97%, respectively). The BDL was higher in patients presenting with systemic inflammatory response syndrome and in patients with bacteremia. Positive correlations were observed for the BDL with WBC, CRP level, and length of stay. We conclude that the BDL supports the diagnosis of S. pneumoniae infection in patients with CAP and provides a putative marker of the severity of disease.Streptococcus pneumoniae is the most prevalent cause of community-acquired pneumonia (CAP) in adults (1, 10). Identification of S. pneumoniae as a cause of CAP can be achieved with Gram stain microscopy, culture of respiratory tract samples, blood culture, PCR, and antigen detection testing (26). A positive blood culture result provides a definite diagnosis. However, blood cultures have a low sensitivity for the confirmation of the pneumococcal etiology of CAP. This is due to the low prevalence of bacteremia in CAP, especially in uncomplicated cases, and the use of antibiotics prior to drawing blood samples for culture (11, 18, 32). In addition, blood cultures usually require 1 or 2 days before results are available and thus may have limited impact on the initial choice of empirical antimicrobial treatment (6, 29). PCR and antigen detection provide non-culture-based tools for the rapid identification of S. pneumoniae in respiratory tract specimens, pleural aspirate samples, and blood samples (7, 8, 13). These tests can add to culture methods in the etiological diagnosis of patients with CAP, particularly for patients who received antimicrobial treatment prior to sampling for culture (13, 26, 33).Several studies have evaluated the use of real-time PCR assays for the detection of S. pneumoniae DNA in blood samples from patients suspected of infection with invasive pneumococcal disease, with variable results (5, 14, 24, 31). These studies all used blood culture as the reference standard. However, positive PCRs relate not only to bacteremia but also to detection of DNA from nonviable bacteria, i.e., within phagocytes or due to the use of antibiotics. As such, it is understandable that most studies found cases with positive S. pneumoniae DNAemia and negative blood cultures. In this study, we used microbiologically documented S. pneumoniae infection as the gold standard for evaluation of PCR results and hypothesized that the bacterial DNA load (BDL) can be used to discriminate between patients with invasive infection and those with localized infection.The magnitude of bacteremia, as based on quantitative blood cultures, relates to the severity of S. pneumoniae infection (2). A high bacterial count in blood samples from children with bacteremia is associated with an increased risk of development of more-serious disease (27). Also, a shorter amount of time needed to detect positivity for blood cultures, supposedly reflecting a higher initial bacterial load, is associated with higher severity of disease (20). This relation between bacterial concentration and severity of disease may also hold true for the level of S. pneumoniae DNA in blood samples: the S. pneumoniae BDL was higher in human immunodeficiency virus-infected children who died from invasive pneumococcal disease than in survivors (5).In this study, we evaluated the S. pneumoniae BDL as a putative diagnostic tool and clinical marker of infection in adult patients with CAP.     

In Vitro Effect of Ultrasound on Bacteria and Suggested Protocol for Sonication and Diagnosis of Prosthetic Infections

Sonication of implants has been shown to be a promising method for diagnosis of prosthetic infections due to its improved sensitivity, simplicity, and low cost. The aim of the present study was to evaluate the effects of ultrasound performed under different conditions regarding temperature, duration, and composition of sonication tubes on bacterial species often associated with prosthetic infections. We found that ultrasound had an inhibitory effect on bacteria, of which gram-negative bacteria, in particular Escherichia coli, were almost eradicated after 5 min of sonication at 35°C. Gram-positive bacteria were found to be resistant to the effect of ultrasound. Four factors were important for the inhibitory effect of sonication: the type of microorganism, the temperature of the sonication buffer, the duration of exposure to ultrasound (minutes), and the material and composition of the sonication tube in which sonication is performed. On the basis of the results from the present study, we propose a protocol for sonication and recovery of bacteria associated with biofilm on infected implants prior to conventional culture. From the present protocol, we recommend sonication for 7 min at 22°C at the maximum effect which permits survival of gram-negative bacteria.In the United States, 250,000 hip and 400,000 knee replacements are performed annually, and the numbers are expected to double within the next 2 decades due to an increasing number of elderly persons (9). Prosthetic joint infection is second to aseptic failure as a cause of joint replacement and is associated with a substantial morbidity rate and cost (20). The levels of risk for infection within 2 years after insertion are less than 1% and 2% for hip and knee implants, respectively, compared with 5 to 40% after revision surgery (20). The main sources of infection are contamination from the skin flora of patients or hospital staff (12).Diagnosis of prosthetic infections is still a challenge, as clinical signs and a laboratory investigation, including microbiological findings, do not always distinguish aseptic loosening from loosening due to infection (6, 8, 27). Microbiological investigation of implants is complicated due to the sizes of the prosthetic components and problems associated with conventional culture, i.e., presence of intracellular bacteria, microorganisms in biofilm, and small-colony variants of bacteria (1, 5, 9, 14), the last of which may be induced after treatment with gentamicin (26).Due to its simplicity and low cost, sonication appears to be the most promising method among the newer techniques for diagnosis of infected implants (2, 4, 10, 15, 17, 19, 21, 23). Several studies have shown that sonication of implants improves sensitivity compared with that obtained with periprosthetic-tissue culture (2, 8, 19, 24).In medicine, ultrasound transducers are used for diagnosis of abnormalities and fetal, abdominal, and heart diseases. Ultrasound has been applied in microbiology for sonication and inactivation of bacteria in food processing (13). However, the effect of ultrasound has mainly been studied for food- and water-associated bacteria, and data are lacking for clinically important species, such as staphylococci, enterococci, Haemophilus influenzae, and Pseudomonas aeruginosa (3, 13).The aim of the present study was to evaluate the effects of temperature, duration, composition of the sonication buffer, and material in the sonication tube during sonication of bacteria often associated with prosthetic infections. On the basis of the results from these experiments, we propose a protocol for sonication of biofilm on extracted implants prior to conventional culture.     

Evaluation of tcdB Real-Time PCR in a Three-Step Diagnostic Algorithm for Detection of Toxigenic Clostridium difficile

Clostridium difficile is the most common infectious cause of diarrhea in hospitalized patients. The optimal approach for the detection of toxigenic C. difficile remains controversial because no single test is sensitive, specific, and affordable. We have developed a real-time PCR method (direct stool PCR [DPCR]) to detect the tcdB gene encoding toxin B directly from stool specimens and have combined it with enzyme immunoassays (EIAs) in a three-step protocol. DPCR was performed on 699 specimens that were positive for C. difficile glutamate dehydrogenase (GDH) by Wampole C Diff Quik Chek EIA (GDH-Q) and negative for toxins A and B by Wampole Tox A/B Quik Chek EIA (AB-Q), performed sequentially. The performance of this three-step algorithm was compared with a modified “gold standard” that combined tissue culture cytotoxicity (CYT) and DPCR. A separate investigation was performed to evaluate the sensitivity of the GDH-Q as a screening test, and toxigenic C. difficile was found in 1.9% of 211 GDH-Q-negative specimens. The overall sensitivity, specificity, and positive and negative predictive values, respectively, were as follows for an algorithm combining GDH-Q, AB-Q, and DPCR: 83.8%, 99.7%, 97.1%, and 97.9%. Those for CYT alone were 58.8%, 100%, 100%, and 94.9%, respectively. In comparison, the sensitivity and specificity of DPCR were estimated to be 97.5% and 99.7%, respectively, using the same modified gold standard. Neither CYT nor toxin EIA was sufficiently sensitive to exclude toxigenic C. difficile, and combining EIAs with CYT in a three-step algorithm failed to substantially improve sensitivity. DPCR is a sensitive and specific method for the detection of toxigenic C. difficile that can provide same-day results at a cost-per-positive test comparable to those of other methods. A three-step algorithm in which DPCR is used to analyze GDH EIA-positive, toxin EIA-negative specimens provides a convenient and specific alternative with rapid results for 87.7% of specimens, although this approach is less sensitive than performing DPCR on all specimens.Clostridium difficile is the leading cause of infectious diarrhea in hospitalized patients. New challenges have been posed by the emergence of highly virulent C. difficile strains that may be refractory to standard treatment and cause disease even in immunocompetent individuals without prior antibiotic exposure (11, 28). Rapid and accurate laboratory diagnosis is critical to reduce the morbidity from C. difficile infection (CDI) and allow the implementation of specific infection control measures.Methods for the detection of toxigenic C. difficile have long been unsatisfactory. The tissue-culture assay for cytotoxin B (CYT) is often considered the “gold standard” for diagnosis (10, 29), but many reports have documented the failure of CYT to detect symptomatic and even life-threatening cases of CDI (14, 33, 44) and the poor sensitivity of CYT in comparison to toxigenic culture (18, 22, 39, 43). The technical complexity of CYT, as well as a requirement for 24 to 48 h of incubation, has resulted in the widespread replacement of CYT with toxin immunoassays that provide results within minutes. In a 2008 College of American Pathologists report of proficiency results, 95% of laboratories reported using an EIA kit to detect C. difficile toxin (6). C. difficile immunoassays are often adopted as stand-alone assays based on validation against CYT, but studies that have employed more sensitive gold standards have documented that rapid toxin EIAs have unacceptably low sensitivities ranging from 32 to 79% (3, 20, 23, 44, 50).Toxigenic culture, when performed under optimal culture conditions and combined with a sensitive and specific toxin detection method, is regarded as the most sensitive method of toxigenic C. difficile detection (18, 44, 50), but complexity and a prolonged turnaround time have discouraged its routine use. An alternative approach has been to test for the glutamate dehydrogenase (GDH) antigen of C. difficile as a surrogate for culture. GDH EIAs have been reported to be highly sensitive for C. difficile detection, allowing same-day reporting of negative results, but positive results must be followed by a sensitive and specific test to differentiate between toxigenic and nontoxigenic strains (5, 57, 58). In this study, we used the Wampole C Diff Quik Chek EIA (GDH-Q), a rapid GDH assay that requires less technical time and expertise in comparison to microtiter plate immunoassays, followed by the Tox A/B Quik Chek (AB-Q) on GDH-Q-negative specimens. The choices for a second-step assay to detect toxigenic C. difficile in GDH-positive specimens have included toxigenic culture, CYT, or toxin EIAs (20, 23, 46, 57), but the insensitivity of CYT and toxin EIAs potentially leaves many cases of CDI undetected.An alternative, highly sensitive method to detect toxigenic C. difficile is real-time PCR (12, 44, 50, 54, 60), with sensitivity values ranging from 83.6% to 93.4% and specificity from 93.9% to 98.2%, respectively, when compared to toxigenic culture (50, 54, 60). Real-time PCR can be completed on the day of specimen submission, thus providing same-day results. However, PCR techniques have not been not widely used for stool specimens, due primarily to budgetary issues, as well as the challenge of extracting nucleic acids from feces and separating template DNA from potentially interfering substances.We have developed a sensitive and specific real-time PCR assay (direct stool PCR [DPCR]) for the C. difficile toxin B-encoding tcdB gene, which can be performed directly on stool specimens. Nucleic acids can be efficiently extracted from feces with the NucliSENS miniMAG system, a semiautomated magnetic silica bead extraction system to remove inhibitors and purify nucleic acids. Results were compared with the total yield of positive specimens detected by CYT and DPCR. Our observations indicate that a multistep algorithm consisting of GDH EIA, toxin EIA, and selective DPCR can provide a specific and cost-effective approach to the laboratory detection of toxigenic C. difficile that is more sensitive than CYT or EIA alone but not as sensitive as toxigenic culture or DPCR.     

Evaluation of Two Immunoblot Assays and a Western Blot Assay for the Detection of Antisyphilis Immunoglobulin G Antibodies

In the present study, two immunoglobulin G (IgG) immunoblot assays and one IgG Western blot assay were compared to the rapid plasma reagin test (RPR), the fluorescent treponemal antibody absorption test (FTA-ABS), and the Treponema pallidum particle agglutination assay (TP-PA). The agreement levels of the Viramed, Virotech, and MarDx assays were 97.0%, 96.4%, and 99.4%, and the agreements of samples inconclusive by FTA-ABS and resolved by TP-PA were 91.7%, 83.3%, and 69.4%, respectively.Syphilis, a disease caused by Treponema pallidum, is transmitted congenitally or through sexual intercourse (8-9). Non-treponema-based tests such as the rapid plasma reagin test (RPR) are used to detect syphilis infection (6, 9-10). These tests may produce false-positive results in pregnant women and patients with infections (3, 5-6, 9, 11). An algorithm has been developed for the serological diagnosis of syphilis which includes a non-treponema-based screening test and a treponema-based confirmatory assay (1-2, 7, 11). Traditional confirmatory assays include the fluorescent treponemal antibody absorption test (FTA-ABS) and the T. pallidum particle agglutination assay (TP-PA) (9).Western blot-based assays to detect immunoglobulin G (IgG) antibodies may prove useful, especially in cases where the FTA-ABS is inconclusive. In the present study, results of two immunoblot assays and one Western blot assay were compared to FTA-ABS/TP-PA and RPR results, as well as to each other.     

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.)     

Rapid Detection of Trypanosoma cruzi in Human Serum by Use of an Immunochromatographic Dipstick Test

We evaluated a commercially available immunochromatographic dipstick test to detect Trypanosoma cruzi infection in 366 human serum samples with known serological results from Argentina, Ecuador, Mexico, and Venezuela. One hundred forty-nine of 366 (40.7%) and 171/366 (46.7%) samples tested positive by dipstick and serology, respectively. Dipstick sensitivity was calculated to be 84.8% (range between countries, 77.5 to 95%), and specificity was 97.9% (95.9 to 100%).Chagas disease is caused by Trypanosoma cruzi and is found in wildlife, domestic animals, and humans in rural as well as peri-urban areas of Mexico, Central America, and South America; in the United States, T. cruzi is found in wildlife, but human cases are rare (29). Although transmission of T. cruzi can occur orally, congenitally, or transfusionally, most transmission to mammalian hosts is through the feces of blood-feeding triatomine bugs when T. cruzi trypomastigotes in the feces contaminate the bite wound or enter the host through mucosal surfaces (22). By causing the loss of an estimated 670,000 disability-adjusted life years (i.e., a measure that sums years of potential life lost due to premature mortality and years of productive life lost due to disability), Chagas disease is the most important parasitic disease in the Americas; 8 to 10 million people are currently infected with T. cruzi, with up to 100 million at risk of contracting the disease (32).There are several methods to diagnose T. cruzi infection (11), but none are ideal when mass screening of samples is required (e.g., epidemiological surveys, blood unit screening). While comparatively easy to use and sensitive, serological tests (i.e., enzyme-linked immunosorbent assay [ELISA], immunofluorescence antibody test [IFAT], indirect hemagglutination test [IHAT], or radioimmunosorbent assay [RIA]) are of varied specificities (i.e., 60 to 100%) (12, 16, 26). Molecular tests, including PCR-based approaches, are very specific but lack sensitivity (i.e., 30 to 95%) and require technological expertise and specialized, expensive laboratory equipment (11, 21, 23). Hemoculture and xenodiagnosis are the current gold standard for T. cruzi parasitological diagnosis (6, 11, 21). Though these techniques are specific, their sensitivity in the chronic phase of infection is quite variable (e.g., 0 to 50% [6]); they also are labor-intensive and time-consuming (e.g., because of the necessity of mass-rearing bugs for xenodiagnosis and examination of them). Thus, a rapid, sensitive, and specific diagnostic test to detect T. cruzi infection would be extremely valuable for mass-screening surveys and intervention campaigns as well as during the onset of outbreaks; results could be read immediately, and control measures could be implemented in situ.Immunochromatographic dipstick tests have been developed for a range of tropical diseases, including malaria (31), leishmaniasis (7), and schistosomiasis (3); until recently (4, 5, 8, 14, 17, 20, 25, 28, 30), none was available for Chagas disease.Recently, the World Health Organization announced renewed efforts to eliminate Chagas disease (27). For such efforts to succeed, an easy-to-use, sensitive, and specific diagnostic test will be crucial for both detecting and treating cases early as well as monitoring the implementation of elimination efforts and evaluating their impact (18, 24).We evaluated the sensitivity and specificity of a commercially available immunochromatographic dipstick test to detect antibodies to T. cruzi infection in human serum samples with known serological results collected in areas of both Chagas disease endemicity and nonendemicity in four different Latin American countries.     

Use of Dense Granule Antigen GRA6 in an Immunoglobulin G Avidity Test To Exclude Acute Toxoplasma gondii Infection during Pregnancy

The usefulness of a specific immunoglobulin G (IgG) avidity enzyme-linked immunosorbent assay (ELISA) based on recombinant GRA6 antigen for distinguishing between acute and chronic Toxoplasma infection was investigated. Two sets of serum samples obtained from pregnant women with acute, chronic, or no Toxoplasma infection collected in France and Iran were used. Among the French subjects, 19 of 20 (95%) women who experienced seroconversion during the past 4 months before sampling displayed low-avidity IgG antibodies against GRA6, while all 17 (100%) women with chronic infection had high-avidity antibodies. When the Euroimmun IgG avidity ELISA was used, 15 of 19 (78.9%) recently infected women had low-avidity antibodies, and 20 of 22 (90.9%) women with chronic infection displayed high-avidity antibodies. The results suggested better performance of the GRA6 avidity ELISA than the Euroimmun avidity ELISA for exclusion of a recent infection occurring less than 4 months previously. Similarly, all 35 Iranian women with acute Toxoplasma infection had low-avidity antibodies against GRA6, whereas all 34 women with chronic infection displayed IgG antibodies of high avidity, indicating the value of GRA6 avidity testing for ruling out a recent infection. Avidity tests based on lysed whole-cell Toxoplasma gondii antigen are currently used to exclude recently acquired infections; however, the use of recombinant antigen(s) might improve the diagnostic performance of avidity tests and facilitate the development of more standardized assays.Congenital toxoplasmosis may occur when maternal infection is acquired during pregnancy and results in severe fetopathy or miscarriage (28, 38). While the rate of fetal infection with Toxoplasma gondii is extremely low in preconception infections, the transmission rate increases and the severity of fetal infection decreases as gestational age progresses (7, 18). Fortunately, prenatal treatment of the infection is effective for reducing both the incidence of clinical manifestations in infected newborns (8) and the maternal-fetal transmission rate (6, 29, 37). It is, therefore, essential to estimate the gestational age of the primary Toxoplasma infection as precisely as possible for the proper clinical management of pregnant women.Diagnosis of acute toxoplasmosis depends mainly on serological testing, as the infection is asymptomatic in 93% to 97% of pregnant women (10, 27). In countries where a prenatal screening of T. gondii infection is performed, detection of seroconversion or a significant rise in the specific immunoglobulin G (IgG) titer establishes a recently acquired infection. In most countries, however, a single serum sample from a pregnant woman is available to determine whether the infection occurred during gestation. IgM antibodies have been traditionally known as the markers of acute infection; however, persistent specific IgM has been found for up to several years after primary infection (15, 24, 39). Measurement of specific IgG avidity was shown to be an effective confirmatory test to help discriminate between recently acquired and distant infections (4, 16, 20, 22, 30, 31). In fact, it was shown that the combination of a sensitive test for T. gondii IgM and a specific IgG avidity assay is the best tool for determining the time of infection (34). The functional affinity (avidity) of specific IgG antibodies is low in the beginning of infection and usually increases with time by antigen-driven B-cell selection. However, low-avidity antibodies may persist for several months after acute infection, indicating that the avidity test is best used to rule out a recent infection in individuals with high-avidity results (5, 20, 30). Some studies reported the presence of very rare borderline- or high-avidity antibodies in acute infection with T. gondii (11, 20-22). The discordant results of avidity assays are attributed to the presence of antiparasitic treatment and immunodeficiency status; however, the complex nature of lysed whole-cell T. gondii antigen might cause such results (4, 35). Recombinant antigens might improve the performance of avidity assays for distinguishing between acute and chronic infection (1, 32, 33). Furthermore, preparation of recombinant antigens with more constant quality, compared to T. gondii antigen prepared from parasites grown either in cell culture or in mice peritoneal cavities, would facilitate development of better standardized assays (2).In this study, we developed an IgG avidity test based on recombinant GRA6 antigen and evaluated its value for differentiation between recently acquired and distant Toxoplasma infections in pregnant women. The potential usefulness of the GRA6 avidity test was compared with that of the Euroimmun IgG avidity enzyme-linked immunosorbent assay (ELISA; Euroimmun, Lübeck, Germany) for exclusion of a recent Toxoplasma infection that occurred less than 4 months before.     

Comparison of Flocked and Rayon Swabs for Detection of Nasal Carriage of Staphylococcus aureus among Pathology Staff Members

Comparison of flocked swabs (E-swabs; Copan) to the standard rayon swabs (Copan) was undertaken for detection of Staphylococcus aureus nasal carriage among staff at Dorevitch Pathology in Heidelberg, Melbourne, Australia. Among 100 volunteers, 36 were found to be colonized with S. aureus by one or both swab results. The prevalence detected by E-swabs was 35%, and the prevalence through rayon swabs was 34% (95% confidence interval [CI] for the difference in proportions, −12 to 14). Thirty-three volunteers tested positive with both types of swabs, while 2 were detected on E-swabs alone and another on rayon swab testing alone. There was no evidence of a significant difference in carriage detected by E-swabs or rayon swabs.Staphylococcus aureus is a common cause of infections in the community and a major cause of hospital-associated morbidity (18). Colonization is well described, with up to 30% of the population thought to be carriers (7, 16, 18), and is associated with a higher risk of infection in the hospital setting (2, 4, 9, 11, 12, 15, 17, 18). The anterior nares have been shown to be the most frequent site of carriage and therefore a single site for detection (9, 10, 18). Nasal carriage is defined as “persistent” or “intermittent or noncarriage,” with persistent carriers showing an increased risk of infection, compared with intermittent carriers who share the same low risk as noncarriers (13). Given the clinical relevance, it is imperative to use the best swab system which would provide the highest yield in detecting nasal carriage. Flocked swabs have been described as improving uptake of epithelial cells and, therefore, microorganisms and viruses (1, 5, 6, 14), but are more expensive than standard rayon swabs, so it is therefore worth investigating whether there is evidence that E-swabs perform better in detecting nasal carriage.     

Improved Detection of Five Major Gastrointestinal Pathogens by Use of a Molecular Screening Approach

The detection of bacterial and parasitic gastrointestinal pathogens through culture and microscopy is laborious and time-consuming. We evaluated a molecular screening approach (MSA) for the detection of five major enteric pathogens: Salmonella enterica, Campylobacter jejuni, Giardia lamblia, Shiga toxin-producing Escherichia coli (STEC), and Shigella spp./enteroinvasive E. coli (EIEC), for use in the daily practice of a clinical microbiology laboratory. The MSA consists of prescreening of stool specimens with two real-time multiplex PCR (mPCR) assays, which give results within a single working day, followed by guided culture/microscopy of the positive or mPCR-inhibited samples. In the present 2-year overview, 28,185 stool specimens were included. The MSA was applied to 13,974 stool samples (49.6%), whereas 14,211 samples were tested by conventional methods only (50.4%). The MSA significantly increased the total detection rate compared to that of conventional methods (19.2% versus 6.4%). The detection of all included pathogens, with the exception of S. enterica, significantly improved. MSA detection frequencies were as follows: C. jejuni, 8.1%; G. lamblia, 4.7%; S. enterica, 3.0%; STEC, 1.9%; and Shigella spp./EIEC, 1.4%. The guided culture/microscopy was positive in 76.8%, 58.1%, 88.9%, 16.8%, and 18.1% of mPCR-positive specimens, respectively. Of all mPCRs, only 1.8% was inhibited. Other findings were that detection of mixed infections was increased (0.9% versus 0.02%) and threshold cycle (C_T) values for MSA guided culture/microscopy-positive samples were significantly lower than those for guided culture/microscopy-negative samples. In conclusion, an MSA for detection of gastrointestinal pathogens resulted in markedly improved detection rates and a substantial decrease in time to reporting of (preliminary) results.Infectious gastroenteritis (IG) is one of the most common diseases worldwide, killing millions of individuals each year (3, 16). In industrialized countries, IG remains a major public health burden, although mortality is low. In the Netherlands, with a population of 16.5 million, the yearly IG incidence is approximately 4.5 million (42). Although most episodes of IG are brief and do not require medical attention, the economic and social burdens of IG are significant (38).The etiology of IG includes viral, parasitic, and bacterial pathogens. Most medical microbiology laboratories use conventional diagnostic procedures, such as culture and microscopy, for routine detection of enteric pathogens. These procedures include enrichment steps, use of selective culture media, biochemical identification, serotyping, and resistance profiling. Final results are obtained after 3 to 4 days, making these procedures laborious and time-consuming. Furthermore, the detection of pathogens in stool specimens by culture is complicated. For instance, bacteria belonging to the normal gastrointestinal flora can present with the same colony mor- phology as enteric pathogens (13, 30). The resultant misidentification increases hands-on time and delay in reporting of a definite negative result. Other problems are the viable but nonculturable state of Campylobacter jejuni (24, 29) and the limited viability of shigellae outside the human body (35). These may compromise the sensitivity of culture.Conventional laboratory diagnosis of gastrointestinal parasites consists of microscopy and/or stool antigen tests. Microscopy in particular has disadvantages, as the detection and correct identification of parasites depend upon the experience and skills of the microscopist. Also, due to intermittent shedding of protozoa the sensitivity can be low, and therefore examination of multiple samples is required (8).The workload involved in examining stool samples is high. Our laboratory receives around 15,000 stool specimens annually, with daily numbers varying greatly between seasons (42). Furthermore, the majority of stool specimens do not yield a positive result (42). Therefore, methods which quickly identify the negative specimens would facilitate routine screening. Antigen detection is a fast and effective alternative for several enteric pathogens (11, 15, 23, 27, 46). However, this method has a limited sensitivity, requires one test per pathogen, and is not available for all relevant pathogens.Molecular methods provide a means for sensitive and rapid detection of enteric pathogens. However, broad application remains limited due to their assumed high costs, inhibition caused by fecal constituents (22), and the need for specialized laboratories. Due to the high throughput of stool screening and the number of possible enteric pathogens, implementation of a molecular approach which uses multiplexing of targets is mandatory (9, 10, 20, 25, 26, 36, 43, 44, 47). For the detection of enteric pathogens it has been proven feasible to use molecular methods with improved performance and turnaround time (TAT) (7, 25, 32).Since December 2006, we have implemented a molecular screening approach (MSA) for the simultaneous detection of five pathogens: Salmonella enterica, Campylobacter jejuni, Giardia lamblia, Shiga toxin-producing Escherichia coli (STEC), and Shigella spp./enteroinvasive E. coli (EIEC). This MSA, involving two internally controlled real-time multiplex PCRs (mPCRs), is now daily practice. The present report describes our experiences with the MSA over a 2-year period (2007 to 2008).(Part of this work was presented at the 19th European Congress of Clinical Microbiology and Infectious Diseases, Helsinki, Finland, 18 May 2009.)     

Incubation Alone Is Adequate as a Culturing Technique for Cardiac Rhythm Management Devices

There exist no standardized methods for culturing cardiac rhythm management devices. To identify the most optimal culturing method, we compared various techniques that comprise vortex, sonication, and incubation or combinations thereof. Incubation alone yielded bacterial colony counts similar to those of other culturing combinations and is the least labor-intensive.Permanent pacemakers (PPM) and implantable cardioverter-defibrillators (ICD) are being utilized increasingly because of an aging population (5, 9). About 180,284 PPM and 57,436 ICD were inserted into cardiac patients in the United States in 2003 (10), and worldwide there are 3 million functioning PPM and 180,000 indwelling ICD (2). Infection is the most common serious complication of PPM and ICD, which are collectively referred to as cardiac rhythm management devices (CRMD). The rates of infection of CRMD range from 0.13% to 19.9% (3, 7) with an average rate of 4% (4). Staphylococcus epidermidis and Staphylococcus aureus collectively account for 70 to 95% of CRMD infections (1, 6). A National Hospital Discharge Survey from 1996 to 2003 indicated that along with the rising use of cardiac medical devices, there has been a disproportional increase in the number of infections of such devices (10). The average cost of combined medical-surgical treatment of an infected CRMD was estimated at $35,000 (4).There are no standardized methods for culturing explanted cardiac generators and leads of the CRMD. Although it has been demonstrated that sonication increases the yield of bacterial cultures from prosthetic joints (8), the utility of this diagnostic approach for CRMD has not been published. To identify the most optimal culturing method, we compared the bacterial yield of various techniques that comprise incubation, vortex, sonication and combinations thereof.     

Diagnosis of Amebic Liver Abscess and Amebic Colitis by Detection of Entamoeba histolytica DNA in Blood,Urine, and Saliva by a Real-Time PCR Assay

The noninvasive diagnosis of amebic liver abscess is challenging, as most patients at the time of diagnosis do not have a concurrent intestinal infection with Entamoeba histolytica. Fecal testing for E. histolytica parasite antigen or DNA is negative in most patients. A real-time PCR assay was evaluated for detection of E. histolytica DNA in blood, urine, and saliva samples from amebic liver abscess as well as amebic colitis patients in Bangladesh. A total of 98 amebic liver abscess and 28 amebic colitis patients and 43 control subjects were examined. The real-time PCR assay detected E. histolytica DNA in 49%, 77%, and 69% of blood, urine, and saliva specimens from the amebic liver abscess patients. For amebic colitis the sensitivity of the real-time PCR assay for detection of E. histolytica DNA in blood, urine, and saliva was 36%, 61%, and 64%, respectively. All blood, urine, and saliva samples from control subjects were negative by the real-time PCR assay for E. histolytica DNA. When the real-time PCR assay results of the urine and saliva specimens were taken together (positive either in urine or saliva), the real-time PCR assay was 97% and 89% sensitive for detection of E. histolytica DNA in liver abscess and intestinal infection, respectively. We conclude that the detection of E. histolytica DNA in saliva and urine could be used as a diagnostic tool for amebic liver abscess.Entamoeba histolytica is a protozoan parasite that causes amebic diarrhea, colitis, and amebic liver abscess (ALA), mostly in developing countries (5, 7, 22, 25). Eighty percent of infected individuals remain asymptomatic carriers, while the other 20% develop clinically overt disease (7, 9, 22, 25). About 50 million symptomatic cases of amebiasis occur worldwide each year, resulting in 40,000 to 100,000 deaths annually (25). Mortality from amebiasis is mainly due to extra-amebic colitis, of which ALA is the most common.It is difficult to differentiate ALA from pyogenic liver abscess or other space-occupying lesions of the liver. Imaging techniques such as ultrasound, computed tomography, and magnetic resonance have excellent sensitivities for the detection of liver abscess arising from any cause, but there are no findings specific for ALA (13). Further complicating the diagnosis is the fact that most patients with an ALA do not have coexistent intestinal infection with E. histolytica (11). Therefore, detection of E. histolytica antigen or DNA in stool samples is not very helpful for the diagnosis of ALA (1, 6, 8, 12).The current means for diagnosis of ALA is the detection of antiamebic antibody by serological tests combined with aspiration of the abscess. The presence of serum antibodies against E. histolytica and the absence of bacteria in the abscess fluid are consistent with an ALA. A drawback of serologic tests is that the serum antibody levels in people from areas of endemicity remain positive for years after infection with E. histolytica (3, 16, 23). Therefore, antiamebic antibodies in the serum may be due to amebiasis in the past, limiting their specificity for the diagnosis of ALA. A further limitation to the current approach to ALA diagnosis is that collection of liver abscess pus is an invasive procedure that requires technical expertise and can be done only in specialized hospitals.Several groups have reported the detection of E. histolytica DNA in liver abscess pus, stool, and other clinical samples by PCR (14, 15, 18, 19, 21, 24, 26). A real-time PCR assay has also been used for detection of E. histolytica DNA in stool and liver abscess pus specimens (2, 10, 20). Real-time PCR has never been used for detection of E. histolytica DNA in urine, saliva, and blood specimens of ALA patients. In this study, we evaluated a real-time PCR assay to detect E. histolytica DNA in blood, urine, and saliva samples of amebic liver abscess and colitis patients in Bangladesh.