These data suggest that the described assays perform well in this population for enhancing the diagnosis of candidemia. The extent to which they may affect clinical outcomes, prescribing practice, and cost-effectiveness of care remains to be ascertained.

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The limitations of classical diagnostic methods for invasive Candida infections have led to the development of molecular techniques such as real-time PCR to improve diagnosis. However, the detection of low titres of Candida DNA in blood from patients with candidaemia requires the use of extraction methods that efficiently lyse yeast cells and recover small amounts of DNA suitable for amplification. In this study, a Candida-specific real-time PCR assay was used to detect Candida albicans DNA in inoculated whole blood specimens extracted using seven different extraction protocols. The yield and quality of total nucleic acids were estimated using UV absorbance, and specific recovery of C. albicans genomic DNA was estimated quantitatively in comparison with a reference (Qiagen kit/lyticase) method currently in use in our laboratory. The extraction protocols were also compared with respect to sensitivity, cost and time required for completion. The TaqMan PCR assay used to amplify the DNA extracts achieved high levels of specificity, sensitivity and reproducibility. Of the seven extraction protocols evaluated, only the MasterPure yeast DNA extraction reagent kit gave significantly higher total nucleic acid yields than the reference method, although nucleic acid purity was highest using either the reference or YeaStar genomic DNA kit methods. More importantly, the YeaStar method enabled C. albicans DNA to be detected with highest sensitivity over the entire range of copy numbers evaluated, and appears to be an optimal method for extracting Candida DNA from whole blood. INTRODUCTIONBlood culture remains the accepted 'gold standard' method for diagnosis of candidiasis and can be highly sensitive. However, in some settings, culture can fail to detect Candida spp. in more than 50 % of patients with chronic disseminated candidiasis MoreiraOliveira et al., 2005). Molecular methods, particularly realtime PCR, are increasingly used alongside conventional microbiological techniques for the diagnosis of systemic candidiasis (Klingspor & Jalal, 2006; Kasai et al., 2006; Löffler et al., 2000;Schabereiter-Gurtner et al., 2007;Bretagne & Costa., 2005;White et al., 2006;Maaroufi et al., 2003; Moreira-Oliveira et al., 2005). However, two major limitations of PCR are the difficulty associated with breaking yeast cell walls to release Candida spp. DNA suitable for amplification (Maaroufi et al., 2004; Löffler et al., 1997) and limited sensitivity when the assays are adapted for testing clinical specimens, such as blood samples, in which the amounts of fungal DNA may be very low (White et al., 2003; Löffler et al., 2000;Ahmad et al., 2002;Challier et al., 2004). As a consequence, these tests are not yet recognized as part of the consensus diagnostic criteria, in contrast to some antigenaemia detection kits (Ascioglu et al., 2002).The yeast cell is notoriously difficult to lyse due to a highly complex cell wall structure that provides rigidity (Müller et al., 1998). Moreover, it has been suggested that the fungal load in blood from ...

Objective: To determine the frequency of primary cytomegalovirus (CMV) infection in pregnant Egyptian women using CMV IgG avidity testing. Subjects and Methods: A cross-sectional study was conducted at Suez Canal University Hospital, Ismailia, Egypt. A total of 546 pregnant women, presenting for routine antenatal screening, were tested for CMV IgG and IgM using a commercially available enzyme-linked immunosorbent assay (ELISA). Sera from CMV IgM-positive women were tested by CMV IgG avidity assay. Results: All the 546 pregnant women were seropositive for anti-CMV IgG. Of the 546 women, 40 (7.3%) were positive or equivocal for IgM antibodies. All sera from the 40 women (IgG+/IgM+) showed a high or intermediate CMV IgG avidity index. Of the 40 women, 23 (57.5%) were in the second or third trimesters of pregnancy and had their first-trimester blood retrieved, and the tested CMV IgG avidity assay showed a high avidity index. Conclusion: Women who were IgM positive had no primary CMV infection in the index pregnancy as evidenced by the high CMV IgG avidity testing.

In view of both the delay in obtaining identification by conventional methods following blood-culture positivity in patients with candidaemia and the close relationship between species and fluconazole (FLC) susceptibility, early speciation of positive blood cultures has the potential to influence therapeutic decisions. The aim was to develop a rapid test to differentiate FLC-resistant from FLC-sensitive Candida species. Three TaqMan-based real-time PCR assays were developed to identify up to six Candida species directly from BacT/Alert blood-culture bottles that showed yeast cells on Gram staining at the time of initial positivity. Target sequences in the rRNA gene complex were amplified, using a consensus two-step PCR protocol, to identify Candida albicans, Candida parapsilosis, Candida tropicalis, Candida dubliniensis, Candida glabrata and Candida krusei; these are the most commonly encountered Candida species in blood cultures. The first four of these (the characteristically FLC-sensitive group) were identified in a single reaction tube using one fluorescent TaqMan probe targeting 18S rRNA sequences conserved in the four species. The FLC-resistant species C. krusei and C. glabrata were detected in two further reactions, each with species-specific probes. This method was validated with clinical specimens (blood cultures) positive for yeast (n533 sets) and the results were 100 % concordant with those of phenotypic identification carried out concomitantly. The reported assay significantly reduces the time required to identify the presence of C. glabrata and C. krusei in comparison with a conventional phenotypic method, from~72 to ,3 h, and consequently allows optimization of the antifungal regimen at an earlier stage.

The emergence and rapid spread of antibiotic-resistant Klebsiella pneumoniae isolates harbouring the bla KPC gene that encodes for carbapenemase production have complicated the management of patient infections. This study in a tertiary care hospital in Egypt used real-time PCR assay to test ertapenem-nonsusceptible isolates of K. pneumoniae for the presence of the bla KPC gene and compared the results with modified Hodge test. Antibiotic sensitivity was performed by standard methods, and interpreted following both the old CLSI breakpoints (M100-S19) for carbapenems and the revised breakpoints (M100-S22). From the 45 non-duplicate isolates of K. pneumoniae recovered from different clinical specimens, a high prevalence of ertapenem-nonsusceptible isolates (44.4%) was reported using the new lower CLSI breakpoints. The bla KPC gene was confirmed in 14/20 (70.0%) of these isolates. The high prevalence of ertapenem nonsusceptibility at a tertiary care hospital in Egypt was predominantly attributed to K. pneumoniae carbapenemase-mediated resistance mechanisms in K. pneumoniae isolates. RÉSUMÉ L'émergence et la propagation rapide des souches de Klebsiella pneumoniae résistantes aux antibiotiques et porteuses du gène bla KPC codant la production de carbapénèmases ont compliqué la prise en charge des infections des patients. La présente étude menée dans un hôpital de soins tertiaires en Égypte a utilisé la méthode de PCR en temps réel pour évaluer la présence du gène bla KPC dans les isolats de K. pneumoniae non sensibles à l'ertapénème, puis a comparé les résultats à l'aide du test de Hodge modifié. La sensibilité aux antibiotiques a été évaluée à l'aide des méthodes standards, puis a été interprétée selon les anciens seuils du Clinical and Laboratory Standards Institute (M100-S19) pour les carbapénèmes et selon les seuils révisés (M100-S22). Après l'analyse des 45 isolats non-dupliqués de K. pneumoniae prélevés à partir de différents échantillons cliniques, une prévalence élevée d'isolats non sensibles à l'ertapénème (44,4 %) a été rapportée selon les nouveaux seuils plus bas du Clinical and Laboratory Standards Institute. La présence du gène bla KPC a été confirmée dans 14 isolats sur 20 (70,0 %). La forte prévalence de la non sensibilité à l'ertapénème dans un hôpital de soins tertiaires en Égypte était principalement imputable aux mécanismes de résistance médiés par les carbapénèmases dans les isolats de K. pneumoniae.

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Multi-drug resistant (MDR) strains of Acinetobacter baumannii are responsible for an increasing number of opportunistic infections in hospitals. This study determined the prevalence of MDR A. baumannii isolates from intensive care units in a large tertiary-care hospital in Ismailia, Egypt, and the occurrence of different beta-lactamases in these isolates. Biotyping and antimicrobial susceptibility profile was done for isolated strains. Respiratory, urine, burn wound and blood specimens were collected from 350 patients admitted to different units; 10 strains (2.9%) of A. baumannii were isolated. All isolates showed resistance to more than 3 classes of antibiotics. Among the isolates, 6 isolates were carbapenemase producers, 2 were AmpC beta-lactamase producers and no isolates were metallo-betalactamase producers. Despite the low prevalence of A. baumannii infection in this hospital, the antibiotic resistance profile suggests that prevention of health-care-associated transmission of MDR Acinetobacter spp. infection is essential. Parmi ces isolats, six produisaient des carbapénèmases, deux des bêta-lactamases AmpC mais aucun isolat ne produisait de métallo-bêta-lactamases. Malgré une faible prévalence de l'infection à A. baumannii dans cet hôpital, le profil de résistance aux antibiotiques laisse penser que la prévention de la transmission de l'infection à Acinetobacter spp. multirésistante associée aux soins de santé est essentielle.