Detection of methicillin-resistant Staphylococcus aureus directly by loop-mediated isothermal amplification and direct cefoxitin disk diffusion tests.

We evaluated the utility of 2 methods for detection of methicillin-resistant Staphylococcus aureus (MRSA) directly from signal-positive blood culture bottles: loop-mediated isothermal amplification (LAMP) assay, and direct cefoxitin disk diffusion (DCDD) test using a 30 µg cefoxitin disk. In parallel, standard microbiological identification and oxacillin susceptibility testing with MecA PCR was performed. Of 60 blood cultures positive for Gram-positive cocci in clusters, LAMP (via detection of the FemA and MecA genes) showed 100% sensitivity and specificity for identification of MRSA/MSSA. When coagulase-negative staphylococci were tested, sensitivity for detection of methicillin resistance was 91.7% and specificity was 100%. DCDD along with direct tube coagulase assay detected only 80.6% of MRSA/MSSA. LAMP showed higher diagnostic accuracy although DCDD was more cost-effective and did not require additional reagents or supplies.

Genotype characterization of occult hepatitis B virus strains among Egyptian chronic hepatitis C patients.

Chronic hepatitis C virus (HCV) infection combined with occult hepatitis B virus (HBV) infection has been associated with increased risk of hepatitis, cirrhosis and hepatocellular carcinoma. This study aimed to determine the prevalence of occult HBV infection among Egyptian chronic HCV patients, the genotype and occurrence of surface gene mutations of HBV and the impact of co-infection on early response to treatment. The study enrolled 162 chronic HCV patients from Ismailia Fever Hospital, Egypt, who were HBV surface antigen-negative. All patients were given clinical assessment and biochemical, histological and virological examinations. HBV-DNA was detectable in sera from 3 patients out of the 40 patients who were positive for hepatitis B core antibody. These 3 patients were responsive to combination therapy at treatment week 12; only 1 of them had discontinued therapy by week 24. HBV genotype D was the only detectable genotype in those patients, with absence of "a" determinant mutations among those isolates.

Phenotypic characterization of Acinetobacter baumannii isolates from intensive care units at a tertiary-care hospital in Egypt.

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-beta-lactamase 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.

Primary cytomegalovirus infection in pregnant Egyptian women confirmed by cytomegalovirus IgG avidity testing.

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.

Detection of methicillin-resistant Staphylococcus aureus directly by loop-mediated isothermal amplification and direct cefoxitin disk diffusion tests

We evaluated the utility of 2 methods for detection of methicillin-resistant Staphylococcus aureus [MRSA]directly from signal-positive blood culture bottles: loop-mediated isothermal amplification [LAMP]assay, and direct cefoxitin disk diffusion [DCDD]test using a 30 microg cefoxitin disk.In parallel, standard microbiological identification and oxacillin susceptibility testing with MecA PCR was performed.Of 60 blood cultures positive for Gram-positive cocci in clusters, LAMP [via detection of the FemA and MecA genes]showed 100% sensitivity and specificity for identification of MRSA/MSSA.When coagulase-negative staphylococci were tested, sensitivity for detection of methicillin resistance was 91.7% and specificity was 100%.DCDD along with direct tube coagulase assay detected only 80.6% of MRSA/MSSA.LAMP showed higher diagnostic accuracy although DCDD was more cost-effective and did not require additional reagents or supplies

Nous avons évalué l'utilité de deux méthodes de détection de Staphylococcus aureus résistant à la méthicilline directement à partir des flacons d'hémoculture donnant des signaux positifs à l'aide de amplification isotherme induite par boucle ainsi que de tests de diffusion sur disque de 30 micro g de céfoxitine directs.En parallèle, une identification microbiologique normalisée et un test de sensibilité à l'oxacilline par PCR visant l'amplification du gène MecA ont été réalisés.Sur 60 hémocultures positives pour les cocci a Gram positif en grappes, l'amplification isotherme induite par boucle [au moyen du dépistage des gènes FemA et MecA]à montré une sensibilité et une spécificité de 100 % pour l'identification de Staphylococcus aureus résistant et sensible à la méthicilline.Lorsque les staphylocoques a coagulase négative ont été analyses, la sensibilité pour la détection de la résistance à la méthicilline était de 91, 7 % et la spécificité de 100 %.Les tests de diffusion sur disque de céfoxitine directs ainsi que le dosage direct de la coagulase à partir des flacons ont détecté seulement 80, 6 % des Staphylococcus aureus résistants/sensibles à la méthicilline.L'amplification isotherme induite par boucle à montré une exactitude diagnostique supérieure, même si les tests de diffusion sur disque à la céfoxitine directs étaient d'un meilleur rapport coût-efficacité et n'exigeaient ni réactifs ni fournitures supplémentaires

قد قامت الباحثات بتقييم فائدة طريقتين للكشف المباشر للعقديات الذهبية المقاومة للميثيسيلين المستمدة من زجاجة زرع الدم الإيجابية العلامة؛ والطريقتان هما المقايسة بالتضخيم المتساوي الحرارة بواسطة العروة، واختبار الانتشار المباشر لقرص وغرام. وأجرت الباحثات بموازاة ذلك اختبارات للكشف المعياري للمكروبات مع الاستجابة للأوكسيسيلين باستخدام التفاعل السلسلي للبوليمراز على مادة MecA.ومن بين 60 زجاجة زرع دم إيجابي للجراثيم المكورة الإيجابية الغرام، تبين أن هناك 100 % استجابة ونوعية لكشف التضخيم المتساوي الحرارة بواسطة العروة [عبر كشف جينات MecA و FemA]وعند إجراء الاختبار على المكورات العنقودية السلبية لإنزيم التخثير [كوأغيولاز]وجدت الباحثات أن حساسية. كشف المقاومة للميثيسيلين بلغت 91.7 % بينما بلغت النوعية 100 % كما أن استخدام الانتشار المباشر لقرص السيفوكسيتين مع المقايسة المباشرة في الأنبوب لإنزيم التخثير لم يكشف إلا 80.6 % من الجراثيم العنقودية المقاومة للميثيسيلين، والمستجيبة له.وهكذا اتضح أن التضخيم المتساوي الحرارة بواسطة العروة يوفر دقة تشخيصية أعلى مما يوفره الانتشار المباشر لأقراص السيفوكسيتين، وأنه أكثر مردودا ولا يتطلب إمدادات أو كواشف إضافية

Phenotypic characterization of Acinetobacter baumannii isolates from intensive care units at a tertiary-care hospital in Egypt

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.baumanniiwere 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-beta-lactamase 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

إن الذراري المقاومة لأدوية متعددة من جراثيم الراكدة البومانية هي الآن المسؤولة عن تزايد أعداد حالات العدوى الانتهازية في المستشفيات. وقد حددت هذه الدراسة معدلات انتشار الجراثيم المقاومة لأدوية متعددة من جنس الراكدة البومانية المستفردة من وحدات الرعاية المكثفة في أحد المستشفيات الكبرة للرعاية الثالثية في الإسماعيلية، مصر، كما حددت الدراسة معدل وجود إنزيات بيتالاكتاماز المختلفة في هذه المستفردات. وأعد الباحثون ملفا يتضمن الأنماط البيولوجية والاستجابة لمضادات المكروبات للذراري المستفردة. فجمعوا عينات من الدم والقيح من الجروح والحروق ومن البول ومن الجهاز التنفسي لدى 350 مريضا أدخلوا في مختلف الوحدات. واستفردوا 10 ذراري [2.9 %]من الراكدة البومانية، وتبين لهم أن جميع المستفردات لديها مقاومة لأكثر من 3 من أصناف المضادات الحيوية.وكان من بين المستفردات ستة جراثيم منتجة لإنزيم كاربابنياز، وثلاثة جراثيم منتجة لإنزيم بيتا لاكتاماز AmpC، ولم يجدوا أي مستفردة منتجة لإنزيم ميتالوبيتا لاكتاماز. ورغم انخفاض معدل انتشار العدوى بجراثيم الراكدة البومانية في المستشفيات، فإن ملف مقاومتها للمضادات الحيوية يشير إلى ضرورة وأهمية الوقاية من العدوى المرتبطة بالرعاية الصحية بأنواع "الراكدة" المقاومة لأدوية متعددة

Les souches d'Acinetobacter baumannii multirésistantes sont aujourd'hui responsables de l'augmentation du nombre d'infections opportunistes dans les hôpitaux.La présente étude a déterminé la prévalence des isolats d'A.baumannii multirésistants prélevés dans des unités de soins intensifs d'un grand hôpital de soins de Santé tertiaires à Ismaïlia [Egypte], et la fréquence de différentes bêta-lactamases dans ces isolats.Le biotypage et le profil de sensibilité aux antimicrobiens ont été établis pour les souches isolées.Des échantillons des voies respiratoires, d'urine, de blessures par brulure et de sang ont été prélevés sur 350 patients admis dans différentes unités; 10 souches [2, 9 %]d'A.baumannii ont été isolées.Tous les isolats présentaient une résistance à plus de trois classes d'antibiotiques.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

Genotype characterization of occult hepatitis B virus strains among Egyptian chronic hepatitis C patients

Chronic hepatitis C virus [HCV]infection combined with occult hepatitis B virus [HBV]infection has been associated with increased risk of hepatitis, cirrhosis and hepatocellular carcinoma.This study aimed to determine the prevalence of occult HBV infection among Egyptian chronic HCV patients, the genotype and occurrence of surface gene mutations of HBV and the impact of co-infection on early response to treatment.The study enrolled 162 chronic HCV patients from Ismailia Fever Hospital, Egypt, who were HBV surface antigen-negative.All patients were given clinical assessment and biochemical, histological and virological examinations.HBV-DNA was detectable in sera from 3 patients out of the 40 patients who were positive for hepatitis B core antibody.These 3 patients were responsive to combination therapy at treatment week 12; only 1 of them had discontinued therapy by week 24.HBV genotype D was the only detectable genotype in those patients, with absence of [a]determinant mutations among those isolates

إن العدوى بفيروس التهاب الكبد المزمن "سي" المصحوبة بفيروس التهاب الكبد البائي الخفي، يرافقها زيادة في خطر الإصابة بالتهاب الكبد، وتليف الكبد، وسرطانة الخلايا الكبدية. وتهدف هذه الدراسة إلى تحديد معدل انتشار العدوى بفيروس التهاب الكبد البائي الخفي بين المرضى المصريين المصابين بالتهاب الكبد الوبائي "سي"، وتحديد الأنماط الجينية، والطفرات الجينية السطحية لفيروس التهاب الكبد البائي "سي"، وتأثير العدوى المشتركة على الاستجابة المبكرة للمعالجة. وقد أدرج في الدراسة 162 مريضا مصابا بفيروس التهاب الكبد "سي" المزمن من مستشفى الإسماعيلية للحميات بمصر، ممن كانوا سلبيين لمستضد فيروس التهاب الكبد البائي، وتم إجراء تقييم سريري مع اختبارات بيولوجية كيميائية وهيستولوجية وفيروسية لجميع المرضى. وتم اكتشاف دنا فيروس التهاب الكبد البائي في مصول ثلاثة مرضى من بين 40 مريضا إيجابيا للضد اللبي لفيروس التهاب الكبد البائي.وقد استجاب هؤلاء الثلاثة للمعالجة التوليفية في الأسبوع الثاني عشر للمعالجة.وتوقف واحد فقط منهم عن المعالجة في الأسبوع 24 . ولم يكتشف سوى النمط الجيني D لفيروس التهاب الكبد البائي لدى هؤلاء المرضى مع غياب أي طفرة محددة في هذه المستفردات

L'infection chronique par le virus de l'hépatite C associée à une infection occulte par le virus de l'hépatite B a été associée à un risque accru d'autres hépatites, de cirrhose et de carcinome hépatocellulaire.La présente étude visait à déterminer la prévalence des infections occultes par le virus de l'hépatite B chez des patients égyptiens atteints d'une hépatite C chronique, le génotype et la survenue des mutations génétiques du virus de l'hépatite B et l'impact d'une coïnfection par le virus de l'hépatite B sur la réponse précoce du patient au traitement.L'étude a recruté 162 patients atteints d'une hépatite C chronique dans l'établissement Fever Hôpital d'Ismaïlia [Egypte], ayant des résultats négatifs pour l'antigène de surface du virus de l'hépatite B.Tous les patients ont été soumis à un examen clinique ainsi qu'à des analyses biochimiques, histologiques et virologiques.L'ADN du virus de l'hépatite B a été dépisté dans le sérum de trois patients sur 40 dont les résultats étaient positifs pour l'anticorps nucléocapsidique de l'hépatite B.Ces trois patients ont répondu au traitement combiné à la Semaine 12 du traitement; seul un patient a interrompu le traitement à la Semaine 24.Le genotype D du virus de l'hépatite B était le seul détectable chez ces patients, et aucune mutation du déterminant "a" n'a été observée dans les isolats

High prevalence of Klebsiella pneumoniae carbapenemase-mediated resistance in K. pneumoniae isolates from Egypt.

The emergence and rapid spread of antibiotic-resistant Klebsiella pneumoniae isolates harbouring the blaKPC 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 blaKPC 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 blaKPC 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.

High prevalence of Klebsiella pneumoniae carbapenemase-mediated resistance in K. pneumoniae isolates from Egypt

The emergence and rapid spread of antibiotic-resistant Klebsiella pneumoniae isolates harbouring the blaKPC 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 blaKPC 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 blaKPC 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

The impact of a PCR assay for candidemia on antifungal drug prescribing in critical care: an interrupted time series pilot study.

SUMMARY OBJECTIVE: To evaluate the feasibility of conducting a definitive study to assess the impact of introducing a rapid PCR-based test for candidemia on antifungal drug prescribing. METHOD: Prospective, single centre, interrupted time series study consisting of three periods of six months' duration. The assay was available during the second period, during which the PCR assay was available for routine use by physicians Monday-Friday with guaranteed 24-h turnaround time. For each period total antifungal drug use, expressed as treatment-days, was recorded and an adjustment was made to exclude estimated use for proven candidemia. Also, during the intervention period, antifungal prescribing decisions for up to 72 h after each PCR result became available were recorded as either concordant or discordant with that result. RESULTS: While overall antifungal use remained relatively stable throughout, after adjustment for candidemia, there was a 38% reduction in use following introduction of the PCR test; however, this was nonsignificant at the 95% level. During the intervention period overall concordance between the PCR result and prescribing decisions was 84%. CONCLUSIONS: The PCR assay for candidemia was requested, prescribing decisions were generally concordant with the results produced and there was an apparent decrease in antifungal prescription, although this was sustained even after withdrawal of the intervention; these findings should be more thoroughly evaluated in a larger trial.

Comparison of serum and whole-blood specimens for the detection of Candida DNA in critically ill, non-neutropenic patients.

In contrast to the multitude of studies on fungal PCR assay methods, little work has been reported evaluating Candida PCR performance when using whole blood compared with serum in candidaemic patients. Here, a comparison of the performance of whole-blood and serum specimens using a set of real-time PCR Candida species assays is described. Specimens were collected prospectively from non-neutropenic adults who were recruited to a diagnostic clinical trial, the primary purpose of which was to verify the performance of the assays using serum; in all, 104 participants also had whole-blood specimens submitted for analysis in addition to the serum specimen. Of these participants, 10 had laboratory-confirmed candidaemia and 94 were categorized as being 'unlikely' to have invasive Candida infection. PCR results from the whole-blood specimens are presented here and compared with the results from serum specimens in this subgroup among whom both specimen types were obtained contemporaneously. All participants with candidaemia were PCR-positive from serum samples; however, only seven were PCR-positive from whole blood. All specimens from patients in the 'unlikely' category were PCR-negative in both types of specimen. Moreover, DNA extraction from serum required 1 h; extraction from whole blood required approximately 3 h. These data tentatively suggest that, overall, serum is an appropriate specimen for Candida PCR for detection of candidaemia in non-neutropenic adults.

A prospective clinical trial of a real-time polymerase chain reaction assay for the diagnosis of candidemia in nonneutropenic, critically ill adults.

BACKGROUND: Invasive Candida infection among nonneutropenic, critically ill adults is a clinical problem that has received increasing attention in recent years. Poor performance of extant diagnostic modalities has promoted risk-based, preemptive prescribing in view of the poor outcomes associated with inadequate or delayed antifungal therapy; this risks unnecessary overtreatment. A rapid, reliable diagnostic test could have a substantial impact on therapeutic practice in this patient population. METHODS: Three TaqMan-based real-time polymerase chain reaction assays were developed that are capable of detecting the main medically important Candida species, categorized according to the likelihood of fluconazole susceptibility. Assay 1 detected Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida dubliniensis. Assays 2 and 3 detected Candida glabrata and Candida krusei, respectively. The clinical performance of these assays, applied to serum, was evaluated in a prospective trial of nonneutropenic adults in a single intensive care unit. RESULTS: In all, 527 specimens were obtained from 157 participants. All 3 assays were run in parallel for each specimen; they could be completed within 1 working day. Of these, 23 specimens were obtained from 23 participants categorized as having proven Candida infection at the time of sampling. If a single episode of Candida famata candidemia was excluded, the estimated clinical sensitivity, specificity, and positive and negative predictive values of the assays in this trial were 90.9%, 100%, 100% and 99.8%, respectively. CONCLUSIONS: 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.

Improving molecular detection of Candida DNA in whole blood: comparison of seven fungal DNA extraction protocols using real-time PCR.

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.

Trends in candidemia and antifungal susceptibility in a university hospital in Northern Ireland 2001-2006.

OBJECTIVES: To describe the species distribution and antifungal susceptibility trends for documented episodes of candidemia at the Royal Hospitals, Belfast, 2001-2006. METHODS: Laboratory-based retrospective observational study of all episodes of candidemia. RESULTS: There were 151 episodes of candidemia. The species recovered were: 96 C. albicans; 26 C. glabrata; 18 C. parapsilosis; five C. tropicalis; four C. guilliermondii; one C. famata and one C. dubliniensis. We separated the data into two periods 2001-2003 and 2004-2006; contrary to the findings of other investigators, there was a notable trends toward increasing frequency of C. albicans and decreasing frequency of non-albicans species over time. Although the proportion of C. albicans, C. parapsilosis and C. tropicalis isolates susceptible to fluconazole was unchanged over time, a trend of decreased susceptibility of C. glabrata to fluconazole was noted over the six-year period. Overall, 73% and 7.7% of C. glabrata isolates had susceptible-dose-dependent and resistant phenotypes, respectively. The percentage of C. glabrata isolates susceptible to fluconazole (MIC <8 microg/ml) decreased from 36% in 2001-2003 to 0% in 2004-2006. Flucytosine resistance was detected in only 4 (2.7%) isolates. None of the isolates had an amphotericin B MIC <1 microg/ml. CONCLUSION: A shift towards increasing dominance of C. albicans contrasts both with reports from other countries and previous data from Northern Ireland. Upwards fluconazole MIC drift among C. glabrata has important implications for empirical therapeutic decisions.

Rapid differentiation between fluconazole-sensitive and -resistant species of Candida directly from positive blood-culture bottles by real-time PCR.

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 (n=33 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 approximately 72 to <3 h, and consequently allows optimization of the antifungal regimen at an earlier stage.

Clinical assessment of a generic DNA amplification assay for the identification of respiratory adenovirus infections.

BACKGROUND: respiratory adenoviruses are common, often resulting in serious sporadic and epidemic infections and impaired immunity can dramatically increase their severity. They are now thought capable of establishing latency. Diagnosis by culture is slow while direct antigen detection by immunofluorescence lacks sensitivity. Molecular diagnosis can be both rapid and sensitive but the genetic heterogeneity of adenoviruses poses problems. OBJECTIVES: to design a generic adenovirus nested polymerase chain amplification assay designed to be capable of detecting all respiratory adenoviruses. This was achieved through optimised thermal cycling and the development of a generic degenerate primer set targeting the adenovirus hexon gene. STUDY DESIGN: this was a cross-sectional study on 172 respiratory specimens from hospital-based patients, and one from a general practice, in Northern Ireland. A comparison was made between the amplification assay, virus culture and immunofluorescence. RESULTS: the nested polymerase chain reaction (nPCR) assay had a generic capacity for adenovirus detection and an analytical sensitivity of 6.4x10(2) copies/ml. Using an expanded gold standard (defined as a true positive or a true negative where a specimen was positive or negative by at least two of the study assays, respectively), PCR had a clinical sensitivity and specificity of 46/46 (100%) and 15/126 (91.3%), respectively. Patients with acute respiratory adenovirus infections were more likely to be male (chi(2), p=0.005) and to present with a fever (chi(2), p=0.02) than patients diagnosed with another respiratory virus. Co-infection was identified in 12/172 patients. CONCLUSIONS: the nested amplification assay proved highly sensitive in both the analytical and clinical settings for the detection of respiratory adenovirus infections.