Nikolaos Siafakas

Laboratory Diagnosis of Common Herpesvirus Infections of the Central Nervous System by a Multiplex PCR Assay

ABSTRACT A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the method was optimized and carried out and its sensitivity was compared to previously described assays. The assay has been used on a limited number of clinical samples and must be thoroughly evaluated in the clinical context. A total of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were included in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of the highest dilution positive by PCR for HSV-1 and HSV-2 respectively, whereas for VZV, CMV and EBV, 14, 18, and 160 ag of genomic DNA were detected corresponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was detected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%), and CMV was also detected in one neonate (1.16%). None of the samples tested was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification from CSF samples was avoided by the inclusion in the present multiplex PCR assay of α-tubulin primers. The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of the present multiplex PCR assay.

Isolation of polioviruses and other enteroviruses in south Greece between 1994 and 1998.

During the five‐year period between 1994 and 1998, a total of 217 clinical samples were assessed for the isolation of enteroviruses at the Enterovirus Reference Centre for South Greece. Fourteen enterovirus strains belonging to different serotypes were isolated. These field strains were detected by cell culture in appropriate cell lines. They were subsequently identified by neutralizing antibodies with the LBM (Lim‐Benyesh Melnick) mixed antisera pools up to 1995 and RIVM (National Institute of Public Health and the Environment, Bilthoven, The Netherlands) pools from 1996 onwards. The isolated viruses included two strains of poliovirus type 2 Sabin‐like, three strains of poliovirus type 1 non‐Sabin‐like, one Coxsackie B2 (CBV2) strain, one Coxsackie B5 (CBV5) strain, one Echo 5 (ECV5) strain, one Echo 7 (ECV7) strain, three Coxsackie A16 (CAV16) strains, and two currently enteroviral strains unidentified by RIVM pools. Reverse transcription‐polymerase chain reaction (RT‐PCR) using poliovirus‐specific primers or poliovirus type‐specific primers and enterovirus specific primers from the highly conserved 5′‐UTR, the latter followed by RFLP, was also applied in 6 clinical isolates (3 strains of poliovirus type 1 non‐Sabin‐like, 1 polio type 2 Sabin‐like, and 2 non‐identified by RIVM pools enteroviruses). The advantages and the drawbacks of these assays against the conventional ones are discussed here. The isolations and the subsequent identification of the strains were carried out from fecal samples of clinical cases that included hand‐foot‐and‐mouth disease, meningitis, and acute flaccid paralysis. The reappearance of non‐Sabin‐like poliovirus strains in Greece in 1996 after 14 years is considered to have an important medical and clinical value. J. Clin. Lab. Anal. 14:157–163, 2000.

Development of a quadriplex polymerase chain reaction for human cytomegalovirus detection.

The development of a quadriplex PCR method with amplification of HCMV in a single‐step procedure using primers taken from four different regions of the viral genome is described. Different concentrations of dNTPs and MgCl2 were assayed in order to optimize the constitution of the buffer for the multiplex PCR. The specificity of the PCR was tested with 100ng, 10ng, and 1ng of genomic MRC‐5 cell DNA infected with CMV in the presence of 10μg of uninfected MRC‐5 cell DNA. The sensitivity of the PCR was evaluated by the amplification of various amounts (100ng, 10ng, 1ng, and 0.1ng) of genomic MRC‐5 cell DNA infected with CMV. The specificity and sensitivity assays were performed for each pair of primers and for the combined four primer pairs in the multiplex PCR. CMV was consistently detected from 10ng of genomic MRC‐5 cell DNA with each primer pair. When all four sets of primers were combined in a single reaction tube, the sensitivity of the assay was equivalent to 10ng of genomic MRC‐5 cell DNA, whereas amplification from 1ng genomic MRC‐5 cell DNA produced only a subset of the amplimers. By amplifying four target‐sequences of HCMV simultaneously with minimum incubation time at each temperature, a quadriplex, highly sensitive PCR assay was performed. The use of four primer sets designed in different genomic regions of HCMV allowed the detection of variants and achieved maximal sensitivity and specificity which are essential for a diagnostic utilization. J. Clin. Lab. Anal. 13:99–105, 1999.

fbl gene as a species-specific target for Staphylococcus lugdunensis identification.

Staphylococcus lugdunensis is an unusually virulent coagulase‐negative species, associated with severe infections. The present report describes the development of a single‐step, species‐specific PCR protocol for S. lugdunensis identification. fbl gene, encoding a fibrinogen‐binding adhesin, was exploited and assessed as a suitable nucleic acid target. The gene was detected in all 17 S. lugdunensis isolates examined, while no amplification product was obtained from 98 isolates representing 11 staphylococcal and 17 nonstaphylococcal species. Forty‐seven percent of the S. lugdunensis strains produced a positive slide coagulase reaction, which is consistent with varying levels of Fbl protein expression within the species. J. Clin. Lab. Anal. 24:119–122, 2010.

Classification and Structure of Echovirus 5′-UTR Sequences

Enteroviruses are classified into two genetic clusters on the basis of 5′-UTR and all echoviruses (ECV) are classified together with coxsackie B viruses (CBV), coxsackie A viruses (CAV) types 2–10, 12, 14 and 16, and enteroviruses (EV) 68, 69, 71 and 73. During the present study, 5′-UTR-derived sequences constituting the largest part of the Internal Ribosome Entry Site (IRES) of ECVs were studied with respect to their possible secondary structures, which were predicted following the phenomenon of “covariance”, i.e. the existence of evolutionary pressure in favour of structural conservation in the light of nucleotide sequence variability. In this and previous studies, no correlation between overall 5′-UTR identity and the currently recognised Human Enterovirus species was found, implying that notwithstanding their divergent protein-encoding regions, these species are free to exchange 5′-UTRs by recombination. Secondary structure features which are known to be highly conserved amongst enteroviruses and specifically the GNRA tetraloop in secondary structure domain IV, involved in long-term tertiary interactions and loop B in secondary structure domain V with an as yet unknown function were also conserved in ECVs. In contrast, the C(NANCCA)G motif, which is considered to be important in virus transcription and translation, was not conserved in all ECVs and sequence patterns observed in other enterovirus groups and rhinoviruses were recorded.

Qualitative and Quantitative Detection of Protein and Genetic Traits in Genetically Modified Food

Abstract Due to the market introduction of genetically modified organisms (GMOs) in crops, foods, and ingredients, legislation worldwide came face to face with the question of the use and labeling requirements on GMO crops and their derivatives. In this review, protein- and DNA-based methods, such as enzyme-linked immunosorbent assay, western blots, and qualitative and quantitative polymerase chain reaction PCR (Q-PCR) are reviewed. Qualitative detection methods for genetically modified (GM) sequences in foods have evolved rapidly during the past years. The sensitivity of these systems is extremely high, even for processed foodstuffs. However, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. The recently introduced labeling threshold for GMOs in food ingredients by the European Union has forced official food control laboratories to apply quantitative PCR methods. Taking the precision of quantitative PCR detection methods into account, suitable sample plans and sample sizes for GMO analysis are discussed. As quantitative GMO detection methods measure GMO contents of samples in relation to reference material, priority must be given to international agreements and standardization on certified reference materials. The rapidly increasing number of GM foods on the market demands the development of more advanced multidetection systems, such as microarray technology. Challenges and problems arising from the inability to detect GM foods for which the modified sequence is unknown, the lengthy standardization procedures, and the need to continuously update databases comprising commercially available GM foods and the respective detection strategies are also discussed.

Single-Dose Pharmacodynamics of Amphotericin B against Aspergillus Species in an In Vitro Pharmacokinetic/Pharmacodynamic Model

ABSTRACT Conventional MIC testing of amphotericin B results in narrow MIC ranges challenging the detection of resistant strains. In order to discern amphotericin B pharmacodynamics, the in vitro activity of amphotericin B was studied against Aspergillus isolates with the same MICs by using a new in vitro pharmacokinetic/pharmacodynamic (PK/PD) model that simulates amphotericin B human plasma levels. Clinical isolates of Aspergillus fumigatus, A. terreus, and A. flavus with the same Clinical and Laboratory Standards Institute modal MICs of 1 mg/liter were exposed to amphotericin B concentrations following the plasma concentration-time profile after single-bolus administration with Cmax values of 0.6, 1.2, 2.4, and 4.8 mg/liter. Fungal growth was monitored for up to 72 h based on galactomannan production. Complete growth inhibition was observed only against A. fumigatus with amphotericin B with a Cmax of ≥2.4 mg/liter. At the lower Cmax values 0.6 and 1.2 mg/liter, significant growth delays of 34 and 52 h were observed, respectively (P < 0.001). For A. flavus, there was no complete inhibition but a progressive growth delay of 1 to 50 h at an amphotericin B Cmax of 0.6 to 4.8 mg/liter (P < 0.001). For A. terreus, the growth delay was modest (up to 8 h) at all Cmaxs (P < 0.05). The Cmax (95% confidence interval) associated with 50% activity for A. fumigatus was 0.60 (0.49 to 0.72) mg/liter, which was significantly lower than for A. flavus 3.06 (2.46 to 3.80) mg/liter and for A. terreus 7.90 (5.20 to 12.29) mg/liter (P < 0.001). A differential in vitro activity of amphotericin B was found among Aspergillus species despite the same MIC in the order A. fumigatus > A. flavus > A. terreus in the in vitro PK/PD model, possibly reflecting the different concentration- and time-dependent inhibitory/killing activities amphotericin B exerted against these species.