Molecular Characterization of High-Level Mupirocin Resistance in Staphylococcus pseudintermedius

The genetic analysis of high-level mupirocin resistance (Hi-Mup^r) in a Staphylococcus pseudintermedius isolate from a dog is presented. The Hi-Mup^rileS2 gene flanked by a novel rearrangement of directly repeated insertion sequence IS257 elements was located, together with the aminoglycoside resistance aacA-aphD determinant, on a conjugative plasmid related to the pSK41/pGO1 family plasmids.     

Rapid Detection of Several Mycobacterial Species Using a Polymerase Chain Reaction Reverse Hybridisation Assay

The ability of a polymerase-chain-reaction-based technique combined with a reverse hybridisation line-probe assay to detect and identify eight of the most clinically significant mycobacterial species directly from cultures in liquid medium was evaluated. The line-probe assay allows simultaneous identification of eight different mycobacterial species. Ninety-seven mycobacterial strains belonging to 22 different species were evaluated. All strains were previously inoculated into MB/BacT bottles (Organon Teknika, USA). The sensitivity and specificity of the test when applied on positive MB/BacT liquid cultures containing isolates from mycobacterial species included as specific probes on the line-probe assay strip was 100% and 100%, respectively. These results further support the potential clinical usefulness of this technique in the diagnosis of mycobacterial infections. Electronic Publication     

Acquisition of High-Level Mupirocin Resistance and Its Fitness Cost among Methicillin-Resistant Staphylococcus aureus Strains with Low-Level Mupirocin Resistance

We investigated whether methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) isolates with low-level mupirocin resistance can serve as recipients of a pSK41-like plasmid conferring high-level mupirocin resistance without substantial fitness cost. Our results suggest that acquisition of the plasmid conferring high-level mupirocin resistance was not necessarily associated with fitness cost in some MRSA recipients with low-level mupirocin resistance.Mupirocin is an antibacterial agent with a unique mechanism of action, the selective binding to bacterial isoleucyl-tRNA synthetase (IleRS) (8). Resistance to mupirocin is phenotypically divided into two groups, low-level resistance, with MICs of 8 to 256 μg/ml, and high-level resistance, with MICs of ≥512 μg/ml. Low-level resistance is mediated by point mutations of ileS. High-level resistance results from the acquisition of a plasmid containing ileS-2 (or mupA) encoding an altered IleRS which has no affinity for mupirocin (7).Mupirocin has been used in an attempt to decolonize methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) carriers as a strategy to prevent subsequent MRSA infections and control the spread of this pathogen. Several studies have recognized the benefit associated with mupirocin use (16, 18, 20). At the same time, it is important to acknowledge the problem of mupirocin resistance. The effect of mupirocin use on the prevalence of mupirocin resistance among staphylococci varies considerably. Studies have shown a significant increase in mupirocin resistance despite low mupirocin use (11) or following widespread or routine use of mupirocin (21). These observations suggest that acquisition of the plasmid conferring high-level mupirocin resistance may not impose a substantial cost to the bacteria.In clinical isolates of S. aureus expressing mupirocin resistance, low-level resistance was more prevalent than high-level resistance (5, 12, 19). As a result of selective pressure imposed by the increased use of mupirocin, plasmid acquisition of resistance determinants would undoubtedly be a potential survival strategy. Defining the impact of antimicrobial resistance on bacterial fitness has implications for microbial ecology and enables researchers to predict the pace of resistance development. This study was aimed at investigating the effect of isolates with low-level mupirocin resistance on their capacity to serve as recipients of the plasmid conferring high-level mupirocin resistance and assessing the fitness trade-off.A total of seven MRSA isolates from a mupirocin surveillance study (14) were used in this study and are listed in Table ​Table1.1. Community-associated MRSA (CA-MRSA) 859, an isolate containing type IV staphylococcal cassette chromosome mec (SCCmec) and exhibiting high-level mupirocin resistance (MIC > 512 μg/ml), was used as the plasmid donor. Six MRSA isolates selected as conjugal recipients were clinical isolates containing type II SCCmec and exhibiting low-level resistance to mupirocin (MICs, 16 to 64 μg/ml). PCR amplification to detect the ileS-2 gene (24) was performed. The ileS-2 gene was detected in CA-MRSA 859 but was not detected in any isolates with low-level mupirocin resistance. PCR amplification of the native ileS gene using primers ileS54L and ileS2798U (2) and sequencing analysis were performed on all isolates. All low-level mupirocin-resistant isolates possessed a G-to-T point mutation at position 1762 (G₁₇₆₂T), causing a Val-to-Phe substitution (V₅₈₈F). No ileS mutation was detected in the CA-MRSA 859 isolate.

TABLE 1.

MRSA strains used in conjugative transfer experiments and their relevant properties

改良聚合酶链反应快速诊断结核性脑膜炎

目的：建立改良的聚合酶链反应的实验方法，快速诊断结核性脑膜炎。方法：应用二步控温洽酶链反应技术检测脑脊液结核杆菌微量ＤＮＡ。结果：经是 铲的４０例结核性脑膜炎聚合酶链反应扩增脑脊液ＤＮＡ全部阳性，２４例不明原因的中枢神经系统感染患者，１６例为阳性，而其它病原体引起的中枢神经系统感染和正常对照组均无扩增反应。结论：二步控温聚合酶链反应技术快速诊断结核性脑膜炎与传统的检验方法相比较，具有快速、敏感、高     

Assessment of Efficacy of Palm Polymerase Chain Reaction with Microscopy,Rapid Diagnostic Test and Conventional Polymerase Chain Reaction for Diagnosis of Malaria

Purpose: Sensitive, specific, rapid and cost-effective technique for malaria diagnosis is need of the hour. Microscopy has been the gold standard for malaria diagnosis, but its interpersonnel variability and lack of sensitivity make it subjective test. Conventional polymerase chain reaction (cPCR) has proven to be sensitive technique, but costly and time-consuming. Considering these factors, we have compared microscopy and cPCR with newly derives ultra-fast, portable PCR machine called Palm PCR. Materials and Methods: Palm PCR is arranged with three heat blocks precisely made for three stages of PCR cycles with 34 min for 1100 bp Plasmodium genus outer primer to amplify and 10 min each for Plasmodium falciparum and Plasmodium vivax inner primers of 120 bp and 205 bp, respectively. A total of 191 suspected samples were processed and evaluated using receiver operating characteristic (ROC) curve analysis. Results: The area under ROC curve analysis for Palm PCR with reference standard microscopy for P. falciparum, P. vivax and Plasmodium was 0.8969, 0.9121 and 0.9116, respectively, and with reference standard cPCR was 1.0 for all of them. ROC curve area close of suggests that Palm PCR can be as significant as cPCR in malaria diagnosis. In fact, ultra-rapid amplification with same precision makes Palm PCR better technique than cPCR. Conclusion: Palm PCR is sensitive, rapid and works on battery with simple laboratory facility requirements. Portable electrophoresis and transilluminator combined with Palm PCR could be implemented as an important diagnostic tool in resource-limited and rural areas. Similar studies with wider parameters in rural areas will help us evaluate and maybe establish Palm PCR as PCR platform of choice for such specific set-ups.     

Evaluation of mupA EVIGENE Assay for Determination of High-Level Mupirocin Resistance in Staphylococcus aureus

Mupirocin is widely used to decolonize patients carrying Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA). The aim of this study was to determine the presence of high-level mupirocin resistance by a new commercially available mupA genotypic diagnostic product, mupA EVIGENE assay (AdvanDx).Mupirocin is a naturally occurring antibiotic produced by Pseudomonas fluorescens. Mupirocin exerts its antimicrobial effect by inhibiting protein synthesis through irreversible and specific binding to bacterial isoleucyl-tRNA synthetase encoded by a chromosomal gene, ileS (8). Mupirocin is widely used, as it is established as the best topical antimicrobial available for both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) (9) and is an important agent in preventing transmission of health care-associated staphylococcal infection, surgical-site infection (12), and bloodstream infection in patients undergoing hemodialysis (2, 14).Unfortunately, bacterial resistance to mupirocin has been seen for both MSSA and MRSA. Mupirocin resistance occurs as two phenotypes: “high level” and “low level.” Low-level resistance is due to a genetic mutation, producing MICs in the range of 8 to 256 μg/ml, whereas high-level resistance (with an MIC of >256 μg/ml) is caused by presence of the plasmid gene mupA (or ileS-2) that encodes an additional isoleucyl-tRNA synthetase that is not inhibited by mupirocin (5, 7). Since application of mupirocin ointment into the external nares results in concentrations of ≥20,000 μg/ml, only high-level resistance is potentially significant in this setting (15).High-level mupirocin resistance occurs in a variety of community-associated MRSA (CA-MRSA) and health care-associated MRSA (HA-MRSA) strains (13). Currently, isolates of the USA300 (ST8) lineage are the most prevalent CA-MRSA strain in United States, and they have been confirmed to carry the conjugative plasmid (pUSA03) expressing the mupA high-level mupirocin resistance gene (6). This indicates the need for susceptibility testing or detection of the mupA gene to guide therapeutic and prophylactic use of mupirocin.Differentiation between high- and low-level resistances requires methods such as broth microdilution for determination of MIC, Etest (which is not cleared by the U.S. Food and Drug Administration [FDA]), or detection of the mupA gene associated with high-level resistance (in-house PCR methods) (5, 15). Genotypic tests offer the advantage of detecting the genetic marker itself rather than phenotypic characteristics. Furthermore, genotypic tests can be performed directly using the clinical isolate without the need for incubation and culturing overnight. The aim of this study was to compare a new genotypic test for the detection of high-level mupirocin resistance, mupA EVIGENE assay (AdvanDx), with PCR for the mupA gene followed by bidirectional sequence analysis, the Clinical and Laboratory Standards Institute (CLSI)-recommended phenotypic mupirocin resistance testing (signle-broth dilution test [256 μg/ml] and 200-μg disk diffusion test [3, 4]), and the mupirocin Etest.The mupA EVIGENE assay is a sandwich hybridization assay that provides rapid (3.5-h) detection of the mupA gene from clinical isolates of S. aureus compared to the 18 to 24 needed for phenotypic tests. The EVIGENE plate allows testing in large batches (94 isolates) or testing of one sample at a time. The hands-on time is approximately 10 min for one sample and 30 min for 50 samples. The only equipment needed is a heating block and heated shaker (55°C, 400 rpm), and because the signal can be read by eye, this assay should be easy to implement for most laboratories.The mupirocin gene has been associated with 46 different plasmid profiles (13). To make the collection of tested isolates as broad and diverse as possible, a total of 156 S. aureus isolates were collected and tested at one U.S. site and one European Union site (Denmark). In addition, a study was performed at the Centers for Disease Control and Prevention (CDC), Atlanta, GA, with 105 unrelated S. aureus isolates with known mupirocin MICs in the range of 8 to >1,024 μg/ml.All 156 isolates were analyzed for the presence of mupA by using a PCR assay (11). As a control for the presence of PCR-inhibitory components in the template preparation, a second PCR was employed (1). Compared with PCR, the mupA EVIGENE assay exhibited 100% sensitivity (i.e., results by the two methods were the same for 16 out of 16 isolates tested) and 100% specificity (140 out of 140 isolates) (Table ​(Table11 ).

TABLE 1.

mupA EVIGENE performance data versus genotypic testing (mupA PCR) data

Detection of Clarithromycin-Resistant Helicobacter pylori by Polymerase Chain Reaction Using Residual Samples from Rapid Urease Test

Background: Approximately 50% of the world population is infected with Helicobacter pylori, which corresponds to a high infection rate. Furthermore, the incidence of antibiotic-resistant H. pylori has increased with the recent rise in use of antibiotics for H. pylori elimination, suggesting growing treatment failures. Aim: The study was aimed to assess the use of residual samples from rapid urease test (RUT) for biomolecular testing as an effective and accurate method to detect antibiotic-resistant H. pylori. Settings and Design: This study was a retrospective study performed using data obtained from medical records of previously isolated H. pylori strains. Materials and Methods: RUT was conducted for 5440 biopsy samples from individuals who underwent health examination in South Korea. Subsequently, 469 RUT residual samples were randomly selected and subjected to polymerase chain reaction (PCR) to detect antibiotic-resistant H. pylori. Statistical Analysis Used: The Chi-square test was used to analyse categorical data. P < 0.05 was considered statistically significant. Results: The results showed a concordance between the results of PCR and conventional RUT in 450 of 469 samples, suggesting that the H. pylori PCR test is a time- and cost-effective detection method. Conclusions: This study demonstrated that PCR test can aid physicians to prescribe the appropriate antibiotics at the time of diagnosis, thus preventing the reduction in H. pylori eradication due to antibiotic resistance, averting progression to serious diseases and increasing the treatment success rate.     

Rapid Detection of Ethambutol-Resistant Mycobacterium tuberculosis in Clinical Specimens by Real-Time Polymerase Chain Reaction Hybridisation Probe Method

Background: Early diagnosis of drug resistance (DR) to ethambutol (EMB) in tuberculosis (TB) remains a challenge. Simple and reliable method (s) are needed for rapid detection of DR Mycobacterium tuberculosis (MTB) in clinical specimens. Objectives: The aim of this study was to design fluorescence resonance energy transfer hybridisation probe-based real-time polymerase chain reaction (PCR) method for the early detection of EMB-resistant MTB direct from clinical sputa. Materials and Methods: Primers and probes were designed against 306 codon of embB gene which is commonly associated with EMB resistance. A comparative study was done between Lowenstein–Jenson (L–J) proportion and hybridisation probe-based real-time PCR method for susceptibility testing. DNA sequencing was used in nine representative isolates to validate the efficiency of real-time PCR method to detect emb306 mutation of MTB. Results: A total of 52 clinical sputum samples and corresponding culture isolates (from category II pulmonary TB cases) were included in this study. Out of 52 MTB isolates, 32 and 20 were resistant and susceptible to EMB, respectively, as determined by L–J proportion method. Real-time PCR showed 95% specificity, 75% sensitivity and 82.69% accuracy when compared with L–J proportion method. A 100% of concordance was observed by validating the real-time PCR results with DNA sequencing. Conclusions: Our real-time PCR hybridisation probe method promises for rapid detection of EMB-resistant MTB directly from clinical specimens. However, future studies and modifications of method by incorporating other potential loci along with targeted mutation (emb306) are still required to increase the sensitivity of method.     

High-Sensitivity Detection of Human Malaria Parasites by the Use of Rapid Diagnostic Tests and Nested Polymerase Chain Reaction in Burdened Communities of North East India

Background and Objectives: The study aimed to evaluate the diagnostic performance of malaria through microscopy and rapid diagnostic test (RDT) analysis performed locally and the accuracy evaluated by nested polymerase chain reaction (PCR) for diagnosis of Plasmodium falciparum from hotspot regions of North East (NE) India. Materials and Methods: One thousand one hundred and seventy-three blood samples were collected for identification of P. falciparum infection using microscopy and RDT analysis. DNA was extracted from whole blood using QIAamp DNA blood mini kit, and nested PCR was performed to confirm P. falciparum for evaluating sensitivity and specificity from various epidemiological surveys and geographical areas of NE India. Results: Of 1173 symptomatic malaria suspected patients, 15.6% (183/1173) patients were diagnosed as malaria positive by RDT and 67.94% cases (53/78) with microscopy. Of 183 malaria-positive patients, 42.62% (78/183) were diagnosed with P. falciparum and 84.61% (66/78) further confirmed to be P. falciparum positive by nested PCR. High sensitivity (97.9%) and low specificity (2.03%) of the RDT and high sensitivity (99.1%) and low specificity (0.9%) in microscopy against nested PCR results was statistically significant (P < 0.05). Epidemiological comparisons expressed highest incidences in Manipur (51.11%) followed by Meghalaya (48.93%) and Assam (35.16%). Overall incidence rate among the genders was observed to be higher in males than in females. Conclusions: Our findings suggest that PCR, RDT and microscopy can potentially determine hotspots at moderate transmission intensities, but PCR testing has a diagnostic advantage as transmission intensity falls. Therefore, malaria control programs should consider PCR testing when the prevalence of infection is low.     

Evaluation of mupA Evigene in Comparison to Disk Diffusion for Detection of High-Level Mupirocin Resistance in Clinical Isolates of Staphylococcus aureus

High-level mupirocin resistance (H-Mu^r) in S. aureus is associated with the mupA gene. The mupA Evigene test rapidly identifies this gene. This study assessed the performance of mupA Evigene compared to that of susceptibility disk testing. mupA Evigene detected H-Mu^r in 6/179 S. aureus isolates, and the results were concordant with those of susceptibility disk testing.Staphylococcus aureus infections are increasing worldwide (7). S. aureus colonizes multiple body surfaces, especially the anterior nares of asymptomatic individuals. Colonization frequently precedes invasive disease and is a risk factor for infection in high-risk populations (5, 14). Active detection of S. aureus colonization followed by decolonization with mupirocin (Bactroban; GlaxoSmithKline) has emerged as an option for control of staphylococcal infections. Mupirocin inhibits protein biosynthesis by binding to the bacterial isoleucyl-tRNA synthetase, encoded by chromosomal gene ileS (3).The eradication rates achieved with mupirocin may reach 90% (11). At least two forms of mupirocin resistance are known to occur in S. aureus. Low-level resistance, characterized by an MIC of 8 to 256 μg/ml, is mediated by point mutations in the chromosomally encoded ileS gene (6). High-level resistance, characterized by an MIC of >256 μg/ml, is mediated by a plasmid-borne mupA gene encoding an additional synthetase that is mupirocin resistant (2). Chromosomally located mupA has also been reported (12). High-level mupirocin resistance (H-Mu^r) may be detected either by the agar-based susceptibility disk assay (200-μg mupirocin disk) or with the broth microdilution assay (single well containing 256 μg/ml mupirocin) (4). Further, H-Mu^r may be detected using mupA-targeted PCR or the mupirocin Etest. Disk susceptibility, microdilution, and Etest each requires 24 h. Mupirocin PCR is faster but requires 6 to 8 h (1, 8). Rapid detection of mupirocin resistance could facilitate decolonization strategies. mupA Evigene (AdvanDx, Woburn, MA) is a signal-amplification nucleic acid hybridization test currently approved for in vitro diagnostic use in the European Union. It has been demonstrated to have the ability to detect the mupA gene in S. aureus isolates in as little as 3 h (9, 13).We evaluated mupA Evigene''s ability to detect H-Mu^r among 179 clinical isolates of S. aureus with unknown mupirocin susceptibility. Isolates were obtained from the clinical microbiology laboratory at the Washington Hospital Center and consisted of fresh, second-passage isolates (less than 3 days old and never frozen) grown on blood agar plates. The isolates were identified as S. aureus using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH; AdvanDx, Woburn, MA) and automatic phenotypic testing (BD Phoenix; Becton Dickinson, Franklin Lakes, NJ). Isolates were randomly selected and obtained both from cultures of samples from sites for clinical testing and from nasal surveillance swab specimens from patients located throughout the hospital from July to September 2008. The tested isolates included both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus strains. mupA Evigene and mupirocin susceptibility disk tests were performed in the our laboratory by an investigator who was blinded to the results of the other test. mupA Evigene was performed according to the manufacturer''s recommendations (1). First, fresh colonies were buffered with lysis solution and incubated with mupA detection probes. Second, the hybridization process was done by adding conjugate and developing the substrates. Finally, the results were read both visually and by measurement of the absorbance at 490 nm using a fixed-wavelength MRX II photometer (Dynex Technologies, Chantilly, VA). Pink or clear wells were considered negative for the mupA gene, whereas red wells were positive. Likewise, an absorbance of >0.5 was considered a positive result. A negative control (a mupA-negative MSSA strain) and a positive control (a mupA-positive MSSA strain) were included in each run to assess quality. Both controls were provided by AdvanDx.Isolates were tested by agar disk diffusion using a 200-μg mupirocin disk, according to the manufacturer''s instructions (Oxoid, Hampshire, United Kingdom) (4). Susceptibility testing was performed using inoculums equivalent to a 0.5 McFarland standard. Agar disk diffusion was performed using Mueller-Hinton agar plates (Becton Dickinson). For agar disk diffusion, isolates were defined as having H-Mu^r when there was no inhibition zone after 24 h of incubation at 37°C.A total of 179 isolates were analyzed by mupA Evigene and agar disk diffusion. Six of the 179 (3.35%) isolates were mupirocin resistant by mupA Evigene (Table ​(Table1).1). There was 100% concordance between the visual reading and the absorbance at 490 nm. For H-Mu^r detection, there was 100% concordance between mupA Evigene and agar disk diffusion. Using disk diffusion as the standard of reference, mupA Evigene achieved 100% sensitivity and specificity. Four isolates with known high-level mupirocin resistance (confirmed by PCR) and one with known susceptibility (PCR negative) were used to assess the reproducibility of the mupA Evigene assay. All isolates were accurately identified as resistant or susceptible by the Evigene assay at least four times throughout the study. The optical densities (ODs) at 490 nm ranged from 0.580 to 1.273 for the resistant isolates, and the OD was 0.224 for the mupirocin-susceptible isolate. There was 100% concordance between the visual and OD results.

TABLE 1.

Diagnostic performance of mupA Evigene

Review of Detection of Brucella sp. by Polymerase Chain Reaction

Here we present a review of most of the currently used polymerase chain reaction (PCR)-based methods for identification of Brucella bacteria in biological samples. We focused in particular on methods using single-pair primers, multiplex primers, real-time PCRs, PCRs for marine Brucella, and PCRs for molecular biotyping. These methods are becoming very important tools for the identification of Brucella, at the species level and recently also at the biovar level. These techniques require minimum biological containment and can provide results in a very short time. In addition, genetic fingerprinting of isolates aid in epidemiological studies of the disease and its control. PCR-based methods are more useful and practical than conventional methods used to identify Brucella spp., and new methods for Brucella spp identification and typing are still being developed. However, the sensitivity, specificity, and issues of quality control and quality assurance using these methods must be fully validated on clinical samples before PCR can be used in routine laboratory testing for brucellosis.Brucellosis is caused by Brucella spp which is composed of seven terrestrial species and at least two marine species. Terrestrial Brucella spp. include B. abortus, B. melitensis, B. suis. B. ovis, B. canis, B. Neotomae, and a new species, B. microti. Brucella isolated from marine mammals are B. ceti and B. pinnipedialis (1). The first 3 terrestrial species include several biovars. The terrestrial Brucella species display a high degree of DNA homology based on DNA-DNA hybridization studies. Nevertheless, DNA polymorphism sufficient to differentiate the first 6 Brucella species and some of their biovars has been shown to exist (2). Brucella isolated from marine mammalian species is still under investigation. According to the classical criteria of host preference and DNA polymorphism at their outer membrane protein 2 (omp2) locus, at least 2 species that infect marine mammals exist (3).The gold standard for the diagnosis of brucellosis is isolation of Brucella bacteria. However, to isolate Brucella bacteria is time- and resource-intensive; it requires level 3 biocontainment facilities and highly skilled technical personnel to handle samples and live bacteria for eventual identification and biotyping. Handling all live Brucella involves risk of laboratory infection and very strict biosafety rules must be observed. In order to avoid these disadvantages, methods based on the polymerase chain reaction (PCR) are becoming very useful and considerable progress has been made recently to improve their sensitivity, specificity, and technical ease and to lower costs. To date, at least 400 reports have been published dealing with various PCR-based methods for Brucellosis detection. In this review, we discuss extraction of DNA and various PCR methods using different primers and reaction conditions.     

Detection of MYCN Amplification in Serum DNA Using Conventional Polymerase Chain Reaction

Neuroblastoma (NB) is the most common extra-cranial solid tumor of childhood and is characterized by a wide range of clinical behaviors. Amplification of MYCN is a well-known poor prognostic factor in NB patients. As the MYCN amplification status is usually tested using tumor specimens, lengthy and invasive procedures are unavoidable. To evaluate the possibility of detecting MYCN amplification without invasive procedure, we performed conventional polymerase chain reaction (PCR) analysis to identify MYCN amplification using the preserved serum DNA. PCR of serum DNA was done in 105 NB patients whose MYCN status had been confirmed by fluorescence in situ hybridization. MYCN amplification was evaluated as the ratio of signal intensities between MYCN and NAGK (M/N ratio). When regarding the tissue FISH results as a reference, 10 patients had MYCN-amplified (MNA) NB, and 95 had non-MNA NB. The M/N ratio of the MNA group (median 2.56, range 1.01-3.58) was significantly higher than that of the non-MNA group (median 0.97, range 0.67-5.18) (P < 0.001). In the receiver operating characteristic curve analysis, the area under the curve was 0.957 (95% confidence interval 0.898–1.000; P < 0.001), and it showed 90.9% sensitivity and 97.9% specificity with the selected cut-off value set as 1.6. The detection of MYCN amplification using conventional PCR analysis of serum samples seems to be a simple and promising method to evaluate the MYCN status of NB patients. Further study with a larger set of patients is needed to confirm the accuracy of this result.     

Polymerase Chain Reaction Detection of Foodborne Salmonella spp. in Animal Feeds

Abstract

Foodborne salmonellosis continues to be a public health issue of considerable concern. Animal feed has been a major link in preharvest food animal production. Although monitoring systems and control measures are available to limit Salmonella spp. contamination on animal feeds detection methodology is relatively time consuming in the context of time inputs for feed processing and mixing. Current cultural methods of Salmonella spp. detection in feeds require several days for confirmation. This amount of time represents significant problems if control measures are to be effectively implemented in a fashion that keeps feed processing costs low. Molecular methods offer improved sensitivity and potential reduction in assay time. In particular, several commercial polymerase chain reaction (PCR) assays, and combined PCR–hybridization assays have been suggested as possible means to implement more rapid detection of Salmonella spp. extracted from animal feeds. It has now become possible to rapidly detect and confirm the presence of foodborne Salmonella spp. in feed matrices by commercial amplification detection systems. The primary challenges remaining are to develop more reliable recovery and extraction procedures for routine processing of samples from a wide variety of feed matrices and apply molecular techniques for assessing physiological status of Salmonella spp. contaminants in animal feeds.     

Evaluation of the mecA femB Duplex Polymerase Chain Reaction for Detection of Methicillin-Resistant Staphylococcus aureus

 This study systematically evaluated a recently described duplex polymerase chain reaction test for methicillin-resistant Staphylococcus aureus with 25 different German epidemic strains of methicillin-resistant Staphylococcus aureus and 66 staphylococci other than methicillin-resistant Staphylococcus aureus, including 17 different coagulase-negative staphylococcal species and subspecies, that were either oxacillin susceptible or oxacillin resistant. The results were compared with those of conventional cultural identification and susceptibility testing. Of the 91 isolates tested, all 25 confirmed strains of methicillin-resistant Staphylococcus aureus were identified correctly. None of the remaining strains of methicillin-susceptible Staphylococcus aureus was misidentified as methicillin-resistant Staphylococcus aureus. It was concluded that the duplex polymerase chain reaction appears to offer a time-saving and accurate method of detection of methicillin-resistant Staphylococcus aureus.     

Polymerase Chain Reaction Assays for the Detection of Cytomegalovirus in Organ and Bone Marrow Transplant Recipients

Cytomegalovirus (CMV) infection is ubiquitous and results in a wide spectrum of clinical manifestations ranging from asymptomatic infection to severe life threatening disease. Infection in normal children and adults usually causes no symptoms but in the immuncompromised host, CMV may result in severe opportunistic infections with high morbidity and mortality. Historically, virus detection was dependent on culture of the virus or on a centrifugation culture system referred to as a shell vial assay. The shell vial assay frequently lacked sensitivity and was unable to detect infection in its early phase. Also, as with culture assays, the results were affected by antiviral therapy. The CMV antigenemia assay was developed to provide more rapid results and has gained wide usage. This assay is limited to detection of the virus in white blood cells and is more sensitive than culture or the shell vial assay.

Application of the polymerase chain reaction (PCR) to these problems has resulted in the development of assays for CMV which are more sensitive than previously available methods. This method employs liquid hybridization with ³²P labeled probes and gel retardation analysis for detection of amplified DNA specific for each virus.

A comparison of the detection of CMV by an antigenemia assay or the PCR method in the leukocytes of renal transplant patients revealed that the PCR assay detects cytomegalovirus earlier and more consistently than the antigenemia assay.

Finally, the application of a fluorescent dye detection system and image analysis of the acrylamide gel with a laser scanner provides additional sensitivity to the detection of cytomegalovirus, as well as avoiding the use of radioactivity, making the assay more adaptable to the clinical laboratory.     

Multiplex Polymerase Chain Reaction for the Detection of High-risk-Human Papillomavirus Types in Formalin-fixed Paraffin-embedded Cervical Tissues

Detecting high-risk-human papillomavirus (HPV) types has become an integral part of the cervical cancer screening programmes. This study aimed to develop a multiplex polymerase chain reaction (PCR) for identification of HPV types 16 and 18 along with the beta globin gene in formalin-fixed and paraffin-embedded cervical biopsy specimens. A total of 59 samples from patients with cervical abnormalities were tested. HPV 16 positivity was 50% in cervical cancers and 52.9% in cervical intraepithelial neoplasia. Our multiplex PCR protocol can be used as a simple and cost-effective tool for high-risk-HPV detection in cervical cancer screening programmes.     

Comparison of Nested Polymerase Chain Reaction and Real-Time Polymerase Chain Reaction Targeting 47kDa Gene for the Diagnosis of Scrub Typhus

Introduction: Scrub typhus is a zoonotic infection caused by Orientia tsutsugamushi which is transmitted by Leptotrombidium mites. The disease manifests as a mild-to-severe illness with non-specific clinical symptoms. Rapid diagnosis and prompt treatment are essential for patient management. Both serological and molecular methods are used for the diagnosis of scrub typhus. The present study assessed the usefulness of detection of the gene encoding the 47kDa outer-membrane protein (OMP) for the laboratory diagnosis of scrub typhus. Materials and Methods: Nested polymerase chain reaction (nPCR) and real-time PCR targeting 47 kDa OMP antigen gene of O. tsutsugamushi were performed on ethylenediaminetetraacetic acid blood samples. Results: Six of the 103 (5.8%) patients showed the presence of 47kDa gene by nPCR. Seventy of 103 (67.9%) cases showed the presence of 47kDa gene by qPCR. Among the 70 positive cases, the majority of them were females (40/70, 57.1%). The highest number of positive cases was observed during October–February. Conclusion: Real-time PCR targeting O. tsutsugamushi-specific 47-kDa gene is more sensitive than nPCR and may be the assay of choice for the detection of the organism in patients with suspected scrub typhus.