Reverse Line Blot Macroarray for Simultaneous Detection and Characterization of Four Biological Warfare Agents

The need for a rapid detection and characterization of biowarfare (BW) agents cannot be over emphasized. With diverse array of potential BW pathogen available presently, rapid identification of the pathogen is crucial, so that specific therapy and control measures can be initiated. We have developed a multiplex polymerase chain reaction based reverse line blot macroarray to simultaneously detect four pathogens of BW importance viz. Bacillus anthracis, Yersinia pestis, Brucella melitensis and Burkholderia pseudomallei. The multiplex PCR utilizes 14 pairs of primers targeting 18 specific markers. These markers include genes which are genus specific, species-specific chromosomal sequences and virulence markers of plasmid origin. The assay was evaluated on various human, environment and animal isolates. The assay w successful in simultaneous detection and characterization of isolates of the four pathogens on as a single platform with sensitivity ranging from 0.3 pg to 0.3 ng of genomic DNA. The assay was able to detect 5 × 10² cfu/ml for B. anthracis, 8 × 10² cfu/ml for Yersinia sp., 1.4 × 10² cfu/ml for B. melitensis and 4 × 10² cfu/ml for B. pseudomallei.     

High Prevalence of Exfoliative Toxins Among Carrier Isolates of Staphylococcus aureus from Healthy Individuals from Various Communities in Chennai, South India

Staphylococcus aureus causes infections both in community and hospital settings, nasal carriage is the important source of these infections. A total of 103 carrier isolates of S. aureus from 352 asymptomatic individuals were screened for methicillin-resistant S. aureus (MRSA) and exfoliative toxins (A, B and D) by two sets of multiplex PCRs. The overall nasal carriage of MRSA was found to be 13/352 (3.7 %), of which 4 were found to be positive for Panton valentine leucocidin (PVL). Twelve (11.65 %) strains were found to carry exfoliative toxins and belonged to one of the following spa types t159, t209 and t1515. High prevalence of exfoliative toxins, pvl and MRSA pose a major threat to public health, since the isolates were from the healthy in various community settings.     

DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR

PCR detection of intestinal protozoa is often restrained by a poor DNA recovery or by inhibitors present in feces. The need for an extraction protocol that can overcome these obstacles is therefore clear. QIAamp® DNA Stool Mini Kit (Qiagen) was evaluated for its ability to recover DNA from oocysts/cysts directly from feces. Twenty-five Giardia-positive, 15 Cryptosporidium-positive, 15 Entamoeba histolytica-positive, and 45 protozoa-free samples were processed as control by microscopy and immunoassay tests. DNA extracts were amplified using 3 sets of published primers. Following the manufacturer''s protocol, the kit showed sensitivity and specificity of 100% towards Giardia and Entamoeba. However, for Cryptosporidium, the sensitivity and specificity were 60% (9/15) and 100%, respectively. A series of optimization experiments involving various steps of the kit''s protocol were conducted using Cryptosporidium-positive samples. The best DNA recoveries were gained by raising the lysis temperature to the boiling point for 10 min and the incubation time of the InhibitEX tablet to 5 min. Also, using a pre-cooled ethanol for nucleic acid precipitation and small elution volume (50-100 µl) were valuable. The sensitivity of the amended protocol to Cryptosporidium was raised to 100%. Cryptosporidium DNA was successfully amplified by either the first or the second primer set. When applied on parasite-free feces spiked with variable oocysts/cysts counts, ≈ 2 oocysts/cysts were theoretically enough for detection by PCR. To conclude, the Qiagen kit with the amended protocol was proved to be suitable for protozoan DNA extraction directly from feces and support PCR diagnosis.     

Detection of Malaysian methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (MRSA) clinical isolates using simplex and duplex real-time PCR

The aim of this study was to develop a methicillin-resistant Staphylococcus aureus (MRSA) detection method based on the melting temperature analysis profiling of S. aureus clinical isolates from three different hospitals in Malaysia. Simplex and duplex real-time PCR assay was used for the simultaneous detection of nuc (species-specific) and mecA (methicillin-resistance) genes in a single SYBR Green I real-time PCR tube assay. Evaluations were based on the melting temperature (T _m) analysis of the amplicons using 23 S. aureus clinical isolates including three ATCC S. aureus standard strains. Real-time PCR amplification products with melting peaks at 78.39 ± 0.4°C and 74.41 ± 0.6°C were detected for nuc and mecA genes, respectively. Each real-time PCR assay was completed within two hours. This rapid genotypic method is useful for the detection of resistant determinant (mecA) and identification of S. aureus (nuc) clinical isolates, thus benefiting patient therapy in hospitals.     

Azithromycin Reduces the Production of α-hemolysin and Biofilm Formation in Staphylococcus aureus

Staphylococcus aureus causes a broad range of life-threatening diseases in humans. This bacterium produces a large number of extracellular virulence factors that are closely associated with specific diseases which are controlled by quorum sensing. In this study, we show that azithromycin was active against methicillin-resistant Staphylococcus aureus (MRSA) strains with MICs ranged from 32 to 64 μg/mL. Azithromycin at subinhibitory concentration, markedly reduced the production of α-hemolysin at (1/16MIC, 1/8MIC) and biofilm formation at (1/16MIC, 1/8MIC), respectively. The results indicated that sub-inhibitory concentrations of azithromycin decreased the production of α-hemolysin and biofilm formation in MRSA in a dose-dependent manner. Therefore, azithromycin may be useful in the treatment of α-hemolysin producing and biofilm formation MRSA infections.     

The Challenge of Antibiotic-Resistant Staphylococcus: Lessons from Hospital Nurseries in the mid-20th Century

In the late 1940s, epidemics of antibiotic-resistant strains of Staphylococcus aureus began to plague postpartum nurseries in hospitals across the United States. Exacerbated by overcrowding and nursing shortages, resistant S. aureus outbreaks posed a novel challenge to physicians and nurses heavily reliant on antibiotics as both prophylaxis and treatment. This paper explores the investigation of the reservoir, mode of transmission, and virulence of S. aureus during major hospital outbreaks and the subsequent implementation of novel infection control measures from the late 1940s through the early 1960s. The exploration of these measures reveals a shift in infection control policy as hospitals, faced with the failure of antibiotics to slow S. aureus outbreaks, implemented laboratory culture routines, modified nursery structure and layout, and altered nursing staff procedures to counter various forms of S. aureus transmission. Showcasing the need for widespread epidemiologic surveillance, ultimately manifesting itself in specialized “hospital epidemiology” training promoted in the 1970s, the challenges faced by hospital nurses in the 1950s prove highly relevant to the continued struggle with methicillin-resistant Staphylococcus aureus (MRSA) and other resistant nosocomial infections.     

Phylogenetic Relationships Based on Ribosomal DNA Data for Four Species of Cyst Nematodes from Italy and One from Syria

Phylogenetic analysis of new ribosomal DNA (rDNA) data for Heterodera mediterranea, H. hordecalis, H. carotae, and H. fici from Italy and H. ciceri from Syria, along with published data for other species, showed high bootstrap support for the following relationships: (((((H. carotae H. cruciferae) H. goettingiana) (((H. trifolii H. ciceri) H. mediterranea) ((H. avenae H. latipons) H. fici))) (Cactodera betulae H. hordecalis)) (Globodera rostochiensis G. pallida)). The rDNA sequence data were for the two internal transcribed spacers (ITS1 and ITS2) plus the 5.8S gene between them. These inferred relationships support the classic ''''Goettingiana Group'''' of H. carotae, H. cruciferae, and H. goettingiana. A clade comprised of Cactodera betulae and H. hordecalis is only distantly related to the other species in the analysis.     

Evaluation of BacLiteRapidMRSA, a rapid culture based screening test for the detection of ciprofloxacin and methicillin resistantS. aureus(MRSA) from screening swabs

Background  

Methicillin-resistantStaphylococcus aureus(MRSA) is a major nosocomial pathogen worldwide. The need for accurate and rapid screening methods to detect MRSA carriers has been clearly established. The performance of a novel assay, BacLiteRapidMRSA (Acolyte Biomedica, UK) for the rapid detection (5 h) and identification of hospital associated ciprofloxacin resistant strains of MRSA directly from nasal swab specimens was compared to that obtained by culture on Mannitol salt agar containing Oxacillin (MSAO) after 48 h incubation.     

Serological and Molecular Characteristics of the First Korean Case of Echinococcus multilocularis

In December 2011, we reported an autochthonous case of Echinococcus multilocularis infection in a 42-year-old woman in Korea. The diagnosis was based on histopathological findings of the surgically resected liver cyst. In the present study, we evaluated the serological and molecular characteristics of this Korean E. multilocularis case. The patient''s serum strongly reacted with affinity-purified native Em18 and recombinant Em18 antigens (specific for E. multilocularis) but negative for recombinant antigen B8/1 (reactive for Echinococcus granulosus). In immunoaffinity chromatography, the serum also strongly reacted with E. multilocularis and only weakly positive for E. granulosus. We determined the whole nucleotide sequence of cox1 (1,608 bp) using the paraffin-embedded cystic tissue which was compared with E. multilocularis isolates from China, Japan, Kazakhstan, Austria, France, and Slovakia. The Korean case showed 99.8-99.9% similarity with isolates from Asia (the highest similarity with an isolate from Sichuan, China), whereas the similarity with European isolates ranged from 99.5 to 99.6%.     

Characterization of the substitution pattern of cellulose derivatives using carbohydrate-binding modules

Background

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA).

Results

The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively.

Conclusions

The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.     

Sensitive and rapid detection of Schistosoma japonicum DNA by loop-mediated isothermal amplification (LAMP)

In this study, a loop-mediated isothermal amplification (LAMP) assay was established to detect Schistosoma japonicum DNA in faecal and serum samples of rabbits, and serum samples of humans infected with S. japonicum. This LAMP assay was based on the sequence of highly repetitive retrotransposon SjR2, and was able to detect 0.08 fg S. japonicum DNA, which is 10⁴ times more sensitive than conventional PCR. The LAMP assay was also highly specific for S. japonicum and able to detect S. japonicum DNA in rabbit sera at 1 week p.i. Following administration of praziquantel, detection of S. japonicum DNA in rabbit sera became negative at 12 weeks post-treatment. These results demonstrated that LAMP was effective for early diagnosis of, and evaluation of therapy effectiveness for, S. japonicum infection. Both PCR and LAMP assays were then used to detect S. japonicum DNA in 30 serum samples from S. japonicum-infected patients and 20 serum samples from healthy persons. The percentage sensitivity of LAMP was 96.7%, whereas that of PCR was only 60%, indicating that LAMP was more sensitive than conventional PCR for clinical diagnosis of schistosomiasis cases in endemic areas. The established LAMP assay should provide a useful and practical tool for the routine diagnosis and therapeutic evaluation of human schistosomiasis.     

Sensitive and Rapid Detection of Giardia lamblia Infection in Pet Dogs using Loop-Mediated Isothermal Amplification

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from 10^-1 to 10^-5 ng/µl for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of 63℃ by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha (EF1α) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.     

The MAK16 Gene of Entamoeba histolytica and Its Identification in Isolates from Patients

To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher''s exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient''s symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage.     

Loop-mediated isothermal amplification assays for identification of antiseptic- and methicillin-resistant Staphylococcus aureus

A method for rapid identification of antiseptic- and methicillin-resistant Staphylococcus aureus (MRSA) based on 3 loop-mediated isothermal amplification (LAMP) assays was developed. LAMP targeting the femB gene identified S. aureus with 100% specificity, and LAMP targeting the mecA gene associated with methicillin resistance identified methicillin-resistant staphylococci with 100% specificity. LAMP targeting the qacA/B gene encoding an efflux pump responsible for antiseptic resistance identified high-acriflavine-resistant (MIC ≥ 100 mg/L) MRSA (92.5% positive) and acriflavine-susceptible (MIC < 25 mg/L) MRSA (100% negative). They were performed under the same reaction conditions within 60 min at 63 °C. The combined LAMP assays will be useful for rapid identification of S. aureus isolates and determination of their antibiotic and antiseptic resistance patterns with regard to methicillin and organic cationic substrates.     

Genomic Differences among Pathotypes of Bursaphelenchus xylophilus

Total genomic DNA from Bursaphelenchus xylophilus pathotypes MPSy-1 and VPSt-1 and from B. mucronatus was digested with restriction endonucleases. DNA fragments were electrophoretically separated, Southern blotted to nitrocellulose, and hybridized to genomic DNA from one of the isolates. The resulting hybridization patterns indicate genomic differences in repetitive DNA sequences among these populations. Greatest differences were seen between B. xylophilus and B. mucronatus, but genomic differences were also apparent between B. xylophilus pathotypes MPSy-1 and VPSt-1 and between a population from P. nigra in New Jersey and a population of a mucronate form from Abies balsamea in Quebec, Canada.     

Lateral transfers of insertion sequences between Wolbachia,Cardinium and Rickettsia bacterial endosymbionts

Various bacteria live exclusively within arthropod cells and collectively act as an important driver of arthropod evolutionary ecology. Whereas rampant intra-generic DNA transfers were recently shown to have a pivotal role in the evolution of the most common of these endosymbionts, Wolbachia, the present study show that inter-generic DNA transfers also commonly take place, constituting a potent source of rapid genomic change. Bioinformatic, molecular and phylogenetic data provide evidence that a selfish genetic element, the insertion sequence ISRpe1, is widespread in the Wolbachia, Cardinium and Rickettsia endosymbionts and experiences recent (and likely ongoing) transfers over long evolutionary distances. Although many ISRpe1 copies were clearly expanding and leading to rapid endosymbiont diversification, degraded copies are also frequently found, constituting an unusual genomic fossil record suggestive of ancient ISRpe1 expansions. Overall, the present data highlight how ecological connections within the arthropod intracellular environment facilitate lateral DNA transfers between distantly related bacterial lineages.     

Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species

A PCR-based diagnostic assay was developed for early detection and identification of Aphelenchoides fragariae directly in host plant tissues using the species-specific primers AFragFl and AFragRl that amplify a 169-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA. These species-specific primers did not amplify DNA from Aphelenchoides besseyi or Aphelenchoides ritzemabosi. The PCR assay was sensitive, detecting a single nematode in a background of plant tissue extract. The assay accurately detected A. fragariae in more than 100 naturally infected, ornamental plant samples collected in North Carolina nurseries, garden centers and landscapes, including 50 plant species not previously reported as hosts of Aphelenchoides spp. The detection sensitivity of the PCR-based assay was higher for infected yet asymptomatic plants when compared to the traditional, water extraction method for Aphelenchoides spp. detection. The utility of using NaOH extraction for rapid preparation of total DNA from plant samples infected with A. fragariae was demonstrated.     

Characterization and evaluation of an arbitrary primed Polymerase Chain Reaction (PCR) product for the specific detection of Brucella species

Laboratory detection of Brucella is based largely on bacterial isolation and phenotypic characterization. These methods are lengthy and labor-intensive and have been associated with a heightened risk of laboratory-acquired infection. Antibody based indirect detection methods also suffer from limitations in proper diagnosis of the organism. To overcome these problems, nucleic acid amplification has been explored for rapid detection and confirmation of the presence of Brucella spp. PCR-based diagnostics is useful for screening large populations of livestock to identify infected individuals and confirms the presence of the pathogen. Random Amplification of Polymorphic DNA (RAPD) was performed and identified a 1.3 kb PCR fragment specifically amplifiable from DNA isolated from Brucella. A BLAST search revealed no significant homology with the reported sequences from species other than the members of Brucella. The isolated fragment seems to be a part of d-alanine–d-alanine ligase gene in Brucella sp. Translational BLAST revealed certain degree of homology of this sequence with orthologs of this gene reported from other microbial species at the deduced amino acid level. The sequence information was used to develop PCR based assays to detect Brucella sp. from various samples. The minimum detection limit of Brucella from blood and milk samples spiked with Brucella DNA was found to be 1 ng/ml and 10 ng/ml, respectively. In conclusion, we demonstrated that the PCR based detection protocol was successfully used for the detection of Brucella from various organs and spiked samples of diseased sheep. Diagnosis of Brucellosis by PCR based method reported in this study is relatively rapid, specific and simple.     

Detection of methicillin-resistant <Emphasis Type="BoldItalic">Staphylococcus aureus</Emphasis> (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers

In this study, we reported on the design of a multiplex real-time PCR assay based on SYBR Green I, incorporating dual priming adenine-thymine (AT)-rich primers for direct detection of MRSA from nasal samples. The multiplex real-time polymerase chain reaction (RT-PCR) assay reported in this study is based on SYBR Green I with incorporation of six dual priming AT-rich primers designed from the SCCmec/orf junction. A string (4–6 bp) of low-melting bases, such as adenine and thymine, was incorporated into the primers, which virtually divided a single primer in two functional regions, thus decreasing non-specific PCR products. The analytical sensitivity and specificity of the RT-PCR assay was determined with genomic DNA of reference strains (MRSA, MSSA, and MRCoNS). RT-PCR assay was performed for analysis of 72 nasal swab specimens, and the results were confirmed by use of a culture method. Furthermore, the results of RT-PCR were compared with LightCycler MRSA advance test. The multiplex RT-PCR assay reproducibly detected a minimum of 1 pg genomic DNA (31.5 copy of genome) of MRSA reference strains and clinical isolates, with a specific melting peak at 83.5 ± 1.5°C, and neither fluorescence nor a melting peak was detected in non-target isolates. The concordance rate between RT-PCR assay and culture method was 87.5% with Cohen's kappa value (κ) 0.75, which showed good agreement between the two assays. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 93.5%, 82.9%, 80.5%, and 94.4%, respectively. In a comparative study for the detection of 72 nasal samples, the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex RT-PCR assay with respect to LightCycler MRSA advance test was 84.2%, 88.2%, 89%, and, 83.3%, respectively. The results of RT-PCR assay demonstrated high specificity (88.2%) and positive predictive value (89%) for the direct detection of MRSA from nasal samples.