Affirm DNA Probe

Becton Dickinson and Company (Sparks, MD, USA) provides a DNA probe-based test, the Affirm VPIII, which uses complementary sequences of DNA that hybridize with the targeted organisms and can detect and differentiate three major agents with cause vaginitis Candida, Gardnerella, and Trichomonas. The test uses two distinct single-stranded nucleic acid probes for each organism, a capture probe and a color development probe, which are complementary to unique genetic sequences of target organsims....

Real Time PCR in Infectious Disease Diagnosis

Molecular diagnostic tools and detection methods such as nucleic acid amplification are being used increasingly in the clinical microbiology laboratory to enhance the diagnosis of microbial pathogens (Lanciotti, 2001 Mackay, 2004). Nucleic acid-based technology is also used to assess drug resistance and epidemiological surveillance (Piatek, 1998 Makinen, 2001 Huletsky, 2004 Sloan, 2004). The principle of the real-time PCR is primarily used to detect and amplify a unique gene or a signature...

Molecular Differential Diagnostic System for Gram Positive Cocci in Clusters

Rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) is critical for the effective treatment of patients and to control the spread of the pathogen (Farr et al., 2001). An ideal MDD test should be able to distinguish coagulase-positive Staphylococcus (Staphylococcus aureus) from coagulase-negative Staphylococcus (CoNS) methicillin-sensitive Staphylococcus aureus (MSSA) from MRSA and hospital-acquired MRSA (HA-MRSA) from community-acquired MRSA (CA-MRSA). An added benefit...

Molecular Methods for MRSA Identification

Rapid detection of MRSA by standard clinical microbiological procedures appears then tedious and time consuming, as it first requires identification of isolated S. aureus colonies from mixed flora samples before assessing their antibiotic susceptibility profile. Direct or indirect particle agglutination assays using antibody-coated beads offer a rapid alternative to oxacillin susceptibility testing. For example, MRSA-Screen (Denka Seiken, Tokyo, Japan) provides sensitive and specific...

Staphylococcus aureus with Decreased Susceptibility to Vancomycin

Clinical emergence of vancomycin intermediate or resistant S. aureus has been reported, even though the prevalence of the strains with reduced vancomycin susceptibility is still low (Srinivasan et al., 2002 Liu and Chambers, 2003). Although the molecular mechanism of the intermediate resistance in staphylococci is not yet established, studies have suggested that a novel mechanism that differs from the one used by enterococci are employed by staphylococci (Walsh and Howe, 2002) Currently, CLSI...

Limitations of Malditof Ms for Bacterial Fingerprinting

As with all identification techniques, there are limitations that need to be considered when interpreting the analytical results. For the MicrobeLynx system, these limitations are as follows 1. Protocol dependence. The technique is protocol dependent, and therefore comparison of spectral data with database entries is only valid when the same protocols are adopted, especially in terms of the culture conditions. This applies to all protein-based MALDI-TOF MS analysis. 2. Knowledge of Gram stain....

References

Electrophoresis of nucleic acids. In Brown, T. A., ed. Essential Molecular Biology A Practical Approach. IRL Press at Oxford University Press, Oxford, pp. 89-126. Arshad, M. F., Dunn, F. J., Vega, R., Valvano, J. W., & Serwer, P. (1993). Progress in developing improved programs for pulsed field agarose gel electrophoresis of DNA. Electrophoresis, 14, 344-348. Bloch, K., & Grossmann, B. (1995). Digestion of DNA with restriction endonucleases. In Current Protocols in...

Sequence Based Bacterial Genome Typing

Many techniques are available to differentiate S. aureus, and specifically MRSA, isolates. Conventionally, isolates were distinguished by phenotypic methods, including antibiotic susceptibility testing and bacteriophage typing. Both methods have limitations, as genetically unrelated isolates commonly have the same antibiogram, and many S. aureus isolates are nontypeable by phage typing. With the advancement of molecular biology, strain typing focused on DNA-based methods restriction...

Molecular Differential Diagnostic System for Health Care Associated Infections

According to the Centers for Disease Control and Prevention (CDC), 90,000 Americans die from health Care-associated infections (HAIs) and another Table 27.8. Detection results of the Respiratory Infections II panel. No. Samples_SARS1 SARS2 INFA INFB RSVA RSVB PIV1 PIV2 PIV3 PIV4 hMPV RhV CVEV Table 27.8. Detection results of the Respiratory Infections II panel. No. Samples_SARS1 SARS2 INFA INFB RSVA RSVB PIV1 PIV2 PIV3 PIV4 hMPV RhV CVEV * Boldface indicate values above cut-off. * Boldface...

Signal Mediated Amplification of RNA Technology

Signal-mediated amplification of RNA technology (SMART) is a novel isothermal amplification technology that uses a three-way junction (3WJ) structure to facilitate target-dependent production of multiple copies of a RNA product (Wharam et al., 2001). The 3WJ structure is composed of two target-specific single-stranded DNA probes (the template probe and the extension probe) and a target sequence. Both probes have a longer region that hybridizes to the target at adjacent sites and a shorter...

Hybrid Capture Technology

The hybrid capture 2 (HC2) technology is the platform for signal-amplified, nucleic acid tests (for review, seeLorincz and Anthony, 2001). HC2 systems areavail-able to detect human papillomavirus (HPV), cytomegalovirus (CMV), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC), and hepatitis B virus (HBV). An assay for herpes simplex virus (HSV) is in development. Cervical cancer is one of the few malignancies for which the cause has been identified the human papillomavirus, a small DNA...

Methicillin Resistance in Staphylococci

Methicillin resistance in clinical isolated staphylococci is mostly mediated through acquisition of mecA gene encoding a mutant penicillin binding protein (PBP)2a by bacterial genome. PBPs are the enzymes that catalyze the reaction that crosslinks the peptidoglycan of the bacterial cell wall. Binding of PBP to (-lactam antimicrobials inhibits the enzyme activity and prevents bacteria growth by interfering with cell wall formation. In contrast to the PBPs in methicillin-susceptible strains,...

Disk Diffusion Testing

Like agar dilution and broth dilution methods of susceptibility testing, the disk diffusion method tests the inhibitory effect of antimicrobial agents against microorganisms. The test is carried out by placing filter paper disks with a known concentration of an antimicrobial agent on the surface of agar plates inoculated with a test organism. The drug on disks diffuses through the agar, creating a concentration gradient decreasing along the distance from the center of the disk (Barry, 1991)....

Qbeta Replicase Amplification

Q-beta replicase is an RNA-dependent RNA polymerase derived from the bacteriophage Q-beta (Haruna and Spiegelman, 1965). It comprises four different subunits with only one polypeptide (i.e., subunit II) encoded in the Q-beta phage genome. The other subunits are generated by the host protein synthesizing apparatus (30S ribosomal protein S1, elongation factor EF-Tu and EF-Ts) (Blumenthal and Landers, 1976). Q-beta replicase has stringent specificity for its templates (Wu et al., 1992). Only a few...

Inducible Clindamycin Resistance in Staphylococci and Streptococci

Some staphylococcal and streptococcal strains that are resistant to erythromycin and susceptible to clindamycin may have inducible clindamycin resistance, referred to as macrolide-lincosamide-streptogramin B (MLSB) resistance, due to the presence of erythromycin ribosomal methylase erm(A) or erm(B) (Hamilton-Miller and Shah, 2000). This family of enzymes methylates the N-amino group of adenine residue 2058 in 23S rRNA, which prevents access of the antimicrobial to its binding site on the...

Source Tracking of Pathogens in Outbreak and Clonal Spread

Rapid typing can significantly reduce costs associated with treatment, containment, and decontamination. The Centers for Disease Control and Prevention (CDC) estimates that 2 million patients acquire nosocomial infections each year, and 90,000 of those patients die as a result of their infections. Infection control programs that include DNA fingerprinting allow outbreaks to be tracked and controlled in real-time. As a laboratory tool, typing is used to assist in tracking sources of...

Molecular Detection of Drug Resistance

Rapid and accurate determination of drug susceptibility of a clinical isolate can be useful for various aspects of patient therapy. The presence of resistance markers can also help distinguish ambiguous break points associated with susceptibility testing. Well-characterized resistant genes can be used to monitor their epidemiological spread in the community or hospital. Despite the fact that there still remains much to learn about these markers, the application of molecular diagnostic methods...

Overnight Biochemical Tests

The overnight biochemical tests are a group of tests that require inoculating one or more culture media containing specific substrates and chemical indicators that detect pH change or specific microbial by-product. Similar to rapid tests, the choice of overnight tests is based on Gram-stain morphology and the results of preliminary testing with rapid enzyme tests. These tests are also inexpensive and easy to perform and may be used in three different ways. They may be used to obtain important...