ABSTRACT
Introduction ........................................................................................................ 184 Phenotyping versus Genotyping ........................................................................ 185 Molecular Methods in the Diagnostic Laboratory ............................................ 187
Introduction ............................................................................................ 187 Hybridization .......................................................................................... 187 Polymerase Chain Reaction .................................................................... 188 Microarrays ............................................................................................. 192 DNA Sequencing .................................................................................... 192 Final Remarks on Technology Principles ............................................... 193
Mycobacterium tuberculosis ............................................................................. 193 Introduction ............................................................................................ 193 Probe-Based Technology for the Detection of Resistance in
Mycobacterium tuberculosis ....................................................... 194 PCR-Based Methods for the Detection of Antibiotic Resistance in
Mycobacterium tuberculosis ....................................................... 197 Microarrays for the Detection of Resistance in Mycobacterium
tuberculosis .................................................................................. 201 Staphylococcus aureus ...................................................................................... 202
The Problem of MRSA ............................................................................. 202 MRSA Detection by Probe-Based Methods ........................................... 203 Real-Time PCR Detection of MRSA ...................................................... 204 Microarrays for the Detection of MRSA ................................................ 206 Detection of Other Resistance Determinants in Staphylococcus aureus ... 206
Helicobacter pylori ............................................................................................ 207 Introduction ............................................................................................ 207 Probe-Based Technologies ...................................................................... 207 PCR-Based Detection of Mutations Associated with Antibiotic
Resistance in Helicobacter pylori ................................................ 209
Microarrays for the Detection of Resistance in Helicobacter pylori ..... 210 Final Remarks for the Detection of Resistance in Helicobacter pylori ... 210
Neisseria gonorrhoea ........................................................................................ 210 Enterobacteriaceae ............................................................................................. 213 Campylobacter spp. ........................................................................................... 215 Pneumococci and Streptococci .......................................................................... 215 Other Bacterial Species ..................................................................................... 216 Concluding Remarks .......................................................................................... 216 References .......................................................................................................... 218
The increase in antibiotic resistance and the increasing possibilities and availability of molecular techniques make it only natural to use these techniques to study and detect the genes involved. Current molecular techniques are derived from a limited number of basic principles: probes, polymerase chain reaction (PCR), DNA sequencing, and microarrays. Probe technology is based on the interaction between two DNA strands, which are either identical or show mismatches due to mutations. The polymerase chain reaction (PCR) has nearly become a household name. With widely available computer programs, it is rather easy to design a PCR to detect a single gene, but more complex assays have been developed, which include the concomitant use of probes in a process called real-time PCR. DNA sequencing is long known, but developments in fl uorescent dyes and the new process of pyrosequencing have revolutionized this technique. Microarrays are a form of large-scale hybridization that is becoming increasingly popular because of its ability to examine many genes simultaneously. Although the principles behind molecular techniques are simple, the design and implementation of a more advanced assay may be problematic. Potential problems involve contamination, lack of a gold standard to validate the assay, poor analytical sensitivity, and a large number of genes or mutations that need to be covered in order to correctly predict resistance. In addition, the importance for therapy of the presence of a mutation or resistance gene has to be considered. In this review, the principles of the techniques and the problems will be discussed. The (im)possibilities of molecular assays will be illustrated by examples intended for routine diagnostics, one of the most challenging environments for molecular techniques. The examples will focus especially, but not exclusively, on the detection of antibiotic resistance in Mycobacterium tuberculosis, methicillin-resistant Staphylococcus aureus (MRSA), and antibiotic-resistant Helicobacter pylori.
