**1. Introduction**

#### **1.1. Diagnosis**

Several infectious diseases caused by microorganisms, including protozoans (malaria, leishmaniasis, trypanosomiasis, amoebiasis, etc.), bacteria (cholera, gastritis and gastric ulcers, meningitis, tuberculosis, leprosy, etc.), and viruses (dengue fever, yellow fever, influenza, chikungunya fever, ebola, human immunodeficiency syndrome, etc.), are threats to public health. In order to control outbreaks, emergence, and reemergence of these infectious diseases, diagnosis, correct identification, treatment, and notification of pathogen‐ ic agents are necessary.

Classical clinical microbiological diagnostics for protozoan and bacteria rely on microscop‐ ic examination with different staining methods, culture isolation, morphological and physiological/biochemical characterization. For viruses, the conventional diagnosis is based on culture isolation of cell monolayer, serological assays, and electronic microscopic examination. These standard diagnostic methods are very useful, and culture isolation associated with other analytical procedures to identify microorganisms continues to be the gold standard method since it enables drug sensitivity tests. However, these diagnostic techniques are unsuitable for several microorganisms presenting fastidious growth characteristics, low morphological and physiological specificity, and requirement of specific biosafety infrastructure. The same is applied to viruses that, even after culture isolation, can only be visualized by electronic microscopy, which is expensive and needs special‐ ized personnel to maneuver. Thus, nucleic acid detection by hybridization and amplifica‐ tion technologies opened a new and innovative period for microbial diagnosis. After the first report on the application of polymerase chain reaction (PCR) in clinical diagnosis of the human immunodeficiency virus (HIV) [1], several other infectious organisms were detected by the same technique and its variations (Section 1.1.1).

In all molecular detection techniques, the gene target is the main device, and its choice depends on the infectious agent and the host genomic and epidemiological characteristics. For a specific diagnosis, the gene of choice has to be specific to the infectious agent and should not crosshybridize with the host genome and other organisms living in the same microhabitat. A sensitive diagnosis depends on the amount of gene target copies in the biological sample and to the physico-chemical characteristics of the constituents implicated in the detection and amplification of gene target. A discussion about the use of single- and multiple-copy genes for specific gene target amplification will be presented in Section 1.1.2.

The detection of drug resistance is dependent on sensitivity tests performed on the isolated microorganism, which is time consuming; however, for several uncultivable pathogenic agents, it is not feasible. The investigation of nucleotide mutations associated to drug resistance allows the development of gene target amplification and post-amplification analytical techniques, such as enzyme restriction analysis and sequencing, to be used directly on the biological infected sample, thereby enabling fast detection of drug resist‐ ance and consequently an efficient treatment. The same strategy can be used to identify organisms from closed biological groups, with identical morphological characteristics on microscopic examination and with different genetic features. Examples of nucleic acid drug resistance detection techniques used in microbiological clinical laboratories will be present‐ ed in Section 1.1.3.
