PCR and Infectious Diseases

2.1 Multiplex PCR

In diagnostic laboratories, the use of PCR is often limited by its cost and sometimes by the availability of adequate sample volume. To overcome these issues and also to increase the diagnostic capacity of PCR, there is a type termed multiplex PCR (mPCR). The mPCR refers to the use of different pairs of primers to simultaneously amplify multiple regions of the nucleic acid of the sample with visualization of the amplified products by gel electrophoresis. The use of multiple primer pairs in mPCRs is an innovation that offers significant benefits in cost, time, and exact diagnosis. The main advantage of this technology is to minimize the number of separate reactions, for example, to detect several pathogens at the same time in a single specimen such as sexually transmitted pathogens [6, 7, 8]. This technique makes it possible to diagnose several diseases with a single diagnostic test, with sensitivity, specificity, and speed, indispensable values in diagnostic tests.

This technique has become a mainstay of research and clinical diagnostic applications, such as sexually transmitted infections (STIs). Considering a major public health problem, the STIs are common everywhere from developed countries and developing countries. It is estimated that each year more than 340 million new cases of bacterial STI arise, including gonorrhea, chlamydia, and syphilis, and the incidence is increased worldwide in adults of 15–49 years of age [9]. The susceptibility to sexually transmitted infections, including the human immunodeficiency virus (HIV), and the high cost of treatment have led to the need for fast and reliable laboratory techniques for the identification of pathogens. Procedures for nucleic acid amplification to detect sexually transmitted pathogens have been developed, especially mPCR methods [10].

The multiplex PCR has the potential to analyze many samples in a single reaction, and it is useful for diagnostic of multipathogenic infection. However, it has some limitations such as the nonspecific products generated through primer-primer interactions that may interfere with the amplification of targets, decreasing sensitivity, and selectivity of reactions.

2.2 Loop-mediated isothermal amplification

Nucleic acid amplification is commonly used in the field of life science research. With the development of molecular biology, many new molecular diagnostic technologies have been developed subsequently [11].

The loop-mediated isothermal amplification (LAMP-PCR) was first developed over 15 years ago, and it has emerged as powerful method to concurrently detect multiple pathogens [12, 13]. The method employs a DNA polymerase with strand displacement activity and a set of four inner and outer primers that recognize a total of six distinct sequences of the target DNA. Moreover, the method involves two successive steps of amplification, with the first step comprising mPCR and the second step LAMP. Amplicons of the first step serve as templates in the second step. The amplification protocol requires only a single temperature for the reaction, and the amplification is diagnosed without the need for electrophoretic techniques, using in situ detection process with colorimetric dye or with a fluorescent dye. The final products are the accumulation of 10⁹ copies of target DNA in less than an hour. The LAMP-PCR has been regarded as an innovative technology and emerged as an alternative to PCR-based methodologies in clinical laboratory with significant increase of detection limits, efficiency, selectivity, and specificity over single-stage.

With more and more scientists focusing their attention on the application of LAMP technology, the range of its use is not limited to the bacteria detection and identification any more [14]. The LAMP-PCR was developed and employed to detect species that cause chorioamnionitis and premature labor, Ureaplasma parvum, and Ureaplasma urealyticum [15].

It was also applied to the parasite and virus detection [16, 17, 18]. Recently, Kurosaki et al. in their study [19] in 2017 developed a LAMP-PCR assay for the detection of Zika virus plasma, serum, and urine samples collected from 120 suspected cases of arbovirus infection in Brazil.

2.3 Real-time PCR

According to [20], clinical diagnostic approaches rely on quantitative PCR (qPCR) as a method to detect and quantify infectious agents. Fluorescence chemistry-based methods have revolutionized molecular diagnostics and become the gold standard for viral load quantification and detection of bacterial and viral pathogens. During qPCR, the nucleic acid is amplified until it produces a certain level of signal which is supplied through a DNA intercalating dye or sequence-specific fluorescent probe. The cycle threshold (Cq), defined as the number of amplification cycles required to reach that signal level, is used to calculate the number of target molecules originally present based on a standard curve [20]. In qPCR, the targets are detected in real time from the sealed PCR plate, and there is no post-PCR processing required; therefore, the risk of false-positive results due to amplicon carryover is substantially decreased compared to conventional [21].

During the past decade, advantages on PCR have become gold standard procedure in the diagnosis of infectious diseases, particularly the diagnosis of viral diseases, such as arboviruses. Arboviruses are causing an unprecedented health calamity in world, especially in Latin American countries, with rising statistics on a daily basis. The diagnosis of these diseases is difficult to establish only by clinical features. A substantial proportion of these infections are asymptomatic, but some patients may also present clinical symptoms similar to the arboviruses such as those caused by the Dengue, Zika, and Chikungunya virus. The qPCR technique is more sensitive and specific than serological tests. Besides that, in serum samples, it is only possible to perform the diagnosis in the acute phase of the disease that lasts up to 4–7 days after the onset of symptoms. In relation to the detection of Zika virus (ZIKV), for example, recent data from the literature indicate that in human urine, viral RNA was found longer compared to serum, up to 20 days after the onset of the first symptoms. In semen, studies show that the genetic material of ZIKV was found for weeks and even months after infection [22]. However, the determination of viral load presents some particular challenges when using qPCR methodology. This is because reliable absolute concentration results of qPCR are dependent on assay efficiency, instrument calibration metrics, and comparison with a known reference sample to convert the Cq measurements to a sample unknown.

2.4 Digital PCR

In contrast to qPCR, the digital PCR (dPCR) uses an alternative method that is not dependent upon the determination of the amplification cycle that the reporter dye signal exceeds a threshold. Instead, prior to amplification, the samples to be subjected to dPCR are divided into thousands of independent PCR reactions and are scored as either positive or negative for amplification of the viral sequence of interest. The positive wells are counted and converted to a target concentration in the original sample. This binary assignment of each reaction greatly minimizes the dependence of measurement on parameters such as the efficiency of the assay and the calibration of the instrument. Therefore, dPCR is the absolute quantification methodology with the greatest potential for quantification of low-load viral nucleic acids.

In the diagnostic routine, it is very common to obtain positive qPCR results obtained at the detection limit of this methodology, which may generate uncertainty of the result. dPCR is a complementary methodology that works beyond the limit of detection of qPCR, since it is based on the Poisson distribution. Consequently, this methodology has a significant impact on research as well as on diagnostic applications [23, 24, 25].

Some benefits of digital PCR in virology can be cited [25]:

• Quantification of viral genomes in samples without the need to use a standard curve;

• Detection of viruses that have a very low viral load;

• Use of low concentration of samples;

• Reduction of the impact of inhibitors present on complex samples.

Table 1 shows a comparison between the two methodologies: qPCR and dPCR.

A great number of studies use dPCR to diagnose infectious disease-related viruses, including hepatitis B virus (HBV), cytomegalovirus, human influenza, and HIV [26, 27, 28]; bacterial infections (Mycobacterium tuberculosis, Chlamydia trachomatis, and Staphylococcus aureus) [29, 30, 31]; and parasitic infectious such as detection of Plasmodium falciparum and Plasmodium vivax [32].