**Abstract**

In clinical, research, and public health laboratories, many diagnostic methods are used to detect the coronavirus. Some tests directly detect infection by detecting viral RNA, while others detect the disease indirectly by detecting host antibodies. Several studies on SARS-CoV-2 diagnostic methods have found varying throughput, batching capacity, infrastructure requirements, analytical efficiency, and turnaround times ranging from minutes to hours. Serosurvey studies have been conducted for antibodies to understand, model, and forecast the prevalence of the disease in an area. While on the research and predictive modeling side, sampling and analysis of sewage have been conducted to determine the number of RNA copies and hence the prevalence. Certain studies indicate usefulness of GIS (Geographic Information System) for understanding the pervasiveness of COVID-19 in an area as well. The current chapter deals with the evolution of diagnostic techniques for COVID-19 and discusses use of specific techniques and appropriateness in certain specified conditions. It also focuses on understanding the methods used for assessing the prevalence of COVID-19 in a particular region to extract mitigative strategies from it, either by prediction or management of the affected area.

**Keywords:** COVID-19, SARS-CoV-2, RT-PCR, GIS, wastewater treatment, biosensors, CRISPR

### **1. Introduction**

Different testing methods are used in clinical, academic, and public health laboratories to diagnose the coronavirus. These methods have different output, batching capacity, analytical result performance, specific requirement of infrastructure setting and worktime. Some tests, such as direct tests, detect viral RNA directly to determine infection, while indirect tests diagnose infection indirectly by measuring host antibodies. The methods that are used for the diagnosis of coronavirus should have enough accuracy and sensitivity to make proper clinical decisions quickly in this pandemic so that the spread of the virus can be controlled [1–3]. A number of experiments were carried out to determine economic loss as well as the urban microclimate [4, 5].

A number of methods that are used for diagnosis have been given an approval from World Health Organization (WHO) and by the US Food and Drug Administration (FDA), while due to the rapid spread of the virus, the Emergency Use Authorization (EUA) has granted conditional approval to several new methods [1–6]. Several studies on sewage sampling and analysis as well as use of Geographic Information System (GIS) have also been conducted to understand the cause of epidemics, its spread pattern and to predict the occurrence of disease in an area. GIS acts as a useful tool in easing the fight against coronavirus with its advanced features such as mapping, location intelligence and spatial analysis providing a way to the government or public authorities in the determination of active COVID - 19 cases, recoveries, fatalities and even creating containment/hotspots zones [7]. On the other hand the surveillance of wastewater with the help of water based epidemiology [WBE] detects the RNA of the viral genome of SARS-CoV-2 enabling the further mitigation of the virus. The samples of the wastewater are collected and tested from the sewer lines indicating the more accurate location of coronavirus outbreak leading to the reorganization of area of concern [8]. It was also discovered that air plays a significant role in the spread of the SARS-CoV-2 virus, as it is transmitted through air [9].

Apart from the equipment and the method used, the result also depends on collection of sample, use of reagents, and probability of cross-contamination and storage requirements for samples/reagents. While selecting any reliable and fast diagnostic method all these factors should be considered so that a proper decision and immediate action to public health can be made. This chapter focuses on the various types of COVID-19 diagnosis methods presently in use in a comprehensive manner and also the working efficiency of the different methods by checking various parameters such as sensitivity, time of detection, specificity etc. in comparison to other methods. An attempt has been made to discuss the prediction methods used for COVID-19 prevalence detection and analysis. The broad areas focused in the chapter includes diagnosis of COVID-19 and surveillance system for disease prevalence.

## **2. Diagnosis of COVID-19**

Coronavirus is detected by reviewing the affected person's medical history, beginning with the point of contact and progressing through the findings of certain clinical examinations. Various respiratory problems and symptoms like pneumonia also comes under COVID-19 symptoms. Diagnosis methods like reverse transcription – polymerase chain reaction (RT-PCR) are being used now a days. Day by day with passing time many more methods are being developed but are pending for the approval from the regulatory authorities. The diagnostic methods that are studied and discussed in the chapter are shown in **Figure 1** and **Table 1**. These methods have been discussed in detail in the subsequent sections.

#### **2.1 Reverse transcription – polymerase chain reaction (RT-PCR)**

RT–PCR is currently the most commonly used laboratory methods for the detection of SARS-CoV-2. This method uses a technique derived from nuclear material to determine the existence of unique genetic material in any pathogen, including viruses. It's also being used to identify other diseases including the Ebola virus and the Zika virus. This method necessitates the collection of samples from body parts where the virus has accumulated, such as the nose or the throat [10]. To extract only the RNA present in the sample, it is treated with different chemicals to remove substances such as proteins and fats. This RNA is a combination of the person's genetic material and, if present, the virus's RNA. The procedure continues with the technique of merging reverse transcription of RNA into complementary

*Evolution of Diagnostic Methods and Prevalence Detection of COVID-19: A Review DOI: http://dx.doi.org/10.5772/intechopen.99241*

**Figure 1.** *Diagnosis approaches for COVID-19.*

DNA or cDNA, followed by polymerase chain reaction amplification of particular DNA (PCR) [11]. According to various studies, there are several advantages of the real time RT-PCR such as it is very highly sensitive, needs only a small amount of DNA and gives fast results in a duration of three hours as compared to other methods, which usually consumes six to eight hours [12, 13]. It is also the most precise method and gives accurate results after detection. This method, however, does not detect past infection, necessitating the use of other methods to detect, monitor, and study past infections, especially those that may have evolved and spread without causing symptoms. Other disadvantages includes its higher cost due to use of expensive apparatus, which makes it quite uneconomical [14, 15]. The flow process for virus detection using RT-PCR technique is given in **Figure 2**.

### **2.2 Next generation sequencing (NGS)**

The method of determining the nucleic acid sequence – the order of nucleotides in DNA, i.e. the order of the four bases: adenine, guanine, cytosine, and thymine – is known as DNA sequencing [16]. There are several DNA sequencing approaches, one of which is NGS, also known as High-throughput sequencing (HTS). By NGS, in a single experiment it is possible to determine the genomic sequencing of more than 1 million base pairs and hence this method is used for diagnosing inheritable diseases, cancer, and infectious diseases [17, 18]. NGS technology employs arraybased sequencing, which utilitizes Sanger sequencing techniques to process millions of reactions in parallel, resulting in extremely high speed and throughput at a lower cost [19]. The first step in NGS is library preparation, which involves randomly fragmenting DNA to build libraries, followed by ligation with custom linkers. Amplification is the second step, in which the library is amplified using clonal amplification methods, and PCR Sequencing is the third step, in which DNA is sequenced by using one of the several strategies. This method for diagnosis is specified as it provides all related information and is also highly sensitive. It is helpful in


**Table 1.**

*Different methods for diagnosis of COVID-19.*

#### *Fighting the COVID-19 Pandemic*

**320**

*Evolution of Diagnostic Methods and Prevalence Detection of COVID-19: A Review DOI: http://dx.doi.org/10.5772/intechopen.99241*

**Figure 2.** *Working process of RT-PCR method.*

identifying secondary infections and has potential tracing. However it is expensive and requires sophisticated laboratory for conducting test.

## **2.3 Computed tomography (CT)**

A computed tomography scan (CT scan) is a medical imaging technique that uses computer-processed combinations of several X-ray measurements taken from various angles to create cross-sectional images of the body, enabling the patient to see inside the body without cutting it open. COVID-19 is a respiratory disease that affects the parenchyma, but several studies claim that extreme cases are linked to a pro-inflammatory cytokine storm that leads to systemic inflammation and sepsis, as well as involvement in other organs such as the cardiovascular system [20]. An integrated Computed Tomography (CT) method may provide useful information on the diagnosis of COVID-19 patients in such circumstances. The expression of acute interstitial lung damage and the subsequent parenchymal changes induced by the cytokine storm triggered by the virus's internalization into the pneumocytes are normal CT findings in patients with COVID-19 [21–23]. During the early stages of the pandemic, CT was commonly used in China to diagnose COVID-19. Although the National Health Commission of China's current recommendations do not include imaging findings in diagnosis of this disease [24]. Furthermore, the American College of Radiology does not consider using a chest CT scan to test for COVID-19 pneumonia as a first-line imaging modality. Patients with symptoms like pulmonary embolism, empyema, or co-infection should get a CT scan, according to the recommendations. Using RT-PCR as a reference standard, several studies have demonstrated the sensitivity of CT. CT scan is being appreciated for its accuracy in results however; extreme precaution must be taken with respect to COVID-19 disease because of a negative CT scan. When compared to RT-PCR, a CT scan of the chest has a sensitivity of 89% and a Likelihood Ratio (LR) of 0.16, according to a study. With an LR+ of 2.81, specificity was moderate (68%) [25].

#### **2.4 Loop mediated isothermal amplification (LAMP)**

For the diagnosis of SARS–CoV-2, isothermal polymerase chain reactions methods such as loop-mediated isothermal amplification (LAMP) are supposedly a replacement for the RT-PCR process [26]. As compared to RT - PCR, LAMP is a powerful nucleic acid amplification method that works under isothermal temperature conditions and thus does not involve frequent temperature changes. To allow rapid amplification, this method involves designing assay primers and using a strand-displacing polymerase. LAMP reaction mix includes six primers that target eight different areas of the bacterial or viral genome. Currently RT-LAMP technique is being used for detecting COVID-19. RT- LAMP is a mechanism for auto cycling strand displacement DNA synthesis in which a polymerase uses one pair of inner and one pair of outer primers to carry out a reaction with high strand displacement operation. This method uses six independent sequences at the start and four independent sequences at the end to identify the target sequences. Primer identification of the target genome leads to a strong colorimetric reaction. The nucleic acid sample, 4 (or 6) specially formulated primers, and the best DNA polymerase are all incubated in the same test tube at 60 to 65 degrees Celsius, depending on the optimum LAMP temperature [27]. The ORF1ab gene, S gene, and N gene are among the main areas of coronavirus genomes where the primers are built for this process. ORF1ab is responsible for viral genome replication, while the S gene is required for coronavirus to bind to human ACE2 protein, and the N gene is a nucleocapsid protein found in many coronaviruses [28]. RT- LAMP completes the detection just within 25–30 minutes hence making it more reliable & suitable as compared to the RT-PCR for monitoring. Although it projects lot of gains, it has limitations such as slightly lower sensitivity of RT-LAMP as compared to RT - PCR. Some ongoing research recommended that the addition of guanidine could improve the sensitivity of detection with RT-LAMP [29]. RT-LAMP has a sensitivity of 75% as compared to RT-PCR, but unlike RT-PCR, it does not produce false-positive results, and when the results of RT-PCR and RT-LAMP are combined, diagnostic sensitivity increases to 92–100% [30], proving it to be a good technique.
