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\def\RunningAuthor{Abrar A. M. Almehmadi and Faten A. S. Alsulaimany}
\firstPage{2757}
\articleType{Original Article}
\receivedDate{18 Mar 2020}
\acceptedDate{16 Apr 2020}
\revisedDate{13 Apr 2020}
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\title{A DNA Sequence Analysis of Helicobacter pylori in Jeddah City, Western Saudi Arabia}
\author{Abrar~A.~M.~Almehmadi\textsuperscript{*}, 
            Faten~A.~S.~Alsulaimany~\\[5pt]{Department of Biology, Faculty of Science, King Abdulaziz University, Jeddah 21442, Saudi Arabia}}

\begin{abstract}
Helicobacter pylori are the type of Gram-negative bacteria which colonize the mucous lining of the human stomach. These bacteria have two major virulence factors: (vacuolating cytotoxin A gene) and (cytotoxin-associated A gene). This study aimed to provide data to determine the prevalent virulence factors (vacA and cagA genes) in Jeddah city, western Saudi Arabia, by sequence analysis. This study included 60 patients with symptoms similar to H. pylori infection. H. pylori were identified by using the 16s rRNA sequence. Then, the screening for specific genes in H. pylori (vacA and cagA) was done by using automated DNA sequencing analysis, and the DNA sequences were compared by BLAST and sequence alignment of the vacA nucleotides that are present in all H. pylori strains using those already reported in GeneBank from various studies. Results indicated that H. pylori infection was detected in 13.3\%, while 86.7\% were negative samples in our study patients. Interestingly, the vacA gene was found in 8.3\%, while the cagA gene was not appear in patient. Also, the female prevalence rate was higher than males (11.7\% female versus 1.7\% males), and the highest infection was between age 40-49 by 6.7\%. In conclusion, this study revealed that the vacA gene was spread in the patients infected with H. pylori in Jeddah, while the cagA gene was not appear in any isolate.
\end{abstract}\def\keywordstitle{Keywords}

\begin{keywords}Helicobacter pylori,\newline cagA,\newline vacA,\newline gastric biopsies
\end{keywords}
\twocolumn[ \maketitle {\printKwdAbsBox}]
\makeatletter{\raggedright\textsuperscript{*}Corresponding Author\par Name:\ Abrar~A.~M.~Almehmadi~\\ Phone:\ ~\\  Email:\ a\_Abrar\_22@hotmail.com
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\section{Introduction}
\textit{H. pylori }were considered one of the most types of pathogenic bacteria in humans and are the major cause of most diseases of the digestive system \unskip~\citep{789325:18797844,789325:18797846}. The infection with \textit{H. pylori} can cause gastrointestinal diseases like ulcer diseases, gastritis, and stomach cancer \unskip~\citep{789325:18797849,789325:18797861}. There is a variation in the spread of bacterial infection among countries, races, and socioeconomic status \unskip~\citep{789325:18797848}. Although \textit{H. pylori} is not as lethal as some bacteria, it is considered to be one of the most successful pathogens in human history because it affects 50\% of the world population \unskip~\citep{789325:18797850,789325:18797857}. This type of bacteria has been classified in humans (1994) because 1\% of those infected with the bacteria develop stomach cancer \unskip~\citep{789325:18797859,789325:18797842}. \textit{H. pylori }are Gram-negative bacteria, and the cellular morphology for these bacteria may be either curved, spiral, or fusiform. \textit{H. pylori }are microaerophilic but require oxygen at a lower concentration in order to survive \unskip~\citep{789325:18797851,789325:18797858}.  Warren and Marshall (1983) discovered \textit{H. pylori} and since that time it has been considered one of the pathogen bacteria in the world \unskip~\citep{789325:18797862,789325:18797841,789325:18797845}. 

\textit{H. pylori} possess two virulence factors: (the vacuolating cytotoxin A gene - \textit{vacA} gene) and (cytotoxin-associated gene A - \textit{cagA} gene) \unskip~\citep{789325:18797859,789325:18797854}. The \textit{vacA} gene stimulates formatting in anion channels and pores in epithelial cell membranes. Gene\textit{vacA}is already found in every strain of\textit{ H.pylori }around the world but is expressed differently. This gene includes several polymorphisms: signal region (s), the intermediate region (i)/ middle (m) region. Each of these polymorphic regions have two major types/alleles \unskip~\citep{789325:18797855}. Also, in genome of \textit{H. pylori}, the \textit{cagA} gene is one of several genes in \textit{cag}-PAI. This gene encodes a protein that causes many cellular changes, and \textit{cagA} gene is not found in all \textit{H. pylori}. The risk of developing duodenal ulcers, stomach cancer, and severe inflammation of the mucosa in the stomach may increase with positive strains of \textit{cag}-PAI \unskip~\citep{789325:18797842,789325:18797853}. Further, the \textit{cagA} gene is associated with severe clinical outcomes due to it being strong connection with expression of the vacuolating cytotoxin. Moreover, most of the strains that possess \textit{cagA} have a more virulent vacuolating form, \textit{vacA}\unskip~\citep{789325:18797842,789325:18797839}. The \textit{H. pylori} genotypes differ in the acuteness of disease in different geographic regions \unskip~\citep{789325:18797854}.

Most infected individuals present no symptoms (about 85\%) at all stages of life \unskip~\citep{789325:18797855,789325:18797838}. The rapid and correct diagnosis of \textit{H. pylori} helps to choose the right treatment and to prevent potential complications \unskip~\citep{789325:18797847}. The most common treatment is triple therapy, which entails two antibiotics and a proton pump inhibitor (PPI) \unskip~\citep{789325:18797860}. Globally, the percentage of infection with \textit{H. pylori} among developed countries was 25\% and in developing countries was 80\% \unskip~\citep{789325:18797852}. Numerous studies have proven the spread of \textit{H. pylori} in Saudi Arabia.  Hence, the aim of this studied to estimate prevalence of \textit{H. pylori} among a group of patients and determine prevalent virulence factors (\textit{vacA} and \textit{cagA} genes) in Jeddah city by sequence analysis.
    
\section{Materials and Methods}
\textbf{Sample Collection}

Biopsy samples were collected through a period of four months from patients selected for endoscopy in the stomach area in King Fahad hospital in Jeddah city, Saudi Arabia. Sixty patients with symptoms similar to \textit{H. pylori} infection were selected, aged between 19 and 69 years old. One sample was taken (biopsy sample) from each patient by an endoscope and then placed in transport media; then, it was cultured in the laboratory. To reduce the negative outcomes induced by using antibiotics, all individuals who took antibiotics within two weeks before to the test were excluded.

\textbf{Gastric Biopsy Culture}

The bacteria were cultured on \textit{H. pylori} Selective Supplement (DENT) medium and then incubated (three days) under microaerophilic conditions using CampyGen CN025 (Atmosphere Generation Systems) at 37 \ensuremath{^\circ}C[9]. 

\textbf{A Rapid Urease Test for H. pylori}

A single colony of \textit{H. pylori} colonies was taken by sterile loop and then placed into rapid urease tests for \textit{H. pylori} secretes a urease enzyme that hydrolyses urea to ammonia, that raises the pH, thus changing the sample color from (yellow - negative) to (pink - positive) \unskip~\citep{789325:18797856}.

\textbf{Genomic DNA Extraction }

After the \textit{H. pylori} culture from the biopsy samples, DNA extracted according to manufacturer's protocol for the Gene JET Genomic DNA Purification Kit Bacteria obtained from the Technolab solutions company. Extracted samples of DNA were stored at \ensuremath{-}20 \ensuremath{^\circ}C until used.

\textbf{Amplification of \textit{vacA} and \textit{cagA }Genes}

DNA was used from all positive samples of \textit{H. pylori} for detecting cagA and \textit{vacA} genes responsible for formation of CagA and VacA proteins using a primer and product size (bp) (Table~\ref{tw-bb20e6bd14bf}) \unskip~\citep{789325:18797845,789325:18797840}. The PCR tubes contain 12 \ensuremath{\mu }L of the master mix, 2 \ensuremath{\mu }L of the forward primer and reverse primer of the \textit{vacA} and \textit{cagA} genes, and 50 ng/\ensuremath{\mu}L of the DNA sample (9 \ensuremath{\mu }L).

The standard PCR protocol of \textit{vacA} and \textit{cagA} genes included an initial denaturation step at 94 \ensuremath{^\circ}C for 5 min and then 35 cycles followed by 1 min at 94 \ensuremath{^\circ}C, 1 min / 58 \ensuremath{^\circ}C, and at 72 \ensuremath{^\circ}C /1 min. Last step done for 10 min / 72 \ensuremath{^\circ}C. A PCR reaction without a DNA template was used as a negative control during each run. The samples were placed in a thermal cycler and were run for 2 h. The resulting products were separated on an agarose gel (Figure~\ref{f-698fb54dae74}).


\begin{table*}[!htbp]
\caption{\boldmath {PCR primers for \textit{cagA} and \textit{vacA} sequences } }
\label{tw-bb20e6bd14bf}
\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.10469999999999999\linewidth-2\tabcolsep}p{\dimexpr.17210000000000008\linewidth-2\tabcolsep}p{\dimexpr.43169999999999995\linewidth-2\tabcolsep}p{\dimexpr.2915\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Gene  & Primer & Primer Sequence (5'\ensuremath{\rightarrow}3') & PCR Product Size (bp)\\
\tblmidrule 
\multicolumn{1}{p{\dimexpr(.10469999999999999\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{CagA }} &
  CagA-F &
  F: 5'-GATAACAGGCAAGCTTTTGAGG-3' &
  \multicolumn{1}{p{\dimexpr(.2915\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{349}}\\
 &
  CagA-R &
  R: 5'-CTGCAAAAGATTGTTTGGCAGA-3' &
  \\
\multicolumn{1}{p{\dimexpr(.10469999999999999\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{VacA}} &
  VacA-F &
  F: 5'-GCCGATATGCAAATGAGCCGC-3' &
  \multicolumn{1}{p{\dimexpr(.2915\linewidth-2\tabcolsep)}}{\multirow{2}{\linewidth}{  678 }}\\
 &
  VacA-R &
  R: 5'-CAATCGTGTGGGTTCTGGAGC-3' &
  \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {\textit{H. pylori} infection rate} }
\label{tw-cb0eaa05e895}
\def\arraystretch{1.1}
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\begin{tabulary}{\linewidth}{p{\dimexpr.484\linewidth-2\tabcolsep}p{\dimexpr.30230000000000004\linewidth-2\tabcolsep}p{\dimexpr.2137\linewidth-2\tabcolsep}}
\tbltoprule \rowcolor{kwdboxcolor}Characteristics  & \multicolumn{2}{p{\dimexpr(.5160000000000001\linewidth-2\tabcolsep)}}{\cAlignHack H. pylori Infection }\\
\rowcolor{kwdboxcolor} & Positive  & Negative \\
\tblmidrule 
Age groups  &
   &
  \\
19{\textendash}29 Years  &
  3 (5.0\%)  &
  14 (23.3\%) \\
30{\textendash}37 Years  &
  0 (0.0\%)  &
  12 (20.0\%) \\
40{\textendash}49 Years  &
  4 (6.7\%)  &
  9 (15.0\%) \\
50{\textendash}59 Years  &
  1 (1.7\%)  &
  12 (20.0\%) \\
60{\textendash}69 Years  &
  0 (0.0\%)  &
  5 (8.3\%) \\
Total   &
  8 (13.3\%)  &
  52 (86.7\%) \\
Nationality  &
   &
  \\
Saudi  &
  7 (11.7\%)  &
  45 (75.0\%) \\
Non-Saudi  &
  1 (1.7\%)  &
  7 (11.7\%) \\
Total   &
  8 (13.3\%)  &
  52 (86.7\%) \\
Gender  &
   &
  \\
Male  &
  1 (1.7\%)  &
  18 (30.0\%) \\
Female  &
  7 (11.7\%)  &
  34 (56.7\%) \\
Total  &
  8 (13.3\%)  &
  52 (86.7\%) \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\begin{table*}[!htbp]
\caption{\boldmath {Phylogenetic identification of \textit{vacA} gene of \textit{H.pylori}} }
\label{tw-216a1dd80ff4}
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\tbltoprule \rowcolor{kwdboxcolor}No. & Samples   & Closest Matches Identification   & Sequence Identity  \\
\tblmidrule 
1  &
  VacA-22 gene  &
  VacA gene-isolate: BH70  &
  96.8\% \\
2  &
  VacA-27 gene  &
  VacA gene-isolate: M18660  &
  93.6\% \\
3  &
  VacA-29 gene  &
  VacA gene-isolate: BH111  &
  96\% \\
4  &
  VacA-35 gene  &
  VacA gene-isolate: AFN4124  &
  97.7\% \\
5  &
  VacA-47 gene  &
  VacA gene-isolate: MV040A  &
  96.9\% \\
\tblbottomrule 
\end{tabulary}\par 
\end{table*}

\bgroup
\fixFloatSize{images/880ba55c-7c19-420a-bd4f-e7b9882f68fe-upicture1.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/880ba55c-7c19-420a-bd4f-e7b9882f68fe-upicture1.png}{\includegraphics{images/880ba55c-7c19-420a-bd4f-e7b9882f68fe-upicture1.png}}{}
\makeatother 
\caption{\boldmath {PCR amplification of the patient's samples of \textit{vacA} gene. Lanes 1{\textendash}5 represent the specific DNA fragments of the \textit{vacA} gene at approximately 678 bp}}
\label{f-698fb54dae74}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/1ce22b93-0173-4c01-ba38-13080e71a539-upicture2.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/1ce22b93-0173-4c01-ba38-13080e71a539-upicture2.png}{\includegraphics{images/1ce22b93-0173-4c01-ba38-13080e71a539-upicture2.png}}{}
\makeatother 
\caption{\boldmath {The presence rate of \textit{vacA} gene in \textit{H. pylori} infected patients}}
\label{f-2c896cc8082a}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/c4511258-6652-42ef-9140-af5cf06a5914-upicture3.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/c4511258-6652-42ef-9140-af5cf06a5914-upicture3.png}{\includegraphics{images/c4511258-6652-42ef-9140-af5cf06a5914-upicture3.png}}{}
\makeatother 
\caption{\boldmath {The phylogenetic tree for backbone sequences of \textit{vacA-}22 gene by using MEGA7}}
\label{f-74aa2863f072}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/002261c8-654c-4aa2-84d2-b3520746e607-upicture4.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/002261c8-654c-4aa2-84d2-b3520746e607-upicture4.png}{\includegraphics{images/002261c8-654c-4aa2-84d2-b3520746e607-upicture4.png}}{}
\makeatother 
\caption{\boldmath {The phylogenetic tree for backbone sequences of the \textit{vacA-}27 geneby using MEGA7}}
\label{f-d95a0045fbe2}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/3d27ccef-a4d1-48e4-bf20-99913ee5e2ee-upicture5.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/3d27ccef-a4d1-48e4-bf20-99913ee5e2ee-upicture5.png}{\includegraphics{images/3d27ccef-a4d1-48e4-bf20-99913ee5e2ee-upicture5.png}}{}
\makeatother 
\caption{\boldmath {The phylogenetic tree for the backbone sequences of the \textit{vacA}-29 gene by using MEGA}}
\label{f-4903054332aa}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/f77f69ef-f601-472a-98b0-5d9a4f309193-upicture6.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/f77f69ef-f601-472a-98b0-5d9a4f309193-upicture6.png}{\includegraphics{images/f77f69ef-f601-472a-98b0-5d9a4f309193-upicture6.png}}{}
\makeatother 
\caption{\boldmath {The phylogenetic tree for backbone sequences of the \textit{vacA}-35 gene by using MEGA7}}
\label{f-b9dafa27d880}
\end{figure}
\egroup

\bgroup
\fixFloatSize{images/42451a3e-d379-4755-bf88-55c81309dc25-upicture7.png}
\begin{figure}[!htbp]
\centering \makeatletter\IfFileExists{images/42451a3e-d379-4755-bf88-55c81309dc25-upicture7.png}{\includegraphics{images/42451a3e-d379-4755-bf88-55c81309dc25-upicture7.png}}{}
\makeatother 
\caption{\boldmath {The phylogenetic tree for backbone sequences of the \textit{vacA}-47 geneusing MEGA7}}
\label{f-bfb4987365f2}
\end{figure}
\egroup
\textbf{DNA Sequencing Protocol}

The sequencing analysis involved two main steps,

1) purification of the PCR products and cycle sequencing 

2) purification of cycle sequencing products and drying and denaturing. 

The purified sequencing samples were then loaded into a 96-Well microtiter sequencer plate and run on a genetic analyzer for data interpretation (3500). The nucleotide sequence data were examined for comparison with a reference sequence retrieved from the National Center for Biotechnology Information and the Basic Local Alignment Search Tool database.

\textbf{Statistical Analysis }

By using SPSS version, 24.0, data were entered and analyzed. Also, a bivariate analysis performed for statistically differences among (\textit{H. pylori}-positive or negative) and (\textit{vacA}/\textit{cagA} positive or negative genes) with individuals in relation to the specific variables.
    
\section{Results}
\textbf{Prevalence of \textit{cagA} and \textit{vacA} Genes}

\textit{H. pylori} infection was diagnosed in 13.3\%. Further, the female spread rate was higher than that of males, and the highest infection rate was between ages 40 and 49 (6.7\%). Those aged between 19 and 29 had a 5.0\% prevalence rate and those between 50 and 59 had a rate of 1.7\%, as shown in Table~\ref{tw-cb0eaa05e895}. The \textit{vacA} gene was obtained in 8.3\%, and 91.7\% were negative samples, as shown in Figure~\ref{f-2c896cc8082a}, while \textit{cagA} gene was not detected in any isolate.

\textbf{DNA Sequencing and Phylogenetic Analysis}

DNA sequencing for all positive samples of \textit{vacA} gene done and compared to the DNA sequences through BLAST and the alignments of the \textit{vacA} nucleotides that were present in the \textit{H. pylori} strain already present in GenBank. The percentage of sequence identity of the \textit{vacA} was also determined (Table~\ref{tw-216a1dd80ff4}). The phylogenetic relationships of the experimental isolates (\textit{vacA} gene) and closely related species were analyzed by using MEGA7, and results were presented in phylogenetic trees, as shown in (Figures~\ref{f-74aa2863f072}, \ref{f-d95a0045fbe2}, \ref{f-4903054332aa}, \ref{f-b9dafa27d880} and~\ref{f-bfb4987365f2}).
    
\section{Discussion}
\textit{H. pylori} infection occurs worldwide, but its spread among countries is very different and also differs among ethnic groups in the same country and age groups \unskip~\citep{789325:18797844}. Infection with \textit{H. pylori }leads to ulcers and develop to stomach cancer, which contributes to a large number of cancer related deaths worldwide. In the late 1980s, Saudi Arabia began to study gastritis after the discovery of this pathogen (\textit{H. pylori}), and several studies have been carried out to determine the effectiveness of different methods of detecting and isolating \textit{H. pylori}. Further, several studies since 1980 have aimed to understand the cause of this pathogen. Moreover, Saudi Arabia has taken the lead among the countries of the Middle East in the study of \textit{H. pylori}'s pathogenesis, the sequence of the whole genome, and molecular characterization, in order to understand more about its methods of transmission, determine the different strains of \textit{H. pylori }bacteria in Middle East, and elucidate interaction among the host and pathogen \unskip~\citep{789325:18797843}.

\textit{H. pylori} infection is widespread in Saudi Arabia among men and women, as well as children. Hence, in our study, to estimate the spread of \textit{H. pylori} among a group of patients and to determine its prevalent virulence factor (\textit{vacA} and \textit{cagA} genes) in Jeddah city by sequence analysis we used 60 patients with similar symptoms of \textit{H. pylori} infection. One sample was taken (biopsy sample) from each patient by an endoscope, and age (19: 69) years old. 

\textit{H. pylori} were identified by using a 16s rRNA sequence, and \textit{H. pylori} infection found in 13.3\%, while 86.7\% were negative isolates. This result is similar to that of another study conducted by Jaber in 2006, where the spread of \textit{H. pylori} in patients was about 23.6\%. However, the number of samples in this study was bigger than that in our study \unskip~\citep{789325:18797852}. Furthermore, the incidence rate with \textit{H. pylori} among Saudi patients was higher than that among non-Saudi patients, and the highest infection of \textit{H. pylori} was between the ages of 40 and 49 years. According to our study, it seems that gender does not affect the acquisition of \textit{H. pylori}. Moreover, the infection rate in females was higher than that among males. However, Abo-Shadietal.found, a higher risk of infection among males than among females \unskip~\citep{789325:18797847}.

The study of \textit{H. pylori} genome focused on trying to understand disease and its ability to cause illness \unskip~\citep{789325:18797859}. Growing evidence to genetic variation of \textit{H. pylori }could be clinical significance \unskip~\citep{789325:18797845}. Also, the clinical results of infection with the bacteria are linked with the virulence factors of \textit{H. pylori}\unskip~\citep{789325:18797842}. Virulence factors of \textit{H. pylori} are responsible for many illnesses of the stomach. The prevalence of genes (\textit{cacA} and \textit{vagA}) are significantly different between countries \unskip~\citep{789325:18797840}.  There is usually variation in the DNA sequence between the isolates, even in essential genes, by a rate of 3\% to 5\%. This diversity may reflect a set of factors, such as recombination between different strains, mutation, and transport between members of the family and people in close contact with them. There is a difference in certain traits from person to person, like the strength of the immune system, specificity, which leads to variations between \textit{H. pylori} strains \unskip~\citep{789325:18797850}. Interestingly, the \textit{vacA} gene was present in 8.3\%, while the \textit{cagA} gene was not detected in any isolate.

DNA sequencing for all our positive samples for the \textit{vacA} gene was done and compared to the published \textit{vacA} gene DNA sequences from \textit{H. pylori} through BLAST. The percentage of sequence identity of \textit{vacA} gene was 96.8\% for \textit{vacA}-22, 93.6\% for \textit{vacA}-27, 96\% for \textit{vacA}-29, 97.7\% for \textit{vacA}-35, and 96.9\% for \textit{vacA}-47, with the closest matches strains. The geographical distribution of the distinct genotypes of \textit{H. pylori} and the extent of spread of virulent genotypes is still unknown in several regions of Saudi Arabia \unskip~\citep{789325:18797839}. This study suggests the need for a larger and more comprehensive study of adults and children to detect the virulence factors in patients infected with \textit{H. pylori} in the Saudi population in Jeddah city.
    
\section{Conclusions}
This study detects that the \textit{vacA} gene was distributed in patients had\textit{H. Pylori }(Jeddah/ Saudi Arabia), while the \textit{cagA} gene was not detected in any isolate A large national epidemiological study is needed to determine prevalence of bacteria. This will open many new avenues for many studiesabout the virulence factors of \textit{H. pylori} (\textit{vacA} and \textit{cagA}) genes. According to our results, there is a need for a larger and more comprehensive study of adults and children to detect the virulence factors in patients infected with \textit{H. pylori} in Jeddah city.

\textbf{Funding source}

Funding was through King AbdulazizCity for Science and Technology [1-17-01-009-0014]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of manuscript.

\textbf{Conflicts of Interest}

The authors declare no conflict of interest.

\textbf{Ethical approval}

This study was approved by Directorate of Health Affairs-Jeddah (protocol number: A00548).

\textbf{Author Contributions}

A.A.A.; sample collection, methodology, analysis, writing, and F.A.A.; review and editing. All authors have read and agreed to publish version of manuscript. 
\section*{Acknowledgement}Thanks and appreciation to King AbdulazizCity for Science and Technology for its financial support for our project (1-17-01-009-0014), which enabled me to provide the requirements and substances for this research.\textbf{\space }
    

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