1 INTRODUCTION

The comma-shaped bacteria Vibrio cholerae is the cause of cholera, an acute diarrheal disease.¹ V. cholerae is a water-borne pathogen and can be transferred through vector and reservoir organisms such as flies, algae, and some crustaceans.^{2, 3} In coastal regions and estuaries, V. cholerae grows best in salt water.^4-6 Fecal samples of human cholera carriers can also carry high concentrations of V. cholerae.⁷ Through such mechanisms, V. cholerae can contaminate water sources and food. Once ingested through the fecal–oral route, the disease can lead to symptoms such as diarrhea and dehydration.⁸ In 1817, cholera appeared in the Ganges Delta and expanded to seven pandemics that killed millions of people across the world.^4-6 V. cholerae is constantly evolving, with novel phenotypes and genotypes arising as a consequence of outbreaks and growing drug resistance.⁹ In Africa, cholera occurs in two different ways: as an endemic condition that recurs every 2–3 years during specific seasons, or as acute epidemics that start suddenly, endanger thousands of lives, and strain the healthcare infrastructure.¹⁰ Cholera outbreaks are difficult to predict and therefore can occur in both endemic and nonendemic areas based on environmental changes, with refugee camps being especially vulnerable.¹¹

Globalization and travel increase the spread of cholera, as was the case in Haiti following the 2010 earthquake, where imported cases from Nepal were the source of the infection. Cholera was brought to Haiti from a single source and then rapidly, widely, and continuously spread.^{12, 13} The spreading of cholera across countries' borders has been proposed between Iraq-Iran and Cameroon-Nigeria.^{14, 15} Since 2019, the COVID-19 pandemic has greatly increased mortality rates and put a burden on medical facilities all around the world. In Africa and the Middle East, COVID-19 has hindered the fight against infectious diseases that can be fatal, such as cholera, tuberculosis, HIV/AIDS, and several tropical diseases.^16-18 Numerous nations in Asia, Africa, and Europe experienced cholera outbreaks during the pandemic,^19-22 whereas no cases were recorded in the Americas in 2022. Due to inadequate monitoring and surveillance, it is anticipated that the actual number of cholera cases may be greater than those that have been released.^19-22 In 2022, isolated cholera cases were associated with damage to the sewage and water systems due to the fighting in Mariupol, Ukraine, which resulted in several health issues.^23-25

This study reviews recent cholera outbreaks in Africa and the Middle East, the geographical distribution of cholera cases in 2022 and 2023, the different serogroups and biotypes of V. cholerae causing pandemics and outbreaks, and the causes of cholera outbreaks raging in Africa and the Middle East. We also describe different manifestations and complications of cholera, methods of diagnosis, available oral cholera vaccines (OCVs), and antimicrobial drug resistance. Finally, we discuss management strategies and promising cholera treatments. We carried out a systematic search in multiple databases, including PubMed, Web of Science, Google Scholar, Scopus, MEDLINE, and Embase, for studies on cholera using the following keywords: ((Cholera) OR (Vibrio cholera) and (Coronavirus) OR (COVID-19) OR (SARS-CoV2) OR (The Middle East) OR (Africa)).

2 EPIDEMIOLOGY AND GEOGRAPHICAL DISTRIBUTION OF CHOLERA IN AFRICA AND THE MIDDLE EAST IN 2022 AND 2023

Since 2021, there has been an upsurge in cholera cases in Africa (e.g., Cameroon, the Democratic Republic of the Congo, Ethiopia, Kenya, Malawi, Mozambique, Nigeria, South Sudan, the United Republic of Tanzania, Zimbabwe, Niger, Burundi, and Somalia), as well as the Middle East (e.g., Syria, Lebanon, Yamen, and Iraq).²⁶ Some countries fail to report cholera cases or deaths because of inadequate or nonexistent monitoring systems. Others delay reporting cholera outbreaks to reduce losses in commerce and tourism as well as societal hysteria. However, early detection of cholera outbreaks may lead to shorter epidemic durations and terminate any potential epidemics.²⁷ Figure 1 shows the geographical distribution of cholera in Africa and the Middle East in 2022.

In Table 1, we collected the recently reported numbers of cholera cases, deaths, and case fatality ratios (CFRs) in Africa and the Middle East in 2022. According to our research, Malawi had the largest number of cholera cases reported in Africa in 2022, with 28,132 confirmed cases, 916 fatalities, and a CFR of 3.3%. While in the Middle East, Syria had the largest number of cases, with 77,561 suspected cases, 1893 confirmed cases, 100 deaths, and a CFR of 0.13%.

3 CHOLERA SEROLOGY

V. cholerae strains are defined by the structure of their O antigen, which has been classified into more than 200 O serogroups.^36-39 Despite this large number of serogroups, only the V. cholerae serogroups O1 and O139 are toxin-producing and have been reported as the cause of disease in epidemics.^{39, 40} The O1 serogroup has three serotypes based on the antigenic factors A, B, and C: Inaba, Ogawa, and Hikojima, and two biotypes, classical and E1 Tor.³⁹ 99% of the worldwide cases are caused by O1 serogroup.⁴¹ Stool samples in the current outbreak in Yemen were positive for the O1 serogroup, Ogawa serotype.^{42, 43} In early October 2022, there was a rapid spread of cholera caused by Serotype O1 El-Tor Ogawa in Syria and Lebanon.^{44, 45}

Usually, V. cholerae strains have a prophage, CTXФ, where the gene encoding V. cholerae toxins is present, the most important factor in its virulence. Atypical El Tor strains have the ctxB allele, which is a characteristic allele of the classical CTXФ strain (CTX^ClassФ).⁴⁶ In the 2004 Mozambique outbreak, there were two types of prophages nearly identical to CTX^ClassФ, but they had the El Tor phenotype and were otherwise genotypically El Tor, so they were named Mozambique EI Tor.^46-48 Other atypical El Tor strains are called hybrid EI Tor because they have classical ctxB and rstR genes. All of these variations are caused by the transfer of mobile genetic elements.⁴⁶

Serogroups other than O1 and O139 arise less frequently and are likely under-reported due to the lack of diagnosis. Currently, 1%–3.4% of cases of acute diarrhea cases are due to the non-O1/non-O139 serogroup (NOVC).⁴⁹ In a study of 58 hospitalized patients with acute diarrhea in Italy, 3.4% of the cases were due to NOVC.⁵⁰ 27.4% of hospitalized cholera patients with acute diarrhea at a hospital in India carried non-O1/non-O139 serogroup bacteria.⁵¹ The O5 serogroup was responsible for cases of cholera in Czechoslovakia in the 1960s; the O37 serogroup caused cholera infections in Sudan in 1968.^52-54 After July 2015, there was a reported case of NOVC cholangitis and septicemia in Beirut, which was the third reported case of NOVC bacteremia in Lebanon and the first after the 2015 Lebanese waste crisis.⁵⁵

Six pandemics caused by the O1 serogroup arising from India have spread worldwide from 1817 to 1923.^{56, 57} A seventh pandemic, which arose in Indonesia in 1961 due to the El Tor biotype, spread into Asia, Africa, and Latin America, including the devastating epidemic that occurred in Haiti in 2010. Other El Tor biotype outbreaks occurred in several African countries from 1997 to 1999, from 2012 to 2014 in Mozambique, and 2010 in Cameroon.^{39, 58-60} O139 epidemics have occurred less frequently but were responsible for infections among the elderly in Pakistan between 1995 and 2010,⁶¹ in Thailand,⁶² and sporadically in China⁶³ and Bangladesh.⁶⁴ Serotype O139 may be responsible for the probable eighth cholera pandemic, which emerged successfully between 1992 and 2015 before dying out, according to a study by Ramamurthy et al.⁶⁵ They included 330 O139 isolates in their study and reported that two key genomic evolutionary changes are responsible for the eventual epidemiological decline of O139. First, there was a transformation from the homogenous toxin genotype in wave-A to heterogeneous genotypes, followed by gradual loss of antimicrobial resistance due to the progression of waves.⁶⁵

Table 2 depicts the classification of V. cholerae into serogroups and biotypes, as well as major pandemics and outbreaks over the years.^{46, 54} Pulsed-field gel electrophoresis (PFGE), polymerase chain reaction (PCR)-based methods, ribotyping, and sequencing-based methods such as multi-locus sequence typing (MLST), variable number of tandem repeats (VNTR) analysis, and multi-locus variable tandem repeat analysis (MLVA) are molecular tools for studying V. cholerae.⁵⁴

4 WHY ARE AFRICA AND THE MIDDLE EAST FERTILE ENVIRONMENTS FOR CHOLERA?

The key factors contributing to Africa's ongoing and repeated cholera outbreaks include poverty, inadequate sanitation, poor water supply, and overpopulation. The situation is exacerbated by the human conflicts that occur throughout Africa as well as the Middle East.^{2, 8, 77, 78} Transmission becomes simpler in crowded environments like refugee camps, prisons, and war zones.^{10, 79} Additionally, climate-related factors such as rainfall, flooding, droughts, and strong winds also propagate cholera (Figure 2).^{2, 77}

Several sociological factors have impacted cholera epidemics in Africa and the Middle East. In developing countries, high-density populations lead to sanitation issues such as unsuitable living conditions; clean water, sewage treatment, and clean, warm food may be limited or not available at all. These living conditions often do not allow for basic sanitation strategies with which the prevention of cholera can be achieved.^{80, 81} Water wells and rivers are common sources of water for displaced communities. Sewage treatment allows for the maintenance of a clean, non-contaminated water supply for the population and is lacking in many impoverished areas.⁷⁸ Sanitation applies to not only water sources but nutrition as well. Cold meals, lack of nutrition, and roadside food vendors all increase the likelihood of contracting cholera.⁷⁷ Helicobacter infection, which is common in young children and adolescents in developing countries in Africa and the Middle East, is another risk factor for cholera.^82-84 Helicobacter pylori plays a significant role in the pathophysiology of diarrheal illnesses like cholera by causing hypochlorhydria, gastric ulcers, and gastric cancer.^{82, 84} Clemens et al. found that infection with H. pylori was related to a considerable increase in the probability of life-threatening cholera. However, this was only observed in people who lacked natural immunity against V. cholerae.⁸⁵

As of 2023, there were about 5.4 million refugees from Syria in neighboring countries.^{8, 86} The prolonged civil war within Yemen has led to the immigration and emigration of individuals who may have been carriers of cholera.² Due to the country's institutional gridlock and economic crisis, cholera affected Lebanon in 2022.⁷⁸ In Ethiopia, the conflict in the Tigray region has led to nearly 53,000 refugees entering Sudan to be housed in refugee camps.^{8, 87} Political conflicts have not only displaced people but have also prevented vaccines from reaching the targeted host population.⁸⁰ Cholera vaccinations are limited in impoverished countries due to factors such as political strife, vaccine costs, and the world's focus on other pathogens.⁸⁰

Cases of cholera outbreaks brought on by climate change, such as those associated with droughts, heavy rainfall, and the propagation of V. cholerae reservoirs and vector organisms have occurred in Africa.^{46, 77} Climate change is likely to worsen cholera outbreaks in Africa and the Middle East. Increases in air temperature were linked to increases in cholera cases in Africa.^88-93 Extreme weather events are predicted to increase with climate change⁹⁴ and both abnormally wet and drought events have been associated with increased cholera cases in Africa^95-98 and elsewhere.^{90, 91, 99} Droughts can lead people to use poor-quality water for drinking, whereas flooding can cause sewage systems to overflow into drinking water.^{92, 100} The obtainment of clean water can be expensive; water wells are often sought after, which may have cholera propagating within them.^{46, 77} Droughts are often accompanied by times of economic hardship for individuals who partake in agriculture. Obtaining vaccines, sanitation supplies, water, and food may be difficult, and cheaper alternatives may be sought, which may contain V. cholerae.⁷⁷ Increased frequency of El Niño events can lead to more climatic extremes that increase cholera cases.^{96, 101-103} Changing ocean conditions favor the growth of V. cholerae bacteria through increased temperature⁹² and higher zooplankton populations that are fed by higher phytoplankton populations that benefit from nutrient-rich runoff from agriculture and human waste.^{88, 99, 100} Because V. cholerae is a temperature-sensitive pathogen, its populations grow within reservoirs at the ideal temperature. This in turn increases the likelihood of the pathogen increasing within the population.⁷⁷ Droughts can allow brackish water up into streams allowing cholera carrying plankton inland.^{90, 91, 99}

The COVID-19 pandemic exacerbated cholera outbreaks in different countries in Africa and the Middle East.^16-22 As of February 2023, approximately 750 million confirmed cases and 6.7 million confirmed deaths have been attributed to COVID-19 globally.¹⁰⁴ The pandemic caused a shift in the focus of governments, organizations, and the media away from cholera.^{81, 105} The worst consequences of the COVID-19 pandemic were suffered by many developing countries in Africa and the Middle East.¹⁰⁶ The pandemic may negatively impact crucial cholera control measures like surveillance, a crucial defense against outbreaks and related mortality.¹⁶ However, an increase in hand washing and sanitization in response to the COVID-19 pandemic is expected to reduce food and waterborne infections. Researchers believe that the restricted availability of clean water and poor waste disposal in many rural and peri-urban slums in Sub-Saharan Africa make efficient hand hygiene implementation difficult.¹⁶

5 THE CHARACTERISTIC CLINICAL MANIFESTATIONS AND COMPLICATIONS OF CHOLERA

V. cholerae infection causes a range of symptoms, from asymptomatic intestinal colonization to severe diarrhea. Other typical symptoms include abdominal pain, borborygmi, and vomiting, especially in the early stages of the illness. The severe illness caused by cholera is attributed to the significant loss of electrolytes caused by diarrhea. The clinical signs of V. cholerae O1 versus O139 are similar.¹⁰⁷ A normal incubation period for cholera is 1–2 days; however, the duration of cholera incubation varies depending on the host's susceptibility and the amount of the inoculum and can range from a few hours up to 3–5 days.^108-110 While mild V. cholerae infection may be clinically indistinguishable from other causes of diarrhea, severe cholera is distinguished by rapid fluid and electrolyte loss. Cholera stools may contain fecal debris and bile in the early stages of the illness.⁶ Nevertheless, the passing of copious “rice-water” stool, a watery stool with mucous particles, is a defining feature of severe cholera (cholera gravis) and usually has a fishy odor. The diarrhea is normally painless and does not cause tenesmus. In the most severe instances, stool production in adults may approach 1 L per hour. The maximum rate of stool excretion in children with severe cholera is frequently between 10 and 20 cc/kg/h, and this rate of fluid loss is unusual in other types of diarrheal sickness.¹¹¹ Additionally, cholera patients’ feces include a greater quantity of sodium, as well as considerable levels of potassium and bicarbonate, when compared to other causes of pediatric diarrheal disease.^{112, 113} Diarrhea is most severe in individuals treated with appropriate rehydration during the first 2 days and subsides after 4–6 days, with total volume loss of up to 100% of body weight occurring throughout the disease.¹¹⁴ Vomiting, which is sometimes accompanied by watery emesis, is common and can occur before or after the onset of diarrhea. Patients may experience abdominal cramps, but not the intense abdominal pain associated with dysentery.¹¹⁴

The most common sequelae of acute cholera diarrhea are hypovolemia and electrolyte abnormalities due to rapid fluid and electrolyte loss. Severe hypovolemia can develop within hours of the onset of symptoms. The median time between the onset of symptoms and death in those who died before being admitted to a cholera treatment facility during the early stages of the Haitian cholera outbreak was 12 h.¹¹⁵ Severe hypovolemia might cause sunken eyes, dry mouth, chilly, clammy skin, reduced skin turgor, or wrinkled hands and feet. The majority of patients exhibit apathy. Kussmaul breathing could develop from lactic acidosis brought on by inadequate perfusion in addition to acidosis from loss of stools' bicarbonate (Figure 3). Pneumonia is a common comorbidity in children with cholera, possibly as a result of aspiration during vomiting, and has been linked to death.¹¹⁶ Because fever is uncommon, the presence of a high temperature should raise the possibility of a concomitant infection or consequence. In the absence of diarrhea, “Cholera sicca” is a unique type of illness in which fluid collects in the intestinal lumen; circulatory collapse and even death may follow.¹¹⁷

Most cholera cases with neurologic complications involve infants and neonates and manifest as meningitis,¹¹⁸ but adults with a higher risk of intracranial infection have experienced neurological effects from cholera. A 58-year-old fisherman with left-sided paresis and a Babinski sign had an intracerebral abscess and bacteremia caused by V. cholerae that did not produce cholera toxin. He had a craniotomy 5 years before presentation for a subdural hematoma, which could have allowed the organism intracranial access.¹¹⁹ There were no risk factors for a 10-day-old boy with V. cholerae meningitis, but recent water studies in his rural Indian village found non-O1 non-O139 V. cholerae in 41% of drinking water samples and 100% of water, sediment, and plankton samples from three nearby lakes.^{118, 120}

Cholera is particularly dangerous to pregnant women and their fetuses. Despite early research suggesting a high risk of fetal death linked to cholera during pregnancy (up to 50% during the third trimester), more recent research has revealed a decreased but still high risk (~8%).^{114, 121-123} According to one study from Guinea, most pregnant women affected by a 2012 cholera outbreak were in their third trimester. The classic clinical manifestations were associated with obstetric difficulties and maternal-fetal hazards. In addition to experiencing choleriform diarrhea in 100% of the women, dehydration was mild in 16%, moderate in 45%, and severe in 39% of the women. There were two threatened abortions, one preterm birth, four intrauterine deaths, and one maternal death.¹²⁴

6 DIFFERENT METHODS OF CHOLERA DIAGNOSIS

In developing countries and poor settings where advanced laboratory diagnostic techniques are limited, cholera is diagnosed by clinical symptoms and manifestations. After the incubation period of V. cholerae (median = 1.4 days), outcomes range from symptomless intestinal colonization to mild or severe diarrhea.¹²⁵ Identification of endemic species of V. cholerae O1 and O139 by isolation and culture of a stool specimen in alkaline peptone water (APW) then subcultured in thiosulfate-citrate-bile salts sucrose (TCBS) agar or taurocholate tellurite-gelatin (TTG) agar is the gold standard in the diagnoses with Cary Blair media as the ideal media for transport.^126-128 Stool analysis is valuable for V. cholerae diagnosis as it enables further molecular phenotypic analysis of the specimen for antibiotic resistance and molecular characterization.¹²⁸ External factors such as improper sampling and shipment can affect the efficacy of stool analysis as a primary diagnostic tool.¹²⁸ Molecular detection and PCR have been developed for V. cholerae species identification and characterization. PCR requires a sample to be grown in pure culture without requiring viable strains. PCR can also detect the pathogen in food, water, and rectal swabs, it does not require a stool sample as most of the tests.¹²⁹

Methods of diagnosis have been developed to replace complicated laboratory investigations in developing countries or endemic settings with poor healthcare quality.¹³⁰ Rapid diagnostic tests (RDTs) are used directly on rectal swabs, fresh stool only, or after enrichment of the specimen by alkaline peptone water for (4–6 h).^{126, 131, 132} RDTs detect the pathogen qualitatively within a maximum of 30 min, which appears as a colored band that can be read with the naked eye.¹³⁰ Almost all RDTs work by detection of antigens specific for lipopolysaccharide (LPS) of serotypes O1 and O139 of V. cholerae using monoclonal antibodies.¹³³ These tests are developed to detect epidemics early to start cholera treatment for patients in their early stages to prevent the disease's progress. RDTs do not require experienced technicians to perform, are low-cost, and do not require cold storage.^{126, 134} These advantages of RDTs lead us to believe that they are more appropriate for use in refugee camps and impoverished regions of Africa and the Middle East. Table 3 shows the characteristics of the most common RDTs, including their sensitivity and specificity.^{135, 143}

7 CURRENT AVAILABLE OCVS

There are four main vaccines for the prevention of cholera in the market. Vaxchora®, Dukoral®, Shanchol™, and Euvichol®/Euvichol-Plus have been prequalified by the WHO (Table 4).

8 CHOLERA VACCINE SHORTAGE CRISIS AND NEW PROMISING OCVS

The ICG managed the distribution of emergency vaccines and drugs to nations during significant outbreaks and received the global cholera vaccine stockpile from Shantha Biotechnics and EuBiologics. The WHO, UNICEF, and the International Federation of Red Cross all have representatives on the ICG. In 2022, outbreaks have been recorded from 30 countries, compared to an average of less than 20 during the past 5 years.^{183, 184} Cholera vaccines are in critically short supply, according to the WHO.^{183, 184} There is no short-term way to enhance production because vaccine manufacturers were already operating at full capacity. As of October 2022, a total of 24 million of the 36 million doses anticipated to be produced in 2022 have already been shipped for vaccination campaigns, and an additional 8 million doses have been approved by the ICG for a second round of emergency vaccination in four countries, highlighting the severe lack of the vaccine.¹⁸⁵

The ICG had been forced to temporarily halt the standard two-dose vaccination regimen in cholera outbreak response campaigns in favor of a single-dose strategy that may allow the doses to be used in more countries, during a period of unprecedented increase in cholera outbreaks globally. Despite the paucity of data on the precise period of protection and the fact that protection seems to be substantially lower in children, the one-dose approach has been successful in controlling outbreaks. Despite the temporary interruption of the two-dose strategy causing a decrease and shortening of immunity, the advantage of administering one dosage still trumps administering none.¹⁸⁶ A recent modeling study demonstrated that rapid vaccination campaigns with a single dose of OCV are likely to prevent more cases and fatalities than a two-dose pro-rata campaign in an epidemic environment with a limited vaccine supply.¹⁸⁷

The COVID-19 pandemic caused collateral damage by affecting the production of Vaxchora. Due to a significant decline in international travel as a result of the COVID-19 pandemic, the vaccine manufacturer informed the FDA in May 2021 that they had decided to temporarily stop the production and distribution of Vaxchora in the United States.¹⁸⁸ In addition, Shantha Biotechnics announced that the production of Shanchol, which comprises 15% of the global stockpile, would stop in 2022, and the distribution by the end of 2023.^{189, 190}

A licensing and technology transfer agreement has been signed by the South African biopharmaceutical company Biovac and the IVI for the development and production of OCVs. This will help enable Biovac to create the antigen/raw materials needed for manufacturing vaccines. The goal of this collaboration is to expand the production rates to alleviate the severe worldwide vaccine shortage required to prevent cholera. This knowledge transfer will also create and show off capability for good manufacturing process scale-up, local manufacturing of clinical trial products, and end-to-end vaccine production in Africa as the production of the first trial batches is scheduled to begin in 2024.¹⁹¹

9 MANAGEMENT, ANTIMICROBIAL DRUG RESISTANCE, AND PROMISING CHOLERA TREATMENTS

Most cholera cases will be treated with oral rehydration therapy, which will be used to correct fluid and electrolyte imbalances¹⁹² (Figure 4). The primary treatment for cholera is rehydration therapy. Antibiotic therapy is also recommended for patients with severe illness and high-risk patients, such as those who are immunocompromised, pregnant, or have comorbid conditions (Table 5).^193-195

10 CONCLUSION

V. cholerae is a complex bacterium that can persist in both human host populations and aquatic environments. In humans, the bacteria can cause severe diarrhea and death. The Middle East and Africa have suffered the brunt of the recent outbreaks of cholera and the purpose of this study was to review the causes and potential solutions for cholera in this region. The study's narrow temporal and geographical focus on cholera outbreaks in Africa and the Middle East may not provide a comprehensive understanding of cholera dynamics globally and limits the generalizability of findings. The impact of COVID-19 likely exacerbated local outbreaks of cholera in Africa and the Middle East as well as led to an undercounting of the number of cases. Going forward there are headwinds and promises toward combating cholera in Africa and the Middle East. Poverty, inadequate sanitation, inadequate water supply, overpopulation, and conflict continue to create environments susceptible to cholera. Conflicts in nations such as Syria, Ethiopia, and Sudan have led to crowded living conditions with reduced access to clean drinking water and cholera treatment. Climate change threatens to exacerbate cholera conditions by increasing the occurrence of flooding and drought in the region.

RDTs that do not rely on expensive laboratory materials will help detect outbreaks sooner, leading to more rapid population-level interventions. Vaccination efforts have been hindered by reduced production and increased demand for vaccines. Eubiologics' Euvichol/Euvichol-Plus will be increasingly relied upon as a relatively inexpensive and effective vaccine for Middle Eastern and African nations. As Africa and the Middle East emerge from the COVID-19 pandemic, renewed efforts will need to be made to address the increasing threat of cholera. Prevention and treatment strategies will likely require a multi-pronged combination of prevention and treatment strategies.

AUTHOR CONTRIBUTIONS

Abdulrahman K. Ahmed: Conceptualization; writing—original draft; validation; data curation. Victor Coll Sijercic: Investigation; writing—original draft; visualization; writing—review and editing; resources. Mahad S. Akhtar: Writing—original draft; methodology; visualization; software. Ahmed Elbayomy: Conceptualization; investigation; resources; data curation; formal analysis. Mohamed A. Marouf: Investigation; writing—original draft; data curation; methodology. Mahlet S. Zeleke: Conceptualization; writing—original draft; project administration; data curation; validation. Reem Sayad: Investigation; writing—original draft; formal analysis. Abdelrahman Abdelshafi: Writing—original draft; resources. Nicholas J. Laird: Writing—original draft; investigation; formal analysis; data curation. Mohamed A. El-Mokhtar: Supervision; project administration; writing—original draft. Gregory R. Ruthig: Conceptualization; writing—original draft; writing—review and editing; visualization; supervision; project administration. Helal F. Hetta: Writing—original draft; project administration; supervision.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

TRANSPARENCY STATEMENT

The lead author Mahlet S. Zeleke affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned (and, if relevant, registered) have been explained.