Exploratory Research and Hypothesis in Medicine

eISSN 2472-0712 A Biomedical Open Access Journal

EXPLORATORY RESEARCH AND HYPOTHESIS IN MEDICINE

Volume 5 Issue 1, March 2020

XIA & HE PUBLISHING INC. EXPLORATORY RESEARCH AND HYPOTHESIS IN MEDICINE

CONTENTS 2020 5(1):1–38

Editorials The Coronavirus Disease 2019 Epidemic Situation in China Jin Wang, Zhihui Li, Jiahai Lu ...... 1 The Ongoing Outbreak and Challenges of Novel Coronavirus (COVID-19) in China Bohao Chen ...... 3

Opinions Blind Spots in Fighting the Outbreak of Coronavirus Disease 2019 Lanjing Zhang ...... 6. . . . . Characteristics of COVID-19 During the Onset Stage and Considerations for Disease Control Lili Wang ...... 8 . . . . .

Original Article Echocardiography Effectiveness in Improving Diagnosis of Rheumatic Heart Disease in North Dar- fur: A Hospital-based Study Mohammed Elmujtba Adam Essa Adam, Sherihan Mohammed Elkundi Osman, Daralsalam Ishag Ateem Abdalrasoul, Ibrahim Adam Osman Yagoup, Mustafa Mohamed Ali Hussein, Mutwaly Defealla Yousif Haron, Ziryab Imad Taha Mahmoud, Abdelkareem A . Ahmed ...... 11

Review Articles Microbial Biomarkers for Colorectal Cancer Identified with Random Forest Model Weili Sun, Lili Wang, Qiuyue Zhang, Quanjiang Dong ...... 19 The Effect of Honey as a Treatment for Oral Ulcerative Lesions: A Systematic Review Maddison Hunter, Jane Kellett, Nathan M . D’Cunha, Kellie Toohey, Andrew McKune, Nenad Naumovski . . . .27

Corrigendum Corrigendum: Acute Soft Skull Syndrome in an Adult Male with Sickle Cell Anemia in Sudan: A Case Report Ziryab Imad Taha, Sulafa Eisa Mohammed, Mohammed Elmujtba Adam Essa, Walaa Mohamed Elsid, Mustafa Mohamed Ali Hussein, Sherihan Mohammed Elkundi Osman, Hussein Osman Ahmed, Mutwaly Defealla Yousif, Abdelkareem A . Ahmed ...... 38 Editorial

The Coronavirus Disease 2019 Epidemic Situation in China

Jin Wang, Zhihui Li and Jiahai Lu*

Department of Epidemiology, School of Public Health, Sun Yat-sen University, Guangzhou, Guangdong Province, 510080, China

It has been 17 years since the outbreak of severe acute respiratory is a β CoV of group 2B, sharing 79.5% of genetic sequence with syndrome (SARS) in 2003, which causes at least 8096 infections SARS-CoV and has 96.2% homology to a bat coronavirus.9,10 and 774 deaths worldwide. History repeats itself! In Dec 2019, However, the possibility of direct transmission from bats to hu- China reported to the World Health Organization (WHO) a cluster mans is small, and an intermediate host is often needed to mediate of patients with pneumonia of unknown etiology in Wuhan, a ma- zoonoses from natural reservoir to terminal hosts. It is speculated jor hub of transportation with a population of 11 million people. that SARS-CoV-2 may come from a certain wild animal and spread On Jan 9, 2020, a novel coronavirus (officially named as SARS- rapidly in the Huanan Seafood Wholesale Market in Wuhan, where CoV-2 on Feb 11) was identified as the cause of this outbreak. the viruses were originally isolated. Homology modelling analy- The number of infected cases increased sharply, and the epidemic sis showed structural similarity between the receptor-binding do- rapidly spread throughout the country. The WHO on Jan 30 de- mains of SARS-CoV and SARS-CoV-2 to the human angiotensin- clared the outbreak a Public Health Emergency of International converting enzyme-2 (ACE-2) receptor.10 SARS-CoV-2 has been Concern. By Feb 15, 2020, the cumulative number of confirmed considered to link to game consumption, a habit obsessive by some cases had reached 66,577, far exceeding the number of SARS Chinese people.11 After the outbreak, the Chinese Government cases in 2003. According to the released news, the case rate fatal- has closed the Huanan Seafood Wholesale Market in Wuhan and ity is 2.3% (1,524/66,577). Geographically, the disease has spread banned all forms of wild animal transaction. beyond China to over 30 countries, with Japan having the most Since the outbreak, geographical spread of the disease raises confirmed cases outside China. grave concerns around the world. The Chinese health authorities The SARS-CoV-2 is mainly transmitted through respiratory have taken unprecedented measures to control the source of infec- droplets. After infection, patients showed lymphopenia and bilat- tion.12 From Jan 23, Chinese authorities have imposed travel bans eral ground-glass opacity or consolidation in chest CT scans, along on Wuhan and several cities near Wuhan. Individuals are popular- with common symptoms that include fever, dry cough, and short- ized the knowledge to take self-protection measures, such as good ness of breath, at the onset of illness.1,2 In severe cases, dyspnea, personal hygiene, fitted masks, ventilation and avoiding crowded respiratory distress syndrome or septic shock may develop.2 Early places. There is likelihood that some mild or asymptomatic pa- observation of infections of health-care workers as well as fam- tients do not seek health care but as a source transmitting the virus ily members has suggested that human-to-human transmission has to other humans, which may complicate or delay the effectiveness occurred among close contacts.3,4 The epidemic doubled in size of infection-control measures.13 At present, there are no specific every 6.4–7.4 days in its early stage, with the basic reproductive antivirals or vaccines although clinical trial of Lopinavir/ritonavir 3,5,6 number (R0) estimated to be 2.2–2.68. has been launched for SARS-CoV-2 (ChiCTR2000029308), and Coronavirus is an enveloped, positive-strand RNA virus, be- the management of infection is largely supportive. Thus, molecular longing to Coronavirinae subfamily within Coronaviridae family. diagnostic platforms for rapidly identifying SARS-CoV-2 as well The coronaviruses can infect human, livestock, avian, bat, mouse as effective vaccine strategies are urgently needed to develop. and other wild animals. They can cause respiratory, gastrointesti- In this century, the coronaviruses attacked China twice, each nal, hepatic and central nervous system diseases of varying sever- time causing public panic, heavy deaths and huge economic losses. ity, sometimes fatal. The two types of coronaviruses that we are It seems that people have not yet established a rapid, effective re- currently familiar with are SARS-CoV in 2003 and Middle East sponse to public health threats. Although scientific research on the respiratory syndrome coronavirus (MERS-CoV) broken out in etiology and structural features of unknown pathogens is impor- 2012. The SARS-CoV-2 is the seventh known coronavirus that can tant, it is more practical to establish a strong and effective public infect humans.7 This virus seems to have greater infectivity (e.g., a health system that can assure the nation's health and safety. The higher R0) but a lower case fatality rate.8 investment in core public-health systems and infrastructure will Bats might be the original host of this virus.9 The SARS-CoV-2 no doubt be critical for preventing or controlling this kind of bios- ecurity. Local health departments need to strengthen their ability to mount an effective health emergency response, including iden- Abbreviations: ACE-2, angiotensin-converting enzyme-2; CoV, coronavirus; COV- tification of suspicious cases as well as individuals with high-risk ID-19, coronavirus disease 2019; CT, computed tomography; MERS-CoV, Middle East respiratory syndrome coronavirus; SARS, severe acute respiratory syndrome; exposures at the early stage, even in a sudden outbreak of unknown WHO, World Health Organization. pathogens. Coordination and collaboration among surveillance Received: February 17, 2020; Revised: March 03, 2020; Accepted: March 04, 2020 departments, health sectors and laboratories are particularly im- *Correspondence to: Jiahai Lu, Department of Epidemiology, School of Public portant. In addition, both MERS and SARS nosocomial outbreaks Health, Sun Yat-sen University, Guangzhou, Guangdong Province, 510080, China. are characterized by early nosocomial super-spreading events. Al- Tel: +86-20 87332438, E-mail: [email protected] though so far no evidence indicates a super transmission event in How to cite this article: Wang J, Li Z, Lu J. The Coronavirus Disease 2019 Epidemic Situation in China. Exploratory Research and Hypothesis in Medicine 2020;5(1):1–2. the COVID-19 outbreak, the situation of nosocomial infection is doi: 10.14218/ERHM.2020.00009. still serious. As of Feb 11, 1,716 medical staffs had been infected

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2 DOI: 10.14218/ERHM.2020.00009 | Volume 5 Issue 1, March 2020 Editorial

The Ongoing Outbreak and Challenges of Novel Coronavirus (COVID-19) in China

Bohao Chen*

Section of Pulmonary and Critical Care Medicine, Department of Medicine, University of Chicago, Chicago, IL, USA

In December 2019, a cluster of unexplained cases of pneumonia two infect avian species. The genus Alphacoronavirus contains the was first reported by the Health Commission of Hubei Province, human viruses HCoV-229E, HCoV-NL63, and many other animal China. Most of cases were epidemiologically linked to a seafood viruses. The genus Betacoronavirus includes the human viruses and wet animal wholesale market in Wuhan, Hubei Province, HCoV-OC43 and HCoV-HKU1 along with a number of animal China, which was officially closed on January 1, 1 2020. As of coronaviruses. Some coronaviruses that infect animals can evolve January 3, the national authorities in China had reported a total to acquire efficient human transmission, making people sick and of 44 patients with pneumonia to WHO. On January 8, virologists becoming a new human coronavirus, such as SARS-CoV, MERS- and other public health investigators identified a new coronavirus, CoV, and now COVID-19.4 In the 2003 SARS pandemic, investi- now known as COVID-19, as the cause of the pneumonia clusters gators found SARS-CoV viral RNA in both palm civets and rac- and posted the genetic sequence of COVID-19 on January 12.2 coon dogs in wet markets. It is very likely that SARS-CoV was The clusters of new cases among families and the infection of 16 transmitted from bats to those animals which served as interme- healthcare professionals indicate human-to-human transmission diary reservoirs and then finally infected humans.5 MERS-CoV of the virus.3 Chinese public health authorities have quarantined is also a zoonotic virus with possible origins in bats and camels. travel from Wuhan to limit the spread of the virus since January The ongoing outbreak of COVID-19 is associated with the Wuhan 23, and since then, more Chinese cities have also been isolated. Huanan Seafood Market where snakes, birds, marmots, pangolin, As new cases and death toll from COVID-19 in China escalate and other small mammals were sold, suggesting animal-to-person rapidly and with COVID-19 spreading to other countries, WHO spread. However, as the outbreak progressed, most cases were con- Director-General Tedros Adhanom Ghebreyesus declared the firmed without exposure to animal markets, and cases were identi- COVID-19 outbreak a public health emergency of international fied among healthcare professionals and other contacts of patients concern on January 30, noting the potential spread of the virus to with COVID-19 infections, indicating either human to human countries with weak health systems. The Trump administration transmission or a more widespread animal source.3 COVID-19 is announced on January 31 that it would temporarily bar foreigners highly contagious and the current epidemic has become a serious from entering the US if they had been to China within the past 14 public health threat to China as well as to other countries. It re- days. Compared to prior SARS-CoV in 2003 and MERS-CoV in quires governments to take emergency and mandatory measures 2013, the ongoing outbreak of COVID-19 presents an urgent and to treat the patients, isolate the suspicious cases, protect healthcare huge health threat since it is far more transmittable and far more professionals, track the spread of the virus, and advise individuals damaging economically and socially. Therefore, governments for public health. and partners should take immediate actions in response to the So far, the source of COVID-19 has not been identified yet. COVID-19 outbreak and bring the scientific community together Since the sequencing of COVID-19 genome by Dr. Zhang’s group to accelerate research and innovation against the global epidemic at Fudan University was published on January 12, 44 COVID-19 of COVID-19. sequences have been deposited in the NCBI Virus website. Ge- Coronaviruses are single-stranded positive-sense RNA viruses nome comparison of all these strains found that they are almost and classified into four main sub-groupings, known as alpha, beta, identical with >99% sequence conservation. The low variability gamma, and delta, based on antigenic relationships and viral ge- suggests a likely single emergence of epidemic viruses from a netic phylogeny. The former two genera primarily infect the res- common animal reservoir. The COVID-19 genome (MN908947.3) piratory and gastrointestinal tract of mammals, whereas the latter shows similarities to Bat SARS-like coronavirus (CoVZC45) with sequence identity of 89.12% and to Bat SARS-like coronavirus (CoVZXC21) with sequence identity of 88.68%. However, COV- Abbreviations: ACE2, angiotensin-converting enzyme 2; COVID-19, novel corona- ID-19 is genetically distinct from the human SARS genome with virus; FDA, Food and Drug Administration; HCoV, human coronavirus; MERS-CoV, 82.3% sequence identity, supporting the claim that human COV- Middle East Respiratory Syndrome Coronavirus; RBD, receptor binding domain; ID-19 is a zoonotic virus with possible origins from bats to hu- SARS-CoV, Severe Acute Respiratory Syndrome Coronavirus; WHO, World Health man.6 However, no bats were sold at the Huanan seafood market, Organization. Received: February 17, 2020; Revised: February 21, 2020; Accepted: February 22, suggesting that another yet-to-be-identified animal acted as an in- 2020 termediate to transmit the virus to humans. It is urgently requested *Correspondence to: Bohao Chen, Section of Pulmonary and Critical Care Medi- that researchers clarify the source of 2019-nCoV including pos- cine, Department of Medicine, University of Chicago, 5841 S. Maryland Ave., Chi- sible intermediate animal vectors in order to end the COVID-19 cago, IL 60637, USA. Tel: 001-773-834-7476, Fax: 001-773-702-4736, spread. E-mail: [email protected] How to cite this article: Chen B. The Ongoing Outbreak and Challenges of Novel Coronaviruses encode five structural proteins in their genomes. Coronavirus (COVID-19) in China. Exploratory Research and Hypothesis in Medi- These are the spike surface glycoprotein (S), membrane protein cine 2020;5(1):3–5. doi: 10.14218/ERHM.2020.00008. (M), nucleocapsid protein (N), small envelope protein (E), and

Exploratory Research and Hypothesis in Medicine 2020 vol. 5 | 3–5

Hemagglutinin Esterase protein (HE). All envelope proteins and peutics, including antibodies, nucleoside analogues, and protease N protein are present in all virions, but HE is only present in some inhibitors for repurposing under FDA Emergency Use Authori- beta coronaviruses. The spike surface glycoprotein plays an essen- zation. Chinese experts are considering a flexible design to offer tial role in engaging with the host cell receptor ACE2 and mediat- investigational therapeutics to those infected with COVID-19 and ing fusion between the viral and host cell membranes. The align- evaluate the safety and efficacy of these drugs on an emergency ment analysis found S protein of COVID-19 shares 97.41% of basis. The government and the private sectors should provide amino acid identity with Bat coronavirus (QHR63300.1), 80.32% more financial assistance for the scientific community to design or with Bat SARS-like coronavirus (AVP78042.1), and 76.12% with develop novel and specific antiviral countermeasures for emerg- SARS coronavirus (PC4-137 AAV49720.1). The modeling and ing coronaviruses. subsequent reports provide strong evidence that the S protein of The COVID-19 epidemic is still going on in China and is COVID-19 has sufficient affinity with the SARS ACE2 receptor far from over. As of February 15, China had reported more than to infect bronchial epithelial cells and type II pneumocytes.7 How- 68,594 confirmed cases, 1,716 of which are medical profession- ever, there is much more to learn about the association of affinity als. The death toll from COVID-19 had risen to 1,667 with 11,272 of the ACE2 receptor with the transmissibility and pathogenesis of critically ill patients. Under the transportation shutdown of Wuhan COVID-19, and more investigations will need to be done to block City and severe shortages of medical resources, Chinese authori- the virus invasion. ties face daunting challenges to provide appropriate medical treat- For SARS-CoV, the S protein is the main antigenic component ment for more than 68,000 patients, protect thousands of health- that is responsible for inducing host immune responses, neutraliz- care professionals from nosocomial infection, quarantine millions ing antibodies and/or protective immunity against virus infection. of people, and deal with the socioeconomic impacts of the epi- S protein has therefore been selected as a major target for vaccine demic. This problem requires international engagement and coop- development and anti-viral therapy. Such efforts include using full- eration against possible COVID-19 global pandemic. length (Novavax, Phase III) or recombinant S protein (Vaxine Pty Ltd, Australia, Phase I) to induce effective neutralizing-antibodies and protective immunity, RBD-ACE2 blockers to block RBD- Acknowledgments ACE2 binding and S protein-mediated infection, small interfering RNAs to reduce virus replication and/or silence S gene expression, inactivated or live attenuated vaccine, etc.8 Although the feasibil- The author thanks all investigators and clinicians who have con- ity of using the above approaches is partially limited by their low tributed to our understanding of COVID-19 infections. antiviral potency, studies and clinical trials on the SARS vaccine provide important information for designing novel strategies for Funding prophylaxis and therapies against emerging infections caused by COVID-19.7 We urge the government and private industry to grant additional funding to advance anti-coronavirus vaccines into clini- None to declare. cal trials. The symptoms and signs of COVID-19 infection are recently described in multiple clinical observations and include fe- Conflict of interest ver, cough, nasal congestion, fatigue, dyspnea, and significant changes visible through chest X-rays and computer tomography The author has no conflict of interests related to this publication. techniques (ground glass abnormalities, patchy consolidation, al- veolar exudates, and interlobular involvement). Cases with gastrointestinal symptoms and asymptomatic infections have also References been reported recently, especially among young children.9 Sta- tistical analysis showed that the highest incidence of COVID-19 [1] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of pa- was observed among adults older than 50 years, with the lowest tients infected with 2019 novel coronavirus in Wuhan, China. Lancet incidence in the age group younger than 20 years. Furthermore, 2020;395(10223):497–506. doi:10.1016/S0140-6736(20)30183-5. males experienced a higher incidence than females. The deaths [2] Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A Novel Coro- also were concentrated in male patients over 60 years of age with navirus from Patients with Pneumonia in China, 2019. N Engl J Med severe pneumonia at diagnosis.10 It is unclear yet why some in- 2020;382(8):727–733. doi:10.1056/NEJMoa2001017. fections have no symptoms while others show severe pneumonia [3] Phan LT, Nguyen TV, Luong QC, Nguyen TV, Nguyen HT, Le HQ, et al. and even death. More research should be conducted on the inter- Importation and Human-to-Human Transmission of a Novel Corona- virus in Vietnam. N Engl J Med 2020;382(9):872–874. doi:10.1056/ actions among viral virulence determinants, the density and the NEJMc2001272. affinity of ACE2 receptor on cell surface, and the host’s immune [4] Wang Q, Qi J, Yuan Y, Xuan Y, Han P, Wan Y, et al. Bat origins of response. MERS-CoV supported by bat coronavirus HKU4 usage of human re- Clinical treatment of infections with COVID-19 is still based ceptor CD26. Cell Host Microbe 2014;16(3):328–337. doi:10.1016/j. on supportive care, like oxygen therapy and fluid management, chom.2014.08.009. which has proven to be very effective. Encouraging preliminary [5] Kan B, Wang M, Jing H, Xu H, Jiang X, Yan M, et al. Molecular evolu- trials indicate that infusions of blood plasma from people who tion analysis and geographic investigation of severe acute respiratory have recovered from COVID-19 could help the critically ill pa- syndrome coronavirus-like virus in palm civets at an animal mar- tients improve clinical symptoms. Although antiviral drugs have ket and on farms. J Virol 2005;79(18):11892–11900. doi:10.1128/ JVI.79.18.11892-11900.2005. made great progress in recent years, currently there are no thera- [6] Gralinski LE, Menachery VD. Return of the Coronavirus: 2019-nCoV. peutic agents licensed and available for COVID-19. In theory, Viruses 2020;12(2):E135. doi:10.3390/v12020135. some anti-SARS-CoV and anti-Ebola virus drugs in clinical trials [7] Wan Y, Shang J, Graham R, Baric RS, Li F. Receptor recognition by should have activity against COVID-19. In the current outbreak novel coronavirus from Wuhan: An analysis based on decade-long of COVID-19, the WHO prioritized a panel of candidate thera- structural studies of SARS. J Virol 2020;JVI.00127-20. Epub 2020 Jan

4 DOI: 10.14218/ERHM.2020.00008 | Volume 5 Issue 1, March 2020 Chen B.: Outbreak and challenges of COVID-19 in China Explor Res Hypothesis Med

29. doi:10.1128/JVI.00127-20. Wuhan, China. Radiology 2020;200269. Epub Feb 7. doi:10.1148/ra- [8] Du L, He Y, Zhou Y, Liu S, Zheng BJ, Jiang S. The spike protein of SARS- diol.2020200269. CoV—a target for vaccine and therapeutic development. Nat Rev Mi- [10] Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, et al. Epidemiologi- crobiol 2009;7(3):226–236. doi:10.1038/nrmicro2090. cal and clinical characteristics of 99 cases of 2019 novel corona- [9] Shi H, Han X, Zheng C. Evolution of CT Manifestations in a Patient virus pneumonia in Wuhan, China: a descriptive study. Lancet Recovered from 2019 Novel Coronavirus (2019-nCoV) Pneumonia in 2020;395(10223):507–513. doi:10.1016/S0140-6736(20)30211-7.

DOI: 10.14218/ERHM.2020.00008 | Volume 5 Issue 1, March 2020 5 Opinion

Blind Spots in Fighting the Outbreak of Coronavirus Disease 2019

Lanjing Zhang*

Department of Pathology, Princeton Medical Center, Plainsboro, NJ, USA; Department of Biological Sciences, Rutgers University, Newark, NJ, USA; Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA; Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers University, Piscataway, NJ, USA

Abstract

The recent outbreak of 2019 novel coronavirus infection has involved more than 110,000 people and 105 countries. Many efforts have been made to prevent, contain and treat the related disease (named as coronavirus disease 2019). However, many blind spots might not yet receive needed attention. I here discuss eight blind spots that may interest related parties. If these issues remain outstanding, they will likely lead to many severe harms to the public, healthcare providers and the economy. Additional research is therefore needed to better understand and address these blind spots in fighting the outbreak of coronavirus disease 2019.

Recently, the outbreak of 2019 novel coronavirus infection in- lance, prevention and control of COVID-19 on the global scale. volved more than 113,702 people and 109 countries in the word, Several key questions have been asked regarding the patho- and has led to a public health emergency of international concern.1,2 genicity and transmissibility characteristics of COVID-19.11 Many Many efforts were rightly and timely made to fight the outbreak of works have been focused on the trends and characteristics of the 2019 novel coronavirus in China, which was recently named as disease/viral infection.8,12,13 However, several blind spots in my Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) view are noteworthy during the fight against the outbreak of COV- by the Coronavirus Study Group,3 and Coronavirus disease 2019 ID-19. If not prevented or insufficiently prepared for, those blind (COVID-19) by the World Health Organization (WHO).4 A group spots might cause unexpected long-lasting harms, and consume of virologists in China also proposed to name the virus as human significant amount of resources. coronavirus 2019 (HCoV-19).5 Despite the differences in name, Eight issues might be of our particular interest and concern: we observed orchestral actions from governments, health care pro- 1. Distrust and isolated actions among (potentially) involved viders, non-profit organizations and individual citizens in China countries. Due to reasonable worries about the international and the other part of the world. As a result, the daily incidence of spread of the COVID-19, there is significant disruption of inter- infection has been decreasing since February 1, 2020 according to national trades, academic/educational exchanges and political the official websites6,7 and a trend analysis on the epidemiology collaborations.14,15 Such disruption may escalate in the near fu- of COVID-19.8,9 On the other hand, the rising incidence in Japan, ture if the spread of COVID-19 continues involving more coun- Korea and Iran is alarming.2 A modelling study also shows that tries. However, it is probably also the time when international Africa might be vulnerable to the pandemic of COVID-19.10 In- collaborations are urgently and most needed. The hard-learned deed, there were 10,566 COVID-19 cases in WHO member states lessons and practical experiences in China in my view are of outside of China on March 3, 2020, which was developed within ultimate values in fighting this novel virus around the world. the past 2 weeks.2 On March 3 alone, 8 new member states of the Probably more importantly, proper coordination among all (in- WHO (namely Andorra, Jordan, Latvia, Morocco, Portugal, Saudi volved) countries should be encouraged and could result in bet- Arabia, Senegal, and Tunisia) reported their first confirmed new ter utilization of available resources at the global scale. case.2 The daily incidence of COVID-19 outside of China has been 2. Performance of the diagnostic tests. We would need tests like HIV tests, which include a screening test of high sensitivity and higher than that in China since February 26, 2020 (459 versus 412). 16,17 Thus, we should continue our undivided attention on the surveil- a confirmatory test of high specificity. 3. Changes and updates of the diagnostic criteria might be necessary as we better understand the dynamic and characteristics of Keywords: COVID-19; Viral infection; Epidemic; Coronavirus; Epidemiology. COVID-19. But these changes and update should be kept as few Abbreviations: COVID-19, coronavirus disease 2019; SARS-CoV-2, severe acute as possible for the continuity of disease surveillance, and con- respiratory syndrome coronavirus 2; WHO, World Health Organization. sistency in treatment protocols and outcome. One recent change Received: February 26, 2020; Revised: March 04, 2020; Accepted: March 05, 2020 of including clinically diagnosed cases in confirmed cases led *Correspondence to: Lanjing Zhang, Department of Pathology, Princeton Medical to a significant increase in daily incidence of COVID-19 in Wu- Center, Plainsboro, NJ, USA. Tel: +1-609-853-6833, Fax: +1-609-853-6841, han City and Hubei Province.6,7,9 It caused much confusion and E-mail: [email protected] anxiety among the health care providers and the public. How to cite this article: Zhang L. Blind Spots in Fighting the Outbreak of Coronavi- rus Disease 2019. Exploratory Research and Hypothesis in Medicine 2020;5(1):6–7. 4. Cautions in rushing into approving drugs for 2019-nCoV. We doi: 10.14218/ERHM.2020.00012. should follow the preset stand protocols in assessing and approv-

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ing new drugs. The due diligence must not be jeopardized despite References the urgent needs for these drugs because an approval of such drug will have long-lasting and profound effects in the world. [1] Zarocostas J. What next for the coronavirus response? Lancet 5. The care for non-infectious diseases may be deprived of needed 2020;395(10222):401. doi:10.1016/S0140-6736(20)30292-0. resources. Largely due to lack of manpower and resources, one [2] WHO. Coronavirus disease (COVID-2019) situation reports. Available should anticipate and prepare for a significant rise in the deaths from: https://www.who.int/emergencies/diseases/novel-coronavi- due to cardiovascular, pulmonary and other chronic diseases. rus-2019/situation-reports/. Accessed Feb. 26, 2020. 6. Psychologic issues of the general public, patients, patient-fami- [3] Gorbalenya AE, Baker SC, Baric RS, de Groot RJ, Drosten C, Gulyaeva lies, local officials and healthcare providers. A recent comment AA, et al. Severe acute respiratory syndrome-related coronavirus: 18 The species and its viruses – a statement of the Coronavirus Study rightfully raise such a concern. If not timely and properly Group. bioRxiv 2020. doi:10.1101/2020.02.07.937862. addressed, such a psychological issue will exhaust healthcare [4] WHO. Coronavirus disease (COVID-19) outbreak. Available from: htt- manpower, decrease morale and cause long-term psychological ps://www.who.int/emergencies/diseases/novel-coronavirus-2019. diseases among impacted individuals including patients, fami- Accessed February 23, 2020. lies and health care providers. [5] Jiang S, Shi Z, Shu Y, Song J, Gao GF, Tan W, et al. A distinct name is 7. Documentation of related policies, implemented approaches needed for the new coronavirus. Lancet 2020. doi:10.1016/s0140- and observed outcomes. This practice will be critical for track- 6736(20)30419-0. ing records, learning the lessons, enforce accountability and [6] Wuhan Municipal Health Commission. Briefings on the viral disease improving efficiency. [Chinese]. Available from: http://wjw.wuhan.gov.cn/front/web/lis- t3rd/no/802. Accessed Feb. 23, 2020. 8. Information overload and errors in reporting. It is inevitable that [7] National Health Commission of China. Briefings on the viral outbreak the conventional media and social media will pour unprecedent [Chinese]. Available from: http://www.nhc.gov.cn/xcs/yqtb/list_gzbd. amount of information to the public, who are eager to better shtml. Accessed Feb. 24, 2020. understand the disease and current situation. The important and [8] Xu J, Cheng Y, Yuan X, Li WV, Zhang L. Trends in epidemiology of 2019 accountable information, however, might be buried in the infor- novel coronavirus. Available from: https://github.com/thezhanglab/ mation overload. Related parties should proactively reach out coronavirus. Accessed Feb. 23, 2020. to the public to “advertise” credible information through new [9] Xu J, Cheng Y, Yuan X, Li WV, Zhang L. Trends and prediction in daily media. Errors in reporting or rumors are very difficult to iden- incidence of novel coronavirus infection in China, Hubei Province and Wuhan City: an application of Farr law. medRxiv 2020:20025148. tify and handle since they could be proven wrong only upon doi:10.1101/2020.02.19.20025148. rigorous, long and time-consuming validation studies. [10] Gilbert M, Pullano G, Pinotti F, Valdano E, Poletto C, Boëlle PY, et al. Another question of significant consequences is how to prop- Preparedness and vulnerability of African countries against impor- erly deal with the aftermath of the COVID-19 outbreak. Hopefully, tations of COVID-19: a modelling study. Lancet 2020. doi:10.1016/ the end of the COVID-19 outbreak will soon come. Many issues S0140-6736(20)30411-6. will emerge. Thus, experts in public health, socioeconomical, geo- [11] Munster VJ, Koopmans M, van Doremalen N, van Riel D, de Wit E. political and biomedical studies should work together to proac- A Novel Coronavirus Emerging in China - Key Questions for Impact tively articulate the related issues, discuss possible solutions and Assessment. N Engl J Med 2020;382(8):692–694. doi:10.1056/NE- formulate feasible, yet impactful, action plans. JMp2000929. [12] Zhang R, Liu H, Li F, Zhang B, Liu Q, Li X, et al. Transmission and epidemi- In summary, the eight blind spots may interest related parties. ological characteristics of Novel Coronavirus (2019-nCoV) Pneumonia If these issues remain outstanding, they will likely lead to many (NCP): preliminary evidence obtained in comparison with 2003-SARS. severe harms to the public, healthcare providers and the economy. medRxiv 2020:20019836(v3). doi:10.1101/2020.01.30.20019836. Additional research is therefore needed to better understand and [13] Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, et al. Early Transmission address these blind spots in fighting the outbreak of coronavirus Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumo- disease 2019. nia. N Engl J Med 2020. doi:10.1056/NEJMoa2001316. [14] DHS. 2020 Travel/Visa Restrictions. Available from: https://www.dhs. gov/news/2020/01/31/2020-travelvisa-restrictions. Accessed March Acknowledgments 3, 2020. [15] Government UK. Policy paper: Coronavirus action plan: a guide to what you can expect across the UK. Available from: https://www.gov. The opinions expressed here are solely those of the author, and not uk/government/publications/coronavirus-action-plan/coronavirus- of his affiliations. action-plan-a-guide-to-what-you-can-expect-across-the-uk. Accessed March 3, 2020. [16] Branson BM. HIV Diagnostics: Current Recommendations and Oppor- Funding tunities for Improvement. Infect Dis Clin North Am 2019;33(3):611– 628. doi:10.1016/j.idc.2019.04.001. [17] Chen DJ, Yao JD. Comparison of turnaround time and total cost of None. HIV testing before and after implementation of the 2014 CDC/APHL Laboratory Testing Algorithm for diagnosis of HIV infection. J Clin Vi- rol 2017;91:69–72. doi:10.1016/j.jcv.2017.04.004. Conflict of interest [18] Xiang YT, Yang Y, Li W, Zhang L, Zhang Q, Cheung T, et al. Timely men- tal health care for the 2019 novel coronavirus outbreak is urgently needed. Lancet Psychiatry 2020;7(3):228–229. doi:10.1016/S2215- The author has no conflict of interest related to this publication. 0366(20)30046-8.

DOI: 10.14218/ERHM.2020.00012 | Volume 5 Issue 1, March 2020 7 Opinion

Characteristics of COVID-19 During the Onset Stage and Considerations for Disease Control

Lili Wang*

Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA

Abstract

COVID-19 outbreak in December 2019 has evolved into a world level pandemic. To cope with an emerging pathogen like SARS-CoV-2, there is no vaccine or specific drug available to treat the disease. However, get to know the characters of the onset stage of the new disease may help us to control the spread of COVID-19. This short article aims to introduce COVID-19 briefly, getting you to know the clinical character, the treatment, public procedurals and researches on COVID-19 quickly but comprehensively.

The first pneumonia case of unknown origins was identified in Epidemiological characteristics Wuhan, China in early December 2019.1 The symptoms of it re- minded people of severe acute respiratory syndrome coronavirus According to the first published descriptive study based on 99 pa- (SARS-CoV), which had shocked China 17 years ago in 2003. tients from Jinyintan Hospital in Wuhan,4 49% of the 2019-nCoV High-throughput sequencing confirmed the new virus is the sev- infected patients had a history of exposure to Huanan Seafood enth member of enveloped RNA coronavirus (subgenus sarbeco- Wholesale Market. 2019-nCoV infection was of clustering onset, virus, Orthocoronavirinae subfamily). Currently, the new virus was more likely to affect older men with comorbidities, and could was named as severe acute respiratory syndrome coronavirus 2, result in severe and even fatal respiratory diseases such as acute SARS-CoV-2), which was also called 2019 novel coronavirus respiratory distress syndrome (ARDS). A later study published on (2019-nCoV) earlier. And the disease caused by it was named cor- Jan 29th with a sample size of 425 cases indicates the majority of onavirus disease in 2019 (COVID-19).2,3 Until now, COVID-19 cases (55%) with onset before Jan 1st, 2020 were linked to the Hua- outbreaks emerged in Wuhan have caused more than 80,000 con- nan Seafood wholesale Market, while after that date only 8.6%. firmed infections; including more than 2,800 deaths (December The mean incubation period was 5.2 days (95% confidence inter- 1st, 2019-Feb 29th 2020, 3 months). On January 30, 2020, the val, 4.1 to7.0) and the basic reproductive number was estimated to International Health Regulations Emergency Committee of the be 2.2 (95% confidence interval, 1.4 to 3.9).5 These data indicate World Health Organization declared the outbreak a “public health human-to-human transmission has occurred among close contacts emergency of international concern”. To cope with an emerging since the middle of December 2019. pathogen with serious outcomes, the first thing to do is get to know the enemy. Scientists and doctors in the frontier responded quickly to this emergency and provided valuable epidemiological and Clinical characteristics clinical data sharing with the rest of the world before its further spread. Knowing what happened during the onset of the diseases, Early reports from Huang et al. on Jan 24, 2020, provided clinical and what test method, diagnosis and treatment was performed is characteristics of 41 patients confirmed with 2019-nCoV infection important for us to assess the procedurals, and further evaluate and in Wuhan Jinyintan Hospital. These data indicated that patients’ control the disease. clinical manifestations included fever, nonproductive cough, dyspnea, myalgia, fatigue, Lymphopenia, and radiographic evidence of pneumonia. Organ dysfunction (e.g., shock, acute respiratory dis- Keywords: COVID-19; SARS-CoV-2. tress syndrome (ARDS), acute cardiac injury, and acute kidney in- Abbreviations: SARS-CoV, severe acute respiratory syndrome coronavirus; 2019- jury) and death can occur in severe cases.1 138 patients confirmed nCoV, 2019 novel coronavirus; COVID-19, coronavirus disease in 2019; SARS- with 2019-nCoV infection from Zhongnan Hospital of Wuhan CoV-2, severe acute respiratory syndrome coronavirus 2; ARDS, acute respiratory distress syndrome. University showed similar clinical characteristics based on the re- 6 Received: February 14, 2020; Revised: February 29, 2020; Accepted: March 02, port on Feb 7 from Wang et al. In this report, one thing to notice is 2020 that infection for affected health professionals and hospitalized pa- *Correspondence to: Lili Wang, Department of Medicine, Division of Infectious tients take 29% and 12.3% respectively. This suggests among the Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. Tel: 138 patients, 41.3% got infected inside the hospital. Case reports +1 212-241-2503, E-mail: [email protected] confirmed human-to-human transmission of 2019-nCoV.7,8 On How to cite this article: Wang L. Characteristics of COVID-19 During the Onset Stage and Considerations for Disease Control. Exploratory Research and Hypothesis the national scale, data of 1,099 patients from 552 hospitals in 31 in Medicine 2020;5(1):8–10. doi: 10.14218/ERHM.2020.00005. provinces were assessed.9 Based on this study preprinted online on

Exploratory Research and Hypothesis in Medicine 2020 vol. 5 | 8–10

Table 1. The summary of the most commonly seen symptom from studies Sample size Median age Fever Fatigue Dry cough Dyspnea Lymphopenia Jinyintan Hospital1 41 49 98% 44% 31% 22% 63% Zhongnan Hospital6 138 56 98.6% 69.6% 59.4% 31.2% 70.3% National9 1,099 47 87.9% 38.1% 67.7% 18.6% 82.1%

Feb 6, 31.30% of the patients had been to Wuhan and 71.80% had COVID-19. The vaccines are still under development. A cryo-EM contact people from Wuhan. This paper also indicated the median study on 2019-nCoV spike protein revealed the prefusion con- incubation period was 3.0 days (range, 0 to 24.0 days). The most formation of spike glycoprotein of 2019-nCoV and found it can commonly seen symptom from these studies was summarized in bind to the receptor ACE2 with higher affinity than its SARS-CoV Table 1.1,6,9 According to the 1,099 people study, 18.6% of the counterpart.14 As the spike glycoprotein is a key target for vac- patients were severe cases (based on American Thoracic Society cines, therapeutic antibodies and diagnostics, the discoveries in guideline), and 5% of them were treated in ICU. The death rate is the details of spike glycoprotein structure will contribute to the around 2% based on these reports. drug and vaccine development. Drugs against coronavirus RNA- dependent RNA polymerase is also under clinical trial. Due to the high infectivity of SARS-CoV-2, the optimal strategies against Treatment spreading remains to be early detection and early quarantine. The early detection relies on a quick and accurate detection method, As the SARS-CoV-2 is a novel coronavirus that just been identi- which is also under clinical test. Until Feb 29, 2020, the number fied, no specific drug has been found to treat COVID-19. Based of newly diagnosed people in China has declined for over a week, on the experiences gained on SARS-CoV infection, the early pa- which is a good sign of COVID-19 under control. However, new tients were treated with antibiotics and oseltamivir. Corticosteroid patients number confirmed in other countries is increasing.15 Rigid therapy was given as a combined regimen if severe pneumonia control by the government would be crucial under these public was diagnosed. Oxygen support was administered to patients ac- emergency circumstances. In this article, I hope all the endeavors cording to the severity of hypoxemia.1,6,9 In vitro studies on anti- we made will shorten the course of this modern plague, with the SARS-CoV-2 drugs have shown high efficacy of remdesivir.10 The least spreading to the other part of the world. first confirmed COVID patient in the U.S. was cured by remdesivir.11 At present at least two phase-3 clinical trials on remdesivir were carried out from early February 2020. With more patients Acknowledgments recovered from COVID-19 and released from the hospital, donat- ed blood plasma from recovered patients with antibodies against I thank the colleges from Icahn School of Medicine for meaningful SARS-CoV-2 is also used to treat severe cases. Other remedies are discussion. also performed to reduce the damage of vital organs by cytokine storm induced by the immune response.12 Funding Comments This article is not fund-supported. A confirmed case with COVID-19 was defined as a positive result to real-time reverse-transcriptase polymerase-chain-reaction (RT- Conflict of interest PCR) assay or high-throughput sequencing for nasal and pharyngeal swab specimens.1 However, when an individual was infected but with low viral load, the test result could be negative due to The author has no conflict of interest related to this publication. the relatively high false-negative rate of the PCR based tests. This might potentially lead to the spread of SARS-CoV-2. There are References cases with negative results in the early days of COVID-19 when the symptom is not severe but later confirmed as positive SARS- CoV-2 infection.13 Self-quarantine for 14 days when has suspi- [1] Huang C, Wang Y, Li X, Ren L, Zhao J, Hu Y, et al. Clinical features of pa- cious syndrome is a safe strategy. SARS-CoV-2 spread rapidly tients infected with 2019 novel coronavirus in Wuhan, China. Lancet 2020;395(10223):497–506. doi:10.1016/S0140-6736(20)30183-5. through human-to-human routes, accumulating tens of thousands [2] Lu R, Zhao X, Li J, Niu P, Yang B, Wu H, et al. Genomic characterisa- of patients within a single month in Wuhan. The huge number of tion and epidemiology of 2019 novel coronavirus: implications for vi- patients that emerged during such a short time saturated the medi- rus origins and receptor binding. Lancet 2020;395(10224):565–574. cal resources. How to properly isolate and treat patients has be- doi:10.1016/S0140-6736(20)30251-8. come a great challenge to the public health system. [3] Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, et al. A Novel Coro- navirus from Patients with Pneumonia in China, 2019. N Engl J Med 2020;382:727–733. doi:10.1056/NEJMoa2001017. Perspectives [4] Chen N, Zhou M, Dong X, Qu J, Gong F, Han Y, et al. Epidemiologi- cal and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study. Lancet Although several promising trials are carried out in China and 2020;395(10223):507–513. doi:10.1016/S0140-6736(20)30211-7. America, at present no specific drugs have been confirmed to cure [5] Li Q, Guan X, Wu P, Wang X, Zhou L, Tong Y, et al. Early Transmission

DOI: 10.14218/ERHM.2020.00005 | Volume 5 Issue 1, March 2020 9 Explor Res Hypothesis Med Wang L.: COVID-19 onset

Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumo- virus (2019-nCoV) in vitro. Cell Res 2020;30:269–271. doi:10.1038/ nia. N Engl J Med 2020. doi:10.1056/NEJMoa2001316. s41422-020-0282-0. [6] Wang D, Hu B, Hu C, Zhu F, Liu X, Zhang J, et al. Clinical Character- [11] Holshue ML, DeBolt C, Lindquist S, Lofy KH, Wiesman J, Bruce H, et istics of 138 Hospitalized Patients With 2019 Novel Coronavirus- al. First Case of 2019 Novel Coronavirus in the United States. N Engl J Infected Pneumonia in Wuhan, China. JAMA 2020. doi:10.1001/ Med 2020;382:929–936. doi:10.1056/NEJMoa2001191. jama.2020.1585. [12] Xu Z, Shi L, Wang Y, Zhang J, Huang L, Zhang C, et al. Pathological [7] Chan JF, Yuan S, Kok KH, To KK, Chu H, Yang J, et al. A familial cluster findings of COVID-19 associated with acute respiratory distress syn- of pneumonia associated with the 2019 novel coronavirus indicat- drome. Lancet Respir Med 2020. doi:10.1016/S2213-2600(20)30076- ing person-to-person transmission: a study of a family cluster. Lancet X. 2020;395(10223):514–523. doi:10.1016/S0140-6736(20)30154-9. [13] Zhou Y, Yang L, Han M, Huang M, Sun X, Zhen W, et al. Clinical Reports [8] Phan LT, Nguyen TV, Luong QC, Nguyen TV, Nguyen HT, Le HQ, et al. on Early Diagnosis of Novel Coronavirus (2019-nCoV) Pneumonia in Importation and Human-to-Human Transmission of a Novel Coro- Stealth Infected Patients. Preprints 2020:2020020156. doi:10.20944/ navirus in Vietnam. N Engl J Med 2020;382:872–874. doi:10.1056/ preprints202002.0156.v1. NEJMc2001272. [14] Wrapp D, Wang N, Corbett KS, Goldsmith JA, Hsieh CL, Abiona O, et [9] Guan WJ, Ni ZY, Hu Y, Liang WH, Ou CQ, He JX, et al. Clinical Charac- al. Cryo-EM structure of the 2019-nCoV spike in the prefusion con- teristics of Coronavirus Disease 2019 in China. N Engl J Med 2020. formation. Science 2020. doi:10.1126/science.abb2507. doi:10.1056/NEJMoa2002032. [15] Hodcroft EB. Preliminary case report on the SARS-CoV-2 cluster in [10] Wang M, Cao R, Zhang L, Yang X, Liu J, Xu M, et al. Remdesivir and the UK, France, and Spain. Swiss Med Wkly 2020;150:w20212. chloroquine effectively inhibit the recently emerged novel corona- doi:10.4414/smw.2020.20212.

10 DOI: 10.14218/ERHM.2020.00005 | Volume 5 Issue 1, March 2020 Original Article

Echocardiography Effectiveness in Improving Diagnosis of Rheumatic Heart Disease in North Darfur: A Hospital-based Study

Mohammed Elmujtba Adam Essa Adam1,2*, Sherihan Mohammed Elkundi Osman3, Daralsalam Ishag Ateem Abdalrasoul1, Ibrahim Adam Osman Yagoup1,2, Mustafa Mohamed Ali Hussein1,2, Mutwaly Defealla Yousif Haron1,2, Ziryab Imad Taha Mahmoud4,2 and Abdelkareem A. Ahmed2,5*

1Department of Medicine, Faculty of Medicine, Al-Fashir University, Al-Fashir, Sudan; 2Department of Clinical Medicine, Medical and Cancer Research Institute (MCRI), Nyala, Sudan; 3Department of Molecular Medicine, Institute of Endemic Diseases, University of Khar- toum, Khartoum, Sudan; 4Department of Internal Medicine, Faculty of Medicine, University of Bahri, Khartoum, Sudan; 5Department of Physiology and Biochemistry, Faculty of Veterinary Science, University of Nyala, Nyala, Sudan

Abstract

Background: Acute rheumatic fever (ARF) is an inflammatory disease caused by autoimmune responses to bacterial infection. Rheumatic heart disease (RHD) damages one or more heart valves through recurrent episodes of ARF. We aimed to determine the changes in sensitivity, specificity and predictive values in RHD Jones diagnostic guidelines following the inclusion of echocardiograph as an additional diagnostic tool for RHD.

Methods: This is a retrospective cross-sectional study done in the echocardiography center of Al-Fashir teaching hospital. We included a total of 1,103 patients who presented at our hospital and had a diagnosis of RHD, ischemic heart disease or congestive heart disease during 2011–2017.

Results: Among the RHD patients, screening with echocardiography was associated with increases of the sensitivity value, positive predictive value and specificity value by 18.1%, 8.1% and 1%, as compared to their initial diagnoses by Jones criteria alone, which were primarily based on clinical presentations. Mitral stenosis was the most common RHD abnormality, followed by aortic and tricuspid valve regurgitation. North Darfur state was found to have the lowest prevalence of RHD in all geographical parts of Sudan that have been studied. The female to male ratio was 3:1.

Conclusions: Our data highlight the important role of echocardiography in diagnosing RHD complications through improved diagnostic sensitivity, positive predictive value and specificity.

Introduction

Keywords: Early diagnosis; Echocardiography; Mitral stenosis; North Darfur state; Acute rheumatic fever (ARF) is a type of disease caused by bac- Rheumatic heart disease. Abbreviations: ARF, acute rheumatic fever; RHD, rheumatic heart disease; RF, rheu- terial infection, commonly involving groups with a β hemolytic matic fever. Streptococci and leading to an autoimmune inflammatory response. Received: August 27, 2019; Revised: October 16, 2019; Accepted: November 07, Most of the body’s organs, such as the heart, brain, joints and skin, 2019 can also be affected by the disease. The recurrent episodic valve *Correspondence to: Abdelkareem Abdallah Ahmed, Department of Physiology and damage to the heart in ARF are known as rheumatic heart disease Biochemistry, Faculty of Veterinary Science, University of Nyala, Nyala, PO Box: 1,2 155 Nyala, Sudan. Fax: +249711833123; E-mail: [email protected]; Moham- (RHD). Both diseases are common in low socioeconomic com- 3 med Elmujtba Adam Essa Adam, Faculty of Medicine, Al-Fashir University, Al-Fa- munities. More than 0.5% of school-age children are suffering shir, Sudan. Tel: +249907009378; E-mail: [email protected] from RHD across the globe, and the incidence is increased in the How to cite this article: Adam MEAE, Osman SME, Abdalrasoul DIA, Yagoup IAO, sub-Saharan region of Africa by nearly half a million patients an- Hussein MMA, Haron MDY, Mahmoud ZIT, Ahmed AA. Echocardiography Effec- nually.4–6 This disease causes damage to the heart valves and the tiveness in Improving Diagnosis of Rheumatic Heart Disease in North Darfur: A Hos- 7,8 pital-based Study. Exploratory Research and Hypothesis in Medicine 2020;5(1):11–18. most common consequent abnormality is mitral regurgitation. doi: 10.14218/ERHM.2019.00020. There is a growing concern for patients with severe conditions

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Copyright: © 2020 Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 International License (CC BY-NC 4.0), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Explor Res Hypothesis Med Adam MEAE. et al: Echocardiography in improving diagnosis of rheumatic heart disease without access to cardiac surgery, which could lead to a potential were confirmed by echocardiography (SusKo, CH5-2Transducer; risk of death being imminent. Reportedly, 20% of patients die be- Siemens), which established the diagnosis. The severity was deter- fore the age of 5 years and 80% before the age of 25 years.9 mined by the American Society of Echocardiography (commonly In regard to the pathology and pathogenesis of RHD, the calci- known as ASE) modified criteria, and the valve lesion was defined fication in the mitral valve is not merely an inactive “dystrophic” according to the jet width and camper to the jet ratio (5 mm and process but it is a regulated inflammatory process with osteoblast more to the left atrium ratio of 20% was considered severe mitral and neo-angiogenesis markers’ expression.10 The World Health regurgitation; and if the jet-valve ratio with the jet width increased Organization (commonly referred of as WHO) Consultant’s Re- severity of the valve became more intense, a 3 mm jet width with port stated that any patient diagnosed by echocardiography with 25% left ventricular outflow tract as confirmed by aortic regurgita- a silent rheumatic valve should be considered and treated as RHD tion echocardiography). The Doppler mean gradient and valve ap- until other proof is available.11 Accurate diagnosis is important pearance were both used to calculate the mitral stenosis. The clini- to avoid long-term treatment for those who have been diagnosed cal examinations included several processes, and careful cardiac and also to prevent further heart damage and the need for surgi- examination was performed in supine and left lateral decubitus po- cal intervention (for those who are underdiagnosed). Currently, sition by an experienced physician. Patients with organic murmur there is no diagnostic test for RHD, and the diagnosis is based were detected clinically and confirmed by echocardiography, then solely on the clinical recognition of the major and minor criteria classified as patients with clinically-detected RHD. of Jones (guidelines published in 1944 by Dr T. Duckett Jones, modified in 2015) with evidence of Streptococcal throat infec- Statistical analysis tion.12 The estimated risk for developing rheumatic fever (RF) after a streptococcal pharyngitis episode is 0.3–3%,13 and a small per- The statistical software package SPSS (version 17.0) was used to centage of RF develops as RHD. The primary risk factor is Strep- analyze all the collected data of RHD as percentages, categories tococcal throat infection. Other types of infections, such as of and p-values for statistical significance testing. Evaluation data group A Streptococci, may lead to RF, as in pyoderma (skin infec- were analyzed by one-way analysis of variance (referred to as tion); however, age, sex, urban residence, overcrowding, poverty, ANOVA). The level of statistical significance was set as p<0.05. offspring of working mothers, and mother illiteracy are also potential risk factors.14 The objective of this study was to determine the incidence, prevalence and risk factors of RHD among children and Results adults in North Darfur State, and to estimate the specificities, sen- sitivities, and positive predictive values of adding echocardiograph The study included in total 1,103 patients who were confirmed to the routine of diagnosing RHD patients. by echocardiography from January 2011 to December 2017. All patients who were screened by echocardiography were confirmed with the following diagnoses: RHD, ischemic heart disease, con- Methods gestive heart disease and cardiomyopathy, (35%, 29.2%, 20% and 15.8% respectively) (Fig. 1). We found that the most affected age Study area groups among all the patients were those above 25 years, and that both the age groups between 15–25 years and under 15 years have North Darfur State is one of the five States composing the Darfu- an equal incidence of RHD (38%, 31%,31% respectively) (Fig. rian region, with an area of 296,420 Km2 and a total population 2). The incidence of RHD was found to be higher in females than of 2,296,068 in 2017 (Central Statistical Agency of Sudan, 2017 males, with a ratio of 3:1 (Fig. 3) (Table 1). report). Al-Fashir, its capital, had 264.734 residents in 2006; how- Most of the patients in the year 2011 had three-valve involve- ever, that number decreased nearly to half in the last 5 years due to ments, double valve involvements and an isolated valve (55.9%, conflict in the area. 26.41% and 14.71% respectively). This contrasted the following years, which showed a semi-steady decrease of the three-valve involvements in the RHD patients and that those patients with Study design three-valve lesions became the fewest among the collective patients with different valves lesions, followed by the isolated valve This is a retrospective study; the data was collected from Al-Fashir involvement patients and dominated by the double valve involve- Teaching Hospital. Scientific and ethical approvals were granted ment patients (4%, 38.8% and 57.2% respectively in 2015). In by the Scientific and Ethics Committees of the Institute ofEn- total, double valve involvement had the greatest incidence, fol- demic Diseases, University of Khartoum. Addition approvals were lowed by an isolated valve then the three-valve involvement obtained from the North Darfur State Ministry of Health and Al- (40.9%, 35.6% and 23.5% respectively) (Table 2). The peak inci- Fashir Teaching Hospital Management Board. The data covered dence of the three-valve involvement was the year 2011 (55.9%), the period from January 2011 to December 2017. The data collect- and the lowest incidence year was 2015 (4.1%) (Fig. 4). The most ed from the hospital files included the clinical records of patients affected valve in our RHD patients was found to be the mitral with confirmed RHD, RF and other cardiac diseases. Demographic valve, followed by the aortic then the tricuspid valve (56.9%, and clinical data were extracted along with each patient’s age, sex, 27.2% and 15.9% respectively) (Table 3). The highest incidences address, clinical presentations, valve involvement, and final diag- of the mitral valve were in 2014 and 2017, in which 100% of nosis. All the studied patients had been selected to be confirmed by patients had mitral valve lesion with or without other affected echocardiography after eliciting clinical suspicion. valves (Table 4). The steps of the disease diagnosis begun from the outpatient When comparing the same patients, who were diagnosed by yard, where the disease history and clinical examinations were per- Jones criteria before and after the screened echocardiography to formed for all patients. After diagnostic suspicion, all of the patients confirm the diagnosis, we found that the echocardiography screen-

12 DOI: 10.14218/ERHM.2019.00020 | Volume 5 Issue 1, March 2020 Adam MEAE. et al: Echocardiography in improving diagnosis of rheumatic heart disease Explor Res Hypothesis Med

Fig. 1. Jones criteria for diagnosing rheumatic fever. Data are hospital diagnoses from the period of 2011–2017.

ing adds better sensitivity, positive predictive value and specificities (18.1%, 8.1% and 1%) (Table 5). The peak incidence year with RHD patients was 2011 (17.6%) and the least affected year was 2017 (11.6%). The years 2012, 2013, 2014, 2015 and 2016 had a fluctuating range of incidence (14.7%, 16.3%, 13.3%, 12.7% and 13.9%) (Table 6, Fig. 5). We also found that mitral stenosis is more common than mitral regurgitation, by a 12.4% difference. The mitral stenosis represents 56.2% of all the mitral involvement patients, in comparison to 43.8% represented by the mitral regur-

Table 1. Male, female differences with rheumatic heart disease

Frequency % Valid % p-value Valid Male 127 32.8 32.8 <0.05 Fig. 2. Age groups of rheumatic heart disease in North Darfur State patients. Female 260 67.2 67.2 <0.05 Total 387 100 100

Table 2. Number and percentage of valve involvement in rheumatic heart disease patients from 2011–2017

Year Isolated valve Double valve Three valves 2011 14.71% 26.41% 55.88% 2012 35.10% 28% 36.90% 2013 44.40% 44.40% 11.20% 2014 53% 39.20% 7.80% 2015 38.80% 57.20% 4% 2016 37.10% 50% 12.90% 2017 31.20% 42.20% 26.60% Fig. 3. Incidence rate between male/female patients with rheumatic heart disease. Total 35.60% 40.90% 23.50%

DOI: 10.14218/ERHM.2019.00020 | Volume 5 Issue 1, March 2020 13 Explor Res Hypothesis Med Adam MEAE. et al: Echocardiography in improving diagnosis of rheumatic heart disease

Fig. 4. Prevalence of valvular abnormality in patients with rheumatic heart disease. gitation. Discussion The most common aortic type of lesion has been aortic regurgitation, found in 84.7% of the patients with aortic valve involve- RF was first detected by Wig in 1935 and since then most of the ment, and the least common type of aortic involvement has been studies conducted in ARF and RHD patients have been identified aortic stenosis. We also found all the patients with tricuspid lesion by many risk factors associated with the disease,12 such as poverty had tricuspid regurgitation with zero tricuspid stenosis. The most and low health access.13–20 common clinical presentation of RHD patients has been arthritis, Most global studies show an inconsistent association of the male followed by carditis, arthralgia, chorea and erythema marginatum to female ratio. The epidemiological data indicate there is the same (62%, 41.1%, 33.5%, 11.4% and 1%). Most of the patients were prevalence of RHD in both sexes,21 which is in contrast to our study, diagnosed with more than one feature (Fig. 6). which demonstrated a higher incidence in females than males (being Table 3. Accumulative percentages and frequencies of the three-valve Table 5. Sensitivity, specificity and positive predictive value changes be- involvements in all the rheumatic heart disease patients fore and after addition of echocardiograph to rheumatic heart disease diagnosis Frequency Percent Valid Percent p-value Valid Jones criteria Jones criteria with alone echocardiography Mitral 377 56.9 56.9 <0.05 Sensitivity 51.2% 69.3% Aortic 180 27.2 27.2 <0.05 Specificity 97% 98% Tricuspid 105 15.9 15.9 <0.05 Positive predictive value 88.3% 96.4% Total 662 100 100 p-value <0.05 <0.05 Table 4. Percentages of cardiac valve lesions’ involvement in rheumatic heart disease patients from 2011–2017 Table 6. Percentage of rheumatic heart disease from 2011–2017

Year Mitral Aortic Tricuspid Year Frequency Percent Valid percent p-value 2011 91.1% 51.4% 39.7% Valid 2012 98.2% 47.3% 14% 2011 68 17.6 17.6 <0.05 2013 98.4% 44.4% 23.8% 2012 57 14.7 14.7 <0.05 2014 100% 37.2% 17.6% 2013 63 16.3 16.3 <0.05 2015 98% 53% 14.2% 2014 51 13.2 13.2 <0.05 2016 98.1% 40.7% 37% 2015 49 12.7 12.7 <0.05 2017 100% 51.1% 42.22% 2016 54 14 14 <0.05

. The table demonstrates the percentage of each valve involvement annually. As the 2017 45 11.6 11.6 <0.05 patients can have all three valves lesions at the same time, the percentage are over- lapping. Total 387 100 100

14 DOI: 10.14218/ERHM.2019.00020 | Volume 5 Issue 1, March 2020 Adam MEAE. et al: Echocardiography in improving diagnosis of rheumatic heart disease Explor Res Hypothesis Med

Fig. 5. Prevalence of rheumatic heart disease in Al-Fashir Teaching Hospital from 2011–2017.

3:1). Our findings are in line with previous studies conducted in Su- The incidence of RHD in North Darfur was found in our study dan and some parts of Asia, such as India and Pakistan, which gave to be less than in any other area in Sudan which had been studied, similar results but showed a lower female to male ratio.21–23 This such as South Darfur, which showed 3.3% in Nyala camps during could be explained by women seeking medical care more than men, the year 2016. Khartoum, being the capital city of North Darfur, especially during the antenatal care period, or the opposite, where was estimated to have 0.03%, and that prevalence are dominated women are less likely to seek medical care when they have a strep by the North Kordofan state (with 6.1% of RHD).23–25 On the oth- throat infection; however, no clear explanation has been identified. er hand, our study revealed that North Darfur has only 0.03% of In addition, there is an elevated infection risk of group A strepto- RHD, which reduced to 0.019% in 2017 (the population density of coccal infection, and RHD may develop if not treated properly; this North Darfur was obtained from the Central Statistical Organiza- constitutes 60% of heart disease during pregnancies.24,25 tion report). Our study found a higher rate of sensitivity and specificity in Another study showed the age groups from 11–30 years to be using the echocardiography to diagnose RHD compared by tradi- the most affected with RHD.23 Similarly, our study confirmed that tional means of screening, such as auscultation. There have been age groups above 25 years have higher incidence. This may be re- several studies in the literature that produced the same finding.26,27 lated to the lack of health services, timely diagnosis and treatment. Other studies have also detected the sensitivity rate of 73% and the There are general agreements on mitral valve, which has been the positive predictive value of 92% in using echocardiography align- valve disease most associated with RHD, and the mitral regurgi- ing with the reference criteria.28 tation, as the most common type of lesion in the valve.24,29 Our

Fig. 6. Clinical presentation of the rheumatic heart disease patients.

DOI: 10.14218/ERHM.2019.00020 | Volume 5 Issue 1, March 2020 15 Explor Res Hypothesis Med Adam MEAE. et al: Echocardiography in improving diagnosis of rheumatic heart disease

Fig. 7. Projected decrease in age-specific mortality from rheumatic heart disease based on country trends from 2000 to 2015. The x-axis demonstrates the total percentage reduction in mortality for those aged below 25 years (WHF) target between 2013 and the year 2025, if 2000 to 2015 trends continue. The y-axis illustrates all percent reduction in mortality for those aged 30–69 years (Sustainable Development Goal 3 [SDG3] target) between 2015 and 2030, if 2000 to 2015 trends continue. analyses also revealed the mitral valve to be the highest involved (Fig. 7).37 but also found mitral stenosis to be more common when compared There are some hypotheses about the role of RHD in being a to mitral regurgitation. The inconsistency of those findings may risk factor for some neurological diseases, such as with stroke, as indicate the higher prevalence of elderly patients, as mitral stenosis it has been detected that patients with mitral stenosis and atrial occurs in advanced ARF.30 fibrillation have increased 4% liability to have stroke.38 Yet, only To control both RHD and RF, there are many factors that should a few studies are being done in this matter, but future studies will be considered, starting with benzathine penicillin G administra- likely clarify the exact role. tion,31 vaccine availability, primordial, overcrowding, and improving primary and secondary prevention against the disease, according to the WHO.31,32 The secondary prevention to RF is the Conclusions continuous provision of some type of antibiotic to patients with an 31 exposed history of ARF and documented RHD, with considera- This was a retrospective hospital-based study which concluded tion to treatment availability and no surgical access. In valvular 33,34 that females were more vulnerable to RHD. Moreover, over 25 disease patients this is particularly important. The last expan- years-old is the most affected age group, and the incidence of valve sion of the echocardiography screening programs led to a reduc- involvement in RHD decreases with time. The sensitivity, specific- tion in the burden of RHD and it will continue to better identify ity and positive predictive value have improved after the addition patients while they are asymptomatic, like in silent RHD. The con- of echocardiography to the Jones criteria. tinuous data provided will help in estimating the prevalence of the diseases and their complications with annual bases. The study presented herein carries some limitations, such as Acknowledgments having no updated population census in Sudan, no available data in regard to patient follow-up, and so on. The authors are very grateful to the Al-Fashir Hospital administration staff for their support in conducting this research. Future research directions, prospectives, predictions and hypothesis Ethics approval and consent to participate In 2013, the World Heart Federation called for a 25% reduction in RHD mortality amid individuals aged <25 years before the year Obtained. 2025.35 Recently, the United Nations Sustainable Development Goal 3 (referred to as SDG3) prospected a 1/3 reduction in prema- ture death due to non-communicable disease by the year 2030.36 Consent for publication Predicting that trends in mortality over the last 15 years hold, many endemic regions are in line to achieve either one of or both targets Not applicable.

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Funding [15] Kumar R, Vohra H, Chakraborty A, Sharma YP, Bandhopadhya S, Dhanda V, et al. Epidemiology of group A streptococcal pharyngitis & impetigo: across-sectional & follow up study in a rural community of No funds have been received in relation to this study or its publica- northern India. Indian J Med Res 2009;130(6):765–771. tion. [16] Carapetis JR, Currie BJ, Kaplan EL. Epidemiology and prevention of group A streptococcal infections: acute respiratory tract infections, skin infections, and their sequelae at the close of the twentieth cen- Conflict of interest tury. Clin Infect Dis 1999;28(2):205–210. doi:10.1086/515114. [17] Nordet P, Lopez R, Duenas A, Sarmiento L. Prevention and control of rheumatic fever and rheumatic heart disease: the Cuban experience All authors declare that they have no conflicts of interest. (1986-1996-2002). Cardiovasc J Afr 2008;19(3):135–140. [18] Allen LB, Allen M, Lesa RF, Richardson GE, Eggett DL. Rheumat- ic fever in Samoa: education as prevention. Pac Health Dialog Author contributions 2011;17(1):107–118. [19] Orün UA, Ceylan O, Bilici M, Karademir S, Ocal B, Senocak F, et al. Acute rheumatic fever in the Central Anatolia Region of Turkey: a Performing the cardiovascular examination, echocardiography 30-year experience in a single center. Eur J Pediatr 2012;171(2):361– screening and confirming the diagnosis (DIAA), scientific writing 368. doi:10.1007/s00431-011-1555-x. and data analysis (MEAEA, SMEO, ZITM, MDYH), collecting [20] Carapetis JR. Rheumatic heart disease in developing countries. N the data from the hospital records (IAOY, MMAH), supervising Engl J Med 2007;357(5):439–441. doi:10.1056/NEJMp078039. the study (AAA). [21] Rizvi SF, Khan MA, Kundi A, Marsh DR, Samad A, Pasha O. Status of rheumatic heart disease in rural Pakistan. Heart 2004;90(4):394– 399. doi:10.1136/hrt.2003.025981. References [22] Agarwal AK, Yunus M, Ahmad J, Khan A. 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Rheumatic fe- 2014;66(3):320–326. doi:10.1016/j.ihj.2014.03.010. ver: neglected again. Science 2009;324(5923):37. doi:10.1126/ [25] Ahmed K, Elfil A, Hamid HM, Saeed A, Ali A. Epidemiology of cardiac science.324.5923.37b. disease during pregnancy in Khartoum Hospital, Sudan. J Women’s [4] Gordis L. The virtual disappearance of rheumatic fever in the United Health Care 2015;4(2):227. doi:10.4172/2167-0420.1000227. States: lessons in the rise and fall of disease. T. Duckett Jones me- [26] Marijon E, Celermajer DS, Tafflet M, El-Haou S, Jani DN, Ferreira B, morial lecture. Circulation 1985;72(6):1155–1162. doi:10.1161/01. et al. Rheumatic heart disease screening by echocardiography: the cir.72.6.1155. inadequacy of World Health Organization criteria for optimizing the [5] Carapetis JR, McDonald M, Wilson NJ. Acute rheumatic fever. Lancet diagnosis of subclinical disease. Circulation 2009;120(8):663–668. 2005;366(9480):155–168. doi:10.1016/S0140-6736(05)66874-2. doi:10.1161/CIRCULATIONAHA.109.849190. [6] Carapetis JR, Steer AC, Mulholland EK, Weber M. The global burden [27] Marijon E, Ou P, Celermajer DS, Ferreira B, Mocumbi AO, Jani D, et of group A streptococcal diseases. Lancet Infect Dis 2005;5(11):685– al. Prevalence of rheumatic heart disease detected byechocardio- 694. doi:10.1016/S1473-3099(05)70267-X. graphic screening. N Engl J Med 2007;357(5):470–476. doi:10.1056/ [7] Chockalingam A, Gnanavelu G, Elangovan S, Chockalingam V. Clinical NEJMoa065085. spectrum of chronic rheumatic heart disease in India. J Heart Valve [28] Mirabel M, Celermajer DS, Ferreira B, Tafflet M, Perier MC, Karam N, Dis 2003;12(5):577–581. et al. Screening for rheumatic heart disease: evaluation of a simpli- [8] Tissier R, Chetboul V, Moraillon R, Nicolle A, Carlos C, Enriquez B, et fied echocardiography-based approach. Eur Heart J Cardiovasc Imag- al. Increased mitral valve regurgitation and myocardial hypertrophy ing 2012;13(12):1024–1029. doi:10.1093/ehjci/jes077. in two dogs with long-term pimobendan therapy. Cardiovasc Toxicol [29] Olson LJ, Subramanian R, Ackermann DM, Orszulak TA, Edwards WD. 2005;5(1):43–51. doi:10.1385/ct:5:1:043. Surgical pathology of the mitral valve: a study of 712cases spanning [9] Remenyi B, Carapetis J, Wyber R, Taubert K, Mayosi BM, World 21 years. Mayo Clin Proc 1987;62(1):22–34. doi:10.1016/s0025- Heart F. Position statement of the World Heart Federation on the 6196(12)61522-5. prevention and control of rheumatic heart disease. Nat Rev Cardiol [30] Hall P, Biorck G. The natural history of rheumatic valvular heart 2013;10(5):284–292. doi:10.1038/nrcardio.2013.34. disease and its bearing upon the results of surgery for mitral ste- [10] Viswanathan V. Acute rheumatic fever. Indian Journal of Rheumatol- nosis. Acta Rheumatol Scand 1958;4(2):70–78. doi:10.3109/ ogy 2012;7(5):36–43. doi:10.1016/S0973-3698(12)60027-2. rhe1.1958.4.issue-1-4.09. [11] Guilherme L, Kalil J. Rheumatic fever: How streptococcal throat in- [31] Moser M, Leo O. Key concepts in immunology. Vaccine 2010;28(Sup- fection triggers an autoimmune disease. In: Shoenfeld Y, Rose NR. pl 3):C2–C13. doi:10.1016/j.vaccine.2010.07.022. Infection and autoimmunity. Amsterdam, Elsevier. 2004:321–330. [32] Guilherme L, Kohler KF, Postol E, Kalil J. Genes, autoimmunity and [12] Gewitz MH, Baltimore RS, Tani LY, Sable CA, Shulman ST, Carape- pathogenesis of rheumatic heart disease. Ann Pediatr Cardiol tis J, et al. Revision of the Jones criteria for the diagnosis of acute 2011;4(1):13–21. doi:10.4103/0974-2069.79617. rheumatic fever in the era of Doppler echocardiography: ascien- [33] Cilliers AM. Rheumatic fever and its management. BMJ tific statement from the American Heart Association. 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2013;10(5):284–292. doi:10.1038/nrcardio.2013.34. doi:10.1016/j.jacc.2018.06.063. [36] United Nations. Sustainable Development Goals: 17 Goals to Trans- [38] Oldgren J, Healey JS, Ezekowitz M, Commerford P, Avezum A, form Our World. Goal 3: Ensure Healthy Lives and Promote Well- Pais P, et al. Variations in cause and management of atrial fibrilla- Being For All at All Ages. New York, United Nations. 2016. tion in a prospective registry of 15,400 emergency department [37] Watkins DA, Beaton AZ, Carapetis JR, Karthikeyan G, Mayosi BM, patients in 46 countries: the RE-LY Atrial Fibrillation Registry.- Cir Wyber R, et al. Rheumatic heart disease worldwide: JACC sci- culation 2014;129(15):1568–1576. doi:10.1161/CIRCULATIONA- entific expert panel. J Am Coll Cardiol 2018;72(12):1397–1416. HA.113.005451.

18 DOI: 10.14218/ERHM.2019.00020 | Volume 5 Issue 1, March 2020 Review Article

Microbial Biomarkers for Colorectal Cancer Identified with Random Forest Model

Weili Sun1, Lili Wang2, Qiuyue Zhang2 and Quanjiang Dong1,2*

1The Affiliated Hospital of Dalian Medical University, Shandong, China;2 Department of Central Laboratories and Gastroenterology, Qingdao Municipal Hospital Affiliated to Qingdao University, Shandong, China

Abstract

Colorectal cancer (CRC) is one of the most common cancers and a leading cause of cancer-related death. Gut microbiota are part of a complex microbe-based ecosystem of the human body, and changes in the microbiota can lead to a variety of diseases. All currently used CRC detection methods, including endoscopy, guaiac-based fecal occult blood test and fecal immunochemical test, have many limitations. Therefore, establishing novel screening methods which are accurate, inexpensive and non-invasive is indicated. Random forest models, as a superiority machine learning model, are increasingly used in research to select biomarkers. In this review, we summarized progressions of the diagnoses of CRC based on the random forest model of gut microbiota. We concluded that some cancer-associated bacteria in gut microbiota could be used as biomarkers for detecting early CRC. We also aimed to discuss how to select possible markers of colorectal diseases based on gut microbiota using the random forest model.

Introduction agnosis. Colonoscopy, the gold standard for the diagnosis of CRC, can facilitate early prevention through adenoma resection; how- Colorectal cancer (CRC) is one of the most common cancers and ever, it is an invasive endoscopy, which causes patient discomfort and reduces their compliance to clinical examinations, relies on a leading cause of cancer-related death. The global annual inci- 5,6 dence of CRC is approximately 1.2 million, and the annual death endoscopist skill, and requires patient bowel cleansing. The tra- ditional guaiac-based FOBT, which is inexpensive and noninva- toll reaches 600,000.1 CRC is characterized by a long preclinical sive, is the first-choice screening test for CRC. Both guaiac-based phase, progressing over years from early adenoma to invasive FOBT and fecal immunochemical test (FIT) can improve the pos- cancer.2,3 Diagnosis of early-stage CRC can significantly improve itive rate of diagnosis by repeated detection over a long period patient prognosis, with a survival rate higher than 80%,4 which of time. However, cancer bleeding is usually intermittent, which makes early diagnosis of CRC more critical for better patient out- leads to lower sensitivity, specificity, and false-negative results comes. with these two tests.7 As the abovementioned methods have many Screening methods for CRC, including fecal occult blood test limitations, it is necessary to establish novel screening methods (referred to as FOBT), colonoscopy, determination of genetic that are accurate, inexpensive, and noninvasive. mutation and gut microbiota tests, have received widespread at- The human microbiota is a very large and complex microbial tention; although, these methods significantly differ in specificity, system. Gut microbiota includes bacteria, viruses, archaea, and accuracy, convenience, and universality with regard to clinical di- fungi, but the main component is bacteria; thus, most studies of the microbiota focus on bacteria, as it is composed of approximately 1014 bacteria, which equates to 10 times the total number of hu- Keywords: Gut microbiota; Colorectal cancer; Colorectal adenoma; Random forest 8 model. man cells. The gut microbiota contains nearly 11,500 common 9 Abbreviations: AUC, area under the curve; CRA, colorectal adenoma; CRC, colo- bacteria. The total number of bacterial genes in the gut microbiota rectal cancer; CEA, carcinoembryonic antigen; FIT, fecal immunochemical tests; is 150 times the total number of human genes.10–12 Coincidentally, FOBT, fecal occult blood test; OTU, operational taxonomic unit; RF, random forest; the gut microbiota has been called a “forgotten organ”.10–12 TNM, tumor-node-metastasis. Dysbacteriosis plays an important role in the pathogenesis of Received: October 24, 2019; Revised: November 21, 2019; Accepted: December 25, 2019 several diseases. For example, the gut microbiota is associated *Correspondence to: Quanjiang Dong, Department of Central Laboratories and Gas- with many diseases, such as obesity, type 2 diabetes, and athero- troenterology, Qingdao Municipal Hospital Affiliated to Dalian Medical University, sclerosis.13–17 Moreover, it is well-known that genomic alterations Shandong 266071, China; Present Address: Donghai Zhong Road 5, Qingdao, Shan- of the APC/Wnt pathway can potentially lead to carcinoma.18 Re- dong, China. Tel: +86-05328890589; E-mail: [email protected] cent studies have increasingly indicated that the gut microbiota is How to cite this article: Sun W, Wang L, Zhang Q, Dong Q. Microbial Biomarkers 19 for Colorectal Cancer Identified with Random Forest Model. Exploratory Research closely related to CRC, with many differences in gut microbiota and Hypothesis in Medicine 2020;5(1):19–26. doi: 10.14218/ERHM.2019.00026. between healthy people and patients with CRC. Studies have also

Exploratory Research and Hypothesis in Medicine 2020 vol. 5 | 19–26

Copyright: © 2020 Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 International License (CC BY-NC 4.0), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Explor Res Hypothesis Med Sun W. et al: Microbial biomarkers of CRC shown that biomarkers from gut microbiota may be used for the cellular and physiologic features. In the proximal colon, Bacte- diagnosis of CRC.20–23 This review focuses on the relationship roides, Actinomyces, Pseudomonas, and Enterobacteriaceae show between gut microbiota and CRC as well as the possibility that differential abundance between the lumen and mucosa.42 Simi- gut microbiotarelated markers may be novel biological screening larly, Enterobacteriaceae, Bacteroides, and Pseudomonas enrich markers for CRC. the proximal colonic mucosa, whereas there is increased relative abundance of Finegoldia, Murdochiella, Peptoniphilus, Porphy- romonas, and Anaerococcus in the distal colon.42 The abundance Random forest model of Turicibacter, Finegoldia, Peptoniphilus, and Anaerococcus was found to be different between the lumen and mucosa microbiota 42 Gut microbiota can be used as a marker to screen for CRC, but in the distal colon. Furthermore, there are differences in the mi- the screening capacity of a single flora is limited, and multibacte- crobiota compositions between different anatomical parts of the 42 rial models are needed to increase the screening capacity. If too colon. many bacterial species are present, this model will be difficult to Colorectal adenoma (CRA) confers a high risk for the develop- apply in the clinical setting; therefore, we need to screen for the ment of CRC but the gut microbiota is necessary for the formation most discriminating bacteria. As an integrated machine-learning of intestinal adenoma, and healthy gut microbiota are associated algorithm, random forest (RF) is a classifier with multiple decision with a reduced risk of advanced CRA.43 In cases of CRA, the rela- trees that are established through randomly repeated sampling, and tive abundance of two bacterial genera (Enterococcus and Strep- a final voting result is obtained.24 The current classifier algorithms tococcus) increased, whereas that of three genera (Clostridium, mainly include, Bayes net, simple logistic, TB-line (TB pedigree), Roseburia, and Eubacterium) decreased.44 Another study found machine learning, sequential minimum optimization algorithm for that some species belonging to Ruminococcaceae, Clostridium, support vector, and RF.25–32 Pseudomonas, and Porphyromonadaceae showed increased In many applications, the RF algorithm, by far, has the best ac- numbers in patients with CRA, whereas other species belonging curacy. Moreover, compared to other technologies, RF has many to Bacteroides, Lachnospiraceae, Clostridiales, and Clostridium advantages, such as anti-interference, simple optimization, and ef- decreased.20 Analyses of fecal microbiota from 95 patients with ficient parallel processing, in processing highly nonlinear biologi- CRA revealed substantial changes in the microbiota compositions. cal data.33,34 The RF model considers the interaction between non- In CRA, the Proteobacteria phylum was found to have enriched linear data and characteristics of the model, and can also carry out the microbiota. These bacteria are associated with precancerous internal cross-validation within the model to prevent overfitting.35 lesions.45 Lachnospiraceae, a potentially beneficial bacteria genus, A recent article reported that an RF model successfully screened was depleted, and the relative abundance of this genus had high microbial markers for early cirrhotic liver cancer.36 Another study accuracy in differentiating patients with CRA from normal indi- on hepatocellular carcinoma used the same method to obtain pre- viduals.23,46 dictive biomarkers for advanced hepatocellular carcinoma.37 An Dysbiosis of the gut microbiota and its substantial composition- RF model based on intestinal gut microbiota was established to al alterations are closely related to the development of CRC.47 The successfully predict the variation of Bifidobacteria after probiotic microbial communities in tumor microhabitat are different from treatment, and it revealed the effects of probiotics on intestinal those in tumor-adjacent healthy tissue.48 Moreover, antibiotic in- flora.38 Thus, RF models, as a superior machine-learning model, tervention in the microbiota can significantly reduce the burden are increasingly used in research for the selection of biomarkers. of colonic tumors.19 Virulence-associated genes in tumors may Therefore, we reviewed the recent progress in the diagnosis of potentially depend upon the genomes Fusobacterium and Provi- colorectal diseases by using an RF model based on gut microbiota dencia.48 A study on fecal microbiota from 120 patients with CRC as well as the selection of possible predictive markers of colorectal showed the increased abundance of a number of species, includ- diseases. ing Pophyromonas assaccharolytica, Fusobacterium nucleatum, Parvimonas micra, Peptostreptococcus stomatis, Gemella spp., and Prevotella spp.23 Furthermore, in a mouse model of CRC, the Dysbiosis of gut microbiota in CRC abundance of Lactobacillus negatively correlated with the number of colonic tumors.49 Moreover, fecal microbiota from patients The gut microbiota and its hosts coparticipate in the establish- with CRC can promote tumorigenesis in both germ-free mice and 50 ment of a symbiotic relationship to maintain homeostasis in the conventional mice. Taking these findings together, it appears that digestive system. In healthy individuals, at the phyla level, the some cancer-associated bacteria in gut microbiota can serve as bio- Firmicutes and Bacteroidetes phyla are predominant in the gut markers to detect CRC. microbiota, despite remarkable interindividual differences.39 At the genus level, a meta-analysis indicated that a high abundance of the genera Barnesiella, Ruminococcaceae UCG-005, Alistipes, RF model for identification of microbial biomarkers for CRC Christensenellaceae R-7 group, and an unclassified member of the Lachnospiraceae family correlated with the healthy state in their Alterations in the relative abundance of bacteria in CRC indicate subjects.40 However, the most abundant bacterial genera in the gut they are potential predictive or diagnostic biomarkers for CRC or microbiota include Prevotella, Bacteroides, and Ruminococcus. CRA. Escherichia coli, Bacteroides fragilis, and Fusobacterium Based on genus compositional variations, the gut microbiota could nucleatum have been shown to directly influence tumor develop- be classified into different enterotypes, namely the Prevotella pre- ment in the colon.19 A recent small-sample study on the quantifi- dominant enterotype, Bacteroides predominant, and Ruminococ- cation of this bacterium in fecal samples found a great increase cus-related enterotype, respectively. Simultaneously, this research in the number of these bacteria in CRC; the conclusive findings shows that the human intestinal microbiota has commonalities.41 of the study supported the use of Fusobacterium nucleatum as As usual, the features of bacterial populations are specified by biomarker of CRC as well as a marker of early CRC.51–53 The su- tissue, colonic lumen, and feces, which themselves have different pernatant of a Fusobacterium nucleatum culture exhibited strong

20 DOI: 10.14218/ERHM.2019.00026 | Volume 5 Issue 1, March 2020 Sun W. et al: Microbial biomarkers of CRC Explor Res Hypothesis Med bactericidal activity against some probiotics, such as Faecalibac- (OTU87), Lachnospiraceae (OTU60), Lachnospiraceae (OTU32), terium prausnitzii and Bifidobacterium strains, that may cause Lachnospiraceae (OTU88), Lachnospiraceae (OTU44), Lachno- disease.54 spiraceae (OTU14), Bacteroides (OTU7), Bacteroides (OTU3), Bac- A study found a stepwise increase in the abundance of teroides (OTU2), Ruminococcus (OTU11), Ruminococcus (OTU16), Clostridium from normal tissues to adenoma and, finally, colonic Ruminococcaceae (OTU29), Blautia (OTU13), Blautia (OTU9), cancer.55 Therefore, Clostridium symbiosum can be used singly as Collinsella (OTU19), Firmicutes (OTU282), Enterobacteriaceae a biomarker for detecting CRC. The results showed that a stepped (OTU28), Parabacteroides (OTU49), Roseburia (OTU5), Clostridi- increase in Clostridium abundance outperformed all other con- ales (OTU10), Faecalibacterium (OTU6), Anaerostipes (OTU8), ventional screening methods, such as carcinoembryonic antigen Porphyromonas (OTU105), and FIT with a 100 ng/mL cutoff. We (referred to as CEA) and FIT, both of which were known to have infer that 16 of these were members of the Firmicutes phylum.23 The greater sensitivity (area under the curve (AUC) = 0.73 vs. 0.38– multitarget microbiota test detected 91.7% of cancers and 45.5% of 0.54 for other methods). In combination with FIT, the predicting adenomas, compared to 75.0% and 15.7% by FIT, respectively.23 accuracy of Clostridium symbiosum increased significantly, with Thus, screening methods for colorectal lesions need to be continu- an AUC of 0.803. Moreover, with the combination of Clostridium ally optimized to find the optimal screening program. symbiosum and Fusobacterium nucleatum, FIT (200 ng/mL) and A recent study analyzed a total of 969 fecal mate genomes, in- CEA (3.3 ng/mL) achieved a performance an AUC of 0.876.55 cluding 5 publicly available data sets, 2 new cohorts, and 2 valida- A meta-analysis of a publicly available dataset showed that the tion cohorts.58 Twenty-four species with high RF accuracy features depletion of Faecalibacterium, Bacteroides, and Romboutsis could were selected; these were: Actinomyces graevenitzii, Alistipes spp., be a potential biomarker for CRC.52 A Chinese study supported the Anaertuncus colihominis, Bifidobacterium longum, Clostridium identification of CRC and differentiation from the healthy group via hathewayi, Clostridium leptum, Clostridium symbiosum, Dialis- 76 fecal potential biomarkers; the CRC group was enriched with ter invisus, Eubacterium eligens, Escherichia coli, Fusobacterium 18 operational taxonomic units (OTUs); moreover, fecal metabo- nuleatum, Gemella morbillorum, Lachnospiaceae 3157FAA CT1, lites in healthy patients and cancer groups are different.56 Another Lachnospiaceae 8157FAA, Lachnospiaceae5163FAA, Parvimonas meta-analysis of eight studies from different countries and regions spp., Peptostreptococcus stomatis, Porphyromonas assccharo- identified 29 species as biomarkers of CRC.57 Furthermore, the mi- lyica, Pravimonas micra, Prevotella copri, Ruminococcus gnavus, crobial species can predict taxonomic and functional microbiome Subdoligranulum spp., Streptococcus parasanguinis, and Strepto- CRC signatures as a basis for future diagnostics. (These data are coccus salivarius. The predictive microbiome signatures trained summarized in Table 1.)49,52–55,57–59 on different data sets consistently showed high accuracy. Nonethe- There have been attempts to explore whether a combination of less, it appears their model has lower sensitivity and specificity bacterial markers could increase the AUC value in predicting CRC. values for predicting CRA.58 Zackular et al.20 analyzed fecal microbiota from healthy subjects Despite significant advances in the study of the effects of gut and patients with CRA or CRC; they found substantial alterations microbiota on colorectal lesions, few studies have investigated in the gut microbiome of patients with CRA or CRC compared to the gut microbiota after the treatment of patients with colorectal healthy controls, with a classification accuracy for CRC of 0.798 lesions. The tumor-node-metastasis (commonly known as TNM) AUC. Therefore, combining microbial markers with known clini- international staging system has always been considered the gold cal risk factors can significantly improve the differentiation ability standard to determine CRC prognosis. In addition, findings of ane- of the tests.21 uploidy, tumor-infiltrating lymphocytes, allelic loss inDCC , TP53, By using a LASSO logistic regression classifier, a model con- APC and MCC genes, TP53 gene mutations, CD44 protein expres- structed with fecal microbiota could predict CRC with an accura- sion, high levels of thymidylate synthetase, microsatellite instabili- cy of 0.82.22 Another study from China demonstrated that a model ty, and gene studies of both RAS and BRAF are independent, strong constructed with microbiota showed better value than FOBT.60 prognostic factors. In addition, C-reactive protein, overexpression Sze et al.49 constructed an RF classification model using 8 taxa of the CEA in tumors, and circulating free DNA are considered based on significant odds ratios obtained in a meta-analysis of to be associated with the prognosis of patients with CRC.19 Ai et 14 studies from various geographical regions. Their analysis in- al.59 analyzed the composition of fecal microbiota in 124 samples cluded 1,737 fecal samples and 492 tissue samples. These encom- from France and 99 samples from Austria. They excluded unre- passed Fusobacterium, Parvimonas, Porphyromonas, Peptostrep- lated and redundant features during feature selection by mutual tococcus, Clostridium XI, Enterobacteriaceae, Ruminococcus, information, and trained an RF classifier on a large mate genomic and Escherichia. The combined model had an AUC of 0.75 based data set of patients with CRC and healthy individuals. The RF clas- on fecal samples. Similarly, the AUC was 0.77 in tissue samples sifier assembled from published reports as well as extracted and used in a combined model trained by Dorea, Blautia, and Weis- analyzed information from learned decision trees. Porphyromonas sella.49 Their model could successfully classify CRC with high asaccharolytica, Peptostreptococcus stomatis, Fusobacterium, accuracy when models trained using one data set were tested on Parvimonas spp., Streptococcus vestibularis, and Flavonifractor other data sets.49 plautii were determined to be key microbial species associated Nonetheless, a test that objectively reflects the early gut changes with CRCs.59 in CRA or CRC fully is needed. New noninvasive screening meth- By using an RF model based on fecal microbiota, Sze et al.61 ods are needed to increase the sensitivity and specificity for CRC found significant differences between the pre- and post-treatment detection. Baxter et al.23 established an RF classification model by samples of 67 individuals, including those with adenoma (n = 22), using the relative abundance of gut microbiota and FIT from stool advanced adenoma (n = 19), and carcinoma (n = 26). Fusobac- samples of 490 patients. They observed that the sensitivity and speci- terium, Porphyromonas, and Parvimonas were significantly de- ficity of a combination model of bacterial abundance and FIT, which creased in the post-treatment samples.61 Furthermore, in a mouse they obtained by incorporating data on hemoglobin concentration model, interventions of microbiota with antibiotics led to a dra- (determined by FIT), and bacterial relative abundances (multitarget matic decrease in the tumor burden in the colon.19 In addition, as microbiota test) for CRC and CRA were better than those with FIT an important probiotic, Bifidobacterium has been shown to enrich alone.23 Their model used 23 OTUs, including Lachnospiraceae the gut microbiome in healthy individuals.61 Moreover, studies

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Table 1. Characteristics of the bacteria species as potential biomarkers for CRC Phyla Genus Abundance Reference Firmicutes Clostridiales Increased Xie YH (2017)55 Unknown Clostridiales Increased Wirbel J et al. (2019)57 Clostridium bolteae/clostridioforme Increased Wirbel J et al. (2019)57 Clostridium symbiosum Increased Wirbel J et al. (2019)57; Thomas AM et al. (2019)58 Clostridium leptum Increased Thomas AM et al. (2019)58 Clostridium hathewayi Increased Thomas AM et al. (2019)58 Unknown Clostridiales Increased Wirbel J et al. (2019)57 Subdoligranulum spp. Decreased Thomas AM et al. (2019)58 Unknown Peptostreptococcaceae Increased Wirbel J et al. (2019)57 Peptostreptococcus stomatis Increased Thomas AM et al. (2019)58; Ai D et al. (2019)59 Anaerococcusobesiensis/vaginalis Increased Wirbel J et al. (2019)57 Anaertuncus colihominis Increased Thomas AM et al. (2019)58 Gemella morbillorum Increased Wirbel J et al. (2019)57; Thomas AM et al. (2019)58 Unknown Dialister Increased Wirbel J et al. (2019)57 Hungatellahathewayi Increased Wirbel J et al. (2019)57 Parvimonas species Increased Wirbel J et al. (2019)57; Ai D et al. (2019)59 Parvimonas spp. Increased Thomas AM et al. (2019)58 Pravimonas micra Increased Thomas AM et al. (2019)58 Ruminococcus torques Increased Wirbel J et al. (2019)57 Ruminococcus gnavus Decreased Thomas AM et al. (2019)58 Uubdoligranulum species Increased Wirbel J et al. (2019)57 Lachnospiaceae 3157FAA CT1 Increased Thomas AM et al. (2019)58 Lachnospiaceae 8157FAA Decreased Thomas AM et al. (2019)58 Lachnospiaceae5163FAA Decreased Thomas AM et al. (2019)58 Alistipes spp. Decreased Sze MA et al. (2018)49 Dialister invisus Decreased Thomas AM et al. (2019)58 Eubacterium eligens Decreased Thomas AM et al. (2019)58 Streptococcus parasanguinis Increased Thomas AM et al. (2019)58 Streptococcus salivarius Decreased Thomas AM et al. (2019)58 Streptococcus vestibularis Increased Ai D et al. (2019)59 Bacteriodetes Bacteroides Decreased Mangifesta M et al. (2018)53 Unknown Porphyromonas Increased Wirbel J et al. (2019)57 Porphyromonas uenonis Increased Wirbel J et al. (2019)57 Porphyromonas somerae Increased Wirbel J et al. (2019)57 Porphyromonas asaccharolytica Increased Ai D et al. (2019)59; Thomas AM et al. (2019)58 Prevotella intermedia Increased Wirbel J et al. (2019)57 Prevotellan igrescens Increased Wirbel J et al. (2019)57 Prevotella copri Increased Thomas AM et al. (2019)58 Flavonifractor Increased Ai D et al. (2019)59 plautii Proteobacteria Faecalibacterium Decreased Mangifesta M et al. (2018)53 Escherichia coli Increased Thomas AM et al. (2019)58

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Table 1. Characteristics of the bacteria species as potential biomarkers for CRC - (continued) Phyla Genus Abundance Reference Fusobacteria Fusobacterium nucleatum Increased Tunsjø HS et al. (2019)52; Mangifesta M et al. (2018)53; Xie YH (2017)55; Bullman S et al. (2017)54; Thomas AM et al. (2019)58; Ai D et al. (2019)59 F. nucleatum subspecies animalis Increased Wirbel J et al. (2019)57 F. nucleatum subspecies nucleatum Increased Wirbel J et al. (2019)57 F. nucleatum subspecies vincentii Increased Wirbel J et al. (2019)57 Fusobacterium species Increased Wirbel J et al. (2019)57 oral taxon 370 Actinobacteria Actinomyces graevenitzii Increased Thomas AM et al. (2019)58 Bifidobacterium longum Decreased Thomas AM et al. (2019)58 Tenericutes Solobacterium moorei Increased Wirbel J et al. (2019)57 have shown that Bifidobacterium can inhibit the growth of intes- spite the close relation of bacteria to colorectal lesions, gut mi- tinal carcinogenic bacteria and protect intestinal mucosa, which crobes can interact with each other. Through RF modeling, Hanni- makes it an important probiotic for clinical application (Table gan et al.63 found that viruses indirectly affect cancer progression 2).20,23,42,44,45,49,59,61,62 by altering bacterial host communities. Nakatsu et al.64 conducted The gut microbiota includes bacteria, viruses, and fungi. De- a study on the survival prediction of CRC by viruses. Their study

Table 2. Important studies in dysbiosis of gut microbiota in CRC, bacterial features, detection methods and models Sample Bacteria Participants Models Bacteria, genus sources features Flynn et al. Healthy (n = 20) Mucosal, Random forest Enterobacteriaceae, Bacteroides Enriched in (2018)42 feces and classification and Pseudomonas the proximal luminal models contents Finegoldia, Murdochiella, Peptoniphilus, Colonic mucous Porphyromonas and Anaerococcus increased in the distal colon Chen et al. Healthy (n = 344), Feces NA Enterococcus and Streptococcus Increased (2013)44 A-CRA groups (n = 344) in A-CRA; Clostridium, Roseburia, and Eubacterium Decreased in A-CRA Zackular et Healthy (n = 30), Feces NA Ruminococcaceae, Clostridium, Increase in al. (2014)20 colonic adenoma (n Pseudomonas, and Porphyromonadaceae CRA patients = 30), and colonic adenocarcinoma (n = 30) Bacteroides, Lachnospiraceae, Decreased in Clostridiales, and Clostridium CRA patients Goedert et Normal patients (n Feces Random forest Proteobacteria Enriched in CRA al. (2015)45 = 24), CRA (n = 20), CRC (n = 2), and other conditions (n = 15) Ai D et al. France (n = 124); Feces Random forest Porphyromonas asaccharolytica, Enriched in CRC (2019)59 Austria (n = 99) Eubacterium hallii, Parvimonas spp., Fusobacterium 7, Prevotella melaninogenica, Streptococcus vestibularis, Prevotellacopri, Peptostreptococcus stomatis, Fusobacterium nucleatum, Parvimonas micra, Gemella morbillorum, Flavonifractor plautii, Fusobacterium, Clostridium SS2

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Table 2. Important studies in dysbiosis of gut microbiota in CRC, bacterial features, detection methods and models- (continued) Sample Bacteria Participants Models Bacteria, genus sources features Baxter et CRC (n = 120), CRA Feces Random forest Pophyromonas assaccharolytica, Increased in CRC al. (2016)23 (n = 198), no colonic Fusobacterium nucleatum, Parvimonas lesions (n = 172) micra, Peptostreptococcus stomatis, Gemella spp. and Prevotella spp. Sze MA et Control (n = 1145), CRA Feces and Random forest Fusobacterium, Parvimonas, Significant ORs al. (2018)49 (n = 521), CRC (n = 536) tissue Porphyromonas, Peptostreptococcus, Clostridium XI, Enterobacteriaceae, Ruminococcus and Escherichia, Dorea, Blautia and Weissella Sze MA et Adenoma (n = 22), Feces Random forest Fusobacterium, Porphyromonas, Decrease in al. (2017)61 advanced adenoma Parvimonasare post-treatment (n = 19), CRC (n = 26)

CRC, colorectal cancer; CRA, colorectal adenoma; NA, not available; OR, odds ratio. found a combination of four classification markers that reduce Funding patient survival in CRC.43 Further research on CRC-related viral group characteristics could lead to the development of new tools to This work was supported by funding from the National Natural identify individuals with CRC or to predict outcomes. Science Foundation of China (No. 81602144, 31870777) There are some limitations of our meta-analysis. Despite the available data on CRC detection through the gut microbiome, there is a lack of consensus on which features are most informative. Conflict of interest The contradictory reports from some studies could be attributed to differences inherent among study populations, procedures for fecal collection and storage, DNA extraction and amplification, The authors have no financial interests or any conflict of interests sequencing, and bioinformatics processing methods. Moreover, to disclose. recent studies used their models only to differentiate CRC from CRA or healthy individuals. Further studies are required to identify methods that can differentiate CRC from other colonic diseases, Author contributions such as inflammatory bowel diseases. Manuscript writing (WLS, QJD); critical revision of the manuscript for important intellectual content (WLS, LLW, QYZ); administra- Future research directions tive, technical, or material support, study supervision (QJD).

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0177-8. [57] Wirbel J, Pyl PT, Kartal E, Zych K, Kashani Al, Milanese A, et al. Meta- [49] Sze MA, Schloss PD. Leveraging existing 16S rRNA gene surveys to analysis of fecal meta genomes reveals global microbial signatures identify reproducible biomarkers in individuals with colorectal tu- that are specific for colorectal cancer. Nat Med 2019;25(4):679–689. mors. mBio 2018;9(3):e00630–18. doi:10.1128/mBio.00630-18. doi:10.1038/s41591-019-0406-6. [50] Wong SH, Zhao L, Zhang X, Nakatsu G, Han J, Xu W, et al. Gavage of fe- [58] Thomas AM, Manghi P, Asnicar F, Pasolli E, Armanini F, Zolfo M, et al. cal samples from patients with colorectal cancer promotes intestinal Metagenomic analysis of colorectal cancer datasets identifies cross- carcinogenesis in germ-free and conventional mice. Gastroenterol- cohort microbial diagnostic signatures and a link with choline deg- ogy 2017;153(6):1621–1633.e6. doi:10.1053/j.gastro.2017.08.022. radation. Nat Med 2019;25(4):667–678. doi:10.1038/s41591-019- [51] Guo SH, Li LF, Xu BL, Li MH, Zeng QY, Xiao H, et al. A simple and novel 0405-7. fecal biomarker for colorectal cancer: ratio of to probiotics popula- [59] Ai D, Pan H, Han R, Li X, Liu G, Xia LC. Using decision tree aggrega- tions, based on their antagonistic effect. Clin Chem 2018;64(9):1327– tion with random forest model to identify gut microbes associated 1337. doi:10.1373/clinchem.2018.289728. with colorectal cancer. Genes (Basel) 2019;10(2):E112. doi:10.3390/ [52] Tunsjø HS, Gundersen G, Rangnes F, Noone JC, Endres A, Bemanian genes10020112. V. Detection ofFusobacterium nucleatum in stool and colonic tissues [60] Ai L, Tian H, Chen Z, Chen H, Xu J, Fang JY. Systematic evaluation of from Norwegian colorectal cancer patients. Eur J Clin Microbiol Infect supervised classifiers for fecal microbiota-based prediction of colo- Dis 2019;38(7):1367–1376. doi:10.1007/s10096-019-03562-7. rectal cancer. Oncotarget 2017;8(6):9546–9556. doi:10.18632/onco- [53] Mangifesta M, Mancabelli L, Milani C, Gaiani F, de’Angelis N, target.14488. de’Angelis GL, et al. Mucosal microbiota of intestinal polyps reveals [61] Sze MA, Baxter NT, Ruffin MT4th, Rogers MAM, Schloss PD, et al. putative biomarkers of colorectal cancer. Sci Rep 2018;8(1):13974. Normalization of the microbiota in patients after treatment for co- doi:10.1038/s41598-018-32413-2. lonic lesions. Microbiome 2017;5(1):150. doi:10.1186/s40168-017- [54] Bullman S, Pedamallu CS, Sicinska E, Clancy TE, Zhang X, Cai D, et 0366-3. al. Analysis of Fusobacterium persistence and antibiotic response in [62] Pinzone MR, Celesia BM, Di Rosa M, Cacopardo B, Nunnari G. colorectal cancer. Science 2017;358(6369):1443–1448. doi:10.1126/ Microbial translocation in chroni liver diseases. Int J Microbiol science.aal5240. 2012;2012:694629. doi:10.1155/2012/694629. [55] Xie YH, Gao QY, Cai GX, Sun XM, Zou TH, Chen HM, et al. Fecal [63] Hannigan GD, Duhaime MB, Ruffin MT4th, Koumpouras CC, Schloss Clostridiumsym biosum for noninvasive detection of early and ad- PD. Diagnostic potential and interactive dynamics of -the colo vanced colorectal cancer: test and validation studies. EBio Medicine rectal cancer virome. mBio 2018;9(6):e02248–18. doi:10.1128/ 2017;25:32–40. doi:10.1016/j.ebiom.2017.10.005. mBio.02248-18. [56] Yang Y, Misra BB, Liang L, Bi D, Weng W, Wu W, et al. Integrated [64] Nakatsu G, Zhou H, Wu WKK, Wong SH, Coker OO, Dai Z, et al. Al- microbiome and metabolome analysis reveals a novel interplay be- terations in enteric virome are associated with colorectal cancer tween commensal bacteria and metabolites in colorectal cancer. and survival outcomes. Gastroenterology 2018;155(2):529–541.e5. Theranostics 2019;9(14):4101–4114. doi:10.7150/thno.35186. doi:10.1053/j.gastro.2018.04.018.

26 DOI: 10.14218/ERHM.2019.00026 | Volume 5 Issue 1, March 2020 Review Article

The Effect of Honey as a Treatment for Oral Ulcerative Lesions: A Systematic Review

Maddison Hunter1,2, Jane Kellett1, Nathan M. D’Cunha1, Kellie Toohey1,2,3, Andrew McKune1,3,4 and Nenad Naumovski1*

1Faculty of Health, University of Canberra, Canberra, Australia; 2Prehabilitation, Cancer, Exercise and Survivorship (PACES) Research Group, University of Canberra, Canberra, Australia; 3Research Institute for Sport and Exercise (UCRISE), Faculty of Health, University of Canberra, Canberra ACT, Australia; 4Discipline of Biokinetics, Exercise and Leisure Sciences, School of Health Sciences, University of KwaZulu-Natal, Durban, KwaZulu-Natal, South Africa

Abstract

Background and objectives: A healthy oral environment features a rapid turnover rate of epithelium cells capable of regeneration and repair, with the oral epithelium contributing as a physical barrier and immune defense. How- ever, the oral cavity can be subjected to unique damage, such as ulcerations. Honey is reported as a therapeutic agent for wound healing, due to its antioxidant, antibacterial and anti-inflammatory properties.

Methods: A systematic review was performed following the PRISMA 2015 Guidelines, to assess the efficacy and safety of the therapeutic use of honey in the oral cavity. Four electronic databases were searched (PubMed, Cochrane Library, Scopus, and Web of Science) for randomized controlled trials examining the effect of honey on oral cavity conditions.

Results: In total, 2,832 records were identified, and after applying exclusion criteria, 13 studies were included. Honey was applied topically throughout, for chemotherapy or radiotherapy-induced oral mucositis (n = 11), dental wounds (n = 1), and recurrent aphthous stomatitis (n = 1), all of which are ulcerations with different pathologies. In the majority of studies (12/13), honey reduced the severity and/or duration of the condition compared with control groups (all p<0.05). However, a group treated with Manuka honey (n = 1) experienced adverse effects and considerable participant attrition.

Conclusions: Honey is an effective treatment for a range of oral ulcerative conditions. Future research should focus on compositional analysis of honeys to determine those with optimal beneficial properties, and whether Manuka honey is safe to use in the oral cavity.

Introduction teeth, tongue, gingiva, and hard and soft palettes, with the latter being composed of stratified squamous cells.1 A healthy oral environment is characterized by a rapid cellular turnover rate, with the soft The oral cavity consists of a range of structures, including the and hard palettes averaging between 14 and 24 days, respectively.2 One of the main purposes of the rapid turnover of the oral epithe- Keywords: Honey; Oral health; Ulcer; Oral cavity. lium is as a defense mechanism, reducing the rate of pathogenic 2 Abbreviations: ROS, reactive oxygen species; PRISMA, preferred reporting items microbial colonization by removing the region they bind to. It for systematic reviews and meta-analyses; PICOS, population, intervention, compara- also serves multiple additional functions, such as a physical barrier tor, outcomes and setting; OM, oral mucositis; RAS, recurrent aphthous stomatitis; and immune defense to potential pathogens and toxins and in pro- MGO, methylglyoxal; GSH, glutathione. viding an environment that is optimal for the established healthy Received: November 01, 2019; Revised: December 10, 2019; Accepted: December 1 16, 2019 microbiome. *Correspondence to: Nenad Naumovski, Faculty of Health, University of Canberra, The turnover of the mucosal cells in the oral cavity is dependent P.O. Box 5018, Bruce, ACT 2617, Australia. Tel: +61-(0)2-6206-8719; E-mail: nenad. on a balance between cell differentiation and desquamation, which [email protected] primarily acts as a pathogen defense mechanism.3 However, this How to cite this article: Hunter M, Kellett J, D’Cunha NM, Toohey K, McKune A, can become unbalanced and lead to the development of several Naumovski N. The Effect of Honey as a Treatment for Oral Ulcerative Lesions: A Sys- tematic Review. Exploratory Research and Hypothesis in Medicine 2020;5(1):27–37. conditions, including hyperplasia and dysplasia, and a reduced doi: 10.14218/ERHM.2019.00029. rate of proliferation, which can lead to the development of ulcera-

Exploratory Research and Hypothesis in Medicine 2020 vol. 5 | 27–37

Copyright: © 2020 Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 4.0 International License (CC BY-NC 4.0), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Explor Res Hypothesis Med Hunter M. et al: Honey as a treatment for oral ulcerations tions.2 Additionally, the oral mucosa is subject to masticatory and Methods abrasive damage, with this oral epithelium disruption having the 1 possibility of leading to the development of oral diseases ; some This systematic review has been completed in accordance with of these common oral conditions include dental caries, denture 4 the Preferred Reporting Items for Systematic Reviews and Meta- wounds, and ulcerations. Analyses (PRISMA) 201520 Guidelines and was registered in the The treatment of adverse health conditions in the oral cavity International Prospective Register of Systematic Reviews (PROS- is focused on therapies that simultaneously eradicate pathogenic PERO Number: CRD42019128480). The systematic review was microbial growth, stimulate the wound healing process, and reduce completed following the Population, Intervention, Comparator, the sensation of pain. One possible treatment option that fulfils Outcomes and Setting (PICOS) approach as follows: these criteria is honey, which has been demonstrated to be effective 5 Population: Human populations with an existing oral health in the treatment of a range of wounds, such as burns and ulcers. condition, or at risk of developing an oral health condition. These potentially beneficial effects can be attributed to its antioxi- 5–7 Intervention: Randomized controlled trials utilizing topically- dant, antibacterial, and anti-inflammatory properties. applied undiluted honey from any floral source as a treatment or The composition of honey, in particular the exact proportions preventative agent without the concurrent use of other treatments. of each of its constituents, is dependent on the floral source of the 8 Only trials containing sample sizes over 12 participants were in- pollen used to produce the honey. Overall, honey is a mixture of cluded; however, there was no time limit placed on study duration. several different categories of compounds, including polyphenols, Comparator: Human comparators being treated with a placebo, ascorbic acid, carotenoids, organic acids, enzymes and other pro- control or routine standard oral care. 9 teins, all of which contribute to honey being a viable nutritional Outcomes: Effects of honey treatment on severity of oral health 10 source of antioxidants. Honey displays strong antioxidant activity conditions, improvements in healing time, and any adverse effects and has the ability to reduce the effects of oxidative reactions, which to participants caused by the use of honey during and following the 8 produce free radicals and reactive oxygen species (ROS). In addi- intervention protocol. tion, honey is also reported to exhibit strong antibacterial effects, Setting: Any. which can be attributed to its physicochemical properties, such as high osmolarity (due to high sugar levels)5 and relatively low pH11 due to the presence of several organic acids. Additionally, activation Search strategy of glucose oxidase during the dilution of honey causes the production of hydrogen peroxide from glucose metabolism, providing an The following four electronic databases were searched; Scopus, Pub- undesirable environment for growth and proliferation of bacterial Med, Cochrane Library and Web of Science, for manuscripts pub- cells12 and potentially mediating the wound healing process.13 lished since journal inception until June 2019. Furthermore, searches The concomitant effects of the antioxidant and antibacterial were also conducted to identify any new publications prior to sub- properties of honey also contribute to its anti-inflammatory ef- mission of this manuscript (end of October 2019). The searches were fects of reducing excessive inflammation, possibly resulting in a limited to studies that were conducted on humans, were randomized wound-healing effect.6 As the presence of ROS has been deter- controlled trials, and were published in peer-reviewed journals in the mined to lead to the production of inflammation,14 the antioxidant English language. The reference lists of included articles were also activity of honey can also contribute to a reduction of an excessive searched to locate any additional articles relating to the topic. The inflammatory response.7 Moreover, honey’s capability to prevent search terminology used included “honey*” and “oral”, “mouth”, “in- the development of bacterial infections (through providing an en- traoral”, “cavity”, “bucc*”, “tooth”, “orthodont*”, “saliv*”, and “dis- vironment that cannot support bacterial growth and proliferation) ease”, “condition”, “lesion”, “wound”, “carie*”, “plaque”, “ulcer”, can further assist with the inhibition of inflammation.5 Further- “bacter*”, “micro”, “count”, with the terms AND and OR between more, the application of honey to a wound has been demonstrated every term. to stimulate the production and release of pro-inflammatory cy- tokines that assist in the wound healing process, such as interleu- kin-1 and tumor necrosis factor-alpha.5 The topical application of Selection criteria honey to various injured tissues has also been shown to stimulate wound repair through the stimulation of growth of epithelial cells, Studies were included if they were original research, randomized reduction of edema, and wound debridement.6,7 controlled trials published in English and in peer-reviewed jour- It is well established that an individual’s oral health impacts their nals. Articles were only considered if they utilized undiluted quality of life.4,15,16 Poor oral health is shown to affect physical honey without any other interventions as a treatment against a and psychological wellbeing through condition-related reductions pre-existing condition of the mouth or as a preventative agent in a in functionality, including the inability to consume adequate nutri- group more susceptible to developing an oral condition. Further- tion and communicate, undesirable effects on physical appearance, more, articles were required to have a control or placebo group in addition to causing pain.15 These conditions, particularly those as a comparator. Any articles that did not meet these criteria were related to the soft and hard palette such as ulcerations, have been excluded from this review. There were no time limitations posed reported as treated using various types of honey in several different on the duration of the studies, as oral conditions have different clinical populations.17–19 These collective data suggest honey as a durations and pathological severities. For the purpose of this sys- reliable food product, being both financially affordable and readily tematic review, a condition of the mouth was defined as a symp- available, that could be used in the treatment of inflammatory and tomatic problem unique to the oral cavity, such as ulcers or dental microbial damage of the epithelial lining of the oral cavity. There- wounds. fore, this systematic review aimed to assess the efficacy of honey as a therapeutic agent used for the treatment of epithelial damage due to the inflammatory and microbial response in the oral cavity. Data extraction Additionally, this review will aim to evaluate the safety of honey in different clinical populations. All articles identified in the initial electronic search were imported

28 DOI: 10.14218/ERHM.2019.00029 | Volume 5 Issue 1, March 2020 Hunter M. et al: Honey as a treatment for oral ulcerations Explor Res Hypothesis Med into an EndNote library (vX8.2; Clarivate Analytics, USA) and Study characteristics duplicates removed. We excluded articles that did not investigate honey or oral health conditions. Following this, articles were ex- The characteristics of the included studies are presented in Table cluded based on study design, and only if pure undiluted honey 1.22–34 The total number of participants recruited in the included was utilized in the absence of any other treatments, with the excep- studies was 634, with sample sizes ranging from 20 to 106 par- tion of patient-required analgesics. ticipants. Of these, 11 studies investigated oral mucositis (OM) Two authors (MH and JK) independently screened each of the in 579 cancer patients,22–32 1 investigated 35 participants with articles based on the selection criteria. The titles were screened denture-induced ulcers,33 and 1 investigated 20 participants with to determine relevance, followed by screening of abstracts to de- recurrent aphthous stomatitis (RAS).34 Although the selection termine studies suitable for full-text analysis. Any disagreements criteria of this review was designed to include a broad range of regarding the selection criteria were resolved through discussion oral health conditions, the intervention was common across stud- until consensus was reached or in consultation with a third author ies, with pure, undiluted honey being applied topically to affected (NN). At this stage, the reference lists of the articles considered for areas in the mouths of patients in the treatment groups. The type full-text review were searched for additional articles. The extrac- of honey and their floral sources used in the included studies was tion of relevant data was completed by two authors (MH and JK) largely unspecified26,28,30,33,34; however, two studies stated that the working independently in predesigned tables including the infor- honey was sourced from a commercial supermarket.23,27 Five of mation on author name, year of publication, condition assessed, the studies identified the local flora that was used in the production honey application protocol, control or placebo, number of partici- of the honey used,22,24,29,31,32 and one study specified their use of pants, and outcomes. Manuka honey.25 In the included articles, participants were instructed to main- Assessment of bias tain good oral hygiene (n = 6)22,24–26,31,32 or prescribed the use of antibacterial, antifungal or analgesic solutions (n = 3).22,23,32 However, this was not specified in six studies.27–30,33,34 For trials The risk of bias was independently assessed according to the investigating OM (n = 11), the studies investigated head and neck Cochrane Collaboration’s Tool for Assessing Bias, which utilizes cancer patients in adult populations24–32 and pediatric patients re- the domains of selection bias through random sequence genera- ceiving treatment for a range of cancers, including leukemia.22,23 tion and allocation concealment, performance bias through blind- Participants with radiotherapy-induced OM were included in six ing of participants and personnel, detection bias through blinding studies,24,25,27–29,31 with 1 study having investigated chemother- of outcome assessment; attrition bias through the addressing of apy-induced OM22 and 4 having determined the effect of honey any incomplete data, reporting bias through selective reporting, on concurrent chemo-radiotherapy-induced OM.23,26,30,32 These and any other sources of bias.21 The risk of bias was assessed studies utilized routine oral care,23,24,30,32 saline rinses of differ- independently by two authors (MH and JK), with a third author ent concentration ranges (0.9%,27 0.09%,31 and unspecified26), (NN) introduced in the case of any disagreements, where appro- 28 priate. water, and a variety of anesthetic and analgesic solutions (7.5% benzocaine gel,22 15% benzydamine hydrochloride,27 lignocaine gel29) serving as controls, while only one study utilized a placebo Data analysis gel25 and another developed their own mixture.22

Due to the broad inclusion criteria used for this review to include Interventions a number of ulcerative lesions, which includes a variety of population groups and intervention protocols, this review consists of con- 24 siderable heterogeneity. As such, a meta-analysis was not deemed In a study by Biswal et al., patients receiving radiotherapy for appropriate. head and neck cancer were provided with 20 mL of honey to smear on the inside of their mouths, and instructed to swallow it slowly in order to coat both the oral and pharyngeal mucosa, with ap- Results plication occurring 15 min before and after they received radiotherapy and 6 h after receiving radiotherapy, for the duration of the treatment cycle. This study design was also replicated by other Study selection studies included in this review,27,29–32 with Jayalekshmi et al.28 and Howlader et al.26 with slight modifications, namely use of 15 The initial search of the electronic databases (Fig. 120) resulted mL of honey28 and consumption of additional honey to potentially in 2,838 articles, with the secondary searches identifying 1 contribute to serum antioxidant levels.26 article. Following removal of articles due to duplicates (n = The remaining studies investigating OM utilized study designs 1,181), study design (n = 115), absence of honey investigation similar to those above, topically coating the oral cavity with hon- (n = 1,416) and absence of oral health intervention (n = 80), ey; however, the amounts of honey used and timing of applica- 46 studies were subjected to full text-analysis. Articles were tion were different. In trials by Al Jaouni et al.23 and Hawley et further excluded due to use of diluted honey with or without al.25 durations were similar of treatments used to Biswal et al.24 the use of additional therapies (n = 14), unsuitable oral health and participants were followed throughout the duration of their conditions (n = 12) and study design (n = 8), with 13 studies oncology treatments. In addition, Hawley et al.25 maintained the fitting the inclusion criteria.22–34 An article investigating the use intervention for 7 days following final radiotherapy treatment, with of honey to assist in the recovery of dental extraction surgery35 all studies applying the honey intervention four times daily. The was considered but it was determined that since this wound is volumes of honey and subsequent saline rinse were not specified due to surgery, and not a condition unique to the oral cavity, this by Al Jaouni et al.23 but Hawley et al.25 specified the prescription article was excluded. of 5 mL of applied honey followed by a fluoride rinse. Abdulrhman

DOI: 10.14218/ERHM.2019.00029 | Volume 5 Issue 1, March 2020 29 Explor Res Hypothesis Med Hunter M. et al: Honey as a treatment for oral ulcerations

Fig. 1. Search strategy and article selection process according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) Guidelines.20 et al.22 included participants with established OM using undiluted honey in the treatment of RAS in healthy adults. Following an ini- honey (0.5 g/kg) and having two control groups (receiving a cus- tial assessment, participants in the treatment group (n = 10) were tom-made mixture (placebo) or an analgesic control). All groups given honey and control groups (n = 10) were given salicylate gel, applied their mixtures three times daily for 10 days or until healing a standard prescribed treatment for RAS, to apply to their ulcers 3 of ulceration. times daily for 5 days. Ulcer size was determined using calipers, The study by Ceylan et al.33 investigated the use of honey in and a visual analog score was used to assess pain before and after the treatment of traumatic ulcers that were caused by newly fit- the intervention. ted dentures in 35 healthy, previously edentulous participants. Of these, 25 participants were provided with honey and instructed to smear directly onto the ulcers three times daily for 3 days follow- Study results ing denture fitting. The control group (n = 10) were provided with a saline rinse to use via the same protocol as the treatment group. The majority of studies investigating the use of honey in the treat- The ulcer areas were measured in all participants using planimetry ment of OM in an oncology population were successful in alleviat- on the first and third day following initial denture fitting. ing negative symptoms, including reducing the severity or duration The study by Halim et al.34 investigated the effectiveness of of the condition (all p<0.05) (Table 1).22–24,26–29,31,32 In these stud-

30 DOI: 10.14218/ERHM.2019.00029 | Volume 5 Issue 1, March 2020 Hunter M. et al: Honey as a treatment for oral ulcerations Explor Res Hypothesis Med p < 0.05) p < 0.00058) p < p < 0.01) p < 0.01) p < 0.05) week post radiotherapy ( p = 0.0001) radiotherapy post week th Reduced Grade 3 and 4 OM in honey group group 3 and 4 OM in honey Grade Reduced hospitalization 55%) and reduced (20% vs. bodyweight, ( p < 0.05), increased pain decreased onset, delayed Reduction in OM in week 4 ( in OM Reduction 0.05) and 5 6 ( 3 OM. Grade 61.54% developed Control: 3 OM Grade 9.09% developed Treatment: Difference in G3 and G4 mucositis was 20% in G3 and G4 mucositis Difference ( and 75% control (treatment) Recovery time reduced for Grade for Grade reduced time Recovery 1 2 OM (Group 3 OM for Grade reduced time Recovery 2 ( 1 and Group in Group 3, 2 and Group Combining Group healing had fastest honey HOPE: burning sensation Results Delayed onset for Group 1 and reduced severity severity 1 and reduced Group for onset Delayed 3 ( 2 and Group Group to compared Difference between groups in the groups between Difference ( p < 0.05) assessment patients’ earlier than control symptoms relieved Honey and salicylate honey between No difference ( p > 0.05); and ulcer size pain score for gel in pain and ulcer size improvement observed 3 ( p = 0.77) for OM Grade groups between No difference nausea; 57% to and placebo contributed Honey rate and 52% placebo dropout treatment 0.05) in mucositis grade weeks weeks grade ( p > 0.05) in mucositis No difference improvements administered); 1–4 (radiotherapy in 4 n = 14 n = 30 n = 30 n = 20 n = 20 n = 20 15 mL water rinsed 15 min before and rinsed 15 min before 15 mL water radiotherapy; after and 6 h after, Routine oral care; care; oral Routine Comparator Group 2: 15mL 0.15% benzydamine 2: 15mL 0.15% benzydamine Group rinsed 15 min hydrochloride after and 6 h and after, before 5 min; n = 20 for radiotherapy 3: 20 mL 0.9% saline rinsed 15 Group after and 6 h and after, min before 5 min; n = 20 for radiotherapy Salt water rinse three times daily; n = 10 rinse three Salt water applied three gel Salicylate n = 10 for 5 days; times daily times applied four 5mL placebo gel saline rinse; fluoride daily following bedtime; n = 52 at treatment Group 2: 0.25 g/kgGroup of 4:2:1 mixture extract oil-propolis olive of honey, (HOPE) applied topically and beeswax for healing or times daily until three care; oral and routine 10 days care; oral Routine Saline rinsed in mouth 15 min before radiotherapy; and after Group 3: benzocaine 7.5% gel 7.5% gel 3: benzocaine Group times three mucosa oral applied to care; oral daily and routine n = 14 n = 20 n = 20 n = 20 n = 20 n = 30 Intervention Group 1: 20 mL honey 1: 20 mL honey Group rinsed 15 min before after and 6 h and after, radiotherapy; rinsed 15 min 15 mL honey and 6 h and after, before radiotherapy; after Group 1: 0.5 g/kgGroup applied the affected to topically times three mucosa oral healing or daily until and routine 10 days for care; oral applied topically Honey followed mucosa, oral to saline rinse alkaline by six times daily and to four care; oral routine applied to 20 mL honey immediately mouth before, after and 6 h after, radiotherapy; applied to Honey 3 times daily wound n = 25 3 days; for times applied three Honey n = 10 5 days; daily for applied four 5 mL honey saline following times daily rinse; fluoride treatment bedtime; n = 54 at applied to 20 mL honey and mouth 15 min before after and 6 hours after, radiotherapy; n = 60 n = 28 n = 40 n = 106 n = 40 Population Oral cancer cancer Oral receiving patients radiotherapy; Head and neck cancer receiving patients radiotherapy; Lymphoblastic Lymphoblastic receiving leukemia Grade chemotherapy; 2 and 3 OM; n = 90 cancer Pediatric chemo/ receiving radiotherapy; Head neck cancer receiving patients radiotherapy n = 40 denture New n = 35 wearers; Minor recurrent stomatitis aphthous n = 20 patients; Head and neck cancer receiving patients radiotherapy; Head and neck patients cancer chemo/ receiving radiotherapy; et

22 27

et 28 23 25 26 33 et al. et et al. et 24 34 et al. et (2012) (2012) (2017) (2014) (2019) (2016) (2003) Biswal Biswal Ceylan Ceylan (2010)* Al Jaouni al. Jayachandran Jayachandran al. et Study Abdulrhman al. et Halim (2013) et Hawley al. et Howlader al. Jayalekshmi Jayalekshmi al. Table 1. Summary of included study characteristics 1. Summary of included study Table

DOI: 10.14218/ERHM.2019.00029 | Volume 5 Issue 1, March 2020 31 Explor Res Hypothesis Med Hunter M. et al: Honey as a treatment for oral ulcerations

ies, OM was assessed using a grading scale, with Grade 1 repre- senting less severe cases and Grade 4 being the most severe. In these studies, the onset of OM was delayed in the honey-treatment group compared to the control group, and fewer participants in the honey group of each study developed higher gradings of the condition. One OM study did not report any significant changes from p < 0.05) pre- to post-treatment nor any difference between the two groups (treatment and control); however, there was a self-reported observed difference in the OM.30 All of these studies utilized standard honey from no particular floral source, with no adverse reactions reported throughout the duration of the honey intervention, except- ing one study31 that reported the exclusion of participants who reported a stinging sensation, though the number of exclusions and effect on the study was not disclosed. A single study by Hawley et al.25 utilized Manuka honey and reported no significant difference 0.001) in treatment group; p < 0.001) in treatment in the severity or duration of OM between the honey and placebo groups (p = 0.77); however, this study experienced considerable dropout rate (57% in the honey group, 52% in the placebo group), mainly attributed to the sensation of nausea caused by the application of the honey. It was determined that honey significantly (p value unreported) Honey delayed onset in about 80%; 63% onset delayed Honey OM had severe group in control control. arm, 11/27 in 3 OM: 5/28 in intervention Grade control arm, 5/27 in 4: 1/28 in intervention Grade 4 3/20 Grade group: in honey severity Reduced arm) ( 4 (control arm), 9/20 Grade (honey Honey associated with less severe OM ( p < 0.0001) with less severe associated Honey OM intolerable developed 15/20 2: Group 1/20; 1: Group interruptions had treatment group Control ( lower OM score symptoms OM develop not did group intervention in 20% Results relieved the symptoms of the denture-induced ulcers earlier than in the control group, and (as assessed by a visual analogue score) reduced pain, with no adverse effects.33 Finally, while no participants with RAS reported adverse effects, no significant difference was found between the honey group and control group of a currently utilized treatment for ulcer size (p = 0.879) and pain (p = 0.514); however, self-reported improvements were observed.34 n = 20

n = 27 n = 20 Risk of bias in included studies n = 20

The risk of bias was assessed according to the Cochrane Risk of Bias Tool and is outlined in Table 2.22–34 The risk of selection bias through allocation concealment was high for the included studies, which was due to placebo products being utilized by 2/13 22,25 28 20 mL lignocaine gel rinsed 15 gel 20 mL lignocaine radiotherapy, and after min before bedtime; and at care; oral Standard 20 mL 0.09% saline rinsed before radiotherapy; and after care; oral Standard Comparator studies. Additionally, Jayalekshmi et al. reported the use of similar bottles for both their treatment and control groups, resulting in a low risk of selection bias. Similarly, the performance bias was high or unclear, which was attributed to the common

n = 20 n = 20 utilization of controls rather than a placebo. The detection bias for the included studies was relatively unclear but there was an overall low risk of attrition and reporting bias (Table 2). No other bias was detected. n = 20 n = 28

- (continued) Discussion Intervention 20 mL honey rinsed 15 20 mL honey and after min before and at radiotherapy, bedtime; rinsed 15 20 mL honey and after min before and at radiotherapy, bedtime; rinsed 15 min 20 mL honey and 6 h and after, before radiotherapy; after rinsed 15 min 20 mL honey and 6 h and after, before radiotherapy; after Effectiveness of honey

The primary aim of this review was to determine the effectiveness n = 40 n = 55 n = 40 n = 40 of topically-applied honey in the treatment of a range of oral health conditions. Of the 13 studies included in this review, 10 reported statistically significant reductions in the severity of the oral health condition compared to a control group, including 9 studies investigating OM22–24,26–29,31,32 and 1 investigating denture wounds.33 Population Oral cancer cancer Oral receiving patients radiotherapy; Head and neck patients cancer chemo/ receiving radiotherapy; Head and neck cancer receiving patients radiotherapy; Head and neck patients cancer chemo/ receiving radiotherapy; While one study did not report any significance30 and another re- 34

31 ported no significant difference in the improvement of RAS, the

32 oral condition was improved in both studies, demonstrating that et 30 et al. et

29 honey was just as effective as the standard prescribed treatments et al. et (2008) for these conditions. Only one study, by Hawley et al.,25 did not (2009) report any improvements in oral condition. Motallebnejad Motallebnejad al. et (2012)* al. Maiti Rashad Study Khanal (2010)

Table 1. Summary of included study characteristics characteristics 1. Summary of included study Table mucositis. OM, oral provided. *No p value It has been proposed that honey possesses wound healing abili-

32 DOI: 10.14218/ERHM.2019.00029 | Volume 5 Issue 1, March 2020 Hunter M. et al: Honey as a treatment for oral ulcerations Explor Res Hypothesis Med

ties due to its antioxidant and antibacterial properties,5–7 which includes the ability to heal ulcerations. All conditions included in this review culminated in the development of an oral ulcer, which had formed due to the loss and necrosis of epithelial tissue and result- Unclear Unclear Unclear Unclear Unclear Unclear Unclear Unclear Unclear Unclear Unclear Unclear Unclear Other bias ing in breaks in the skin or mucous membrane36; each condition

- formed due to different pathologies. Oral mucositis is a condition that develops as a toxicity during oncology treatments (chemotherapy and radiotherapy) and is characterized by inflammation, erythema, microbial colonization, and ulceration.37 Similarly, RAS is a condition of unknown cause that affects the lining of the mouth, causing erythema, burning and ulceration,34 and denture-induced Low Low Low Low Low Low Low Low Low Low Low Low Low Selective report Selective ing bias) (reporting ulcers are caused by the introduction of ill-fitting dentures causing abrasive damage to the lining of the mouth, resulting in ulcerative lesions.33 The antibacterial and antioxidant properties of honey contribute to honey’s wound-healing abilities. In the case of OM, the initiation phase is characterized by the production of ROS,37 which could potentially cause further damage to the affected areas. A study investigating participants with RAS determined that those Low Low Low Low Low Low Low Low Low Low Low Low Low Incomplete data data Incomplete addressed bias) (attrition with the condition had significantly lower salivary antioxidant levels compared to healthy controls,38 demonstrating that the ul-

- cerations potentially produced ROS, causing a reduction in the antioxidant compounds present in the mouth. This highlights a possible mechanism for the effectiveness of honey as a treatment for the ulcerations discussed in this review. Potentially, the presence of the antioxidant compounds provided by the topically-applied honey can reduce the effects of ROS activity during the de- Unclear Unclear Unclear Unclear Unclear Low Low Unclear Unclear Low Unclear Low Unclear Blinding of out assessment come bias) (detection velopment of ulcerations. Furthermore, the consumption of honey increases total systemic plasma antioxidant levels, and swallow- - ing of the topically-applied honey may contribute to longer lasting antioxidant effects,10 further contributing to the wound healing process.

Adverse effects Unclear High Unclear Unclear Unclear Low High Unclear High High Unclear Low High Blinding of partici pants and personnel and personnel pants bias) (performance The secondary aim of this systematic review was to determine any

- adverse effects during the topically-applied honey interventions. Only one of the included studies reported an adverse effect due to the honey intervention.25 Namely, Hawley et al.25 reported considerable dropouts due to nausea, which could be attributed to the treatments being associated with radiotherapy; however, it was Low High High High High Low High High High High High High High Allocation con Allocation cealment (selection bias) reported that the Manuka honey that was used worsened nausea symptoms and encouraged a retching motion. A study by Bardy et al.39 also used Manuka honey and reported a low compliance rate due to the honey being difficult to use, along with an undesirable taste and texture. A study by Parsons et al.40 initially had to be discontinued due to the Manuka honey not being well-tolerat- ed by participants, and in that study all participants withdrew due to a perceived stinging pain and nausea following application. In- terestingly, Abdulrhman et al.22 reported no adverse side effects Low Unclear Low Unclear Unclear Low Low Unclear Low Low Unclear Unclear Unclear Random sequence generation (selection bias) in the honey intervention arm of their study, which used honey from a different floral source to Manuka honey; however, in their 31 22 27 comparative group using their own formulation, discomfort was 28 reported, which was proposed to be due to the addition of propo- 26

23 33 34

(2008) lis. The studies investigating denture wounds and RAS did 25 32 29 (2012) 33 (2012) 24 34 30

(2016) not report any adverse effects. This further supports evidence that (2019) et al. et the application of honey to the inside of the oral cavity is effec- (2017) et al. et (2014) (2009) et al. et (2010) (2010)

(2003) tive in reducing the pathogenic bacteria that cause dental caries, (2013) (2012) 17,41,42

et al. et and has a positive effect on plaque levels and gingivitis. et al. et et al. et et al. et et al. et

et al. et These findings demonstrate that the use of honey in future studies et al. et is safe for the dental health of participants. It was also proposed that similar trials comparing two intervention groups of Manuka Study Al Jaouni Biswal Ceylan Halim al. et Hawley al. et Howlader al. et Jayachandran Jayalekshmi Khanal Maiti Motallebnejad Rashad Abdulrhuman 18 Table 2. Assessment of risk bias in included studies. 2. Assessment Table and conventional honey should be completed ; however, given

DOI: 10.14218/ERHM.2019.00029 | Volume 5 Issue 1, March 2020 33 Explor Res Hypothesis Med Hunter M. et al: Honey as a treatment for oral ulcerations the evidence of the adverse effects of Manuka honey, this may served beneficial health effects of honey, are largely unexplored. not be feasible. Four of the included studies, all investigating OM, completed in vitro analysis of the potential wound healing properties, including pH,22,24,31,32 moisture content,22,24,31,32 phytochemical profile,24,32 Issues with use and development of placebo and microbiological analysis.24,31 However, none of the studies utilized these results in their discussion on the effectiveness of The majority of the studies included in this review (n = 12) uti- honey as a treatment. A trial of this nature should be completed to lized a control or ‘standard’ prescribed care in the place of a pla- determine the individual in vitro wound healing properties of dif- cebo. Due to the nature and progression of oral health conditions ferent types of honey, so that recommendations can be offered to included in this review, particularly OM in oncology patients, it participants experiencing these oral health conditions about what would be unethical to provide no treatment to the control group, type of honey should be used to provide optimal benefits. Due to resulting in most studies in this review receiving standard care or the overall lack of analysis of the honey utilized in these trials, it a commonly-used treatment. This is also partially reflected by the is difficult to draw conclusions about the best type/s of honey and reported bias (Table 2), where many studies were graded as having the properties that are most appropriate for use in the treatment of a “high risk” of selection and performance bias, with the exception these oral conditions. of the study by Jayalekshmi et al.28 who utilized similar packaging for their honey treatment and control. If a placebo were to be used in future studies to assess the effec- Limitations tiveness of honey as a topical treatment, it would be best practice to use products which share certain physicochemical properties The inclusion criteria for this review evaluated only articles that with honey to support undetectability by participants, such as its utilized pure, undiluted honey as a treatment, as the concurrent properties of osmolarity and acidity.31 The use of such a placebo utilization of other compounds or medications has the possibility could have confounding effects, as the physicochemical properties to contribute to any potential beneficial effect provided by just the of honey, including its osmolarity, contribute to honeys wound- honey in isolation. As a result, studies that utilized honey and no healing abilities.19 One such example is brought forth by the study other treatments were included, though the use of standard care was by Bardy et al.,39 which had been excluded (during full-text re- allowed. It would be unethical to request that patients discontinue view) from our review due to not having used undiluted honey. their normal oral care routines for the duration of an intervention, In that study, golden syrup which has a similar appearance and due to the potential impact on their dental health. Additionally, the texture to honey, was assigned as a placebo alongside a Manuka conditions investigated in this review can cause considerable pain, honey treatment for OM. While there was no significant differ- which could lead to the requirement of the additional analgesics. ence observed in the severity or duration of the OM between the This may provide a reduction in the effectiveness of the honey; treatment and control groups (p > 0.05), the pathogenic bacteria however, the use of honey in isolation to routine oral care in in- present in both groups was consistent with baseline throughout the terventions such as the ones included in this review is impractical. study period, demonstrating that both honey and the placebo controlled the bacterial growth. This further supports the notion of the importance of osmolarity action (shared by the honey and golden Future research proposals syrup) on the antibacterial activity of honey. The only study in this review that utilized a placebo was completed by Hawley et al.,25 where the product was developed to look The adverse effects caused by Manuka honey highlighted in this re- like honey. Similar to the study by Bardy et al.,39 this study did not view should be taken into consideration of future research for oral result in a significant reduction in the severity or duration of the health conditions. Manuka honey contains relatively high levels of OM experienced by participants; however, this could be attributed methylglyoxal (MGO), which has been identified to be unique to to the possible non-suitability of Manuka honey for this type of this type of honey and directly contributes to Manuka honey’s an- treatment, rather than the activity of the placebo. Additionally, a tibacterial abilities.43,44 This compound also occurs in humans as a study by Abdulrhman et al.22 developed a honey-containing prod- by-product to metabolic processes, such as glycolysis45; however, uct, which they assessed as a treatment for OM alongside a treat- it has been shown to exhibit potentially toxic side effects, includ- ment group that received pure, undiluted honey, in addition to the ing the ability to modify DNA and other macromolecules.46 Addi- use of a standard care control (Table 1). tionally, in healthy human cells, the tripeptide glutathione (GSH) is It is evident that the utilization of a placebo in studies of this capable of suppressing the activity of MGO47; however, once a cell nature is not practical, and so further care should be taken in future becomes damaged, such as during the oral conditions discussed in studies to ensure that both participants and researchers are blinded this review, there is a reduction in the expression of GSH.48 This to the allocation of honey or control. This could potentially be can result in further damage, with GSH depletion leading to MGO achieved through disclosing that the intervention is designed to accumulation.49 This is depicted in Figure 2, where, in a healthy improve the oral health condition, while not specifying that honey oral cavity, GSH is able to suppress the activity of MGO, thereby is the treatment. preventing any cellular modifications. Although, when Manuka honey is applied to the damaged oral cavity, the reduced levels of GSH allow the levels of MGO to increase, and to potentially cause Compositional properties of used honeys further cellular damage. As previously discussed, one study included in this review It is well established that several honey properties can contribute reported considerable adverse effects following the topical appli- to its wound healing effects, including a combination of antioxi- cation of Manuka honey.25 Studies of similar interventions also dant, physicochemical and antimicrobial properties. However, the reported adverse effects,39,40 which could potentially be due to the optimal levels of compounds or categories of compounds, includ- proposed interactions between MGO and reduced levels of GSH. ing their interaction with each other, that can contribute to the ob- These adverse reactions were not observed in studies utilizing hon-

34 DOI: 10.14218/ERHM.2019.00029 | Volume 5 Issue 1, March 2020 Hunter M. et al: Honey as a treatment for oral ulcerations Explor Res Hypothesis Med

Fig. 2. Effect of honey on the oral cavity. (a) GSH present in the healthy oral cavity suppresses the activity of MGO from Manuka honey. (b) The use of honey does not cause adverse effects in a healthy oral cavity, and is effective in treating a damaged oral cavity. (c) Reduced levels of GSH in the damaged oral- cav ity do not suppress the activity of MGO from Manuka honey, with the MGO potentially causing further damage, and Manuka honey not being an effective treatment. GSH, tripeptide glutathione; MGO, methylglyoxal. ey from other floral sources, with the topical application of honey be completed to determine optimal levels of MGO that can be tol- to a damaged oral cavity being demonstrated to result in improve- erated. ments to the oral condition (Fig. 2). While Manuka honeys have demonstrated substantial potential wound healing abilities in vitro and in vivo,11,44,50 there is currently Conclusion a lack of relevant clinical trials utilizing this product for wound healing treatments. Due to its compositional properties, it should The use of honey as a therapeutic treatment of oral conditions has be considered that Manuka honey be utilized in clinical trials for been identified to be effective in reducing the duration and sever- site-specific topical application in wound sites other than the oral ity of conditions in comparison to standard treatments and con- cavity, such as a laceration or burn on the skin to test for effective- trols. Specifically, it has been demonstrated that honey is effective ness and tolerability. Similarly, further trials in the treatment of against a range of oral ulcers with multiple pathologies and, in oral conditions or investigating alternative application sites could particular, in the treatment of oral mucositis. Any adverse reactions

DOI: 10.14218/ERHM.2019.00029 | Volume 5 Issue 1, March 2020 35 Explor Res Hypothesis Med Hunter M. et al: Honey as a treatment for oral ulcerations that were reported throughout the duration of the included studies foodchem.2013.02.015. could be attributed to the type of honey used and were not due to [9] Escuredo O, Seijo MC, Salvador J, González-Martín MI. Near infra- the type of oral health condition, demonstrating that the topical red spectroscopy for prediction of antioxidant compounds in the application of honey, other than Manuka honey, is relatively safe honey. Food Chem 2013;141(4):3409–3414. doi:10.1016/j.food- for use across a range of oral health conditions. The use of honey chem.2013.06.066. 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DOI: 10.14218/ERHM.2019.00029 | Volume 5 Issue 1, March 2020 37 Corrigendum

Corrigendum: Acute Soft Skull Syndrome in an Adult Male with Sickle Cell Anemia in Sudan: A Case Report

Ziryab Imad Taha1,2*, Sulafa Eisa Mohammed3, Mohammed Elmujtba Adam Essa1,4*, Walaa Mohamed Elsid2, Mustafa Mohamed Ali Hussein1,4, Sherihan Mohammed Elkundi Osman4,5, Hussein Osman Ahmed4, Mutwaly Defealla Yousif1,4 and Abdelkareem A. Ahmed1,6*

1Department of Clinical Medicine, Medical and Cancer Research Institute (MCRI), Nyala, Sudan; 2Department of Internal Medicine, Faculty of Medicine, University of Bahri, Khartoum, Sudan; 3Department of Internal Medicine, National Ribat University, Khartoum, Sudan; 4Department of Internal Medicine, Faculty of Medicine, AlFashir University, AlFashir, Sudan; 5Department of Molecular Medi- cine, Institute of Endemic Diseases, University of Khartoum, Khartoum, Sudan; 6Department of Physiology and Biochemistry, Faculty of Veterinary Science, University of Nyala, Nyala, Sudan

Corrigendum on: Taha ZI, Mohammed SE, Essa MEA, Elsid WM, Hussein MMA, Osman SME, et al. Acute Soft Skull Syndrome in an Adult Male with Sickle Cell Anemia in Sudan: A Case Report. Explor Res Hypothesis Med. 2019;4(4):90–93. doi: 10.14218/ERHM.2019.00024.

The original version of the article contained an error in the last line of Table 1. It should appear as: Date Investigation Result Reference value 21/5/2019 Creatinine 1.5 mg/dL 0.7–1.4 mg/dL Instead of: Date Investigation Result Reference value 21/5/2019 Creatinine 0.5 mg/dL 0.7–1.4 mg/dL

The authors apologize for this error and state that the update does not change the results and conclusions originally reported. The original article has been updated.

*Correspondence to: Mohammed Elmujtba Adam Essa Adam, Medical and Cancer Research Institute (MCRI), Department of Clinical Medicine, Faculty of Medicine, AlFashir University, AlFashir, Sudan. Tel: +249907009378, E-mail: Awadali818@yahoo.com; Ziryab Imad Taha, Department of Internal Medicine, Faculty of Medicine, University of Bahri, Khartoum, Sudan, Tel: +249912129921, E-mail: Ziryab2008@ yahoo.com; Abdelkareem Abdallah Ahmed, Department of Physiology and Biochem- istry, Faculty of Veterinary Science, University of Nyala, Nyala, P.O. Box: 155 Nyala, Sudan. Fax: +249711833123, E-mail: [email protected] How to cite this article: Taha ZI, Mohammed SE, Essa MEA, Elsid WM, Hussein MMA, Osman SME, Ahmed HO, Yousif MD, Ahmed AA. Corrigendum: Acute Soft Skull Syndrome in an Adult Male with Sickle Cell Anemia in Sudan: A Case Re- port. Exploratory Research and Hypothesis in Medicine 2020;5(1):38. doi: 10.14218/ ERHM.2019.00024C.

Exploratory Research and Hypothesis in Medicine 2020 vol. 5 | 38