Respiratory Tract Infection Viruses (Measles Virus, Mumps Virus, Respiratiry-Syncytial Virus, Coronaviruses)

(Prepared by Inzhevatkina S.M., Department of Microbiology and Virology of Russian National Research Medical University NI Pirogov) Causative Agent of Measles

Order: Mononegavirales Family: Paramyxoviridae Genus: Morbillivirus Species : Measles virus (only one serotype) Measles Virus by Electron Microscopy Structure of Measles Virus • Roughly spherical but often pleomorphic particle.

• 120-250 nm in diameter.

• The tightly coiled helical nucleocapsid is surrounded by lipoprotein envelope.

• Two of the membrane envelope proteins are important in its pathogenesis. The F (fusion) protein is responsible for fusion of virus and host cell membranes, viral penetration and hemolysis. The H (hemagglutinin) protein is responsible for adsorption of the virus to cells. Structure of Measles Virus Transmission of Measles Humans are the only known natural host of measles. Measles is spread by respiratory droplets (air-borne) or by direct contact with nasal or throat secretions of infected persons, and less commonly, by articles contaminated with nose and throat secretions. Measles is one of the most highly communicable infectious diseases known. The patient can pass the disease to other persons from beginning of the disease to 4 days after appearance of the rash. The incubation period before the disease varies from 7 to 18 days, usually around 14 days. Cytopathic Effect of Measles Virus Measles virus can cause fusion of infected cells, leading to the formation of syncytia. Thus, the virus can spread directly from cell to cell and escape antibody control. Inclusions can occur commonly in the cytoplasm of host cells and are composed of incomplete viral particles. Persistent infection without lysis can occur in certain cell types (e.g., human brain cells). Cytopathic Effect of Measles Virus Syncytial formation caused by measles virus in cell culture GIANT CELL WITH INTRACYTOPLASMIC INCLUSIONS (MEASLES) Pathogenesis of Measles The virus enters the body through the respiratory tract or the conjunctiva, infects monocytes and lymphocytes and multiplies locally in the adjoining lymph nodes. The virus then spread to the reticuloendothelial system through the blood. After multiplication, a secondary viremia transport the virus to the epithelial surfaces including the skin, mouth, respiratory tract urinary tract, small blood vessels, lymphatic system, conjunctiva and central nervous system. Pathogenesis of Measles

measles virus ↓respiratory tract epithelial cells(multiply) ↓lymphoid tissue blood (first virusemia) ↓ MPS(multiply) ↓ blood (second virusemia) ↓ general toxic symptoms Clinical Manifestations of Measles Clinical Manifestations of Measles Measles is a serious febrile illness. The disease presents with cough, runny nose, fever, red eyes and white spots (Koplick’s spots) inside the mouth. This is followed 3 to 7 days later by a red blotchy skin rash, which spreads from the face to the rest of the body. The rash usually lasts 4 - 7 days but can persist for up to 3 weeks. The characteristic maculopapular rash is caused by immune T-cells targeted to measles-infected endotelial cells lining small blood vessels. Measles rash Picture of a baby with measles Measles Rash on Back and Legs Measles Rash and Bloodshot Eyes Picture of Koplik’s Spots Koplick’s spots are a characteristic of measles used in diagnosis. They appear opposite the molars as red spots with blue white centers. Symptoms of measles Symptoms usually begin 8 - 12 days after you are exposed to the virus • Bloodshot eyes • Rash which usually appears 3 - 5 • Cough days after the first signs of being sick and may last 4 - 7 days; • Fever • Rash usually starts on the head and spreads to other areas, • Light sensitivity (photophobia) moving down the body • Muscle pain • Rash may appear as flat, discolored areas (macules) and • Redness and irritation of the solid, red, raised areas (papules) eyes (conjunctivitis) that later join together • Rash is itchy • Runny nose • Tiny white spots inside the mouth • Sore throat (Koplik's spots) Complications of Measles Measles is frequently complicated by middle ear infection or diarrhea. The disease can be severe, with bronchopneumonia or brain inflammation (meningoencephalitis) leading to death in approximately 2 of every 1,000 cases in developed countries. Bacterial superinfection leads to pneumonia. In the developing world, case-fatality rates often exceed 150 deaths per 1000 cases . A rare late complication is subacute sclerosing panencephalitis (SSPE). Immunity Against Measles Three kinds of antibodies are produced after infection, that is 1. complement combining antibody; 2. hemagglutinin inhibiting antibody 3. neutralizing antibody Recovery follows the rash in most people, immunity is lifelong, but in rare cases the measles can occur in humans with impaired immune memory the second time (usually at 45 years old). Subacute Sclerosing Panencephalitis (SSPE)

SSPE occurs in 7 in 106 patients years after a measles infection. The SSPE virus acts as slow virus, and causes cytopathologic effect in neurons many years after acute disease. Infection spreads directly from cell to cell without mature virus release. Laboratory Diagnosis of Measles Clinical specimen: respiratory tract secretions, blood, urine, brain tissue; serum. Virological method. Measles virus can be cultivated on human or monkey kidney and human amnion cultures. Isolates can be adapted for growth on continuous cell lines (HeLa,Vero) and in the amniotic sac of hen’s eggs. The presence of measles virus is indicated by formation of syncytia. On Romanowsky-Giemsa-stained cells characteristic cytopathic effects,including multinucleated giant cells (Wartin-Finkeldey cells) with cytoplasmic and nuclear inclusion bodies, can be seen. Indication is done by hemagglutination and hemadsorption of monkey’s erythrocytes. Identification of virus is based on hemagglutination, hemadsorption and cytopathic effect neutralization. Laboratory Diagnosis of Measles Immunofluorescent method (indirect immunofluorescence for detection of virus in clinical specimen) in pharyngeal cells or urine sediment. Serological method can be used to identify a response to acute infection (IgM) or previous infection (IgG) in serum. Paired serums are used for detection of increase in titer by ELISA and RIA. Molecular-genetic method: RT-PCR is used for analysis of respiratory secretions. Prophylaxis of Measles Measles vaccine contains live attenuated measles virus. It is available as a single-antigen preparation or combined with live, attenuated mumps or rubella vaccines, or both. Combined measles, mumps, and rubella (MMR) vaccine is recommended whenever one or more of the individual components are indicated. Initial vaccination at 12 months old, booster immunization at 6 years old. Treatment of Measles

Normal human gamma globulin given within six days of exposure can prevent or modify the disease depending on the dose. This is valuable in children with immunodeficiency, pregnant women and others at special risk. There is no specific antiviral therapy for measles, and the basic treatment consists of providing necessary supportive therapy such as hydration and antipyretics and treating complications such as pneumonia. Causative Agent of Mumps Taxonomy Order: Mononegavirales

Family: Paramyxoviridae

Genus: Rubulavirus

Species: Mumps Virus (only one serotype) Mumps Virus by Electron Microscopy Mumps Virus • Envelopes virus • Shape: Spherical • Capsid: Helical • Genetic makeup: Linear single stranded molecule of ss “-” RNA • Rapidly inactivated by chemical agents, heat and ultraviolet light Structure of Mumps Virus • Possesses HN and F spikes. • Growth in Chick Embryos, in the Amniotic cavity, Adopts in allantoic cavity, • Cell cultures – Primary Monkey kidney, • Typical Paramyxoviruses, produce cytopathic effects (formation of giant multinuclear cells due fusion of infected host cells). MUMPS Mumps is an acute viral infection of the paramyxoviruses family. As its alternative name (infectious parotits) suggests, the infection is characterized by swelling more commonly bilateral than unilateral of the parotid salivary glands. The incubation period is 14-21 days and is communicable from 6 days before to 9 days after facial swelling is apparent. It can lead to brain inflammation, deafness or sterility. Pathogenesis of Mumps

Respiratory transmission of virus and direct person to person contact. (Note! Saliva and urine of mumps patient are infective. ) Initial replication occurs in nasopharynx and regional lymph nodes. Viremia 12-25 days after exposure with spread to tissues Multiple tissues infected during viremia (parotid glands, testes, ovary, pancreas, thyroid, salivary glands, eye, peripheral nerves, central nervous system., etc.) Mumps: Main Clinical Manifestation Involvement of Parotid Gland a Major Manifestation • Swelling of the salivary glands follows these symptoms. Swelling of the glands near the jaw line below the ears may give you "chipmunk cheeks” Involvement of Salivary Glands

• Painful swelling of the salivary glands (classically the parotid gland is the most typical presentation) Painful testicular swelling (orchitis) and rash may also occur Mumps: Clinical Manifestations

• Nonspecific prodrome of myalgia, malaise, headache, low- grade fever • Parotitis in 30%- 40% • Up to 20% of infections asymptomatic Complications of Mumps • Epididymoorchids which may lead to atrophy, sterility, low sperm counts. • CNS involvement in 60% cases which may manifest with aseptic meningitis • Pancreatitis • Deafness • Arthritis • Oopharitis • Nephritis • Myocarditis The Most Severe Complications of Mumps • Orchitis. This inflammatory condition causes swelling of one or both testicles. Orchitis is painful. • Pancreatitis, which can lead to juvenile diabetes . • Encephalitis. A viral infection, such as mumps, can lead to inflammation of the brain (encephalitis). Although it's serious, encephalitis is a rare complication of mumps. Complications of Mumps

• Meningitis. Meningitis is infection and inflammation of the membranes and fluid surrounding your brain and spinal cord. • Inflammation of the ovaries. Pain in the lower abdomen in women may be a symptom of this problem. Fertility doesn't seem to be affected. • Hearing loss. • Miscarriages. Immunity against Mumps

• Immunity is lifelong • Mumps rare before 6 months of age. Laboratory Diagnosis of Mumps Clinical specimen: pharynx secretions, saliva (5 days after onset of infection), urine (two weeks after onset of infection), cerebrospinal fluid; serum. Virological method. Mumps virus can be cultivated on human or monkey kidney and human amnion cultures. Isolates can be adapted for growth on continuous cell lines (HeLa,Vero) and in the amniotic sac of hen’s eggs. The presence of mumps virus is indicated by formation of syncytia. On Romanowsky-Giemsa-stained cells characteristic cytopathic effect of multinucleated giant cells can be seen. Indication is done by hemagglutination and hemadsorption of guinea pig’s erythrocytes. Identification of virus is based on hemagglutination, hemadsorption and cytopathic effect neutralization. Laboratory Diagnosis of Mumps Immunofluorescent method (indirect immunofluorescence for detection of virus in clinical specimen) in pharyngeal cells or urine sediment. Serological method can be used to identify a response to acute infection (IgM) or previous infection (IgG) in serum. Paired serums are used for detection of increase in titer by CFT, ELISA and RIA. Molecular-genetic method: RT-PCR is used for analysis of respiratory secretions. Hemadsorption of guinea pig erythrocytes on the surface of cells infected with mumps virus Vaccination against Mumps • Live attenuated vaccine Jernyl Lynn Strain Grown in chick embryo fibroblasts Vaccine as MMR vaccine A single dose protects for 10 years (95% lifelong immunization protects with a single dose). Prophylaxis of Mumps Mumps vaccine contains live attenuated measles virus. It is available as a single-antigen preparation or combined with live, attenuated measles or rubella vaccines, or both. Combined measles, mumps, and rubella (MMR) vaccine is recommended whenever one or more of the individual components are indicated. Initial vaccination at 12 months old, booster immunization at 6 years old. Immune globulin ineffective for post exposure prophylaxis. Treatment: antiviral agents are not available. Causative Agent of Respiratory Syncytial Infection Taxonomy

• Family: Pneumoviridae • Genus: Orthopneumovirus • Species: Human respiratory syncytial virus (it has two antigenic subgroups). Structure of Respiratory- Syncytial Virus (RSV)

RSV is a enveloped virus with ss “-” RNA as the family Paramyxoviridae, which includes common respiratory viruses such as those causing measles and mumps . It acquired name from the fact that F protein on the surface of the virus cause the cell membranes on nearby cells to fuse, forming syncytia (multinucleated giant cells). Structure of RSV Proteins of RSV (1) • NS1 and NS2 inhibit type I interferon activity. • N encodes nucleocapsid protein that associates with the genomic RNA forming the nucleocapsid. • M encodes the Matrix protein required for viral assembly. • SH, G and F form the viral coat. The G protein is a surface protein that is heavily glycosylated. It functions as the attachment protein. The F protein is another important surface protein; F mediates fusion, allowing entry of the virus into the cell cytoplasm and also allowing the formation of syncytia. The F protein is homologous in both subtypes of RSV; antibodies directed at the F protein are neutralizing. In contrast, the G protein differs considerably between the two subtypes. Proteins of RSV (2)

• M2 is the second matrix protein also required for transcription and encodes M2-1 (elongation factor) and M2-2 (transcription regulation). M2 contains CD8 epitopes. • L encodes the RNA polymerase. • The phosphoprotein P is a cofactor for the L protein. RSV by Electron Microscopy Life Cycle of RSV Pathogenesis of RSV Infection Syncytial formation caused by RSV in cell culture This is respiratory syncytial virus (RSV) in a child. Note the giant cells which are part of the viral cytopathic effect. The inset demonstrates a typical giant cell with a round, pink intracytoplasmic inclusion . RSV Infection A major and most common cause of serious respiratory illness in children under the age of two. It causes severe infections of the lungs and breathing passages. RSV to be the "most common cause of bronchiolitis (inflammation of the small airways in the lung) and pneumonia in children under 1 year of age. RSV is spread by either direct contact with respiratory secretions or by indirect contact through toys or objects from a person that is infected. Incubation period of RSV infection is 4- 5 days. RSV Infection Bronhiolitis is inflammation of bronchiole, there is air trapping and decreased ventilation. RSV infections can lead to other serious illnesses in premature babies and other children that already have diseases effecting the lungs, heart and immune system. It is so common that nearly all children will become infected by the age of three. Clinical Manifestations of RSV Infection • Stuffy nose and nasal flaring • Deep cough • Shortness of breath • Wheezing • Low grade fever • Rapid breathing • Ear Infection • Panting or Retractions of the chest wall( the visible pulling in of the chest wall) • Rattling of the chest that may be felt through the child's back or chest Comparison of RSV Infection and Influenza RSV Infection RSV binding and triggering of cellular responses. RSV is bound by surface glycosaminoglycans, and the F protein binds to TLR4, while G glycoprotein (virus associated or secreted) binds fractalkine receptor CX3CR1. The interaction with TLR4 leads to upregulation of NF-κB via MyD88. RSV upregulates NF-κB via I κB and STAT1 and -3 via reactive oxygen species (ROS), and RSV RNA activates protein kinase R. Viral NS proteins inhibit the interferon response factor (IRF3) pathway. Overview of the sequence of immune events in viral clearance and disease. In primary infection of nonvaccinated individuals (A), virus peaks on about day 4, associated with recruitment of NK cells, which make IFN-γ. Virus is eliminated between days 5 and 8, during which time activated CD4 and CD8 T cells are recruited and produce local cytokines. The peak of disease coincides with this phase. Anti-RSV serum antibody appears relatively late. In previously vaccinated or sensitized individuals (B), the virus titer is typically 100- to 1,000-fold less than in primary infection and peaks earlier (e.g., day 2). However, the rapid and potent cellular response enhances disease severity, which is usually much greater than in primary infection. Laboratory Diagnosis of RSV Infection Clinical specimen: Nasopharyngeal swab, wash, or aspirate, oropharyngeal swab, bronchial wash; serum. Virological method is not used because RSV is difficult to isolate in culture. Immunofluorescent method (indirect immunofluorescence for detection of virus in clinical specimen) in pharyngeal cells. Serological method can be used with paired serums. The paired serums are used for detection of increase in titer by CFT, ELISA and RIA. Molecular-genetic method : RT-PCR is used for analysis of respiratory secretions. Immunofluorescent Method for Detection of RSV Immunofluorescent Method for Detection of RSV Prophylaxis and Treatment of RSV Infection No vaccine is available now. A vaccine trial in 1960s using a formalin-inactivated vaccine (FI-RSV), increased disease severity in children who had been vaccinated. Palivizumab, a moderately effective prophylactic drug is available for infants at high risk. Palivizumab is a monoclonal antibody directed against RSV surface fusion protein. It is given by monthly injections, which are begun just prior to the RSV season and are usually continued for five months. RSV prophylaxis is indicated for infants that are premature or have either cardiac or lung disease. Ribavirin is used, but its efficacy is limited. Treatment of RSV Infection Coronaviruses Taxonomy Order: Nidovirales Family: Coronaviridae Subfamily: Orthocoronavirinae Genera : Alphacoronavirus (alphaCoV) , Betacoronavirus (betaCoV) , divides into five sub - genera or lineages (examples are COVID-19 virus, SARS virus, MERS virus) Gammacoronavirus (gammaCoV) Deltacoronavirus (deltaCoV) Genomic characterization has shown that probably bats and rodents are the gene sources of alphaCoVs and betaCoVs. Structure of Coronaviruses Coronaviruses are large enveloped spherical viruses with bulbous spikes. The average diameter of the virus particles is around 125 nm. The diameter of the envelope is 85 nm and the spikes are 20 nm long. The lipid bilayer has the membrane (M), envelope (E) and spike (S) structural proteins are anchored. On average a coronavirus particle has 74 surface spikes. Betacoronaviruses also have a shorter spike-like surface protein called hemaglutininesterase. The coronavirus surface spikes are homotrimers of the S protein, which is composed of an S1 and S2 subunit. The homotrimeric S protein mediates the receptor binding and membrane fusion between the virus and host cell. The S1 subunit forms the head of the spike and has the receptor binding domain (RBD). The S2 subunit forms the stem which anchors the spike in the viral envelope and on protease activation enables fusion. The E and M protein are important in forming the viral envelope and maintaining its structural shape. Nucleocapsid is formed from multiple copies of the nucleocapsid (N) protein, which are bound to the “+” single-stranded RNA genome in a continuous besds on a string type conformation. Structure of Coronaviruses Structural Proteins of Coronaviruses Genome of Coronaviruses Full-genome sequencing and phylogenic analysis indicated that the coronavirus that causes COVID-19 is a betacoronavirus in the same subgenus as the severe acute respiratory syndrome (SARS) virus (as well as several bat coronaviruses), but in a different clade. The structure of the receptor-binding gene region is very similar to that of the SARS coronavirus, and the virus has been shown to use the same receptor, the angiotensin-converting enzyme 2 (ACE2), for cell entry. The Coronavirus Study Group of the International Committee on Taxonomy of Viruses has proposed that this virus be designated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) In a phylogenetic analysis of 103 strains of SARS-CoV-2 from China, two different types of SARS-CoV-2 were identified, designated type L (accounting for 70 percent of the strains) and type S (accounting for 30 percent). Replication of Coronaviruses Cultivation of Coronaviruses

1. Coronaviruses are cultivated through organ culture of human embtyonic trachea. 2. The isolated virus when intranasally inoculated into volunteers (!). Epidemiology of Coronavirus Infection

Since the first reports of cases from Wuhan, a city in the Hubei Province of China, at the end of 2019, more than 80,000 COVID-19 cases have been reported in China, with the majority of those from Hubei and surrounding provinces. A joint World Health Organization (WHO) -China fact -finding mission estimated that the epidemic in China peaked between late January and early February 2020. Cases have been reported in all continents, except for Antarctica, and have been steadily rising around the world. Globally, millions of confirmed cases of COVID-19 have been reported. Transmission of Coronavirus Infection Transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurs mainly via respiratory droplets, resembling the spread of influenza. With droplet transmission, virus released in the respiratory secretions when a person with infection coughs, sneezes, or talks can infect another person if it makes direct contact with the mucous membranes; infection can also occur if a person touches an infected surface and then touches his or her eyes, nose, or mouth. Droplets typically do not travel more than two meters and do not linger in the air. SARS-CoV-2 has been detected in non-respiratory specimens, including stool, blood, and ocular secretions, but the role of these sites in transmission is uncertain Transmission of Coronavirus Infection Transmission of SARS-CoV-2 from asymptomatic individuals (or individuals within the incubation period) has been documented. Presymptomatic transmission occurs one to three days prior to symptom development. How long a person remains infectious is also uncertain. The duration of viral shedding is variable; there appears to be a wide range, which may depend on severity of illness: patients with mild to moderate illness (no hypoxia), 90 percent had repeated negative viral RNA tests on nasopharyngeal swabs by 10-24 days after the onset of symptoms; tests were positive for longer in patients with more severe illness from naso- or oropharyngeal specimens, and the longest was 42 days. However, as mentioned above, detectable viral RNA does not always correlate with isolation of infectious virus, and there may be a threshold of viral RNA level below which infectivity is unlikely. In the study of nine patients with mild COVID-19 described above, infectious virus was not detected from respiratory specimens when the viral RNA level was <10 6 copies/mL. Risk of Transmission of COVID-19 The risk of transmission from an individual with SARS-CoV-2 infection varies by the type and duration of exposure, use of preventive measures, and likely individual factors (e.g., the amount of virus in respiratory secretions). Most secondary infections have been described among household contacts, in congregate or health care settings when personal protective equipment was not used (including hospitals and long-term care facilities), and in closed settings (e.g., cruise ships). However, reported clusters of cases after social or work gatherings also highlight the risk of transmission through close, non-household contact. Pathogenesis of Coronavirus Infection Clinical Manifestations of Coronavirus Infection Incubation period — The incubation period for COVID-19 is thought to be within 14 days following exposure, The median incubation period was 5.1 days e exposure, 97.5 percent of infected individuals within 11.5 days. Spectrum of illness severity and case fatality rates — The spectrum of symptomatic infection ranges from mild to critical; most infections (80 percent of infected individuals) are not severe. Clinical Manifestations of Coronavirus Infection Clinical Manifestations of Coronavirus Infection 1.Mild (no or mild pneumonia) was reported in 80 percent. 2.Severe disease (e.g., with dyspnea, hypoxia, or >50 percent lung involvement on imaging within 24 to 48 hours) was reported in 15 percent. 3.Critical disease (e.g., with respiratory failure, shock, or multiorgan dysfunction) was reported in 5 percent. Typical Symptoms of COVID-19 Risk Factors for Severe Coronavirus Illness • Severe illness can occur in otherwise healthy individuals of any age, but it predominantly occurs in adults with advanced age or underlying medical comorbidities. Comorbidities that have been associated with severe illness and mortality include: • Cardiovascular disease • Diabetes mellitus • Hypertension • Chronic lung disease • Cancer (in particular hematologic malignancies, lung cancer, and metastatic disease) • Chronic kidney disease • Obesity Coronavirus Pneumonia Pneumonia appears to be the most frequent serious manifestation of infection, characterized primarily by fever, cough, dyspnea, and bilateral infiltrates on chest imaging. However, other features, including upper respiratory tract symptoms, myalgias, diarrhea, and smell or taste disorders, are also common. There are no specific clinical features that can yet reliably distinguish COVID-19 from other viral respiratory infections, although development of dyspnea several days after the onset of initial symptoms is suggestive. Chest computer tomography demonstrates consolidation and ground glass opacification with or without consolidative abnormalities, with bilateral, peripheral, and lower lung zone distributions ; lung involvement increased over the course of illness, with a peak in severity at 10 to 12 days after symptom onset. Laboratory Diagnosis of Coronavirus Infection Clinical specimen: Nasal swab, nasopharyngeal swab, oropharyngeal swab, nasal or nasopharyngeal wash/aspirate, expectorated sputum, lower respiratory tract aspirate or bronchoalveolar lavage - collected by a health care professional ; serum. Virological method is not used because coronavirus is difficult to isolate in culture (see cultivation). Molecular -genetic method : RT -PCR tests. Currently, most tests have low sensitivity and specificity, thus, a lot of false- negative results. Serological method can be used with paired serums. The paired serums are used for detection of increase in titer by ELISA. Fourfold increase is typical in 10-14 days. Specific IgM appears from the second week of disease and present in convalescent individuals, then IgG appears (15 days the onset of illness. The titer varies greatly of severity of infection. The sensitivity and specificity of many serologic tests are uncertain Treatment of Coronavirus Infection

No specific antiviral treatments for COVID-19 exist right now. A lot of schemes are tested. The main therapy is symptomatic treatment. Scientists are working hard to develop effective treatments. Therapies that are under investigation include drugs that have been used to treat malaria (e.g., hydroxychloroquine, chloroquine) and autoimmune diseases; antibiotics (e.g.,azithromycin has some anti- inflammatory action) are recommended; antiviral drugs (e.g., remdesivir, lopinavir/ritonavir, interferon beta) that were developed for other viruses; and passive immunization by convalescent plasma from people who have recovered from COVID-19. Treatment of Coronavirus Infection Remdesivir specifically targets key viral proteins involved in making new copies of the virus and prevents them from working. Only large clinical trials on patients with COVID-19 will be able to reveal precisely whether these interventions are safe and effective. Unfortunately, these kinds of large trials take time to carry out, but they are ongoing.