Rhesus monkey
Researchers found that 5 days post-reinfection (dpr), the re-exposed monkeys (Rhesus monkey Macaca mulatta) tested negative. Image Credit: Gulf News / https://www.biorxiv.org/content/10.1101/2020.03.13.990226v1.full.pdf+html

Dubai: Knowledge regarding SARS-CoV-2 virology is rapidly expanding, thanks to a fairly open-source science.

This also provides a significant number of potential drug targets. Currently, there are hundreds of active clinical treatment trials underway, including the use of repurposed drugs or experimental new treatments for COVID-19.

Various randomized trials on HIV and malaria drugs to provetheir safety and efficacy are being done.

The WHO, meanwhile, named three candidate (experimental) vaccines, even as many states have also approved a protocol for the use of convalescent plasma therapy to treat COVID-19 patients. These treatments had, in various forms, been reported to have led to alleviating the severity of COVID-19 infection.

Now, however, there had been concerns raised over reported COVID-19 “reinfections”. On February 28, 2020, Reuters reported that a number of discharged patients in China and elsewhere are testing positive after "recovering".

Here’s a lowdown on the reported reinfection story:

Q: What triggered such reinfection concerns?

Following unspecified reports of "reinfection", on April 11, 2020 (Saturday), the World Health Organization (WHO) said that it was looking into reports of some COVID-19 patients testing positive again — after initially testing negative for the disease while being considered for discharge.

Q: What’s prompted reports of “reinfection”?

Beside the February reports in China, on April 10 (Friday), South Korean officials also reported 91 patients thought cleared of COVID-19 had “tested positive again”. Korean health officials said, however, that they are trying to find out if this is the case.

Q: What accounts for the supposed “reinfections”, as in the 91 reported cases in South Korea?

Experts said it’s possible it was a relapse, instead of a reinfection, as some SARS-CoV-2 tests are not rigorous enough, that they could yield “false negative” results for the virus.

The discovery of immunity in monkeys 28 days following the primary infection sheds some light on puzzling cases in which patients appeared to relapse, or be re-infected. Some discharged after testing negative for coronavirus later again developed symptoms, even testing positive.

Jeong Eun-kyeong, director of the Korea Centers for Disease Control and Prevention, told a briefing that the virus may have been “reactivated” — rather than the patients being “re-infected”.

South Korean health officials said that it remains unclear what is behind the trend, with epidemiological investigations still under way.

Q: What did the WHO say about the reported “re-infection’ report from Seoul?

“We are aware of these reports of individuals who have tested negative for COVID-19 using PCR (polymerase chain reaction) testing and then after some days testing positive again,” the WHO said in a statement to Rueters.

“We are closely liaising with our clinical experts and working hard to get more information on those individual cases. It is important to make sure that when samples are collected for testing on suspected patients, procedures are followed properly,” it said.

Q: What are the WHO guidelines on discharging COVID-19 recovered cases?

According to the WHO's guidelines on clinical management, a patient can be discharged from hospital after two consecutive negative results in a clinically-recovered patient. The tests should be done at least 24 hours apart.

Q: How long does it take between infection and recovery among COVID-19 patients?

Based on current studies, there is a period of about two weeks (14-15 days) between the onset of symptoms and clinical recovery of patients with mild COVID-19 disease, according to the WHO.

“We are aware that some patients are PCR positive after they clinically recover, but we need systematic collection of samples from recovered patients to better understand how long they shed live virus,” it said.

“As COVID-19 is a new disease, we need more epidemiological data to draw any conclusions of virus shedding profile,” the WHO said.

The CDC has also published an interim guidance on precautions and the discontinuation of isolation for persons with COVID-19.

Viral shedding
It rfers to the expulsion or release of virus progeny (offspring) following successful reproduction during host-cell infection.

People who contract COVID-19 emit high amounts of the virus very early on — in the first 5 days — in their infection, a study (pre-print, not peer-reviewed) from Germany suggests.
Coronavirus Early on
People who contract COVID-19 emit high amounts of the virus very early on — in the first 5 days — in their infection, a study (pre-print, not peer-reviewed) from Germany suggests. Image Credit: Gulf News / Jay Hilotin Sources: https://bit.ly/3dndRvl https://bit.ly/2Up12b3

Q: What does the COVID-19 study on the reinfection of monkeys show?

There’s no reinfection of SARS-CoV-2 based on a study on monkeys.

The preprint version of the study, conducted by a team of Chinese researchers, showed reinfection could not occur in SARS-CoV-2 infected monkeys (rhesus macaques).

The researchers found no “viral loads” as well as viral replication in all primary tissue compartments at 5 days post-reinfection (dpr) in the “re-exposed” monkeys.

The study, however is a pre-print, or preliminary, one published on bioRxiv. The site is a preprint server for biology.

(Note: A preliminary report means it has not been peer-reviewed. They should not be regarded as conclusive, guide clinical practice/health-related behaviour, or be reported in news media as established information.)

In the customary disclosure notes, the researchers stated their study was supported by the CAMS initiative for Innovative Medicine of China, National Mega projects of China for Major Infectious Diseases and National Key Research and Development Project of China.

Q: What did the researchers do?

The researchers tracked re-exposure of the rhesus monkeys after the disappeared symptoms of the SARS-CoV-2-infected monkeys.

Here’s what they found in monkeys after the primary infection:

  • Weight loss in some monkeys
  • viral replication mainly in nose, pharynx, lung and gut
  • moderate interstitial pneumonia at 7 days post-infection (dpi)

Q: Did they deliberately infect the monkeys and then reinfected the recovered primates with the same SARS-CoV-2 strain?

Yes. After the symptoms were alleviated and the specific antibody tested positively, half of infected monkeys were re-challenged with the same dose of SARS-CoV-2 strain.

Researchers found that “neither viral loads in nasopharyngeal and anal swabs along timeline nor viral replication in all primary tissue compartments at 5 days post-reinfection (dpr) was found in re-exposed monkeys.”

Combined with the follow-up virologic, radiological and pathological findings, the monkeys with re-exposure showed no recurrence of COVID-19, similarly to the infected monkey without re-challenge.

Q: How many monkeys were used in the study?

Four. They were 3- to 5-year old rhesus macaques, named as M1 to M4. They were housed and cared for in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC).

Figure 1
EXPERIMENTAL DESIGN AND SAMPLE COLLECTION: Four monkeys were initially challenged with 1×106 TCID50 SARS-CoV-2 with the intratracheal route. To investigate the influence of reinfection, M3 and M4 after recovery were intratracheally rechallenged with the same dose of SARS-CoV-2 at 28 days post-infection (dpi). Two animals (M1 and M3) were sacrificed at 7 dpi and 5 days post-rechallenge (dpr), respectively. M2 with single infection and M4 with primary infection followed by 168 secondary challenge were longitudinally monitored during the entire observation. Body weight, body temperature and nasal/throat/anal swabs were measured along the timeline at a short interval. Two measurements of virus distribution and histopathology (HE/IHC stain) were carried out at 7d dpi (M1) and 5 dpr (M3). The specific antibodies against SARS-CoV-2 were detected seven times and X-ray was examined three times. Image Credit: https://www.biorxiv.org/content/10.1101/2020.03.13.990226v1.full.pdf+html

Researchers said all animal procedures and experiments were carried out in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (BLL20001).

All animals were anesthetized with ketamine hydrochloride (10 mg/kg) prior to sample collection, and the experiments were performed in the animal biosafety level 3 (ABSL3) laboratory.

Q: How were the animal subjects infected with SARS-CoV-2?

For primary (first) infection, all animals were challenged intra-tracheally (through what is commonly called the “windpipe’) with SARS-CoV-2 stock virus at a dosage of 106 TCID50/1 mL inoculum volume.

The specific viral strain used in the study was the SARS-CoV-2/WH-09/human/2020/CHN isolated from the researchers’ laboratory.

To investigate the influence of reinfection, M3 and M4 after recovery were intratracheally re-challenged (virus reinserted) with the same dose of SARS-CoV-2 at 28 days post-infection (dpi).

Two animals (M1 and M3) were sacrificed at 7 dpi and 5 days post-rechallenge (dpr), respectively.

M2, with single infection, and M4 with primary infection followed by secondary challenge, were monitored daily during the entire observation.

Body weight, body temperature and nasal/throat/anal swabs were measured along the timeline at a short interval.

Two measurements of virus distribution and histopathology (HE/IHC stain) were carried out at 7 days post infection (for M1) and 5 days post recovery (for M3).

The specific antibodies against SARS-CoV-2 were detected seven times and X-ray was examined three times.

Q: What did the researchers find out in terms of clinical signs, viral replication and immune response in the monkeys?

After the initial infection, all animals were monitored along the timeline to record body weights, body and rectal temperature, clinical signs, nasal/throat/anal swab, X-ray and specific antibody.

The same work was done followed by the virus rechallenge (reinfection).

Detection of viral RNA was done through nasal, throat and anal swabs. SARS-CoV-2 RNA was detected by qRT-PCR in the swabs from four monkeys at different time points.

Compared to M1 with primary infection at 7 dpi, viral replication tested negatively in the indicated tissues from M3 (at 5 days post rechallenge) with the virus rechallenge.

monkey stdy
Image Credit: Screengrab www.biorxiv.org/content/10.1101/2020.03.13.990226v1

Q: What did the scientists find out about the presence of antibodies after the re-challenge (reintroduction of SARS-CoV-2)?

The levels of specific IgG (immunoglobulins) against the “spike protein” were measured. The levels of anti-viral antigen specific IgG from each monkey were detected at 3, 7, 14, 21, 28 days post infection.

What is anti-viral antigen? How do we know their presence?
A virus antigen is a toxin or other substance given off by a virus which causes an immune response in its host.

A viral protein is an antigen specified by the viral genome that can be detected by a specific immunological response.

Virus antigen detection tests can be performed directly on a variety of specimen types and are highly specific and rapid. https://www.sciencedirect.com/topics/neuroscience/virus-antigen

Q: What does this mean?

It shows the anti-viral antigens against the specific strain of SARS-CoV-2, started to kick in from as early as three days following infection. The presence of antigens in the blood samples taken from the monkey is amplified with each passing day.

Researchers found that "the level of specific IgG at 14 dpi (days post infection), 21 dpi or 28 dpi was significantly higher than that at 3 dpi or 7 dpi." 

What is IgG?
Immunoglobulin G (IgG) is a type of antibody. Representing approximately 75% of serum antibodies in humans, IgG is the most common type of antibody found in blood circulation. (Definition from Frontiers in Immunology, https://www.frontiersin.org/articles/10.3389/fimmu.2014.00520/full)

IgG molecules are created and released by plasma B cells. Each IgG has two antigen binding sites.

A class of immunoglobulins including the most common antibodies circulating in the blood are the only immunoglobulins to cross over the placenta from mother to fetus.

Q: Does the SARS-CoV-2 virus attack the brain of monkeys?

Yes.

One of the startling findings of the researchers is detection the presence of SARS-CoV-2 (through viral RNA) in the main organs of the monkey — such as the brain, eye, nose, pharynx, lung and gut.

The study showed the presence of viral loads in the brain at 7 days post infection. But the most severe viral loads were detected in the nose, pharynx, lung and gut.

Q: Study limitations?

With only four (4) subjects, this is not a statistical study. Rather, it covers the anatomical effects of the SARS-CoV-2 virus and the immunity of the macaque rhesus monkeys to them.

It was also not immediately clear what were the sexes of the monkeys.

Future studies could expand it to establish with greater confidence through running statistics that may tell us it may hold over to humans and at much bigger numbers.

A study humans are being conducted, but also on the test kits. Still, the results from this study are encouraging. Chimps and humans are 96 per cent the same, according to one gene study.

Q: What are the implications of this study?

Taken together, results indicated that the primary SARS-CoV-2 infection could protect from subsequent exposures.

But being "pre-print" version, it needs to be peer reviewed. If validated, researchers said, could then be used as the reference of prognosis of the disease and has important implications for the development of a vaccine.

viral loads monkey study
Viral loads table on the monkeys.

Q: Who are the researchers and where do they work?

Most of them are from the Beijing Key Laboratory for Animal Models of Emerging and Remerging Infectious Diseases, NHC Key Laboratory of Human Disease Comparative Medicine, Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences and Comparative Medicine Center, Peking Union Medical College, Beijing, China.

Another member of the research team came from Department of Radiology, Bejing Anzhen Hospital, Capital Medical University.

Q: Medical experts say: Follow advice of medical authorities 

Marc Lipsitch, a Harvard epidemiologist who consults for the United States’ Centres for Disease Control (CDC) believes immunity in a recovered patient could last "for about a year".

Another doctor said there's not enough data to support the reinfection theory, but cited the case with chickenpox.

“People who have been vaccinated against chickenpox have been noted to contract it after many years, but in such cases, the symptoms are less severe. We may find a similar trend occurring with the novel coronavirus. As of now however, we simply do not have enough data,” said Dr Rasha Alani, family medicine specialist at Medcare Medical Centre – Al Khawaneej.

“Protecting against possible re-infection is the same as protecting against infection. It’s imperative that people follow local authority guidelines, practise physical distancing, stay at home as much as possible, wash their hands frequently and avoid touching their face,” said Dr Maher Balkis, associate staff physician for infectious diseases at Cleveland Clinic Abu Dhabi.

References:

www.biorxiv.org/content/10.1101/2020.03.13.990226v1

https://www.biorxiv.org/content/10.1101/2020.03.13.990226v1.full.pdf+html

htts://www.cdc.gov/coronavirus/2019-ncov/hcp/disposition-hospitalized-patients.html

https://www.cdc.gov/coronavirus/2019-ncov/hcp/disposition-in-home-patients.html

https://bit.ly/3et2Uc2

https://bit.ly/3akb6YE

https://bit.ly/2xD2Y8o