Articles

Virome: The role of precision medicine in the diagnosis and treatment of viral infectious diseases

MSc. Deyvid Amgarten

A look back at the HIV/AIDS epidemic

Infectious and rapidly spreading diseases caused by viruses (epidemics) have been constant companions of humanity throughout our history; from the most common and frequent, such as the flu and colds, to the largest pandemics worldwide, such as the black plague in the 14th century, Spanish flu in 1920 and H1N1 influenza in 2009.

To tackle diseases that afflicted humanity more recently, let’s approach the global HIV / AIDS pandemic. It is interesting to discuss its beginning, and the reaction of the medical community when faced with this new type of disease. Perhaps the oldest documented case of what would be Acquired Immunodeficiency Syndrome (AIDS) is that of a patient in Congo, dated from 1959 [1]. After this, cases were also documented in Norway in 1966, throughout the 1970s and 1980s in the USA and finally with the identification of the virus causing the syndrome in the 1980s. Two groups of researchers independently identified a new retrovirus in 1983, publishing his findings in the same issue of the journal Science [2]; [3].

This retrospective on the history of HIV shows us the time that passed between the first reports of the disease and the identification of the virus: more than 20 years. And since sequencing of viral genomes was time-consuming and unusual at that time, there was a lot of confusion about the virus’s identification and origin for at least another 5 years. Meanwhile, the virus was spreading around the world, without researchers and health officials being able to do anything significant to control its replication and dispersal.

By sequencing and making hundreds to thousands of HIV genomes available in public databases, researchers were able to characterize the virus’s mechanisms of infection, its tropism (which cells they infect), its likely zoonotic origin (from ape monkeys in Africa ), and which proteins they encode with their respective roles in the infection. More than that, they were able to develop drugs that target specific proteins in the viral cycle and prevent their replication. This is fantastic results were attained mainly due to the advancement of new generation molecular sequencing techniques and bioinformatics.

Epidemics continue to emerge, what is changing is our response time

Another example to put 20 years of HIV in perspective. The Zika virus (ZIKV), was isolated for the first time from rhesus monkeys in the Zika forest, Uganda, in 1947. Since then, researchers have been studying the virus and characterizing it, without much urgency, as the virus apparently remains restricted to small, localized outbreaks. In 2015, when the first febrile cases with a negative result for dengue appeared in Brazil, researchers were able to set up molecular tests based on the complete sequences already available in public databases and to identify the first cases of ZIKV transmission in Brazil [4].

However, creating a new molecular test is not trivial, requiring expensive equipment and highly specific knowledge by laboratories. The consequence is that these types of testing are often restricted to major research centers of excellence in the country. Perhaps this was one of the reasons that contributed to the delay in associating the various cases of microcephaly breaking out in the northeast with the Zika virus that was already known to circulate in the region.

The crucial point of this text is illustrated by the scenario above, in which it was necessary a single doctor (Dr. Adriana Melo) to have the brilliant idea to test patients for the Zika virus and confirm her hypothesis of association of the virus with previously observed cases of microcephaly treated by herself in the Brazilian hinterland. Its understood that the medical teams worked with the tools available in their context and were extremely competent in attracting the attention of the health authorities to the problem and in providing a quick response to stop the cases of microcephaly in the region. But there is undoubtedly room to shorten this response time in future outbreaks.

Real-time PCR tests are the gold standard for diagnosing viral infections

The standard molecular tests for virus diagnosis today in Brazil and in the world are called real-time PCR (RT-PCR). These are tests developed with prior knowledge of the genomic sequence of the virus and work specifically on the targeted virus. If the region chosen for the test is highly changeable, the test can be specific to a strain of the virus (for example, being able to recognize a circulating genotype, but not the vaccine). The results can be qualitative (positive or negative) and in some cases quantitative (viral load). However, there is a problem with these techniques: They are highly specific and targeted, that is, if a patient has feverish symptoms, red spots on the skin and headache, there is a handful of possibilities for clinicians to test. The physician would then have to order several PCR tests or serologies (they have different sensitivities and prices) in order to discard each of their so-called diagnostic hypotheses. This process can be time-consuming and costly for the single health system or for the patient himself. It can also be flawed, because it is dependent on the doctor’s ability to remember all the possible diagnostic hypotheses.

Virome as a highly informative tool to assist clinicians to issue an accurate and unambiguous diagnosis

This is where bioinformatics and genome sequencing come in together in precision medicine, being capable of bringing valuable information and providing a “wildcard tool” to help infectious disease clinicians to reach a diagnosis in an accurate and unambiguous way. This is the metagenomics new generation sequencing, or mNGS. Metagenomics is a term used by researchers to study all microorganisms contained in a sample. If the interest is for all viruses present in a sample, the term “Virome” is used. And this is where things get interesting, as a virome test would be able to identify any virus present in a clinical sample (urine, saliva, plasma, etc.) without the need for multiple targeted PCR tests. 

Better than that, given that prior knowledge of the sequence of a given virus or genotype is not necessary, since the genomic fragments are sequenced by the equipment and generate sequences regardless of that. There are still challenges to this test, such as the experimental standardization of a test with sensitivity similar to that of RT-PCR tests and bioinformatics analyzes, which are responsible for the precise identification of the viral pathogen and validation of the finding. Nevertheless, many clinicians and researchers believe that this is the future of diagnosing infectious diseases.

Virome at Albert Einstein Hospital and other institutions

Some clinical research groups in the USA, Germany and England have shown very interesting results in the identification of agents that cause neuro encephalitis, in the diagnosis of arboviruses and in the various types of hepatitis. Early in 2020, Albert Einstein Hospital, through internal research and development, created an RNA virus Virome exam available as one of its routine exams [5]. The exam was validated internally following the conventions of the College of American Pathologists (CAP), of which the laboratory is credited. The accuracy in samples previously tested with RT-PCR reached 100%, and some samples considered negative were demonstrated by the virome test as containing viruses for which no previous RT-PCR result was found. Subsequently, the respective RT-PCR tests were performed on samples considered positive by the virome test and the result was confirmed. Even in the validation process, the test has proved to be much more comprehensive than conventional techniques. In addition to validating accuracy, the hospital team also performed reproducibility and sensitivity validations for the test. All with results similar to those of standard RT-PCR tests. And perhaps most interestingly, the test is capable of recovering fragments or complete genomes of the viral pathogens in the samples. With the genome in hand, the hospital, together with surveillance agencies, can map the emergence of diverse etiologic agents and their variations, as well as to carry out highly sophisticated molecular epidemiology studies. It is worth remembering that as this is a totally new exam, all results are carefully analyzed by a panel of laboratory analysts, bioinformatics and infectious disease physicians before the report is released to the patient.

In addition to the test developed by Albert Einstein Hospital, other diagnostic companies are also making efforts to enter the market and offer good tools in precision infectious medicine. DASA, for example, closed an international partnership with the American company Kairus, which performs a metagenomic test to identify viral and bacterial DNA pathogens (RNA viruses are not identified) . According to the latest information available in the news media, the test is not yet carried out in Brazil and biological samples are sent to the USA for analysis.

And now? What are the next steps?

There are infinite possibilities Virome tests, but we will only have a good overview of what is to come after routine performance of many of these tests are concluded. It is reasonable to predict that the genomic and epidemiological information generated by the data from these examinations will allow physicians to provide increasingly accurate and personalized results for each sample, patient or set of symptoms. The world of machine learning is a whole universe to be explored, and one that can provide even more tools to assist clinicians in making accurate and unequivocal diagnoses.

Moreover, a friendly interface, in tools such as Varstation, for analysts and infectologists to view the results is essential and extremely important for the development and wide use of metagenomic tests. Another point to consider is prices, which tend to decrease sharply in the short-term future. The test developed by Albert Einstein Hospital, for instance, already has prices comparable to RT-PCR techniques. In addition, the institution recently acquired state-of-the-art equipment from the sequencing company Illumina, whose expectations are for prices to drop to even lower values.

Precision medicine has indeed arrived for the diagnosis of infectious diseases. More than that, it has arrived once and for all in the areas of clinical microbiology, molecular epidemiology and many other areas. Epidemics of the most diverse viruses will continue to occur due to factors that we are not completely able to control. The difference now is that we have powerful tools to win this fight. In that way, we hope that stories like HIV and ZIKV will not be repeated in our history. We hope that they will be promptly identified and fought so that they never stop being small, self-contained outbreaks.

About the author: 

Deyvid Amgarten is a bioinformat specialized in viral genomics with a bachelor’s and master’s degree from the University of São Paulo. He is currently linked to the clinical laboratory of the Hospital Albert Einstein and writes in partnership with Varstation.

References:

[1] An African HIV-1 sequence from 1959 and implications for the origin of the epidemic. Nature. 1998 Feb 5;391(6667):594-7

[2] Isolation of human T-cell leukemia virus in acquired immune deficiency syndrome (AIDS). Science. 1983 May 20;220(4599):865-7.

[3] Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science. 1983 May 20;220(4599):868-71.

[4] Zanluca, Camila et al. “First report of autochthonous transmission of Zika virus in Brazil.” Memórias do Instituto Oswaldo Cruz vol. 110,4 (2015): 569-72. doi:10.1590/0074-02760150192.

[5] Pesquisa de Vírus de RNA e Genotipagem – PLASMA (EDTA- GEL). Disponível em: <https://www.einstein.br/exames/info/#!6811>.

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