BR102021012552A2 - IN VITRO METHOD, KIT AND USE OF A PRIMER COMBINATION FOR THE DIFFERENTIAL DIAGNOSIS OF HERPESVIRUS INFECTIONS OF THE CENTRAL NERVOUS SYSTEM - Google Patents

Abstract

The present invention relates to an in vitro method for the differential diagnosis of infections of the central nervous system by herpesviruses, comprising the steps of extracting DNA from a sample of human cerebrospinal fluid; multiplex real-time PCR amplification using primers as defined in any one of the sequences SEQ ID NO: 1 to 12; and identification of the amplicon(s) produced according to the dissociation temperature (Tm) from dissociation curves with high resolution (HRM). The present invention further relates to a kit for the differential diagnosis of infections of the central nervous system by herpesvirus comprising a combination of primers selected from the group SEQ ID NO: 1 to 12; commercial human DNA and water as negative controls; and plasmid DNAs containing the viral sequences as positive controls; and reagents for the multiplex real-time PCR reaction. The present invention also refers to the use of a combination of primers selected from the group SEQ ID NO: 1 to 12, for the differential diagnosis of infections of the central nervous system by herpesviruses.

Description

IN VITRO METHOD, KIT AND USE OF A PRIMER COMBINATION FOR THE DIFFERENTIAL DIAGNOSIS OF HERPESVIRUS INFECTIONS OF THE CENTRAL NERVOUS SYSTEM FIELD OF THE INVENTION

[001] The present invention is in the field of molecular biology. The present invention relates to an in vitro method for the differential diagnosis of herpesvirus infections of the central nervous system. The present invention also refers to a kit for the differential diagnosis of infections of the central nervous system by herpesvirus. The present invention further relates to the use of a combination of primers for the differential diagnosis of herpesvirus central nervous system infections.

BACKGROUND OF THE INVENTION

[002] Infections in the central nervous system (CNS) are a challenge for clinical diagnosis, since different disorders, such as encephalitis, myelitis and meningitis, may be associated with infections by a diverse spectrum of pathogens. However, the Herpesviridae family has a central etiological role in such manifestations (CALVARIO et al., 2002).

[003] The Herpesviridae family is composed of more than 100 different viruses that infect a wide variety of animals (SOARES; PROVENZALE, 2016). Viruses belonging to this family have a genome composed of a double-stranded DNA molecule, which varies from 80 to 240kb, containing 60 to 120 genes, and a high incidence of regions of inverted repeated sequences. The replication process of these viruses occurs in the nucleus of infected cells and differences between the various herpesviruses are found especially in the specific glycoproteins present in the viral envelope (FERREIRA, 2013).

[004] Among the herpesviruses, only eight are capable of infecting varicella zoster (VZV), the Epstein-Barr virus (EBV), the cytomegalovirus (CMV), and the human herpesviruses (HHV) 6, 7 and 8 (SOARES; PROVENZALE, 2016). Of these, HSV-1, HSV-2, VZV, CMV, EBV and HHV-6 represent the main herpesviruses associated with CNS infections in immunocompetent or immunosuppressed individuals (MINJOLLE et al., 1999).

[005] In cases of high viremia or conditions favorable to viral replication, such as periods of immunosuppression, for example, viral particles can invade the CNS through cerebral capillaries, choroid plexuses or peripheral nerves, causing a variety of clinical manifestations. Thus, changes in the CNS, such as encephalitis and meningitis, often have an undefined origin, especially when clinical signs are not specific enough to attribute the infection to a particular virus (CALVARIO et al., 2002).

[006] Encephalitis is an inflammation of the brain parenchyma that generates neurological dysfunction, and can result from infectious or non-infectious causes, or even after infectious processes (BRADSHAW; VENKATESAN, 2016), its cause being identified in less than half of cases, and with an annual incidence, when the infectious origin is presumed, estimated at 1.5 to 7 cases per 100,000 inhabitants, excluding epidemics.

HSV-1/2 infections

[007] HSV infections are among the most common worldwide, with seropositivity observed in 50% to 90% of adult populations (WHITLEY, 2015). In general, primary infection by HSV-1/2 is associated with the production of lesions on the skin and on the oral and genital mucosa. In general, HSV-1 infections are commonly associated with lesions in the oral mucosa and in the ocular region, being one of the main etiological agents of sporadic encephalitis, known as herpetic encephalitis (MENENDEZ; CARR, 2017). On the other hand, HSV-2 infections, responsible for most cases of genital herpes, when symptomatic, is characterized by the periodic reappearance of painful genital ulcers. However, genital herpes is asymptomatic, or unidentified, in about 80-90% of individuals. Thus, although individuals with HSV-2 infection have a greater potential for transmission when symptomatic, it is believed that most transmission occurs when the source partner is asymptomatic (LOOKER et al., 2017).

[008] Epithelial cells constitute the main cell type involved in HSV infections. However, sensory ganglia neurons, as well as other CNS cells, are infected and constitute a reservoir of these viruses during the latency periods of the infection (TYLER, 2004). HSV infection triggers an intense response from the innate immune system until adaptive immunity is able to help clear the infection.

[009] Events that cause disturbances in the homeostasis of the immune system may favor the spontaneous recurrence of the infection, which has a chronic nature. However, the development of complications and the duration of symptoms depend on the individual's immune competence (KLEINSCHMIDT-DEMASTERS; GILDEN, 2001). According to MENENDEZ and CARR (2017), the association between HSV-1 and neurological disease also raises the possibility of latent infection influencing the development of neuronal deficits or CNS disorders.

[0010] After the neonatal period, most cases of HSV encephalitis are associated with HSV-1 reactivation (STEINER; BENNINGER, 2013). Patients usually present with a progressive decline in the level of consciousness, with or without fever, and seizures (SOARES; PROVENZALE, 2016). HSV-2 infection is also considered one of the main causes of neonatal encephalitis, which occurs from perinatal transmission due to maternal genital herpes, with CNS involvement being observed in approximately 35% of these cases (FONSECA-ATEN et al., 2005). However, HSV-2 neonatal neuroinfections respond well to prolonged antiviral therapy with acyclovir, whereas cases of HSV-1 infection do not show the same benefit (WHITLEY, 2015).

[0011] In young adults, HSV-2 neuroinfections are also related to cases of recurrent viral meningitis, called Mollaret's meningitis (TYLER, 2004). Although HSV encephalitis is considered a rare event (WHITLEY, 2015), mortality rates can reach rates of 70% in the absence of therapy (TYLER, 2004). Therefore, since the introduction of new drugs for the treatment of HSV infections has advanced slowly in the last three decades, efforts have focused on establishing an early diagnosis, with CRP in the cerebrospinal fluid (CSF) currently considered the gold standard. , allowing better application of antiviral therapy (WHITLEY, 2015).

VZV infections

[0012] VZV infections are associated with the development of Varicella, characterized by a generalized and pruritic vesicular rash, highly contagious, which occurs more frequently in children, and Zoster, which manifests itself after a relapse of the infection, especially in individuals above 60 years old (SOARES; PROVENZALE, 2016). Zoster is characterized by the appearance of papulo-vesicular lesions following the nerve ramifications, mainly the facial and thoracic ones, usually unilaterally (ŞALÇINI et al., 2015), and affects 15% of immunocompetent patients and 50% of immunosuppressed ones. Serological studies show that 98% of adults have already been exposed to this virus, and that factors such as physical and/or psychic stress, immunosuppression due to AIDS, transplants, neoplasms, autoimmune diseases, old age and immunosuppressive treatments are considered triggers for its occurrence. do Zoster (BOLLEA-GARLATTI et al., 2017).

[0013] In primary infection, VZV particles are produced by cells of the upper respiratory tract and, after viremia, remain latent in sensory ganglia. Acute varicella (chicken pox), once a common rash illness, has gradually disappeared in areas with active vaccination programs. Even when chickenpox was more common, CNS complications associated with this virus were rare, occurring in less than 1% of children. However, in cases of Zoster, especially in patients over 60 years of age, the occurrence of postherpetic neuralgia is observed in 40% to 45% of cases, being defined by pain with dermatomal distribution, which persists for more than six weeks after the rash disappears (KLEINSCHMIDT-DEMASTERS; GILDEN, 2001).

[0014] When clinical manifestations involve more than two contiguous dermatomes, with more than 20 vesicles outside the initial dermatome and systemic involvement, the disease is called disseminated zoster, being infrequent and affecting mainly immunosuppressed patients (BOLLEA-GARLATTI et al., 2017). Immunosuppressed individuals may also present more serious complications of viral reactivation, such as greater depth of tissue penetration and higher viremia (GILDEN et al., 2000).

[0015] Neurological involvement by VZV is more frequent in immunosuppressed and elderly patients, and cerebellitis may occur, which presents as ataxia and vomiting weeks after the onset of infection, encephalitis or meningoencephalitis, vasculitis, or even myelitis, often more insidious and progressive in HIV-infected individuals, in which extensive necrosis is observed, often being fatal (GILDEN et al., 2000; KLEINSCHMIDT-DEMASTERS; GILDEN, 2001; LEVIN; WEINBERG; SCHMID, 2016; SOARES; PROVENZALE, 2016). In immunocompetent patients, Zoster can complicate the myelitis, usually one to two weeks after the development of a skin rash, with clinical features such as paraparesis with sensory alteration and sphincter impairment being observed (GILDEN et al., 2000).

[0016] A retrospective study conducted by BOLLEA-GARLATTI et al. (2017), from 2010 to 2015, showed that more than half of patients with disseminated Zoster had thrombocytopenia, and that leukopenia, anemia and changes in liver biochemistry are also frequent. Furthermore, it was observed that such laboratory alterations are also present in primary VZV infection, emphasizing the importance of monitoring such parameters in association with the clinical picture.

[0017] Advances in molecular biology have allowed important considerations on the pathogenesis of VZV infection. The detection of these viruses in blood vessels and other tissues by methods based on the polymerase chain reaction (PCR) has expanded the clinical spectrum of associated acute and chronic disorders (GILDEN et al., 2000). Sensitive methods are also desirable for an adequate diagnosis, since the load of VZV in the CSF is considered low (KÖLLER et al., 2016).

EBV infections

[0018] EBV infection, as well as HSV-1/2, has a worldwide distribution, with a seroprevalence of up to 90% in adults (NOWALK; GREEN, 2016). Transmission is by contact, especially through saliva (on hands or toys, or by kissing). EBV is present in male and female genital secretions, although there is little evidence of sexual spread of the virus. The infection can also be transmitted by blood transfusion and organ transplantation (MAZUR-MELEWSKA et al., 2016).

[0019] In a retrospective study that analyzed 194 children with EBV infection, from 2010 to 2015, it was observed that EBV transmission has two peaks of infection rate as a function of age, the first being in children aged between one and five years, which accounted for 62% of cases, and the second in adolescents, aged between 13 and 17 years, which accounted for 25% of cases (MAZUR-MELEWSKA et al., 2016). This dynamic is associated with socioeconomic factors, being illustrated by the higher frequency of infection in the first years of life, in which seroconversion is frequently observed among children aged three to four years in developing countries, while in developed countries, the infection often it is postponed until adolescence (MAZURMELEWSKA et al., 2016).

[0020] The infection by this virus can persist throughout the individual's life, remaining quiescent in circulating B lymphocytes and/or in the nasopharyngeal lymphoid tissue (VOLPI, 2004). Latently infected B lymphocytes circulate systemically and serve as lifelong viral reservoirs; these lymphocytes transiently express only a highly restricted set of EBV genes and therefore are largely undetectable by immune surveillance mechanisms. Intense responses to EBV by CD4+ and CD8+ T lymphocytes occur in patients with infectious mononucleosis, and evidence suggests that these cellular responses limit primary infection and control chronic infection, although they may contribute to the symptoms of infectious mononucleosis. .

[0021] Thus, EBV infection has been associated with upper respiratory tract infection accompanied by fever in children, and with infectious mononucleosis in young adults, which is characterized by drowsiness, fatigue, fever, pharyngitis, hepatosplenomegaly and adenopathy (NOWALK; GREEN, 2016).

[0022] Neurological involvement in EBV infection is observed in up to 5% of cases of infectious mononucleosis (LUZURIAGA; SULLIVAN, 2010; MAZUR-MELEWSKA et al., 2016), and clinical features include altered levels of consciousness, visual hallucinations, status epilepticus and psychosis. Most patients with EBV encephalitis have nonspecific prodromal symptoms, including fever and headache, which last for an average of seven days, and it is quite uncommon for all the classic signs to appear together with the symptoms of mononucleosis. ).

[0023] In immunosuppressed patients, EBV infection is also associated with the development of different types of tumors, including nasopharyngeal carcinoma, Hodgkin's and non-Hodgkin's lymphomas, and Burkitt's lymphoma (SOARES; PROVENZALE, 2016). Therefore, the oncogenic potential of EBV has been shown to be an important factor in the development of primary CNS lymphomas in HIV patients and in post-transplant lymphoproliferative disorder (KLEINSCHMIDTDEMASTERS; GILDEN, 2001; NOWALK; GREEN, 2016).

CMV infections

[0024] CMV infection has proven to be a major cause of morbidity and mortality in transplanted individuals, despite advances in preventive strategies. Two strategies can be employed to prevent CMV infection in transplant patients: universal prophylaxis, which involves administration of antivirals at the start of transplantation, and preventive antiviral therapy, in which antivirals are prescribed only for patients with active CMV replication. 2016).

[0025] CMV is considered one of the most widespread viruses of the Herpesviridae family, being able to infect humans at all stages of life, especially pregnant women (SOARES; PROVENZALE, 2016), in which it can lead to congenital malformations. Affected neonates are usually microcephalic and present systemic involvement in the form of hemolytic anemia, jaundice, purpura, hearing loss and chorioretinitis, with a permanent deficit being observed in up to 20% of cases (NETO et al., 2004).

[0026] Oral transmission is predominant among children, while the sexual route is considered epidemiologically relevant in adulthood. Transmission of CMV can occur through the transfusion of blood products, from mother to child, through the placenta, and by organ transplantation. After primary infection, which is usually mild or asymptomatic in immunocompetent individuals, CMV establishes latent/persistent infection of CD14+ peripheral mononuclear cells, and CD34+ and CD33+ myeloid precursors of the bone marrow, and can be detected in different organs and mucous membranes, and in different cell types, including macrophages, epithelial and endothelial cells, which reveals the difficulty of the immune system in eliminating the infection (NAVARRO, 2016).

[0027] Most cases of CMV infection are asymptomatic, and retinitis is the most common manifestation, accounting for 85% of cases with symptoms (DIOVERTI; RAZONABLE, 2016). However, in cases of acute infection, both in children and adults, hepatitis and symptoms similar to mononucleosis may occur.

[0028] In children, CMV can lead to immunosuppression and secondary viral infection, such as some respiratory viruses, and respiratory symptoms are observed in about 50% of infected children (MEDOVIĆ et al., 2016). On the other hand, CMV infections associated with a mononucleosis-like picture in immunocompetent adults are characterized by fever, lymphadenopathy and lymphocytosis, while in immunosuppressed individuals they may be associated with pneumonia, esophagitis, encephalitis, hepatitis, pancreatitis, gastritis, enteritis and colitis. (DIOVERTI; RAZONABLE, 2016). In these individuals, such as carriers of HIV infection, for example, CMV retinitis may be followed by CNS infection, resulting in encephalitis, polyradiculitis and ventriculitis (KLEINSCHMIDT-DEMASTERS; GILDEN, 2001; SILVA et al., 2010).

HHV-6 infections

[0029] HHV-6 was initially described in 1986, in patients with lymphoproliferative disorders, and later divided into two distinct groups, called variants A and B. Like other herpesviruses, HHV-6 are capable of infecting different cell types and of constitute latent infection (AGUT; BONNAFOUS; GAUTHERET-DEJEAN, 2016)

[0030] HHV-6B infection is related to sudden exanthema, and is estimated to be responsible for half of fever episodes in children under two years of age, being transmitted mainly through saliva, and establishing latency in the salivary gland, in leukocytes, and in the CNS (DE BOLLE; NAESENS; DE CLERCQ, 2005). The main complication of primary HHV-6 infection is epilepsy during the febrile period. With the exception of the manifestation of fever without rash, and febrile seizures in very young children, immunocompetent individuals rarely present with CNS infection (KLEINSCHMIDT-DEMASTERS; GILDEN, 2001).

[0031] Symptomatic HHV-6 infection predominates among immunosuppressed patients, especially transplanted individuals (PHAN et al., 2018) and carriers of HIV infection (KLEINSCHMIDT-DEMASTERS; GILDEN, 2001). The role of opportunistic pathogen is well described for HHV-6 and less clear for HHV-7. It mainly concerns the CNS, bone marrow, lungs, gastrointestinal tract, skin and liver. Thus, HHV-6 infections are associated with both the manifestation of exanthema subitum, a benign disease associated with the primary infection, and severe encephalitis after reactivation (AGUT; BONNAFOUS; GAUTHERET-DEJEAN, 2016).

[0032] The clinical presentation of HHV-6 encephalitis is characterized by temporal lobe seizures, fever, and changes in mental status, including insomnia, and resembles HSV-1 encephalitis. Currently, the diagnosis of HHV-6 infection can be defined by CSF PCR, and rapid therapeutic intervention with ganciclovir or foscarnet offers significant benefits, while untreated cases can present mortality rates greater than 50% (SOARES; PROVENZALE, 2016 ), which makes it essential to discriminate the encephalitis associated with HHV-6 and HSV-1.

Laboratory diagnosis of herpesvirus neuroinfections

[0033] The laboratory diagnosis of CNS herpesvirus infections proves to be a challenge. The conventional method of viral isolation has a limited role in the investigation of neurological diseases, as it has low sensitivity and a prolonged turnaround time, requiring 1 to 28 days to obtain results and, therefore, has little help in diagnosis (DEBIASI et al., 2002). Likewise, serological tests for the detection of specific antibodies have a low positive predictive value (CALVARIO et al., 2002; RAMAMURTHY et al., 2011). The increase in antibody titers may take two to four weeks after the acute infection and, therefore, are of little help in deciding whether to start treatment (CALVARIO et al., 2002; DEBIASI et al., 2002; RAMAMURTHY et al. , 2011).

[0034] Thus, PCR has played a key role in the diagnosis of infectious diseases, as it allows the identification of infectious agents that are difficult or unfeasible to isolate (DEBIASI et al., 2002). Thus, PCR has been recognized as the reference method in the diagnosis of human herpesvirus infections, in a highly sensitive and specific way (MCIVER et al., 2005). In contrast to serological tests, PCR produces positive results during acute infection, especially in the first days of infection, when viral replication is observed, allowing better therapeutic management of patients (CALVARIO et al., 2002; DEBIASI et al., 2002 ; LEVEQUE et al., 2011). In addition, performing PCR from cerebrospinal fluid DNA is considered less invasive and has a lower cost when compared to brain biopsy, previously considered the gold standard for the diagnosis of many herpesvirus infections (DEBIASI et al., 2002).

[0035] Despite the great potential of PCR, in the past, it was necessary to carry out individual tests for each virus, making the diagnosis time-consuming and expensive (TAFRESHI et al., 2005). With the development of recombinant DNA polymerases with high enzymatic activity, it was possible to establish multiplex PCR, in which two or more DNA sequences are amplified simultaneously, using multiple primers (TAFRESHI et al., 2005). VANDEVANTER et al. (1996) described a PCR method with consensus primers for the detection and analysis of several herpesvirus species (eight humans and 14 animals). This assay, which has a nested-PCR format, aims at the amplification of the viral DNA polymerase region using degenerate primers, directed to highly conserved regions, with approximately 800-bp. However, the transposition of this method to real-time PCR approaches with dissociation curve analysis (melting) would be hampered by the use of degenerate primers, whose intrinsic variation in their sequences would directly influence the dissociation dynamics of the amplified fragments (amplicons). Furthermore, this amplicon size is inappropriate for real-time PCR.

[0036] In the development of multiplex PCR assays, the presence of different pairs of primers increases the probability of formation of unspecific amplification products, in particular of primer dimers. As the performance of primers in multiplex PCR is not presumable, it is necessary to carry out empirical tests, in which a matrix of concentrations is used to determine the optimal concentration of each primer in the reaction. Thus, in order for there to be similarity in sensitivity between individual and multiple tests, it is essential that the test conditions are optimized, in order to maintain the performance of multiple tests in relation to each of the individual tests that constitute it (COMPSTON et al. ., 2008).

[0037] Different human herpesvirus real-time PCR assays are described in the literature, although most are limited to the detection of up to three distinct viral types (ZERR et al., 2002; COHRS; GILDEN, 2007; HABBAL; MONEM; GÄRTNER, 2009; HONG et al., 2014; LO et al., 1999; NAGEL et al., 2014). In general, these assays apply the SYBR Green dye to reveal the DNA amplification, which generates variation in the dissociation temperature (Tm, melting temperature) inter-assays and hinders the reproducibility of viral identification, or they use TaqMan probes , which make the assay more expensive and restrict the number of DNA target sequences.

[0038] In addition, studies that proposed to establish PCR assays for simultaneous amplification of more than three types of herpesviruses present the revelation of the test by electrophoresis in agarose gel, or even, by performing Southern blot (hybridization with specific probes of DNA) (CALVARIO et al., 2002; MINJOLLE et al., 2002).

[0039] The development of new double-stranded DNA fluorescent dyes, such as EvaGreen®, LCGreen® and SYTO-9®, which have saturating DNA binding activity, has enabled the improvement of dissociation curve analysis, which suffers variation with the use of SYBRGreen®. With this, it is currently possible to carry out analysis of high resolution melting dissociation curves (HRM), which have advantages such as better cost-effectiveness in relation to tests based on fluorescent probes of the TaqMan® type, as well as as in relation to conventional PCR, as it does not require post-amplification manipulation, which always represents a risk of contamination of reagents and the work area, leading to the generation of false-positive results (TONG; GIFFARD, 2012).

[0040] The analysis of the dissociation curve is performed after the conclusion of the PCR, without the need to open the reaction tube, and is characterized by the analysis of the behavior of the transition from the double-stranded to single-stranded state (dissociation) of the generated amplicons . The gradual increase in temperature at the end of the PCR, with increments ranging from 0.2 to 0.5°C per second, results in the separation of the DNA strands and, consequently, the turning off of the double-stranded DNA dye molecules, causing the reduction of fluorescence. Denaturation of DNA fragments with increasing temperature defines the dissociation curve, and the Tm can be calculated by determining the peak of the first derivative of the dissociation curve (dF/dT). In the case of HRM, the decay of fluorescence generates a sigmoidal curve (TONG; GIFFARD, 2012).

[0041] Thus, the dynamics of the fluorescence decay evaluated in the HRM allows a more accurate discrimination between PCR products than the simple comparison of Tm. Tm values are defined by the size of the DNA sequences and the percentage of GC (%GC). The correlation of Tm with %GC is due to the stronger bond between the DNA double-stranded chains provided by the presence of three hydrogen bonds between GC pairs, compared to the two bonds between AT pairs (TONG; GIFFARD, 2012). In HRM these characteristics are added to the nucleotide substitutions in the definition of the dissociation curves. Thus, it is possible to identify genetic polymorphisms and epigenetic DNA alterations.

[0042] Ptaszyńska-Sarosiek et al. (2019) demonstrate the determination of 6 viruses HSV-1, HSV-2, VZV, EBV, CMV and HHV-6 using the multiplex PCR technique. From 47 samples of deaths with independent and random causes in Poland, it was possible to detect CNS infections in 30/47 (63.8%) of the samples. More specifically, among the results, HHV-6 was the most prevalent herpesvirus in the samples, being found in almost half of them (46.8%), followed by HSV-1 (14.9%), EBV (8.5%) , HSV-2 (4.3%) and CMV (2.1%). The Seegene® PCR kit (DPOTM) and the one-tube multiplex PCR method were used. However, that document does not teach the multiplex PCR procedure combined with HRM analysis.

[0043] Leveque et al., 2011 reveal the simultaneous detection by multiplex PCR of 9 diseases including HSV-1, HSV-2, VZV, CMV, EBV, HHV-6, HHV-7, HHV-8 and HEV (human enterovirus ). A total of 106 samples were used (78 from cerebrospinal fluid, CSF, and 28 from CNS). Discrepancies were observed for samples with low concentration of HSV-2 and HEV. The multiplex test was able to detect infections in 27 out of 28 CNS samples. However, that document does not teach the multiplex PCR procedure combined with HRM analysis. On the contrary, they make use of real-time PCR combined with microarray.

[0044] WO2019/117701 discloses a method of multiple and simultaneous detection of EBV, CMV, HHV6, HHV7 and KSV (Kaposi's sarcoma virus). For this, multiplex real-time PCR is used, using primers obtained from the literature. It is important to point out that the aforementioned document does not address all viruses in the present study, in this case HSV-1, HSV-2 and VZV

[0045] WO2009122201 describes a method and kit for detection and identification of Herpesviruses in an HSV-1, HSV-2, VZV, CMV, EBV, HEV, HHV-6, HHV-7 and HHV-8 sample. The technique employed is real-time PCR with dot blot assay and heteroduplex mobility shift assay (HSMA). Therefore, said document does not teach the multiplex PCR procedure combined with HRM analysis. Rather, they make use of real-time PCR combined with HSMA.

[0046] WO2004016219 reveals a kit and methods for identifying herpesviruses in a sample, allowing the detection of ten different types by multiplex PCR technique using consensus primers to amplify conserved regions of the viral DNA. However, that document does not teach the multiplex PCR procedure combined with HRM analysis.

[0047] US 2007/0207453 discloses a multiplex PCR detection method capable of identifying CMV, HSV-1 and VZV. It is important to point out that the aforementioned document does not address all the viruses in the present study, in this case HSV-2, EBV and HHV-6.

[0048] KR101099041 reports a method of diagnosing different types of herpesvirus by multiplex PCR. CMV, EBV, HHV-6 and HVV-7 were amplified. The multiplex PCR reaction medium generated the amplicons corresponding to the HHV-7, EBV, CMV and HHV-6 viruses, separated by agarose gel electrophoresis. It is important to point out that the aforementioned document does not address all the viruses in the present study, in this case HSV-1, HSV-2 and VZV.

[0049] CN105385787 presents a PCR multiple detection kit for 12 encephalitis viruses, nucleic acids used in the kit and the diagnostic method. However, that document does not teach the multiplex PCR procedure combined with HRM analysis.

[0050] The invention described has the advantage of high sensitivity, allowing to differentiate herpeviruses responsible for infections of the central nervous system more quickly and more conveniently. In addition, the primers used allow for greater reliability in the result obtained.

SUMMARY OF THE INVENTION

[0051] Currently, there is a growing demand for diagnostic methods that provide fast, accurate answers and that allow a wide detection of pathogens, especially those associated with similar or nonspecific clinical manifestations, or that present similar epidemiological characteristics.

[0052] The etiological diagnosis of herpesvirus infections is often complicated, as clinical and laboratory findings are often nonspecific or even similar between infections by different viral types (TAFRESHI et al., 2005). Furthermore, the purely clinical diagnosis is unreliable, mainly because a wide variety of viruses are capable of infecting CNS cells. Thus, laboratory diagnosis is essential, both for confirmation and for exclusion of possible etiological agents in clinical suspicion.

[0053] The Herpesviridae family is responsible for most cases of meningoencephalitis and sporadic viral myelitis in immunocompetent individuals of all ages. The viruses of this family are considered important human pathogens, as they have a high global prevalence (WONG et al., 2016). Its importance is even greater in patients with different levels of immunosuppression, such as individuals infected with HIV or undergoing organ transplantation, with hereditary immunodeficiencies and neoplasms. In these groups, CNS infections by herpesvirus are related to higher morbidity and mortality and can occur atypically, with significant overlapping of signs and symptoms, making clinical diagnosis difficult, which highlights the need for sensitive and specific laboratory methods for diagnosis.

[0054] Currently, there are effective antiviral drugs against different herpesviruses, such as acyclovir, ganciclovir, foscarnet, cidofovir, valaciclovir, valganciclovir, famciclovir, among others (NATH; TYLER, 2013). However, in immunocompromised patients, the main risk factor for viral dissemination is the time elapsed between the onset of symptoms and therapeutic intervention, with the greatest risk being increased when the initiation of therapy is postponed.

[0055] Laboratory diagnosis by serological methods or direct detection of antigens has limitations (AGUT; BONNAFOUS; GAUTHERET-DEJEAN, 2016). A retrospective study aimed at determining clinical and laboratory differences between EBV and CMV in children with infectious mononucleosis demonstrated that 40% of patients with acute EBV infection also had increased IgM titers for CMV. However, CMV IgG avidity tests demonstrated that such cases were a previous CMV infection, and the results were then interpreted as an acute EBV infection (MEDOVIĆ et al., 2016). Such results, with a high frequency of IgM reactivity for both viruses, represent a basis that highlights the need for further research to establish diagnostic methods. The most prominent technique is the quantification of viral DNA in blood and other body fluids and tissues using real-time PCR (AGUT; BONNAFOUS; GAUTHERETDEJEAN, 2016). In addition, PCR can be used to amplify DNA sequences in the CSF of patients, especially in the first days of infection, and studies have described the simultaneous detection of different viral types in single PCR tests.

[0056] Thus, the detection of pathogens by multiplex PCR, followed by sequence identification by dissociation curve analysis, conventional or HRM, can be considered a valuable approach for several reasons, such as, reduction in sample manipulation and in the chance of laboratory contamination, since the tests can be performed in a single step and without opening the reaction tube, reduction in expenses with reagents, in addition to favoring cases in which the amount of sample is limited or difficult to access (BRISSON, MARNI et al., 2004; TONG; GIFFARD, 2012).

[0057] Therefore, the establishment of a multiplex PCR method with specific primers for the simultaneous detection of six different herpesviruses would improve the cost-effectiveness of the test and reduce the diagnostic and decision-making time for implementing specific antiviral therapy, especially in individuals with AIDS or on immunosuppressive therapy as a result of organ transplantation.

[0058] It is also worth noting that there is no description in the literature of a real-time multiplex PCR methodology with such a detection range for these viruses.

[0059] The present invention aims to develop a real-time polymerase chain reaction (PCR) method aimed at the simultaneous detection and discrimination of six herpesviruses (HSV-1, HSV-2, VZV, EBV, CMV and HHV-6) commonly associated with central nervous system infections in humans.

[0060] More specifically, the present invention aims to:
• Evaluate the specificity of PCR primers for the amplification of human herpesvirus genome fragments HSV-1, HSV-2, VZV, EBV, CMV and HHV-6;
• Evaluate the compatibility of PCR primers for carrying out tests for the detection of multiple DNA target sequences, both in the formation of dimers and in the generation of nonspecific products in relation to human DNA;
• Establish the optimal real-time PCR conditions, in single and multiple reactions, for the detection of human herpesviruses, with confirmation of the identity of the amplified sequences by conventional and high-resolution dissociation curve analysis (HRM);
• Define the amplification efficiency of each of the herpesviruses evaluated in the individual and multiplex PCR assays;
• Determine the interference that the simultaneous amplification of two DNA target sequences causes in the detection efficiency of each of the herpesviruses by multiplex PCR;
• Define the detection limits, in number of viral genomic DNA copies per reaction, of real-time multiplex PCR.

[0061] It is important to clarify that although the selection of primers for these six viruses occurs from the literature, such a strategy presents some difficulties. First, there is a lack of bibliography in the area, because almost all of the studies described are either directed to conventional PCR, or use TaqMan-type probes to reveal DNA amplification.

[0062] Additionally, another part of the difficulty is associated with the detection and identification of six distinct viruses in a single reaction, which is a major challenge due to the selection of specific primers. A person skilled in the art recognizes that the greater the number of primers present in the reaction, the greater the chance of forming nonspecific products, particularly primer dimers. Therefore, maintaining the same performance levels of primers in individual tests is another challenge in multiplex PCR development and optimization.

[0063] In one aspect, the present invention relates to an in vitro method for the differential diagnosis of herpesvirus central nervous system infections, which consists of the following steps: extracting DNA from a sample of human cerebrospinal fluid; amplifying by multiplex real-time PCR using primers as defined in any one of the sequences SEQ ID NO: 1 to 12; and identify the amplicon(s) produced according to the dissociation temperature (Tm) from dissociation curves with high resolution (HRM). In one embodiment, the amplicon is from one or more of the following viral types HSV-1, HSV-2, VZV, EBV, CMV and HHV-6. In another embodiment, the main disorders associated with said central nervous system infections can be selected from encephalitis, cerebellitis, vasculitis, retinitis, polyradiculitis, ventriculitis, myelitis and meningitis. In another embodiment, the subject is a human subject. In another embodiment, further optionally quantitative diagnosis by individual assays can be performed.

[0064] In another aspect, the present invention relates to a kit for the differential diagnosis of herpesvirus central nervous system infections, comprising a combination of primers selected from the group SEQ ID NO: 1 to 12; commercial human DNA and water as negative controls; and plasmid DNAs containing the viral sequences, as positive controls; and reagents for the multiplex real-time PCR reaction. In one embodiment, the kit is for diagnosing and identifying one of the following herpesviruses: HSV-1, HSV-2, VZV, EBV, CMV and HHV-6.

[0065] In another aspect, the present invention relates to the use of a combination of primers selected from the group SEQ ID NO: 1 to 12 for differential diagnosis of central nervous system herpesvirus infections.

BRIEF DESCRIPTION OF THE FIGURES

[0066] The purpose of the invention, together with additional advantages thereof, may be better understood by referring to the attached figures and the following descriptions:

[0067] Figure 1 shows a workflow.

[0068] Figure 2 shows the vector map pGEM-T Easy (Promega). Confirmation of the cloning of the amplicons of each of the evaluated herpesviruses was performed by PCR and/or sequencing using primers for the M13 region, called M13 fwd and M13 rev.

[0069] Figure 3 shows PCR dissociation curve for HSV-1 with primers at 17.5 pmoles. The reactions were carried out with annealing/extension temperature at 60°C. The curves showed the specific peaks of HSV-1 in the positive controls (CP), unspecific amplifications from human DNA (DNA H) and dimer formation in the negative control containing only distilled water (WATER), and the dissociation temperatures (Tm ) presented in the associated table through the color legend.

[0070] Figure 4 shows PCR dissociation curve for EBV, CMV and HHV-6. Reactions were carried out with primers at a concentration of 17.5 pmol and annealing/extension temperature at 60°C. The curves show the specific peaks of EBV, CMV and HHV-6 in the positive controls (CP), unspecific amplifications from human DNA (DNA H), and the dissociation temperatures (Tm) are presented in the associated table through the color legend . WATER, negative control containing only distilled water.

[0071] Figure 5 displays PCR dissociation curve for HSV-2 and VZV. Reactions were carried out with primers at a concentration of 17.5 pmol and annealing/extension temperature at 60°C. The curves show the peaks of HSV-2 and VZV in the positive controls (CP), with peaks with different dissociation temperatures (Tm) being observed for VZV, according to the table with the color legend. WATER, negative control with distilled water.

[0072] Figure 6 shows PCR dissociation curve for HSV-1 with primers at 10 pmoles. The reactions were carried out with annealing/extension temperature at 60°C. The curves show the specific peaks of HSV-1 in the positive controls (CP), the unspecific amplifications from human DNA (DNA H) and the formation of dimers in the negative control containing water (WATER), with the dissociation temperatures (Tm ) presented in the associated table through the color legend.

[0073] Figure 7 shows PCR dissociation curve for EBV and CMV. Reactions were carried out with primers at a concentration of 10 pmoles and annealing/extension temperature at 60°C. The curves show the specific peaks of EBV and CMV in the positive controls (CP) and the unspecific amplifications from human DNA (DNA H), with the dissociation temperatures shown in the associated table through the color legend. WATER, negative control with distilled water.

[0074] Figure 8 displays PCR dissociation curve for HSV-2, VZV and HHV-6. Reactions were carried out with primers with a concentration of 10 pmoles and annealing/extension temperature at 60°C. The curves show the peaks of HSV-2, VZV and HHV-6 in the positive controls (CP), being observed for VZV peaks with different dissociation temperatures (Tm), in addition to nonspecific amplification from human DNA (DNA H) to HSV-2 and VZV, according to the chart with color legend. WATER, negative control with distilled water.

[0075] Figure 9 shows PCR dissociation curve for EBV with annealing/extension at 60°C. Reactions were carried out with primers at a concentration of 17.5 pmoles. The curves show the EBV-specific peaks in the positive controls (CP) and the unspecific amplifications from human DNA (DNA H). Dissociation temperatures (Tm) shown in the table by color legend. WATER, negative control with distilled water.

[0076] Figure 10 shows PCR dissociation curve for VZV and CMV. Reactions were performed with primers at 17.5 pmoles and an annealing/extension step at 60°C. The curves show the specific peaks for VZV and CMV in the positive controls (CP), and the absence of dimers and unspecific amplification with human DNA (DNA H), according to the associated table through the color legend. WATER, negative control with distilled water.

[0077] Figure 11 shows PCR dissociation curve for EBV with annealing/extension at 70°C. Reactions were carried out with primers at a concentration of 17.5 pmoles. The curves show the specific peaks for EBV in the positive controls (CP), and the absence of dimer formation and unspecific amplification from human DNA (DNA H) and water, as shown in the associated table through the color legend.

[0078] Figure 12 shows PCR dissociation curve for HSV1/2. Reactions were carried out with primers at a concentration of 17.5 pmol and annealing/extension temperature at 70°C. The curves show the specific peaks for HSV-1 and HSV-2 and nonspecific amplification in positive controls (CP), dimer formation and nonspecific amplification from human DNA (DNA H), as shown in the associated table through the color legend . CN, negative control.

[0079] Figure 13 shows primer combinations for modified HSV-1/2.

[0080] Figure 14 shows PCR dissociation curve with modified primers for HSV-1/2. The reactions were carried out with combination 6 primers at a concentration of 17.5 pmoles and an annealing/extension step at 70°C. The curves show the specific peaks for HSV-1 and HSV-2 and nonspecific amplification in positive controls (CP), absence of dimer formation and nonspecific amplification from human DNA (DNA H), as shown in the associated table through the color legend. CN, negative control.

[0081] Figure 15 shows PCR dissociation curve with modified primers for HSV-1/2 and Vero cell DNA. Reactions were carried out with combination 6 primers at a concentration of 17.5 pmoles and an annealing/extension step at 70°C. The curves show the peaks for HSV-1 and HSV-2 and unspecific amplification in positive controls (CP), absence of dimers and unspecific amplification with human DNA (DNA H), and Vero cells, as shown in the associated table through the color legend. CN, negative control.

[0082] Figure 16 shows PCR dissociation curve for HSV1/2 with modified primers from Hong et al. (2014). Reactions were carried out with primers at a concentration of 17.5 pmoles and an annealing/extension step at 70°C. The curves show the specific peaks for HSV-1 and HSV-2 in the positive controls (CP), absence of dimer formation and unspecific amplification from human DNA (DNA H), as shown in the associated table through the color legend . CN, negative control.

[0083] Figure 17 shows multiplex PCR dissociation curve for VZV, CMV and HHV-6 with annealing/extension step at 60°C. Reactions were carried out with primers at a concentration of 17.5 pmoles. The curves show the specific peaks of VZV, CMV and HHV-6 in positive controls (CP), dimer formation in negative controls (CN) and unspecific amplification from human DNA (DNA H). The dissociation temperatures are presented in the associated table, through the color legend.

[0084] Figure 18 shows multiplex PCR dissociation curve for VZV, CMV and HHV-6 with annealing/extension step at 62°C. Reactions were carried out with primers at a concentration of 17.5 pmoles. The curves show the specific peaks of VZV, CMV and HHV-6 in the positive controls (CP), the absence of dimers in the negative controls (CN) and unspecific amplification in one of the replicates from human DNA (DNA H). The dissociation temperatures are presented in the associated table, through the color legend.

[0085] Figure 19 shows multiplex PCR dissociation curve for VZV, CMV and HHV-6 with annealing/extension step at 64°C. Reactions were carried out with primers at a concentration of 17.5 pmoles. The curves show the specific peaks of VZV, CMV and HHV-6 in positive controls (CP), absence of dimers in negative controls (CN) and unspecific amplification in one of the replicates from human DNA (DNA H). The dissociation temperatures are presented in the associated table, through the color legend.

[0086] Figure 20 shows multiplex PCR dissociation curve for VZV, CMV and HHV-6 with annealing/extension step at 68°C. Reactions were carried out with primers at a concentration of 17.5 pmoles. The curves show the specific peaks of VZV, CMV and HHV-6 in positive controls (CP), absence of unspecific amplification from human DNA (DNA H) and dimers in negative controls (CN). The dissociation temperatures are presented in the associated table, through the color legend.

[0087] Figure 21 shows multiplex PCR dissociation curve for VZV, CMV and HHV-6 with annealing/extension step at 70°C. Reactions were carried out with primers at a concentration of 17.5 pmoles. The curves show the specific peaks of VZV, CMV and HHV-6 in positive controls (CP), absence of dimers in negative controls (CN) and unspecific amplification in one of the replicates from human DNA (DNA H). The dissociation temperatures are presented in the associated table, through the color legend.

[0088] Figure 22 shows multiplex PCR dissociation curve for VZV, EBV, CMV and HHV-6. Reactions were carried out with primers at a concentration of 17.5 pmoles and an annealing/extension step at 70°C. The curves show the specific peaks of VZV, EBV, CMV and HHV-6 in the positive controls (CP), and absence of dimer formation and nonspecific amplification from human DNA (DNA H). The dissociation temperatures are presented in the associated table, through the color legend.

[0089] Figure 23 shows multiplex PCR dissociation curve for HSV-1/2, VZV, EBV, CMV and HHV-6, with primers modified for HSV-1/2. Reactions were carried out with primers at a concentration of 17.5 pmoles and an annealing/extension step at 70°C. For the detection of HSV-1/2, primers from combination 6 were used. The curves show the specific peaks of HSV-1, HSV-2, VZV, EBV, CMV and HHV-6 in the positive controls (CP), and absence of dimer formation and unspecific amplification from human DNA (DNA H). Non-specific peaks can be seen in HSV-1/2 and HHV-6 positive controls. Dissociation temperatures are shown in the associated box with color legend.

[0090] Figure 24 shows HRM curve analysis performed with six viral types. The curves demonstrate the dissociation dynamics in a temperature range from 78ºC to 93ºC for the different viruses. The difference in the decay curves allows the identification and discrimination of six viral types, with a confidence level greater than 96%. The genotype and confidence level are shown in the associated box with a color legend.

[0091] Figure 25 shows HRM analysis performed for HSV-1 and HSV-2 discrimination. The curves demonstrate the dissociation dynamics in a temperature range from 88.5ºC to 93.5ºC. The difference in the decay curves allows the identification and discrimination of HSV-1 and HSV-2, with a confidence level greater than 99%. The genotype and confidence level are shown in the associated box with a color legend.

[0092] Figure 26 shows multiplex PCR dissociation curve for HSV-1/2, VZV, EBV, CMV and HHV-6, with primers for HSV-1/2 modified from HONG (2014). Reactions were carried out with primers at a concentration of 17.5 pmol and annealing/extension temperature at 70°C. The curves show the specific peaks of HSV-1, HSV-2, VZV, EBV, CMV and HHV-6 in the positive controls (CP), and dimer formation and non-specific amplification from human DNA (DNA H) occurring from random way. The dissociation temperatures are presented in the associated table, through the color legend.

[0093] Figure 27 shows confirmation of sequences in recombinant plasmid controls. Alignment with the sequences of the viral lineages available in GenBank, using the online tool nucleotide-BLAST, showed that all cloned sequences presented 100% identity for VZV, CMV and HHV-6.

[0094] Figure 28 shows single PCR efficiency for HSV-1 with primers at 17.5 pmoles/reaction. Reactions were performed in triplicate, with an annealing/extension step at 70ºC, with base 10 dilutions (1:100 to 1:10-3) of recombinant plasmid containing the target sequence. Specific peaks of HSV-1 were observed in all reactions except the negative control (CN). The dissociation temperatures and the average of the cycle in which the reaction became positive (Ct) are presented in the associated table. Efficiency (E) and data fit values to the generated standard curve (R2) are also presented.

[0095] Figure 29 shows single PCR efficiency for HSV-2 with primers at 17.5 pmoles/reaction. Reactions were performed in triplicate, with an annealing/extension step at 70ºC, with base 10 dilutions (1:100 to 1:10-3) of recombinant plasmid containing the target sequence. Specific peaks of HSV-2 were observed in all reactions except the negative control (CN). The dissociation temperatures and the average of the cycle in which the reaction became positive (Ct) are presented in the associated table. Efficiency (E) and data fit values to the generated standard curve (R2) are also presented.

[0096] Figure 30 shows single PCR efficiency for HSV1 with primers at 10 pmoles/reaction. Reactions were performed in triplicate, with an annealing/extension step at 70ºC, with base 10 dilutions (1:100 to 1:10-3) of recombinant plasmid containing the target sequence. HSV-1 specific peaks were observed in all reactions except the negative control (CN). The dissociation temperatures and the average of the cycle in which the reaction became positive (Ct) are presented in the associated table. Efficiency (E) and data fit values to the generated standard curve (R2) are also presented.

[0097] Figure 31 shows single PCR efficiency for HSV-2 with primers at 10 pmoles/reaction. Reactions were performed in triplicate, with an annealing/extension step at 70ºC, with base 10 dilutions (1:100 to 1:10-3) of recombinant plasmid containing the target sequence. Specific peaks of HSV-2 were observed in all reactions except the negative control (CN). The dissociation temperatures and the average of the cycle in which the reaction became positive (Ct) are presented in the associated table. Efficiency (E) and data fit values to the generated standard curve (R2) are also presented.

[0098] Figure 32 shows single PCR efficiency for VZV. Reactions were performed in triplicate, with primers at a concentration of 17.5 pmoles and an annealing/extension step at 70ºC, with base 10 dilutions (1:10-5 to 1:10-9), of a recombinant plasmid containing the sequence- target. VZV-specific peaks were observed in all reactions except the negative control (CN). The dissociation temperatures and the average of the cycle in which the reaction became positive (Ct) are presented in the associated table. Efficiency (E) and data fit values to the generated standard curve (R2) are also presented.

[0099] Figure 33 shows individual PCR efficiency for EBV. Reactions were performed in triplicate, with primers at a concentration of 17.5 pmoles and an annealing/extension step at 70ºC, with base 10 dilutions (1:10-4 to 1:10-8), of a recombinant plasmid containing the sequence- target. VZV-specific peaks were observed in all reactions except the negative control (CN). The dissociation temperatures and the average of the cycle in which the reaction became positive (Ct) are presented in the associated table. Efficiency (E) and data fit values to the generated standard curve (R2) are also presented.

[00100] Figure 34 shows single PCR efficiency for CMV. Reactions were performed in triplicate, with primers at a concentration of 17.5 pmoles and an annealing/extension step at 70ºC, with base 10 dilutions (1:10-6 to 1:10-9), of a recombinant plasmid containing the sequence- target. CMV-specific peaks were observed in all reactions except the negative control (CN). The dissociation temperatures and the average of the cycle in which the reaction became positive (Ct) are presented in the associated table. Efficiency (E) and data fit values to the generated standard curve (R2) are also presented.

[00101] Figure 35 shows single PCR efficiency for HHV-6. Reactions were performed in triplicate, with primers at a concentration of 17.5 pmoles and an annealing/extension step at 70ºC, with base 10 dilutions (1:10-4 to 1:10-8), of a recombinant plasmid containing the sequence- target. CMV-specific peaks were observed in all reactions except the negative control (CN). The dissociation temperatures and the average of the cycle in which the reaction became positive (Ct) are presented in the associated table. Efficiency (E) and data fit values to the generated standard curve (R2) are also presented.

[00102] Figure 36 shows the standard curve for analyzing the efficiency of multiplex PCR for HSV-1. Reactions were performed in triplicate, with primers for HSV-1 and HSV-2 at a concentration of 10 pmoles, and primers for VZV, EBV, CMV and HHV-6 at a concentration of 17.5 pmoles. The annealing/extension step was performed at 70ºC, and base 10 dilutions (1:100 to 1:10-4) of recombinant plasmid containing the HSV-1 target sequence. HSV-1 specific peaks were observed in all reactions except the negative control (CN). Dissociation temperatures, efficiency values (E) and data fit to the generated standard curve (R2) are presented in the associated table.

[00103] Figure 37 shows standard curve for efficiency analysis of multiplex PCR for HSV-2. Reactions were performed in triplicate, with primers for HSV-1 and HSV-2 at a concentration of 10 pmoles, and primers for VZV, EBV, CMV and HHV-6 at a concentration of 17.5 pmoles. The annealing/extension step was performed at 70ºC, and base 10 dilutions (1:100 to 1:10-3) of recombinant plasmid containing the HSV-2 target sequence. Specific peaks of HSV-2 were observed in all reactions except the negative control (CN). Dissociation temperatures, efficiency values (E) and data fit to the generated standard curve (R2) are presented in the associated table.

[00104] Figure 38 shows the standard curve for analysis of efficiency of multiplex PCR for VZV. Reactions were performed in triplicate, with primers for HSV-1 and HSV-2 at a concentration of 10 pmoles, and primers for VZV, EBV, CMV and HHV-6 at a concentration of 17.5 pmoles. The annealing/extension step was performed at 70ºC, and base 10 dilutions (1:10-6 to 1:10-9) of recombinant plasmid containing the VZV target sequence. VZV-specific peaks were observed in all reactions except the negative control (CN). Dissociation temperatures, efficiency values (E) and data fit to the generated standard curve (R2) are presented in the associated table.

[00105] Figure 39 shows the standard curve for the efficiency analysis of multiplex PCR for EBV. Reactions were performed in triplicate, with primers for HSV-1 and HSV-2 at a concentration of 10 pmoles, and primers for VZV, EBV, CMV and HHV-6 at a concentration of 17.5 pmoles. The annealing/extension step was performed at 70ºC, and base 10 dilutions (1:10-4 to 1:10-8) of recombinant plasmid containing the EBV target sequence. Specific EBV peaks were observed in all reactions except the negative control (CN). Dissociation temperatures, efficiency values (E) and data fit to the generated standard curve (R2) are presented in the associated table.

[00106] Figure 40 shows standard curve for efficiency analysis of multiplex PCR for CMV. Reactions were performed in triplicate, with primers for HSV-1 and HSV-2 at a concentration of 10 pmoles, and primers for VZV, EBV, CMV and HHV-6 at a concentration of 17.5 pmoles. The annealing/extension step was performed at 70ºC, and base 10 dilutions (1:10-6 to 1:10-9) of recombinant plasmid containing the CMV target sequence. CMV-specific peaks were observed in all reactions except the negative control (CN). Dissociation temperatures, efficiency values (E) and data fit to the generated standard curve (R2) are presented in the associated table.

[00107] Figure 41 shows the standard curve for efficiency analysis of multiplex PCR for HHV-6. Reactions were performed in triplicate, with primers for HSV-1 and HSV-2 at a concentration of 10 pmoles, and primers for VZV, EBV, CMV and HHV-6 at a concentration of 17.5 pmoles. The annealing/extension step was performed at 70ºC, and base 10 dilutions (1:10-4 to 1:10-8) of recombinant plasmid containing the HHV-6 target sequence. Specific peaks of HHV-6 were observed in all reactions except the negative control (CN). Dissociation temperatures, efficiency values (E) and data fit to the generated standard curve (R2) are presented in the associated table.

[00108] Figure 42 shows multiplex PCR dissociation curve for HSV-1, HSV-2, VZV, EBV, CMV and HHV-6. Reactions were performed with primers for HSV-1 and HSV-2 at a concentration of 10 pmoles and primers for VZV, EBV, CMV and HHV-6 at a concentration of 17.5 pmoles, and an annealing/extension step at 70ºC. The curves show specific peaks for all viruses tested in the positive controls (CP), and dimer formation and unspecific amplification in the negative controls (CN) containing water or human DNA (DNA H). Dissociation temperatures are shown in the associated table with color legend

[00109] Figure 43 shows the dissociation curve of the multiplex PCR in the simultaneous detection of HSV-1 and HSV-2. Reactions were performed in triplicate, with primers for HSV-1 and HSV-2 at a concentration of 10.0 pmoles, and primers for VZV, EBV, CMV and HHV-6 at a concentration of 17.5 pmoles. The annealing/extension step was performed at 70°C, and base 10 dilutions (1:100 to 1:10-3) of recombinant plasmids containing HSV-1 and HSV-2 target sequences were used. In all reactions, only HSV-2 specific peaks were clearly evident. The number of dilution points tested and the dissociation temperatures (°C) of the HSV-1 and HSV-2 peaks are shown in the associated table.

[00110] Figure 44 shows the multiplex PCR dissociation curve in the simultaneous detection of EBV and CMV. Reactions were performed in triplicate, with primers for HSV-1 and HSV-2 at a concentration of 10.0 pmoles, and primers for VZV, EBV, CMV and HHV-6 at a concentration of 17.5 pmoles. The annealing/extension step was performed at 70ºC, and base 10 dilutions of recombinant plasmids containing the EBV (1:10-4 to 1:10-7) and CMV (1:10-6 to 1) target sequences were used. :10-9). In all reactions, specific peaks of EBV and CMV were observed. The four dilution points tested and the dissociation temperatures (°C) of the CMV and EBV peaks are shown in the associated table.

[00111] Figure 45 shows the multiplex PCR dissociation curve in the simultaneous detection of VZV and HHV-6. Reactions were performed in triplicate, with primers for HSV-1 and HSV-2 at a concentration of 10.0 pmoles, and primers for VZV, EBV, CMV and HHV-6 at a concentration of 17.5 pmoles. The annealing/extension step was performed at 70ºC, and base 10 dilutions of recombinant plasmids containing the target sequences of VZV (1:10-6 to 1:10-9) and HHV-6 (1:10 -4 to 1:10-7). In all reactions, only the VZV-specific peaks were very evident. The number of dilution points tested and the dissociation temperatures (ºC) of the VZV and HHV-6 peaks are presented in the associated table.

[00112] Figure 46 shows conventional single and multiplex PCR dissociation curve for defining the detection limit for HSV-1. Reactions were performed in triplicate, with an annealing/extension step at 70ºC, using dilutions containing 10,000, 1,000, 100, 10 and 1 copy/reaction of HSV-1 genomic DNA. The associated table shows the five points of the tested dilutions, with the results in the individual PCR (I) and in the multiplex PCR (M). Positive results for HSV-1 are represented by (+) and negative by (-). The presence of dimers and/or unspecific amplification is indicated as yes (Y) or no (N). Ct, cycle in which the reaction became positive; ND, not detectable; N.A., not applicable.

[00113] Figure 47 shows limit of detection for HSV-1 in single and multiplex PCR with HRM curve. The HRM curve analysis was performed with an initial normalization range of 81°C to 81.4°C and a final normalization range of 90°C to 90.4°C. Positive samples with peak dissociation temperature compatible with HSV-1 were evaluated. The HRM curves for HSV-1 are presented, and the associated table with the single and multiplex PCR results, including the identification confidence level. N.R., not performed.

[00114] Figure 48 shows the conventional dissociation curve of single and multiplex PCR to define the limit of detection for HSV-2. Reactions were performed in triplicate, with an annealing/extension step at 70°C, using dilutions containing 10,000, 1,000, 100, 10, and 1 copy/reaction of HSV-2 genomic DNA. The associated table shows the five points of the tested dilutions, with the results in the individual PCR (I) and in the multiplex PCR (M). Positive results for HSV-2 are represented by (+) and negative by (-). The presence of dimers and/or unspecific amplification is indicated as yes (Y) or no (N). Ct, cycle in which the reaction became positive; ND, not detectable; N.A., not applicable.

[00115] Figure 49 shows limit of detection for HSV-2 in single and multiplex PCR with HRM curve. The HRM curve analysis was performed with an initial normalization range of 80°C to 81.4°C and a final normalization range of 90°C to 90.4°C. Positive samples with peak dissociation temperature compatible with HSV-2 were evaluated. The HRM curves for HSV-2 are presented, and the associated table with the single and multiplex PCR results, including the identification confidence level. N.R., not performed.

[00116] Figure 50 shows conventional single and multiplex PCR dissociation curve for defining the limit of detection for VZV. Reactions were performed in triplicate, with an annealing/extension step at 70ºC, using dilutions containing 10,000, 1,000, 100, 10 and 1 copy/reaction of VZV genomic DNA. The associated table shows the five points of the tested dilutions, with the results in the individual PCR (I) and in the multiplex PCR (M). Positive results for VZV are represented by (+) and negative results by (-). The presence of dimers and/or unspecific amplification is indicated as yes (Y) or no (N). Ct, cycle in which the reaction became positive; ND, not detectable; N.A., not applicable.

[00117] Figure 51 shows the limit of detection for VZV in single and multiplex PCR with HRM curve. The HRM curve analysis was performed with an initial normalization range of 81°C to 82°C and a final normalization range of 90°C to 90.4°C. Positive samples with peak dissociation temperature compatible with VZV were evaluated. The HRM curves for VZV are presented, and the associated table with the single and multiplex PCR results, including the identification confidence level. N.R., not performed.

[00118] Figure 52 shows conventional single and multiplex PCR dissociation curve for defining the limit of detection for EBV. Reactions were performed in triplicate, with an annealing/extension step at 70ºC, using dilutions containing 10,000, 1,000, 100, 10 and 1 copy/reaction of EBV genomic DNA. The associated table shows the five points of the tested dilutions, with the results in the individual PCR (I) and in the multiplex PCR (M). Positive results for EBV are represented by (+) and negative by (-). The presence of dimers and/or unspecific amplification is indicated as yes (Y) or no (N). Ct, cycle in which the reaction became positive; ND, not detectable; N.A., not applicable.

[00119] Figure 53 shows the limit of detection for EBV in single and multiplex PCR with HRM curve. The HRM curve analysis was performed with an initial normalization range of 81°C to 82°C and a final normalization range of 90°C to 90.4°C. Positive samples with peak dissociation temperature compatible with EBV were evaluated. The curves of HRM for EBV are presented, and associated table with the results of the single and multiplex PCR, including the confidence level of the identification. N.R., not performed.

[00120] Figure 54 shows the conventional dissociation curve of single and multiplex PCR to define the limit of detection for HHV-6. Reactions were performed in triplicate, with an annealing/extension step at 70°C, using dilutions containing 10,000, 1,000, 100, 10, and 1 copy/reaction of HHV-6 genomic DNA. The associated table shows the five points of the tested dilutions, with the results in the individual PCR (I) and in the multiplex PCR (M). Positive results for HHV-6 are represented by (+) and negative by (-). The presence of dimers and/or unspecific amplification is indicated as yes (Y) or no (N). Ct, cycle in which the reaction became positive; ND, not detectable; N.A., not applicable.

[00121] Figure 55 shows the limit of detection for HHV-6 in individual PCR with HRM curve. The HRM curve analysis was performed with an initial normalization range of 75°C to 75.4°C and a final normalization range of 81°C to 81.4°C. Positive samples with peak dissociation temperature compatible with HHV-6 were evaluated. The HRM curves for HHV-6 are presented, and the associated table with the individual PCR results, including the identification confidence level. N.R., not performed.

DETAILED DESCRIPTION OF THE INVENTION

[00122] Although the present invention may be amenable to different embodiments, a preferred embodiment is shown in the drawings and the following detailed discussion, with the understanding that the present description is to be considered an exemplification of the principles of the invention and is not intended to limit the present invention to what has been illustrated and described here.

DEFINITIONS

[00123] “Diagnosis”, as used in the present invention, refers to the determination of a medical condition, generally by investigating its history, etiology and symptoms and applying tests. This method involves a series of steps that lead to the identification of a clinical condition, which include steps of analysis and interpretation of the data obtained.

[00124] The term "nucleic acid", according to the present invention, refers to any type of nucleic acid, such as DNA and RNA, and variants thereof, as well as combinations thereof, modifications thereof, including modified nucleotides etc. The terms "nucleic acid" and "oligonucleotide" and "polynucleotide" are used interchangeably in the context of the present invention. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, and optionally purified, chemically synthesized, etc. Where appropriate, for example in the case of chemically synthesized molecules, the nucleic acids may comprise nucleoside analogues such as analogues having chemically modified bases or sugars, backbone modifications, and the like. A nucleic acid sequence is represented in the 5'-3' direction unless otherwise indicated. As used herein, the symbols for nucleotides and polynucleotides are those recommended by the IUPAC-IUB Commission of Biochemical Nomenclature (Biochem. 9:4022, 1970).

[00125] The term "individual" is defined herein to include animals, such as mammals. In a preferred embodiment, the subject is a human.

[00126] The term “Polymerase Chain Reaction” or the acronym in English, PCR, refers to a method in which a fragment of nucleic acid is amplified. Primers or primers are designed that have sequences complementary to the sequence to be amplified. PCR can be used to amplify RNA, DNA or cDNA sequences.

[00127] The term “Body Fluid” or “Biological Fluid” refers to liquids originating from bodies. Body fluid samples are selected from cerebrospinal fluid (cerebrospinal fluid), urine, blood, saliva, urethral secretions, seminal fluid, and vaginal secretions.

[00128] The term “Amplicon” refers to a piece of DNA or RNA that is a product of amplification or replication events. It can be formed artificially, using various methods, including polymerase chain reactions (PCR) or ligase chain reactions (LCR), or naturally through gene duplication. As it refers to the product of an amplification reaction, amplicon is used interchangeably with the term "PCR product".

[00129] The term “HRM”, from English “High Resolution Melting” refers to a post-PCR analysis method to identify variations in nucleic acid sequences. The method is based on the detection of small differences in PCR melting (dissociation) curves.

In vitro method for the differential diagnosis of herpesvirus infections of the central nervous system

[00130] In a first embodiment, the present invention relates to an in vitro method for the differential diagnosis of central nervous system infections by herpesvirus, which consists of the following steps: extracting DNA from a sample of human cerebrospinal fluid; amplifying by multiplex real-time PCR using primers as defined in any one of the sequences SEQ ID NO: 1 to 12; and identify the amplicon(s) produced according to the dissociation temperature (Tm) from dissociation curves with high resolution (HRM). In one embodiment, the amplicon is from one or more of the following viral types HSV-1, HSV-2, VZV, EBV, CMV and HHV-6. In another embodiment, the main disorders associated with said central nervous system infections can be selected from encephalitis, cerebellitis, vasculitis, retinitis, polyradiculitis, ventriculitis, myelitis and meningitis. In another embodiment, the subject is a human subject. In another embodiment, further optionally quantitative diagnosis by individual assays can be performed.

Kit for the differential diagnosis of herpesvirus central nervous system infections

[00131] In a second embodiment, the present invention relates to a kit for the differential diagnosis of infections of the central nervous system by herpesviruses, comprising a combination of primers selected from the group SEQ ID NO: 1 to 12; commercial human DNA and water as negative controls; and plasmid DNAs containing the viral sequences, as positive controls; and reagents for the multiplex real-time PCR reaction. In another embodiment, the kit is for diagnosing and identifying one of the following herpesviruses: HSV-1, HSV-2, VZV, EBV, CMV and HHV-6.

Use of initiators

[00132] In a third embodiment, the present invention refers to the use of a combination of primers selected from the group SEQ ID NO: 1 to 12 for differential diagnosis of infections of the central nervous system by herpesviruses.

EXAMPLES

[00133] The study represents the development of a laboratory diagnosis method, using commercially obtained HSV-1, HSV-2, VZV, EBV, CMV and HHV-6 DNA samples (Vircell, Spain). Selection and evaluation of specific PCR primers

[00134] First, specific PCR primers for HSV-1, HSV-2, VZV, EBV, CMV and HHV-6 were selected from studies published in indexed journals, by searching the PubMed database (NCBI) ( Figure 1). The descriptor “real-time PCR” was used together with the descriptors of each of the six herpesviruses mentioned. Considering that in the optimization of real-time PCR assays, the size of the amplicon is a critical factor (BRISSON, MARNI et al., 2004), studies were selected describing primers that generated amplicons between 50 and 250 base pairs (bp) . Then, the primers obtained were evaluated in silico for dimer formation, an important interference in real-time DNA amplification analysis, with the Multiple Primer Analyzer tool (THERMO FISHER SCIENTIFIC, 2017), in tests containing specific primers for the six herpesviruses evaluated, simulating a reaction in multiplex format. Primers with a strong possibility of dimer formation were excluded and replaced by new ones at each test.

[00135] The analysis of the primers was also performed with the primer-BLAST tool (NCBI, 2017), in order to identify the complementarity with sequences of the human genome that would allow the amplification of fragments smaller than 300-bp, thus generating nonspecific products. Initiators meeting these criteria were also excluded. With this, the primers shown in Table 1 were selected.

[00136] The real-time PCR assays were performed with the Type-it HRM PCR kit reagent (Qiagen), which contains the EvaGreen® dye, in 25 μL reactions, containing 10 or 17.5 pmoles of each primer, depending on indicated, and in Rotor-Gene Q 5-plex HRM equipment (Qiagen). The amplification cycle consisted of two steps: denaturation at 95ºC for 10 seconds and annealing/extension at 60ºC, 62ºC, 64ºC, 68ºC or 70ºC for 30 seconds, as indicated, with fluorescence detection at the end of this step, with a total of 45 cycles. At the end of the PCR, a dissociation curve ranging from 70ºC to 95ºC was performed with a fluorescence reading after each temperature increment of 0.2ºC per second.

[00137] To assess the specificity of the selected primer pairs, individual tests were first performed against the corresponding DNA of each virus (Table 2), and negative controls consisting of distilled water (Invitrogen) and commercial sample of human DNA (Promega) . The primers that did not present unspecific amplification in the individual assays containing human DNA, and that did not present dimer formation, were submitted to a reaction in multiplex format under the conditions previously described (Figure 1).

Construction of plasmids for amplification control

[00138] Amplification controls for the real-time PCR assays were constructed by cloning the PCR products obtained with the primers that proved to be specific, using the pGEM-T Easy Vector System kit (Promega).

[00139] For the construction of these controls, a conventional PCR was initially performed with the enzyme Platinum Taq DNA Polymerase (Invitrogen), 10 pmoles of primers, in a final volume of 25 μL, with the commercial DNA of each virus. After confirmation of a single band by electrophoresis on a 2% agarose gel in 1xTBE with 0.5x GelRed at 100 V for 90 minutes, the binding reaction of the amplicons to the pGEMT vector was performed, according to the manufacturer's recommendations: 5 μL of buffer ligation (2x), 1 µL of pGEM-T vector (50 ng), 3 µL of amplicon, and 1 µL of T4 DNA ligase. The reactions were incubated at 4°C overnight in order to increase efficiency in obtaining colonies after transformation.

Competent Escherichia coli transformation

[00140] The transformation of competent Escherichia coli (E. coli) was performed according to the pGEM-T Easy Vector kit manufacturer's recommendations (Promega). Briefly, 2 μL of the ligation reaction was transferred to a sterile 1.5 mL microtube, and then 50 μL of competent E. coli was added. After slight homogenization, the samples were incubated in an ice bath for 20 minutes.

[00141] Subsequently, the cells were subjected to thermal shock by incubation in a dry bath at 42ºC for 50 seconds, and then in an ice bath for 2 minutes. Afterwards, 450 μL of SOC medium were added, and the cells were incubated at 37ºC for 90 minutes with agitation at 150 rpm. Then, 100 μL of the culture was sown in LB medium containing X-GAL, IPTG and 50 μg/mL ampicillin, and the plates were incubated at 37ºC overnight.

[00142] Subsequently, three to four white colonies corresponding to the transformation reactions with the recombinant plasmids containing the amplicon of each virus were selected, which were inoculated in 1 mL of LB broth with ampicillin, and incubated for 24 hours. Afterwards, the cultures were centrifuged, with the supernatant discarded and the sediment resuspended in sterile distilled water. Then, the cells were boiled for 15 minutes and frozen at -20°C to break the cell wall and membrane and release the DNA.

[00143] Confirmation of cloning of amplicons was performed by PCR with the Platinum Taq DNA polymerase kit (Invitrogen), with 100 ng of primers for the M13 region of the pGEM-T vector (M13-fwd: 5'- CGCCAGGGTTTTCCCAGTCACGAC-3' and M13-rev: 5'-GTCATAG CTGTTTCCTGTGTGA-3') ( Figure 10 ). Samples of recombinant plasmids, obtained after boiling the transformed E. coli cultures, were centrifuged at 13,000 rpm for 1 minute, and 2 μL were applied to the reaction mixture with a final volume of 25 μL. The results were revealed by electrophoresis in 2% agarose gel in 1x TBE with 0.5x GelRed at 100V for 90 minutes and photodocumented in L-Pix system (Loccus Biotecnologia).

[00144] Samples containing bands of the expected size were purified with the QIAQuick gel extraction kit (Qiagen) and sent for DNA sequencing by the Sanger method with primers M13-fwd and M13-rev to confirm the sequences of the cloned fragments. The alignment of the sequences obtained with sequences of reference viral strains available on GenBank was performed using the online tool nucleotide-BLAST.

Real-time PCR setup with conventional and high-resolution dissociation curve analysis (HRM) Individual amplification efficiency tests

[00145] The optimal concentration of primers for amplification of human herpesvirus genome sequences was evaluated, in individual reactions, through the construction of standard curves, with at least four points, of serial dilution at base 10 of sample of recombinant plasmids containing the target sequences.

[00146] The PCR efficiency is related to the slope (slope) of the standard curve that is obtained with the equation: Efficiency = (10−1/slope)-1, where the slope is derived from a graph with the values of the cycle in which the reaction became positive (Ct, Cycle threshold) as a function of the DNA concentration inoculated in the reaction, in logarithm at base 2. Thus, the slope of -3.32 indicates an efficiency of 100%.

[00147] The analysis of the indicators of the individual standard curves was performed with the Rotor-Gene equipment program (Qiagen), and an amplification efficiency close to 100% was considered optimal (Figure 1). Efficiency values greater than 90% are desirable in individual reactions before being combined in a multiplex reaction. Furthermore, coefficients greater than 110% are suggestive of nonspecific amplification, such as, for example, the formation of primer dimers or secondary products (BRISSON, MARNI et al., 2004).

Multiplex amplification efficiency tests

[00148] To evaluate the PCR amplification efficiency in a multiplex assay, the optimal concentrations of the pre-defined primers in individual reactions were maintained, as this enables a precise amplification of DNA in a wide range of concentrations in PCR in multiplex format. For this purpose, standard curves were made, with at least four points, of serial dilution in base 10 of sample of recombinant plasmids containing the target sequences. The same dilutions performed in the individual assays were reassessed in multiplex assays, in order to compare the average values of Ct in both assays and the efficiency values of the reactions. The analysis of the indicators of the standard curves in a multiplex assay was performed using the Rotor-Gene equipment program, and an amplification efficiency close to 100% was considered optimal (Figure 1).

Simultaneous amplification of two viral types and limit of detection

[00149] The possibility of simultaneous amplification of two DNA sequences altering the reaction detection sensitivity in multiplex assays was evaluated with recombinant plasmids combined in pairs. To determine the combination of viral DNA pairs, the dissociation curves of each virus were considered, and those whose specific peaks were separated, that is, without overlapping curves, were combined, in order to allow a better identification of the viruses during amplification simultaneous. Thus, the following pairs were formed: HSV-1 and HSV-2, EBV and CMV, VZV and HHV-6. The starting points for the serial dilution of each virus were defined according to concentrations that showed a positive result in cycle 20, approximately, as follows: HSV1 and HSV-2, sample without dilution (1:100); EBV, 1:10-4 dilution; CMV, 1:10-6 dilution; VZV, 1:10-5 dilution; HHV-6, 1:10-4 dilution. Subsequently, the dilutions of each predefined pair were combined, in equal volume, and then serial dilutions were performed in base 10, with at least four dilution points.

[00150] In addition, real-time multiplex PCR detection limits for herpesviruses, expressed in the number of viral DNA copies per reaction, were determined by serial limiting dilution of DNA controls. However, due to the unavailability of CMV genomic DNA, it was not possible to determine the limit of detection for this viral type. Thus, the assays were performed only with DNA from HSV-1, HSV-2, VZV, EBV and HHV-6.

[00151] The tests to determine the limit of detection were performed in triplicate, with five dilution points containing 10,000, 1,000, 100, 10 and 1 copy of viral DNA per reaction. Viral detection and identification in at least two replicates of the triplicate was established as a criterion for defining the limit of detection. Initially, the identity of the amplicons was evaluated by the dissociation temperature (Tm) of the amplification peak defined in the conventional dissociation curve performed at the end of the PCR, ranging from 70ºC to 95ºC with fluorescence reading after each temperature increment of 0.2ºC per second. In addition, in all assays, in order to define the best approach for confirming the identity of amplification products, HRM curve analysis was also performed, with initial and final normalization ranges ranging from 81.0°C at 81.4°C and from 90.0°C to 90.4°C, respectively.

Analysis plan

[00152] The specificity of the primers was evaluated in order to exclude false-positive results. The amplification efficiency of individual and multiplex PCR assays, Tm values of different amplicons in different concentrations of viral DNA, and the detection limit of the technique developed, were evaluated in order to establish a sensitive and specific diagnostic method. For this purpose, intra- and inter-assay variations in the mean values of Ct and Tm obtained were evaluated.

EXAMPLE Evaluation of primer specificity in individual assays

[00153] Initially, the specificity of primers 1 to 12 (Table 1) was evaluated in individual assays, at a concentration of 17.5 pmoles, under an annealing/extension temperature of 60ºC. The primers were evaluated for dimer formation, in controls with distilled water, and for unspecific amplification, in reactions with human DNA. Primers for HSV-1 (Table 1, primers 1 and 2) generated dimers and unspecific amplification from human DNA (Figure 3), whereas primers for EBV (Table 1, primers 7 and 8), CMV (Table 1, primers 9 and 10), and HHV-6 (Table 1, primers 11 and 12) generated only nonspecific amplification from human DNA (Figure 4). Primers for HSV-2 (Table 1, primers 3 and 4) and VZV (Table 1, primers 5 and 6) did not generate dimers or unspecific amplification. However, the dissociation curve did not show a single peak in VZV positive controls (Figure 5).

[00154] To reduce the formation of dimers and unspecific amplification due to a possible excessive concentration of the tested primers, new tests were performed with primers at a concentration of 10 pmoles. In reactions with human DNA, unspecific amplifications were observed with primers for HSV-1 (Table 1, primers 1 and 2) (Figure 6), EBV (Table 1, primers 7 and 8) and CMV (Table 1, primers 9 and 10) (Figure 7). Therefore, these initiators were excluded, and a search for new initiators was carried out.

[00155] Primers for HSV-2 (Table 1, primers 3 and 4) and for VZV (Table 1, primers 5 and 6) did not form dimers in the negative control. However, nonspecific amplification and different Tm peaks were observed in their positive controls, as shown in Figure 8. Thus, these primers were also excluded from the study and, therefore, a search for new primers was initiated. Of the primers cited in Table 1 (1 to 12), only the primers for HHV-6 did not generate dimers or unspecific amplification from human DNA, thus fulfilling the selection criteria in individual assays (Figure 8).

[00156] During the search for new works describing primers for human herpesviruses, a study was selected containing primers for HSV-1/2, VZV, EBV, CMV, HHV-6A/B and HHV-7 designed for a conventional multiplex PCR ( TANAKA et al., 2009), from which primers for VZV, EBV and CMV were selected (Table 1, primers 13 to 18).

[00157] The selected primers for VZV (Table 1, primers 13 and 14), EBV (Table 1, primers 15 and 16) and CMV (Table 1, primers 17 and 18) were tested at 17.5 pmoles and at temperature of annealing/extension at 60ºC. The results showed that only the EBV primers generated dimers and nonspecific amplification (Figure 9), while the VZV and CMV primers showed single peaks in the positive controls and no dimers and nonspecific amplification (Figure 10).

Reassessment of primers for EBV with annealing/extension at 70°C

[00158] Initially, when tested at an annealing temperature of 60ºC, the primers for EBV described by TANAKA et al. (2009) (Table 1, primers 15 and 16) were excluded from the study, as they had allowed the generation of dimers and unspecific amplification, and only primers for VZV and CMV were selected for assays in multiplex format. However, since the study by TANAKA et al. (2009) uses a conventional PCR with an annealing step starting at 70ºC with progressive reduction at each PCR cycle, which reduces the probability of dimer formation and/or nonspecific amplification, the primers were reassessed for EBV ( Table 1, primers 15 and 16) in amplification cycle with annealing/extension step at 70°C. The results showed single peaks in the positive controls and absence of dimers and unspecific amplification (Figure 11).

[00159] Thus, after evaluating nine pairs of primers for six human herpesviruses, primers for VZV, EBV, CMV and HHV-6 were selected for testing in multiplex PCR with amplification cycle with annealing/extension step at 70ºC (Table 3).

Search and evaluation of new primers for HSV-1 and HSV-2

[00160] As demonstrated, the primers for VZV, EBV and CMV described by TANAKA et al. (2009) performed well in real-time PCR. Therefore, the HSV-1/2 primers from this same study (Table 1, primers 19 and 20) were also evaluated at a concentration of 17.5 pmoles in an annealing/extension reaction at 70°C (Figure 12).

[00161] The results demonstrated secondary amplifications in the positive controls, the formation of dimers in the negative controls with water, in addition to nonspecific amplification from human DNA. However, since the primers for HSV-1/2 presented different Tm values in relation to the other viral types (VZV, EBV, CMV and HHV-6), new tests were performed with these primers before definitive exclusion.

[00162] In silico tests were carried out to evaluate the possibility of dimer formation and pairing with human genome sequences, and subsequently the alteration of the sequence of primers for HSV-1/2 described by TANAKA et al. (2009), with the removal of nucleotides in the 3' region, as shown in Table 1, in order to eliminate the amplification of nonspecific products.

[00163] The modified HSV-1/2 primers (Table 1, primers 21 to 26) were tested under the same conditions as the original primers (Table 1, primers 19 and 20), in a total of 16 different combinations between sense primers (F ) and antisense (R) (Figure 13). The results of these combinations showed nonspecific amplifications in all reactions with the positive controls. However, in six combinations (1, 3, 6, 7, 9 and 11) it was possible to eliminate the formation of dimers and nonspecific amplifications from human DNA (Figure 14).

[00164] To clarify the origin of the second sequence amplified in the positive controls, the combination 6 of the primers for HSV-1/2 was used in assays with controls of viral DNA and human DNA, and with a sample of DNA from Vero cells, used in the replication of HSV-1 and HSV-2 in the manufacture of viral controls (Table 2).

[00165] The results showed unspecific amplification only in the HSV-1/2 positive controls, with no amplification from the DNA of Vero cells (Figure 15), suggesting that a second sequence of the HSV-1 and HSV-2 genome itself would be amplified. However, in all tests performed with primer combination 6 for HSV1/2, no dimer formation or unspecific amplification with human DNA was observed. Considering that such amplification would be associated with the HSV DNA itself, these primers were included for evaluation in multiplex assays.

[00166] Primers for HSV-1 described by HONG et al. (2014) (Table 1, primers 1 and 2), initially evaluated at annealing/extension temperature of 60°C and excluded by dimer generation and unspecific amplification, were modified. The extension of the 3' region of the original primers was carried out taking into account the complementarity with the sequences of each virus, in order to reduce the chance of amplification of nonspecific products, and to enable the stability of the hybridization of the primers with the target sequences at 70ºC.

[00167] The primers for HSV-1 (Table 1, primers 27 and 28) and HSV-2 (Table 1, primers 29 and 30) were tested at the concentration of 17.5 pmoles, in individual reactions, and amplification cycle with annealing/extension step at 70ºC. Single peaks were obtained in positive controls and absence of dimers and nonspecific amplification (Figure 16). Therefore, these primers were also included for evaluation in multiplex assays.

EXAMPLE 2 Evaluation of primer specificity in multiplex assays Multiplex PCR for VZV, CMV and HHV-6

[00168] The primers for VZV (Table 1, primers 13 and 14), CMV (Table 1, primers 17 and 18) and HHV-6 (Table 1, primers 11 and 12), selected in the individual trials, underwent testing in multiplex format, initially, at a concentration of 17.5 pmoles and annealing/extension temperature at 60ºC, in order to define the influence of the reaction temperature on the specificity of the test and on the functioning of the primers. Reactions with the positive controls of these three viruses showed results that confirmed the specificity of the primers. However, dimer formation and unspecific amplification with different Tm were observed in replicates of reactions containing human DNA (Figure 17). Therefore, the temperature of the annealing/extension step was successively raised to 62ºC (Figure 18), 64ºC (Figure 19), 68ºC (Figure 20) and 70ºC (Figure 21), thus increasing the stringency of the reaction. Although the occurrence of dimers was eliminated, the generation of nonspecific amplification in reactions containing only human DNA was not completely excluded, which apparently occurred randomly, since peaks with different Tm were observed in replicates and between different assays, indicating it is not a single amplified sequence.

Multiplex PCR for VZV, EBV, CMV and HHV-6

[00169] After the reassessment and subsequent approval of the primers for EBV (Table 1, primers 15 and 16) in individual assays, they were tested in a multiplex reaction, together with the primers for VZV, CMV and HHV-6, at the concentration of 17.5 pmoles and annealing/extension temperature at 70ºC, as shown in Figure 22. The results obtained with the positive controls of these four viruses confirmed the specificity of the primers, with no dimer formation or unspecific amplification from human DNA being observed, indicating that dimers and non-specific amplification occur randomly.

Multiplex PCR for HSV-1, HSV-2, VZV, EBV, CMV and HHV-6

[00170] The primers for HSV-1/2 modified from TANAKA and collaborators (2009) (combination 6) were included in the multiplex PCR, containing the primers for VZV, EBV, CMV and HHV-6, and evaluated at the temperature of annealing/extension at 70ºC.

[00171] The results revealed specific peaks of the six viruses, without unspecific amplification from human DNA, however, the formation of dimers was observed only in the HHV-6 control (Figure 23). The occurrence of secondary amplification in HSV-1 and HSV-2 positive controls was also observed (Figure 23).

[00172] The initial analysis by HRM curve performed in the temperature range from 78ºC to 93ºC (Figure 24), enabled the identification of the six viral types through the decay curves, with a confidence level greater than 98% in the identification of HSV -2, VZV, EBV, CMV and HHV-6; for HSV-1 the confidence level was 96.84%.

[00173] A second analysis of the HRM curve, performed with a narrower temperature range, ranging from 88.5ºC to 93ºC (Figure 25), allowed raising the confidence level in the identification of HSV-1 to 99.81%. In this sense, it was verified that even in the presence of two distinct peaks in the positive controls of HSV-1 and HSV-2, the analysis with HRM curve allowed to differentiate all the targets. In addition, because they have higher dissociation temperatures than the other viruses, performing a second analysis band on the HRM curve for HSV-1 and HSV-2 allowed a more accurate identification of these viruses.

[00174] In parallel, primers for HSV-1 and HSV-2 modified from the description by HONG et al. (2014) were also tested under the same conditions. The results showed single peaks of HSV-1 and HSV-2 in the positive controls, with dimer formation and non-specific amplification from human DNA occurring at random in the negative control replicates (Figure 26). It is noteworthy that the Tm values of amplicons generated in negative controls were lower than those of specific amplicons, indicating the possibility of excluding these interfering elements from the analysis of conventional dissociation curves and HRM, thus not compromising the identification of viral types.

[00175] Therefore, since assays with modified primers from the primers described by HONG et al. (2014) showed better results than those modified from the study by TANAKA et al. (2009), the first ones were selected to compose the multiplex PCR. In Table 4, the binding regions of the selected primers are indicated.

EXAMPLE 3 Construction of plasmids for use as amplification controls

[00176] For the optimization of multiplex PCR, including the evaluation of amplification efficiency rates in individual reactions, recombinant plasmids containing the amplicons of HSV-1, HSV-2, VZV, EBV, CMV and HHV-6 were constructed for use as controls.

[00177] Confirmation of competent E.coli transformation with the recombinant plasmids was performed by real-time PCR, in individual assays containing specific primers for each virus, and identical Tm values were obtained in the reactions with the plasmids and with the controls commercials for HSV-1, HSV-2, VZV, EBV, CMV and HHV-6.

[00178] The sequences of VZV, CMV and HHV-6 in the recombinant plasmids was also confirmed by DNA sequencing, as demonstrated by alignment with sequences from the reference viral strains available on GenBank (Figure 27).

EXAMPLE 4 Individual PCR amplification efficiency Amplification efficiency of single PCRs for HSV-1 and HSV-2

[00179] In the preparation of the PCR standard curve for HSV-1 and HSV -2, four points were performed, in triplicate, the first without dilution of the recombinant plasmid samples, followed by dilutions from 1:100 to 1:10- 3. The efficiency rates obtained in reactions containing 17.5 pmoles of each primer for HSV-1 and HSV-2 were 112% (Figure 28) and 116% (Figure 29), respectively. The Tm values did not show significant variation, with the minimum and maximum values being 84.16ºC and 84.36ºC for HSV-1, and 87.14 ºC and 87.26 ºC for HSV-2. However, following the recommendation of establishing individual PCR assays with efficiency rates between 90% and 110% before combining in a multiplex reaction, a new standard curve was performed with the primers at a concentration of 10 pmoles/reaction. The amplification rates obtained for HSV-1 and HSV-2 were, respectively, 105% (Figure 30) and 110% (Figure 31), with minimum and maximum Tm values of 84.06ºC and 84.20ºC for HSV- 1, and 86.90ºC and 87.04ºC for HSV-2. The results revealed good intra-assay reproducibility, even at different concentrations of the target sequences. Furthermore, all Tm values in the curve performed with 10 pmoles of primers were similar to those obtained with primers at 17.5 pmoles. Thus, in individual assays, a concentration of 10 pmoles of primers for HSV-1 and HSV-2 was selected.

Amplification efficiency of single PCRs for VZV and EBV

[00180] Standard curves of individual PCR assays for VZV and EBV were performed by serial dilution at the base 10 of the recombinant plasmids, in triplicate, containing five points (1:10-5 to 1:10-9) for VZV, four points (1:10-4 to 1:10-8) on EBV. Efficiency rates of 96% were obtained for both viruses (Figures 32 and 33). The Tm values of the peaks did not show significant variation, with minimum and maximum values of 82.13ºC and 82.33ºC for VZV, and 88.20ºC and 88.30ºC for EBV, indicating good intra-assay reproducibility in viral identification even in different concentrations of the target sequences.

Amplification efficiency of single PCRs for CMV and HHV-6

[00181] In the evaluation of PCR for CMV and HHV-6, standard curves were performed containing four point dilutions of control plasmids for CMV ranging from 1:10-6 to 1:10-9, and with five point dilutions of the HHV-6 plasmid ranging from 1:10-4 to 1:10-8, in triplicate. Efficiency rates of 104% (Figure 34) and 93.5% (Figure 35) were observed, respectively. The Tm peaks were also reproducible, with minimum and maximum values of 85.33ºC and 85.43ºC for CMV, and 79.65ºC and 79.77ºC for HHV-6.

EXAMPLE 5 Multiplex PCR efficiency for HSV-1, HSV-2, VZV, CMV and HHV-6

[00182] The optimal concentrations of the primers, defined in the previous assays, were used in the preparation of the multiplex real-time PCR assay. Standard curves, with at least four points of base 10 serial dilutions of recombinant plasmids containing HSV-1, HSV-2, VZV, EBV, CMV and HHV-6 target sequences, were performed in triplicate in all assays .

[00183] In the amplification of HSV-1 and HSV-2, the efficiency rates obtained were 107% (Figure 36) and 102% (Figure 37), respectively, and as in the individual reactions, no significant variation in the Tm of their respective amplicons, with minimum and maximum values of 84.16°C and 84.30°C for HSV-1, and 86.84°C and 86.90°C for HSV-2. In reactions with VZV and EBV, rates of 101% (Figure 38) and 97% (Figure 39) were obtained, respectively. The amplicon dissociation temperatures also did not show great variation, with minimum and maximum Tm of 82.26ºC and 82.50ºC for VZV, and from 88.10ºC to 88.24ºC for EBV. Finally, in the amplification of CMV and HHV-6, the rates were 100% for both viruses (Figures 40 and 41), with Tm showing little variation, with minimum and maximum values of 85.60ºC and 85.70ºC for CMV, and 79.84ºC and 80ºC for HHV-6.

[00184] Thus, the individual and multiplex PCR tests showed amplification efficiency rates close to 100% (Table 5), showing that the concentrations of predefined primers in individual assays were compatible with PCR in multiplex format. However, the mean Ct values obtained in the multiplex assays with each of the viruses were higher than those obtained in their respective individual assays (Table 6), with the highest differences observed in the tests with VZV and HHV-6, although not a decrease in the efficiency rate of the reaction has been observed.

EXAMPLE 6 Evaluation of multiplex PCR after adjustment of primer concentration

[00185] After carrying out efficiency tests, concentrations of 10 pmoles of primers for HSV-1 and HSV-2 and 17.5 pmoles of primers for VZV, EBV, CMV and HHV-6 were defined in the multiplex PCR. Therefore, assays using such concentrations were carried out to evaluate the generation of dimers and unspecific amplification, and to confirm the detection and identification of virial types according to the Tm in dissociation curves.

[00186] The results showed that it was not possible to completely eliminate the formation of dimers and/or unspecific amplification, which occurred randomly, as it was not present in all replicas of negative controls containing water or human DNA (Figure 42), similarly to that seen in the reaction containing all primers at 17.5 pmol (Figure 26).

[00187] To identify the primers involved in the formation of dimers, tests were performed with all possible combinations between sense (F) and antisense (R) primers, in reactions containing only water. It was observed that the highest frequency of dimer formation occurred with the HSV-1F primer (Table 1). Dimers also occurred in reactions with primers HSV-1R and EBV-R, HSV-2R and EBV-R, HSV-2F and CMV-R, and HHV-6F and CMV-F.

[00188] Considering that the presence of dimers in the PCR can be monitored and excluded from the dissociation curve analyzes by selecting the temperature range above which they occur, without compromising, however, the analysis of specific amplicons, we proceeded to the evaluation of multiplex PCR in simultaneous detection and limit of detection assays.

EXAMPLE 7 Simultaneous amplification tests in multiplex PCR

[00189] Reactions containing combinations of HSV-1 and HSV2, EBV and CMV, and VZV and HHV-6 DNA were evaluated, in triplicate, considering four points of serial dilutions at base 10 resulting in Ct values between 20 and 34. In addition to dilutions containing two viral types, dilutions of each viral DNA were also evaluated in individual reactions, which were used as amplification controls.

[00190] In the HSV-1 and HSV-2 simultaneous amplification assay, only the peaks for HSV-2 were very evident at all dilution points, indicating that HSV-1 DNA amplification was compromised in the presence of a second viral type in equivalent concentration (Figure 43). The individual assays carried out as HSV-1 and HSV-2 amplification controls showed their respective specific peaks at different DNA concentrations, with average Ct values within the pre-established range (Table 7). The minimum and maximum Tm values for HSV-1, in the presence of a second viral type, were 83.70ºC and 84.00ºC, respectively, slightly below the averages obtained in triplicates of individual reactions, which ranged from 84, 17°C to 84.22°C (Table 8). On the other hand, the Tm values for HSV-2 observed in the multiple amplification reactions were slightly higher than those obtained in individual reactions, with amplitudes from 86.80ºC to 86.83ºC (Figure 43) and from 86.86ºC to 86, 90ºC (Table 8), respectively.

[00191] In the simultaneous amplification of EBV and CMV, specific peaks were observed for both viruses in all evaluated concentrations, indicating that there is no mutual interference in DNA amplification (Figure 44). In the individual EBV and CMV amplification controls, unique and specific peaks were observed for each viral type, at the different concentrations tested, and with Ct averages within the predefined range (Table 7). The minimum and maximum Tm values for EBV in the simultaneous amplification were 88.10ºC and 88.20ºC, respectively; for CMV, the values were 85.10ºC and 85.30ºC, respectively (Figure 44). However, when comparing Tm values from individual assays, it was evident that simultaneous amplification can result in slightly lower Tm values (Table 8).

[00192] Regarding the results of the simultaneous amplification assays of VZV and HHV-6, results similar to those of HSV-1 and HSV-2 were obtained. At all dilution points evaluated, well-defined specific peaks were observed only for VZV, indicating preferential detection of this target over amplification of the HHV6 sequence (Figure 45). The individual assays for VZV and HHV-6 showed unique and specific peaks for each of these viruses, at different DNA concentrations, and the Ct averages were within the predefined range (Table 7). The minimum and maximum Tm values for VZV in simultaneous amplification (Figure 53: 82.24ºC and 82.30ºC, respectively) were slightly lower than the range of 82.32ºC to 82.35ºC observed in the individual assays (Table 8). On the other hand, the peak Tm values for HHV-6 in the individual reactions and in the competition with VZV were significantly different, with variation ranges from 79.90ºC to 79.92ºC and from 79.50ºC to 79.70ºC, respectively. .

EXAMPLE 8 Multiplex PCR detection limits

[00193] Real-time multiplex PCR detection limits for HSV-1, HSV-2, VZV, EBV and HHV-6 were determined by serial limiting dilution of quantified viral DNA controls containing 10,000 copies to 1 copy per reaction in triplicate. The identification of targets to define the limits was performed using conventional and HRM dissociation curves, in order to define the best approach for viral detection and identification.

Limit of detection for HSV-1

[00194] Conventional dissociation curve analysis allowed detecting and discriminating HSV-1 DNA in reactions containing at least 100 copies/reaction, both in individual assays and in multiplex PCR (Figure 46). Although one of the reaction replicates containing 10 copies was positive in the multiplex, the limit of detection was set at 100 copies, as two replicates of the triplicate should be positive. In individual assays, only one of the reaction replicates containing 10 viral copies showed non-specific dimer/amplification. In the multiplex assay, the presence of dimers was observed more frequently in dilutions containing concentrations equal to or less than 1,000 viral copies (Figure 46).

[00195] However, the generation of dimers did not preclude viral identification by the conventional dissociation curve (Figure 46). On the other hand, analysis of the HRM curve of positive samples, that is, with a Tm peak compatible with HSV-1, identified with a confidence level greater than 90% only reactions with more than 1,000 genomic copies of HSV-1, both in single assays and in multiplex PCR (Figure 47).

Limit of detection for HSV-2

[00196] Conventional dissociation curve analysis enabled the identification of HSV-2 DNA up to the limit of 10 copies/reaction in multiplex PCR, even in the presence of dimers in some of the reactions. In individual assays, HSV-2 DNA was only identified in one of the reactions containing 10 genomic copies and, therefore, the limit of detection in individual PCR was set at 100 copies/reaction (Figure 48). The analysis of the HRM curve performed with positive samples in individual and multiplex PCR identified, with a confidence level greater than 90%, the samples with the highest concentration of HSV-2 DNA (above 1,000 copies/reaction) (Figure 49). However, this identification was only possible with the exclusion of the temperature range of occurrence of nonspecific dimers/amplification.

Limit of detection for VZV

[00197] VZV DNA discrimination by conventional dissociation curve analysis was possible up to the limit of 100 copies/reaction, even in the presence of dimers (Figure 50). In individual assays, HRM curve analysis also allowed identification in reactions containing at least 100 genomic copies, with a confidence level greater than 90%. However, in multiplex PCR, only reactions with the highest concentrations of VZV DNA (above 1000 copies/reaction) were identified with the same level of confidence after excluding the temperature range of occurrence of dimers (Figure 51).

Limit of detection for EBV

[00198] By analyzing the conventional dissociation curve, it was possible to discriminate EBV DNA up to the limit of 100 copies/reaction, both in individual PCR and in the multiplex format. In individual assays, it was also possible to detect and identify EBV DNA in one of the triplicate reactions containing 10 copies, or even 1 genomic copy. The presence of dimers and/or unspecific amplifications in the multiplex PCR did not prevent the identification of EBV by the analysis of the conventional dissociation curve (Figure 52). However, in the analysis of the HRM curve, using a confidence level of 90%, it was possible to identify only some of the samples previously identified in the conventional dissociation curve (Figure 53).

Limit of detection for HHV-6

[00199] Conventional dissociation curve analysis allowed identifying HHV-6 DNA in reactions with at least 10 copies/reaction in individual PCR (Figure 54). On the other hand, in multiplex PCR, the formation of dimers and/or unspecific amplifications generated peaks in a temperature range very close to the Tm of the HHV-6 target sequence, which hindered accurate identification (Figure 54). With the results of the individual PCR, in the analysis of the HRM curve with the positive samples in the conventional dissociation curve, only the reactions with at least 1000 genomic copies showed identification of the sequence as HHV-6 with a confidence level greater than 90% (Figure 55).

EXAMPLE 8

[00200] The laboratory diagnosis of viral neuroinfections has shown recent advances, although it still represents a challenge. According to DEBIASI and collaborators (2002), PCR has become the reference method in the diagnosis of CNS infections, especially when considering the limitations of other previously used methods, such as viral isolation and serological tests. Therefore, the main objective of the present application was to develop a real-time PCR with HRM curve analysis for the detection of HSV-1, HSV-2, VZV, EBV, CMV and HHV-6, which are human herpesviruses commonly involved in neuroinfections.

[00201] The selection of primers for these six viruses from the literature presented some difficulties, since most of the studies described are directed to conventional PCR, or use probes of the TaqMan type to reveal the amplification of DNA (ZERR et al., 2002; COHRS; GILDEN, 2007; HABBAL; MONEM; GÄRTNER, 2009; HONG et al., 2014; LO et al., 1999; NAGEL et al., 2014). However, it was possible to perform the selection of primers, which, after in silico evaluations, were tested to define specificity in the amplification of target sequences, since the generation of nonspecific products influences the efficiency of PCR.

[00202] The results of evaluating the specificity of primers selected from the COHRS studies; GILDEN, 2007; HABBAL; MONEM; GÄRTNER, 2009; HONG et al., 2014; LO et al., 1999; NAGEL et al., 2014; TANAKA et al., 2009 showed the formation of dimers and/or unspecific amplification with human DNA, despite the lack of description of such interferents in the original works. Primers for EBV described by TANAKA and collaborators (2009), for example, showed nonspecific amplifications with human DNA and dimer formation when tested with annealing temperature at 60ºC (Figure 17). However, the use of these primers at 70ºC (Figure 19) significantly reduced the generation of dimers and unspecific amplification, returning results compatible with the agarose gel electrophoresis images of the original study, which revealed amplification of a single sequence. In this study, TANAKA et al. (2009) used an amplification cycle with high stringency, with an initial annealing temperature of 70ºC, and a reduction of 1ºC/cycle in the first ten cycles, subsequently maintained at 60ºC until the end of the reaction. Thus, the use of a higher annealing temperature favored specificity in real-time PCR.

[00203] Performing multiplex real-time PCR with primers for VZV, EBV and CMV described by TANAKA and collaborators (2009) was only possible after increasing the temperature in the annealing/extension step, which proved to be one of the decisive factors for PCR optimization. Thus, after adding the primers for HHV-6 to the previous set, both in individual and multiplex assays, the selection of new primers for HSV-1 and HSV-2 became even more difficult, since, in addition to the difficulties with regard to the formation of nonspecific products, it was necessary to consider the Tm of specific amplicons of these viruses, as they could not present values similar to those of the others. The detection and identification of six different viruses in a single reaction represents a great challenge regarding the selection of specific primers, since the greater the number of primers present in the reaction, the greater the chance of forming nonspecific products, particularly primer dimers. (COMPSTON et al., 2008). TANAKA et al. (2009) opted for a single pair of primers for HSV-1 and HSV-2, in order to reduce the formation of these interferents in multiplex PCR, since the therapeutic decision dispenses with the identification of the HSV subtype.

[00204] Real-time PCR in individual assays performed with primers for HSV-1/2 (Figure 20) described by TANAKA and collaborators (2009) showed secondary amplifications in positive controls, dimer formation in negative controls with water, in addition to unspecific amplification with human DNA. Since the hybridization of the 3' region of primers with DNA sequences is decisive in nonspecific amplification, as it allows the initiation of synthesis by DNA polymerase, even without complete complementarity between the primer and the DNA template strand, the removal of nucleotides from the 3' end, in order to eliminate the generation of dimers and unspecific amplification from human DNA. However, this did not eliminate amplification of more than one sequence in the positive controls.

[00205] The HRM analysis performed in multiplex real-time PCR with primers for HSV-1/2 described by TANAKA and collaborators (2009) made it possible to identify and differentiate all viruses, even in the presence of more than one amplicon in positive controls of HSV-1 and HSV-2, since the amplicons generated for each of the viruses had different sizes and sequences (Figure 32). The analysis of the HRM curve allowed the distinction of each one of them, as this technique allows the differentiation of fragments containing even a single point mutation A→T, which generates a difference of approximately 0.2°C in the Tm (TONG; GIFFARD, 2012). The results obtained with the analysis of the secondary amplification observed in reactions with HSV controls suggest that, probably, this amplification would only occur in positive cases for HSV-1/2, which did not prevent the identification of these viruses by HRM analysis. However, the proximity of the Tm values of the secondary amplification of HSV-2 and EBV would make it difficult to define a possible co-infection by these two viruses.

[00206] In the search for other primers for HSV-1 and HSV-2, the study by HONG and collaborators (2014) was reassessed and, after in silico analysis, nucleotides were added to the 3' end of the original primers, considering the complementarity with the genome of these viruses. These primers, when tested individually, showed specific peaks in positive controls, without dimer formation and unspecific amplification with human DNA (Figure 24). However, when inserted in the multiplex PCR, the occurrence of dimers and nonspecific amplification was observed, corroborating the statement by (COMPSTON et al., 2008), who highlight that maintaining the same performance levels of the primers in individual tests is a of the challenges in the development and optimization of multiplex PCR. In this perspective, the pairs of primers, described in Table 1, for HHV-6 (11 and 12), VZV (13 and 14), EBV (15 and 16), CMV (17 and 18), HSV-1 (27 and 28), and HSV-2 (29 and 30) were specific in individual assays, indicating the need to optimize the reaction in the multiplex format to eliminate dimer formation.

[00207] BRISSON, MARNI and collaborators (2004) advise that during PCR optimization, efficiency tests must be performed individually, and that all tests must be previously repeated before performing multiplex reactions, since the amplification of DNA sequences in the presence of competition between different primers can alter the amplification efficiency coefficients obtained in individual reactions. Therefore, the optimal concentration of primers for each of the six herpesviruses was evaluated in individual reactions, with the construction of standard curves containing the sequences of HSV-1, HSV-2, VZV, EBV, CMV and HHV-6.

[00208] The results obtained with the primers for HSV-1 and HSV-2 showed that the reduction in the concentration of primers from 17.5 pmoles to 10 pmoles favored the efficiency of the reaction, because at the highest concentration, the efficiency rates showed values greater than 110%, suggesting possible problems in adjusting the reactions. Therefore, in the multiplex reactions, the concentration of 10 pmoles established in the individual assays for these viruses was maintained, and the efficiency values remained above 90%.

[00209] When comparing the results of multiplex and individual PCR assays, BRISSON, MARNI and collaborators (2004) point out that it is essential that there is no accentuated difference in Ct between the two formats. Thus, when comparing the means of Ct of the individual and multiplex assays with different concentrations of viral DNA (Table 6), an average increase of 1.5 cycles was observed for HSV-1, HSV-2, EBV and CMV in the multiplex reactions , while for VZV and HHV-6 the differences were greater than 4 cycles, indicating a possible competition with the primers in binding by DNA polymerase, which reduced the sensitivity of the reaction in the presence of multiple primers. However, such differences in Ct values did not influence the detection limit of the targets, which did not change between individual and multiplex assays, except for HHV-6, which cannot be evaluated in multiplex PCR due to the presence of dimers and unspecific amplifications .

[00210] In addition, targets with greater amplification efficiency have an advantage in competition with the others for the reaction components (BRISSON, MARNI et al., 2004). Therefore, in the simultaneous amplification of two or more targets, impairment in the detection of those with lower amplification efficiency can be observed. However, the analysis of the results of simultaneous detection assays showed a reduction in the amplification of the HSV-1 target sequence in relation to that of HSV-2, and in the amplification of the HHV-6 DNA against the VZV DNA, even with values of equivalent amplification efficiencies. On the other hand, in the simultaneous amplification of EBV and CMV, no interferences between the targets were observed. Thus, similar levels of PCR efficiency do not necessarily guarantee balanced competition between different targets during DNA amplification.

[00211] The formation of dimers in the assays for determining the limits of detection occurred more frequently in triplicates with DNA concentrations equal to or less than 1,000 copies/reaction, corroborating BRISSON, MARNI and collaborators (2004), who claim that in the presence of a high number of copies of the target DNA (above 2,000 copies), there is inhibition of the formation of the primer dimer by competition. That is, at low target DNA copy numbers, dimer formation occurs more rapidly, competing for the reaction components, and depleting available primers for target amplification. Thus, preventing the formation of dimers and nonspecific amplifications in a multiplex PCR contemplating six different targets is something extremely complex. However, with the current format, multiplex PCR is capable of detecting and identifying five out of eight herpesviruses that infect humans.

[00212] The HRM curve analysis in the interpretation of the results was not favorable, because, despite the high degree of conservation of the amplified regions, variations in fluorescence intensity, especially in reactions containing few copies of DNA, can affect the level of confidence in the identification of amplicons. Furthermore, possible nucleotide substitutions in clinical isolates may influence the HRM curve, thus compromising identification. Therefore, the characterization of the viral type using only the conventional dissociation curve showed more consistent results. Therefore, even if variation in Tm is observed due to nucleotide substitutions, amplification intensity, or even the simultaneous amplification of two viral types, there is an acceptable margin of variation guaranteed by a difference of more than 1°C between the Tm values of the amplicons specific to each virus.

[00213] Therefore, due to the variation in the Tm of amplicons, especially in the simultaneous detection assays, in which a slight reduction in the Tm values was observed in relation to the individual reference assays (Table 8), real-time multiplex PCR with clinical samples should be used qualitatively, allowing the screening of positive samples. In a second moment, the confirmation of the results, and cases of co-infection, can be performed by individual tests, in addition to the quantification of the viral DNA with the construction of a standard curve with quantified controls, thus guaranteeing more accurate and reliable results.

[00214] Thus, the use of multiplex PCR in the diagnosis of CNS infections by members of the Herpesviridae family, which are responsible for meningoencephalitis and sporadic viral myelitis in immunocompetent individuals and in patients with different levels of immunosuppression, may contribute to the clinical diagnosis, especially in atypical cases, with significant overlapping of signs and symptoms.

[00215] In addition, obtaining results in a shorter period of time, promoted by real-time multiplex PCR, can also contribute to the reduction in the time elapsed between the onset of symptoms and therapeutic intervention, reducing the risk of complications of the condition clinical. In the case of CMV and EBV infections, especially in transplanted individuals, quantification of viral DNA in blood and other body fluids is essential, as it helps in monitoring the appropriate moment for therapeutic intervention or preemptive therapy.

[00216] Real-time PCR has the advantage of reducing sample handling and the chance of laboratory contamination, as the tests can be performed in a single step and without opening the reaction tube, and the multiplex format favors cases in which the amount of sample is limited or difficult to access. Therefore, the application of real-time multiplex PCR method in the identification of HSV-1, HSV-2, VZV, EBV, CMV and HHV-6 infections improves the cost-effectiveness of the diagnosis, and the post-PCR analysis by Conventional dissociation curve allows test execution by a wider range of equipment.

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Claims (8)

In vitro method for the differential diagnosis of herpesvirus infections of the central nervous system, characterized in that it consists of the following steps:
- extract DNA from a sample of human cerebrospinal fluid;
- amplifying by multiplex real-time PCR using the primers as defined in any one of the sequences SEQ ID NO: 1 to 12; and
- identify the amplicon(s) produced according to the dissociation temperature (Tm) from dissociation curves with high resolution (HRM). Method according to claim 1, characterized in that the amplicon comes from one or more of the following viral types HSV-1, HSV-2, VZV, EBV, CMV and HHV-6. Method according to claim 1 or 2, characterized in that the main disorders associated with said central nervous system infections can be selected from encephalitis, cerebellitis, vasculitis, retinitis, polyradiculitis, ventriculitis, myelitis, and meningitis. Method according to any one of claims 1 to 3, characterized in that the subject is a human subject. Method according to any one of claims 1 to 4, characterized in that quantitative diagnosis is optionally carried out by individual tests. Kit for the differential diagnosis of infections of the central nervous system by herpesvirus, characterized by the fact that it comprises:
- a combination of primers selected from the group SEQ ID NO: 1 to 12;
- commercial human DNA and water, as negative controls, and plasmid DNAs containing viral sequences, as positive controls; and
- reagents for the multiplex real-time PCR reaction. Kit according to claim 6, characterized in that it is used to diagnose and identify the following herpesviruses: HSV-1, HSV-2, VZV, EBV, CMV and HHV-6. Use of a combination of primers selected from the group SEQ ID NO: 1 to 12, characterized by the fact that it is for the differential diagnosis of infections of the central nervous system by herpesviruses.

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