The elusive nature of the oligoclonal bands in multiple sclerosis.

Intrathecal immunoglobulin G (IgG) and oligoclonal bands (OCBs) detected in both the brain and cerebrospinal fluid (CSF) are seminal features of multiple sclerosis (MS). The presence of OCBs correlates with elevated disease burden and severity and supports the diagnosis of MS. Despite numerous investigations into the potential viral and autoantigen targets, the precise antigenic specificity of OCBs has remained elusive. We have little knowledge of the nature regarding these oligoclonal IgG bands. Here, we present compelling evidence highlighting the key findings that both OCBs and intrathecal IgG antibodies are under genetic control and that OCBs originate from clonal B-cells in both the periphery and CNS. We propose that MS OCBs are IgG immune complexes composed of IgG1 and IgG3 antibodies and that the pathological role of OCB stems from the IgG effector functions of these complexes, leading to demyelination and axonal injuries. We present additional evidence regarding the nature of MS OCBs: (1) disease-modifying therapies have been shown to affect CSF OCB; (2) OCBs have also been detected in several neuroinfectious diseases; (3) Epstein-Barr virus (EBV) has been particularly linked with MS pathogenesis, and its association with OCB is an important area of study. Although OCBs are closely associated with MS, more meticulously planned research is necessary to clarify the precise role of OCB in MS, both in terms of disease pathogenesis and diagnosis.

The Spectrum of Neurological Manifestations of Varicella-Zoster Virus Reactivation.

Varicella-Zoster virus (VZV) is a pathogenic human alpha herpes virus that causes varicella (chicken pox) as a primary infection and, following a variable period of latency in different ganglionic neurons, it reactivates to produce herpes zoster (shingles). The focus of this review is on the wide spectrum of the possible neurological manifestations of VZV reactivation. While the most frequent reactivation syndrome is herpes zoster, this may be followed by the serious and painful post-herpetic neuralgia (PHN) and by many other neurological conditions. Prominent among these conditions is a VZV vasculopathy, but the role of VZV in causing giant cell arteritis (GCA) is currently controversial. VZV reactivation can also cause segmental motor weakness, myelitis, cranial nerve syndromes, Guillain-Barre syndrome, meningoencephalitis, and zoster sine herpete, where a neurological syndrome occurs in the absence of the zoster rash. The field is complicated by the relatively few cases of neurological complications described and by the issue of causation when a neurological condition is not manifest at the same time as the zoster rash.

Phenotype and Neuronal Cytotoxic Function of Glioblastoma Extracellular Vesicles.

Glioblastoma (GBM) is the most aggressive and lethal form of brain tumor. Extracellular vesicles (EVs) released by tumor cells play a critical role in cellular communication in the tumor microenvironment promoting tumor progression and invasion. We hypothesized that GBM EVs possess unique characteristics which exert effects on endogenous CNS cells including neurons, producing dose-dependent neuronal cytotoxicity. We purified EVs from the plasma of 20 GBM patients, 20 meningioma patients, and 21 healthy controls, and characterized EV phenotypes by electron microscopy, nanoparticle tracking analysis, protein concentration, and proteomics. We evaluated GBM EV functions by determining their cytotoxicity in primary neurons and the neuroblastoma cell line SH-SY5Y. In addition, we determined levels of IgG antibodies in the plasma in GBM (n = 82), MMA (n = 83), and controls (non-tumor CNS disorders and healthy donors, n = 50) with capture ELISA. We discovered that GBM plasma EVs are smaller in size and had no relationship between size and concentration. Importantly, GBM EVs purified from both plasma and tumor cell lines produced IgG-mediated, complement-dependent apoptosis and necrosis in primary human neurons, mouse brain slices, and neuroblastoma cells. The unique phenotype of GBM EVs may contribute to its neuronal cytotoxicity, providing insight into its role in tumor pathogenesis.

The Possible Role of Neural Cell Apoptosis in Multiple Sclerosis.

The etiology of multiple sclerosis (MS), a demyelinating disease affecting the central nervous system (CNS), remains obscure. Although apoptosis of oligodendrocytes and neurons has been observed in MS lesions, the contribution of this cell death process to disease pathogenesis remains controversial. It is usually considered that MS-associated demyelination and axonal degeneration result from neuroinflammation and an autoimmune process targeting myelin proteins. However, experimental data indicate that oligodendrocyte and/or neuronal cell death may indeed precede the development of inflammation and autoimmunity. These findings raise the question as to whether neural cell apoptosis is the key event initiating and/or driving the pathological cascade, leading to clinical functional deficits in MS. Similarly, regarding axonal damage, a key pathological feature of MS lesions, the roles of inflammation-independent and cell autonomous neuronal processes need to be further explored. While oligodendrocyte and neuronal loss in MS may not necessarily be mutually exclusive, particular attention should be given to the role of neuronal apoptosis in the development of axonal loss. If proven, MS could be viewed primarily as a neurodegenerative disease accompanied by a secondary neuroinflammatory and autoimmune process.

Variable Gene Expression in Human Ganglia Latently Infected with Varicella-Zoster Virus.

Varicella-Zoster virus (VZV) is a pathogenic human herpes virus that causes varicella ("chicken pox") as a primary infection, following which it becomes latent in neuronal cells in human peripheral ganglia. It may then reactivate to cause herpes zoster ("shingles"). Defining the pattern of VZV gene expression during latency is an important issue, and four highly expressed VZV genes were first identified by Randall Cohrs in 1996 using cDNA libraries. Further studies from both his and other laboratories, including our own, have suggested that viral gene expression may be more widespread than previously thought, but a confounding factor has always been the possibility of viral reactivation after death in tissues obtained even at 24 h post-mortem. Recent important studies, which Randall Cohrs contributed to, have clarified this issue by studying human trigeminal ganglia at 6 h after death using RNA-Seq methodology when a novel spliced latency-associated VZV transcript (VLT) was found to be mapped antisense to the viral transactivator gene 61. Viral gene expression could be induced by a VLT-ORF 63 fusion transcript when VZV reactivated from latency. Prior detection by several groups of ORF63 in post-mortem-acquired TG is very likely to reflect detection of the VLT-ORF63 fusion and not canonical ORF63. The contributions to the VZV latency field by Randall Cohrs have been numerous and highly significant.

Higher Levels of IgG3 Antibodies in Serum, But Not in CSF, Distinguish Multiple Sclerosis From Other Neurological Disorders.

Increased intrathecal IgG and oligoclonal bands (OCB) are seminal features of multiple sclerosis (MS). Although no such differences in MS blood total IgG antibodies have been reported, serum OCB are a common and persistent finding in MS and have a systemic source. Recent studies showed that IgG3+ B cells and higher levels of serum IgG3 are linked to the development of MS. Additionally, intrathecal IgG synthesis in MS is associated with IgG3 heavy chain gene single nucleotide polymorphisms, and there is a strong relationship between susceptibility to MS and an IgG3 restriction fragment length polymorphism. These studies support the role of IgG3 in disease pathogenesis. Using multiple immunoassays, we investigated levels of total IgG, IgG1, and IgG3 in sera and CSF of 102 MS patients (19 paired CSF and sera), 76 patients with other neurological disorders (9 paired CSF and sera), and 13 healthy controls. We show that higher levels of total IgG and IgG3 antibodies were detected in MS serum, but not in CSF, which distinguishes MS from other inflammatory and non-inflammatory neurological disorders, with Receiver Operating Characteristic (ROC) Curves 0.79 for both IgG3 & total IgG. Our data support the notion that IgG3 antibodies may be a potential candidate for MS blood biomarker development.

Aberrant Immunoglobulin G Glycosylation in Multiple Sclerosis.

A hallmark of the inflammatory response in multiple sclerosis (MS) is the presence of intrathecal Immunoglobulin G (IgG) antibodies and oligoclonal bands (OCBs). The biological activity of IgGs is modulated by changes in glycosylation. Using multiple immunoassays with common lectins for sialylation and galactosylation, we investigated levels of IgG glycosylation in 28 MS and 37 control sera as well as paired CSF and serum. We demonstrated the presence of significantly lower levels of IgG sialylation in MS CSF compared to paired serum. Further, we showed that in MS there was no correlation between sialylated IgG and total IgG antibodies, or between sialylated IgG in CSF and serum. ELISA with native IgG antibodies showed significantly higher levels of sialylated and galactosylated IgG in MS compared to other neurological disorders and normal healthy controls. We conclude that lower levels of sialylated intrathecal IgG and higher levels of serum IgG galactosylation in MS may play significant role in disease pathogenesis. The unique IgG glycosylation profiles suggest a complexed nature of the IgG antibodies which may influence its effector functions in MS.

Microarray profiling predicts early neurological and immune phenotypic traits in advance of CNS disease during disease progression in Trypanosoma. b. brucei infected CD1 mouse brains.

Human African trypanosomiasis (HAT), also known as sleeping sickness, is a major cause of mortality and morbidity in sub-Saharan Africa. We hypothesised that recent findings of neurological features and parasite brain infiltration occurring at much earlier stages in HAT than previously thought could be explained by early activation of host genetic programmes controlling CNS disease. Accordingly, a transcriptomal analysis was performed on brain tissue at 0, 7, 14, 21 and 28dpi from the HAT CD1/GVR35 mouse model. Up to 21dpi, most parasites are restricted to the blood and lymphatic system. Thereafter the trypanosomes enter the brain initiating the encephalitic stage. Analysis of ten different time point Comparison pairings, revealed a dynamic transcriptome comprising four message populations. All 7dpi Comparisons had by far more differentially expressed genes compared to all others. Prior to invasion of the parenchyma, by 7dpi, ~2,000 genes were up-regulated, denoted [7dpi↑] in contrast to a down regulated population [7dpi↓] also numbering ~2,000. However, by 14dpi both patterns had returned to around the pre-infected levels. The third, [28dpi↑] featured over three hundred transcripts which had increased modestly up to14dpi, thereafter were significantly up-regulated and peaked at 28dpi. The fourth, a minor population, [7dpi↑-28dpi↑], had similar elevated levels at 7dpi and 28dpi. KEGG and GO enrichment analysis predicted a diverse phenotype by 7dpi with changes to innate and adaptive immunity, a Type I interferon response, neurotransmission, synaptic plasticity, pleiotropic signalling, circadian activity and vascular permeability without disruption of the blood brain barrier. This key observation is consistent with recent rodent model neuroinvasion studies and clinical reports of Stage 1 HAT patients exhibiting CNS symptoms. Together, these findings challenge the strict Stage1/Stage2 phenotypic demarcation in HAT and show that that significant neurological, and immune changes can be detected prior to the onset of CNS disease.

Recent Issues in Varicella-Zoster Virus Latency.

Varicella-zoster virus (VZV) is a human herpes virus which causes varicella (chicken pox) as a primary infection, and, following a variable period of latency in neurons in the peripheral ganglia, may reactivate to cause herpes zoster (shingles) as well as a variety of neurological syndromes. In this overview we consider some recent issues in alphaherpesvirus latency with special focus on VZV ganglionic latency. A key question is the nature and extent of viral gene transcription during viral latency. While it is known that this is highly restricted, it is only recently that the very high degree of that restriction has been clarified, with both VZV gene 63-encoded transcripts and discovery of a novel VZV transcript (VLT) that maps antisense to the viral transactivator gene 61. It has also emerged in recent years that there is significant epigenetic regulation of VZV gene transcription, and the mechanisms underlying this are complex and being unraveled. The last few years has also seen an increased interest in the immunological aspects of VZV latency and reactivation, in particular from the perspective of inborn errors of host immunity that predispose to different VZV reactivation syndromes.

The Role of Autophagy in Varicella Zoster Virus Infection.

Autophagy is an evolutionary conserved cellular process serving to degrade cytosolic organelles or foreign material to maintain cellular homeostasis. Autophagy has also emerged as an important process involved in complex interactions with viral pathogens during infection. It has become apparent that autophagy may have either proviral or antiviral roles, depending on the cellular context and the specific virus. While evidence supports an antiviral role of autophagy during certain herpesvirus infections, numerous examples illustrate how herpesviruses may also evade autophagy pathways or even utilize this process to their own advantage. Here, we review the literature on varicella zoster virus (VZV) and autophagy and describe the mechanisms by which VZV may stimulate autophagy pathways and utilize these to promote cell survival or to support viral egress from cells. We also discuss recent evidence supporting an overall antiviral role of autophagy, particularly in relation to viral infection in neurons. Collectively, these studies suggest complex and sometimes opposing effects of autophagy in the context of VZV infection. Much remains to be understood concerning these virus-host interactions and the impact of autophagy on infections caused by VZV.

Varicella-Zoster Virus Infection of Neurons Derived from Neural Stem Cells.

Varicella-Zoster virus (VZV) is a human herpesvirus that causes varicella (chickenpox) as a primary infection, and, following a variable period of ganglionic latency in neurons, it reactivates to cause herpes zoster (shingles). An analysis of VZV infection in cultures of neural cells, in particular when these have been obtained from induced pluripotent stem cells (iPSCs) or neural stem cells consisting of highly purified neuronal cultures, has revealed much data that may be of neurobiological significance. Early studies of VZV infection of mature cultured neural cells were mainly descriptive, but more recent studies in homogeneous neural stem cell cultures have used both neuronal cell markers and advanced molecular technology. Two general findings from such studies have been that (a) VZV infection of neurons is less severe, based on several criteria, than that observed in human fibroblasts, and (b) VZV infection of neurons does not lead to apoptosis in these cells in contrast to apoptosis observed in fibroblastic cells. Insights gained from such studies in human neural stem cells suggest that a less severe initial lytic infection in neurons, which are resistant to apoptosis, is likely to facilitate a pathological pathway to a latent state of the virus in human ganglia.

An overview of viral infections of the nervous system in the immunosuppressed.

Several viruses have the capacity to cause serious infections of the nervous system in patients who are immunosuppressed. Individuals may be immunosuppressed because of primary inherited immunodeficiency, secondary immunodeficiency due to particular diseases such as malignancy, administration of immunosuppressant drugs or organ or bone marrow transplantation. The viruses capable of such opportunistic infection of the nervous system include herpes simplex virus (HSV), Varicella-Zoster virus (VZV), Cytomegalovirus (CMV), Epstein -Barr virus (EBV), Human Herpes virus type 6 (HHV-6), JC virus (JCV), enterovirus, measles virus and Covid-19. In most cases it seems likely that immunological defence mechanisms in the immunosuppressed are deficient which creates a suitable environment for certain viruses to become opportunistic in the nervous and other systems. Further research is required both to understand these opportunistic mechanisms in more detail and also to determine how many virus infections are modified by specific inborn errors of immunological responses.

The Role of Antibodies in the Pathogenesis of Multiple Sclerosis.

The presence of persistent intrathecal oligoclonal immunoglobulin G (IgG) bands (OCBs) and lesional IgG deposition are seminal features of multiple sclerosis (MS) disease pathology. Despite extensive investigations, the role of antibodies, the products of mature CD19+ B cells, in disease development is still controversial and under significant debate. Recent success of B cell depletion therapies has revealed that CD20+ B cells contribute to MS pathogenesis via both antigen-presentation and T-cell-regulation. However, the limited efficacy of CD20+ B cell depletion therapies for the treatment of progressive MS indicates that additional mechanisms are involved. In this review, we present findings suggesting a potential pathological role for increased intrathecal IgGs, the relation of circulating antibodies to intrathecal IgGs, and the selective elevation of IgG1 and IgG3 subclasses in MS. We propose a working hypothesis that circulating B cells and antibodies contribute significantly to intrathecal IgGs, thereby exerting primary and pathogenic effects in MS development. Increased levels of IgG1 and IgG3 antibodies induce potent antibody-mediated cytotoxicity to central nervous system (CNS) cells and/or reduce the threshold required for antigen-driven antibody clustering leading to optimal activation of immune responses. Direct proof of the pathogenic roles of antibodies in MS may provide opportunities for novel blood biomarker identification as well as strategies for the development of effective therapeutic interventions.

The Actigraphy Sleep Score: A New Biomarker for Diagnosis, Disease Staging, and Monitoring in Human African Trypanosomiasis.

Human African trypanosomiasis (HAT) remains a serious public health problem with diagnostic and treatment challenges in many African countries. The absence of a gold-standard biomarker has been a major difficulty for accurate disease staging and treatment follow-up. We therefore attempted to develop a simple, affordable, and noninvasive biomarker for HAT diagnosis and staging. Simultaneous actigraphy and polysomnography as well as cerebrospinal fluid (CSF) white blood cell (WBC) count, trypanosome presence, and C-X-C motif ligand (CXCL)-10 cytokine levels were performed in 20 HAT patients and nine healthy individuals (controls) using standard procedures. The International HIV Dementia Scale (IHDS) was scored in some patients as a surrogate for clinical assessment. From actigraphic parameters, we developed a novel sleep score and used it to determine correlations with other HAT markers, and compared their performance in differentiating between patients and controls and between HAT stages. The novel actigraphy sleep score (ASS) had the following ranges: 0-25 (healthy controls), 67-103 (HAT stage I), 111-126 (HAT intermediate), and 133-250 (HAT stage II). Compared with controls, stage I patients displayed a 7-fold increase in the ASS (P < 0.01), intermediate stage patients a 10-fold increase (P < 0.001), and HAT stage II patients an almost 20-fold increase (P < 0.001). CXCL-10 showed high interindividual differences. White blood cell counts were only marked in HAT stage II patients with a high interindividual variability. The International HIV Dementia Scale score negatively correlated with the ASS. We report the development and better performance of a new biomarker, ASS, for HAT diagnosis, disease staging, and monitoring that needs to be confirmed in large cohort studies.

Recombinant antibodies derived from laser captured single plasma cells of multiple sclerosis brain identified phage peptides which may be used as tools for characterizing intrathecal IgG response.

Oligoclonal bands and increased IgG antibody levels can be detected in the cerebrospinal fluid in vast majority of patients with Multiple Sclerosis (MS). However, the antigenic specificity of oligoclonal IgG has yet to be determined. Using laser capture microdissection, we isolated single CD38+ plasma cells from lesion areas in two autopsy MS brains, and generated three recombinant antibodies (rAbs) from clonally expanded plasma cells. Panning phage-displayed random peptide libraries was carried out to determine peptide antigen specificities of these MS brain rAbs. We identified 25 high affinity phage peptides from which 5 peptides are unique. Database searches revealed that they shared sequence homologies with Epstein-Barr nuclear antigens 4 and 6, as well as with other viral proteins. Significantly, these peptides were recognized by intrathecal IgG and oligoclonal IgG bands in other MS patients. Our results demonstrate that functional recombinant antibodies can be generated from clonally expanded plasma cells in MS brain lesions by laser capture microdissection, and that these MS brain rAbs have the potential for determining the targets of intrathecal IgG and oligoclonal bands.

Generation of neuroinflammation in human African trypanosomiasis.

Human African trypanosomiasis (HAT) is caused by infection due to protozoan parasites of the Trypanosoma genus and is a major fatal disease throughout sub-Saharan Africa. After an early hemolymphatic stage in which the peripheral tissues are infected, the parasites enter the CNS causing a constellation of neurologic features. Although the CNS stage of HAT has been recognized for over a century, the mechanisms generating the neuroinflammatory response are complex and not well understood. Therefore a better understanding of the mechanisms utilized by the parasites to gain access to the CNS compartment is critical to explaining the generation of neuroinflammation. Contrast-enhanced MRI in a murine model of HAT has shown an early and progressive deterioration of blood-CNS barrier function after trypanosome infection that can be reversed following curative treatment. However, further studies are required to clarify the molecules involved in this process. Another important determinant of brain inflammation is the delicate balance of proinflammatory and counterinflammatory mediators. In mouse models of HAT, proinflammatory mediators such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and CXCL10 have been shown to be crucial to parasite CNS invasion while administration of interleukin (IL)-10, a counter inflammatory molecule, reduces the CNS parasite burden as well as the severity of the neuroinflammatory response and the clinical symptoms associated with the infection. This review focuses on information, gained from both infected human samples and animal models of HAT, with an emphasis on parasite CNS invasion and the development of neuroinflammation.

Update on human African trypanosomiasis (sleeping sickness).

Human African trypanosomiasis (HAT), also known as sleeping sickness, is one of the Africa's 'neglected diseases' and is caused by infection with protozoan parasites of the Trypanosoma genus. Transmitted by the bite of the tsetse fly, it puts 70 million people at risk throughout sub-Saharan Africa and is usually fatal if untreated or inadequately treated. In this brief overview, some important recent developments in this disease are outlined. These cover various aspects including a reduction in disease incidence, newly recognised parasite reservoir sites in humans, disease outcome, novel diagnostic methods, new and improved treatment, and disease neuropathogenesis.

Clinical and Neuropathogenetic Aspects of Human African Trypanosomiasis.

Trypanosomiasis has been recognized as a scourge in sub-Saharan Africa for centuries. The disease, caused by protozoan parasites of the Trypanosoma genus, is a major cause of mortality and morbidity in animals and man. Human African trypanosomiasis (HAT), or sleeping sickness, results from infections with T. brucei (b.) gambiense or T. b. rhodesiense with T. b. gambiense accounting for over 95% of infections. Historically there have been major epidemics of the infection, followed by periods of relative disease control. As a result of concerted disease surveillance and treatment programmes, implemented over the last two decades, there has been a significant reduction in the number of cases of human disease reported. However, the recent identification of asymptomatic disease carriers gives cause for some concern. The parasites evade the host immune system by switching their surface coat, comprised of variable surface glycoprotein (VSG). In addition, they have evolved a variety of strategies, including the production of serum resistance associated protein (SRA) and T. b. gambiense-specific glycoprotein (TgsGP) to counter host defense molecules. Infection with either disease variant results in an early haemolymphatic-stage followed by a late encephalitic-stage when the parasites migrate into the CNS. The clinical features of HAT are diverse and non-specific with early-stage symptoms common to several infections endemic within sub-Saharan Africa which may result in a delayed or mistaken diagnosis. Migration of the parasites into the CNS marks the onset of late-stage disease. Diverse neurological manifestations can develop accompanied by a neuroinflammatory response, comprised of astrocyte activation, and inflammatory cell infiltration. However, the transition between the early and late-stage is insidious and accurate disease staging, although crucial to optimize chemotherapy, remains problematic with neurological symptoms and neuroinflammatory changes recorded in early-stage infections. Further research is required to develop better diagnostic and staging techniques as well as safer more efficacious drug regimens. Clearer information is also required concerning disease pathogenesis, specifically regarding asymptomatic carriers and the mechanisms employed by the trypanosomes to facilitate progression to the CNS and precipitate late-stage disease. Without progress in these areas it may prove difficult to maintain current control over this historically episodic disease.

Clinical Features of Varicella-Zoster Virus Infection.

Varicella-zoster virus (VZV) is a pathogenic human herpes virus that causes varicella (chickenpox) as a primary infection, following which it becomes latent in peripheral ganglia. Decades later, the virus may reactivate either spontaneously or after a number of triggering factors to cause herpes zoster (shingles). Varicella and its complications are more severe in the immunosuppressed. The most frequent and important complication of VZV reactivation is postherpetic neuralgia, the cause of which is unknown and for which treatment is usually ineffective. Reactivation of VZV may also cause a wide variety of neurological syndromes, the most significant of which is a vasculitis, which is treated with corticosteroids and the antiviral drug acyclovir. Other VZV reactivation complications include an encephalitis, segmental motor weakness and myelopathy, cranial neuropathies, Guillain⁻Barré syndrome, enteric features, and zoster sine herpete, in which the viral reactivation occurs in the absence of the characteristic dermatomally distributed vesicular rash of herpes zoster. There has also been a recent association of VZV with giant cell arteritis and this interesting finding needs further corroboration. Vaccination is now available for the prevention of both varicella in children and herpes zoster in older individuals.

The challenging problem of disease staging in human African trypanosomiasis (sleeping sickness): a new approach to a circular question.

Human African trypanosomiasis (HAT), also known as sleeping sickness, puts millions of people at risk in sub-Saharan Africa and is a neglected parasitic disease that is almost always fatal if untreated or inadequately treated. HAT manifests itself in two stages that are difficult to distinguish clinically. The problem of staging in HAT is extremely important since treatment options, some of which are highly toxic, are directly linked to the disease stage. Several suggested investigations for disease staging have been problematic because of the lack of an existing gold standard with which to compare new clinical staging markers. The somewhat arbitrary current criteria based on the cerebrospinal fluid (CSF) white blood cell (WBC) count have been widely used, but the new potential biomarkers are generally compared with these, thereby making the problem somewhat circular in nature. We propose an alternative 'reverse' approach to address this problem, conceptualised as using appropriate statistical methods to test the performance of combinations of established laboratory variables as staging biomarkers to correlate with the CSF WBC/trypanosomes and clinical features of HAT. This approach could lead to the use of established laboratory staging markers, potentially leading to a gold standard for staging and clinical follow-up of HAT.