Cerebrospinal fluid inflammatory cytokine profiles of patients with neurotropic parasitic infections

@#The pathogenesis of chronic parasitic central nervous system (CNS) infections, including granulomatous amoebic meningoencephalitis (GAE), cerebral toxoplasmosis (CT), and neurocysticercosis (NCC), is primarily due to an inflammatory host reaction to the parasite. Inflammatory cytokines produced by invading T cells, monocytes, and CNS resident cells lead to neuroinflammation which underlie the immunopathology of these infections. Immune molecules, especially cytokines, can therefore emerge as potential biomarker(s) of CNS parasitic infections. In this study, cerebral spinal fluid (CSF) samples from suspected patients with parasitic infections were screened for pathogenic free-living amoebae by culture (n=2506) and PCR (n=275). Six proinflammatory cytokines in smear and culture-negative CSF samples from patients with GAE (n = 2), NCC (n = 7), and CT (n = 23) as well as control (n = 7) patients were measured using the Multiplex Suspension assay. None of the CSF samples tested was positive for neurotropic free-living amoebae by culture and only two samples showed Acanthamoeba 18S rRNA by PCR. Of the six cytokines measured, only IL-6 and IL-8 were significantly increased in all three infection groups compared to the control group. In addition, TNFa levels were higher in the GAE and NCC groups and IL-17 in the GAE group compared to controls. The levels of IL-1b and IFNg were very low in all the infection groups and the control group. There was a correlation between CSF cellularity and increased levels of IL-6, IL-8, and TNFa in 11 patients. Thus, quantifying inflammatory cytokine levels in CSF might help with understanding the level of neuroinflammation in patients with neurotropic parasitic diseases. Further studies with clinico-microbiological correlation in the form of reduction of cytokine levels with treatment and the correlation with neurological deficits are needed.

Role of skin punch biopsy in diagnosis of small fiber neuropathy-A review for the neuropathologist

Over the last three decades, skin punch biopsy has become the gold standard for diagnosis of small fiber neuropathies, including autonomic neuropathies commonly seen in diabetics, patients with HIV, and children with hereditary sensory autonomic neuropathies and toxin-induced neuropathy. Clinical, biochemical, electrophysiological tests are inconclusive, making it difficult to diagnose and initiate treatment. A skin punch biopsy is easy to perform in the outpatient clinic, is highly sensitive, and provides an objective diagnosis. Importantly, it helps avoid performing invasive nerve biopsy in patients with small fiber neuropathy, thereby preventing complications such as non-healing of the biopsy site, which is common in these patients. Secondly, the greatest advantage of skin punch biopsies is that they can be repeated any number of times, unlike a nerve biopsy, and are useful to evaluate disease progression and therapeutic response. More recently, its use has been expanded to the diagnosis of large fiber neuropathies, inherited demyelinating neuropathies, etc., obviating the need for a nerve biopsy. The European Federation of Neurological Societies has published guidelines for evaluation to ensure uniformity with regard to the site of biopsy, processing, and quantification. The evaluation of the skin biopsy involves morphometric assessment of the intraepidermal nerve fiber density using PGP 9.5 immunostained sections by bright-field microscopy. This review focuses on the practical aspects of skin punch biopsy and its utility for the practicing pathologist.

Recent advances in the diagnosis of immune mediated demyelinating neuropathies

Inflammatory neuropathies are a group of acquired neuropathies which could be due to autoimmune, infectious, paraneoplastic, or paraproteinemic etiology. The etiological diagnosis of inflammatory neuropathy is not simple, and often requires combination of clinical, electrophysiological, and histopathological findings to arrive at a precise diagnosis which is important for management of the disorder. Whereas there are comprehensive and sensitive panel of serological tests available for diagnosis of the infectious, paraneoplastic, paraproteinemic neuropathies, the diagnosis of immune-mediated demyelinating neuropathies remain a considerable challenge as there is both clinical and pathological overlap. Newer non-invasive methodologies such as high-resolution ultrasound, magnetic resonance imaging (MRI), and importantly, serological testing for antibodies are emerging, and it is essential for the practicing pathologist to be up-to-date with emerging modalities. In this review, we focus on the approach to diagnosis of immune-mediated demyelinating neuropathies.

Brain banking in India: Relevance in current day practice

Biobanks are set to become the norm. The explosion of new and powerful technologies like genomics and other multiomics has catapulted research from individual laboratories to multi-institutional and international partners. Today, with increasing life span, and the rising incidence of brain diseases, Brain Banks have become an invaluable source for unravelling the pathogenesis of several brain disorders, and develop effective therapies. The article briefly reviews the evolution of brain banking, rise of global networks, with a brief overview of steps involved from donor recruitment, protocols of processing, storage, annotation, and tissue distribution. The ethics of biobanking is one of the most controversial issues in bioethics, the key issues being consent, confidentiality, and commercialisation. Regulatory authorities in different countries and in India, the Indian Council of Medical Research has taken a lead to formulate new ethical guidelines for research involving human participants protecting rights, and well-being of research participants. Although brain banks have been established in the 1960s, in India, the first Brain Bank was established in 1995 at the National Institute of Mental Health and Neurosciences, Bengaluru. Now a network with two more Brain banks is being established in the country. The challenges and benefits of establishing the first Brain Bank as a National Research Facility in India is shared. For optimising available resources and promote brain banking, it is essential for medical professionals, and the public to perceive the crucial advantage in conversion of biological waste into invaluable resources for neuroscience. This will be the greatest “gift of hope” that we can offer for the future generations to overcome hitherto untreatable disorders such as dementias.

Role of the pathologist in the diagnosis of autoimmune encephalitis

Autoimmune encephalitis is a group of non-infectious immune-mediated inflammatory disorders manifesting with epilepsy and encephalitis syndromes that are associated with autoantibodies in the serum and/or cerebrospinal fluid (CSF). Pathogenic autoantibodies have been discovered against intracellular onconeural antigens, surface neuronal, or synaptic antigens with distinctive pathogenesis that underlie differences in response to immunotherapy. The onconeural antigens incite cytotoxic T-cell-mediated neuronal destruction, whereas surface antigens trigger direct damage by autoantibodies via complement mediated pathways, and hence respond well to immunomodulatory therapy, in contrast to poor response in the former. Neuroimaging, electroencephalogram, and CSF findings being non-specific, detection of autoantibodies is essential for a confirmatory diagnosis. Detection methods available include tissue-based assay, cell-based assays, immunoblot, cell culture, flow cytometry, and enzyme-linked immunosorbent assays. In this review, we discuss the various testing modalities available for onconeural and cell surface antibodies, their sensitivity and specificity and the emerging role of the pathologist in the diagnosis of autoimmune encephalitis. Early diagnosis is crucial for instituting treatment and preventing morbidity and mortality.

Non invasive tests for diagnosis of parasitic infections of CNS

Central nervous system (CNS) infections are among the most devastating diseases with high mortality and morbidity. In the pre-human immunodeficiency virus (HIV) era, the occurrence of CNS infections was very infrequent. However, in the past four decades or so, with a global increase in the immunocompromised population, the incidence of opportunistic infections of the CNS has changed. This includes a global increase in the incidence of parasitic infections such as Toxoplasma gondii. Infections such as neurocysticercosis and cerebral malaria are quite prevalent in developing countries. Early diagnosis of these infections is crucial for instituting accurate therapy and preventing mortality and morbidity. Despite advances in neuroimaging techniques, laboratory diagnosis remains the mainstay for confirmation of diagnosis. We present an update on the noninvasive tests available for laboratory diagnosis of parasitic infections of the CNS.

Non-granulomatous inflammatory lesions of CNS: Approach to diagnosis

Infections constitute an important and common category of diseases, particularly in less developed countries. Infections present with a broad spectrum of clinical and radiologic features dictated by the cell and tissue tropism and host response elicited, posing a considerable diagnostic challenge. Early diagnosis and treatment are crucial in preventing mortality and morbidity. Recourse is often made to biopsy for ascertaining the diagnosis, and hence the pathologist plays a vital role in patient management. Therefore, knowledge of the histopathologic changes is necessary to recognize the histological changes and guide the diagnostic workup and management. Each microbial agent elicits a distinctive pattern of inflammatory tissue response, which can serve as a clue to the etiological agent. Based on the causative organism, microbial, and host factors, the inflammatory response may be acute or chronic, necrotic or non-necrotic. The inflammation can be of varied patterns – lymphohistiocytic, granulomatous, inflammatory demyelinating, fibrosing, or showing minimal inflammation. The pattern of necrosis also differs based on the causative organism. Typically, pyogenic bacteria are associated with suppurative inflammation, tuberculosis with caseous granulomatous, and fungi with suppurative granulomatous inflammation. Viral infections are associated with lymphohistiocytic non-necrotizing inflammation and, based on cell tropism, can cause demyelination (e.g., JCV) and/or viral inclusions. Parasitic infections (protozoal or metazoal) display a broad spectrum of inflammatory changes that overlap with other types of infections. This review briefly describes pathological patterns and associated pathogens and provides an algorithmic approach based on pattern recognition that may be useful for the practicing pathologist.

Identification of microbial agents in culture-negative brain abscess samples by 16S/18S rRNA gene PCR and sequencing

@#Despite clinical suspicion of an infection, brain abscess samples are often culture-negative in routine microbiological testing. Direct PCR of such samples enables the identification of microbes that may be fastidious, non-viable, or unculturable. Brain abscess samples (n = 217) from neurosurgical patients were subjected to broad range 16S rRNA gene PCR and sequencing for bacteria. All these samples and seven formalin-fixed paraffin-embedded tissue (FFPE) samples were subjected to species-specific 18S rRNA PCR for neurotropic free-living amoeba that harbour pathogenic bacteria. The concordance between smear and/or culture and PCR was 69%. One-third of the samples were smear- and culture-negative for bacterial agents. However, 88% of these culture-negative samples showed the presence of bacterial 16S rRNA by PCR. Sanger sequencing of 27 selected samples showed anaerobic/fastidious gram negative bacteria (GNB, 38%), facultative Streptococci (35%), and aerobic GNB (27%). Targeted metagenomics sequencing of three samples showed multiple bacterial species, including anaerobic and non-culturable bacteria. One FFPE tissue revealed the presence of Acanthamoeba 18S rRNA. None of the frozen brain abscess samples tested was positive for 18S rRNA of Acanthamoeba or Balamuthia mandrillaris. The microbial 16/18S rRNA PCR and sequencing outperformed culture in detecting anaerobes, facultative Streptococci and FLA in brain abscess samples. Genetic analyses of 16S/18S sequences, either through Sanger or metagenomic sequencing, will be an essential diagnostic technology to be included for diagnosing culture-negative brain abscess samples. Characterizing the microbiome of culture-negative brain abscess samples by molecular methods could enable detection and/or treatment of the source of infection.

Central nervous system infections caused by pathogenic free-living amoebae: An Indian perspective

@#Pathogenic free-living amoebae (FLA), namely Acanthamoeba sp., Naegleria fowleri and Balamuthia mandrillaris are distributed worldwide. These neurotropic amoebae can cause fatal central nervous system (CNS) infections in humans. This review deals with the demographic characteristics, symptoms, diagnosis, and treatment outcomes of patients with CNS infections caused by FLA documented in India. There have been 42, 25, and 4 case reports of Acanthamoeba granulomatous amoebic encephalitis (GAE), N. fowleri primary amoebic meningoencephalitis (PAM), and B. mandrillaris meningoencephalitis (BAE), respectively. Overall, 17% of Acanthamoeba GAE patients and one of the four BAE patients had some form of immunosuppression, and more than half of the N. fowleri PAM cases had history of exposure to freshwater. Acanthamoeba GAE, PAM, and BAE were most commonly seen in males. Fever, headache, vomiting, seizures, and altered sensorium appear to be common symptoms in these patients. Some patients showed multiple lesions with edema, exudates or hydrocephalus in their brain CT/MRI. The cerebrospinal fluid (CSF) of these patients showed elevated protein and WBC levels. Direct microscopy of CSF was positive for amoebic trophozoites in 69% of Acanthamoeba GAE and 96% of PAM patients. One-fourth of the Acanthamoeba GAE and all the BAE patients were diagnosed only by histopathology following autopsy/biopsy samples. Twenty-one Acanthamoeba GAE survivors were treated with cotrimoxazole, rifampicin, and ketoconazole/amphotericin B, and all eleven PAM survivors were treated with amphotericin B alongside other drugs. A thorough search for these organisms in CNS samples is necessary to develop optimum treatment strategies.

Pigmented variant of pleomorphic xanthoastrocytoma - A rare long-term epilepsy associated neoplasm

Pleomorphic xanthoastrocytoma (PXA) is an uncommon, long-term epilepsy associated tumor of young adults. Its pigmented variant is exceedingly rare, with only five previously reported cases on record. We report the sixth case of pigmented PXA in a 24-year-old lady presenting with long-standing seizures. The MRI revealed a solid cystic lesion located in the right medial temporal lobe. Histopathologically, the superficially located tumor showed typical features of PXA with melanin-laden astrocytic component and was negative for V600E-mutant BRAF. The histogenesis is discussed.

Successful treatment of primary cerebral mucormycosis: Role of microbiologist.

Fungal brain abscess is rare with a rapidly progressive disease with fulminant course and invariably fatal outcome, unless diagnosed early and treated rapidly. We report a 56‑year‑old woman diagnosed to have fungal abscess who recovered completely following amphotericin B treatment. She presented with weakness of the right hand, deviation of mouth to left and aphasia for 2 days. Computed tomography of the brain revealed a left frontal capsuloganglionic hypodense lesion. Stereotactic biopsy was performed, and microbiological confirmation of non‑septate fungal hyphae from pus from aspirate within 2 h helped initiate timely and appropriate treatment leading to cure. Histopathology and culture later confirmed mucormycosis.

Nocardial brain abscess in an immunocompetent host.

Nocardial infection of the central nervous system is rare and usually manifests as brain abscess. Here we describe an elderly gentleman who presented with signs and symptoms of an intracranial mass lesion localising to the frontal lobe. Clinical examination and CT scan suggested neoplasia as the probable diagnosis. A biloculated abscess was seen at surgery. Aspiration of the contents and examination of pus revealed Nocardia asteroides . Treatment included total excision and prolonged antibiotic therapy which resulted in an excellent outcome.

Toxoplasma granuloma of brainstem: A rare case.

Toxoplasmosis is a common opportunistic infection in patients with AIDS in whom it frequently presents as intracranial space-occupying lesions. In the immunocompetent patient the most common manifestation is as asymptomatic cervical lymphadenopathy which may be associated with vague systemic manifestations such as fever or myalgia. In very rare cases people with normal immunity may present with meningoencephalitis polymyositis or myocarditis. It is very rare to encounter a brainstem granuloma due to toxoplasma infection in such patients. We report a non-immunocompromised man who presented with multiple cranial nerve palsies due to a brainstem lesion, which turned out to be a toxoplasma granuloma. He recovered completely after a four-week course of Pyrimethamine and Sulphadoxine. An extensive search of the literature failed to reveal any prior reports of a similar nature. This case is being reported because of its rarity and the complete recovery made by the patient.

Dysmyelinating neuropathy in benign form of megalencephalic leukoencephalopathy with subcortical cysts: A novel observation from south India.

A 37-year-old gentleman presented with macrocephaly since early childhood and progressive impairment of motor and cognitive functions. Magnetic resonance imaging revealed extensive white matter involvement and frontotemporal subcortical cysts. Absent ankle jerk and abnormal nerve conduction study raised a possibility of associated peripheral neuropathy. Sural nerve biopsy was suggestive of dysmyelinating neuropathy. This report serves to expand the clinical spectrum of this rare leukodystrophy.

Toxoplasma seroprevalence in healthy voluntary blood donors from urban Karnataka.

BACKGROUND & OBJECTIVES: Although many infections can be transmitted through blood transfusion, it is not possible to carry out screening tests for all. Among the protozoal diseases transmitted by blood transfusion in India the most important is malaria, followed by toxoplasmosis. Screening for malaria is mandatory in India. We evaluated the seroprevalence of Toxoplasma gondii in healthy adult population of blood donors in Karnataka, south India. METHODS: A total of 1000 serum samples collected in two batches (500 each) in the years 2004 and 2005 from healthy voluntary blood donors were tested for T. gondii antibodies by ELISA method, in addition to the other five mandatory tests. RESULTS: Overall 20.3 per cent were positive for T. gondii IgG antibody, of which, 63 per cent had high and 7 per cent low avidity, 3.6 per cent IgM positive. IgG titre ranged from 18-362 IU/ml. INTERPRETATION & CONCLUSION: Our study showed a high prevalence of T. gondii antibodies in healthy voluntary blood population. It may be appropriate to include screening for T. gondii also in the pretransfusion blood testing schedule.

Cryptococcal meningitis: Clinical, diagnostic and therapeutic overviews.

Cryptococcal meningitis has emerged as a leading cause of infectious morbidity and mortality in patients with AIDS. Among the human immunodeficiency virus (HIV)-seropositive subjects, cryptococcal meningitis is the second most common cause of opportunistic neuro-infection. Current trends are changing due to the marked improvement of quality and length of life produced by highly active antiretroviral therapy (HAART). The introduction of generic HAART in India has resulted in an increase in the number of individuals getting treatment for HIV infection, as the cost of highly active antiretroviral therapy (HAART) has decreased 20- fold. Cryptococcal meningitis occurs in non-HIV patients who are immunodeficient due to diabetes, cancer, solid organ transplants, chemotherapeutic drugs, hematological malignancies etc and rarely in healthy individuals with no obvious predisposing factors. Diagnosis of cryptococcal meningitis is fairly straightforward once the diagnosis is considered in the differential diagnosis of chronic meningitis. Treatment of a patient with cryptococcal infection is a challenge for both the physician and the patient, but rewarding, as many would recover with timely and adequate antifungal therapy.

Pathobiology of fungal infections of the central nervous system with special reference to the Indian scenario.

Ubiquitously present fungi in the environment find a nidus in the human body and adopt its metabolic machinery to be in symbiosis or become pathogenic. Immunocompromised states like human immunodeficiency virus (HIV) / acquired immunodeficiency syndrome (AIDS), systemic neoplasia and organ transplantation have enhanced the frequency of fungal infections. High-risk behavior, IV drug abuse and air travel have led to the emergence of new fungal infections hitherto geographically localized. The pathology in the central nervous system (CNS) is dictated largely by the size of the fungus - the yeast forms, by virtue of their small size enter the microcirculation to cause meningitis and microabscesses, while hyphal forms invade the vasculature to manifest as large pale or hemorrhagic infarcts. The growth kinetics of fungi, the antigenic character of the capsule. the proteases secreted by the mycelial forms and the biochemical milieu in the host also determine clinical manifestations. A hospital-based analysis of the available information from India suggests that in the non-HIV patient population, hyphal forms like Aspergillosis and Zygomycosis are the most common pathogens, while yeast forms like Cryptococcus and Candida are the prime pathogens in cases of HIV/AIDS, the altered macrophage function acting in synergy with suppressed cell-mediated immunity. In Northeastern states, systemic infection by Penicillium marneffei is reported in association with HIV though CNS involvement is not recorded. Although fungal infections of the CNS are reported from various hospitals in India, studies are limited by non-availability of relevant microbiological studies and the reported prevalence data is biased by the surgical practices, availability of postmortem and microbiology and laboratory support. Detailed clinical and mycological investigations related to the interaction between the fungus and host environment is a fertile area of research to understand the basic pathogenetic mechanisms.

Fatal Group A Streptococcal meningitis in an adult.

Despite the recent resurgence in reports of invasive Group A Streptococcal (GAS) infections worldwide, it remains a rare cause of pyogenic meningitis both in children and adults. We report a case of fatal GAS meningitis in a healthy adult emphasizing the need for clinicians to be aware of its fulminant course, prompting early diagnosis and treatment. There is also a need to consider postexposure chemoprophylaxis in close contacts of such cases.

Kindling & mossy fibre sprouting in the rat hippocampus following hot water induced hyperthermic seizures.

BACKGROUND & OBJECTIVES: Hot water epilepsy (HWE) is well recognized reflex epilepsy with possible genetic susceptibility. Rat model and human experimentation had proven that HWE is a type of hyperthermic seizure with possible kindling on repeated stimulation in animals. The present study was undertaken to investigate kindling associated with hyperthermic seizures induced by repeated hot water stimulation in the rat model and to prove hyperthermic kindling. METHODS: Epileptic seizures were induced in 36 male Wistar albino rats by means of hot water sprays at 48 h time intervals. Progression of seizure activity was investigated by studying the behaviour, severity and duration of the seizure. Threshold of rectal temperatures and timed latency for seizure induction were studied. Seizure discharges (EEG) were recorded from ventral hippocampus in six of these rats. Timm's staining was used to study the neuronal sprouting as a consequence of kindling. Studying the seizure threshold, latency, duration of seizure discharge and behavioural seizure following a stimulus-free interval of 30 days tested permanence of kindling. RESULTS: Following 8-12 episodes of hot water stimulations there was progressive epileptic activity manifested in the form of lowering of rectal temperature thresholds from 41.5 to 40.0 degrees C, drop in latency for developing seizures from 185 to 118 sec, increase in duration of hippocampal seizure discharge from 15 to 140 sec, along with progressive increase in complexity of EEG after discharges, increase in behavioural seizure severity from Grade 1 to 5 in all the rats, and neuronal sprouting observed in supragranular molecular layer and in stratum lacunosum. INTERPRETATION & CONCLUSION: Our study covered all aspects of kindling and provided a useful animal model for human hot water epilepsy. Hyperthermic seizures induced by hot water in the rat model kindle as demonstrated by Timm's staining.