cerebrospinal fluid

Cerebrospinal Fluid Offers Clues to Post-COVID Brain Fog

Posted by

0


Summary: Patients who experienced cognitive impairments, or brain fog, following COVID-19 infection had abnormalities in their cerebrospinal fluid. Researchers say the overstimulation of the immune system as a result of COVID may be the cause of cognitive deficits.

Source: UCSF

Some patients who develop new cognitive symptoms after a mild bout of COVID have abnormalities in their cerebrospinal fluid similar to those found in people with other infectious diseases. The finding may provide insights into how SARS-CoV-2 impacts the brain.

In a small study with 32 adults, comprising 22 with cognitive symptoms and 10 control participants without, researchers from UC San Francisco and Weill Cornell Medicine, New York, analyzed the cerebrospinal fluid of 17 of the participants who consented to lumbar puncture. All participants had had COVID but had not required hospitalization. 

They found that 10 of 13 participants with cognitive symptoms had anomalies in their cerebrospinal fluid. But all four of the cerebrospinal samples from participants with no post-COVID cognitive symptoms were normal. The research publishes on Jan. 18, 2022 in Annals of Clinical and Translational Neurology. 

The average age of the participants with cognitive symptoms was 48, versus 39 for the control group. Participants with these symptoms presented with executive functioning issues, said senior author Joanna Hellmuth, MD, MHS, of the UCSF Memory and Aging Center. “They manifest as problems remembering recent events, coming up with names or words, staying focused, and issues with holding onto and manipulating information, as well as slowed processing speed,” she said.   

“Brain fog” is a common after-effect of COVID, affecting some 67 percent of 156 patients at a post-COVID clinic in New York, a study published this month shows. In the current study, patients were enrolled in the Long-term Impact of Infection with Novel Coronavirus (LIINC) study that evaluates recovery in adults with confirmed SARS-CoV-2.

Examinations of the cerebrospinal fluid revealed elevated levels of protein, suggesting inflammation, and the presence of unexpected antibodies found in an activated immune system. Some were found in the blood and cerebrospinal fluid, implying a systemic inflammatory response, or were unique to the cerebrospinal fluid, suggesting brain inflammation. While the targets of these antibodies are unknown, it is possible that these could be “turncoat” antibodies that attack the body itself. 

Immune System Runs Amok Months After COVID

“It’s possible that the immune system, stimulated by the virus, may be functioning in an unintended pathological way,” said Hellmuth, who is principal investigator of the UCSF Coronavirus Neurocognitive Study and is also affiliated with the UCSF Weill Institute for Neurosciences. “This would be the case even though the individuals did not have the virus in their bodies,” she said, noting that the lumbar punctures took place on average 10 months after the participants’ first COVID symptom. 

This shows a person pulling string out of a brain in a cartoon
They found that 10 of 13 participants with cognitive symptoms had anomalies in their cerebrospinal fluid. Image is in the public domain

The researchers also found that the participants with cognitive symptoms had an average of 2.5 cognitive risk-factors, compared with an average of less than one risk factor for participants without the symptoms. These risk-factors included diabetes and hypertension, which can increase the risk of stroke, mild cognitive impairment and vascular dementia; and a history of ADHD, which may make the brain more vulnerable to executive functioning issues. Other risk factors included anxiety, depression, a history of  heavy alcohol or repeated stimulant use, and learning disabilities. 

Testing May Fall Short in Diagnosing Mild Cognitive Disorders

All participants underwent an in-person cognitive testing battery with a neuropsychologist, applying equivalent criteria used for HIV-associated neurocognitive disorder (HAND). Surprisingly, the researchers found that 13 of the 22 participants (59 percent) with cognitive symptoms met HAND criteria, compared with seven of the 10 control participants (70 percent). “Comparing cognitive performance to normative references may not identify true changes, particularly in those with a high pre-COVID baseline, who may have experienced a notable drop but still fall within normal limits,” said Hellmuth.

“If people tell us they have new thinking and memory issues, I think we should believe them rather than require that they meet certain severity criteria.”

Cognitive symptoms have been identified in other viruses, in addition to COVID and HIV. These include the coronaviruses SARS and MERS, hepatitis C and Epstein-Barr virus. 

Co-Authors: First author is Alexandra C. Apple, PhD, of the UCSF Memory and Aging Center and the Weill Institute for Neurosciences. For a complete list of co-authors, please refer to the paper. 

Cerebrospinal Fluid May Be Able to Identify Aggressive Brain Tumors in Children

Posted by

0


--
Ranjan Perera, Ph.D.

It may be possible to identify the presence of an aggressive brain tumor in children by studying their cerebrospinal fluid, according to new research led by Johns Hopkins Kimmel Cancer Center investigators.

Comparing cerebrospinal fluid samples from 40 patients with medulloblastoma — the most common malignant brain tumor in children, accounting for 10% to 15% of pediatric central nervous system tumors — and from 11 healthy children without the disease, investigators identified 110 genes, 10 types of RNA–the machinery that translates proteins–called circular RNAs, 14 lipids and several metabolites that were expressed differently between the two groups. While these details were not specific enough to distinguish among the four subtypes of medulloblastoma, they could be used to identify the presence of cancer versus normal fluid.

A description of the work was published Feb. 24 in the journal Acta Neuropathologica Communications.

“We believe this is the first comprehensive, integrated molecular analysis of the cerebrospinal fluid in medulloblastoma patients,” says senior study author Ranjan Perera, Ph.D., director of the Center for RNA Biology at Johns Hopkins All Children’s Hospital (JHACH) in St. Petersburg, Florida. Perera is also a senior scientist at the JHACH Cancer & Blood Disorders Institute and an associate professor of oncology at the Johns Hopkins University School of Medicine. He has a secondary affiliation with the JHACH Institute for Fundamental Biomedical Research.

“Our study provides proof of principle that all three molecular approaches — studying RNA, lipids and metabolites — can be successfully applied to cerebrospinal fluid samples, not only to differentiate medulloblastoma patients from those without the disease, but also to provide new insights into the pathobiology of the disease,” Perera adds.

“This study provides data for novel biomarkers to detect and track medulloblastoma, which are very much needed to enable improved patient outcomes,” says Chetan Bettegowda, M.D., Ph.D., Jennison and Novak Families Professor of Neurosurgery at Johns Hopkins. “This work also forms the theoretical basis for examining similar biomarkers for other types of brain cancers and other neurological disorders.”

Current diagnosis is based on clinical assessment, imaging and biopsies from tumor tissue. There is an unmet need for diagnostic tests to detect the disease sensitively during the initial presentation and especially during any recurrences, because recurrences are not always seen on magnetic resonance imaging (MRI), Perera says.

Liquid biopsy — the molecular analysis of biofluids — is a minimally invasive method that shows promise for disease detection and monitoring by measuring circulating tumor cells, DNA, RNA or other substances in the urine, cerebrospinal fluid and blood samples. Because cerebrospinal fluid bathes the brain and spinal cord, it was considered a way to provide a window to tumors arising in the central nervous system and disseminating in the fluid, Perera says.

During the study, Perera and colleagues used gene sequencing, metabolic and lipid profiling laboratory techniques to tease out the differences in RNA, metabolites and lipids in cerebrospinal fluid samples from patients with medulloblastoma and healthy controls. Patients with medulloblastoma were found to have a unique RNA metabolic and lipid landscape in their fluid that might be helpful for diagnosis and monitoring, and that reflects biological changes consistent with the presence of medulloblastoma in the central nervous system, Perera says. The metabolite and lipid profiles both contained indicators of tumor hypoxia — a condition in which tumor cells were deprived of oxygen.

More studies in larger patient populations are necessary to confirm the findings, Perera says. The analysis provides several biomarkers that can be studied further.

Infant MR images show autism linked to increased cerebrospinal fluid

Posted by

0


A national research network led by UNC School of Medicine’s Joseph Piven, MD, found that many toddlers diagnosed with autism at two years of age had a substantially greater amount of extra-axial cerebrospinal fluid (CSF) at six and 12 months of age, before diagnosis is possible. They also found that the more CSF at six months – as measured through MRIs – the more severe the autism symptoms were at two years of age.

“The CSF is easy to see on standard MRIs and points to a potential biomarker of autism before symptoms appear years later,” said Piven, co-senior author of the study, the Thomas E. Castelloe Distinguished Professor of Psychiatry, and director of the Carolina Institute for Developmental Disabilities (CIDD). “We also think this finding provides a potential therapeutic target for a subset of people with autism.”

The findings, published in Biological Psychiatry, point to faulty CSF flow as one of the possible causes of autism for a large subset of people.

“We know that CSF is very important for brain health, and our data suggest that in this large subset of kids, the fluid is not flowing properly,” said Mark Shen, PhD, CIDD postdoctoral fellow and first author of the study. “We don’t expect there’s a single mechanism that explains the cause of the condition for every child. But we think improper CSF flow could be one important mechanism.”

Until the last decade, the scientific and medical communities viewed CSF as merely a protective layer of fluid between the brain and skull, not necessarily important for proper brain development and behavioral health. But scientists then discovered that CSF acted as a crucial filtration system for byproducts of brain metabolism.

Every day, brain cells communicate with each other. These communications cause brain cells to continuously secrete byproducts, such as inflammatory proteins that must be filtered out several times a day. The CSF handles this, and then it is replenished with fresh CSF four times a day in babies and adults.

In 2013, Shen co-led a study of CSF in infants at UC Davis, where he worked with David Amaral, PhD, co-senior author of the current Biological Psychiatry study. Using MRIs, they found substantially greater volumes of CSF in babies that went on to develop autism. But they cautioned the study was small – it included 55 babies, 10 of whom developed autism later – and so it needed to be replicated in a larger study of infants.

When he came to UNC, Shen teamed up with Piven and colleagues of the Infant Brain Imaging Study (IBIS), a network of autism clinical assessment sites at UNC, the University of Pennsylvania, Washington University in St. Louis, and the University of Washington.

In this most recent study of CSF, the researchers enrolled 343 infants, 221 of which were at high risk of developing autism due to having an older sibling with the condition. Forty-seven of these infants were diagnosed with autism at 24 months, and their infant brain MRIs were compared to MRIs of other infants who were not diagnosed with autism at 24 months of age.

The six-month olds who went on to develop autism had 18 percent more CSF than six-month olds who did not develop autism. The amount of CSF remained elevated at 12 and 24 months. Infants who developed the most severe autism symptoms had an even greater amount of CSF – 24 percent greater at six months.

Also, the greater amounts of CSF at six months were associated with poorer gross motor skills, such as head and limb control.

“Normally, autism is diagnosed when the child is two or three years old and beginning to show behavioral symptoms; there are currently no early biological markers,” said David G. Amaral, director of research at the UC Davis MIND Institute. “That there’s an alteration in the distribution of cerebrospinal fluid that we can see on MRIs as early as six months, is a major finding.”

The researchers found that increased CSF predicted with nearly 70 percent accuracy which babies would later be diagnosed with autism. It is not a perfect predictor of autism, but the CSF differences are observable on a standard MRI. “In the future, this sort of CSF imaging could be another tool to help pediatricians detect risks for autism as early as possible,” Shen said.

Piven added, “We can’t yet say for certain that improper CSF flow causes autism. But extra-axial CSF is an early marker, a sign that CSF is not filtering and draining as it should. This is important because improper CSF flow may have downstream effects on the developing brain; it could play a role in the emergence of autism symptoms.”

Cerebrospinal Fluid Test Given OK from FDA

Posted by

0


The first cerebrospinal fluid (CSF) nucleic acid-based test that can detect pathogens affecting the central nervous system may now be marketed after getting a green light from the FDA.

The FilmArray Meningitis/Encephalitis (ME) Panel tests CSF for various pathogens — like bacteria, viruses, or yeast — in patients at risk of meningitis or encephalitis. According to the FDA, which made the announcement yesterday on its website, FilmArray is designed to be used as a diagnostic tool in conjunction with other clinical and laboratory tools.

The test claims to be able to identify 16 targets with 200 µl of CSF and with only 2 minutes of hands-on time. Within an hour, it can produce results, “which may enable clinicians to make informed treatment decisions earlier” for a population that needs to be treated quickly, according to the FDA.

“Testing one sample for many pathogens and potentially having test results sooner should allow physicians to use this information, along with other clinical findings and test results, to provide improved diagnosis and treatment for these very serious illnesses,” said Alberto Gutierrez, who works in diagnostics at the Center for Devices and Radiological Health at the FDA.

According to the drug agency, the bacterial and yeast pathogens that are identifiable by the test include Escherichia coli K1, Haemophilus influenzae, Listeria monocytogenes, Neisseria meningitidis, Streptococcus agalactiae, Streptococcus pneumoniae, and Cryptococcus neoformans/gattii. And identifiable viruses include Cytomegalovirus, Enterovirus, Herpes simplex virus 1, Herpes simplex virus 2, Human herpesvirus 6, Human parechovirus, and Varicella zoster virus.

But the FDA added that the FilmArray Panel is limited in that it doesn’t detect all the causes of an infection, and it doesn’t note which antimicrobial drugs should be used to treat the infection.

“Physicians should continue to perform standard CSF bacterial and fungal cultures in conjunction with the FilmArray ME Panel because false negative and false positive results are possible with the FilmArray ME Panel, and bacterial growth is needed for drug susceptibility testing when results are positive,” they added. “False negative results could potentially occur when the concentration of organisms in the CSF specimen is below the limit of detection for the FilmArray ME Panel.”

The permission to market was granted by the de novo classification process, which the FDA uses for devices that have a low to medium risk and are different from any other products on the market. The drug agency analyzed data from a trial in which CSF samples were taken from more than 1,500 patients and the FilmArray ME Panel was used in conjunction with other methods. In addition, two separate studies looked at clinical CSF samples that had already been determined to contain microorganisms or bacteria and viruses.

“Study results demonstrated high agreement between the FilmArray ME Panel, comparator methods and expected results,” wrote the FDA.

FilmArray also has a respiratory panel that can be used to detect bacteria.

The FilmArray ME Panel is manufactured by BioFire Diagnostics, in Salt Lake City, Utah.

Brain Clears Toxins During Sleep.

Posted by

1


Scientists have long wondered why sleep is restorative and why lack of sleep impairs brain function.

Now, new animal research suggests how the sleep state may help clear the body of potentially toxic central nervous system (CNS) metabolites.

Proteins linked to neurodegenerative diseases, including β-amyloid (Aβ), are present in the interstitial space surrounding cells in the brain. In a series of experiments, researchers tested the hypothesis that Aβ clearance is increased during sleep and that the sleep-wake cycle regulates the glial cell–dependent glymphatic system, which is responsible for clearing waste from the brain and spinal cord.

“Basically, we found a new function of sleep,” said study lead author Lulu Xie, PhD, Division of Glial Disease and Therapeutics, Center for Translational Neuromedicine, Department of Neurosurgery, University of Rochester Medical Center, New York.

“When mice are awake, the brain cells continuously produce toxic waste. This waste can build up in the spaces between the brain cells and damage them. However, during sleep, the spaces between brain cells increase, which may help the brain flush out the toxic waste. Therefore, a good sleep can clear the brain.”

“Sleep changes the cellular structure of the brain. It appears to be a completely different state,” Maiken Nedergaard, MD, DMSc, codirector of the Center for Translational Neuromedicine at the University of Rochester Medical Center, who is a leader of the study, said in a statement from the National Institute of Neurological Disorders and Stroke, which supported the study.

The new research was published October 18 in Science.

Sleeping vs Awake Brain

The researchers infused fluorescent dye into the cerebrospinal fluid (CSF) of mice and observed it flow through the brain. At the same time, they monitored electrical brain activity and wakefulness with electrocorticography (EcoG) and electromyography (EMG)..

“In the sleeping brain, we found the CSF flushed into the brain very quickly and broadly,” said Dr. Xie. “After half an hour, we woke the mice up by gently touching their tails, and injected another color of dye. But what we saw is that CSF barely flowed when the same mice were awake.”

These results suggest that the awake brain may have more resistance to CSF influx, which leads to the assumption that the path of CSF flow into the brain is smaller in the awake brain, said Dr. Xie.

Next, the scientists inserted electrodes into the brain to directly measure the space between brain cells, and found that it increased by around 60% when the mice were asleep.

“Theoretically, big spaces lead to easier fluid influx,” said Dr. Xie. “So we presumed that the clearance of the toxic protein between cells will become more efficient.”

To test this assumption, they infused radio-labeled Aβ into the brain and measured how long it stayed in both the sleeping brain and the awake brain.

We found Aβ disappeared 2-fold faster in the sleeping mice brains as compared with awake mice,” noted Dr. Xie. “Based on this experiment, we can see that the sleeping brain is more capable of clearing out the toxic protein.”

Technically, it might be relatively easy to study these processes in humans, possibly using magnetic resonance imaging. However, Dr. Xie said she does not know when human trials, which involve “a lot more concerns” than animal experiments, might come about.

“These results may have broad implications for multiple neurological disorders,” said Jim Koenig, PhD, a program director at the National Institute of Neurological Disorders and Stroke (NINDS), which funded the study, in a statement. “This means the cells regulating the glymphatic system may be new targets for treating a range of disorders.”

Antibody Index Test May Aid PML Diagnosis on Natalizumab.

Posted by

0


A new test that compares JC virus (JCV) antibody levels in cerebrospinal fluid (CSF) with those in serum might be a useful complementary tool in the diagnostic workup for progressive multifocal leukoencephalopathy (PML) in patients with multiple sclerosis (MS) treated with natalizumab (Tysabri, Biogen Idec), a new study suggests.

The study was presented by Clemens Warnke, MD, Heinrich Heine University, Düsseldorf, Germany, at the recent 29th Congress of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS). 

Dr. Warnke explained that PML caused by JCV can be difficult to diagnose because early symptoms can be mistaken for a relapse in MS.

“At present, JCV-DNA detection by polymerase chain reaction (PCR) in cerebrospinal fluid is used for diagnosis of PML. However, false-negatives often occur, leading to delayed diagnosis and poor patient outcome in some cases,” he said. “So we need more tests to allow earlier diagnosis.”

“Our main findings are when you have clinical suspicion of PML — changes in behavior, personality, and motor function untypical for MS in patients treated with natalizumab, we might suspect it to be PML,” he told Medscape Medical News.

He and his colleagues have proposed using the anti-JCV antibody specificity index (ASI-JCV) as an additional test for JCV DNA detection. This involves measuring the JCV antibody level in both serum and CSF and calculating the proportion of antibodies in CSF compared with serum.

In this study, Dr. Warnke and colleagues calculated the anti-JCV antibody specificity index in 25 patients who had developed PML while receiving natalizumab and in 47 patients also taking natalizumab who had not developed PML. Results showed that none of the control patients had JCV antibodies in CSF, whereas CSF antibodies were detected in more than 60% of the patients with PML.

And while all patients with natalizumab-associated PML exhibited an ASI-JCV of 0.47 or higher, this was seen in none of the 47 patients with MS treated with natalizumab who did not develop PML (P < .0001).

Dr. Warnke stressed that this test would not replace the PCR test for JCV DNA. “But levels of JCV DNA detection and the antibody index tests do not exactly correlate, so here is an argument for using the antibody index alongside the DNA test for additional diagnostic value,” he suggested.

“If we assume an estimate of 70% to 80% for the early diagnosis of PML using PCR, maybe we can increase this up to 85% using this antibody index test as well,” he added.

The antibody index test is not yet commercially available.

“We have developed our own test and now use it at our hospital,” he noted. “But it is not an established procedure yet. It is still a clinical research tool which needs further validation.”

 

Diagnostic Accuracy of Quantitative PCR (Xpert MTB/RIF) for Tuberculous Meningitis in a High Burden Setting: A Prospective Study.

Posted by

0


Background

Tuberculous meningitis (TBM) is difficult to diagnose promptly. The utility of the Xpert MTB/RIF test for the diagnosis of TBM remains unclear, and the effect of host- and sample-related factors on test performance is unknown. This study sought to evaluate the sensitivity and specificity of Xpert MTB/RIF for the diagnosis of TBM.

Methods and Findings

235 South-African patients with a meningeal-like illness were categorised as having definite (culture or Amplicor PCR positive), probable (anti-TBM treatment initiated but microbiological confirmation lacking), or non-TBM. Xpert MTB/RIF accuracy was evaluated using 1 ml of uncentrifuged and, when available, 3 ml of centrifuged cerebrospinal fluid (CSF). To evaluate the incremental value of MTB/RIF over a clinically based diagnosis, test accuracy was compared to a clinical score (CS) derived using basic clinical and laboratory information.

Of 204 evaluable patients (of whom 87% were HIV-infected), 59 had definite TBM, 64 probable TBM, and 81 non-TBM. Overall sensitivity and specificity (95% CI) were 62% (48%–75%) and 95% (87%–99%), respectively. The sensitivity of Xpert MTB/RIF was significantly better than that of smear microscopy (62% versus 12%; p = 0.001) and significantly better than that of the CS (62% versus 30%; p = 0.001; C statistic 85% [79%–92%]). Xpert MTB/RIF sensitivity was higher when centrifuged versus uncentrifuged samples were used (82% [62%–94%] versus 47% [31%–61%]; p = 0.004). The combination of CS and Xpert MTB/RIF (Xpert MTB/RIF performed if CS<8) performed as well as Xpert MTB/RIF alone but with a ~10% reduction in test usage. This overall pattern of results remained unchanged when the definite and probable TBM groups were combined. Xpert MTB/RIF was not useful in identifying TBM among HIV-uninfected individuals, although the sample was small. There was no evidence of PCR inhibition, and the limit of detection was ~80 colony forming units per millilitre. Study limitations included a predominantly HIV-infected cohort and the limited number of culture-positive CSF samples.

Conclusions

Xpert MTB/RIF may be a good rule-in test for the diagnosis of TBM in HIV-infected individuals from a tuberculosis-endemic setting, particularly when a centrifuged CSF pellet is used. Further studies are required to confirm these findings in different settings.

Discussion

Although the utility profile and accuracy of Xpert MTB/RIF has been well characterised in sputum samples, there are hardly any data to guide its utility and implementation for TBM. This is critical as the rollout of Xpert MTB/RIF means that quantitative PCR is now available in many high burden settings, and data are urgently required to guide appropriate and relevant usage of this technology in biological fluids other than sputum. That Xpert MTB/RIF performs poorly in fluids from some compartments, e.g., the pleural space, highlights the need for such data [27]. The key findings of this study were as follows: (1) Xpert MTB/RIF is likely a good rule-in test for the diagnosis of TBM in HIV-infected patients; (2) centrifugation of the sample improved sensitivity in this context to almost 80%; (3) among HIV-infected patients, Xpert MTB/RIF performed significantly better than the widely available same-day alternative tests, i.e., smear microscopy, which suggests that prompt diagnosis of TBM is potentially achievable in the majority of patients in this setting; (4) the diagnostic value of Xpert MTB/RIF for HIV-infected patients is clinically meaningful given that it performed significantly better than hypothetical decision-making based on clinical characteristics and basic laboratory data (the CS); and (5) when combined with the CS, Xpert MTB/RIF test usage could be reduced by only a modest ~10% whilst retaining similar sensitivity and specificity compared to using Xpert MTB/RIF alone. This last finding informs clinical practice in resource-poor settings. Finally, we quantified the limit of detection of the assay, its relationship to bacterial load, and the impact of PCR inhibition. These data require reproduction in HIV-uninfected and non-TB-endemic populations.

There are limited data about Xpert MTB/RIF performance in TBM [28]. Published data include only small numbers of microbiologically proven TBM cases (range of 0 to 23) [29]–[32], often in a case-control design with a non-uniform reference standard, and often CSF-associated data were published as part of a laboratory-based evaluation of extrapulmonary TB samples, usually including samples from countries with low TB prevalence. Furthermore, there are no studies from high burden settings, and technical performance evaluations, including bacterial load studies, threshold level of detection, and impact of PCR inhibition, have hitherto not been undertaken.

Xpert MTB/RIF sensitivity was as high as 80% when a centrifuged CSF sample from an HIV-infected patient was used. This suggests that Xpert MTB/RIF, at least in an HIV-endemic environment, represents a possible new standard of care for the diagnosis of TBM. Sensitivity was considerably better than in previous studies using commercially available or non-standardised PCR tools [9],[32]–[34]. The ostensibly better performance is likely related to a combination of centrifugation (and hence concentration of bacilli) and technical aspects, including a more efficient standardised extraction protocol, fractionation of mycobacteria by a pre-sonication step, and a nested PCR protocol, thus maximising amplification. However, possibly higher bacterial loads in HIV-infected patients may have also played a role. Our findings have practical relevance because they imply that at least 3 ml of CSF should be set aside and centrifuged, and re-suspended in phosphate-buffered saline, before being run on the Xpert MTB/RIF. This high-sensitivity and potentially rapid diagnosis in most cases is likely to benefit HIV-infected patients suspected of having M.tb., as diagnostic and treatment delay is associated with higher mortality [35]–[37]. Impact-related studies are now required to verify this hypothesis. It is noteworthy that a second sample improved sensitivity minimally. These data suggest that, at least in an HIV-endemic setting, using a second cartridge is unlikely to give further benefit. However, larger studies are required to confirm this possibility.

Similar to the findings when using sputum, the level of detection of Xpert MTB/RIF was between 80 and 100 colony forming units per millilitre. This explains the sub-optimal sensitivity of Xpert MTB/RIF compared to culture, where the detection threshold is as low as 1–10 organisms per millilitre [38]. We did not find a correlation between TTP and Xpert MTB/RIF CT values, as has been shown in sputum [39]. In contrast to previous PCR-based studies [40],[41], we found that CSF had a minimal inhibitory effect on the PCR reaction when compared to sputum. This may be due to the wash step incorporated into the assay that removes extracellular debris. We did not find a difference in TTP between the Xpert MTB/RIF–positive samples from centrifuged versus uncentrifuged CSF. This may be due to a type two statistical error, as the sample numbers were small.

There were three patients who were culture negative but Xpert MTB/RIF positive, i.e., Xpert MTB/RIF positive in the non-TB group. Our previous work has shown that such cases (Xpert MTB/RIF positive but culture negative) are likely to be true TB positives, and this is corroborated by high specificity obtained in large sputum-based studies where a significant minority of the patients had had previous TB [11]. If these culture-negative Xpert MTB/RIF–positive individuals are hypothetically designated definite-TB cases, then the overall case detection rate improves by a further ~10%.

The proper and meaningful value of a test lies in its ability to influence patient management through its incremental value over pre-test probability, or to have an impact on decision-making based on logical clinical judgement (based upon clinical features and basic laboratory parameters). We therefore derived a CS, hitherto unavailable for HIV-endemic settings, to evaluate Xpert MTB/RIF utility in clinical practice. Xpert MTB/RIF had significantly better performance outcomes than the clinical prediction rule (using a rule-in cut point, so appropriate comparisons could be made). Furthermore, hypothetically combining the CS with Xpert MTB/RIF resulted only in a modest ~10% reduction in test usage, but still maintained high sensitivity and specificity. These data suggest that clinical algorithms or scoring systems to limit test usage are unlikely to be significantly useful in resource-poor settings.

There are several limitations of our study. We could not determine the impact of Xpert MTB/RIF (time and proportion of patients initiated on treatment) compared to a smear microscopy/empiric treatment-based strategy given our study design and the fact that management decisions were not based on Xpert MTB/RIF results. However, this was because Xpert MTB/RIF had not yet been endorsed by the World Health Organization when the study commenced, had not been validated for use in CSF, and had been used as a research tool only (thus, for ethical reasons, study samples were evaluated only several weeks later). Although the confidence intervals of some of our estimates are wide (because of limited sample numbers), this is to our knowledge the largest diagnostic study undertaken in TBM (based on the number of microbiologically proven TBM cases; n = 59). This reflects the challenge and difficulty in performing such studies in resource-poor settings. It is possible that the Xpert MTB/RIF performs much better in HIV-infected individuals because of a possibly higher bacterial load, and thus our findings need to be confirmed in other settings. Given the small number of HIV-uninfected patients, we were unable to meaningfully compare this sub-group. The CS was developed to assess only incremental value above basic clinical and CSF parameters. The CS and the combination of CS plus Xpert MTB/RIF need prospective and independent validation. The non-significant difference in sensitivity between the paired centrifuged and non-centrifuged samples may reflect a type two statistical error, as the number of culture-positive paired samples was limited. Lastly, there were nine patients who could not be categorised within our defined groups and were excluded from the analysis.

In conclusion, Xpert MTB/RIF may be a good rule-in test for the diagnosis of TBM in HIV-infected individuals in a TB-endemic setting, particularly when a centrifuged CSF pellet is used. A second Xpert MTB/RIF test had minimal incremental benefit. Smear microscopy and the CS, when combined with Xpert MTB/RIF, only modestly minimised test usage in a resource-poor setting. Further studies are now required in non-HIV-endemic settings, and using validated scoring systems, to evaluate the impact of Xpert MTB/RIF on diagnostic accuracy, and morbidity and mortality in patients with TBM.

Source:PLOS

Utility of the Xpert MTB/RIF Assay for Diagnosis of Tuberculous Meningitis.

Posted by

0


Tuberculous meningitis (TBM) is characterized by copious cerebrospinal fluid (CSF) inflammation and yet few Mycobacterium tuberculosis. This combination creates a disease that is notoriously difficult to definitively diagnose. In this week’s issue of PLOS Medicine, Patel and colleagues report the diagnostic performance of the GeneXpert system’s Xpert MTB/RIF assay for the diagnosis of TBM in a cohort of 204 South African, predominantly HIV-infected, adults presenting with suspected meningitis of whom 59 had definitive TBM [1]. The Xpert MTB/RIF assay’s overall sensitivity was 62%, and specificity was 95%. The performance was better using larger volumes of centrifuged CSF among HIV-infected persons with sensitivity of approximately 80% and excellent specificity for microbiologically confirmed TBM. Xpert MTB/RIF performance was less impressive using uncentrifuged CSF with a sensitivity of ≤50%, and Xpert MTB/RIF performance was negligible in HIV-uninfected persons.

What Is GeneXpert?

The GeneXpert System (Cepheid) is a single use cartridge-based real-time PCR fully automated system that performs sample decontamination, sonication, automated nucleic acid amplification, and fluorescence-based quantitative PCR [2]–[4]. The Xpert MTB/RIF assay, developed by David Alland, detects M. tuberculosis DNA in approximately 2 hours with minimal hands-on time[3]. This new technology was endorsed by the World Health Organization in December 2010, and as of June 30, 2013, a total of 1,402 GeneXpert instruments and over 3 million Xpert MTB/RIF cartridges have been procured in the public sector in 88 countries [5]. The concessional pricing is US$9.98 per cartridge for 145 low- and middle-income countries [6]. The same GeneXpert platform also can be used for a variety of US Food and Drug Administration (FDA)-approved testing (e.g., influenza, Clostridium difficile, methicillin-resistant Staphylococcus aureus).

What Is the Performance of Xpert MTB/RIF?

There is a rapidly emerging literature regarding the performance of the Xpert MTB/RIF assay. Fundamentally, the sensitivity depends on the burden of organisms and thereby the target DNA present in the specimen. The published Xpert MTB/RIF detection threshold is approximately 100–130 colony forming units (cfu)/ml of sample [2],[3]. Patel and colleagues observed a similar threshold of 80–100 cfu/ml of CSF in this study [1]. In comparison, the detection threshold is <10 cfu/ml for mycobacterial liquid culture and is >5,000 cfu/ml for Ziehl-Neelsen staining for acid fast bacilli (AFB) via standard microscopy in sputum [7]–[9]. In real world clinical terms, this means 98%–99% detection by Xpert MTB/RIF of AFB smear-positive pulmonary TB, and approximately 75% detection of smear-negative, culture-positive pulmonary TB [3],[10],[11].

The threshold of detection is a key principle. The Xpert MTB/RIF test performs better when there is a larger burden of infectious organisms present in the specimen being tested. Yet, TB meningitis is a paucibacillary condition with few organisms. More organisms are likely present when the host is immunocompromised, or when a larger input volume is used for the test. Thus specimen centrifugation can compensate and should improve diagnostic yield, as demonstrated by a 35% improvement in sensitivity in this study [1].

The prior data on Xpert MTB/RIF testing of CSF are limited. In India, the Xpert MTB/RIF assay detected two of seven culture-positive specimens using an input volume of ~1 ml [12]. In an Italian study, 11 of 13 TBM patients were detected by Xpert MTB/RIF using an input volume of 2 ml into the cartridge without the standard N-acetyl-L-cysteine-sodium hydroxide (1% NALC-NaOH) decontamination and mucolytic step (i.e., GeneXpert Sample Reagent) [13]. This sample reagent was designed for sputum samples. Numerous commercially available PCR assays exist for other pathogens (e.g., herpes simplex PCR) without such a decontamination step [13], and the necessity of using the sample reagent for non-bloody CSF is unclear.

Public Health Significance

Although two commercial TB PCR tests previously existed [14], the innovation is that the GeneXpert platform is fully automated and is being rolled out in low- and middle-income countries. Thus, GeneXpert is an actual technology that can be—and is being—widely used globally. However, immediate implementation of a US$10 Xpert MTB/RIF assay for all cases of meningitis is unwise and unsustainable. Further research is needed on how best to incorporate the Xpert MTB/RIF test into diagnostic testing for meningitis, to ensure that it is a cost-effective intervention that improves health and does not waste resources.

Patel and colleagues modeled a clinical score to predict who had such high pretest probability of TBM that Xpert MTB/RIF was unnecessary to perform [1]. Yet health systems also need the opposite, a clinical score or algorithm to identify who has such low pretest probability that they do not require testing. Several investigators have developed meningitis diagnostic algorithms, yet broader validation is needed [15]–[17]. Ordering comprehensive testing of all the available diagnostic tests for every patient with suspected meningitis, including Xpert MTB/RIF testing, is 3–4-fold more expensive, without any better diagnostic yield than a targeted stepwise approach[15].

Key Principles of TB Meningitis Diagnosis

Despite molecular diagnostics, there remain a number of key pieces of information that inform clinicians as to the likelihood of a TBM diagnosis, so as to target testing in a cost-effective manner. The first is history. TBM is a subacute illness. Symptoms <6 days are atypical for TBM, yet near universal for bacterial meningitis [16]. Second is the immunology of the patient. Immunosuppression due to HIV/AIDS or age (e.g., infants, elderly) are key drivers of TBM, and immunosuppression increases the bacillary burden of M. tuberculosis organisms, likely increasing the diagnostic yield of molecular testing. In the current study, the GeneXpert performed poorly among HIV-uninfected persons, and the performance in children is unknown. Third is the CSF profile. TBM is classically a lymphocytic meningitis (i.e., >30%–50% lymphocytes in >90% of persons [16],[18]); with a low CSF glucose of <60% of serum glucose or an absolute CSF glucose concentration <2·2 mmol/l (<40 mg/dl) in >92%–95% [18]–[20].

In HIV-infected adults, the clinical history and CSF profile overlap extensively with meningitis due to Cryptococcus neoformans, and cryptococcal meningitis is overall the most common meningitis etiology in adults in sub-Saharan Africa [15]. Thus before a US$10 Xpert MTB/RIF test is performed for a less common condition, a US$2 cryptococcal antigen lateral flow assay should likely be performed for a more frequent condition [15].

If there is insufficient CSF volume available for testing (i.e., ❤ ml), in a clinically stable patient treated presumptively for bacterial meningitis a repeated lumbar puncture in 48 hours is likely a better strategy than sub-optimal Xpert MTB/RIF testing using a limited volume. A repeat lumbar puncture can collect a sufficiently large volume as well as reassess CSF glucose. At 48 hours, the CSF glucose should have risen by >100% of the initial level in treated bacterial meningitis[16]. Persistently low CSF glucose levels at 48 hours coupled with excluding cryptococcal meningitis should prompt Xpert MTB/RIF testing and/or empiric anti-TB therapy [16].

Xpert MTB/RIF appears to be a highly useful test to “rule in” the diagnosis of TBM, yet the clinical acumen of physicians remains a necessity for the wise use of any new diagnostic test. Careful application of these new diagnostic tools should improve clinicians’ ability to deliver timely, cost-effective care to patients with suspected TBM throughout the world, an approach that future studies should systematically evaluate.

Source:PLOS

To Sleep, Perchance to Clean.

Posted by

0


Study reveals brain ‘takes out the trash’ while we sleep.

In findings that give fresh meaning to the old adage that a good night’s sleep clears the mind, a new study shows that a recently discovered system that flushes waste from the brain is primarily active during sleep. This revelation could transform scientists’ understanding of the biological purpose of sleep and point to new ways to treat neurological disorders.

“This study shows that the brain has different functional states when asleep and when awake,” said Maiken Nedergaard, M.D., D.M.Sc., co-director of the University of Rochester Medical Center (URMC) Center for Translational Neuromedicine and lead author of the article. “In fact, the restorative nature of sleep appears to be the result of the active clearance of the by-products of neural activity that accumulate during wakefulness.”

The study, which was published today in the journal Science, reveals that the brain’s unique method of waste removal – dubbed the glymphatic system – is highly active during sleep, clearing away toxins responsible for Alzheimer’s disease and other neurological disorders. Furthermore, the researchers found that during sleep the brain’s cells reduce in size, allowing waste to be removed more effectively.

Image shows cerebral spinal fluid (in blue) entering the brain via a “plumbing system” that piggybacks on the brain’s blood vessels.

The purpose of sleep is a question that has captivated both philosophers and scientists since the time of the ancient Greeks. When considered from a practical standpoint, sleep is a puzzling biological state. Practically every species of animal from the fruit fly to the right whale is known to sleep in some fashion. But this period of dormancy has significant drawbacks, particularly when predators lurk about. This has led to the observation that if sleep does not perform a vital biological function then it is perhaps one of evolution’s biggest mistakes.

While recent findings have shown that sleep can help store and consolidate memories, these benefits do not appear to outweigh the accompanying vulnerability, leading scientists to speculate that there must be a more essential function to the sleep-wake cycle.

The new findings hinge on the discovery last year by Nedergaard and her colleagues of a previously unknown system of waste removal that is unique to the brain. The system responsible for disposing cellular waste in the rest of the body, the lymphatic system, does not extend to the brain. This is because the brain maintains its own closed “ecosystem” and is protected by a complex system molecular gateways – called the blood-brain barrier – that tightly control what enters and exits the brain.

The brain’s process of clearing waste had long eluded scientists for the simple fact that it could only be observed in the living brain, something that was not possible before the advent of new imaging technologies, namely two-photon microscopy. Using these techniques, researchers were able to observe in mice – whose brains are remarkably similar to humans – what amounts to a plumbing system that piggybacks on the brain’s blood vessels and pumps cerebral spinal fluid (CSF) through the brain’s tissue, flushing waste back into the circulatory system where it eventually makes its way to the general blood circulation system and, ultimately, the liver.

The timely removal of waste from the brain is essential where the unchecked accumulation of toxic proteins such as amyloid-beta can lead to Alzheimer’s disease. In fact, almost every neurodegenerative disease is associated with the accumulation of cellular waste products.

One of the clues hinting that the glymphatic system may be more active during sleep was the fact that the amount of energy consumed by the brain does not decrease dramatically while we sleep. Because pumping CSF demands a great deal of energy, researchers speculated that the process of cleaning may not be compatible with the functions the brain must perform when we are awake and actively processing information.

Through a series of experiments in mice, the researchers observed that the glymphatic system was almost 10-fold more active during sleep and that the sleeping brain removed significantly more amyloid-beta.

“The brain only has limited energy at its disposal and it appears that it must choice between two different functional states – awake and aware or asleep and cleaning up,” said Nedergaard. “You can think of it like having a house party. You can either entertain the guests or clean up the house, but you can’t really do both at the same time.”

Another startling finding was that the cells in the brain “shrink” by 60 percent during sleep. This contraction creates more space between the cells and allows CSF to wash more freely through the brain tissue. In contrast, when awake the brain’s cells are closer together, restricting the flow of CSF.

The researchers observed that a hormone called noradrenaline is less active in sleep. This neurotransmitter is known to be released in bursts when brain needs to become alert, typically in response to fear or other external stimulus. The researchers speculate that noradrenaline may serve as a “master regulator” controlling the contraction and expansion of the brain’s cells during sleep-wake cycles.

“These findings have significant implications for treating ‘dirty brain’ disease like Alzheimer’s,” said Nedergaard. “Understanding precisely how and when the brain activates the glymphatic system and clears waste is a critical first step in efforts to potentially modulate this system and make it work more efficiently.”

Functional Brain Imaging May Spot Alzheimer’s Early.

Posted by

0


Two hallmark cerebrospinal fluid (CSF) biomarkers of early Alzheimer’s disease (AD) correlate with decay in the default mode network (DMN) seen on resting-state functional connectivityMRI (rs-fcMRI), a new study suggests.

Beau M. Ances, MD, PhD, and colleagues from the Knight Alzheimer’s Disease Research Center at Washington University, St. Louis, Missouri, say rs-fcMRI may be an effective noninvasive way to detect early asymptomatic AD.

Earlier research by Dr. Ances and others has shown that AD disrupts connections in the DMN and other brain networks, asreported at that time by Medscape Medical News. “Our study is one the first that links resting-state functional connectivity and CSF biomarkers,” Dr. Ances told Medscape Medical News.

“We are excited about this work,” Dr. Ances said. “We hope in the future that resting-state functional connectivity markers will be added to our arsenal for evaluation of early AD. While these results are only cross-sectional in nature they help place changes in resting-state functional connectivity in context with other measures.”

Still, he added, “we do not advocate for using this technique yet at the individual patient level, but our work creates a stepping stone for helping justify the use of this method. We think that resting-state functional connectivity will provide additional spatial information for areas involved in early AD.”

The study was published online August 19 in JAMA Neurology.

Best Chance of Success

The ability to diagnose AD early is crucial as many researchers now believe that treating patients before symptoms of dementia appear offers the best chances of success.

Patients with symptomatic AD typically have reduced amyloid β42 (Aβ42) and increased phosphorylated tau181 (ptau) in CSF. Cognitively healthy individuals can also exhibit these CSF biomarkers of AD pathology.

Emerging evidence, Dr. Ances and colleagues note, suggests that functional scans of brain networks involved in AD has “great potential” for characterizing pathophysiologic changes during the preclinical phase of AD.

They note that rs-fcMRI abnormalities have been “consistently” observed in the DMN in patients with symptomatic AD. More recent rs-fcMRI investigations have detected DMN changes in asymptomatic individuals.

For their latest study, Dr. Ances and colleagues examined changes in the DMN in relation to CSF Aβ42 and ptau in a cross-sectional longitudinal cohort of 207 older adults with normal cognition (Clinical Dementia Rating, 0).

They found that decreased CSF Aβ42 and increased CSF ptau were each independently associated with reduced DMN integrity, with the most prominent decreases in functional connectivity observed in the posterior cingulate cortex (PCC) and medial temporal lobe (MTL).

These are “2 key hubs that are often affected by AD” and are associated with memory, Dr. Ances said. Memory impairment in the early phases of AD may be attributable to the “convergent effects of both amyloid and tau pathology,” the researchers note in their paper.

They also note that the effects of CSF Aβ42 and ptau on DMN functional connectivity were not due to age or structural atrophy in the PCC and MTL.

Caveats “Substantial”

In an accompanying editorial in JAMA Neurology, William Jagust, MD, from the Helen Wills Neuroscience Institute and School of Public Health at University of California, Berkeley, agrees that this technique has potential but that more study is needed.

“From a clinical perspective,” he writes, “the results suggest that a relatively simple magnetic resonance imaging measure might be a reasonable biomarker for early AD. The caveats, however, are substantial.”

For example, he notes that “technical factors in assessing resting state networks are crucial: small amounts of head motion can produce artifacts that are difficult to detect, there are multiple approaches to data analysis, and reliability over time and across centers has not been extensively established.”

Dr. Jagust also says validating this approach as a biomarker requires much stronger links to disease phenotypes that include the progression to AD dementia. “Nevertheless, the appeal of this technique is that it can be performed on available clinical instruments, requires no particular cognitive task, and can be obtained in a few minutes,” Dr. Jagust writes.

Targeting the PCC and the MTL “could be a simple and widely applicable data-analytic approach to track early disease if the difficult technical issues, predictive value, sensitivity, and specificity can be worked out,” he concludes.

Source: medscape.com