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Lipkin paper investigating previously undetectable pathogens in central nervous system infections

Bob

Senior Member
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England (south coast)
Use of Staged Molecular Analysis to Determine Causes of Unexplained Central Nervous System Infections
Chien-Chin Hsu, Rafal Tokarz, Thomas Briese, Hung-Chin Tsai, Phenix-Lan Quan, and W. Ian Lipkin
September 2013
Emerg Infect Dis
DOI: 10.3201/eid1909.130474
http://wwwnc.cdc.gov/eid/article/19/9/13-0474_article.htm


This seems to be a collaboration between the CDC, Lipin and colleagues of Lipkin.

About lead author, Chien-Chin Hsu, the paper says:
> Dr Hsu is a physician-scientist at Chi-Mei Medical Center, Tainan, Taiwan. A recent graduate of the doctoral program in epidemiology at the Mailman School of Public Health, Columbia University, he is dedicated to advancing infection control in Taiwan through the introduction of molecular diagnostics.

I thought perhaps it might give some clues to some of the technology that Likpin & Hornig are using in the CFS study.


They Investigated cerebrospinal fluid samples from patients with central nervous system (CNS) infections of unknown cause in 1 hospital in Taiwan:

"We determined the infectious agent for 31 (24%) of 131 previously negative samples."


Abstract
No agent is implicated in most central nervous system (CNS) infections. To investigate cerebrospinal fluid samples from patients with CNS infections of unknown cause in 1 hospital in Taiwan, we used a staged molecular approach, incorporating techniques including multiplex MassTag PCR, 16S rRNA PCR, DNA microarray, and high-throughput pyrosequencing. We determined the infectious agent for 31 (24%) of 131 previously negative samples. Candidate pathogens were identified for 25 (27%) of 94 unexplained meningitis cases and 6 (16%) of 37 unexplained encephalitis cases. Epstein-Barr virus (18 infections) accounted for most of the identified agents in unexplained meningitis cases, followed by Escherichia coli (5), enterovirus (2), human herpesvirus 2 (1), and Mycobacterium tuberculosis. Herpesviruses were identified in samples from patients with unexplained encephalitis cases, including varicella-zoster virus (3 infections), human herpesvirus 1 (2), and cytomegalovirus (1). Our study confirms the power of multiplex MassTag PCR as a rapid diagnostic tool for identifying pathogens causing unexplained CNS infections.


(The text that I've bolded seems relevant to CFS/ME.)
 

heapsreal

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interesting, so just to confirm i understand this, prior to this new technique of diagnosing pathogens these patients infections werent picked up in normal blood work so the cause of the meningittis was unexplained until the multiplex tag PCR testing etc was used??
 

Bob

Senior Member
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England (south coast)
interesting, so just to confirm i understand this, prior to this new technique of diagnosing pathogens these patients infections werent picked up in normal blood work so the cause of the meningittis was unexplained until the multiplex tag PCR testing etc was used??

That's my interpretation as well, but I haven't read enough detail to know how new or novel their techniques are.
 

alex3619

Senior Member
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Logan, Queensland, Australia
I don't think the fundamentals of this technology are new. I think its about advances in how to combine various older technologies, and how to do so to enable simultaneous broad analysis. That is what is new(ish).
 

Firestormm

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interesting, so just to confirm i understand this, prior to this new technique of diagnosing pathogens these patients infections werent picked up in normal blood work so the cause of the meningittis was unexplained until the multiplex tag PCR testing etc was used??

I don't think so.

Each of the patients studied had already been diagnosed and although it doesn't say so - let's assume were being treated:

Meningitis was diagnosed by clinical features of fever (>38°C), headache, nuchal rigidity, abnormal CSF profile (CSF protein >45 mg/dL [reference 15–45 mg/dL] and leukocyte >5/μL [reference 0–5/μL]), and/or meningeal involvement noted by brain magnetic resonance imaging (MRI).

Encephalitis was diagnosed when there was evidence of brain parenchyma involvement (e.g., paralysis or focal or generalized seizure) and when the finding of brain MRI or electroencephalogram (EEG) was compatible with encephalitis.

CSF samples from meningitis or encephalitis patients with noninfectious diagnosis, including traumatic, metabolic, malignant, vascular, surgical, hypoxic, and toxic causes, were excluded.

Pathogens were identified for some but not for others:

A total of 212 CSF samples from 212 patients (165 meningitis and 47 encephalitis cases) were obtained during 2006–2008 in Kaohsiung Veterans General Hospital, a tertiary care hospital in southern Taiwan.

Tests conducted in the hospital included bacterial and viral culture, cryptococcal antigen test, VDRL (Venereal Disease Research Laboratory) test and human herpesvirus (HHV) 1 PCR.

Pathogens were identified for 71 meningitis patients and includedCryptococcus neoformans (36 cases), M. tuberculosis (18), Klebsiella pneumonia (7), enterovirus (4), Streptococous pneumoniae (2), Listeria monocytogenes (2), and S. viridians (2).

Pathogens were identified for 6 encephalitis patients and included Japanese encephalitis virus (3 cases) and herpesviruses (3).

The remaining 131 patients with unexplained meningitis or encephalitis with CSF pleocytosis and without a definitive diagnosis were included in this study. CSF samples were stored at –70°C until analysis.

Our investigation confirmed the presence of microbial sequences in 31 (24%) of 131 CSF samples; 25 were identified by MassTag PCR. We detected EBV in 16 samples.

All of the 16 EBV-positive patients in our study had meningitis; none had encephalitis. EBV is an uncommon cause of meningitis (0.9%) (28) and of encephalitis (2.3%) (29). However, given the data reported here, EBV-associated CNS infections in immunocompromised patients may be underestimated. In our study, 56% of EBV-positive patients were HIV infected.

In our study, most of the candidate pathogens were identified by the multiplex MassTag PCR and 16S rRNA PCR. Although this finding may raise questions about the utility of more expensive and sophisticated techniques...This approach may facilitate pathogen discovery for patients with CNS infections of unexplained cause as it becomes more popular in molecular diagnostics.

So 16 out of 131 unidentified cases - what's that around 8%? - had EBV identified as the pathogen for their diagnosis of meningitis. Not sure how this might have changed the patient's treatment protocol - I don't know enough about the condition and the study doesn't seem to comment on the patient's state of health or if the presence of this pathogen is still likely to be affecting their health to a significant (directly related) degree; but:

Rapid and accurate identification of the causative agent of a CNS infection can affect clinical management of individual patients. On the scale of populations, agent identification is crucial for determining the incidence of CNS infections caused by specific agents, enabling prioritization of targets for public health intervention and to prevent outbreaks of disease.

Our study confirms the power of multiplex MassTag PCR as a rapid diagnostic tool for identifying pathogens for patients with CNS infections and shows that viral and bacterial pathogens were detected in CSF from patients with CNS infections of unidentified cause. Additionally, the staged molecular approach incorporating complementary tools may enable detection of pathogens for patients with CNS infections of previously unrecognized causes, which would otherwise be missed. This approach may aid in explaining the observed worldwide high proportion of CNS infections of unknown cause.
 

Bob

Senior Member
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Location
England (south coast)
Thanks for the quotes, Firestormm.

To summarise:

"We determined the infectious agent for 31 (24%) of 131 previously negative samples."

"25 [out of 31] were identified by MassTag PCR."

They excluded cases of meningitis or encephalitis with noninfectious diagnosis:
"CSF samples from meningitis or encephalitis patients with noninfectious diagnosis, including traumatic, metabolic, malignant, vascular, surgical, hypoxic, and toxic causes, were excluded."