Establishment of a digital PCR method for detection of Borrelia burgdorferi sensu lato complex DNA in cerebrospinal fluid.

Direct detection of Borrelia burgdorferi sensu lato bacteria in patient samples for diagnosis of Lyme neuroborreliosis (LNB) is hampered by low diagnostic sensitivity, due to few bacteria in cerebrospinal fluids (CSF) samples. Evaluation of novel molecular methods, including digital PCR (dPCR), as future tools in diagnostics of LNB is desirable. This study aimed to establish a dPCR assay and validate pre-PCR procedures for detection of Borrelia in CSF. Synthetic DNA fragments and cultured Borrelia reference strains were used during optimisation experiments. In addition, 59 CSF specimens from patients examined for LNB were included for clinical validation. The results showed that the pre-PCR parameters with the highest impact on Borrelia-specific dPCR method performance were incubation of the PCR-plate at 4 °C for stabilization of droplets, centrifugation for target concentration, quick-spin for dPCR rain reduction, and PCR inhibition by matrix components. Borrelia DNA in CSF was detected in one out of nine patients with LNB. Diagnostic sensitivity was determined to be 11.1% and specificity 100%. In conclusion, this study reports an optimized Borrelia-specific dPCR method for direct detection of Borrelia in CSF samples. The present study does not support the use of Borrelia-specific dPCR as a routine method for diagnosing LNB.

Cerebrospinal Fluid from Healthy Pregnant Women Does Not Harbor a Detectable Microbial Community.

Cerebrospinal fluid (CSF) circulating in the human central nervous system has long been considered aseptic in healthy individuals, because normally, the blood-brain barrier can protect against microbial invasions. However, this dogma has been called into question by several reports that microbes were identified in human brains, raising the question of whether there is a microbial community in the CSF of healthy individuals without neurological diseases. Here, we collected CSF samples and other samples, including one-to-one matched oral and skin swab samples (positive controls), from 23 pregnant women aged between 23 and 40 years. Normal saline samples (negative controls), sterile swabs, and extraction buffer samples (contamination controls) were also collected. Twelve of the CSF specimens were also used to evaluate the physiological activities of detected microbes. Metagenomic and metatranscriptomic sequencing was performed in these 116 specimens. A total of 620 nonredundant microbes were detected, which were dominated by bacteria (74.6%) and viruses (24.2%), while in CSF samples, metagenomic sequencing found only 26 nonredundant microbes, including one eukaryote, four bacteria, and 21 viruses (mostly bacteriophages). The beta diversity of microbes compared between CSF metagenomic samples and other types of samples (except negative controls) was significantly different from that of the CSF self-comparison. In addition, there was no active or viable microbe in the matched metagenomic and metatranscriptomic sequencing of CSF specimens after subtracting those also found in normal saline, DNA extraction buffer, and skin swab specimens. In conclusion, our results showed no strong evidence of a colonized microbial community present in the CSF of healthy individuals. IMPORTANCE The microbiome is prevalent throughout human bodies, with profound health implications. However, it remains unclear whether it is present and active in human CSF, which has been long considered aseptic due to the blood-brain barrier. Here, we applied unbiased metagenomic and metatranscriptomic sequencing to detect the presence of a microbiome in CSF collected from 23 pregnant women with matched controls. Analysis of 116 specimens found no strong evidence to support the presence of a colonized microbiome in CSF. Our findings will strengthen our understanding of the internal environment of the CSF in healthy people, which has strong implications for human health, especially for neurological infections and disorders, and will help further disease diagnostics, prevention, and therapeutics in clinical settings.

Diagnostic accuracy of Xpert MTB/RIF Ultra for tuberculous meningitis in a clinical practice setting of China.

A comparative performance evaluation of the novel Xpert MTB/RIF Ultra (Xpert Ultra) and MTB/RIF Xpert (Xpert) for tuberculous meningitis (TBM) diagnosis was performed. The cerebrospinal fluids of suspected TBM patients were collected consecutively and subjected to smear microscopy, culture, Xpert, and Xpert Ultra. In total, 160 patients were recruited. Xpert Ultra produced a higher sensitivity (45%, 34 of 76) than Xpert (28%, 21 of 76; P = 0.001) and culture (18%, 14 of 76; P < 0.001), respectively. Inclusion of Xpert Ultra outcomes increased the percentage of definite TBM case from 36% (27 of 76) to 51% (39 of 76). Both Xpert Ultra and Xpert accurately identified the one rifampicin (RIF)-resistant and the 5 RIF-sensitive cases defined by phenotypic drug sensitivity test. The specificities of all of the culture, Xpert and Xpert Ultra were 100% (45 of 45). Xpert Ultra outperformed both Xpert and culture for TBM diagnosis, which may speed up the appropriate treatment of patients in clinical practice.

A comparison of the accuracy of the CapitalBio Mycobacterium real-time polymerase chain reaction and the Xpert MTB/RIF assay for the diagnosis of tuberculous meningitis.

OBJECTIVES: We aimed to compare the efficiency of the CapitalBioMycobacterium real-time polymerase chain reaction (PCR) detection test with the standard Xpert MTB/RIF assay for the diagnosis of tuberculous meningitis (TBM). METHODS: We analyzed cerebrospinal fluid (CSF) from 163 patients with suspected TBM that were collected between January 1, 2018, and December 31, 2019. For both tests, we determined the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and area under the curve (AUC). Next, we compared the diagnostic accuracy of the two techniques using clinical diagnosis as a reference standard. RESULTS: The sensitivity, specificity, PPV, NPV, and AUC, of the CapitalBio Mycobacterium detection test were 48.5%, 100%, 100%, 29.6%, and 0.74, respectively, when used for the diagnosis of TBM. In comparison, the Xpert MTB/RIF assay returned values of 47.0%, 100%, 100%, 29.0%, and 0.74, respectively. Our analysis showed that the diagnostic accuracies of the CapitalBio Mycobacterium detection test and the Xpert MTB/RIF assay were very similar; the accuracy of both tests for detecting mycobacteria was significantly higher than that associated with acid-fast staining. CONCLUSIONS: The CapitalBio Mycobacterium real-time PCR detection test has moderate sensitivity and very high specificity for TBM; results are very similar to those generated by the Xpert MTB/RIF assay. We recommend that the CapitalBio PCR test should be used as an initial screening method for TB.

Hearing loss in individuals at risk for neurosyphilis.

Otosyphilis is a serious complication of syphilis.329 participants enrolled in a study of cerebrospinal fluid (CSF) abnormalities in syphilis underwent portable audiometry (250 Hz to 8000 Hz at 5-75 dB); it was repeated in 33 after otosyphilis treatment. Treponema pallidum spp pallidum (T. pallidum) DNA in blood was quantitated by polymerase chain reaction. Odds ratios (ORs) or hazard ratios (HRs) with 95% confidence intervals (CIs) were determined by logistic, ordinal or Cox regression.166 (50.5%) had normal hearing; 15 (4.6%) had low frequency (LF) loss alone, 93 (28.3%) had high frequency (HF) loss alone, and 55 (16.7%) had both. Adjusted odds of any hearing loss were higher with detectable blood T. pallidum DNA (3.00 [1.58-5.69], p = 0.001), CSF pleocytosis (2.02 [1.12-3.66], p = 0.02), and older age (2.22 per 10-year increase, [1.70-2.91], p < 0.001). HRs of normalization of LF and HF loss were lower for older individuals (0.20 [0.07-0.63, p = 0.005] and 0.22 [0.05-0.94, p = 0.04]), and HRs for normalization of HF loss were lower for those with more severe loss (0.09 [0.02-0.43], p = 0.002), and in those with CSF pleocytosis (0.32 [0.11-0.96], p = 0.04).Older age and CSF pleocytosis increase the likelihood of otosyphilis and impair hearing recovery after otosyphilis treatment.

Usefulness of Nested Polymerase Chain Reaction with Clinical Specimens for Diagnosis of Leptospirosis: a Case Series and a Review of Literature.

A culture of the Leptospira species and the microscopic agglutination test (MAT) are considered as the reference standard for the diagnosis of leptospirosis, but both tests are imperfect for early diagnosis. We describe 4 patients diagnosed with leptospirosis using nested polymerase chain reaction (N-PCR) that targeted the 16S rRNA gene and the passive hemagglutination assay (PHA). In our 4 cases, Leptospira DNA in the urine, plasma, or cerebrospinal fluid (CSF), was detected by N-PCR in the early phase of leptospirosis, except in the sample from the buffy coat. Especially, case 3 showed that N-PCR with the urine and CSF was positive 8 days after symptom onset, but not for the plasma or buffy coat. We report 4 cases of leptospirosis that were diagnosed by N-PCR that targeted the 16S rRNA gene with urine, plasma, or CSF, but not the buffy coat. Three were cured by doxycycline but the case 4 was fatal. Detection of Leptospira DNA by PCR from the urine and CSF, in addition to plasma, may be helpful to confirm the diagnosis.

Rapid Diagnosis of Tuberculosis Meningitis by Detecting Mycobacterium tuberculosis Cell-Free DNA in Cerebrospinal Fluid.

OBJECTIVES: Tuberculosis meningitis (TBM) is one of the most severe forms of tuberculosis. However, TBM diagnosis is quite challenging due to nonspecific clinical presentation and the paucity of the pathogen in cerebrospinal fluid (CSF) samples. In this study, we report a new method for detecting cell-free Mycobacterium tuberculosis DNA (cf-TB) in CSF and evaluate its clinical value for TBM diagnosis. METHODS: Of 68 patients prospectively recruited, 46 were diagnosed as having TBM and 22 as non-TBM. We compared the cf-TB method with CSF smear microscopy, mycobacterial culture, and the Xpert MTB/RIF assay (Xpert) using the consensus case definition for TBM proposed in 2009 as a reference standard. RESULTS: The sensitivity of the cf-TB test was 56.5% (26/46) in patients with TBM, and it was significantly higher than other methods: microscopy (2.2%, 1/46; P < .001), mycobacterial culture (13.0%, 6/46; P < .001), and Xpert (23.9%, 11/46; P = .001). For specificity, none of the four methods reported false-positive results in the non-TBM group. CONCLUSIONS: The new method detecting cell-free M tuberculosis DNA in CSF is rapid and accurate for diagnosis of TBM and easily incorporated into regular laboratory tests.

Molecular studies of meningococcal and pneumococcal meningitis patients in Ethiopia.

Neisseria meningitidis infections in sub-Saharan Africa usually present with distinct symptoms of meningitis but very rarely as fulminant septicemia when reaching hospitals. In Europe, development of persistent meningococcal shock and multiple organ failure occurs in up to 30% of patients and is associated with a bacterial load of >106/ml plasma or serum. We have prospectively studied 27 Ethiopian patients with meningococcal infection as diagnosed and quantified with real-time PCR in the cerebrospinal fluid (CSF) and serum. All presented with symptoms of meningitis and none with fulminant septicemia. The median N. meningitidis copy number (NmDNA) in serum was < 3.5 × 103/ml, never exceeded 1.8 × 105/ml, and was always 10-1000 times higher in CSF than in serum. The levels of LPS in CSF as determined by the limulus amebocyte lysate assay were positively correlated to NmDNA copy number ( r = 0.45, P = 0.030), levels of IL-1 receptor antagonist, ( r = 0.46, P = 0.017), and matrix metallopeptidase-9 (MMP-9; r = 0.009). We also compared the inflammatory profiles of 19 mediators in CSF of the 26 meningococcal patients (2 died and 2 had immediate severe sequelae) with 16 patients with Streptococcus pneumoniae meningitis (3 died and 3 with immediate severe sequelae). Of 19 inflammatory mediators tested, 9 were significantly higher in patients with pneumococcal meningitis and possibly linked to worse outcome.

Development and validation of an advanced fragment analysis-based assay for the detection of 22 pathogens in the cerebrospinal fluid of patients with meningitis and encephalitis.

BACKGROUND: Meningitis and encephalitis (ME) are central nervous system (CNS) infections mainly caused by bacteria, mycobacteria, fungi, viruses, and parasites that result in high morbidity and mortality. The early, accurate diagnosis of pathogens in the cerebrospinal fluid (CSF) and timely medication are associated with better prognosis. Conventional methods, such as culture, microscopic examination, serological detection, CSF routine analysis, and radiological findings, either are time-consuming or lack sensitivity and specificity. METHODS: To address these clinical needs, we developed an advanced fragment analysis (AFA)-based assay for the multiplex detection of 22 common ME pathogens, including eight viruses, 11 bacteria, and three fungi. The detection sensitivity of each target was evaluated with a recombinant plasmid. The limits of detection of the 22 pathogens ranged from 15 to 120 copies/reaction. We performed a retrospective study to analyze the pathogens from the CSF specimens of 170 clinically diagnosed ME patients using an AFA-based assay and compared the results with culture (bacteria and fungi), microscopic examination (fungi), polymerase chain reaction (PCR) (Mycobacterium tuberculosis), and Sanger sequencing (virus) results. RESULTS: The sensitivity of the AFA assay was 100% for 10 analytes. For Cryptococcus neoformans, the sensitivity was 63.6%. The overall specificity was 98.2%. The turnaround time was reduced to 4-6 hours from the 3-7 days required using conventional methods. CONCLUSIONS: In conclusion, the AFA-based assay provides a rapid, sensitive, and accurate method for pathogen detection from CSF samples.

Combined testing for herpes simplex virus and Mycobacterium tuberculosis DNA in cerebrospinal fluid of patients with aseptic meningitis in Burkina Faso, West Africa.

BACKGROUND: Little is known about the involvement of herpes simplex virus (HSV) or Mycobacterium tuberculosis (MTB) as potentially curable causes of central nervous system (CNS) infections in sub-Saharan Africa. OBJECTIVE: In this study, we developed a PCR assay dedicated to simultaneous testing of HSV1/HSV2 and MTB in Burkina Faso, a country where HSV is neglected as a cause of CNS infection and where TB prevalence is high. METHODS: A consensus HSV1/HSV2 set of primers and probe were designed and combined to primers and probe targeting the IS6110 repetitive insertion sequence of MTB. Analytical performances of the assay were evaluated on reference materials. Cerebrospinal fluid (CSF) collected from subjects with aseptic meningitis was tested for HSV1/HSV2 and MTB DNA. RESULTS: The UL29 gene was chosen as a highly conserved region targeted by the HSV1/HSV2 nucleic acid test. The lower limits of detection were estimated to be 2.45 copies/µL for HSV1, 1.72 copies/µL for HSV2, and 2.54 IS6110 copies per µL for MTB. The PCR was used in 202 CSF collected from subjects suspected of aseptic meningitis. Five samples (2.46%) tested positive, including two children positive for HSV1 (0.99%) and three adults tested positive for MTB (1.47%). CONCLUSION: Using an in-house real-time PCR assay, we showed that both HSV and MTB are etiologic pathogens contributing to aseptic meningitis in Burkina Faso. This molecular test may have clinical utility for early diagnosis for those treatable CNS infections.

Testing for Bartonella ssp. DNA in cerebrospinal fluid of dogs with inflammatory central nervous system disease.

BACKGROUND: Neurobartonellosis occurs in people. The role these organisms might play in inflammatory brain disease of dogs is unclear. HYPOTHESIS/OBJECTIVES: That Bartonella spp. DNA would be amplified more commonly from the CSF of dogs with inflammatory disease compared to those with noninflammatory disease. To report the prevalence of Bartonella spp. in dogs with and without inflammatory CNS disease with a commercially available PCR assay. ANIMALS: Cerebrospinal fluid (CSF) samples from 172 dogs from either Washington State University or Colorado State University. METHODS: Retrospective study. A search was performed of all medical records from dogs with CSF samples submitted to CSU's Center for Companion Animal Studies or Veterinary Diagnostic Laboratory from CSU or WSU for Toxoplasma or Neospora PCR assay. Increased CSF nucleated cell counts and an adequate volume of CSF must have been present to evaluate Bartonella spp. by PCR assay. RESULTS: Inflammatory CNS disease was confirmed in 65 dogs, none of which were positive for Bartonella spp. DNA. Of the other 107 dogs, one was positive for B. henselae DNA. The CSF from this dog contained red blood cells. CONCLUSIONS AND CLINICAL IMPORTANCE: Failure to amplify Bartonella spp. DNA from the CSF of the dogs with inflammatory disease suggests the organism was not involved in the etiology of the disease, the organism was in the CNS tissues but not in the CSF, or the organism was present but in quantities undetectable by this PCR assay. The combination of PCR and culture is the most sensitive way to detect Bartonella spp. and the use of that technique should be considered in future studies.

Rapid pathogen identification using a novel microarray-based assay with purulent meningitis in cerebrospinal fluid.

In order to improve the diagnosis of pathogenic bacteria in cerebrospinal fluid (CSF) with purulent meningitis, we developed a DNA microarray technique for simultaneous detection and identification of seven target bacterium. DNA were extracted from 24 CSF samples with purulent meningitis (or suspected purulent meningitis). The specific genes of each pathogen were chosen as the amplification target, performed the polymerase chain reaction (PCR), labeled with a fluorescence dye, and hybridized to the oligonucleotide probes on the microarray. There is no significant cross-hybridization fluorescent signal occurred in untargeted bacteria. There were 87.5% (21/24) positive results in DNA microarray compared with the 58.3% (14/24) of the CSF culture test. Of which 58.3% (14/24) of the patients with culture-confirmed purulent meningitis, 37.5% (9/24) patients who were not confirmed by culture test but were demonstrated by the clinical diagnosis and DNA microarray. Multiple bacterial infections were detected in 5 cases by the microarray. In addition, the number of gene copies was carried out to determine the sensitivity of this technique, which was shown to be 3.5 × 101 copies/µL. The results revealed that the microarray technique which target pathogens of the CSF specimen is better specificity, accuracy, and sensitivity than traditional culture method. The microarray method is an effective tool for rapidly detecting more target pathogens and identifying the subtypes of strains which can eliminate the impact of the different individuals with purulent meningitis for prompt diagnosis and treatment.

Utility of pyrosequencing for rapid detection of tubercular meningitis (TBM) and associated susceptibility directly from CSF specimens.

Tubercular meningitis (TBM) is a serious form of tuberculosis (TB). The diagnosis of TBM & susceptibility/resistance is difficult as TB MGIT culture lacks sensitivity & timeliness. Timely and accurate diagnosis of the TBM is the need of the hour for initiation of appropriate therapy. We have exploited pyrosequencing to detect TB and associated Multi/extensively drug resistant (MDR/ XDR-TB) directly from Cerebrospinal Fluid (CSF) specimens.

Detection of Neisseria meningitidis in a paediatric patient with septic arthritis using multiplexed diagnostic PCR targeting meningitis/encephalitis (ME).

BACKGROUND: Neisseria meningitidis is associated with meningitis and septicemia. Septic meningococcal arthritis is relatively uncommon and its diagnosis associated with clinical and microbiological challenges. Early recognition and treatment is required to prevent joint destruction. PURPOSE: We describe a case of an eleven-year-old boy with septic arthritis and the first reported use of a multiplexed diagnostic PCR test, capable of simultaneous rapid detection of 14 pathogens directly from CSF samples, to determine presence of N. meningitides in a synovial fluid sample. RESULTS: In this case, blood cultures and an aspiration of the joint fluid were negative for microbial growth, but leucocytes were present. Analysis of samples using the multiplexed FilmArray® meningitis/encephalitis panel (MEP) proved positive for N. meningitidis. In parallel, samples forwarded to an accredited reference laboratory confirmed the findings by bacterial 16S rRNA gene amplification and sequencing. Subsequent to these results, empiric treatment with intravenous flucloxacillin was discontinued and oral amoxicillin administered for 1 month. The status of the patient improved with etiology-based antimicrobial therapy. CONCLUSIONS: This case demonstrates difficulties associated with clinical and microbiological diagnosis of primary septic meningococcal arthritis. We describe the first successful use of the FilmArray® MEP assay in detection of N. meningitidis in that context.

Colistin-resistant KPC-2-producing Klebsiella pneumoniae ST423 harboring an IS5-like element in the mgrB gene isolated from cerebrospinal fluid.

We describe colistin-resistant KPC-2-producing Klebsiella pneumoniae isolates from cerebrospinal fluid, belonging to ST423, selected during treatment for neuroinfection. Colistin resistance was related to mgrB gene interruption by an IS5-like.

Direct Molecular Detection and Genotyping of Borrelia burgdorferi Sensu Lato in Cerebrospinal Fluid of Children with Lyme Neuroborreliosis.

The current diagnostic marker of Lyme neuroborreliosis (LNB), the Borrelia burgdorferisensu lato antibody index (AI) in the cerebrospinal fluid (CSF), has insufficient sensitivity in the early phase of LNB. We aimed to elucidate the diagnostic value of PCR for B. burgdorferisensu lato in CSF from children with symptoms suggestive of LNB and to explore B. burgdorferisensu lato genotypes associated with LNB in children. Children were prospectively included in predefined groups with a high or low likelihood of LNB based on diagnostic guidelines (LNB symptoms, CSF pleocytosis, and B. burgdorferisensu lato antibodies) or the detection of other causative agents. CSF samples were analyzed by two B. burgdorferisensu lato-specific real-time PCR assays and, if B. burgdorferisensu lato DNA was detected, were further analyzed by five singleplex real-time PCR assays for genotype determination. For children diagnosed as LNB patients (58 confirmed and 18 probable) (n = 76) or non-LNB controls (n = 28), the sensitivity and specificity of PCR for B. burgdorferisensu lato in CSF were 46% and 100%, respectively. B. burgdorferisensu lato DNA was detected in 26/58 (45%) children with AI-positive LNB and in 7/12 (58%) children with AI-negative LNB and symptoms of short duration. Among 36 children with detectable B. burgdorferisensu lato DNA, genotyping indicated Borrelia garinii (n = 27) and non-B. garinii (n = 1) genotypes, while 8 samples remained untyped. Children with LNB caused by B. garinii did not have a distinct clinical picture. The rate of detection of B. burgdorferisensu lato DNA in the CSF of children with LNB was higher than that reported previously. PCR for B. burgdorferisensu lato could be a useful supplemental diagnostic tool in unconfirmed LNB cases with symptoms of short duration. B. garinii was the predominant genotype in children with LNB.

Neisseria meningitidis Serogroup X ST-5799 (ST-22 complex) in Turkey: A unique pediatric case.

Although outbreaks of Neisseria meningitidis serogroup X occured in a couple of African countries, a limited number of serogroup X meningococcal cases were reported in America and Europe as well as Turkey. Additionally, serogroup X is still not represented in current conjugated meningococcal vaccines. Here, we describe the first pediatric case with meningitis caused by Neisseria meningitidis serogroup X ST-5799 (ST-22 complex) that formed a distinct lineage.

Does more favourable handling of the cerebrospinal fluid increase the diagnostic sensitivity of Borrelia burgdorferi sensu lato-specific PCR in Lyme neuroborreliosis?

BACKGROUND: Tests for direct detection of Borrelia burgdorferi sensu lato (Bb) in Lyme neuroborreliosis (LNB) are needed. Detection of Bb DNA using PCR is promising, but clinical utility is hampered by low diagnostic sensitivity. We aimed to examine whether diagnostic sensitivity can be improved by the use of larger cerebrospinal fluid (CSF) volumes and faster handling of samples. METHODS: Patients who underwent CSF examination for LNB were included. We collected two millilitres of CSF for PCR analysis, extracted DNA from the pellets within 24 h and analysed the eluate by two real-time PCR protocols (16S rRNA and OspA). Patients who fulfilled diagnostic criteria for LNB were classified as LNB cases and the rest as controls. RESULTS: Bb DNA in CSF was detected by PCR in seven of 28 adults with LNB. Two were Bb antibody negative. No Bb DNA was detected in CSF from 137 controls. Diagnostic sensitivity was 25% and specificity 100%. There was a non-significant trend towards larger CSF sample volume, faster handling of the sample, shorter duration of symptoms, and higher CSF cell count in the PCR-positive cases. CONCLUSION: We did not find that optimized handling of CSF increased diagnostic sensitivity of PCR in adults with LNB. However, our case series is small and we hypothesize that the importance of these factors will be clarified in further studies with larger case series and altered study design. PCR for diagnosis of LNB may be useful in cases without Bb antibodies due to short duration of symptoms.