The diagnostic value of metagenomic next-generation sequencing for identifying Streptococcus pneumoniae in paediatric bacterial meningitis.

BACKGROUND: There is currently no research on the diagnostic value of metagenomic next-generation sequencing (mNGS) for a single pathogens in CSF. The aim of this study was to analyse the value of mNGS for identifying Streptococcus pneumoniae (S. pneumoniae) in paediatric bacterial meningitis. METHODS: Bacterial meningitis (BM) cases from October 23, 2014, to December 31, 2016, and December 1, 2017, to July 31, 2018 at Beijing Children's Hospital were reviewed. Clinical features and pathogens were analysed. RESULTS: We diagnosed 135 patients with BM in this study. A total of 43 S. pneumoniae were identified by combination methods. 26/135 (19.3%) patients had positive results in S. pneumoniae by blood and/or cerebrospinal fluid (CSF) culture. Alere BinaxNow®Streptococcus pneumoniae Antigen test was positive in 35/135(25.9%) cases. 32/135 (23.7%) S. pneumoniae were identified by mNGS. Six CSF samples were identified as S. pneumoniae only by mNGS technology. Taking culture as the gold standard, the sensitivity and specificity of mNGS for diagnosing S. pneumoniae meningitis were 73.1 and 88.1%, respectively. The positive predictive value (PPV) and negative predictive value (NPV) of diagnosing S. pneumoniae meningitis by mNGS were 59.4 and 93.2%, respectively. When comparison between mNGS and combined tests (culture and Alere BinaxNow®Streptococcus pneumoniae Antigen test), the sensitivity and specificity of mNGS for S. pneumoniae identification were 70.3 and 93.9%, the PPV and NPV in the identification of S. pneumoniae by mNGS were 81.4 and 89.3%, respectively. The difference in number of unique reads of S. pneumoniaein from CSF sample (< 14 days onset) and CSF sample (> 14 days from onset) was statistically significant (170.5 VS. 13, P = 0.019). The difference in the collected time of CSF for culture and mNGS was statistically significant (4 days VS. 14 days, P < 0.001). CONCLUSIONS: mNGS has high sensitivity and specificity for S. pneumoniae identification. The pathogen load (number of unique reads) of S. pneumonia is related to the CSF collection time. mNGS was less affected than culture by the use of antibiotics before CSF collection.

Rare Meningitis and Epileptic Seizure Infected with Brucella melitensis: a Case Report.

BACKGROUND: Although brucellosis is the most common zoonotic disease worldwide, human meningitis infected with Brucella melitensis is rare and difficult to diagnosis. Herein we describe the clinical aspects of a rare case of Brucella melitensis meningitis accompanied by epileptic seizure. METHODS: Bacterial culture of CSF was utilized to find the pathogen. Serum and CSF agglutination tests were used to detect the capacity of Brucella antigen. Bacterial clone was identified by the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). Magnetic resonance imaging (MRI) was used to monitor the recovery changes for meningitis. RESULTS: The bacterial culture was positive for Brucella app. Antigen-antibody agglutination test was also positive with a titer more than 1/200. A reliable identification score of 2.8 for Brucella melitensis was obtained by MALDI-TOF. MRI showed obviously improved meningitis after therapy. CONCLUSIONS: The results of this study suggest the treatments for Brucella meningitis must be planned depending on the patient's clinical conditions and the laboratory identification of pathogen infection as early as possible.

Epidemiology and emm types of invasive group A streptococcal infections in Finland, 2008-2013.

Invasive Streptococcus pyogenes (group A streptococcus, GAS) infections are a major global cause of morbidity and mortality. We analysed the surveillance data on invasive GAS and the microbiological characteristics of corresponding isolates to assess the incidence and emm type distribution of invasive GAS infections in Finland. Cases defined as patients with isolations of blood and cerebrospinal fluid S. pyogenes are mandatorily notified to the National Infectious Disease Registry and sent to the national reference laboratory for emm typing. Antimicrobial data were collected through the network including all clinical microbiology laboratories. Pulsed-field gel electrophoresis (PFGE) analysis was performed to assess clonality. In total, 1165 cases of invasive GAS were reported in Finland during 2008-2013; the median age was 52 years (range, 0-100) and 54% were male. The overall day 7 case fatality rate was 5.1% (59 cases). The average annual incidence was 3.6 cases per 100,000 population. A total of 1122 invasive GAS isolates (96%) were analysed by emm typing; 72 different emm types were identified, of which emm28 (297 isolates, 26%), emm89 (193 isolates, 12%) and emm1 (132 isolates, 12%) were the most common types. During 2008-2013, an increase of erythromycin resistance (1.9% to 8.7%) and clindamycin (0.9% to 9.2%) was observed. This resistance increase was in parallel with the introduction of a novel clone emm33 into Finland. The overall incidence of invasive GAS infections remained stable over the study period in Finland. We identified clonal spread of macrolide-resistant invasive emm33 GAS type, highlighting the importance of molecular surveillance.

Diagnostic accuracy of intracellular mycobacterium tuberculosis detection for tuberculous meningitis.

RATIONALE: Early diagnosis and treatment of tuberculous meningitis saves lives, but current laboratory diagnostic tests lack sensitivity. OBJECTIVES: We investigated whether the detection of intracellular bacteria by a modified Ziehl-Neelsen stain and early secretory antigen target (ESAT)-6 in cerebrospinal fluid leukocytes improves tuberculous meningitis diagnosis. METHODS: Cerebrospinal fluid specimens from patients with suspected tuberculous meningitis were stained by conventional Ziehl-Neelsen stain, a modified Ziehl-Neelsen stain involving cytospin slides with Triton processing, and an ESAT-6 immunocytochemical stain. Acid-fast bacteria and ESAT-6-expressing leukocytes were detected by microscopy. All tests were performed prospectively in a central laboratory by experienced technicians masked to the patients' final diagnosis. MEASUREMENTS AND MAIN RESULTS: Two hundred and eighty patients with suspected tuberculous meningitis were enrolled. Thirty-seven had Mycobacterium tuberculosis cultured from cerebrospinal fluid; 40 had a microbiologically confirmed alternative diagnosis; the rest had probable or possible tuberculous meningitis according to published criteria. Against a clinical diagnostic gold standard the sensitivity of conventional Ziehl-Neelsen stain was 3.3% (95% confidence interval, 1.6-6.7%), compared with 82.9% (95% confidence interval, 77.4-87.3%) for modified Ziehl-Neelsen stain and 75.1% (95% confidence interval, 68.8-80.6%) for ESAT-6 immunostain. Intracellular bacteria were seen in 87.8% of the slides positive by the modified Ziehl-Neelsen stain. The specificity of modified Ziehl-Neelsen and ESAT-6 stain was 85.0% (95% confidence interval, 69.4-93.8%) and 90.0% (95% confidence interval, 75.4-96.7%), respectively. CONCLUSIONS: Enhanced bacterial detection by simple modification of the Ziehl-Neelsen stain and an ESAT-6 intracellular stain improve the laboratory diagnosis of tuberculous meningitis.

Neonatal purulent meningitis in southern Tunisia: Epidemiology, bacteriology, risk factors and prognosis.

OBJECTIVES: To study the epidemiological, clinical and bacteriological aspects and outcome of purulent neonatal meningitis (PNM). METHODOLOGY: Retrospective analysis of 55 cases of PNM hospitalized in the pediatric ward of Hedi Chaker Hospital from 1990 to 2012. Infants less than 29 days of age were included. The diagnosis was made on either the presence of bacteria in the cerebrospinal fluid (CSF) or the combination of pleocytosis >30 cells/mm(3), protein level >1.3 g/l and glucose level <2.2 mmol/l or CSF/blood glucose ratio <0.4. RESULTS: The male:female sex ratio was 1.75. One or more maternal risk factors for infection were found in 24 cases. The main symptoms were fever and poor feeding. Soluble antigen was positive in four cases and cultures had isolated the bacteria in 28 cases. The mortality rate was 40%. The sequelae rate in the survivors was 16.4%. CONCLUSION: This study emphasizes the severity of PNM with high rates of mortality and neurological sequelae.

[A comparative study for adenosine deaminase and anti-antigen A-60 antibodies detection for the diagnosis of tuberculous meningitis]. / Comparación de adenosina deaminasa y detección de anticuerpos anti-antígeno A60 para el diagnóstico de meningitis tuberculosa.

BACKGROUND: Diagnosis of tuberculous meningitis (TBM) is hampered by the lack of rapid and accurate diagnostic tools. We evaluated the immunological response to Mycobacterium tuberculosis anti-A60 antibodies in cerebrospinal fluid (CSF) in comparison to adenosine deaminase (ADA) determination, for the diagnosis of TBM. METHODS: A total of 63 CSF samples were analyzed by indirect ELISA for the detection of anti- A60 IgG, IgM and IgA. These include samples from 17 patients with confirmed TBM and 46 control patients with other infections. RESULTS: The mean individual anti-A60 IgM, IgG and IgA CSF antibody titers were significantly higher in TBM in comparison with control groups (p < 0.01). The best discriminatory CSF antibody for confirming TBM diagnosis was IgM, with an area under the receiver operating characteristic curve of 0.928 (95%CI 0.834-0.978), compared to 0.863 (95% CI: 0.752-0.936) for ADA testing (p = NS). The sensitivity of anti- A60 IgM CSF antibody titers (cutoff > 0.06 U/ml) was 94.1% compared to 88.2% for ADA (cutoff > 6.2 U/ml), p = NS. Both anti A60 IgM and ADA showed the same moderate specificity (80.4%). Two cases of TBM were correctly identified by anti-A60 IgM but missed by ADA. CONCLUSION: The ELISA test for anti-antigen A60 antibodies (IgM) is a rapid and sensitive tool for the rapid diagnosis of TBM that can be a complement to ALDA determination. The specificity of both tests is still a limitation in TBM diagnosis.

Diagnostic Model for Discrimination Between Tuberculous Meningitis and Bacterial Meningitis.

Background: The differential diagnosis between tuberculous meningitis (TBM) and bacterial meningitis (BM) remains challenging in clinical practice. This study aimed to establish a diagnostic model that could accurately distinguish TBM from BM. Methods: Patients with TBM or BM were recruited between January 2017 and January 2021 at Tongji Hospital (Qiaokou cohort) and Sino-French New City Hospital (Caidian cohort). The detection for indicators involved in cerebrospinal fluid (CSF) and T-SPOT assay were performed simultaneously. Multivariate logistic regression was used to create a diagnostic model. Results: A total of 174 patients (76 TBM and 98 BM) and another 105 cases (39 TBM and 66 BM) were enrolled from Qiaokou cohort and Caidian cohort, respectively. Significantly higher level of CSF lymphocyte proportion while significantly lower levels of CSF chlorine, nucleated cell count, and neutrophil proportion were observed in TBM group when comparing with those in BM group. However, receiver operating characteristic (ROC) curve analysis showed that the areas under the ROC curve (AUCs) produced by these indicators were all under 0.8. Meanwhile, tuberculosis-specific antigen/phytohemagglutinin (TBAg/PHA) ratio yielded an AUC of 0.889 (95% CI, 0.840-0.938) in distinguishing TBM from BM, with a sensitivity of 68.42% (95% CI, 57.30%-77.77%) and a specificity of 92.86% (95% CI, 85.98%-96.50%) when a cutoff value of 0.163 was used. Consequently, we successfully established a diagnostic model based on the combination of TBAg/PHA ratio, CSF chlorine, CSF nucleated cell count, and CSF lymphocyte proportion for discrimination between TBM and BM. The established model showed good performance in differentiating TBM from BM (AUC: 0.949; 95% CI, 0.921-0.978), with 81.58% (95% CI, 71.42%-88.70%) sensitivity and 91.84% (95% CI, 84.71%-95.81%) specificity. The performance of the diagnostic model obtained in Qiaokou cohort was further validated in Caidian cohort. The diagnostic model in Caidian cohort produced an AUC of 0.923 (95% CI, 0.867-0.980) with 79.49% (95% CI, 64.47%-89.22%) sensitivity and 90.91% (95% CI, 81.55%-95.77%) specificity. Conclusions: The diagnostic model established based on the combination of four indicators had excellent utility in the discrimination between TBM and BM.

Pneumococcal serotypes causing pediatric meningitis in Turkey: application of a new technology in the investigation of cases negative by conventional culture.

The surveillance of serotypes causing invasive pneumococcal disease (IPD) provides further insight into the pathogenesis of pneumococcal disease and is important in order to track vaccine impact. Although the Quellung reaction has been accepted as the standard method for serotyping, prior antibiotic use causes a gap in studies based on bacterial culture. A total of 31 cerebrospinal fluid (CSF) samples found to be positive for Streptococcus pneumoniae by polymerase chain reaction (PCR) targeting the ply gene during an active surveillance were tested in a Bio-Plex multiplex antigen detection assay capable of detecting 14 serotypes/groups (1, 3, 4, 5, 6A, 6B, 7F/A, 8, 9V, 14, 18, 19A, 19F, and 23F). Twenty-seven CSF samples could be serotyped. The most common serotypes were serotypes 5 (n = 7), 19F (n = 5), 1 (n = 3), and 23F (n = 3). Theoretical coverage rates by the heptavalent (PCV7), 10-valent (PCV10), and 13-valent (PCV13) pneumococcal conjugate vaccines for bacterial meningitis were 48.1, 85.2, and 92.3%, respectively, for all age groups and 71.4, 85.7, and 100.0%, respectively, for those under 2 years of age. We propose that antigen detection assay used in conjunction with a PCR assay can be effectively applied in CSF samples to detect the pneumococcal serotypes, especially when the patient may have already been treated and, therefore, the cultures would be negative.

Detection of bacterial antigen in cerebrospinal fluid in patients with bacterial meningitis: a literature review.

Rapid detection of bacterial pathogen causing meningitis is very important to guide antimicrobial therapy before the standard culture result is available. Other than gram stain, one of the most useful rapid methods is the detection of bacterial antigen in cerebrospinal fluid. This article reviewed the methods of bacterial antigen detection for diagnosis of meningitis as well as a microbiology aspect of this life-threatening disease.

Diagnostic Yield of Pneumococcal Antigen Detection in Cerebrospinal Fluid for Diagnosis of Pneumococcal Meningitis Among Children in China.

OBJECTIVE: To determine the diagnostic accuracy of pneumococcal antigen detection in diagnosis of pneumococcal meningitis in children. METHODS: Purulent meningitis was diagnosed according to European Society for Clinical Microbiology and Infectious Diseases (ESCMID) guideline between July 2014 and June 2016. Along with a cerebrospinal fluid (CSF) culture, pneumococcal antigen detection in cerebrospinal fluid (CSF) was performed, and further identification of pathogens was done with 16S rDNA-PCR and high-throughput sequencing. RESULTS: CSF samples collected from 184 children (median age of 1.92 mo). CSF culture was used as the gold standard. 46 (25%) had positive results for culture and 10 (5.4%) were pneumococci; 34 (18.5%) were pneumococcal antigen positive. The sensitivity and specificity of pneumococcal antigen detection were 100% (95% CI: 89.4%-100%) and 86.2% (95% CI: 96.4%-99.9%), respectively. 92.3% (12/13) were confirmed by nucleic acid detection to be pneumococci. CONCLUSIONS: Pneumococcal antigen detection in CSF has adequate sensitivity and specificity in diagnosing pneumococcal meningitis.

Surveillance of pneumococcal meningitis among children in Sindh, southern Pakistan.

BACKGROUND: Information about the burden of invasive pneumococcal disease among children in Pakistan is limited. METHODS: Surveillance of bacterial meningitis among children aged <5 years was set up at 18 hospitals in southern Pakistan that fulfilled the following criteria: (1) >30 pediatric admissions weekly, (2) skilled personnel to perform lumbar punctures, and (3) close proximity to an Aga Khan University Hospital laboratory collection point. RESULTS: A total of 2690 children were admitted to the hospital with suspected acute bacterial meningitis, and 2646 (98%) underwent lumbar puncture. Of the 2646 cerebrospinal fluid specimens obtained, 412 (16%) were purulent, and pathogens were detected by culture or latex agglutination testing in 83 (20.1%) of the purulent specimens. Of the 83 isolates detected, 48 (57.8%) were Haemophilus influenzae type b, 32 (38.5%) were Streptococcus pneumoniae, and 3 (3.6%) were Neisseria meningiditis. Overall, 81% of the pathogens detected were from children aged <1 year. More than 50% of families reported definite prior antimicrobial use. The minimum detected incidence rates of purulent meningitis in Hyderabad were 112 cases per 100,000 children aged <1 year and 45.3 cases per 100,000 children aged <5 years. After adjustment for limitations in access to care and the low sensitivity of cerebrospinal fluid culture, the adjusted incidence rates of pneumococcal meningitis were 81 cases per 100,000 children aged <1 year (95% confidence interval, 26.2-190.5 cases per 100,000) and 20 cases per 100,000 children aged <5 years (95% confidence interval, 7.3-43.7 cases per 100,000). Of the 32 children with pneumococcal meningitis, 8 (25%) died during hospitalization. CONCLUSIONS: Our surveillance system detected a substantial burden of purulent meningitis among infants and children in southern Pakistan. H. influenzae type b and S. pneumoniae accounted for >90% of detected pathogens. The use of vaccines against these 2 pathogens could prevent a substantial portion of disease and deaths in Pakistan.

Hospital-based surveillance of invasive pneumococcal disease among young children in urban Nepal.

BACKGROUND: Streptococcus pneumoniae is a leading cause of pneumonia and meningitis in young children. Before implementation of the pneumococcal conjugate vaccine in developing countries, there is an urgent need to provide regional epidemiological data on pneumococcal disease. The aims of this study were to determine the prevalence and serotype distribution of invasive pneumococcal disease among young children hospitalized in urban Nepal. METHODS: Children aged 2 months to 5 years who were admitted to Patan Hospital, Kathmandu, with fever and/or suspected pneumonia, meningitis, or bacteremia were recruited. Blood culture specimens were collected from all participants. In cases of suspected meningitis, cerebrospinal fluid specimens were cultured and were tested for S. pneumoniae antigen. RESULTS: A total of 885 children were recruited during the 21-month study period. Of these, 76 (9%) had meningitis and 498 (56%) had pneumonia, on the basis of clinical criteria. Radiographically confirmed pneumonia occurred in 354 (40%), and probable or definite meningitis occurred in 47 (5%). S. pneumoniae was isolated in specimens from 17 (2%) of the children. Serotypes 1 and 12A were isolated most frequently, and only 1 of 17 isolates had a serotype contained in the currently available 7-valent pneumococcal conjugate vaccine. CONCLUSIONS: More than 60% of children aged <5 years who were admitted with fever and/or suspected invasive bacterial disease in urban Nepal had the clinical syndromes of meningitis and/or pneumonia. A new generation of pneumococcal vaccines that prevent infection with a broader range of serotypes may be necessary to most effectively control pneumococcal disease in young children in Kathmandu.

Invasive pneumococcal disease in Kanti Children's Hospital, Nepal, as observed by the South Asian Pneumococcal Alliance network.

BACKGROUND: Pneumonia accounts for approximately 2 million deaths annually among children aged <5 years, with most of these deaths occurring in Africa and southern Asia. The South Asian Pneumococcal Alliance (SAPNA) network in Nepal is generating local epidemiological data to assist in the development of national and regional policies for prevention of pneumococcal and Haemophilus influenzae (Hib) disease. METHODS: Children aged 2 months to 5 years with suspected invasive bacterial disease were recruited from Kanti Children Hospital, Kathmandu, Nepal. Specimens of blood, CSF, and normally sterile body fluids were cultured, and analysis of antimicrobial susceptibility patterns and serotyping of Streptococcus pneumoniae isolates were performed. CSF specimens were also tested for S. pneumoniae and Hib antigens by a latex agglutination test and an immunochromatographic test of pneumococcal antigen (NOW S. pneumoniae Antigen Test; Binax). RESULTS: A total of 2528 children with suspected invasive bacterial disease were recruited, of whom 82% had pneumonia, 9.6% had meningitis, 2% had very severe disease, and 0.4% had bacteremia; the remainder received another diagnosis. Before hospitalization, 26.7% had received antibiotic treatment. Fifty children had S. pneumoniae identified as the etiological agent of invasive disease. Of 2461 blood cultures performed, 22 were positive for S. pneumoniae. Of 33 cases of S. pneumoniae meningitis, 11 were detected by CSF culture, and 21 were detected by latex agglutination and pneumococcal antigen tests. The rate of detection of S. pneumoniae in CSF was 3.6% by culture, compared with 7.8% by latex agglutination and 10% by pneumococcal antigen testing. The rate of detection of H. influenzae in CSF was 1.7% by culture and 6.5% by latex agglutination. The most common serotypes found were 1, 5, 2, and 7F, followed by 12A, 19B, and 23F. Of all the invasive isolates, 3.8% were resistant to penicillin, and 68% were resistant to trimethoprim-sulfamethoxazole. CONCLUSIONS: The SAPNA network has identified Hib and pneumococci as causes of significant disease in Nepal.

Surveillance of invasive pneumococcal disease in Colombo, Sri Lanka.

The South Asian Pneumococcal Surveillance network uses standard recruitment and laboratory procedures for surveillance of invasive pneumococcal disease in India, Nepal, and Sri Lanka. Children aged 2 months to 5 years who were admitted to the sentinel surveillance site, Lady Ridgeway Hospital for Children, in Colombo, Sri Lanka, and who presented with signs and symptoms of meningitis, pneumonia, or very severe disease were studied. Blood culture and CSF culture specimens were analyzed at the microbiology laboratory at Lady Ridgeway Hospital for Children. Specimens were processed by routine conventional methods. Antigen testing was performed on CSF specimens with use of commercially available latex agglutination test kits. From January 2005 to March 2007, we observed 23 isolates of Streptococcus pneumoniae, and the most common serotypes were 19F, 14, 23F, and 6B. Of the serotypes found, 60% are covered by the currently available 7-valent conjugate pneumococcal vaccine. More than 90% of the isolates were penicillin resistant, and the rate of resistance to third-generation cephalosporins was also high.

Field evaluation of rapid diagnostic tests for meningococcal meningitis in Niger.

OBJECTIVE: To evaluate dipstick rapid diagnostic tests (RDTs) for meningococcal meningitis in basic health facilities. METHODS: Health facility staff received a one-day training. During the meningitis season, they performed RDTs on cerebrospinal fluid (CSF) specimens from suspected cases of meningitis. A frozen aliquot of CSF was later tested using polymerase chain reaction (PCR) to establish the reference diagnosis. RDTs used in health facilities were archived to allow checking the concordance between reported diagnosis and observed results. Reported diagnosis was also compared to PCR diagnosis. A second RDT was performed on each CSF specimen at the reference laboratory. RESULTS: Using RDTs, health facilities reported 382 negative results (73.9%), 114 NmA (22.1%), 12 NmW135 (2.3%) and nine uninterpretable results (1.7%), the latter corresponding to the misuse of a reagent by three agents. The agreement between reported diagnosis and archived dipsticks was excellent (kappa = 0.98). The agreement between PCR diagnosis and reported RDTs results was strong (kappa = 0.82). In health facilities, the sensitivity of RDTs for N. meningitidis A was Se = 0.91. The kappa coefficient measuring the agreement between RDTs operated in the reference laboratory and RDTs operated in health facilities was kappa = 0.78. CONCLUSION: We confirmed that dipstick RDTs to identify N. meningitidis serogroups A, C, W135 and Y can be reliably operated by non-specialized staff in basic health facilities. RDTs proved very useful to recommend vaccination in NmA epidemics, and also to avoid vaccination in epidemics due to serogroups not included in vaccines (NmX).

Diagnostic value of early secreted antigenic target-6 for the diagnosis of tuberculous meningitis patients.

BACKGROUND: The early diagnosis of tuberculous meningitis (TBM) is very crucial, since delayed diagnosis can lead to various neurological manifestations. We have previously developed an in-house indirect enzyme-linked immunosorbent assay (ELISA) for TBM diagnosis using the Antigen 85 (Ag 85) complex. It has been suggested that the Ag 85 complex might give false-positive reactions for individuals vaccinated with Bacillus Calmette-Guérin (BCG). OBJECTIVES: In the present study, we describe a prospective evaluation demonstrating that early secreted antigenic target- 6 (ESAT-6), which is absent in Mycobacterium bovis BCG strains, is in the cerebrospinal fluid (CSF) of TBM patients. METHODS: We used an indirect ELISA to detect ESAT-6 antigens in the CSF of TBM patients using polyclonal antibodies against ESAT-6. RESULTS: Using the indirect ELISA method, we demonstrated a sensitivity and specificity of 80% and 94%, respectively, for the diagnosis of TBM. CONCLUSION: The detection of ESAT-6 in the CSF of TBM patients by indirect ELISA is a promising method and can be used to develop an immunodiagnostic assay with increased sensitivity and specificity.

Cerebrospinal fluid Lyme multiplex assay results are not diagnostic in horses with neuroborreliosis.

BACKGROUND: The accuracy of the Lyme multiplex assay for the diagnosis of neuroborreliosis in horses is unknown. HYPOTHESIS/OBJECTIVES: To describe Lyme multiplex results in horses with a postmortem diagnosis of neuroborreliosis. The hypothesis was that paired serum and cerebrospinal fluid (CSF) results and a CSF : serum ratio would allow differentiation of horses with neuroborreliosis from those with other neurologic diseases. ANIMALS: Ninety horses that had neurologic examinations, serum and CSF Lyme multiplex analyses, and postmortem examination of the nervous system performed. METHODS: Retrospective study. Data collected included signalment, ante- and postmortem diagnoses, and serum and CSF Lyme multiplex results. The CSF : serum ratio was calculated by dividing CSF median fluorescent intensity (MFI) by serum MFI for each result. RESULTS: Ten horses had a final diagnosis of neuroborreliosis, 70 were diagnosed with other neurologic diseases, and 10 had no neurologic disease. Not all horses with neuroborreliosis had positive results: 4/10 had at least 1 positive serum result, 5/10 had at least 1 positive CSF result, and 3/10 had at least 1 CSF result 4-fold higher than the corresponding serum result. Results were similar for the 70 horses with other neurologic diseases: 53% had at least 1 positive serum result, 50% had at least 1 positive CSF result, and 16% had at least 1 CSF result 4-fold higher than the corresponding serum result. CONCLUSIONS AND CLINICAL IMPORTANCE: Positive Lyme multiplex results were common in horses with neurologic diseases and did not adequately differentiate horses with neuroborreliosis from horses with other disorders.

Generation and application of DNA aptamers against HspX for accurate diagnosis of tuberculous meningitis.

Tuberculous meningitis (TBM) is the most severe manifestation of tuberculosis and its diagnosis remains a challenge even today due to the lack of an adequate test. HspX antigen of Mycobacterium tuberculosis was previously established as a reliable diagnostic biomarker for TBM in an ELISA test format using anti-HspX polyclonal antibodies. Towards overcoming the limitations of batch-to-batch variation and challenges of scalability in antibody generation, we utilized Systematic Evolution of Ligands by EXponential enrichment (SELEX) to develop high affinity DNA aptamers against HspX as an alternative diagnostic reagent. Post-SELEX optimization of the best-performing aptamer candidate, H63, established its derivative H63 SL-2 M6 to be superior to its parent. Aptamer H63 SL-2 M6 displayed a specific and high affinity interaction with HspX (Kd ∼9.0 × 10-8 M). In an Aptamer Linked Immobilized Sorbent Assay (ALISA), H63 SL-2 M6 significantly differentiated between cerebrospinal fluid specimens from TBM and non-TBM subjects (n = 87, ***p < 0.0001) with ∼100% sensitivity and ∼91% specificity. Notably, ALISA exhibited comparable performance with previously reported antibody-based ELISA and qPCR. Altogether, our findings establish the utility of HspX aptamer for the reliable diagnosis of TBM and pave the way for developing an aptamer-based point-of-care test for TBM.

Rapid diagnosis of tuberculous meningitis: a comparative evaluation of in-house PCR assays involving three mycobacterial DNA sequences, IS6110, MPB-64 and 65 kDa antigen.

A PCR was standardized for amplifying three different mycobacterial--IS6110, MPB-64, 65 kDa DNA sequences. A comparative evaluation of the three PCR assays was carried out for the rapid diagnosis of tuberculous meningitis (TBM) using cerebrospinal fluid (CSF) specimens. While the IS6110 PCR was a single-step amplification reaction, the MPB-64 and 65 kDa antigen PCR assays were nested reactions. A total of 176 cerebrospinal fluid (CSF) samples from 176 patients were subjected to amplification of the three different mycobacterial sequences. Amongst them, 45 samples were obtained from confirmed cases of TBM (culture positive) and 56 samples were obtained from clinically suspected cases of TBM which were culture-negative. The remaining 75 CSF samples were categorized under the non-infectious and infectious illness of the central nervous system (CNS). Against a gold standard of culture, a sensitivity of 98% (NPV=99%) and a specificity of 100% (PPV=100%) was observed with the IS6110 PCR. Among the nested PCRs, a sensitivity of 91% (NPV=94%) and a specificity of 91% (PPV=85%) was observed with the MPB-64 assay, while the 65 kDa protocol had an associated sensitivity of 51% (NPV=76%) and a specificity of 92% (PPV=79%). These findings suggest that among the nested PCR assays, the MPB-64 PCR assay was associated with an enhanced degree of sensitivity and was comparable in terms of specificity. Our study also demonstrates that the IS6110 assay, while being a single-step PCR had the advantage of being a rapid test for the diagnosis of TBM, with increased sensitivity and enhanced specificity as compared to the nested PCR protocols.