Diagnosis of Bacterial Meningitis and AMR Profile Using Molecular and Immunological Techniques

Background: Bacterial meningitis (BM) is severe complication of central nervous system (CNS) and is often associated with high mortality and morbidity rates if not timely diagnosed and treated. Current diagnostic tools for BM and drug resistance suffer from lack of sensitivity due to paucibacillary nature of Cerebrospinal fluid (CSF). Objectives: The objective of the study is to develop rapid and efficacious immunological tools (Enzyme linked immunosorbent assay and Lateral flow) for diagnosis of BM in hospital settings. In addition, in-house molecular assays will be developed for diagnosis of extended spectrum beta lactamases and Carbapenems drug resistance in community and hospital acquired BM infection Methodology: A prospective observational study will be carried out in patients admitted in IPD awards of CIIMS, Nagpur. Diagnosis of BM will be done using conventional gold standards and by in-house designed nested polymerase chain reaction (PCR) for 8 etiological pathogens. An inhouse ELISA assay will be standardized and optimized based on culture filtrates antigen isolated from clinical isolates of etiological pathogens associated with BM for immunological diagnosis. For diagnosis of drug resistance, in-house conventional PCR assay targeting ESBL (TEM, SHV and CTX-M) and Carbapenems (NDM, OXA-48, VIM) resistance genes will be standardized and Study Protocol Parsodkar et al.; JPRI, 33(38A): 89-105, 2021; Article no.JPRI.71217 90 optimized. The sensitivity and specificity of developed test will be determined by standard ROC curve using medical software. The diagnostic accuracy will be determined based on concordance with gold standards. Expected results: A simple, rapid ELISA assay will be developed for etiological diagnosis BM in hospital settings. In addition, in house molecular assay will be developed for rapid identification of ESBL and Carbapenems drug resistance in BM cases. Conclusion: A simple, rapid, efficacious immunological and molecular tools for diagnosis of BM and drug resistance will be developed for improved management of cases in hospital settings as governance of bacterial meningitis in India is poor.


INTRODUCTION
Bacterial meningitis (BM) remains a significant cause of concern worldwide due to associated mortality and lasting neurological sequelae. In spite of the recent development of potent new antibiotics, the mortality rate due to BM is found to be significantly high ranging from 16-32% in India owing to the lack of awareness and diagnosis [1]. Meningococcal disease, in India, is endemic in New Delhi and sporadic cases have occurred in Uttar Pradesh, West Bengal, Haryana, Gujarat, and Orissa [2,3]. Various demographic studies have so far revealed that several pathogens are responsible for causing meningitis depending on the patient's age group [4,5,6].
The diagnosis of bacterial meningitis is however still very low in resource poor settings and developing countries established on basis gram staining and culture [7,8]. The diagnostic accuracy of these investigations is, more often than not, affected by the use of antibiotics administered prior to the lumbar puncture and due to paucibacillary nature of etiological agents in CSF [9,10]. The use of molecular techniques like polymerase chain reaction (PCR) for the expeditious diagnosis of BM has the potential to overcome the poor sensitivity of culture when antibiotics have already been introduced but even so PCR is not available in many parts of developing countries [11,12]. The rapid detection and estimation of the severity of the disease are important factors, both in diagnosis and initiating the treatment [13].
Moreover in recent years, increasing trend in cases of Antimicrobial resistance in etiological agents of BM has added a fatal blow to the already limited treatment leading to increase in morbidity of infected cases [14]. In India, resistance to Carbapenems and β-Lactams has increased due to Extended-spectrum β-Lactamases (ESBL) production among members of gram negative organisms. Moreover, the commercially available Drug susceptibility testing techniques for detection of drug resistance are not only dependent on culture positivity but are also exorbitant for common public making the treatment of this disease even more strenuous and challenging [15,16] .
Simpler yet accurate diagnostic techniques should be introduced that can help overcome this drawback as they do not require a sophisticated lab setup and over the odds instruments so that diagnosis and treatment can reach the low resource setting areas of our country where appropriate facilities are not available.
Rapid and efficacious techniques like ELISA and lateral flow can be developed and introduced in such remote locations in order to help overcome the limitation of diagnosis. This culture secretome of an organism contains cocktail of several bacterial proteins, with broad yet specific immune response. The antibodies against such secretome may be detected using simple ELISA based test which are rapid yet cost effective and even have scale up capacity to be converted into rapid point of care in form of lateral flows. Development of specific yet cost effective inhouse molecular technique can help reduce the burden of AMR and subsequently the challenges faced in treatment of BM infection.
Such assays can aid clinicians in bedside screening for BM and initiate specific treatment in suspected cases to avoid complications associated with empirical regimes.

Study Design
Present prospective study will be carried out at IPD wards of CIIMS, Nagpur. Patients suspected of suffering form BM will be routinely screened using pre designed in house semi-nested PCR by Bhagchandani et al for detection of bacterial meningitis [17].

Nested PCR
DNA Extraction Protocol: The reagents and instruments used in laboratory for DNA extraction are mentioned in Table 1 and Fig. 1 [17].
The schematic flowchart of DNA extraction can be seen in Fig. 2.

16s rDNA PCR
In developing countries, the advanced technique for detection of broad range 16s rDNA gene in bacteria is being introduced in recent years, though not very extensively. [17] Reagents and figures required for 16s rDNA protocol can be seen in Table 2 and Fig. 3 respectively. Table 3 depicts the sequence of primers used while the PCR protocol can be studied in Table 4 and amplification program consecutively in Table 5.

Nested protocol for detection of 8 bacterial species
Using the purified 16s PCR product as DNA template, prepare a PCR reaction mix (20 μL) to perform PCR amplification in thermal cycler (Applied Biosystems). Run the obtained product on 2% Agarose gel containing Ethidium bromide for visualization of bands on a gel documentation system (Bio-Rad Laboratories). [17] Primers used for the Nested PCR have been mentioned in Table 6 while PCR protocol and Amplification protocols of 8 organisms can be studied in Table  6, Table 7, Table 8, Table 9, Table 10, Table 11 and Table 12 respectively. Amplification programs for thermal cycler:

PCR protocol for Antimicrobial Resistance (AMR) in gram negative bacteria
Antibiotic resistance can be termed as the reduction in the overall effectiveness of a drug in treating an infection or a disease. Most common type of resistance is seen in gram negative bacteria against Beta Lactams antibiotics and Carbapenems. Literature based primers for standardization of PCR for diagnosis of antimicrobial resistance genes are mentioned in Table 13.

Primers and Amplification Protocol for PCR:
The inhouse primers for these genes will be designed in later stage of study and optimization will be done by changing concentrations of primers and DNA templates in a standard PCR protocol.

Gel Electrophoresis
Materials and reagents required for visualization of PCR products on 2% agarose gel electrophoresis are mentioned in Table 14. 2 g Agarose will be dissolved in 100 mL of 1X TAE buffer. Boil until a clear solution is obtained. Add 12 uL of 1mg/mL of Ethidium Bromide to it. Pour the gel into casting tray and allow solidifying with casting combs. Mix the PCR products with 6X Gel Loading dye and load into the wells with 5 μL of 100 bp DNA ladder. Allow the samples to run at a constant voltage of 150V and then observe the gel under UV light or in a Gel documentation system.    Alternatively, different protocols for cell lysate extraction will be processed and optimized to develop a clinically suitable protocol for protein extraction. Some of the methods are listed in Table 15.

Protein quantification
Determine the exact concentrations of micro proteins in the whole cell lysates by Nanodrop technique (Thermofisher, USA).

Procedure
1. Start the program on Nanodrop software module 2. Set Blank using distilled water or TE buffer based on diluents used in sample.

Add 2μL of distilled water/TE buffer on the
Nanodrop instrument pedestal and click Blank 4. A 2μL sample size is recommended for protein measurements. Add 2μL extracted whole cell lysate on the Nanodrop pedestal and select the tab labelled Measure 5. The software module measures concentration of protein (mg/mL) along with purity ratio 6. Dilute the lysates to the concentration of 1mg/mL before proceeding to ELISA.

Elisa
Enzyme-linked immunosorbent assay (ELISA) works based on an enzyme system to provide distinct combination of an antibody to its specific antigen. This method is used for quantifying an antigen immobilized on a solid surface like a micro-well tray. ELISA technique uses a very specific secondary antibody which has an enzyme coupled covalently to it. Fig. 6 shows the diagrammatic representation of ELISA technique and Fig. 7 shows the equipments required for ELISA technique in laboratory.

Standardization of ELISA technique
1. Standardization of the ELISA will be conducted by using Checker board method to know the exact concentration of antigen and secondary antibody in which the results can be optimal.

Several dilutions of antigens (WCL) and
antibody (IgM) will be used. Each dilution of antibody should be used against each dilution of antigen prepared as can be studied in Fig. 8     Add 100 μL substrate to each well followed by incubation at RT for 1 minute. 9. Add stop solution after 1 minute and measure OD at 450 nm on an ELISA plate reader. 10. Repeat the same procedure for wells coated with the concentration of 10 ng/mL, 20 ng/mL, 50 ng/mL and 100 ng/mL. 11. Based on the results obtained, the appropriate dilution of antigen and secondary antibody should be decided for further processing of clinical samples.

Coating of ELISA plates
1. Calculate the number of ELISA wells for the samples in duplicate 2. For coating the wells, add 1.2μL of 1mg/mL of cell lysate to 12 mL of 1X PBS 3. Dispense 100 μL of the above dilution in each well to be coated and then incubate for 3 hours at 37˚C 4. Post incubation, wash theses wells with the help of wash buffer for 1 min. 5. For blocking the antigens dispense 0.5% BSA in the sample wells and incubate for 2 hours at 37⁰C. 6. Wash the plates using wash buffer for 1 minute and then dry the wells by gently tapping the ELISA plates on tissue paper. 7. Cover the plate with tissue and ELISA plate cover. Store in inverted manner at 4⁰C.

ELISA protocol for Antibody detection
1. Before starting the procedure, wash the coated wells twice with wash buffer. 2. Dispense the optimized amount of CSF in duplicates for accurate results and incubate for 35 minutes at 37˚C 3. Follow this by washing thrice with wash buffer 4. Add the optimized amount of secondary antibody to all the wells and incubate as above. 5. Wash the wells again 4 times with wash buffer before adding 100μL substrate to each well and incubate for 1 minute 6. Add Stop solution and measure OD at 450nm 7. This protocol will be followed through in order to develop the ELISA technique for the 8 bacteria included in the study individually.

Participants
The recruitments of participants with clinical as well as baseline criteria can be seen in Fig. 9 also depicting clinical inclusion and exclusion criteria designed for the present study.
The reference test in the present study will be Microbiological culture, the universal gold standard for diagnosis of Bacterial meningitis. If in case, the microbiological culturing yields undetermining results, the results of in house Molecular PCR technique developed by Sharda Bhagchandani et al will be considered as reference. For the molecular tests developed to study antibiotic resistance, the reference standard will be automated BD Phoenix system for drug susceptibility testing. The clinical information and results of reference standards and index tests will be available to the performer, reader, assessor alike.
The test positivity cut off for serology based technique can be defined as that value, where above if the result obtained, will be considered positive. There will be no cut off determination required for the molecular tests. For the test that will be developed based on ELISA, test cut-offs, sensitivity and specificity will be determined by ROC using the MedCalc statistical software (10.1.2.0).

Analysis
Test concordance between molecular and serological tests developed will be assessed using the kappa (κ) statistic. Indeterminate test or reference standard will be handled by repeated evaluation of the particular test until valid results are obtained. Missing data for index test or reference standard will be dealt with retrospective follow up via telephonic conversations.
The sample size of the study has been determined by a two-stage sampling approach adopted using R 3.4.3-programming tool, taking into consideration the prevalence of 15% and the confidence interval of 95%, a sample size of 307 was determined.

Test Results
The distribution of BM in patients with suggestive condition will be studied by the effects of the disease observed in the patient as well as by studying the reports of routine analysis performed for CSF after lumber puncture. The higher the amount of total cells present in the CSF more will be the severity of infection. This can further be determined by testing the organisms causing infection for antimicrobial resistance as multidrug resistant bacteria generally tend to cause more severe form of infections due to lack of appropriate treatment. The time interval from the point of patient's admittance to diagnosis will depend from case to case.
The patient after being suspected or diagnosed of a neurological complication will be proceeded for lumbar puncture and the CSF sample will be received for diagnosis of BM using predesigned in-house nested PCR technique at the same time, a portion of this will be sent for CSF routine analysis for studying aspects including Total cell count, Differential cell count, CSF sugar, CSF protein, CSF lactate and more. This can also be helpful in preliminary diagnosis of Bacterial Meningitis. The positivity will be determined based on results obtained on gel electrophoresis following 16s rDNA and Nested PCR amplifications of the extracted DNA.
ELISA technique will be developed using the whole cell lysate obtained by the optimized protocol of protein extraction. The lysate containing antigens will be used for coating the ELISA wells which subsequently help in identifying the antibodies present in the sample. Standardization of the ELISA technique will be performed according to the above mentioned Checkerboard method and the evaluation of the standardized method will be further conducted using the positive and negative samples characterized by nested PCR technique.
The frequency of the demographic, baseline and clinical factors will be measured on a nominal scale. MedCalc statistical software (version 10.1.2.0) will be used to perform statistical analysis. Baseline data will be compared in the fore mentioned groups using Chi square analysis and a difference of P < 0.05 will be considered to be significant. Negative predictive and positive predictive value will be estimated using a 2 × 2

DISCUSSION
In the proposed study, the aim is to aid the diagnosis and treatment of BM in low resource setting areas and tertiary care hospitals by development of rapid, efficacious yet affordable techniques for detection. Initial screening will be conducted on the basis of clinically structured proforma and the patients suspected of BM will be further diagnosed on basis of Nested PCR tool developed by Bhagchandani et al. [17].
Moreover, data of antimicrobial resistance in BM samples is severely lacking in India and needs to be studied. Molecular based PCR techniques that can detect these antimicrobial resistant genes with specifically targeted primers will tremendously help in antibiotic stewardship programs [18][19][20].

CONCLUSION
ELISA for diagnosis of BM will be developed using the whole cell lysates of the organisms which contain an array of bacterial proteins. These proteins will be used for detection of the infection with specific antibodies present in the sample. This semi quantitative test can be performed in matter of few hours thus initiating appropriate treatment to prevent co-morbidities incurred. A few commercial ELISA tests available have a disadvantage of being expensive which cannot be sustained by a common person [21][22][23]. This can be overcome by development of affordable inhouse ELISA that can produce accurate results in a short period of time. The lysates can also be scaled up to development of point of care tests like lateral flow for diagnosis in remote areas where sophisticated laboratory cannot be established [24][25][26][27].

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
Admitted patients suggestive of BM will be recruited after taking written consents and verbal explanation about the study using predefined clinical inclusion and exclusion criteria.

ETHICAL APPROVAL
As per international standard or university standard written ethical approval has been collected and preserved by the author(s).