Latex Agglutination Test for Early Detection of Causative Organism in Acute Bacterial Meningitis

Address for correspondence Dr. Baliram Mishra Head of Department of Paediatrics Janakpur Zonal Hospital Janakpur, Nepal E-mail: balirammishra@yahoo.com How to cite this article ? Mishra B, Mahaseth C, Rayamajhi A. Latex Agglutination Test for Early Detection of Causative Organism in Acute Bacterial Meningitis. J Nepal Paediatr Soc 2013;33(1):34-38. Latex Agglutination Test for Early Detection of Causative Organism in Acute Bacterial Meningitis


Introduction
A cute bacterial meningitis (ABM) is a major public health problem.It is one of the most important causes of mortality and morbidity in children.ABM accounts for an estimated annual 5, 00,000 cases worldwide with at least 50,000 deaths and an equal number of neurological disabilities 1 .Even with best of care and antibiotic therapy, case fatality rates remain at 5% to 10% in industrialized countries and can reach up to 20% in developing countries.Between 10% and 30% of survivors develop permanent neurological sequelae such as paralysis, epilepsy, cognitive defi cit or sensorineural deafness 2 .
Identifi cation of the causative organism in ABM is crucial to its management as it infl uences the choice of antibiotics, duration of therapy as well as the disease outcome.Three organisms namely Neisseria meningitidis, Streptococcus pneumoniae and Hemophilus infl uenza account for more than 80% of cases of ABM 3 .The diagnosis of meningitis in a suspected case is based on the analysis of cerebrospinal fl uid (CSF) obtained by performing a lumbar puncture 4 .The CSF is examined for turbidity, white blood cell (WBC) count, Gram stain and the amount of protein and sugar.Bacterial culture of the CSF has long been the gold standard for the diagnosis of ABM, but due to its poor yield, longer time and unavailability in a resource limited country like ours we decided to conduct this study to see the usefulness of latex agglutination test (LAT) in the early diagnosis of ABM.
LAT relies on the detection of soluble antigen in the CSF.Pre-treated latex beads agglutinate in the presence of the bacterial antigen, allowing rapid visualization of positive agglutination to the naked eye.In contrast to the hours spent on bacterial culture LAT can be completed in approximately 20 min (including the time taken to heat and centrifuge the CSF sample).The LAT can differentiate between N. meningitidis serogroups A, C and Y/W135, N. meningitidis serogroups B/Escherichia coli, Hemophilus infl uenzae, Streptococcus pneumoniae and Group B Streptococcus hence allowing prompt institution of directed therapy that is specifi c for the bacteria detected.Inclusion and exclusion criteria.All children between the age of 2 months to 14 years who fulfi lled the case defi nition of a suspected case of ABM and had no exclusion criteria were included in the study.Children in whom lumbar puncture was contraindicated or refused; those receiving immunosuppressant's; those with a history of having undergone a neurosurgical procedure and those who had received parenteral antibiotics for more than 72 hours were excluded from the study.

Case defi nitions
Suspected case.A child between 2 months to 14 years of age presenting with clinical features of meningitis namely fever and/or headache and/or neck stiffness and/or bulging fontanel and/or altered sensorium and/or convulsions.

Confi rmed case.
A suspected case whose CSF has at least one of the following fi ndings -(1) elevated protein to more than 40 mg/dl, (2) decreased glucose to less than 40 mg/dl, (3) a CSF to serum glucose ratio of less than 0.6, (4) a total leukocyte count of more than 10 cells/mm 3 with neutrophillic predominance, (5) organism in Gram's stain or bacterial culture.
Laboratory methods and data analysis.After obtaining written consent lumbar puncture was done using sterile technique (thorough cleaning of local part using povidone iodine and then alcohol skin preparation; sterile disposable needle and collection tubes).Two millilitres of CSF was collected in three different clear sterile plastic collection tubes and immediately transported to the laboratory for analysis of cells, sugar, protein, Gram's stain, bacterial culture and LAT.For bacterial culture 20 μL of CSF was inoculated into plates of blood agar and chocolate agar and incubated.All plates negative for growth after 48 hours were considered culture negative and discarded.LAT was done using the BD Directigen TM

Results
CSF from 150 consecutive clinically suspected cases of ABM between the age group of 2 months to 14 years were analyzed.Bacterial culture and LAT was done on CSF obtained from all 150 suspected cases of ABM.38 (25.3%) patients were confi rmed cases of ABM as per the case defi nition.Of the 150 CSF samples analysed organisms could be identifi ed in 29 cases as shown in Figure 1.

Discussion
ABM in children is a potentially life threatening condition requiring early diagnosis, prompt identifi cation of the causative organism and institution of effective therapy 5 .The yield of bacterial culture in our country is low, hence there was need to evaluate a test that could perform better and could rapidly identify the causative organism of ABM.Ours is the fi rst study to evaluate LAT against bacterial culture in Nepal.

Streptococcus pneumoniae, Hemophilus infl uenza
and Group B Streptococcus were the commonest causative organisms isolated in our study.This is in accordance to the organisms isolated from CSF in other studies 6,7,8,9 .Seasonal variation in the incidence of meningococcal meningitis and vaccination against it might have accounted for the fact that Neisseria meningitidis was not isolated in any of the CSF samples in our study.Of the 150 CSF samples analysed culture could identify organism in only 4 and LAT in 29 giving an isolation rate of 1.3% and 19.3% respectively.The sensitivity of LAT (60.5%) was higher than that of CSF culture (10.5%).Similar results have been observed in other studies with sensitivity of LAT ranging from 60 to 95% and specifi city from 70 to 98% 10,11,12,13,14 .However there have been confl icting results in the literature regarding the utility of LAT with studies suggesting that it is no better than using Gram's stain and CSF culture together 15,16,17 .These studies are mostly from countries where the yield of CSF culture is high as compared to our country.
One of the drawbacks of our study is the fact that we did not include Gram's stain in our study protocol.Secondly, in the defi nition of confi rmed case only 4 had positive culture and the rest were classifi ed as confi rmed on the basis of CSF criteria's which are not the gold standard for the diagnosis of ABM and this might have affected the sensitivity and specifi city of LAT.Thirdly, we did not look into how early diagnosis using LAT affected disease outcome in terms of mortality, reduction in the duration of hospital stay, cost of therapy and unnecessary use of empirical antibiotic therapy instead of specifi c therapy based on the organism identifi ed.However this study forms a foundation on the basis of which further studies to look into cost effectiveness of using LAT can be performed.Study to see whether early detection of organism and subsequent specifi c therapy brings about a decrease in mortality and morbidity, duration of hospital stay, antibiotic resistance needs to be done.
The strength of our study is that in a country where the yield of culture is low and where culture is not readily available simple and rapid test like the LAT can be used in the peripheral set up to promptly identify the organism and start specifi c therapy.This becomes especially more important as steroids have a role in reducing mortality and morbidity in ABM due to Hemophilus infl uenza type b if they are given for the fi rst 2 days.Secondly, LAT can be extremely useful if there is an outbreak of ABM.It is yet to be studied whether the implementation of LAT in the peripheral hospitals will bring about an effect similar to that brought by the use of Rapid antigen capture test for Plasmodium falciparum malaria.Though the technology for detection of bacterial antigen in CSF by latex agglutination is still imperfect for widespread use, it still can have a major impact in a country like ours.
In conclusion as LAT has a better yield, provides microbiological diagnosis earlier than the traditional CSF culture and is easy to perform, it should be provided by the government as a part of national programme to the peripheral centres.

Fig 1 :
Fig 1: Showing the causative organisms of acute bacterial meningitis.
Meningitis Combo test kit (Becton, Dickinson and company, USA) for Streptococcus pneumoniae, group B Streptococcus, Escherichia coli, Neisseria meningitidis group A,C and Y/W135, and Hemophilus infl uenzae type b.CSF of all suspected cases enrolled in the study underwent bacterial culture and LAT.Data was analysed by using SPSS Version 11.5.Sensitivity, Specifi city, Positive and Negative predictive values were calculated.Fischer's exact test and chi square test with Yate's correction were performed to assess statistical signifi cance of relationship between variables and meningitis.A 'p' value of <0.05 was considered signifi cant.

Table 1 :
Organisms identifi ed by CSF culture and LAT Group B Streptococcus.The sensitivity, specifi city, positive and negative predictive values of bacterial culture and LAT has been shown in table2,3,4.

Table 2 :
Performance of Latex agglutination test

Table 3 :
Performance of bacterial culture of cerebrospinal fl uid.

Table 4 :
Comparison of performance of CSF culture and Latex agglutination test.