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ORIGINAL ARTICLE
Year : 2002  |  Volume : 20  |  Issue : 4  |  Page : 183-186
 

Pneumolysin in urine: A rapid antigen detection method to diagnose pneumococcal pneumonia in children


Department of Microbiology, BP Koirala Institute of Health Sciences, Dharan, Nepal

Correspondence Address:
Department of Microbiology, BP Koirala Institute of Health Sciences, Dharan, Nepal

 ~ Abstract 

PURPOSE: Etiological diagnosis of pneumococcal pneumonia is difficult in small children in whom blood culture cannot be done or who have already been started on antibiotics. A simple technique which can be applied at the bedside or in the outpatient department may help in obviating this problem. Detection of pneumolysin, a product of invasive pneumococci is being exploited as a diagnostic tool. METHODS: An attempt was made to detect this protein in urine of seventy children, clinically suspected and radiologically diagnosed cases of pneumonia. Seventy age and sex matched controls were included in the study. Purified pneumolysin was prepared from clinical isolates of invasive pneumococcal infections. This was used to raise polyclonal antisera in rabbits. The antisera was used to sensitise Cowan 1 Staphylococcus aureus (CoA). A slide agglutination was performed with 25 L urine and equal quantity of the reagent. RESULTS: Results were compared with CoA reagent sensitised with antisera raised against a genetically derived pneumolysoid and capsular polysaccharide for antigen detection in the urine. Pneumolysin could be detected in 42.9% (30/70) urine samples from cases with pneumonia by the genetically derived antigen and in 37.1% samples by the in house prepared antigen, in contrast to 2.1% in healthy controls and 4.2% in children with infections other than pneumonia. The result was statistically significant. Detection of pneumolysin was slightly better than detection of capsular polysaccharide antigen in urine although the result was not statistically significant. Blood culture proved to be positive in only 29.5% cases. CONCLUSIONS: Pneumolysin detection in urine showed promising results and was found to be simple and rapid. It will help in quickening the diagnosis of pneumococcal pneumonia.

How to cite this article:
Rajalakshmi B, Kanungo R, Srinivasan S, Badrinath S. Pneumolysin in urine: A rapid antigen detection method to diagnose pneumococcal pneumonia in children. Indian J Med Microbiol 2002;20:183-6


How to cite this URL:
Rajalakshmi B, Kanungo R, Srinivasan S, Badrinath S. Pneumolysin in urine: A rapid antigen detection method to diagnose pneumococcal pneumonia in children. Indian J Med Microbiol [serial online] 2002 [cited 2020 Oct 31];20:183-6. Available from: https://www.ijmm.org/text.asp?2002/20/4/183/6953


Streptococcus pneumoniae is a leading cause of morbidity and mortality worldwide, especially among age extremes and people with underlying diseases.[1] Despite appropriate antibiotic therapy, mortality from bacteremic pneumococcal pneumonia is high and there is fear that the rates of complication and death may increase with increasing resistance of  S.pneumoniae   to commonly used antibiotics. This factor has led to rekindling of interest in establishment of etiology of the disease in clinical setting. Establishing the etiology of pneumonia has posed a problem. Bacterial agents have been cultured in only 10-30% children with acute respiratory infections, including those hospitalized.[2] More recently, methods of antigen detection and serological tests for antipneumococcal antibody have been used. These tests have incriminated S.pneumoniae as the etiological agent in larger proportion of cases, when compared to conventional laboratory techniques.[3] Attempts for improving the diagnostic procedures are still continuing.
Antigen detection assays have several advantages over bacterial culture. Samples for antigen detection usually do not need any special transport media and the tests are simple, and can be performed directly after sampling. These assays are less influenced by concomitant use of antimicrobial drugs unlike culture.[4]
Pneumolysin is an intracellular 53-kDa protein, elaborated by S.pneumoniae. It is produced by all strains of invasive pneumococci and hence is an ideal target for antigen detection. Less than 5% cross-reaction between streptolysin and pneumolysin has been seen.[5] On the other hand, capsular polysaccharide is a type-specific antigen of pneumococci which cross-reacts with the antigens of viridans streptococci.[6] Pneumolysin has been detected from sputum samples by Western blot and enzyme linked immunosorbent assay (ELISA). Neutralisation tests and ELISA have also been employed in detecting antibodies against pneumolysin.[5],[7],[8],[9] In this study, an attempt was made to evaluate the suitability of pneumolysin as an antigen detection method in urine for diagnosing pneumococcal pneumonia in children, and to compare the results with the detection of capsular polysaccharide antigen.

 ~ Materials and Methods Top

Subjects
Study population included clinically suspected and radiologically diagnosed cases of pneumonia between the age group of 1-10 years. Seventy one such cases admitted to the paediatric ward of JIPMER, a tertiary care hospital, during the study period of December 1998 to December 2000 were included. All the children enrolled had a duration of symptoms of less than 4 weeks, with fever >390C, chest in drawing, refusal to feed and increased respiratory rate above normal. Severity of infection was noted and children were accordingly admitted for intensive care or general management. Exclusion criteria included cases of respiratory infections associated with any other co-morbid conditions such as immunosuppression, chronic infections, diarrhoea and other metabolic or endocrine disorders.
Controls comprised of two groups. First group included seventy age and sex-matched children with nonpneumococcal infections. These were children, clinically diagnosed and laboratory confirmed cases of tuberculosis, enteric fever, malaria, hepatitis and diarrhoea with septicaemia. The second group of healthy controls comprised of 46 children from a nearby school, who did not present with any infection.
Blood and urine samples were collected from cases, and urine alone was collected from healthy controls. Blood was cultured in 5% CO2 and the organisms isolated were identified by standard techniques.[10] Urine samples were centrifuged at 12,000g for 15 minutes and the supernatant was taken for antigen detection. Those samples which could not be tested immediately were stored at -70C till further use.
Preparation of antigens
Pneumolysin (Ply) was prepared from fresh clinical isolates of S.pneumoniae belonging to serotypes 1, 6 and 19 according to the method described by Shumway and Klebanoff.[11] These serotypes were earlier implicated to be predominantly involved in invasive infections in a multicentric study in India.[12] Briefly, the clinical isolates of pneumococci were inoculated in brain heart infusion broth supplemented with glucose and cysteine and incubated at 37C for 18 hours, followed by centrifugation at 18,000g at 4C. The sediment was washed with cold phosphate buffered saline (PBS) and sonicated at 8 amp for 10 minutes. The disrupted microorganisms were centrifuged and the clear supernatant solution was subjected to acid precipitation followed by 60% ammonium sulphate saturation, adsorbed on to diethyl aminoethyl (DEAE) cellulose column, eluted and concentrated by ultrafiltration. The concentrated pneumolysin was then applied to a column of Sephadex G-100 and eluted with PBS. Eluted pneumolysin was once again concentrated by ultrafiltration. The protein content of the derived pneumolysin was found to be 0.4 mg/mL by Lowry's method.
Molecular weight of the purified pneumolysin was determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Twenty micrograms of the purified pneumolysin was heated for 5 minutes in a boiling water bath and then dissolved in the chamber buffer supplemented with SPS and DTT. Molecular weight markers (Sigma, USA) used were phosphorylase b: 97,400; bovine albumin: 66,000; ovalbumin: 45,000 and carbonic anhydrase: 29,000.
To compare our pneumolysin, we also used a genetically derived pneumolysoid (Plo), kindly donated by Dr. James Paton (Adelaide Children's Hospital, Adelaide, Australia), which was also subjected to SDS-PAGE along with the purified pneumolysin (Ply). The molecular weight of the Ply extracted in house, was compared with that of the standard Plo and was found to be approximately 52 kDa. Haemolytic activity of the pneumolysin was determined by the method of Kanclerski and Mollby.[14] Serial two fold dilutions of the pneumolysin were prepared with PBS (pH 7.4) supplemented with 10mM dithiothreitol 0.1% for 30 minutes. Well showing 50% lysis was taken as the end-point and the titre determined which was found to be 256. Capsular polysaccharide antigen (CPS) was the commercially available 23 valent pneumococcal vaccine (Pneumo-23, Pasteur Merieux, Lyon- France). Polyclonal antisera to pneumolysin (Plo and Ply) and capsular polysaccharide were raised in rabbits acccording to the method described by Cima-Cabal et al.[15] Co-agglutination reagent was prepared according to the method described by Lalitha et al.[16] Ten percent (vol/vol) suspension of formalinised, heat killed Cowan -I strain of  S.aureus   was sensitized with optimum sensitizing doses of anti-Plo, anti-Ply and anti-CPS antisera separately.
Test Procedure
Twentyfive microlitres of centrifuged urine sample was mixed with 25 mL of the respective Co-A reagent on a black tile and rotated on a flat surface at room temperature. Positive test was indicated by clumping of the cells with a clear background within 3 minutes and uniform turbidity indicated a negative result. Unsensitized Co-A cell suspension was used as the negative control. Statistical analysis was done by chi-square test for proportions with Yate's correction using Epi info statistical package version 6.0.

 ~ Results Top

Pneumolysoid proved to be slightly better in the antigen detection system with 42.9% (30/70) urine samples being positive with antisera raised against Plo and 37.1% (26/70) with Ply antisera. Capsular polysaccharide was detected in 38.6% (27/70) as shown in [Table - 1]. When we compared two different sources of pneumolysin used to raise antisera for the detection of antigen by Co-A, it was found that the genetically derived pneumolysoid was better than the purified pneumolysin prepared in house (42.9% vs 37.1%). However, the differences were not statistically significant. Of the 46 healthy controls, one (2.1%) was positive for Plo antigen, giving a specificity of 97.8% [Table - 2].
A specificity and sensitivity of 61.2% and 52.3% for Plo and 67.3% and 52.3% for CPS respectively was observed by Co-A in urine [Table - 3]. The tests had an efficiency of 58.5%, 64.2% and 62.8% for Ply, Plo and CPS respectively when compared with culture. Blood culture proved to be positive for S.pneumoniae in only 29.5% of clinically diagnosed cases of pneumonia. The negative predictive value of the tests ranged between 75 -77.2%.

 ~ Discussion Top

Antigen detection has been attempted by various workers to simplify diagnosis of many infectious diseases. Pneumococcal antigen detection too has been widely investigated. Rates of antigen detection varied in different studies. Lower sensitivity (12%) was achieved for co-agglutination of urine samples by Burman et al[17] for CPS antigen. A higher sensitivity at 74% and 79% were achieved by Whitby et al[18] and Burman et al[17] respectively, by co-agglutination in sputum samples. Ortqvist et al[19] have reported a sensitivity of 63% for the detection of CPS by co-agglutination in sputum samples.
In the present study, pneumolysin was detected in greater number of cases than the capsular polysaccharide antigen of S.pneumoniae, in the urine of children with bacterial pneumonia. Detection of pneumolysin antigen, which is common to all pneumococci, appears to be better than the type-specific capsular polysaccharide which shows cross reactions with other streptococcal species.[6]
Employing pneumococcal vaccine to raise antisera was found to be an alternative for the use of omniserum. Most of the invasive serotypes of S.pneumoniae have been included in the currently available pneumococcal vaccine.
This study confirms that although culture is a reliable method in well collected samples taken early in the illness before antibiotic treatment, antigen detection by screening techniques like co-agglutination assay will help in rapid diagnosis of pneumococcal pneumonia. Low sensitivity of our tests may have resulted from a low titre antisera used to sensitise the staphylococci. A high avidity antisera will overcome this difficulty. Presence of immunecomplexes in the urine could be the other reason for the low detection as the test was based on detection of free antigen. The test, however, in the present form, had a high negative predictive value and may be used to rule out pneumococcal pneumonia. 

 ~ References Top

1.Heffron R. Pneumonia with special reference to pneumococcal lobar pneumonia. (Harvard University Press, Cambridge), 1979:342.  Back to cited text no. 1    
2.Selwyn BJ. Coordinated data group of BOSTID researchers: Epidemiology of acute respiratory tract infections in young children: comparison of findings from several developing countries. Rev infect Dis 1990;12:S870-S888.  Back to cited text no. 2    
3.Febson DS, Musher DM, eskola J. Pneumococcal vaccine, In : Vaccines. Plotkin SA, Orenstein WA. (Ed), 3rd edn, (WB Saunders Company, USA) 1999: 553-607.  Back to cited text no. 3    
4.Lankinen KS, Salo P, Rapola S, Salo E, Takala AK, Leinonen M. Pneumococcal capsular antigen detection after enrichment culture : an alternative to culture methods in epidemiologic research. Am J Trop Med 1997;56:211-215.  Back to cited text no. 4    
5.Kalin M, Kanclerski K, Granstrom M, Mollby R. Diagnosis of pneumococcal pneumonia by enzyme-linked immunosorbent assay of antibodies to pneumococcal hemolysin (pneumolysin). J Clin Microbiol 1987;25:226-229.  Back to cited text no. 5    
6.Holmberg H, Danielsson D, Hardie J, Krook A, Whiley R. Cross-reaction between a - streptococci and Omniserum, a polyvalent pneumococcal serum, demonstrated by direct immunofluorescence, immunoelectroosmophoresis and Latex agglutination. J Clin Microbiol 1985;21:745-748.  Back to cited text no. 6    
7.Wheeler J, Freeman R, Steward M, Henderson K, Lee MJ, Piggott NH, Eltringham GJ, Galloway A. Detection of pneumolysin in sputum. J Med Microbiol 1999;48:863-866.  Back to cited text no. 7    
8.Kanklerski K, Blomquist S, Granstrom M, Mollby R. Serum antibodies to pneumolysin in patients with pneumonia. J Clin Microbiol 1988;26:96-100.  Back to cited text no. 8    
9.Bhaskar M, Kanungo R, Mahadevan S, Anil P, Badrinath S. Detection of antipneumolysin in invasive childhood pneumococcal infections. Indian J Med Res 1999;109:90-93.  Back to cited text no. 9    
10.Facklam RA Washington JA. Streptococci and related catalase negative gram positive cocci, In : Manual of Clinical Microbiology, 5th ed. Balows A, Hauser WJ, Herman KL, Isenberg HD, Shadomy HJ, (Ed). (ASM Press, Washington DC) 1991:238-257.  Back to cited text no. 10    
11.Shumway CN, Klebanoff SJ. Purification of pneumolysin. Infect immun 1971;4:388-392.  Back to cited text no. 11    
12.Invasive Bacterial Infection Surveillance (IBIS) Group, International Clinic Epidemiology Network (INCLEN). Prospective multicentric hospital surveillance of Streptococcus pneumoniae disease in India. Lancet 1999;353:1216-1221.  Back to cited text no. 12    
13.Lowery OH, Rosebrough OH, Farr AL, Randll RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-275.  Back to cited text no. 13    
14.Kanclerski K, Molby R. Production and purificaiton of Streptococcus pneumoniae hemolysin (pneumolysin). J Clin Microbiol 1987;25:222-225.  Back to cited text no. 14    
15.Cima-Cabal MD, Vazquez F, Toyos DL, Mendez FJ. Rapid and reliable identification of Streptococcus pneumoniae isolates by pneumolysin-mediated agglutination. J Clin Microbiol 1999;37:1964-1966.  Back to cited text no. 15    
16.Lalitha MK, Sridharan G, John M. Co-agglutination for diagnosis of bacterial infection. Indian J Pediatr 1989;56:327-333.  Back to cited text no. 16    
17.Burman LA, Trollfors B, Andersson B, Henrichsen J, Juto P, Kallings I, et al. Diagnosis of pneumonia by cultures, bacterial and viral antigen detection tests and serology with special reference to antibodies against pneumococcal antigens. J Infect Dis 1991;163:1087-1093.  Back to cited text no. 17    
18.Whitby M, Kristinsson KG, Brown M. Assessment of rapid methods of pneumococcal antigen detection in routine sputum bacteriology. J Clin Pathol 1985; 38:341-344.  Back to cited text no. 18    
19.Ortqvist A, Jonsson I, Kalin N, Krook A. Comparison of three methods for detection of pneumococcal antigen in sputum of patients with community acquired pneumonia. Eur J Clin Microbiol Infect Dis 1989;8:956-961.  Back to cited text no. 19    
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