Coagglutination Technique Research Articles

Increasing Incidence of Penicillin- and Cefotaxime-resistant Streptococcus pneumoniae Causing Meningitis in India: Time for Revision of Treatment Guidelines?

Purpose: Pneumococcal meningitis is a life-threatening infection, requiring prompt diagnosis and effective treatment. Penicillin resistance in pneumococcal infections is a concern. Here, we present the antibiotic susceptibility profile of pneumococcal meningeal isolates from January 2008 to August 2016 to elucidate treatment guidelines for pneumococcal meningitis. Materialsand Methods: Invasive pneumococcal isolates from all age groups, were included in this study. Minimum inhibitory concentrations for the isolates were identified by agar dilution technique and VITEK System 2. Serotyping of isolates was done by co-agglutination technique. Results: Out of 830 invasive pneumococcal isolates, 167 (20.1%) isolates were from meningeal infections. Cumulative penicillin resistance in pneumococcal meningitis was 43.7% and cefotaxime non-susceptibility was 14.9%. Penicillin resistance amongst meningeal isolates in those younger than 5 years, 5–16 years of age and those aged 16 years and older was 59.7%, 50% and 27.3%, respectively, with non-susceptibility to cefotaxime in the same age groups being 18%, 22.2% and 10.4%. Penicillin resistance amongst pneumococcal meningeal isolates increased from 9.5% in 2008 to 42.8% in 2016, whereas cefotaxime non-susceptibility increased from 4.7% in 2008 to 28.5% in 2016. Serotypes 14, 19F, 6B, 6A, 23F, 9V and 5 were the most common serotypes causing meningitis, with the first five accounting for over 75% of resistant isolates. Conclusions: The present study reports increasing penicillin resistance and cefotaxime non-susceptibility to pneumococcal meningitis in our setting. This highlights the need for empiric therapy with third-generation cephalosporins and vancomycin for all patients with meningitis while awaiting results of culture and susceptibility testing.

Pneumococcal serotypes associated with invasive disease in under five children in India & implications for vaccine policy.

Background & objectives:Streptococcus pneumoniae is a major cause of morbidity and mortality especially in children less than five years, particularly in India. We present data on S. pneumoniae infections in children less than five years age group, with response to its serotype distribution, antibiotic resistance profile and available vaccines expected coverage.Methods:Children aged less than five, who were suspected for invasive pneumococcal disease were included in the study and their sterile body fluids were investigated for the presence of S. pneumoniae. Invasive S. pneumoniae isolates from sterile body fluids were identified by bile solubility and optochin susceptibility test. Pneumococcal serotyping was performed with co-agglutination technique and reconfirmed with multiplex PCR.Results:The most common pneumococcal serotypes causing invasive infections in children less than five years of age were 14, 19F, 5, 6A and 6B. Of the 114 S. pneumoniae isolates studied, 110 (96.4%) were non-susceptible to co-trimoxazole and 30 per cent were non-susceptible to erythromycin, 5.2 per cent of the isolates were non-susceptible to penicillin and only 0.8 per cent was non-susceptible to cefotaxime.Interpretation & conclusions:Our results indicate that PCV-10 can protect against 64 per cent of serotypes causing invasive pneumococcal infections. Use of PCV-13 in this region can provide increase in protection upto 74.6 per cent against serotypes causing invasive pneumococcal infections. Incorporating PCV-13 in the Universal Immunization Programme may provide incremental protection against IPD serotypes in the southern region of the country.

Giardia antigen and antibody detection using coagglutination

Giardiasis is a parasitic gastrointestinal infection of world-wide proportions. Yet, its diagnosis remains problematic. Microscopic stool examinations are still the mainstay of diagnosis, but even repeated analyses may miss a large number of patients who harbor the parasite. It has often been necessary to resort to more invasive measures in order to diagnose this disease. Recently, immunological tests have been investigated as non-invasive tests for giardiasis. Staphylococcus aureus particle agglutination (coagglutination) is a simple, rapid and inexpensive immunodiagnostic technique which has rarely been applied to protozoa. We have successfully used coagglutination techniques to identify G. lamblia antigen in pure suspensions, as well as in fecal specimens. Giardia-sensitized Staphylococcus aureus (“coagglutination reagent”) was developed by incubating a 10% suspension of Cowan's strain S. aureus in phosphate buffered saline, with human, rabbit and sheep serum containing anti- Giardia IgG. This coagglutination reagent was able to detect as few as 10 3 G. lamblia trophozoites ml −1 (0·1 μg protein ml −1), or as few as 3 × 10 4 cysts ml −1. When compared to direct microscopy, clinical stool specimens tested with the coagglutination reagent demonstrated an 80% sensitivity and 85% specificity for this test. The coagglutination technique was also used to detect serum antibody, by fixing trophozoites and cysts (antigen source) to microscope slides, incubating with serum, rinsing the slides and then incubating with the S. aureus. Positive serum samples caused the S. aureus to surround the Giardia, presumably being bound by the anti- Giardia IgG. Thus, coagglutination shows promise as an immunodiagnostic procedure for Giardia, as it appears to detect either antigen or antibody. It may also be useful for detecting other protozoa, for which our current diagnostic tests may be inadequate.

Rapid diagnosis of typhoid fever through identification of Salmonella typhi within 18 hours of specimen acquisition by culture of the mononuclear cell-platelet fraction of blood

Detection of Salmonella typhi in blood by culture of the mononuclear cell-platelet layer was compared with other methods currently used for the diagnosis of typhoid fever. Colonies of S. typhi were present in all mononuclear cell-platelet layer-positive cultures within 18 h of plating and were identified within an additional 10 min by a coagglutination technique. In contrast, identification of all positive cultures by conventional blood culture required 3 days.

Rapid diagnosis of acute bacterial meningitis by coagglutination and latex agglutination

Early diagnosis and initiation of appropriate antimicrobial therapy is essential for the reduction of mortality and morbidity in acute pyogenic meningitis. The rapid diagnosis of meningitis by the detection of bacterial antigens in the cerebrospinal fluid (CSF) is now possible with the availability of commercially prepared kits. One hundred and eighty-six CSF samples were cultured for bacteria and examined for antigens of common organisms causing meningitis by a coagglutination technique (COA) and a latex agglutination test (LA). The COA had a concordance rate of 95·7% with culture while that of LA was 92·9%. The detection of Haemophilus influenzae type b antigens by both kits were in complete agreement with culture results ( 15 15 ). With Streptococcus pneumoniae antigens the COA was slightly more sensitive than the LA. With the Streptococcus group B antigen, however, both the tests proved to be oversensitive thus giving rise to false positives. Both kits failed to detect Neisseria meningitidis serogroup B exoantigens in two culture positive cases. Thus, while rapid diagnosis of acute bacterial meningitis by the detection of antigens in the CSF is extremely useful, negative results do not exclude the possibility of meningitis and positive results should be interpreted with the clinical picture and other laboratory parameters.

Coagglutination for diagnosis of bacterial infection.

Among the various immunodiagnostic tests available for rapid diagnosis of infectious diseases, COA test should be ex-ploited because of the relative ease of reagent preparation and use. If good quality antisera are used for making the reagents, the test will give reliable results. In the past, the reagents have been made using polyclonal antisera. This has given rise to problems especially in the rapid diagnosis of enteric fever by antigenic detection in body fluids. Microbial antigens cross-reactions may interfere with the performance of the test. These problems could be over-come by using monoclonal antibody with high specificity for the individual micro-organisms. Like the Gram stain, the coagglutination technique, if exploited well, will prove to be a valuable tool especially in the laboratories of developing countries. It is relatively cheap and the reagents have a satisfactory shelf-life.

Phadebact Monoclonal GC OMNI Test for confirmation of Neisseria gonorrhoeae.

The Phadebact Monoclonal GC OMNI Test (Pharmacia Diagnostics, Piscataway, N.J.) is used for the definitive identification of Neisseria gonorrhoeae. In this test, boiled organisms are examined by using a 1-min coagglutination technique. A total of 776 Neisseria strains, confirmed to the species level by patterns of acid production from carbohydrates incorporated in cysteine-tryptose agar or morphologically consistent with Neisseria meningitidis and fluorescent antibody negative, were tested by the coagglutination technique. Of the 516 isolates of N. gonorrhoeae, 8 (1.6%) were negative with the OMNI Test. Of the 260 isolates of Neisseria spp. other than N. gonorrhoeae, none showed a positive coagglutination reaction. The Phadebact Monoclonal GC OMNI Test provided rapid, accurate identification of N. gonorrhoeae (sensitivity, 98.4%; specificity, 100%).

Serotyping of campylobacters by co-agglutination on the basis of heat-stable antigens.

A new and simple method of serotyping campylobacters has been developed which utilises co-agglutination to detect the presence of heat-stable antigens. Campylobacters are heated at 75 degrees C for 30 min to destroy antigenic protein and allowed to react on a glass slide with staphylococci coated with antibody. Of 74 isolates, 67 gave the same result by co-agglutination and the previously described passive haemagglutination method. The co-agglutination technique may be used as a rapid screening test before serotyping by passive haemagglutination.

Identification of Naegleria fowleri in fresh isolates of environmental amoebae using a staphylococcal coagglutination technique

A coagglutination test using Staphylococcus aureus Cowan strain 1 sensitized with specific antiserum successfully identified Naegleria fowleri in axenic and monoxenic culture by detection of a heat stable antigen released into the culture medium during growth. Strong coagglutination, visible to the naked eye, occurred within 30 sec when the sensitized reagent was mixed on a slide with medium from N. fowleri cultures. N. lovaniensis gave a weak cross-reaction which developed over one min clearly distinguishable from that of N. fowleri. N. gruberi and N. australiensis gave no reaction. The technique is proposed as part of a rapid and economical scheme for detecting N. fowleri in environmental samples very soon after primary isolation, with distinct advantages over conventional methods. Further development of the technique to detect other Naegleria spp. and the early diagnosis of primary amoebic meningoencephalitis are suggested as future applications.

Immunological identification of Vibrioparahaemolyticus: Staphylococcal coagglutination test and enzyme-linked immunosorbent assay

The species-specific antigenicity of the lateral flagella of Vibrio parahaemolyticus was applied to techniques for immunological identification of the vibrio. One was a coagglutination test using staphylococcal cells sensitized with anti-lateral-flagella antibody bound to protein A located in the staphylococcal cell wall. The other technique was an enzyme-linked immunosorbent assay (ELISA) using a four-layer sandwich procedure. By means of the staphylococcal coagglutination technique, about 10 6 vibrio cells or 60 ng of flagellar protein could be detected and the whole procedure was performed within 1 h. The sandwich ELISA procedure was more sensitive, but the time required for the whole procedure was about 8 h. Both techniques were specific for V. parahaemolyticus, because no cross-reactivity was observed with other related vibrios, and the tests were not inhibited by heterologous bacterial cells. These results suggest that both techniques are useful for the rapid identification of V. parahaemolyticus in complex samples.

Rapid Identification of Colonization Factor Antigens (CFA/I and CFA/II) of Enterotoxigenic E. coli (ETEC) and O-Antigens of Enteropathogenic E. coli (EPEC) by Coagglutination Test

Specific antisera for CFA/I (O78), CFA/II (O6) or enteropathogenic (EPEC) somatic Oantigens (serogroups O26, O86a and O111) were adsorbed to Staphylococcus aureus (strain Cowan I) to produce coagglutination reagents. Conventional agglutination tests with bacterial cells showed that antiserum against strain C922a-1 reacted with strain C91f, whereas anti-C91f did not agglutinate cells of strain C922a-1. Antisera raised against strains 4334f and C91f agglutinated their bacterial cells. Anti-303d did not react with strains C922a-1, 91f, nor with 4334f. Similar results were obtained with tests performed with COA-reagents of the above strains. Homologous antisera diluted 1: 1000 gave positive COA reaction, after being adsorbed to Cowan I. Such diluted antisera gave negative agglutination reaction in the conventional agglutination tests. The COA test is at least 100 times more sensitive than the conventional agglutination test. Hence, preparation of COA reagents implies practically economical use of antisera. Furthermore, the COA technique is much more sensitive and rapid (< 5 min) than immundiffusion test which takes ≥ 24 h to read and in which large molecular weight antigens fail to diffuse in to agarose. 30 CFA/I strains (100%) gave COA positive reaction and only 25 CFA/II strains (71,4%) were positive with CFA/II COA reagents. The coagglutination technique was also applicable to identification of enteropathogenic E. coli by using somatic O-antigens (026, 086a and Olli) antisera. Sensitized Staphylococcus aureus (Cowan I) by EPEC O-antigen sera diluted to ≥ 1/1500 could specifically aggregate strains belonging to corresponding O-serog-roup. In conclusion the COA technique is an appropriate, rapid diagnostic tool which can easily be used for epidemiological studies in developing and industrialized countries.

Differentiation of the major flagellar antigens of Pseudomonas aeruginosa by the slide coagglutination technique.

Antisera against the two major flagellar antigens of Pseudomonas aeruginosa were obtained by immunization of rabbits with isolated flagella and absorption of contaminating antisomatic antibodies. In the conventional slide agglutination test, the pure H antisera did not agglutinate the flagellated cells of the homologous strains. The addition of protein A-bearing staphylococci to H antiserum and homologous flagellated cells, the so-called slide coagglutination, results in a rapid development of flaky clumps. H coagglutination tests of reference strains, which formerly have been H typed by long-term tube agglutination and by the indirect fluorescent-antibody technique, yielded exactly the same subdivision of the strains in H type a and H type b as the more laborious and time-consuming methods. O grouping and H typing of 181 isolates from clinical specimens revealed a free combination of the somatic and flagellar antigens. 25 OH serovars were found. The simple and rapid coagglutination technique can promote the serovar determination of P. aeruginosa, particularly for the purpose of hospital infection control.

Rapid screening of fecal samples for Vibrio cholerae by a coagglutination technique.

A coagglutination procedure for detecting Vibrio cholerae was applied directly to 125 watery fecal samples received in the laboratory for bacteriological culture: many of these were from suspected cases of cholera. Of 47 bacteriologically proved cases of cholera, 44 (93.6%) gave positive results by the coagglutination method. There was a good correlation between the coagglutination method, dark-field microscopy, and culture.

Phadebact coagglutination test for the rapid identification of pneumococci

Conventional tests for the presumptive identification of Streptococcus pneumoniae from culturally similar non-pneumococcal alpha hemolytic streptococci include optochin sensitivity, bile solubility and inulin fermentation. These tests require up to 24 h for completion. In this paper we report the evaluation of a simple and rapid coagglutination procedure that requires less than 5 min. Of the 150 clinical isolates of pneumococci that were identified by conventional methods, all were correctly recognized by the Phadebact coagglutination technique within one minute. Forty strains of non-pneumococcal alpha hemolytic streptococci were also tested to determine the specificity of the method. Of these, 32 (80 %) did not react in either the typing or control reagents, whereas 8 (20%) showed coagglutination in the control. The Phadebact coagglutination test for the definitive identification of pneumococci is simple, rapid, and easy to perform and interpret.

Serological identification of group D streptococci using commercial antisera

Three commercial group D streptococcal antisera were tested for the serological identification of 100 group D enterococci; 20 Streptococcus bovis; 5 isolated from each of the following streptococcal groups: A, B, C and G; and 3 isolates from serological group F. Antisera from Difco Laboratories, BBL, and Wellcome Reagents Limited were used in the classic capillary tube precipitin test on extracts prepared using the Rantz and Randall procedure. No false positive precipitin reactions were observed. Of the enterococcal isolates, all 100 reacted with the Wellcome, 99 reacted with the BBL, and 96 reacted with the Difco group D antisera. However, of the 20 S. bovis isolates, only 2 reacted with the BBL, and 1 reacted with both the Difco and the Wellcome antisera. Each antiserum was then used to prepare staphylococcal coagglutination (CoA) reagents and each isolate was subsequently tested. A simple extraction procedure was performed by suspending colonies of an isolate in a loopful of salin on a microscope slide and gently heating the slide directly in the flame of a Bunsen burner. All 100 enterococci and all 20 S. bovis gave positive results with the BBL and the Wellcome CoA reagents. Using the Difco reagent, 2 S. bovis isolates failed to produce postitive results. No false positive results were observed with the non-Group D isolates. Our results indicate that the CoA technique using commercial group D antisera may provide faster and sometimes more sensitive serological identification than the classic capillary tube precipitin test.

Foodborne Illness Caused by Escherichia coli: A Review

Enteropathogenic Escherichia coli (EEC) can be defined as any strain of E. coli that has the potential to cause diarrheal illness. Four major categories of EEC exist. Classical enteropathogenic E. coli (EPEC) commonly refers to serogroups of E. coli historically associated with outbreaks of diarrhea in young children and infants. Facultatively enteropathogenic E. coli (FEEC) are non-EPEC serogroups associated with sporadic diarrhea, and include many serogroups associated with the normal intestinal flora. Enterotoxigenic E. coli (ETEC) is commonly isolated from outbreaks of traveler's diarrhea, and includes those strains which produce a heat-stable enterotoxin (ST) only, a heat-labile enterotoxin (LT) only and those which produce both ST and LT. These organisms adhere to and colonize the epithelial cell surfaces of the proximal small intestine. This colonization is mediated by specific types of fimbriae which are host-specific. Toxigenicity is plasmid-related. Enteroinvasive E. coli (EIEC) exert their pathogenic effect through an invasive infection of the gastrointestinal tract. Many techniques currently exist to determine the presence of enterotoxins produced by a particular strain of E. coli. These include bioassay, tissue culture and in vitro immunological techniques. Of the newer in vitro immunological methods, the staphylococcal coagglutination technique to detect LT seems to have potential for routine use in diagnostic microbiology laboratories. Since large numbers (106 – 109) of EEC are necessary for diarrhea, an unsanitary environment is needed for transmission of illness. Presence of EEC varies geographically; however, E. coli diarrhea is not likely to occur in the more hygenic areas of the world, except in occasional common-source outbreaks where the organism has time to replicate in food or water. The following foods have been implicated in documented E. coli diarrheal outbreaks worldwide: meat and meat products, fish, poultry, milk and dairy products, vegetables, baked products, rice formulations, coffee substitutes and water.

Evaluation of the Phadebact Test for identification of Neisseria gonorrhoeae.

The Phadebact Gonococcus Test (Pharmacia Diagnostics, Piscataway, N.J.) is used for the identification of Neisseria gonorrhoeae. In this test, boiled organisms are examined by using a 3-min coagglutination technique. A total of 313 isolates of Neisseria confirmed by the fluorescent-antibody technique or identified to the species level by the cysteine-tryptose agar utilization technique, were tested by the coagglutination technique. Of 229 isolates of N. gonorrhoeae, 13 (5.7%) were negative by the coagglutination technique on the first testing, 4 (1.7%) of which remained negative on subsequent testing after subculture. Of the 84 isolates of Neisseria other than N. gonorrhoeae, none showed a positive coagglutination reading. The Phadebact Gonococcus Test provides rapid, accurate identification for most isolates of N. gonorrhoeae. As with any new technique, standard procedures (fluorescent-antibody or cysteine-tryptose agar analysis or both) should be performed along with the new technique until laboratory workers are comfortable with the performance and interpretation of the test. We must, however, emphasize that it is very important to follow carefully the procedures outlined on the package insert.

Enrichment culture coagglutination test for rapid, low-cost diagnosis of salmonellosis.

Specific diagnosis of salmonellosis by conventional culture and identification methods usually requires 2 to 4 days. Since Salmonella may be disseminated from infected individuals during this period, this amount of time required for diagnosis may be too slow to aid in epidemic control. To obtain earlier diagnoses of salmonellosis, a coagglutination test was used for rapid, simplified detection of Salmonella oranienburg antigens in enrichment broth cultures of fecal specimens from infants involved in a nursery outbreak. Two selective enrichment broths were used, selenite cystine and dulcitol selenite. These were compared in parallel for efficiency by subculture on deoxycholate lactose sucrose, MacConkey, xylose lysine deoxycholate, and tryptic soy lactose teepol agars. These overnight enrichment broth cultures of stool specimens were also examined by a coagglutination slide test with stabilized protein A-containing staphylococci sensitized with antisera for Salmonella antigens C1, E, and Vi. Of 113 diarrhea stool specimens tested, 86 were positive by conventional culture, 82 were positive by dulcitol selenite-coagglutination, and 55 were positive by selenite cystine-coagglutination. All these tests were negative on 50 stool specimens from infants in a noninfected nursery. Salmonellae were specifically detected in stool cultures within 20 h by the coagglutination technique. This early detection of Salmonella antigens provided a useful adjunct to culture for rapid diagnosis of salmonellosis.

Rapid typing of Escherichia coli K1 containing bacteria using the co-agglutination technique

Rapid typing of Escherichia coli K1 containing bacteria using the co-agglutination technique

Rapid typing of Escherichia coli K1 containing bacteria using the co-agglutination technique

Rapid typing of Escherichia coli K1 containing bacteria using the co-agglutination technique