Coagglutination Research Articles

Improvement of laboratory diagnosis of pertussis infection

To detect pertussis antigens, the authors used a rapid and easy-to-use coagglutination (COAG) test with diagnostic reagents (COA reagents) based on affine purified antibodies. Model experiments using collection strains studied the specificity, sensitivity, and reproducibility of the test using the antibody COA reagents. The specificity of the COAG test was confirmed by the examination of a group of children with diseases of non-pertussis etiology. The results of a comprehensive examination of children for pertussis, by applying COAG, bacteriological, and serological tests, are given in the paper. Comparative studies established the consistency between the results of the COAG test and the data of polymerase chain reaction and indirect immunofluorescence test. The COAG test that is a simple and convenient analytical technique may be used as a rapid method in the complex diagnosis of pertussis.

A serological study of cysticercosis in patients with HIV

Neurocysticercosis (NCC) has attained the importance of one of the most common cause of focal brain lesions in patients infected with HIV (human immunodeficiency virus). Adequate data regarding the rate of this co-infection is lacking. Therefore, the present study was carried out to determine the prevalence of cysticercosis among HIV patients residing in Puducherry or its neighboring districts of Tamil Nadu State, India. A total of one hundred blood samples were collected from HIV seropositive cases visiting JIPMER hospital, Puducherry, between June 2007 and May 2008. Enzyme immunotransfer blot (EITB) and enzyme linked immunosorbent assay (ELISA) were used to demonstrate anti- T. solium larval stage antibodies and Co-agglutination (Co-A) test was used to detect T. solium larval stage antigens in sera. Two HIV seropositive cases were found positive for anti-T. solium larval stage antibody by EITB and four were positive by ELISA. Only one sample was positive by both EITB and ELISA. No serum sample was found positive for T. solium larval stage antigen by Co-A test. The overall seropositivity detected by all the methods was 5% in this study group. The accurate clinical diagnosis of NCC in HIV is difficult due to deranged immunological parameters in the HIV infected patients. The results of this study provides important data on the prevalence of cysticercosis in HIV positive patients in Puducherry and neighboring areas which was previously unknown. This study will also increase awareness among physicians and public health agencies about T. solium cysticercosis in the selected group.

Detection of Cysticercus antigens and antibodies in cerbrospinal fluid of patients with chronic meningitis

Chronic meningitism is a less frequent manifestation of neurocysticercosis caused by Taenia solium cysticerci. In the present study we used Co-agglutination (Co-A), a simple and rapid slide agglutination test to detect specific Cysticercus antigen in the 67 cerebrospinal fluid (CSF) samples from patients with chronic meningitis of unknown etiology. The results were compared with that of ELISA for detection of antibodies. Among these samples four (5.97%) were positive for Cysticercus antigen by Co-A test and six (8.95%) were positive for antibodies by ELISA. Two samples were positive by both Co-A and ELISA, two were positive only by Co-A and four were positive only by ELISA. In the present study, although Cysticercus antigen and antibodies were present in CSF samples from eight (11.94%) patients, we cannot affirm that all the cases of chronic meningitis are due to cysticercosis, but for any case of chronic meningitis of unknown origin, it would be useful to consider the possibility of cysticercal meningitis.

Co-agglutination test for cysticercus antigen detection in the serum for the diagnosis of cysticercosis

The objective of the study was to develop the co-agglutination (Co-A) test, a rapid slide agglutination test for the diagnosis of cysticercosis. The present study included 21 cases of cysticercosis, which comprised seven cases of clinico-radiologically definite cases of neurocysticercosis (NCC) proven with either computed tomography (CT) scan or magnetic resonance imaging (MRI), eight cases of clinically strong NCC, six cases of extraneural cysticercosis in muscle and eye; 40 non-cysticercal parasitic infection controls; and 20 healthy controls. Hyperimmune cysticercus antiserum was raised in rabbits and was used to coat Staphylococcus aureus (Cowan strain-I) bearing protein A (SAPA) cells, and the Co-A was standardized to detect cysticercal antigen in the serum. Serum samples from 12 out of 21 (57%) cases of cysticercosis were positive for cysticercal antigen by the Co-A test. Of the 12 positive samples, eight were from cases of neurocysticercosis and four from cases of extra-neural cysticercosis. Serum samples from seven out of 40 non-cysticercal parasitic infection controls and serum samples from one out of 20 (5%) healthy controls showed a false-positive reaction for the antigen by the Co-A test. There was a statistically significant difference between the antigen detection rates among cysticercosis patients on one hand, and the patients with other parasitic diseases (P = 0.0014), and healthy controls (P=0.0003) on the other. The Co-A test appears to be a moderately sensitive and specific test for the diagnosis of cysticercosis.

Cysticercus antibodies and antigens in serum from blood donors from Pondicherry, India

The aim of the present study was to screen the serum of blood donors, which are apparently healthy and residing in Pondicherry or its neighboring districts of Tamil Nadu State, for specific detection of Cysticercus antigens and antibodies. A total of 216 blood samples were collected from blood donors at the Central Blood Bank, JIPMER Hospital, Pondicherry, India during January and February 2004. Enzyme-linked immunosorbent assay (ELISA) was used to demonstrate anti-Cysticercus antibodies and the Co-agglutination (CoA) was used to detect antigen in sera. 14 (6.48 %) males were positive for either anti-Cysticercus antibodies or antigens. Of these eight sera were positive for anti-Cysticercus antibodies and six were positive for antigens. Results of the present study show that serum Cysticercus antigen detection may be a useful adjunct to antibody testing for seroprevalence studies of cysticercosis in the community. The present study is the first kind of study, carried out to determine both cysticercal antibodies as well as antigens in the serum samples collected from the healthy blood donors.

Detection of specific cysticercus antigen in the urine for diagnosis of neurocysticercosis

Neurocysticercosis (NCC) is an important zoonotic infection, and a significant cause of morbidity and mortality in India. Immunodiagnosis plays an important role in the early, rapid, and specific diagnosis of the condition. The aim of the present study was to detect specific cysticercal antigen excreted in the urine for the diagnosis of NCC. The objective was to develop and evaluate staphylococcal Co-agglutination (Co-A) test for the detection of urinary cysticercal antigen. In the present study, urine samples were collected from 9 cases of clinically suspected NCC, 8 cases of CT/MRI proven cases of NCC, 11 of non-cysticercal CNS infection controls, and 25 healthy control subjects. Polyclonal antisera raised in rabbits against porcine complete cysticercus homogenate antigen, was used in the Co-A test to detect cysticercal antigen. The Co-A detected excreted cysticercal antigen in the urine of five of nine (55.5%) clinically diagnosed NCC and in five of eight (62.5%), CT/MRI proven cases of NCC. A false positive reaction was observed with 1 of 11 (9%) of control urine specimens from non-cysticercal CNS infection controls. No false positivity was demonstrated with 25 urine samples collected from healthy controls. Results of the present study shows that the Co-A test can be employed as a moderately sensitive and specific test for detection of urinary antigen in the diagnosis of NCC in poorly equipped laboratories.

COMPARISON OF LATEX AGGLUTINATION AND CO-AGGLUTINATION FOR THE DIAGNOSIS AND PROGNOSIS OF CRYPTOCOCCAL MENINGITIS

Purpose: To compare a commercially available Latex agglutination test and an in house co-agglutination test for the detection of cryptococcal antigen in cases of chronic meningitis. Methods: One hundred and fifty cerebrospinal fluid (CSF) samples from 150 cases of chronic meningitis were tested for the presence of Cryptococcus neoformans by modified India ink, culture and antigen detection by latex agglutination test (LAT) and co-agglutination (Co-A) test. Results: Thirty-nine cases were positive by one or more tests employed. Antigen detection in CSF by LAT and Co-A was found to be most sensitive (94.9%) while culture was the least (25.6%). Of the two antigen detection methods, Co-A was found to be more sensitive than the LAT, the difference being statistically significant. Initial CSF antigen titres did not have any prognostic significance. Conclusions: Co-A for antigen detection is an inexpensive and useful adjunct to direct microscopy and culture for the diagnosis of cryptococcal meningitis, though its usefulness in prognosis needs to be evaluated further.

Study of antigenic heterogeneity among Actinobacillus pleuropneumoniae serotype 7 strains.

Actinobacillus pleuropneumoniae serotype 7 strains were studied for their antigenic heterogeneity using rabbit polyclonal hyperimmune sera against all the known twelve reference strains of A. pleuropneumoniae and a battery of different serological tests such as coagglutination (COA), immunodiffusion (ID), indirect hemagglutination (IHA), counterimmunoelectrophoresis (CIE), rapid dot-ELISA (RDE), serum soft-agar (SSA) and growth agglutination (GA). Reference serotype 7 strain (WF83) showed cross-reactivity with reference serotype 1B strain but not with other serotypes. Field serotype 7 strains showed cross-reactivities with serotypes 1A, 1B, 4, 9, 10, and 11 in COA, ID, and CIE tests, but not in IHA test. Two field strains of serotype 7 (90-3182 and 86-1411) which appeared to be different from the typical serotype 7 strains were selected for further antigenic characterization by SDS-PAGE, Western blot, and Tricine SDS-PAGE assays, and identified as serotypes 1 and 7, respectively. For serotyping atypical strains, it is suggested to use Western blot assay as a confirmatory test to identify serotype-specific capsular and somatic antigens.

Urinary hydatid antigen detection by coagglutination, a cost-effective and rapid test for diagnosis of cystic echinococcosis in a rural or field setting.

We describe here coagglutination (Co-A), a rapid slide agglutination test for the detection of hydatid antigen in the urine for the diagnosis of cystic echinococcosis (CE). Paired urine and serum samples were collected from 16 patients with surgically confirmed CE, 10 patients with ultrasound-proven CE, 14 patients with clinically diagnosed CE, 24 patients with various parasitic diseases other than CE, and 25 healthy control subjects. Co-A detected excreted hydatid antigen in the concentrated urine of 7 of 16 (43.75%) surgically confirmed cases, 6 of 10 (60%) ultrasound-proven cases, and 8 of 14 (57.14%) clinically diagnosed cases of CE. A false-positive reaction was observed with 12.50% of control urine specimens from patients with parasitic diseases other than CE and 12% of urine samples from healthy controls. The circulating antigen was detected in the serum in 13 of 16 (81.25%) surgically confirmed cases, 6 of 10 (60%) ultrasound-proven cases, and 13 of 14 (92.86%) clinically diagnosed cases of CE. False-positive reactions were observed with three sera (12.5%) from controls with other parasitic diseases. The low sensitivity of Co-A for detection of antigen in the urine of a patient whose serum was positive for the antigen is possibly due to low levels of antigen in the urine. Unlike the collection of blood for serum, which is an invasive procedure and also requires technical expertise and disposable syringes, urine can be collected easily and frequently without causing any inconvenience to the patient. Urine as a clinical specimen alternative to serum would be immensely useful in the diagnosis of CE, particularly in a rural or field setting. In such situations as well as in poorly equipped laboratories, Co-A has the potential to be used as a simple, rapid, and economical slide agglutination test for detection of urinary hydatid antigen in the diagnosis of CE.

Evaluation of a rapid coagglutination (COA) test for the detection of infectious pancreatic necrosis virus (IPNV) in tissue samples of Atlantic salmon, Salmo salar L.

The field use of a staphylococcal coagglutination (COA) test for the detection of infectious pancreatic necrosis virus (IPNV) in tissue samples from Atlantic salmon, Salmo salar L., was evaluated. The COA test was compared with an immunohistochemical (IHC) method for the detection of clinical outbreaks of infectious pancreatic necrosis (IPN). The present paper describes the evaluation of 320 COA test results performed at local fish health laboratories in Norway from 1994 to 1996, and COA test results from two infection trials with IPNV. The agreement between the COA test and the IHC was very good. The agreement beyond chance, measured as kappa values, was 0.74 in individuals and 0.90 in pooled samples. Thus, the COA test was suited for the detection of outbreaks of IPN. Covert infections with IPNV remained undetected by the COA test. The minimum IPNV titre needed to obtain a positive COA test was ≈ 105 TCID50 mL–1.

Co-agglutination test for the detection of circulating antigen in amebic liver abscess.

We report here a simple and economical slide agglutination test, the co-agglutination (Co-A) test, for the detection of circulating amebic antigen in sera for the diagnosis of amebic liver abscess. Fifty serum specimens from cases of amebic liver abscess, 25 from other individuals with parasitic and miscellaneous infections, and 25 from healthy controls were tested for the presence of serum antigen by the Co-A test. Forty-five (90%) amebic liver abscess sera were found to be amebic-antigen positive by the Co-A test. None of 25 sera from healthy controls were positive for the antigen. However, false-positive results were seen with two sera from those with other parasitic and miscellaneous infection controls. These results show that the Co-A test can be used as a sensitive and specific rapid slide agglutination test for the detection of amebic antigen in the sera for diagnosis of cases of amebic liver abscess in a routine parasitology laboratory.

A review of some simple immunoassays in the serodiagnosis of cystic hydatid disease

Laboratory diagnosis of cystic hydatid disease (CHD) in the field or rural health centres catering to the needs of rural populations, especially in the developing countries, is faced with many difficulties. This paper reviews the need for simple and rapid immunoassays for the diagnosis of hydatid disease in such conditions. Simple and rapid immunoassays such as Dot-enzyme-linked immunosorbent assay (ELISA), hydatid antigen dot immunobinding assay (HA-DIA) and Co-agglutination (Co-A) which have the potential for wider use in the field or poorly equipped laboratories for serodiagnosis of hydatid disease are discussed.

Detection of hydatid antigen by co-agglutination in fluid samples from hydatid cysts

The co-agglutination (Co-A) procedure was standardized and evaluated to detect hydatid antigen in fluid samples aspirated from hydatid cysts. Samples were collected from 14 hydatid cysts in different organs (liver, 7; lung, 5; multiple abdominal cysts, 1; and kidney, 1) by surgery. Echinococcus granulosus scolices and booklets were seen in only 6 (42.85%) samples by routine microscopy. In contrast, hydatid antigen was demonstrated in all the samples by Co-A (100% sensitivity). Co-A could be employed as a simple and rapid diagnostic procedure, as an alternative to microscopy, to confirm the hydatid aetiology of a suspected cyst.

Evaluación diagnóstica de las técnicas rápidas del esputo frente a neumococo en la neumonía adquirida en la comunidad. Utilización del teorema de Bayes para su aplicación clínica

This study aimed to quantify the diagnostic value of immunological techniques and methods for rapid analysis of sputum for pneumococcus, using sensitivity and specificity values reported in the literature to calculate positive and negative predictive values (PPV and NPV) according to Bayes formulas. Diagnostic gains of the test are calculated and compared to pretext probability. We located articles reporting sensitivity and specificity of counterimmunoelectrophoresis (CIE), coagglutination (CoA) and latex agglutination (LA) tests. We also calculated the probability ratios for the three tests. LA achieved the best overall diagnostic utility rating. CoA had the highest PPV, whereas LA offered the highest NPV. CIE was the least useful. These three tests are more useful at intermediate levels of prevalence of pneumococcus, which coincide with estimate in our population. We conclude that LA and CoA are of greater diagnostic utility for community acquired pneumonia, as they are useful for determining prevalence as well as for deciding initial antibiotic treatment.

Rapid coagglutination test for the detection and typing of foot and mouth disease virus

Protein A containing Staphylococcus aureus was used to develop a coagglutination (COA) test for the detection and typing of foot and mouth disease virus (FMDV) O, A and C serotypes in infected cells and tissues. Different batches and amounts of guinea pig anti-FMDV sera were assessed to optimize the preparation of COA conjugates. The sensitivity and specificity of the COA Test for the detection of FMDV O, A and C serotypes and heterologous viruses was also characterized. Comparison between the COA Test and complement fixation test for the detection and typing of FMDV obtained from extracts of tongue epithelial tissues from infected cattle revealed high agreement in the results and indicated a potential application of the COA Test for the direct diagnosis of viruses.

Antigenic differences within Actinobacillus pleuropneumoniae serotype 1

Two distinct antigenic subtypes of Actinobacillus pleuropneumoniae serotype 1 were identified via coagglutination (Co-A) and designated as 1A and 1B. The reference strains, ATCC 27088 (1A) and ISU 158 (1B), were used to prepare hyperimmune rabbit sera for Co-A reagents. Of 75 serotype 1 field isolates tested by Co-A, 35 isolates typed as 1A, 12 as 1B and 28 as 1A/1B. Significant cross-reactivity between the 2 subtypes was found in the Co-A and was eliminated in 20/28 1A/1B strains by using Co-A reagents prepared with rabbit sera absorbed with the heterologous reference strain. However, twelve isolates (5 1A and 7 1A/1B; 16%) showed no reaction with Co-A reagents prepared with absorbed sera. Immunoblots of outer membranes (OM) prepared from APP 1A or 1B reference strains and field isolates indicated that antigenic differences between subtypes 1A and 1B were located within the high molecular weight (MW) region of the gels (40–100 kDa). Hyperimmune rabbit sera against 1A or 1B and sera from pigs vaccinated with whole-cell, formalin inactivated 1A or 1B bacterins reacted with the high MW region only in strains of the homologous subtype. In contrast, 4 of 5 sera from 1B infected pigs and 2 of 5 sera from 1A infected pigs reacted with all APP serotype 1 strains regardless of subtype. Apparently, infection exposed cross-reactive antigenic determinants that were not exposed by immunization with killed bacteria preparations. SDS-PAGE gels with LPS purified from APP 1A, 1B, 9 and 11 showed that 1A, 9 and 11 LPS O-antigens had an identical smooth ladder pattern, while 1B LPS was distinctly different. In immunoblots with OM or LPS and in dot-immunobinding assays with LPS, rabbit antiserum against APP 1A reacted with 1A, 9 and 11. In contrast, rabbit antiserum against APP 1B only reacted with APP 1B and weakly with APP 9 in the OM immunoblot and with LPS from APP 1B, 9 and 11 in the LPS immunoblot and dot-immunobinding assay. We conclude that 2 subtypes of APP serotype 1 can be distinguished based on their antigenic differences. These differences are located, at least in part, within the LPS O-antigens from APP 1B appear more antigenically similar to APP 9 LPS than to either APP 11 LPS. There may also be antigenic differences in the capsular polysaccharides of APP 1A and 1B strains. The antigenic differences between APP subtypes 1A and 1B may be sufficient so that vaccination with one subtype would not elicit a protective immune response against the heterologous subtype.

Coagglutination Test for Rapid Detection of Salmonella Enterotoxin

The Staphylococcal Coagglutination (CoA) test using Staphylococcus aureus (Cowan type-1) cells coated with antiserum against purified Salmonella enterotoxin was standardized and carried out to screen Salmonella serotypes for their enterotoxigenicity. Of the 101 cell-free culture supernatants from an equal number of Salmonella strains belonging to 15 different serotypes tested, 76 were found to be enterotoxigenic. All of the 24 strains which gave positive reactions in the rabbit ligated ileal loop test were also positive with this test. As little as 7.5 ng of purified Salmonella enterotoxin could be detected by the CoA test. The presence of enterotoxin in polymyxin B extract of cells and cell-free preparation of a S. typhimurium strain was detected as early as after 1.5 and 3.0 h of incubation, respectively. Besides the test standardized with the antiserum to purified V. cholerae toxin (CT) could detect purified toxin in a quantity as little as 5.0 ng and also enterotoxigenicity in an E. coli strain (LT+). The usefulness of the test as a routine, rapid and economic one for the detection of enterotoxin of Salmonella, Vibrio cholerae and E. coli is discussed.

Cross-reactions between Actinobacillus (Haemophilus) pleuropneumoniae strains of serotypes 1 and 9

Actinobacillus pleuropneumoniae strains of serotypes 1 and 9 were studied for their serological properties by means of agglutination, coagglutination (CoA), indirect hemagglutination (IHA), Co-IHA, ring precipitation (RP), and immunodiffusion (ID) tests. Particulate and soluble antigens of unheated and heat-treated bacterial cells were used in various serological tests. Agglutination, CoA, and RP tests demonstrated common antigens between strains of serotypes 1 and 9. Quantitative estimation of serotype-specific antigenic activity by CoA, RP, and ID tests proved useful in differentiating strains of serotypes 1 and 9. IHA and Co-IHA tests using sheep erythrocytes sensitized with unheated or heat-treated whole-cell saline extract and the ID test using boiled whole-cell saline extract as antigen distinguished the strains of serotypes 1 and 9. In studies of absorption of rabbit antisera with heterologous whole-cell antigens there was no absorption of antibodies in tube agglutination and IHA tests, suggesting that serotype 1 and 9 strains belong to two distinct serogroups. It appears that the cross-reactivity between serotype 1 and 9 strains could be due to common epitopes associated with cell wall antigens.

Rapid diagnosis of bacterial meningitis by an enzyme immunoassay of cerebrospinal fluid

A total of 250 cerebrospinal fluid (CSF) specimens were analyzed using a rapid enzyme immunoassay (Pharmacia Meningitis EIA-Test) (EIA) for the detection of antigens of Haemophilus influenzae type b, Neisseria meningitidis (serogroups A,B,C) and Streptococcus pneumoniae (25 selected types). The test is performed in less than 1 h and read by the naked eye. EIA and coagglutination (CoA) were compared with a constructed reference that comprised samples which were either positive by culture and/or on direct microscopy (DM), or in which there were positive results with both EIA and CoA for the bacteria covered by the assays. Using this reference for CSF samples assayed in a period between two meningococcal meningitis epidemics, the sensitivity was 0.86 for EIA and 0.69 for CoA, the specificity 0.95 (EIA) and 0.97 (CoA), the predictive value for a positive result 0.81 (EIA) and 0.87 (CoA) and, the predictive value for a negative result 0.96 (EIA) and 0.93 (CoA). Antibiotics had been given to 54% of the patients before admission. All of the 56 samples that were positive in any of the tests taken during an epidemic of group A meningococcal disease were detected by EIA; CoA was negative in 45% and culture/DM was negative in 32%. Sequential dilutions of two CSF samples from which H. influenzae type b had been isolated, showed the EIA to be 16-32 times more sensitive than CoA. With both technical feasibility and good sensitivity and specificity, the EIA seems to be useful and reliable for the rapid diagnosis of bacterial meningitis, especially in situations where pretreatment with antibiotics are likely.

Coagglutination (COA) test for the rapid diagnosis of cryptococcal meningitis.

Cryptococcus coagglutination (COA) test reagent was prepared locally and showed no cross reactions with different species of bacteria or yeasts or with 75 control sera including 25 that gave positive results for RA factor. We used the COA test to detect cryptococcus antigen in the CSF and we could confirm the diagnosis of 11 out of 115 suspected cases of fungal meningitis; the titre varied from 4 to 128. A four-fold rise in titre confirmed the diagnostic value and a steady fall in titre in three patients on therapy indicated the prognostic value of the test. The earliest confirmation was in a renal transplant patient on the eighth day after onset of symptoms. The COA test was negative with the CSF of 118 patients with chronic meningitis. Cryptococcal colony forming units (cfu) in CSF varied from 100 to greater than 100,000/ml and correlated well with microscopy and with the COA antigen titre in CSF. Four out of the 11 patients who had cryptococcaemia, had 50,000-100,000 cfu/ml in the CSF. Cryptococcus antigen was detected by COA in the serum of all 11 patients, even in those with only 100 cfu/ml in CSF. In the three post-renal transplant patients, who were being monitored regularly, the diagnosis was made early and all three recovered on antifungal therapy with no relapse to date (1-2 years). All the others, including the two primary CNS infections, succumbed to the disease because they presented late for diagnosis and therapy. The cryptococcus COA test is a simple and specific test that can be used as a rapid test to confirm early diagnosis and permit prompt therapy, which should improve the prognosis in CNS and other forms of systemic cryptococcosis. Moreover, it is reproducible and cost-effective, particularly in countries where the latex and other expensive test reagents are not generally available.