Rapid methods for the immunodiagnosis of infectious diseases: recent developments

Current techniques for rapid diagnosis of microbial infections by direct detection of the microbial agent are compared. The techniques include enzyme immunoassay (EIA) tests, immunofluorescence, latex agglutination assays, and nucleic acid hybridization procedures. It is concluded that, for the near future, the preferred methods for rapid diagnosis will be by (1) EIA tests utilizing monoclonal antibodies and improved enzyme detection systems, and (2) improved latex agglutination procedures for certain antigens. Nucleic acid hybridization techniques, as currently performed, will need to be substantially improved to become the methods of choice.     

Evaluation of a latex agglutination test for rapid detection of rotavirus in faecal specimens

Two methods for detecting rotaviruses (latex agglutination, electron microscopy) have been compared on 80 faecal samples. These samples were obtained from infants between the age of four and 30 months hospitalized for acute gastroenteritis in the Children's Hospital, Karl Marx University at Leipzig, in 1982. Complete agreement among the two techniques was found in 75 specimens. Sensitivity of latex agglutination could be estimated at 95%, the specificity also at 95%. Only one sample reacted nonspecifically. Performance of the latex agglutination proved quite simple. The results indicate that latex agglutination is suitable for rapid screening of rotavirus induced gastroenteritis in clinical practice thus enabling the rate of nosocomial rotavirus infections in children's hospitals to be reduced.     

A Rapid Latex Agglutination Assay for the Detection of Penicillin-Binding Protein 2′

A simple and rapid slide latex agglutination assay was developed to detect penicillin-binding protein 2′ (PBP2′) from isolates of staphylococi. PBP2′ present in the membranes of methicillin-resistant Staphylococcus aureus (MRSA) or methicillin-resistant coagulase negative staphylococci (MRCNS) was rapidly extracted by alkaline treatment and, by combining with a slide agglutination reaction using latex particles sensitized with monoclonal antibodies raised against it, PBP2′ could be detected from a single loopful of cells taken from agar plates not containing beta-lactum antibiotics within 15 min. In a study of clinical isolates previously characterized as either MRSA or methicillin-susceptible Staphylococcus aureus (MSSA) by antibiotic susceptibility testing, 231 specimens of 232 MRSA were PBP2′ positive by latex agglutination, and the 87 specimens of MSSA were all negative. One specimen identified as MRSA by susceptibility testing but PBP2′ negative by latex agglutination was confirmed as mecA gene negative by PCR. This simple and rapid slide latex reagent should be useful in clinical diagnostics.     

Evaluation of the usefulness for the latex agglutination test for serodiagnosis of Mycoplasma pneumoniae infections

The usefulness of latex agglutination test prepared in our laboratory for the diagnosis of M. pneumoniae infections was assessed. A total of 628 serum samples obtained from patients with respiratory tract infections were tested by complement fixation test and by latex test, from among them 274 serum samples were additionally tested by ELISA--Ig A/--IgG/--IgM and by immunoelectroprecipitation test. The highest sensitivity and specificity was displayed by the latex test in relation to ELISA when determining mycoplasmal antibodies of IgM class (respectively 82.1% and 89.6%) and to the complement fixation test (81.0% and 89.0%). Positive latex test results in our investigations were associated only with the presence of IgM antibodies and were not dependent on the IgA and IgG antibody classes. The latex agglutination test may be used in routine serodiagnosis of mycoplasmosis under condition that the results obtained in this test will be confirmed by the complement fixation test or ELISA.     

Release of Vi antigens from Salmonella typhi: implications for virulence and diagnosis

The release of Vi antigens from three clinical isolates of Salmonella typhi was measured by a Vi-specific monoclonal antibody. Large quantities of Vi antigens were detected in the culture supernates from all three strains using either passive latex agglutination or rocket immunoelectrophoresis. Vi antigens were also detected in broth cultures of S. typhi containing about 10(5) cells/ml using the sandwich enzyme linked immunosorbent assay. The significance of this finding in relationship to the virulence and the diagnosis of S. typhi was discussed.     

Development of a Rapid Agglutination Latex Test for Diagnosis of Enteropathogenic and Enterohemorrhagic Escherichia coli Infection in Developing World: Defining the Biomarker,Antibody and Method

Background

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC/EHEC) are human intestinal pathogens responsible for diarrhea in both developing and industrialized countries. In research laboratories, EPEC and EHEC are defined on the basis of their pathogenic features; nevertheless, their identification in routine laboratories is expensive and laborious. Therefore, the aim of the present work was to develop a rapid and simple assay for EPEC/EHEC detection. Accordingly, the EPEC/EHEC-secreted proteins EspA and EspB were chosen as target antigens.

Methodology

First, we investigated the ideal conditions for EspA/EspB production/secretion by ELISA in a collection of EPEC/EHEC strains after cultivating bacterial isolates in Dulbecco’s modified Eagle’s medium (DMEM) or DMEM containing 1% tryptone or HEp-2 cells-preconditioned DMEM, employing either anti-EspA/anti-EspB polyclonal or monoclonal antibodies developed and characterized herein. Subsequently, a rapid agglutination latex test (RALT) was developed and tested with the same collection of bacterial isolates.

Principal findings

EspB was defined as a biomarker and its corresponding monoclonal antibody as the tool for EPEC/EHEC diagnosis; the production of EspB was better in DMEM medium. RALT assay has the sensitivity and specificity required for high-impact diagnosis of neglected diseases in the developing world.

Conclusion

RALT assay described herein can be considered an alternative assay for diarrhea diagnosis in low-income countries since it achieved 97% sensitivity, 98% specificity and 97% efficiency.     

A simple and rapid immunochromatographic test kit for rabies diagnosis

In rabies endemic countries, funds and infrastructure are often insufficient to employ the approved gold standard for the definitive diagnosis of rabies: the direct fluorescent test. In the present study, two types (type 1 and 2) of an ICT kit were evaluated for detection of rabies. These were developed using monoclonal antibodies which recognize epitope II and III of the nucleoprotein of rabies virus. Both kits specifically detected all rabies virus strains and there was no cross reactivity with Lyssaviruses (Lagos, Mokola and Duvenhage), Rhabdovirus (VSV and Oita 296/1972) and other common canine-pathogenic viruses. In type 1, a single type of monoclonal antibody was used. It was capable of detecting recombinant nucleoprotein and showed sensitivity of 95.5% (42/44) and specificity of 88.9% (32/36) using brain samples from rabid dogs. In contrast, type 2 which was made of two different monoclonal antibodies had a lower sensitivity of 93.2% (41/44) and higher specificity of 100% (36/36). These ICT kits provide a simple and rapid method for rabies detection. They need neither cold chain for transportation nor complicated training for personnel. This diagnostic test is suitable for rabies screening, particularly in areas with a high prevalence of rabies and where the fluorescent antibody test is not available.     

Toxoplasma gondii infections in captive black-footed ferrets (Mustela nigripes), 1992-1998: clinical signs, serology, pathology, and prevention

An epizootic of toxoplasmosis occurred among 22 adult and 30 kit black-footed ferrets (Mustela nigripes) maintained under quarantine conditions at the Louisville Zoological Garden (Louisville, Kentucky, USA) in June, 1992. Black-footed ferrets appear to be highly susceptible to acute and chronic toxoplasmosis. Clinical signs were observed in 19 adults and six kits and included anorexia, lethargy, corneal edema, and ataxia. Two adults and six kits died with acute disease. High antibody titers to Toxoplasma gondii were detected by latex agglutination and modified agglutination assay in 10 black-footed ferrets. One adult and six kits that died with acute clinical signs were necropsied and T. gondii-like organisms were found microscopically in multiple organs. Diagnosis of toxoplasmosis was confirmed by immunohistochemical staining with anti-T. gondii antibodies and by ultrastructural examination. Although the source of T. gondii for black-footed ferrets was not identified, frozen uncooked rabbit was the most likely source. Chronic toxoplasmosis resulted in the death of at additional 13 black-footed ferrets that were adults during the epizootic. Affected animals developed chronic progressive posterior weakness and posterior ataxia 6-69 mo after the epizootic began. Meningoencephalitis or meningoencephalomyelitis associated with chronic toxoplasmosis were identified at necropsy in all 13 ferrets. Precautions to prevent introduction of pathogens into the colony were insufficient to exclude T. gondii. Although toxoplasmosis may cause significant mortality in mustelids, the high mortality of black-footed ferrets in this epizootic was of concern due to their endangered status. This is the first detailed report of toxoplasmosis in black-footed ferrets.     

Screening of aquatic samples for Vibrio cholerae serotype O1 by a dot-blot method and a latex agglutination test.

A dot-blot, enzyme-linked immunosorbent method and a latex agglutination test were studied for their abilities to detect Vibrio cholerae serotype O1 in aquatic samples by testing artificially contaminated water as well as samples from natural potential sources. Water samples were preenriched with alkaline peptone and then enriched with Monsur peptone water. For the dot-blot test, enriched cultures of organisms in a small portion of the Monsur peptone water were transferred to a polyvinylidene difluoride membrane with a microfiltration apparatus. The enzyme-linked immunosorbent assay was performed by using biotin-labeled antibodies and avidin-biotin-peroxidase complex; brown dots developed in the wells that contained serotype O1 vibrios. Latex agglutination tests were performed by mixing 1 drop of the culture in Monsur with 1 drop of reagent coated with monoclonal antibody specific for antigen A. The sensitivities and specificities of the methods were compared with those of the colony-blot method, which identified individual colonies of V. cholerae O1 in mixed bacterial cultures on isolation media. Our results indicate that the dot-blot method is as sensitive as the colony-blot method and is useful for screening for V. cholerae serotype O1 even in specimens that are heavily contaminated with non-O1 vibrios.     

Effect of serum sample inactivation on the performance of latex agglutination test for paracoccidioidomycosis serodiagnosis

Paracoccidioidomycosis is diagnosed from the direct observation of the causative agent, but serology can facilitate and decrease the time required for diagnosis. The objective of this study was to determine the influence of serum sample inactivation on the performance of the latex agglutination test (LAT) for detecting antibodies against Paracoccidioides brasiliensis. The sensitivity of LAT from inactivated or non-inactivated samples was 73% and 83%, respectively and the LAT selectivity was 79% and 90%, respectively. The LAT evaluated here was no more specific than the double-immunodiffusion assay. We suggest the investigation of other methods for improving the LAT, such as the use of deglycosylated antigen.     

Release of Vi antigens from Salmonella typhi: implications for virulence and diagnosis

Abstract The release of Vi antigens from three clinical isolates of Salmonella typhi was measured by a Vi-specific monoclonal antibody. Large quantities of Vi antigens were detected in the culture supernates from all three strains using either passive latex agglutination or rocket immunoelectrophoresis. Vi antigens were also detected in broth cultures of S. typhi containing about 10⁵ cells/ml using the sandwich enzyme linked immunosorbent assay. The significance of this finding in relationship to the virulence and the diagnosis of S. typhi was discussed.     

Detection of Clostridium difficile toxin A by reversed passive latex agglutination.

A reversed passive latex agglutination (RPLA) assay for detecting Clostridium difficile toxin A is presented. Purified monoclonal antibody (mAb 37B5) was used for latex sensitization. The culture supernatants of 93 strains of C. difficile were tested by RPLA assay and the results compared with those of a commercially available latex agglutination test, PCR and cytotoxin assay with Vero cells. There was agreement between RPLA, cytotoxicity and PCR assays, but 29 strains were positive in the RPLA assay while 35 were positive in the cytotoxicity test and PCR using primer pair NK3-NK2 directed to the nonrepeating portion of the C. difficile toxin A gene. The 6 cytotoxic but RPLA-negative strains were demonstrated to be toxin A-negative/toxin B-positive strains in the PCR assay by using primer pair NK11-NK9 directed to the repeating portion of the C. difficile toxin A gene. There were no cross-reactions with culture supernatants of the other clostridial strains except for two strains of C. sordelli that produced hemorrhagic toxin (which is immunologically related to C. difficile toxin A).     

酶联免疫吸附试验定量检测血清肝素酶方法的建立及应用

目的：建立一种简便、微创的血清肝素酶酶联免疫吸附（ELISA）定量检测方法,并对肿瘤患者和正常人血清肝素酶进行比较,初步探讨血清肝素酶水平与肿瘤发生、发展的关系。方法：选择鼠抗人肝素酶单克隆抗体和兔抗人肝素酶多克隆抗体,建立双抗夹心ELISA检测方法,并利用此方法检测健康献血者和肿瘤患者血清中的肝素酶水平。结果：组内数据稳定,可重复;肿瘤患者血清中肝素酶D450nm值高于健康献血者。结论：所建立的血清肝素酶ELISA检测方法灵敏、高效,可用于肿瘤的辅助诊断。     

Latex agglutination and hemagglutination tests for the rapid identification of methicillin sensitive and methicillin resistant Staphylococcus aureus

Ten latex agglutination (LA) and hemagglutination (HA) kits for the identification of Staphylococcus aureus were compared with reference methods for their reliability and performance. The ten commercial kits consisted of Accu-Staph, Bacto-Staph, Hemastaph, Staphaurex, Staph-Latex, Staphylochrome, Staphyloslide, Staph-Rapid, Sero-Stat and Veri-Staph. The conventional methods included slide coagulase test, tube coagulase test (4 hr, 24 hr), thermonuclease and growth on mannitol salt agar (MSA). A total of 583 clinical isolates of staphylococci were used and all the kits correlated well with the conventional methods (93.1-99.4% sensitivity) in their ability to identify both methicillin sensitive (MSSA) and methicillin resistant S. aureus (MRSA). Although all were rapid, easy to perform and simple to interpret, Staphaurex and Staphyloslide gave the best sensitivities and specificities.     

A latex agglutination test for the detection of Mycoplasma pneumoniae in respiratory exudates: a comparative study with a commercially available DNA-probe test.

We prepared polyclonal antibody specific to Mycoplasma pneumoniae. Using this antibody, we developed a latex agglutination test (LAT) for detecting the organism in respiratory exudates as rapid diagnosis of M. pneumoniae infection. Further, LAT was compared with DNA-probe test (DP) which was the only commercially available test for the rapid detection of the organism. In LAT, both M. pneumoniae and M. genitalium give positive agglutination, but the titer of M. genitalium was significantly lower than that of M. pneumoniae. The detection limit of LAT was 2 x 10(5) CFU/ml and that of DP was 5 x 10(4) CFU/ml in vitro. It was considered that target molecules in LAT were accumulated in the pharyngeal portion of the patients, because of their long half-life at 37 C. However, ribosomal RNA which was target molecule in DP was destroyed at 37 C much sooner, and the accumulation could not be expected. Actually, positive rate in LAT was higher than that in DP among clinical specimens in which M. pneumoniae was detected by culture method. The procedure of LAT is much easier and more rapid than that of DP in which radioactive isotope is required. LAT could be the choice of test for rapid diagnosis of M. pneumoniae infection.     

Development of ELISAs for quantification of HMFG1-specific human anti-mouse IgG and IgM antibodies

The aim of this study was to develop and validate ELISAs for quantification of HAMA-IgM and HAMA-IgG in serum of patients with ovarian cancer who enrolled in a large international randomized phase III trial of intraperitoneal Yttrium-90-labeled HMFG1 murine monoclonal antibody therapy. The capture antibody of these 2 assays was the murine antibody HMFG1, while mouse anti-human IgM-HRP or mouse anti-human IgG(Fc)-HRP served as tracer antibodies. A pool of HAMA-positive serum samples was used to prepare a series of assay standards and another pool served as reference preparation. The analytical sensitivity of the HAMA-IgM assay was 2.5 arbitrary units per mL (AU/mL) and 4.7 AU/mL for the HAMA-IgG ELISA. Diluted serum samples showed good parallelism with the HAMA-IgM and HAMA-IgG standard dose-response curves. Within-assay coefficient of variation was 7.5% for HAMA-IgM and 6.5% for HAMA-IgG. Between-assay variation was 14.2% for HAMA-IgM and 15.3% for HAMA-IgG. The developed HAMA-IgM and HAMA-IgG ELISAs show satisfactory reliability criteria (sensitivity, parallelism and precision) and are suitable for monitoring of HAMA-IgM and HAMA-IgG responses in ovarian cancer patients. These ELISAs will be used to monitor the development of HAMAs in patients who received radioimmunotherapy with murine HMFG1.     

Rapid detection of varicella-zoster viral antibodies by latex agglutination assay: a practical experience for medical and science undergraduates

From 1993 to 1997, laboratory exercises have been conducted using a latex agglutination assay to detect antibodies to varicella-zoster virus (VZV) among 576 medical and science undergraduates aged 18–25 years. Of 295 students who volunteered a past history of chickenpox, there was good correlation with VZV antibody positivity (89.8%). However, despite a history of chickenpox, 30 (10.2%) tested negative for VZV antibodies, suggesting previous misdiagnosis or false negatives caused by the prozoning phenomenon due to unusually high VZV antibody levels. Indeed, out of 22 initially seronegative sera from selected students with a history of chickenpox, 13 sera were subsequently seropositive upon re-testing the sera at higher dilutions. Although 192 students gave a negative history of chickenpox, 22 (11.5%) tested positive for VZV antibodies (of whom 2 were vaccinated, with the rest reflecting previous subclinical infection). Of 89 subjects unsure of their history of chickenpox, 30 (33.7%) were seropositive. Thus elicitation of a history of symptoms of chickenpox may not be a reliable indicator of past VZV exposure. While 55% of this cohort of this cohort of young adults were seropositive, a significant fraction of seronegative individuals (45%) were still susceptible to VZV infection. This practical study exemplifies latex agglutination as a rapid and simple diagnostic technique, and integrates certain key principles of virology, immunology and medicine.     

D-dimer and CoV-2 spike-immune complexes contribute to the production of PGE2 and proinflammatory cytokines in monocytes

An overreactive inflammatory response and coagulopathy are observed in patients with severe form of COVID-19. Since increased levels of D-dimer (DD) are associated with coagulopathy in COVID-19, we explored whether DD contributes to the aberrant cytokine responses. Here we show that treatment of healthy human monocytes with DD induced a dose dependent increase in production of pyrogenic mediator, Prostaglandin E2 (PGE2) and inflammatory cytokines, IL-6 and IL-8. The DD-induced PGE2 and inflammatory cytokines were enhanced significantly by co-treatment with immune complexes (IC) of SARS CoV-2 recombinant S protein or of pseudovirus containing SARS CoV-2 S protein (PVCoV-2) coated with spike-specific chimeric monoclonal antibody (MAb) containing mouse variable and human Fc regions. The production of PGE2 and cytokines in monocytes activated with DD and ICs was sensitive to the inhibitors of β2 integrin and FcγRIIa, and to the inhibitors of calcium signaling, Mitogen-Activated Protein Kinase (MAPK) pathway, and tyrosine-protein kinase. Importantly, strong increase in PGE2 and in IL-6/IL-8/IL-1β cytokines was observed in monocytes activated with DD in the presence of IC of PVCoV-2 coated with plasma from hospitalized COVID-19 patients but not from healthy donors. The IC of PVCoV-2 with convalescent plasma induced much lower levels of PGE2 and cytokines compared with plasma from hospitalized COVID-19 patients. PGE2 and IL-6/IL-8 cytokines produced in monocytes activated with plasma-containing IC, correlated well with the levels of spike binding antibodies and not with neutralizing antibody titers. Our study suggests that a combination of high levels of DD and high titers of spike-binding antibodies that can form IC with SARS CoV-2 viral particles might accelerate the inflammatory status of lung infiltrating monocytes leading to increased lung pathology in patients with severe form of COVID-19.     

Development of new versions of anti-human CD34 monoclonal antibodies with potentially reduced immunogenicity

Despite the widespread clinical use of CD34 antibodies for the purification of human hematopoietic stem/progenitor cells, all the current anti-human CD34 monoclonal antibodies (mAbs) are murine, which have the potential to elicit human antimouse antibody (HAMA) immune response. In the present study, we developed three new mouse anti-human CD34 mAbs which, respectively, belonged to class I, class II and class III CD34 epitope antibodies. In an attempt to reduce the immunogenicity of these three murine mAbs, their chimeric antibodies, which consisted of mouse antibody variable regions fused genetically to human antibody constant regions, were constructed and characterized. The anti-CD34 chimeric antibodies were shown to possess affinity and specificity similar to that of their respective parental murine antibodies. Due to the potentially better safety profiles, these chimeric antibodies might become alternatives to mouse anti-CD34 antibodies routinely used for clinical application.