Development of a Latex Agglutination Test for Detecting Pathogenic Burkholderia and its Approbation in the Endemic Regions of Vietnam

The aim of the work was development of a monoclonal antibody-based latex agglutination test to identify the causative agent of melioidosis, and the approbation of a freeze-dried experimental preparation for screening of environmental bacterial isolates in Vietnam.Materials and methods. The carriers of specific antibodies were polyacrolein latex particles with active aldehyde groups on the surface. Typical strains of the causative agents of melioidosis and glanders with a full-fledged antigenic structure, as well as the strains Burkholderia thailandensis, Burkholderia cepacia, Pseudomonas aeruginosa, and Pseudomonas putida were used to control the test specificity. The latex agglutination reaction was carried out on plastic Petri dishes with daily bacterial cultures, from which suspensions were prepared at a concentration of 1–2·109 m.c./ml. The results of the reaction were registered visually for 5–8 min using a 4-cross system against a dark background under lighting. The reaction to 3–4 crosses was recorded as positive. Colonies suspected of belonging to pathogenic Burkholderia from primary inoculations were transferred to L-agar with polymyxin B and grown for 36 hours at (37±1) °C. The species of the selected suspicious colonies was determined by multiplex PCR.Results and discussion. With collection strains, latex test demonstrated high sensitivity agglutinating 97.7 % of B. pseudomallei and all B. mallei strains. At the same time, it was negative with B. thailandensis, B. cepacia, P. aeruginos and P. putida. In microbiological screening of bacterial cultures isolated from environmental objects, the latex test had a diagnostic sensitivity of 89.4 %. Using the latex test at the stage of primary screening, it is possible to significantly reduce the time when processing a lot of samples received for analysis, as well as to reduce the consumption of reagents used at the subsequent stages of identification.

Brucellosis Spinal Epidural Abscess: A Case Series of Fourteen Patients

Objective: In the present study, we aimed to describe the clinical features, diagnosis, treatment and prognosis of Brucellosis spinal epidural abscess (BSEA). Methods: The complete clinical data of 14 BSEA patients who were treated in our hospital system from January 2014 to February 2019 were retrospectively analyzed. Moreover, the clinical features, diagnosis, treatment and prognosis of 60 BSEA cases collected from the English literature from 1994 to 2019 were also investigated. Results: 3 cases were positive for blood culture, 6 cases were positive for Brucella latex agglutination test, and 9 cases were positive for tissue culture. All 14 cases showed focal spinal pain, 11 cases showed neurological deficits, and 7 cases showed fever. Of the 14 cases, 12 involved the lumbosacral spine and 2 involved the cervical spine. 13 cases were cured, 1 case left limb numbness, and the follow-up time was 12-20 months. Conclusion: The classic diagnosis of triad (focal spinal pain, neurological deficit and fever) is less specific for the diagnosis of BSEA. MRI examination can find epidural abscess, brucella latex agglutination test, blood culture, tissue culture and biopsy can be used for etiological diagnosis. Brucellosis is an uncommon cause of epidural abscess. For BSEA, early detection, early diagnosis, and early treatment should be performed, and the most suitable treatment method should be selected through comprehensive evaluation.

Comparative evaluation of recombinant LigB based latex agglutination test with microscopic agglutination test for the diagnosis of wildlife leptospirosis

In the present study a comparative evaluation microscopic agglutination test with rLigB protein based latex agglutination test was carried out, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiralserovars. It was employed for serodiagnosis of leptospirosis. The 46 KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in PQE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against Leptospirosis suspected wildlife field sera. A total of 80 wildlife sera samples were collec ted, including 27 wild feline sera samples (18 tigers, 8 lions, and 1 jaguar) obtained from Chhatbir zoo, Chandigarh, 42 sera samples (8 tigers, 4 lions and 6 leopards, 2 cheethals, 1 black buck, 12 buffaloes and 9 zoo staff) were received from Jodhpur zoo Rajasthan, 8 sera samples (4 tigers, 3 leopards, 1 lion) from Van Vihar National park, Bohpal, Madhya Pradesh and 3 sera samples (2 lions and 1 tiger) received from Biwani Mini zoo, Haryana, India. The result showed that sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, rLigB based LAT is a rapid, pen site, reliable diagnostic tool of high sensitivity and specificity, under laboratory and field conditions, for the detection of Leptospirosis. Asian J. Med. Biol. Res. September 2020, 6(3): 440-448

Assessment of different methods for diagnosis of Group B streptococci during pregnancy

Objectives: To compare the different diagnostic techniques used to detect GBS colonization in pregnant women in late third trimester after thirty five weeks and to detect the frequency of GBS colonization among a sample of pregnant Egyptian women. Patients and methods: Vaginal swabs from the lower third of vagina were collected from 100 pregnant women in the late third trimester. Isolation of the organism by culture on selective media and confirmation by latex agglutination test and detection of CAMP factor by conventional PCR were compared. GBS isolates were tested by double disk diffusion method and D-zone test simultaneously for susceptibility to erythromycin and clindamycin and inducible clindamycin resistance for intrapartum antibiotic prophylaxis (IAP). Results: 25 participants (25%) were positive for GBS by culture in Lim broth with subculture onto TSA supplemented with 5% defibrinated sheep blood, while 75 participants (75%) were negative. Of the 25 GBS isolates, 19 (76%) were sensitive to erythromycin, 3 (12%) were intermediate and 3 (12%) were resistant. Of the 25 GBS isolates, 15 (60%) were sensitive to clindamycin, 2 (8%) were intermediate and 8 (32%) were resistant. Fourteen isolates (56%) were sensitive to both erythromycin and clindamycin whereas 3 (12%) were resistant to both (cMLSB). Latex agglutination test for GBS detection from the 24 hours incubated Lim broth was positive in 25 cases (25%). GBS was detected in 9 cases (9%) by the conventional PCR assay done directly from vaginal swabs specimens. Sensitivity, specificity, PPV and NPV for latex agglutination from the inoculated broth and PCR assay are 100%, 100%, 100%, 100% and 36%, 100%, 100%, 82.4% respectively. Latex agglutination test from the inoculated broth showed a statistically significant perfect agreement (100.0%) with culture with Kappa value 1.0 and 95% CI (1.0 – 1.0). PCR assay also showed a statistically significant but moderate agreement (84.0%) with culture with Kappa value 0.458 and 95% CI (0.253 – 0.662). Conclusion: Detection of GBS colonization by latex agglutination test from incubated selective broth directly is comparable to the gold standard (culture) as regards accuracy. PCR offers a rapid and highly specific method for detection of GBS colonization especially in intrapartum settings for administration of IAP in non-screened pregnant females; however, sensitivity is low resulting in a low NPV.

Recombinant leptospiral immunoglobulin like B protein based latex agglutination test for serodiagnosis of human leptospirosis

Humans get leptospirosis by contact with fresh water, damp soil, or vegetation contaminated by the urine of infected animals, swallowing contaminated food or water or while working in contaminated flood plains or at wet agricultural settings. The bacteria enter the body through abrasions in the skin and mucous membranes. In the present study recombinant LigB protein is employed in latex agglutination test, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiral serovars. It was employed for serodiagnosis of leptospirosis. The 46 KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in PQE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against leptospirosis suspected human sera. A total of 28 human sera samples were received from Post Graduate Institute of Medical Education & Research (PGIMER), Chandigarh India, which tested positive by IgM ELISA test kit were subjected to both rLigB based LAT and MAT. All the 28 sera showed seropositivity by both the tests. Icterohaemorrahigae was the predominant serovar followed by Javanica and Grippotyphosa. Six out seven sera samples received from Indian Veterinary research Institute, Human Hospital and City Hospital, Bareilly were tested positive both by rLAT and MAT. The result showed that sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, rLigB based LAT is a rapid, reliable diagnostic tool at resource poor and remote diagnostic centers with high sensitivity and specificity, under laboratory and field conditions, for the detection of leptospirosis. Asian J. Med. Biol. Res. June 2020, 6(2): 229-236

Cloning and Expression of Leptospiral Immunoglobulin Like B Gene and Use of The Recombinant Antigen for The Diagnosis of Bovine and Caprine Leptospirosis

In the present study recombinant LigB protein is employed in latex agglutination test, which is a cross reacting lipoprotein able to detect acute infection caused by any pathogenic leptospiral serovars. It was employed for serodiagnosis of leptospirosis. The 46KDa 6X His tagged LigB protein, obtained by IPTG induction of recombinant E. coli M15 cells containing the N-terminal region of LigB gee in PQE30 expression vector, was purified by Ni-NTA affinity chromatography and adsorbed on latex bead surface for performing latex agglutination test against Leptospirosis suspected field sea. Western blot confirmed that rLigB is an immunodominant protein against which antibodies are produced during active infection. A total of 453 field sera, including 432 bovine sera, 18 caprine sera and three sera samples of buffalo bull collected post-mortem following death the animal from Indian Veterinary Research institute (IVRI) were tested using rLigB based LAT. The result showed that 300 sera were tested positive by rLigB based LAT, which were reconfirmed using microscopic agglutination test (MAT). The results from LAT were in concordance with MAT. In conclusion, under laboratory and field conditions, rLigB based LAT is a rapid, pen site, reliable diagnostic tool of high sensitivity and specificity for the detection of Leptospirosis. Int. J. Appl. Sci. Biotechnol. Vol 8(2): 146-153