CASE SERIES: Here, we reported five cases of this disorder with different clinical presentations from two tertiary hospitals in Kelantan state, Malaysia within a two year-period. Most of them were elderly, except for one who presented at the age of 36 years old. No direct or secondary cause was identified except for one patient who had developed from pregnancy-related at 3 weeks postpartum. These patients presented with spontaneous bleeding typically into skin, muscles, and mucous membranes but also at rare site in the epidural space. All patients denied previous history of bleeding or family history of bleeding disorder. FVIII activities were recorded between <1% to 19%, while the inhibitor titre levels were between 3.9 BU to 340 BU. The treatment approaches especially at presentation were complicated by unfamiliarity of managing this rare condition but all these patients received appropriate medical attention.
DISCUSSION: Prompt diagnosis and management in the right hand are critical. Awareness of this disorder by medical personnel at all levels in the community and in various specialties is important.
METHODS: A total of 1267 suspected cases of Toxoplasma infection were enrolled in this study from January 2016 to December 2016. The cases were screened for anti-Toxoplasma IgM and IgG by electrochemiluminiscence immunoassay (ECLIA) method. Based on the serological profiles, all cases with first seropositive serum samples were considered as suggestive cases of Toxoplasma infection. Thus, second serum samples were obtained after an interval of 2 weeks. The diagnosis was made based on laboratory results and clinical data.
RESULTS: A total of 482 T. gondii seroreactive cases were selected. The patient's records were traced and the data were analysed. Accordingly, 152 cases were diagnosed as clinically confirmed cases; 198 cases were clinically asymptomatic and 132 cases were newborn babies or infants who did not have toxoplasmosis and only acquired passive immunity from their mothers. The paired serum algorithm allowed classifying the seroreactive cases as follows: early (0.6%), acute (1.9%), reactivation (13.5%), recent (1.5%), passive immunity from mother (27.3%) and possible congenital infections (1.2%). In addition, cases of reactivated toxoplasmosis were detected among the pregnant mothers (13/82; 15.8%), children aged above 1 year (2/8; 25.0%) and immunocompetent mothers (5/135; 3.7%). Furthermore, the application of the paired serum analysis resulted in remarkably improved treatment initiation.
CONCLUSIONS: Toxoplasmosis diagnosis and treatment can be improved through the use of paired serum diagnostic algorithm.
Methods: This study aims to develop a recombinant anti-mKRAS scFv-fused mutant Hydra actinoporin-like-toxin-1 (mHALT-1) immunotoxin that is capable of recognizing and eradicating codon-12 mutated k-ras antigen abnormal cells. One G13D peptide mimotope (164-D) and one G12V peptide mimotope (68-V) were designed to elicit antigen specific IgG titres against mutated K-ras antigens in immunised Balb/c mice. The RNA was extracted from splenocytes following ELISA confirmation on post-immunized mice sera and was reverse transcribed into cDNA. The scFv combinatorial library was constructed from cDNA repertoire of variable regions of heavy chain (VH) and light chain (VL) fusions connected by a flexible glycine-serine linker, using splicing by overlap extension PCR (SOE-PCR). Anti-mKRAS G12V and G13D scFvs were cloned in pCANTAB5E phagemid and superinfected with helper phage. After few rounds of bio-panning, a specific mKRAS G12V and G13D scFv antibody against G12V and G13D control mimotope was identified and confirmed using ELISA without any cross-reactivity with other mimotopes or controls. Subsequently, the anti-mKRAS scFv was fused to mHALT-1 using SOE-PCR and cloned in pET22b vector. Expressed recombinant immunotoxins were analyzed for their effects on cell proliferation by the MTT assay and targeted specificity by cell-based ELISA on KRAS-positive and KRAS-negative cancer cells.
Results: The VH and VL genes from spleen RNA of mice immunized with 164-D and 68-V were amplified and randomly linked together, using SOE-PCR producing band sizes about 750 bp. Anti-mKRAS G12V and G13D scFvs were constructed in phagemid pCANTAB5E vectors with a library containing 3.4 × 106 and 2.9 × 106 individual clones, respectively. After three rounds of bio-panning, the anti-mKRAS G12V-34 scFv antibody against G12V control mimotope was identified and confirmed without any cross-reactivity with other controls using ELISA. Anti-mKRAS G12V-34 scFv fragment was fused to mHALT-1 toxin and cloned in pET22b vector with expression as inclusion bodies in E. coli BL21(DE3) (molecular weight of ~46.8 kDa). After successful solubilization and refolding, the mHALT-1-scFv immunotoxin exhibited cytotoxic effects on SW-480 colorectal cancer cells with IC50 of 25.39 μg/mL, with minimal cytotoxicity effect on NHDF cells.
Discussion: These results suggested that the development of such immunotoxins is potentially useful as an immunotherapeutic application against KRAS-positive malignancies.