Phenotypical and Genotypical Comparison of Clostridium difficile Isolated from Clinical Samples: Homebrew DNA Fingerprinting versus Antibiotic Susceptibility Testing (AST) and Clostridial Toxin Genes

Background. Clostridium (Clostridioides) difficile is recognized as the major cause of healthcare antibiotic-associated diarrhea. We surveyed a molecular epidemiological correlation between the clinical isolates from two general hospitals in Iran through clustering toxigenic types and antibiotic susceptibility testing (AST) accuracy. Methods. Study population included 460 diarrhoeic specimens from inpatients with a history of antibiotic therapy. All samples underwent enriched anaerobic culture, confirmed by detection of gluD gene with PCR. Toxin status and AST were assessed by the disk diffusion method (DDM) and minimal inhibitory concentrations (MICs) of metronidazole, vancomycin, and rifampin. C. difficile outbreak was analyzed through conventional PCR by tracing toxin genes and Homebrew pulsed-field gel electrophoresis (PFGE) for characterizing isolates within our healthcare systems. Results. A total of 29 C. difficile strains were isolated by enriched anaerobic culture from the clinical samples. Among them, 22 (4.8%) toxigenic profiles yielded toxins A and B (tcdA, tcdB) and binary toxins (cdtA, cdtB). The minimum inhibitory concentration (MIC) was 18.1% and 9% for vancomycin and metronidazole, and all isolates were susceptible to rifampicin and its minimum inhibitory concentration was at <0.003 μg/mL. The most dominant toxigenic and antibiotic-resistant “pulsotype F” was detected through PFGE combined with multiple Clostridial toxigenic pattern and AST. Conclusions. DNA fingerprinting studies represent a powerful tool in surveying hypervirulent C. difficile strains in clinical settings. Resistance to vancomycin and metronidazole, as first-line antibiotics, necessitate accomplishment of proper control strategies and also prescription of tigecycline as a more appropriate option.

Extrauterine Epithelioid Trophoblastic Tumor (ETT) Arising from an Antecedent Undiagnosed Molar Pregnancy Presenting as an Adnexal mass: A Case Report

Introduction/Objective Epithelioid trophoblastic tumor (ETT) is an extremely rare neoplasm derived from chorionic type intermediate trophoblast. ETT usually follows an antecedent term pregnancy but can also follow spontaneous abortions or molar pregnancy. ETT most often arises from the endometrium, followed by the cervix. Extrauterine ETT are extremely rare, with few cases reported in literature. Methods/Case Report A 41-year-old woman with three term pregnancies presented with abdominal pain, ten years after her last pregnancy. Imaging findings of a 3.5 cm adnexal mass coupled with an elevated serum β-hCG (~ 900 mIU/ml), led to the suspicion of an ectopic pregnancy. Hysterectomy with salpingectomy revealed a 4.7 cm, tan- yellow, necrotic mass in the adnexal region abutting but distinct from the uterine serosa. Histologic evaluation showed a well- circumscribed tumor with pushing borders. The tumor cells were epithelioid with well-defined eosinophilic cytoplasm, monomorphic nuclei, frequent mitosis, and abundant geographic necrosis. The tumor cells were positive for β-hCG, GATA-3, PLAP and inhibin, with focal weak staining squamous markers p63 and p40. DNA fingerprinting analysis, performed to confirm the diagnosis of ETT, revealed a homozygous tumor with two copies of non-maternal genes indicating that the antecedent index gestation giving rise to the tumor was an undiagnosed hydatidiform mole. Following surgery, serum β-hCG levels were normal and the patient is currently on surveillance. Results (if a Case Study enter NA) NA Conclusion We present an extremely rare case of extrauterine ETT arising from a previously undetected molar pregnancy. The diagnosis should be suspected when a mass is observed at extrauterine sites with elevated β-hCG levels in patients with or without vaginal bleeding. Histologic differential of squamous cell carcinoma needs to be ruled out with immunostains. Due to its rarity and highly variable presentation, this entity remains a diagnostic challenge. DNA fingerprinting analysis demonstrating non-maternal genes can help confirm the diagnosis of ETT.

DNA Finger-Printing: Current Scenario and Future

Linearly arranged chemical structure in chromosome is known as DNA. It is a double helix made up of two strands of genetic material spiraled around each other. Each strand has a sequence of bases. There are four types of basis namely adenine, guanine, cytosine and thiamine which are very unique to each individual just like their actual fingerprint. The nitrogen base adenine always binds with thymine and cytosine also always binds with guanine. Thus the DNA profiling unique to each individual is collectively known as DNA fingerprinting. DNA determines individuality or uniqueness of the each human being except in uniovular twins. The chances of complete similarity are one in 30 billion to 300 billion i.e. half the population of world. The technique of DNA fingerprinting was first developed by Dr. Alec Jeffery’s from Britain in 1984. He discovered a minisatellite region close to the human myoglobin gene. He isolated this sequence and used it as a probe to investigate human DNA. He found that the minisatellite probe result was a complex band pattern for each individual. In India, initially it was done at CCMB, Hyderabad by Dr. Lalji Singh. Now there are various centers where DNA fingerprinting is carried out. In Maharashtra it is carried out at Sate Forensic Science Laboratory, Vidya Nagar, Kalina, Mumbai – 400 098 (Phone 022–26670755). Using this technique FBI formally concluded the participation of Mr. Bill Clinton in Monica Lewyninskey case. In India more than 79 cases have been solved by using this technique including important case of Dhanu and Shivarasan alleged assailant of Late Priminister Shr. Rajiv Gandhi, Tandori case, Madhumati murder case etc.

Anti-trypanosomal activity and DNA fingerprinting of fifteen Euphorbia species using ISSR and SCoT markers

Background Euphorbia is the largest genus in family Euphorbiaceae with a great biological and genetic diversity. The anti-trypanosomal activity of the crude extract of 15 Euphorbia species against Trypanosoma brucei brucei was carried out. Furthermore, DNA fingerprinting of the tested species using ISSR and SCoT markers was also investigated. Results The anti-trypanosomal activity of the 15 Euphorbia species revealed the highest activity of E. officinarum L. and E. milli Des Moul. against Trypanosoma brucei brucei with IC50 values < 10 μg/mL after 48- and 72-h incubation. Moreover, the assessment of the genetic diversity among the 15 tested species showed similar correlation coefficients of 0.76–0.98 which classified Euphorbia species into two main groups, one contained two species and the other contained 13 species. Conclusions Some Euphorbia species exhibited significant growth inhibitory activity toward Trypanosoma brucei strain TC221. Results also indicated the suitability of both markers for genetic fingerprinting of the tested Euphorbia species. To our knowledge, this is the first detailed comparison of the performance of two targeted DNA molecular markers (SCoT and ISSR) on the tested 15 Euphorbia species. The results guide future efficient use of these molecular markers in the genetic analysis of Euphorbia.