Diagnosis of Blastocystis sp., Cryptosporidium sp., and Giardia intestinalis by Multiplex PCR: An Optimization Study

Objective: It was aimed to develop a new Multiplex Polymerase Chain Reaction (PCR) protocol with isolates obtained from local patients for the diagnosis of Blastocystis sp., Cryptosporidium sp. and Giardia intestinalis, which can cause severe gastrointestinal system complaints especially in immunocompromised patients and children. Method: DNA isolation was performed with a commercial kit from three stool samples of different patients whose microscopic examination showed dense amounts of Blastocystis sp., Cryptosporidium sp. and Giardia intestinalis. First, a special PCR protocol has been developed for each protozoon. Then, the multiplex PCR protocol, in which these three protozoa can be diagnosed together, was optimized. Results: In the multiplex PCR protocol performed after DNA isolation, bands of 95 bp., 227 bp. and 258 bp. were obtained for Cryptosporidium sp., Blastocystis sp. and G. intestinalis, respectively. Conclusion: Blastocystis sp., Cryptosporidium sp. and Giardia intestinalis were diagnosed by multiplex PCR with the original protocol developed. Due to the difficulties in using different methods in parasitological examination, by adding other protozoa important for public health to this optimized protocol, it will be possible to detect a large number of parasites with a single molecular method.

Investigation of Carbapenemase Genes in Carbapenem Resistant Enterobacterales Isolates: First KPC Report From Dokuz Eylul University Hospital

Objective: In recent years, increasing carbapenem resistance of Enterobacterales bacteria limits treatment options, considerably. The main mechanism of this resistance is the production of carbapenemase enzymes. The aim of this study is to determine carbapenemase gene types in Enterobacterales isolates from our hospitalized patients and assess the clonal associations of the isolates with KPC gene. Method: A total of 48 clinical Enterobacterales isolates resistant to at least one carbapeneme and received between January 2019 and March 2019 were included in the study. Sample types were consisted of urine, blood, tracheal aspirate, wound and sputum. Of these isolates, three were Escherichia coli while 45 were Klebsiella pneumoniae. Types of carbapenemases were investigated by polymerase chain reaction, using specific primers for VIM, IMP, NDM, KPC and OXA-48 genes. PFGE was performed to determine the clonal associations between blaKPC positive K. pnemoniae isolates. Results: According to the results, blaOXA-48 (n=2) and blaKPC (n=1) were found to be present among E. coli isolates. Regarding 45 K. pneumoniae isolates; only blaOXA-48 and only blaNDM were present in 30 and two isolates, respectively. Seven K. pneumoniae isolates were found positive for both blaOXA-48 and blaNDM. Remaining K. pneumoniae isolates (n=6) harboured only blaKPC. None of the isolates were positive for blaIMP and blaVIM. PFGE analysis showed four isolates had the same pulsotype (A), while two had different pulsotypes (B-C). Conclusion: To our knowledge, this is the first report of KPC gene isolated in Dokuz Eylul University Hospital.

Investigation of Cryptosporidium spp. and Giardia spp. in Lake Eğirdir (Isparta)

Objective: The aim of this study was to assess both the presence and seasonal variability of Cryptosporidium spp. and Giardia spp. in Eğirdir Lake within the borders of Isparta province, which is used for drinking, agricultural irrigation and recreational purposes. Method: The research was carried out between July 2016 and January 2017 and water samples were taken from five different stations in three different seasons in Lake Eğirdir. After direct microscopic examination of the samples (Native-Lugol method), they were stained with Modified Acid Fast (MAF), and examined under the light microscope for parasites. Results: Cryptosporidium spp and Giardia spp were detected in 15 water samples in summer months, with an average density of 99.2% and 93.3% respectively, in Lake Eğirdir. In addition, both parasites were also detected intensively in autumn and winter Conclusion: The use of Lake Eğirdir for daily needs of people, agriculture andrecreational purposes cause increase in protozoal density. Thus, it is necessary to conduct parasitological studies on Lake Eğirdir, especially during the periods of swimming tourism, to determine the protozoal epidemiology in humans and animals. In addition, it is important to carry out adequate disinfection processes and plan the necessary control programs in terms of public health in the regions where Lake Eğirdir is used as drinking water.

In silico Analysis of Phylogeny and Virulence Genes in STEC Strains Associated with Outbreaks

Objective: Shiga toxin-producing E. coli (STEC) strains are important foodborne pathogens. Significant outbreaks with STEC strains can be encountered, even if the geography, time or resources were different. The aim of our in silico study was to compare the virulance factors and phylogeny of STEC strains such as EDL933 and Sakai, which have been identified as an agent in important outbreaks in different parts of the world and whole genomic data were in open databases. Method: Genomic NCBI data of eight strains were included in our study, including seven different STEC strains associated with significant epidemics in different parts of the world, and one supershedder strain obtained from cattle feces. Results: According to phylogeny analysis, the most similar strain to EDL933 strain was TW14588, with 96.4% similarity. The most distant similarity was Sakai strains with 79.2%. According to the virulence genes analysis; the presence of 333 genes that constitute virulence factors under nine headings were detected. In the first STEC origin, EDL933, 45% of all virulence genes were found to be active. Adherence genes such as Ecp, Elf, Hcp and toxin genes such as clyA were active in all strains except stx genes. Conclusion: In our in silico study of comparative genomic analysis of STEC strains which are associated with outbreaks, it was determined that STEC strains used different virulence genes besides the stx gene. Indeed, they used certain virulence genes, even their sources, time and locations were different, in the pathogenesis

Spread of Carbapenemase Producing Klebsiella pneumoniae Isolates in Our Hospital: Investigation of Molecular Typing and Clonal Relationship

Objective: Carbapenem resistance has been reported with increasing frequency among members of Enterobacterales, especially in the last 10 years. Screening and detection of carbapenemase-producing isolates is important in terms of both directing the treatment and preventing its spread. In our study, it was aimed to determine the carbapenemase types and molecular epidemiological relationships of carbapenem resistant Klebsiella pneumoniae isolates, which were isolated sequentially from the samples sent to microbiology laboratory of our hospital. Method: A total of 32 carbapenem-resistant K. pneumoniae isolates of the samples sent to microbiology laboratory between July and September 2014, were included in the study. In addition to classical methods, identification of isolates at species level was made with BD Phoenix ID/AST automated system. Carbapenemase types (blaOXA-48, blaNDM, blaIMP, blaKPC, blaVIM and blaGES) of the isolates were investigated by PCR. The clonal relationship between the isolates was assessed with PFGE. Results: It was noted that 18 isolates were obtained from intensive care units, 9 from inpatient and 5 from outpatient departments. The blaOXA48 gene was found in all isolates while the other carbapenemase genes were not found. It was determined that strains were isolated from 32 patients in our hospital had 12 different PFGE pulsotypes, named as A-L. Among these, the most common ones were B (n=18) and closely related B1 pattern (n=2). The remaining isolates were represented by 11 different types. It was observed that the first isolate with B pulsotype was responsible for the spread of the outbreak from General Intensive Care Unit. Conclusion: It has been thought that the spread of carbapenem- resistant K. pneumoniae isolates in the hospital was probably occurred through the transfer of isolates from patients with gastrointestinal colonization to other patients through hospital staff. Therefore, the spread of the isolates in hospitals can be limited by detecting colonization with active surveillance programs and by applying contact isolation and effective infection control measures.

Hope in the Treatment of Candida Vaginitis: Ibrexafungerp

Candida vaginitis (CV) is a neglected but growing public health problem. It is estimated that three out of four women have had at least one CV attack. The diagnosis and treatment of CV is often inadequate due to the tendency of women to self-diagnose and use over-the-counter drugs when they have vaginal or vulvar complaints, and clinicians plan treatment only according to clinical findings. There are limitations regarding the safety and efficacy of oral azoles, which are primarily preferred for the treatment of vaginitis, and these drugs have not revealed the desired levels of clinical or mycological cure. Also, there are a few options for current drugs used for treatment are not numerous the development of new antifungal drugs is thus urgently needed. Ibrexafungerp (IBX) is a semisynthetic triterpenoid glucan synthase inhibitor derived from enfumafungin. IBX has been shown to be a promising oral antifungal in the treatment of acute CV, and its use was approved on June 1, 2021. IBX is remarkable by it’s high oral bioavailability, low risk of side effects, few drug-drug interactions, good tissue penetration, increased activity at low pH in the vagina, and efficacy with regard to multi-drug-resistant fungi. In this review, in vitro and in vivo data on IBX were evaluated and compiled in light of current knowledge.

The Relationship Between the Use of Hydrogel and Silicone Hydrogel Contact Lenses and Coagulase-Negative Staphylococci Population in the Conjunctiva and Biofilm Forming Staphylococcus epidermidis

Objective: The most important bacteria of the conjunctival microbiota are Staphylococcus epidermidis, diphteroid rods, Corynebacterium spp. and Cutibacterium acnes. Especially biofilm formation of S. epidermidis is very important for contact lens related infections. For this purpose, we aimed to examine the changes in the presence of biofilm-forming S. epidermidis and other coagulase-negative staphylococci in conjunctival swabs taken before and after lens usage in 140 patients (90 hydrogel, 50 silicone hydrogel) who were prepared to wear lenses. Methods: Coagulase-negative staphylococci isolated from the conjunctival microbiota identified standard clinical microbiological methods, after identification of S.epidermidis strains with API Staph; Slime production was determined by Congo red agar, standard tube and molecular methods. Results: S.epidermidis was the most frequently isolated species in conjunctival microbiota before and after lens usage. Before lens usage, slime positive S. epidermidis strains were found as 45-50% but after lens usage it was 59% in hydrogel contact lens users and 70.2% in silicone hydrogel contact lens users. For the investigation of slime production, 82 (50.9%) of 161 S. epidermidis strains were found positive by using Congo red agar, 61 (37.8%) by standard tube method and 91 (56.5%) by molecular methods. Conclusion: The result of our study suggests that there are no significant changes in bacterial ratios before and after lens use, but bacteria such as S. epidermidis can predispose to infections by using slime production and contact lens factor. Also; molecular methods and Congo Red Agar method were found to be more reliable than the Standard Tube method.

The Dark Matter of Human Microbiota: Virobiota and Virome

The viral component of the human microbiome is referred as ‘virobiota’. The virobiota is the sum of all viruses found in or on humans. The set of all genes of virobiota is referred as ‘virome’. The human virome consists of virus-derived genetic elements found in human genome constituted of viruses that infect eukaryotic cells, bacteriophages, prokaryotic cells, and, endogenous retroviruses. The development of new sequencing technologies, such as high-throughput sequencing techniques allowed the analysis of the human virome. Many new viruses have been discovered lately, using new generation sequencing technology. In recent years, there has been an increase in the studies of the human virome as changes in virome have been observed in diseases. The alterations in the human virome may be associated with infectious, inflammatory diseases, cancer and autoimmunity. The understanding of how the virome affects human health and disease can provide the development of potential therapeutic approaches that target the members of the virome.