Ülfet Çetinkaya

Stool sample storage conditions for the preservation of Giardia intestinalis DNA

Stool is chemically complex and the extraction of DNA from stool samples is extremely difficult. Haemoglobin breakdown products, such as bilirubin, bile acids and mineral ions, that are present in the stool samples, can inhibit DNA amplification and cause molecular assays to produce false-negative results. Therefore, stool storage conditions are highly important for the diagnosis of intestinal parasites and other microorganisms through molecular approaches. In the current study, stool samples that were positive for Giardia intestinalis were collected from five different patients. Each sample was stored using one out of six different storage conditions [room temperature (RT), +4ºC, -20ºC, 70% alcohol, 10% formaldehyde or 2.5% potassium dichromate] for DNA extraction procedures at one, two, three and four weeks. A modified QIAamp Stool Mini Kit procedure was used to isolate the DNA from stored samples. After DNA isolation, polymerase chain reaction (PCR) amplification was performed using primers that target the β-giardin gene. A G. intestinalis-specific 384 bp band was obtained from all of the cyst-containing stool samples that were stored at RT, +4ºC and -20ºC and in 70% alcohol and 2.5% potassium dichromate; however, this band was not produced by samples that had been stored in 10% formaldehyde. Moreover, for the stool samples containing trophozoites, the same G. intestinalis-specific band was only obtained from the samples that were stored in 2.5% potassium dichromate for up to one month. As a result, it appears evident that the most suitable storage condition for stool samples to permit the isolation of G. intestinalis DNA is in 2.5% potassium dichromate; under these conditions, stool samples may be stored for one month.

The Prevalence of Pediculus humanus capitis in Two Primary Schools of Hacilar, Kayseri

OBJECTIVE Pediculosis capitis is a worldwide public health concern, and today, head lice are seen in all socio-economic levels. The infestation usually occurs by head-to-head contact and children, primarily girls, aged 3-12 years are mostly affected. In the present study a total of 405 pupils (214 boys and 191 girls) from two pre- and primary schools in the Kayseri-Hacılar region were examined for pediculosis capitis during March 2010. METHODS Lice and/or eggs were detected by visual examination of the childrens hair. RESULTS Out of 405 children, 44 (10.9%) were infested with head lice. There were significant differences between the schools and the gender while no significant differences could be found between infestation and childs age, education of the parents, income of the family, housing type, source of water, and the presence or absence of a bathroom. CONCLUSION Head lice remain a public health problem and more emphasis should be given to the education of parents regarding their biology and control.

[Investigation of intestinal parasites among primary school students in Kayseri-Hacılar].

OBJECTIVE Parasitic infections are an important health problem which affect children more than adults. Especially in growth-age children, this leads to problems such as malnutrition, malabsorption, growth retardation and learning disabilities. In this study, 328 students who were investigated in two primary schools between the ages of 6 and 14 in Kayseri-Hacılar region were analyzed for intestinal parasites. METHODS Stool samples were analyzed by light microscopy for the detection of helminths and protozoon using the native-lugol method. Cellophane tape samples were also analyzed by light microscopy for the detection of Enterobius vermicularis and Taenia spp. RESULTS At least one or more intestinal parasite species were found in 116 (35.4 %) children. The distribution of parasites which were detected in stool samples was as follows; Blastocystis hominis, 77 (23.5%); Enterobius vermicularis, 35 (10.7%); Giardia intestinalis, 14 (4.3%); Entamoeba coli, 15 (4.6%); Endolimax nana, 6 (1.8%); Hymenolepis nana, 1 (0.3%); Iodamoeba butschlii, 1 (0.3%). CONCLUSION Parasitic diseases are a major public health problem and we believe that education about personal hygiene, sanitation rules and parasitic diseases is important to overcome this problem.

The high prevalence of Encephalitozoon intestinalis in patients receiving chemotherapy and children with growth retardation and the validity of real-time PCR in its diagnosis

BACKGROUND/AIM The aim of this study was to investigate the presence of Encephalitozoon intestinalis in different patient groups consisting of immunocompromised and immunocompetent individuals. MATERIALS AND METHODS The stool samples of 100 patients consisting of 25 patients receiving chemotherapy and with acute gastrointestinal complaints, 25 with bone marrow transplant and acute gastrointestinal complaints, 25 with urticaria, and 25 with growth retardation were included in the study. As control groups, 25 subjects without any chronic disease but with acute gastrointestinal complaints and 25 healthy volunteers, making a total of 50 subjects, were included in the study. E. intestinalis was investigated by IFA-MAbs and molecular methods. RESULTS Forty percent of patients receiving chemotherapy and with acute gastrointestinal complaints, 24% of patients with bone marrow transplant and acute gastrointestinal complaints, 20% of patients with urticaria, 40% of children with growth retardation, and 28% of patients without any chronic disease but with acute gastrointestinal complaints were determined as positive. CONCLUSION To the best of our knowledge, this is the first report to assess the relationship between E. intestinalis and growth retardation. We think that the reliability of the use of molecular methods, especially real-time PCR, should be improved for the diagnosis of E. intestinalis.

Mikrosporidialar ve Mikrosporidiyozis

All microsporidia are obligate parasites and have no active stages outside their host cells. Microsporidia lack some typical eukaryotic characteristics. There are now over 1200 species identified in 144 genera. The most familiar stage of microsporidia is the small, highly resistant spore, the size of which differs according to the species and is often 1-10 μm. The general life cycle pattern of the microsporidia can be divided into three phases: the infective or environmental phase, the proliferative phase, and the sporogony or spore-forming phase. There are several methods for diagnosing microsporidia: light microscopic, transmission electron microscopy (TEM), immunofluorescence assays (IFA) and molecular methods. The clinical course of microsporidiosis depends on the immune status of the host and site of infection. Microsporidia can cause infections such as diarrhoea, keratitis, myositis, bronchitis and brochiolitis. Human microsporidiosis represents an important and rapidly emerging opportunistic disease, occurring mainly, but not exclusively, in severely immunocompromised patients with AIDS. The treatment of microsporidiosis is generally achieved with medications and supportive care. Depending on the site of infection and the microsporidia species involved, different medications are utilized. The most commonly used medications for microsporidiosis include albendazole and fumagillin.

[Investigation of anti-Echinococcus granulosus antibodies in patients with suspected cystic echinococcosis].

OBJECTIVE Cystic echninococcosis (CE) is an important helmintho-zoonotic disease causing health-threatening and economic losses for developing countries. In this study, anti-Echinococcus granulosus antibodies were evaluated in 1556 CE suspected patients (701 males, 855 females) who applied to the serology laboratory of the Parasitology Department of Erciyes University between June 1999 and July 2010. METHODS Fifty-six (3.6%) patients were evaluated with the three different methods of Indirect Hemagglutination Test (IHA), Indirect Fluorescent Antibody Test (IFAT) and Western blot (WB). 378 (24.3%) were tested with both IHA and IFAT, 123 (7.9%) with both IHA and WB,and 999 (64.2%) were evaluated with one of these three methods. RESULTS In 353 (22.7%) patients, anti-E. granulosus antibodies detected by one of above three methods were considered as positive. CONCLUSION Since some patients were assessed either as negative or positive with one of above test, we believe that it should be safer to use at least two tests together for diagnosis of CE.

Evaluation of four commercial DNA extraction kits for the detection of Microsporidia and the importance of pretreatments in DNA isolation

Microsporidia are obligate intracellular parasitic protozoa infecting the wide variety of hosts and are commonly known as a cause of chronic diarrhea particularly in immunocompromised individuals. Molecular-based tests have high sensitivity and specificity in disease diagnosis. However, these tests’ performance relies on the isolation of DNA in a good concentration. The standard procedures of commercial DNA extraction kits are usually insufficient for this purpose due to the tough walls of spores. This study aimed to test the significance of pretreatments by glass beads and freeze-thawing processes in DNA isolation from microsporidia spores. The parasite was cultured in growing Vero cells and seven serial dilutions were prepared from the collected spores. DNA purification was performed according to different tissue kits and stool kit procedures with and without any pretreatment. Concentration of isolated DNA samples were evaluated by real-time PCR. As a result of this study, the detectable amount of spores is minimum 10 spores in each 100 μl sample according to the different tissue kits’ standard protocols. However, according to the DNA stool mini kit, the detectable amount of spores was found to be 1,000 spores/100 μl of stool sample when pretreated with both the freeze-thawing and glass beads methods.In conclusion, the current study demonstrated that further pretreatments are an essential process for DNA extraction from the stool specimens in order to avoid possible false negativity in the diagnosis of microsporidiosis.

Cryptosporidium parvum Gastroenteritis in a Patient with Renal Transplantation

In this study, a case who starting abundant watery diarrhea on the 14th day of renal transplantation is presented. Stool sample was analyzed for Cryptosporidium spp. by carbol fuchsin staining method, copro-ELISA and nested polimeraze chain reaction (PCR). From sample found positive by Carbol-fuchsin staining method and Copro-ELISA, DNA sequence analysis was performed, gel-purified from amplicon obtained by nested PCR. As a result of DNA sequence analysis was determined to be Cryptosporidium parvum. Although C. parvum is a rare causative agent of gastroenteritis it can be cause serious clinical diarrhea solid organ transplantation patient. As a result, also C.parvum must be considered as a causative agent of diarrhea occurring after organ transplantation.

[Investigation of anti-Toxoplasma gondii antibodies among foreign students in a high school].

OBJECTIVE Toxoplasmosis is a major health problem in both developed and developing countries. Infection is transmitted to humans by consumption of raw or undercooked meat containing tissue cysts and by ingesting foods contaminated with oocysts from the feces of infected cats and felidae. In this study, it was aimed to investigate the incidence of T. gondii antibodies in 347 high school students from 28 different countries studying in Kayseri city. METHODS These students had ages ranging between 15 and 21 (average: 17.52 ± 1.36). Serum samples were researched by IFAT for T. gondii IgG and IgM antibodies. RESULTS 81 (23.3%) students were found to be seropositive for IgG, and 6 (1.72%) of students were positive for both IgG and IgM. There was no IgM seropositivity in IgG negative serum specimens. IgG avidity test was also done for 87 subjects who were found to be seropositive for anti-T. gondii IgG. The results indicated that 8 (9.2%) patients have equivocal range avidity and 79 (90.8%) patients have high avidity. CONCLUSION As a result, this disease, which is important in children and adolescents, can lead to severe disease staes. Therefore, in various regions, especially in the pediatric age group, sero-prevalence tests and necessary measures are needed in high sero-prevalent areas.

The molecular mechanisms of erythrocyte invasion of Plasmodium spp. as a model organism of apicomplexan protozoa.

Apicomplexan protozoa are a phylum of parazites that includes medically and agriculturally important pathogens. They are named for their cell apex which contains a number of organelles (rhoptri, micronemes, conoid, apical polar ring, dense granules and apicoplast), important for their invasion and development within host cells. Among important apicomplexan parasites that affect human health directly or indirectly are Plasmodium spp., Toxoplasma gondii, Cryptosporodium, Eimeria, Babesia, and Theileria. Apicomplexan parasites move and actively enter host cells by substrate-dependent gliding motility. In these parasites, gliding motility and host cell invasion are driven by an actomyosin-based system (Glydeosome). A gliding motor machinery is embeded between the plasma membrane and inner membrane complex (IMC), a unique double membrane layer. A unique actomyosin motor powers both host cell invasion and locomotion of apicomplexan invasive stage. The cytoplasmic motor, a transmembrane bridge, and surface ligants essential for cell invasion are conserved among the main apicomplexan pathogens. In this review, erythrocytet invasion of Plasmodial merozit, which is a model organism of apicomplexan parasites, has been reviewed in detail.