Gut microbiota in vaccine naïve Gabonese children with rotavirus A gastroenteritis.

Background: While the gut microbiome modulates the pathogenesis of enteric viruses, how infections caused by rotavirus A (RVA), with or without diarrhoea, alter the gut microbiota has been sparsely studied. Methods: From a cohort of 224 vaccine naïve Gabonese children with and without diarrhoea (n = 177 and n = 67, respectively), 48 stool samples were analysed: (i) RVA with diarrhoea (n = 12); (ii) RVA without diarrhoea (n = 12); (iii) diarrhoea without RVA (n = 12); (iv) healthy controls without diarrhoea and RVA (n = 12). The 16S rRNA metabarcoding using Oxford Nanopore sequencing data was analysed for taxonomic composition, abundance, alpha and beta diversity, and metabolic pathways. Findings: Alpha diversity showed that children with acute diarrhoea (with and without RVA infection), and children with acute diarrhoea without RVA had low microbial diversity compared to healthy children (p = 0.001 and p = 0.006, respectively). No significant differences observed when comparing children with RVA with or without diarrhoea. Beta diversity revealed high microbial heterogeneity in children without diarrhoea. Proteobacteria (68%) and Firmicutes (69%) were most common in the diarrhoea and non-diarrhoea groups, respectively. Proteobacteria (53%) were most common in children without RVA, while Firmicutes (55%) were most common with RVA. At the genus level, Escherichia (21%), Klebsiella (10%) and Salmonella (4%) were abundant in children with diarrhoea, while Blautia (11%), Clostridium (8%), Lachnoclostridium (6%) and Ruminococcus (5%) were abundant in children without diarrhoea. Metabolites involved in amino acid, carbohydrate, lipid, nucleotide, and vitamin metabolism were quantitatively altered. Interpretation: Although host physiology dictates the intestinal milieu, diarrhoea per se can alter a balanced gut microbiota, whereas infectious diarrhoea disrupts the gut microbiome and reduces its diversity.

Aetiologies and clinical presentation of central nervous system infections in Vietnamese patients: a prospective study.

Knowledge of the clinical presentation of central nervous system (CNS) infections and the causative pathogens is crucial for appropriate diagnosis and rapid initiation of appropriate treatment to prevent severe neurological sequelae. The aim of this study is to understand the aetiology of CNS infections based on the clinical presentation of Vietnamese patients. A prospective hospital-based cohort study was conducted between May 2014 and May 2017. We screened 137 patients with clinically suspected CNS infection for fungal, bacterial and viral pathogens using their cerebrospinal fluid (CSF) and blood cultures. In addition, DNA or RNA extracted from CSF samples were subjected to nucleic acid testing (NAT) with a selective panel of bacterial, viral and fungal pathogens. At least one pathogen could be detected in 41% (n = 56) of the patients. The main pathogens causing CNS infections were Streptococcus suis (n = 16; 12%) and Neisseria meningitidis (n = 9; 7%), followed by Herpes simplex virus 1/2 (n = 4; 3%) and Klebsiella pneumoniae (n = 4; 3%). Other pathogens were only identified in a few cases. Patients with bacterial CNS infections were significantly older, had a worse outcome, a lower Glasgow Coma Scale (GCS), a higher rate of speech impairment and neck stiffness than patients with viral or tuberculous CNS infections. In northern Vietnam, adults are mostly affected by bacterial CNS infections, which have a severe clinical course and worse outcomes compared to viral or tuberculous CNS infections. Clinicians should be aware of the regional occurrence of pathogens to initiate rapid and appropriate diagnosis and treatment.

Longitudinal monitoring of laboratory markers characterizes hospitalized and ambulatory COVID-19 patients.

Early detection of severe forms of COVID-19 is absolutely essential for timely triage of patients. We longitudinally followed-up two well-characterized patient groups, hospitalized moderate to severe (n = 26), and ambulatory mild COVID-19 patients (n = 16) at home quarantine. Human D-dimer, C-reactive protein (CRP), ferritin, cardiac troponin I, interleukin-6 (IL-6) levels were measured on day 1, day 7, day 14 and day 28. All hospitalized patients were SARS-CoV-2 positive on admission, while all ambulatory patients were SARS-CoV-2 positive at recruitment. Hospitalized patients had higher D-dimer, CRP and ferritin, cardiac troponin I and IL-6 levels than ambulatory patients (p < 0.001, p < 0.001, p = 0.016, p = 0.035, p = 0.002 respectively). Hospitalized patients experienced significant decreases in CRP, ferritin and IL-6 levels from admission to recovery (p < 0.001, p = 0.025, and p = 0.001 respectively). Cardiac troponin I levels were high during the acute phase in both hospitalized and ambulatory patients, indicating a potential myocardial injury. In summary, D-dimer, CRP, ferritin, cardiac troponin I, IL-6 are predictive laboratory markers and can largely determine the clinical course of COVID-19, in particular the prognosis of critically ill COVID-19 patients.

A Case Series of Imported Malaria Caused by Plasmodium falciparum in Kayseri and Review of Literature.

Malaria is a major worldwide public health problem. In the last years, no domestic cases of malaria have been detected and cases of imported malaria exist only in Turkey. In this study, clinical and laboratory findings of five Plasmodium falciparum (P. falciparum) malaria patients who were admitted to the emergency department between January 2013 and December 2015 were retrospectively presented. One of the patients was an African student, and the other patients had a history of travelling to Africa. Ring formation was observed when Giemsa staining was performed on the blood smears of all patients, and in three patients, P. falciparum was also detected using multiplex polymerase chain reaction (PCR) (Bio-Rad, United States of America). P. falciparum was not detected by PCR in the other two patients. Malaria should be primarily considered in febrile patients who have a history of travelling to endemic regions, and peripheral blood smears should definitely be examined.

Cloning of the DNA Vaccine Candidate LACK Gene of Leishmania infantum.

OBJECTIVE: Leishmaniasis is caused by an obligate intracellular protozoa belonging to Leishmania genus and listed among major tropical diseases by WHO. Because of the high costs, toxicity, and adverse effects of routinely used compounds in the treatment, alternative treatment and vaccine studies are underway. An effective vaccine has not been developed to date. In this study, we aimed to clone one of the most promising DNA vaccine candidates: the homolog-activated C kinase (LACK) gene of Leishmania infantum. METHODS: L. infantum genomic DNA was isolated from promastigote culture. The LACK gene was placed into plasmid pJET1.2. Then, recombinant plasmids were transformed into competent cells. The presence of recombinant plasmids was determined by PCR screening. Cloning was confirmed by PCR, restriction enzyme assays, and finally, DNA sequence analysis, after making miniprep from positive colonies. RESULTS: After performing PCR with LACK-gene specific primers, 939-bp PCR products were observed. Recombinant plasmids, which were transformed into competent Escherichia coli cells, were verified by PCR screening. It was verified by PCR that the recombinant plasmid contained the LACK gene. DNA sequence analysis was performed to obtain the DNA sequence. CONCLUSION: One of the most promising DNA vaccine candidates against leishmaniasis, the LACK gene, was cloned in this study.

Microbiota: In Health and in Sickness, From Birth to Death.

Microorganisms colonize tissues and organs such as the skin and gastrointestinal, respiratory, and genitourinary systems. These microorganisms are generally called as "human microbiota". Human microbiota mostly consists of commensal microorganisms. The commensal microorganisms located on and in the human body are bacteria, fungi, viruses, archaea, and parasites. The microbiota genome is 100 times bigger in size than the human genome. Although the human genome is stationary, microbial genome has a compatible flexible variability during human life. As well as 2-year-old child and newborn, adult and adolescent, the elderly and pregnant woman have a different microbiota. Microbiota and the microbiota genome can be changed by personal and household diet, antibiotic use, mode of delivery, and hygiene within days or even hours, depending on such as these factors. The human immune system and microbiota grow up, develop, and mature as childhood friends by playing with each other from birth to death. Association between microbiota and human is not just related to childhood-it continues with health and disease, until death separates them. This review focused on the roles of microbiota in parasitology, autoimmune diseases, metabolic diseases, and cancer treatment in detail. In addition, inflammatory and immunoregulatory roles of microbiota on the intestinal immune system and how innate and adaptive immune systems regulate microbiota and its content were explained.

[Investigation of potentially pathogenic free-living amoebae and their in vivo pathogenicity in water supplies of Turkey]. / Türkiye'deki sularda serbest yasayan potansiyel patojen amipler ve patojenitelerinin in vivo olarak arastirilmasi.

Free-living amoebae (FLA) are found widely in soil and water in the nature. Among them in which potentially pathogenic for humans and animals are known as "potential pathogenic free-living amoebae (PPFLA)". PPFLA are characterized as the causes of clinical manifestations leading to death especially in immunosuppressed people. Four genus of PPFLA (Acanthamoeba, Naegleria, Balamuthia and Sappinia) are known to be pathogenic to humans. The aims of this study were to investigate the presence of PPFLA in the water supplies in Turkey and to determine their in vivo pathogenicity. A total of 664 water samples were collected from the ponds, rivers, streams and wells found in provinces located at different regions (central, western, eastern and southeastern regions) of Turkey. These samples were initially inoculated in the monoxenic culture media and evaluated by both microscopy and polymerase chain reaction (PCR) in terms of the presence of FLA. The samples identified as positive were then cultured in axenic media, the growth of amoebae that were confirmed microscopically, were than studied with PCR for molecular characterization. The isolates that were found positive by PCR from axenic cultures were inoculated intranasally to immunocompetent and immunodeficient (athymic) [BALB/c Rag2(-/-) gamma(c)(-/-)] BALB/c mice followed by the evaluation on the 21st day by histopathological and molecular methods to investigate their in vivo pathogenicity. In our study, 143 water samples were detected as positive in monoxenic cultures and 41 of them were detected as positive in axenic cultures. Twenty of 41 samples detected as positive in axenic culture could be continued in culture for three months. As a result of PCR using primers common to SYA, only nine have been identified from 20 samples as positive. According to the result of the PCR with specific primers, all (n= 9) were positive for Acanthamoeba sp., eight for Sappini sp. and five for Balamuthia mandrillaris, while none was observed Naegleria fowleri. Histopathologic examination revealed that both groups of mice that were infected with the nine isolates had normal brain tissue sections; but haemorrhages and mononuclear cell proliferation were determined in four immunocompetent and seven athymic animal lung sections. When the presence of parasites in tissue samples were evaluated by real-time PCR, Balamuthia was detected in at least one blood, lung, brain or nasal mucosa sample of the four immunocompetent mice, Sappinia sp. in four and Acanthamoeba sp. in seven immunocompetent mice infected with nine isolates. Additionally, seven Balamuthia sp., seven Sappinia sp. and eight Acanthamoeba sp. were detected in immunodeficient mice. In this study, B. mandrillaris and Sappinia sp. were the first isolated potentially pathogenic amoebae from water supplies located at different parts of Turkey. As a result awareness and precautions against suspicious water supplies used for drinking, daily use and swimming purposes should be treated more carefully.

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.

[Cryptosporidium parvum Gastroenteritis in a Patient with Renal Transplantation]. / Renal Transplantli Bir Hastada Cryptosporidium parvum Gastroenteriti.

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 the presence of Encephalitozoon intestinalis and Enterocytozoon bieneusi in bone marrow transplant patients by IFA-MAbs method]. / Kemik iligi transplant hastalarinda Encephalitozoon intestinalis ve Enterocytozoon bieneusi varliginin IFA-MAbs yöntemiyle arastirilmasi.

Microsporidian pathogens are obligatory intracellular eukaryotic parasites which can be found worldwide. They have been represented in 144 genera and more than 1200 species that may cause infections in both vertebrate and invertebrate hosts. Enterocytozoon bieneusi and Encephalitozoon intestinalis are the most common species among 14 species of microsporidia identified as human pathogens and they cause infections in the gastrointestinal tract. These species may also cause chronic diarrhea particularly in immunocompromised patients, as well as disseminated infections with severe clinical conditions which can be life-threatening. Since the spores of microsporidia are quite small-sized structures, they frequently may be overlooked in routine stool examinations. Therefore, molecular methods and transmission electron microscopy, if possible, are used as the gold standard methods in laboratory diagnosis. In laboratories in which those methods could not be applied, immunofluorescence assay using monoclonal antibodies (IFA-MAbs) may be advantageous compared to conventional methods. The aim of this study was to investigate the presence of E.intestinalis and E.bieneusi in bone marrow transplant (BMT) patients by using IFA-MAbs method. A total of 200 BMT patients (134 male, 66 female; mean age: 43.2±15.01 years), of them 147 with diarrhea and 80 healthy subjects (43 male, 37 female; mean age: 31.9±11.76 years) as control group were included in the study. All of the stool samples were examined by a commercial IFA-MAbs (Bordier Affinity Products, Switzerland) method as well as conventional (native-lugol and modified acid-fast staining) methods. Of the patients 25.5% (51/200) were positive for E.intestinalis, 4% (8/200) for E.bieneusi and 9.5% (19/200) for both of them, giving a total positivity rate of 39% (78/200). Those rates were 5% (4/80), 2.5% (2/80), 3.8% (3/80) and 11.3% (9/80), respectively for control group. The difference between the patient and control groups in terms of positivity was found statistically significant (39% vs 11.3%, p<0.05). Among 78 positive BMT patients, 67 (85.9%) were suffering from diarrhea. The correlation between the presence of diarrhea and the presence of microsporidia was statistically significant (p<0.05). It was concluded that, BMT patients particularly those with gastrointestinal complaints, have to be evaluated for microsporidian pathogens regularly to improve quality of life and to decrease the problems during the treatment period.

[The Epidemiology of Malaria in Kayseri Between 2001 and 2013]. / 2001-2013 Yillari Arasinda Kayseri'de Sitma Epidemiyolojisi.

OBJECTIVE: Malaria is the primary parasitic cause of morbidity and mortality in the world. As a result of the expansion of international travel in recent years, imported malaria cases especially are increasing in our country. Likewise, while there were more domestic cases earlier in Kayseri, more imported cases were seen in recent years. In our study, the epidemiology of malaria cases between the years of 2001-2013 is intended to be done with the data obtained from the Provincial Health Directorate. METHODS: The data was performed retrospectively. RESULTS: Considering the last 12 years of data; a total of 34,459 blood samples were analyzed and 47 of these cases were found to be malaria, 21 cases were domestic and others were imported cases of malaria. P. vivax was detected in all domestic cases. While one of the imported cases have been identified as P. malariae, others were P. falciparum. CONCLUSION: We believe that our study of the epidemiological data would be beneficial for taking preventive cautions and fight against malaria.

[Prevalence of Encephalitozoon intestinalis and Enterocytozoon bieneusi in cancer patients under chemotherapy]. / Kemoterapi alan kanserli hastalarda Encephalitozoon intestinalis ve Enterocytozoon bieneusi prevalansi

Microsporidia species are obligate intracellular parasites and constitute one of the most important opportunistic pathogens that can cause severe infections especially in immunocompromised patients. Enterocytozoon bieneusi and Encephalitozoon intestinalis are the most common species among 14 microsporidia species identified as human pathogens. The aim of this study was to investigate the prevalence of E.intestinalis and E.bieneusi in cancer patients under chemotherapy by immunofluorescent antibody and conventional staining methods. A total of 123 stool samples obtained from 93 patients (58 male, 35 female) with cancer who were followed in oncology and hematology clinics of our hospital and 30 healthy volunteers (13 male, 17 female) were included in the study. Fifty-one (55%) of the patients had complain of diarrhea. The presence of E.intestinalis and E.bieneusi were investigated by a commercial immunofluorescence antibody test using monoclonal antibodies (IFA-MAbs; Bordier Affinity Products, Switzerland) in all of the samples, and 50 of the samples were also investigated by modified trichrome, acid-fast trichrome and calcofluor staining methods. A total of 65 (69.9%) patients were found positive with IFA-MAbs method, including 43 (46.2%) E.intestinalis, 9 (9.7%) E.bieneusi and 13 (14%) mixed infections. In the control group, 5 (16.7%) subjects were positive with IFA-MAbs method, including 2 (6.7%) E.intestinalis, 1 (3.3%) E.bieneusi and 2 (6.7%) mixed infections. The difference between the positivity rate of the patient and control groups was statistically significant (p< 0.05). Of the patients with diarrhea, 68.6% (35/51) were infected with microsporidia, and the difference between cases with and without (48.6%) diarrhea was statistically significant (p< 0.05). When 50 samples in which all of the methods could be performed were evaluated, the frequency of microsporidia were detected as follows; 66% (n= 33) with IFA-MAbs, 34% (n= 17) with modified trichrome staining, 24% (n= 12) with acid-fast trichrome staining and 42% (n= 21) with calcofluor staining methods. Our data indicated that the use of IFA-MAbs method along with the conventional staining methods in diagnosis of microsporidia will increase the sensitivity. As a conclusion, the prevalence of E.intestinalis and E.bieneusi in cancer patients under chemotherapy was detected quite high (69.9%) in our study, it would be appropriate to screen these patients regularly in terms of microsporidian pathogens.

[Cloning DNA Vaccine Candidate SAG1 Gene of Toxoplasma gondii]. / Toxoplasma gondii DNA Asisi Adayi SAG1 Geninin Klonlanmasi.

OBJECTIVE: Toxoplasma gondii, which is observed in our country and worldwide, can cause mortality and is an important public health problem because of engaging babies from pregnant women. An effective vaccine against toxoplasmosis has not yet been developed. SAG1 protein is released from the bradyzoites and tachyzoites stages of T. gondii and is important at the pathogenesis of the disease. This study aimed to clone a promising DNA vaccine candidate, SAG1 gene, of T. gondii. METHODS: T. gondii genomic DNA was isolated from tachyzoites of T. gondii. SAG1 gene was amplified with specific primers and then cloned into the pJET1.2 vector. Recombinant plasmids were transformed into Escherichia coli cells. The presence of recombinant plasmids was determined by polymerase chain reaction (PCR) screening. Following the purification of the recombinant plasmid from positive colonies, cloning was confirmed by PCR, restriction enzyme assays, and DNA sequence analysis. RESULTS: After PCR with SAG1 gene-specific primers, 1010-bp PCR products were obtained. Recombinant plasmid, which was transformed into competent E. coli cells, was verified by PCR screening. Moreover, PCR verified that the recombinant plasmids contained the SAG1 gene. The DNA sequence was analyzed, and the DNA sequence was obtained. CONCLUSION: One of the promising DNA vaccine candidates against toxoplasmosis, SAG1 gene, has been cloned.

Molecular discrimination of Echinococcus granulosus and Echinococcus multilocularis by sequencing and a new PCR-RFLP method with the potential use for other Echinococcus species.

BACKGROUND/AIM: To develop a novel polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol using a new genomic marker sequence and a novel set of restriction enzymes in order to detect and discriminate 2 Echinococcus species, E. granulosus and E. multilocularis, found in formalin-fixed paraffin-embedded (FFPE) human tissues. MATERIALS AND METHODS: DNA was isolated from 11 FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis. A mitochondrial genomic marker region was amplified and sequenced using a novel primer pair and a new PCR-RFLP protocol was developed for the detection and discrimination of E. granulosus and E. multilocularis using a set of restriction enzymes including AccI, MboI, MboII, and TsoI. RESULTS: The selected marker region was amplified using DNA isolated from FFPE human tissue samples positive for cystic echinococcosis or alveolar echinococcosis and the discrimination of E. granulosus and E. multilocularis was accomplished by use of the novel PCR-RFLP method. CONCLUSION: In this PCR-RFLP protocol, use of any single restriction enzyme is enough for the discrimination of E. granulosus and E. multilocularis. The PCR-RFLP protocol can be potentially used for the discrimination of 5 other Echinococcus species: E. oligarthus, E. shiquicus, E. ortleppi, E. canadensis, and E. vogeli.

[Evaluation of imported Plasmodium falciparum malaria cases: the use of polymerase chain reaction in diagnosis]. / Yurt Disi Kaynakli Plasmodium falciparum Sitmali Olgularin Degerlendirilmesi: Tanida Polimeraz Zincir Reaksiyonunun Yeri.

Malaria affecting almost half of the world population continues to be an important health problem. Although domestic malaria cases have been decreasing in Turkey recently, cases caused by Plasmodium falciparum have increased due to the frequent travelling to Africa. The aims of this study were to evaluate demographic characteristics, clinical and laboratory findings in cases with falciparum malaria who attended to our clinic in 2012-2013 period, and the impact of polymerase chain reaction (PCR) for diagnosis. Nine patients evaluated were all male with a mean age of 34.3 (age range: 18-48) years, with the history of travel to Africa. Six cases did not take prophylaxis against malaria and other three cases used insufficient time. Mean duration of symptoms after return was 18.4 (range: 1-75) days, and the patients were admitted to the clinic within a mean of 5.2 (range: 1-15) days. Two patients had leucopenia, two patients had anemia, and eight patients had thrombocytopenia on admission. Alanine aminotransferase (ALT) levels in four cases and total bilirubin levels of six cases were over upper normal limits. Definitive diagnosis of cases was performed with the detection of ring and/or gametocytes forms of the parasite in Giemsa-stained peripheral blood smears. Furthermore, samples from seven patients were studied by nested PCR by using genus (Plasmodium rPLU 1 and 5) and species (rFAL 1 and 2, rVIV 1 and 2, rMAL 1 and 2, rOVA 1 and 2) specific primers. All of these seven samples yielded positive results with primers specific for P.falciparum ssrRNA. In the treatment, arthemeter/lumefantrin and doxycycline combination was used in seven patients, while intravenous artesunate and doxycycline combination was given to two patients, resulting with complete cure. Mean duration for the resolving of fever was 3.3 days, and mean duration for clearing the parasitemia from peripheral blood was 4.9 days. Initial ALT values and the duration of fever resolution (-796; p= 0.010), as well as the duration of parasitemia and initial thrombocyte counts (-797; p= 0.010) were negatively- correlated. It was concluded that, providing sufficient information on malaria and prophylaxis to people travelling to the endemic areas are crutial for protection. Moreover, in endemic areas for Crimean-Congo hemorrhagic fever, patients with fever and thrombocytopenia should be questioned in detail about the travel history, and peripheral blood smears should be examined in terms of malaria, since their clinical features are similar. Plasmodium PCR should be considered as one of the alternative diagnostic method in malaria, especially in cases with inconclusive microscopy.

[Microsporidia and microsporidiosis]. / 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.

Pronounced transcriptional regulation of apoptotic and TNF-NF-kappa-B signaling genes during the course of thymoquinone mediated apoptosis in HeLa cells.

Thymoquinone (TQ) is the active ingredient extracted from the essential oil of Nigella sativa. A number of studies implicated TQ as an antitumor agent. In this study, cytotoxic effects of the oil of N. sativa and TQ were evaluated on human cervical cancer cell line, HeLa cells. IC50 value was ~0.125 µl/ml for N. sativa oil preparations and 12.5 µM for TQ. TQ strongly inhibited wound healing at all concentrations ranging from 12.5 to 100 µM in a scratch wound healing assay. Additionally, induction of apoptosis by TQ was assessed by Giemsa staining and TQ was found to induce apoptosis in cancer cells especially at concentrations of 50 and 100 µM. TQ-mediated transcriptional regulation of 84 genes involved in apoptosis was studied using a PCR array. At low dose (12.5 µM), TQ was found to induce expression of four pro-apoptotic genes: BIK (~22.7-fold), FASL (~2.9-fold), BCL2L10 (~2.1-fold), and CASP1 (~2-fold). TQ was also found to reduce the expression of an anti-apoptotic gene implicated in NF-kappa-B signaling and cancer: RELA (~8-fold). At high dose (100 µM), TQ mediated the expression of 21 genes implicated directly in apoptosis (6 genes), TNF signaling (10 genes), and NF-kappa-B signaling (3 genes) such as BIK, BID, TNFRSF10A, TNFRSF10B, TNF, TRAF3, RELA, and RELB. In conclusion, this study implicates the role of TQ in the inhibition of cancer cell proliferation and migration. At the same time, our results strongly suggest that TQ intervenes with TNF and NF-kappa-B signaling during TQ-mediated induction of apoptosis in cancer cells.

[Cloning of Blastocystis sp subtype 3 small-subunit ribosomal DNA]. / Blastocystis sp. Subtip 3 Small-subunit Ribozomal Geninin Klonlanmasi

OBJECTIVE: Different sub-types or genetic variations of Blastocystis sp. are thought to play a role in the differential symptoms caused by the parasite or asymptomatic cases. In this study, it was aimed to clone a fragment of SSUrDNA gene of Blastocystis from a patient in order to define its phylogenetic subtype. METHODS: In this study, DNA isolation from the stool of a Blastocystis infected patient was performed. Blastocystis specific primers were used to amplify a SSUrDNA genomic fragment. The amplified DNA fragment was cloned into a plasmid and sequenced using plasmid specific primers. The obtained DNA sequence was analyzed using BLAST and a phylogenetic tree was constructed using the software MEGA. RESULTS: It was found that the Blastocystis isolate in our study is subtype 3. CONCLUSION: Cloning and sequencing of the target genomic region is suggested for phylogenetic analysis studies.

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.

[Investigation of anti-Echinococcus granulosus antibodies in patients with suspected cystic echinococcosis]. / Kistik Ekinokokkozis Ön Tanili Hastalarda Anti-Echinococcus granulosus Antikorlarinin Arastirilmasi.

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.