Real-Time PCR Confirmation of a Fatal Case of Primary Amoebic Meningoencephalitis in Turkey Caused by Naegleria fowleri or Brain-Eating Amoeba.

BACKGROUND: Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis (PAM), is a free-living amoeba. It is a water-borne infection usually detected in children and young people with healthy immune system who swim, dive and perform activities in fresh and hot springs. PURPOSE: In this study, it was aimed to raise awareness in the differential diagnosis of meningitis etiopathogenesis by showing that N. fowleri may also be the causative agent, albeit very rarely, in meningitis cases in Turkey. METHODS: Our case was an 18-year-old male patient whose relatives stated that he has gone to the hot spring; his headache complaint started after 2 to 3 days after return from the hot spring. Cerebrospinal fluid (CSF) sample taken from the patient was investigated by direct microscopic examination, real-time PCR method and sequence analysis. RESULTS: The CSF sample collected was taken into distilled water considering the possibility of transformation of trophozoites to intermediate form and incubated at 37 °C for 1 to 2 h, and pear-shaped non-permanent flagellated forms were observed in the direct microscopic examination, and molecular typing was performed to confirm the diagnosis. This study was a comprehensive case of N. fowleri whose etiological agent was isolated and confirmed by real-time PCR in Turkey. CONCLUSION: Clinician awareness would be the key factor in correctly diagnosing PAM. It is also recommended to investigate all likely environmental water sources in Turkey for more detailed information on the distribution and molecular identification of Naegleria species, ultimately to evaluate the potential pathogenic threat to human health and to develop strategies to combat such threats.

Imported Malaria: A Plasmodium falciparum Malaria Case in Turkey with Rarely Seen Blood Stages of Infection

Malaria is an important public health problem, with about 219 million cases, which account for 435 thousand deaths of children in Africa in 2017. The infection is transmitted by female anopheles mosquito, including Plasmodium species pathogen for human. In Turkey, no domestic cases have been reported; however, according to Annual Health Statistics, 214 imported cases were reported in 2017. Although P. vivax exactly was reported in previous years, imported Plasmodium falciparum (P. falciparum) cases have increased in the last few years. Herein, we report a case of a 41-year-old male patient who returned two weeks ago from a one-week African journey. A limited number of different blood stages of the parasite (mature trophozoite, young and mature schizont) have been seen in the examination of the peripheral smear; however, the gametocyte stage was not seen. In the present case, detecting mature stages beside the early forms at the peripheral smear could not eliminate the probability of P. falciparum. To confirm the diagnosis and regulate the treatment protocol, molecular methods were employed to differentiate the potential mixture of infection. In this case study, we propose how to approach an uncertain case of severe falciparum malaria or mixture of malaria infection combined with another Plasmodium species, as a result of limited number of different blood stages of the infection at the peripheral smear.

Two Imported Plasmodium falciparum Malaria Cases in Sanliurfa

In a 2017 data of World Health Organisation, malaria is still an important medical health care problem by threatening 217 million people and causing 435 thousand deaths. In our country, as a result of successful eradication programmes, any domestic cases were not encountered; however, approximately 200 import cases were seen each year from 2013 to 2017. This study aimed to create awareness for cases caused by P. falciparum that are increasingly seen in rare import cases, which displays more severe clinical course than other Plasmodium species.

[Alternative Approaches in Direct Microscobic Examination of Fresh Fecal Samples]. / Taze Diski Örneklerinde Direkt Mikroskobik Incelemede Alternatif Yaklasimlar.

Aim of the present study was to identify protozoones which are difficult to define through wet slide in fresh fecal samples by using different fixatives with modified Trichrome stain within five minutes. Two different fixatives prepared for the alternative approach. The slides were fixed by two different fixatives, one of them (fixative-1) was based ethylalcohol, formalin, acetic acid, distilled water and the other one (fixative-2) based ethylalcohol, formalin, citric acid, distilled water included a mordant [divalent or polyvalent metals which make coordination complex with some dyes] consisted copper sulphate pentahydrate (CuSO4 .5H2 O). Slides prepared by the two different fixatives were stained by a different modification of Gomori's trichrome stain that we made. Samples fixed by Schaudinn fixative including mercury chloride were stained by Wheatley modification of Gomori's trichrome stain as a gold standard for control and comparison. We worked with 50 fecal samples which we thought included human intestinal protozoones after the wet slide examination. Comparing the methods, slides prepared with the method including citric acid gave almost similar results with the classical method excluded Entamoeba coli cystes. Slides prepared with the methode including acetic acid gave low performance compared with the classical method especially E.coli cystes and Blastocystis spp., Endolimax nana, Iodamoeba bütschlii, E.hartmanni. Both new fixatives gave superior performance at the slides included Dientamoeba fragilis and approximately shorten the procedure process ten times than the classical method. When the both alternative methods compared in each other, the slides prepared with fixative-2 exposed better performance for the protozoones Blastocystis spp., E.nana, I.bütschlii and E.hartmanni while the fixative-1 displayed minimal superiority for D.fragilis including criterias that we based. The fixative-2 and modified stain methode that we used in our study, makes available the diagnostic phase ten times faster than the classical method in human stool parasitological tests excluding the E.coli cystes at parasitology and microbiolgy laboratories. It seems to be a good option to the classical method for routine usage.

Distribution of Intestinal Parasites Detected in Sanliurfa Mehmet Akif Inan Education and Research Hospital Between October 2015 and October 2016.

OBJECTIVE: A total of 7353 patients who referred to the Microbiology Laboratory of Mehmet Akif Inan Education and Research Hospital between October 2015 and October 2016 were evaluated for intestinal parasites. METHODS: All fecal samples were investigated using three methods: wet mount, formalin ethyl acetate concentration, and trichrome staining. The cellophane tape method could only be used in a limited number of patients owing to the absence of a pediatrics outpatient clinic in our hospital. RESULT: One or more intestinal parasites were detected in 2,322 (31.6%) among the 7,353 patients. Blastocystis spp. (1,884, 63.6%), Entamoeba coli (390, 13.2%), and Giardia intestinalis (169, 5.7%) were the most frequently observed parasites in stool samples. Two or more parasites were detected in 528 (22.7%) of the positive cases. The positivity rate was 30.3% among women and 33.4% among men. CONCLUSION: Intestinal parasitosis is still significant for community health care in the Sanliurfa Province.

Alternative Staining Method for Four Parasites that are Difficult to Identify through Direct Inspection.

OBJECTIVE: The aim of the present study was to identify Blastocystis spp. (particularly the granular form), Dientamoeba fragilis, and Entamoeba histolytica/dispar trophozoites, which are difficult to identify using wet mount, as well as Giardia intestinalis cysts, which are difficult to identify by unexperienced researchers, in fresh fecal samples within 5 min using a different fixative employing trichrome staining. METHODS: The slides were fixed by fixatives based ethyl alcohol, formalin, acetic acid, and distilled water including four different mordants (divalent or polyvalent metals that form coordination complex with some dyes) consisted of zinc sulfate hydrate, copper sulfate hydrate, aluminum sulfate hydrate, and ferric sulfate hydrate using a modification of Gomori's trichrome staining. Samples fixed by Schaudinn fixative including mercury chloride were stained using Wheatley modification of Gomori's trichrome staining as gold standard for control and comparison. RESULTS: Regarding D. fragilis and E. histolytica/dispar trophozoites, similar results were obtained among the slides stained by classical fixation/staining method and those stained by the alternative method. However, regarding Blastocystis spp. and Giardia intestinalis cysts, classical fixation/staining method was relatively superior to the alternative method. CONCLUSION: Although the alternative method is slightly inferior to the classical fixation/staining method, it seems to be a good option with respect to the process time.

[An alternative biphasic nutrient medium for the diagnosis of cutaneous leishmaniasis]. / Kutanöz leysmanyazis tanisinda alternatif bifazik nutrient besiyeri.

Cutaneous leishmaniasis (CL) caused by the Leishmania spp. parasites, is a disease characterized by nodulo-ulcerative lesions in the skin. CL is transmitted to humans by infected sandflies during blood sucking, and is endemic in about 98 countries over the world. The demonstration of amastigotes via microscopic examination, and the growth of promastigotes in NNN (Novy-MacNeal-Nicolle) medium are gold standard methods for laboratory diagnosis. The aim of this study was to compare the biphasic NNN medium that is frequently used in routine laboratories with the biphasic nutrient medium that can be prepared easily in microbiology laboratories, for the growth of promastigotes. In the study, the aspiration fluid sample was used as clinical sample which was obtained from the skin lesion of a 47-year-old female patient admitted to Izmir Katip Celebi Ataturk Education and Research Hospital dermatology outpatient clinic and pre-diagnosed as CL. The aspirate sample taken from the lesion was evaluated with microscopy, cultivation in two different media and real-time polymerase chain reaction (Rt-PCR) methods. In microscopic examination Leishmania amastigotes were observed in Giemsa-stained smears prepared from the aspiration fluid. In Rt-PCR performed by using specific primers and probes targeting ITS1 region of Leishmania parasite, a melting-curve compatible with L.tropica was detected. For cultivation, triple inoculations of the aspirate sample into NNN (NNN + RPMI 1640 + 10% fetal calf serum) and nutrient media (nutrient agar + nutrient broth + 10% fetal calf serum) were used. The cultures were incubated at 27°C for 10 days, and the number of propagated promastigotes were counted on the third, seventh and tenth days. The growth of Leishmania promastigotes was detected in both media on the third day. The number of promastigotes grown in NNN medium on the third, seventh and tenth days were 105/ml, 106/ml and 108/ml, respectively. Those values in nutrient medium were 106/ml, 107/ml and 108/ml on the third, seventh and tenth days, respectively. Although the number of promastigotes on the third and seventh days were higher in nutrient medium than NNN medium, the number of cultivated promastigotes were equal on the tenth day. As a result, nutrient medium is considered to have an impact in the diagnosis of CL, by providing an alternative to the routine medium used and can readily be available in microbiology and parasitology laboratories with long shelf-life. It was concluded that biphasic nutrient medium could be used as a supplementary medium for diagnosis in laboratories in the absence of NNN medium or can not be provided.

Comparing the Efficacy of Commercially Available Insecticide and Dimeticone based Solutions on Head Lice, Pediculus capitis: in vitro Trials.

OBJECTIVE: Head lice infestation is a public health and social problem for almost all countries worldwide. For its treatment, insecticide and dimeticone-based solutions are currently available in the markets in many countries. We aimed to compare the efficacy of commercially available anti-head lice shampoos containing insecticide and physically effective products with different percentages of dimeticone using an in vitro technique. METHODS: Head lice specimens were collected from primary school children using special plastic and metal combs. Anti-head lice products were commercially purchased and used directly. The specimens were placed one by one in 5-cm Petri dishes containing a slightly wet filter paper and were kept in a plastic cage at 28±2°C and 50%±20% relative humidity. A standardized protocol was used for testing all the products, and mortality data were obtained after 24 h. Two control tests were performed with each batch of trials. For each product and control, 10-20 head lice specimens were used, and the results were statistically analyzed. RESULTS: Our study demonstrated that among all the tested products, two products containing mineral oils [5.5% dimeticone & silicone (patented product) and dimeticone (no percentage mentioned in the prospectus) & cyclopentasiloxane] were found to be more effective for killing head lice in vitro. CONCLUSION: Physically effective products can be repetitively used because they are non-toxic and resistance to them is not expected. To control the infestation at a public level, the use of these products needs to be encouraged with respect to their cost price.

[The investigation of malaria cases in Manisa between 2008-2012]. / Manisa Ilinde 2008-2012 Yillari Arasinda Saptanan Sitma Olgularinin Degerlendirilmesi.

OBJECTIVE: We aimed to determine the malaria prevalence in a 5-year period by using the data obtained from Malaria Control Center associated with the Manisa Province Public Health Office Infectious Diseases Department in Manisa between 2008 and 2012. METHODS: The data were evaluated according to age, gender, type of parasite, and the places of travel. RESULTS: In this study, six imported malaria cases were detected in blood samples from 86,955 patients by the Malaria Control Center, associated with the Manisa Province Public Health Office Infectious Diseases Department, with active and passive surveillance between 2008 and 2012. Positivity rate was 0.007%. CONCLUSION: Imported malaria cases, due to increasing international travel and migration, have a serious endemic potential based on unsuitable or insufficient prophylaxis, although malaria cases have decreased notably by successful eradication studies in Turkey. This paper was prepared especially in order to point out this subject, because all of the cases were imported.