Clinical practice guidelines for the diagnosis and treatment of cutaneous leishmaniasis in Turkey.

BACKGROUND: Cutaneous leishmaniasis (CL) is a vector-born parasitic disease characterized by various skin lesions that cause disfiguration if healed spontaneously. Although CL has been endemic for many years in the southern regions of Turkey, an increasing incidence in nonendemic regions is being observed due to returning travelers and, more recently, due to Syrian refugees. Thus far, a limited number of national guidelines have been proposed, but no common Turkish consensus has emerged. OBJECTIVES: The aim of this study was to develop diagnostic and therapeutic guidelines for the management of CL in Turkey. METHODS: This guideline is a consensus text prepared by 18 experienced CL specialists who have been working for many years in areas where the disease is endemic. The Delphi method was used to determine expert group consensus. Initially, a comprehensive list of items about CL was identified, and consensus was built from feedback provided by expert participants from the preceding rounds. RESULTS: Evidence-based and expert-based recommendations through diagnostic and therapeutic algorithms according to local availability and conditions are outlined. CONCLUSION: Because CL can mimic many other skin diseases, early diagnosis and early treatment are very important to prevent complications and spread of the disease. The fastest and easiest diagnostic method is the leishmanial smear. The most common treatment is the use of local or systemic pentavalent antimony compounds.

Incidence of Parasitic Diarrhea in Patients with Common Variable Immune Deficiency.

OBJECTIVE: Parasites might cause atypical and severe infections in immunocompromised hosts. The prevalence of diarrhea among common variable immune deficiency (CVID) syndrome patients varies between 20% and 94%, which indicates that diarrhea and gastrointestinal system (GIS) complaints could be the second leading cause of morbidity in CVID patients after respiratory tract infections. This study aimed to assess the prevalence of intestinal parasites in CVID patients with GIS complaints and diarrhea. METHODS: In this study, all cases followed up in the Immunology and Allergy Clinic of Ege University School of Medicine from July 2008 to August 2015 with the diagnosis of CVID were reviewed retrospectively. The stool samples of patients with diarrhea were identified using direct microscopy of native (0.09% NaCl) and Lugol's iodine preparations followed by formol-ethyl acetate concentration to apply modified Kinyoun, trichrome, acid-fast trichrome, and modified trichrome stains for the presence of intestinal parasites. RESULTS: Overall, 26 of 37 CVID patients had diarrhea; white and red blood cells (WBCs and RBCs, respectively) were identified in 11 and 10 of these 26 samples, respectively. Intestinal parasites were found to be present in 7 of the 11 patients with WBCs and 3 of the 10 patients with RBCs. With the addition of patients who neither had WBCs nor RBCs in their stool, a parasitic agent was detected in 13 (50%) of the 26 patients with diarrhea. There was no significant difference between the diarrheic patients with or without intestinal parasites with respect to cramps, fever, nausea and vomiting, tenesmus, bloody feces, and presence of mucus in the stool. Only one patient had malabsorption, which was not associated with intestinal parasites. The most common parasites detected in this study were Cryptosporidium spp. (n=9; 69.2%), Giardia spp. (n=7; 53.8%), and Blastocystis spp. (n=3; 23.1%). We also identified that parasitic diarrhea in CVID patients tended to last longer (M (mean): 16.2 days) than other causes of infectious diarrhea; this is in accordance with previous studies. CONCLUSION: Cryptosporidium spp. was found be the major cause of parasitic intestinal infection in this patient population. It was concluded that parasitic infections may cause chronic diarrhea, which are major causes of morbidity in CVID patients. Therefore, special attention is necessary for the identification of intestinal parasites in CVID patients with diarrhea.

Leishmaniasis in Turkey: Determination of Leishmania Species by Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS).

BACKGROUND: Cutaneous leishmaniasis (CL) is endemic in Southeastern Anatolia, mainly in Sanliurfa and Hatay provinces, and the causative agents are mostly Leishmania tropica and less frequently L. infantum. Here, we report the first MALDI-TOF analyses of Leishmania promastigotes obtained from the cultures of two CL cases from Osmaniye and Hatay provinces who were initially diagnosed by microscopy, culture and identified as L. infantum with Real-Time PCR (RT-PCR). METHODS: Samples obtained from the skin lesions of patients were initially stained with Giemsa and cultivated in NNN medium. Examination of the smears and cultures revealed Leishmania amastigotes and promastigotes, respectively. The promastigotes (MHOM/TR/2012/CBU15 and MHOM/TR/2012/MK05) obtained from the cultures of both patients were used for RT-PCR targeting the ITS-1 region in the SSU of rRNA. The reference strains of four Leishmania species (L. infantum, L. donovani, L. tropica and L. major) were initially assessed with MALDI-TOF and their data were added to MALDI-TOF Biotyper Library. RESULTS: Both RT-PCR and MALDI-TOF analyses indicated that the causative agent in both patient samples was L. infantum. CONCLUSION: Despite disadvantages such as requirement of culture fluid with nothing but promastigotes and high cost, MALDI-TOF analysis may be a fast, sensitive and specific diagnostic tool in especially large-scale research studies, where the cost declines, relatively.

Degree and frequency of inhibition in a routine real-time PCR detecting Pneumocystis jirovecii for the diagnosis of Pneumocystis pneumonia in Turkey.

Routine laboratory diagnosis of Pneumocystis jirovecii is currently achieved by PCR in almost all laboratories with sufficient equipment due to its high sensitivity and specificity compared to staining methods. A current issue that limits the reliability and sensitivity of PCR is the degree of inhibition caused by inhibitory substances in respiratory samples. The present study aimed to analyse the degree and frequency of inhibition in real-time PCR detecting P. jirovecii in respiratory specimens submitted to a Pneumocystis pneumonia (PcP) diagnosis laboratory in Ege University Medical School, Turkey. Between July 2009 and December 2010, 76 respiratory specimens [63 bronchoalveolar lavage (BAL) fluid, 10 sputum samples, two tracheal aspiration fluid and one thoracentesis fluid] obtained from 69 PcP-suspected patients were investigated for the presence of P. jirovecii using real-time PCR targeting the cdc2 gene. Of these samples, 42 of the specimens were stained and examined by microscopy according to the request of the clinicians. PCR was positive in 15 specimens in the initial run. Of the remaining 61 samples, 41 of them were negative with positive internal inhibition controls (i.e. true-negative group). The frequency of inhibition in the initial run was 26.31 % (20/76) as determined by spiked negative controls. All of the inhibited samples were resolved after 1 : 2, 1 : 5, 1 : 10 and 1 : 20 dilutions. P. jirovecii was detected by PCR in two inhibited specimens after retesting with diluted samples which were also positive by microscopy. The incidence of P. jirovecii in respiratory specimens was 22.36 % (17/76) as determined by real-time PCR and 7.14 % (3/42) by microscopy. Overall, the incidence of P. jirovecii in respiratory samples was 23.68 % (18/76) as detected by both methods. In conclusion, inclusion of spiked positive controls in each sample and retesting with diluted samples to resolve inhibition increased the reliability of the real-time PCR assay in terms of determining false-negative results and influencing the treatment of the patient. Furthermore, results of the present study determined for the first time the frequency and degree of inhibition in a real-time PCR detecting P. jirovecii in respiratory specimens during routine diagnosis of PcP.

[The serological and entomological survey of zoonotic visceral leishmaniasis in Ayvacik Region of Canakkale Province, Turkey]. / Canakkale Ili Ayvacik Bölgesinde Zoonotik Visseral Leishmaniasisin Serolojik ve Entomolojik Olarak Arastirilmasi.

The field studies were carried out in Ilyasfaki village belonging to Ayvacik town, Kalabakli village and center of Kepez town belonging to Canakkale province to determine the epidemiological situation of visceral leishmaniasis between June and August 2007. A total of 27 blood samples were taken from the dogs which are known as reservoir of Leishmania infantum causing visceral leishmaniasis in human and dogs. The dogs were also examined physically for clinical symptoms of the disease. A total of 789 sand fly specimens were collected using CDC miniature light traps and examined. Six Phlebotomus and one Sergentomyia species were identified as: Phlebotomus negletus, P. tobbi, P. simici, P. papatisi, P. perfiliewi, P. halepensis and S. theodori. Among these species P. neglectus, was found to be a dominant species in Ilyasfaki village by the rate of 94.4%, while P. tobbi was also found to be a dominant species in city center (including Kepez) and Kalabaki village by the rate of 50% and 48.1%, respectively. No seropositive dogs were found by IFA test. Seropositivity below cut-off level was only observed in two dogs from Kepez, in dilutions of 1/16 and 1/64. The risk level for human and canine visceral is found to be high and it requires more field studies on canine leishmaniasis in and around the area.

In vitro activity of Arbutus unedo against Leishmania tropica promastigotes.

Pentavalent antimonials are the first choice for the treatment of anthroponotic cutaneous leishmaniasis (ACL) in health centers in Turkey, however in rural areas, traditional plants may be preferred for the treatment of lesions. In recent years a number of papers are published related to the natural products especially plant derivates. Our aim is to investigate the antileishmanial effect of Arbutus unedo which is a wild plant mainly grown in maquis and rocky places of the seabord in South Europe. In the present study, the ethanolic, water and n-hexane extracts from the leaves of Arbutus unedo were originally tested in vitro against Leishmania tropica promastigotes. The ethanol extract of Arbutus unedo leaves at the concentrations of 100, 250, 500 microg/ml were found to be more effective than the other extracts (p:0.000). Our study showed that the ethanolic extract of Arbutus unedo leaves can be a promising antileishmanial agent and further experiments are needed.

Serological and entomological survey of zoonotic visceral leishmaniasis in Denizli Province, Aegean Region, Turkey.

A cross-sectional seroepidemiological survey of leishmaniasis was carried out among children and adults from four villages and one district of Denizli province located in the Southern Aegean Region of Turkey where 14 human visceral leishmaniasis (HVL) cases including 4 adults were reported between 1993 and 2000. Blood samples were taken from 329 children, 217 adults and 140 dogs and a physical examination was also done. Indirect fluorescent antibody test and enzyme linked immunosorbent assay were performed for all sera. All 329 sera collected from children were found to be negative while 2 (0.09%) out of 217 adult sera were found to be seropositive. One seropositive adult patient was confirmed parasitologically as HVL after bone marrow aspiration and treated with AmBisome while the other was followed only serologically because of the absence of symptoms. The overall canine leishmaniasis seroprevalence was found to be 20.7%. Sand flies were collected using CDC light traps in three out of five study sites and midguts of females were checked for promastigotes after dissection/identification. Eight Phlebotomus species were found in the region. Phlebotomus neglectus and P. papatasi were determined as dominant species with the ratio of 43.52% and 37.35%, respectively. No promastigotes were found in the midgut specimens. In addition, the results showed the presence of vector sand fly species, as well as a high seroprevalence of anti-Leishmania antibodies among dogs from rural and a suburban area of Denizli province with a large proportion of asymptomatic seropositive dogs.

In vitro activity of Arbutus unedo leaf extracts against Trichomonas vaginalis trophozoites.

Trichomonas vaginalis (T. vaginalis) is a flagellated protozoan commonly causing sexually transmitted disease. T. vaginalis infections are treated with a 5-nitroimidazole derivate. However, drug resistance has been known to occur for a long time and new alternatives are under investigation. Arbutus unedo is a wild plant mainly growing in maquis and rocky places of the seaboard in Southern Europe. In our study, ethanolic, water, hexane and ethyl acetate extracts of Arbutus unedo leaves were tested in vitro against T. vaginalis trophozoites and the ethyl acetate extract of Arbutus unedo leaves was found to be effective (Growth inhibition rate (GI): 100%, at the concentration of 500 microg/ml). It may be a promising anti-trichomonacidal agent in the future and further experiments are needed.

[Case report: two imported Plasmodium falciparum cases]. / Olgu sunumu: yurtdisi kaynakli iki Plasmodium falciparum olgusu.

A 23 year-old Pakistani man presented at the emergency clinic of Tepecik Research and Training Hospital with the symptoms such as fever (39.3 degrees C), blurred consciousness, subicteric sclera and splenomegalia. In the laboratory examination, a slightly increase of the liver enzymes and anemia were detected. The gametocytes and trophozoites of Plasmodium falciparum were seen in a bone marrow aspiration and treatment was started with quinine and doxycycline but since resistance was detected to these medicines, mefloquine was chosen as alternative therapy. He was cured and discharged. The second case was also a Pakistani 20-year old man and he was a friend of first case. He presented at our hospital with the symptoms such as fever (39.4 degrees C), closed consciousness, icteric sclera and systolic soufflé in all cardiac foci. During the laboratory examination, severe anemia and trombocytopenia, and an increase in the liver enzymes were found. P. falciparum gametocytes and young trophozoites were seen in a blood smear stained with Giemsa. For treatment, mefloquine was used but unfortunately the patient died due to an adult respiratory distress syndrome (ARDS) complication. In this study, two cases with different clinical manifestations were presented to emphasize the importance of timing in starting the correct treatment.

The infection risk of visceral leishmaniasis among household members of active patients.

Human visceral leishmaniasis (HVL), caused by Leishmania infantum is mainly observed as sporadic cases in Turkey and dogs are considered as the main reservoir of the disease. The incidence of visceral leishmaniasis among members of households where a HVL infection has already been diagnosed was studied in clusters around the diagnosed cases in different regions in Turkey. A total of 47 serum samples collected from the households of 11 proven visceral leishmaniasis patients were screened for anti-Leishmania antibodies by indirect immunofluorescent antibody test (IFAT). Three and one such household members belonging to the different families were found to be seropositive and borderline, respectively. Diagnosis was confirmed with the presence of amastigotes in bone marrow aspiration samples in all seropositives while the borderline case with slight and indefinitive symptoms of VL was followed only serologically at 3-month intervals and improved spontaneously in 1 year. Household members of individuals with previously confirmed visceral leishmaniasis were found to have higher frequency of the disease suggesting the household members should be included in the risk group for visceral leishmaniasis and serological screening should be performed for the detection of possible infection.