Parasitological Methods Research Articles

The Presence of Potentially Pathogenic Protozoa in Lettuce (Lactuca sativa) Sold in Markets in the Central Peruvian Andes.

Peru is currently one of the world’s leading culinary destinations, whose world-renowned cuisine uses vegetables, mainly lettuce, as frequent ingredients. Vegetable consumption is promoted worldwide as a part of a healthy diet. However, vegetables, more frequently lettuce, have been implicated as a vehicle of infection for several foodborne parasites. This study aimed to determine the presence of potentially pathogenic parasites in lettuce marketed in the Central Andes of Peru. A total of 75 lettuce samples were collected from the two largest wholesale markets and the main open-air market in Jauja province, in the central Peruvian Andes. The province of provenance (coast vs. highlands), lettuce variety (“curly-leaf”, “iceberg”, and “butter”), and type of market were recorded. The samples were microscopically examined for detection of parasitic life forms using standard parasitological methods including direct slide smear, Lugol’s iodine staining, and Modified Ziehl−Neelsen staining. The overall positivity of parasitic contamination in lettuces was 45.3% (CI 95%: 34−56.6%). Cryptosporidium spp., Isospora belli, Giardia lamblia, Balantidium coli, and Entamoeba spp. were detected in twenty-six (34.7%), six (8%), four (5.3%), two (2.7%), and two (2.7%) lettuces, respectively. I. belli was found in a significantly (p < 0.01) lower proportion in the “butter” variety, and significantly (p < 0.05) higher contamination with G. lamblia was found in lettuce sold at the open-air market. B. coli, G. lamblia, and E. histolytica/E. dispar/E. moshkovskii were detected only in lettuce from the highlands (Tarma province). This study provides important data for health authorities to develop food safety programs. This information is also of interest to the international community because of the increased visibility that Peru has gained as a tourist destination.

Performance characteristics of diagnostic assays for schistosomiasis in Ontario, Canada.

Due to lower intensity of infection and greater intervals from last exposure, parasitologic detection methods for schistosomiasis are poorly sensitive in non-endemic areas, challenging accurate diagnosis. We evaluated parasitologic versus indirect detection methods for schistosomiasis. We included specimens submitted for Schistosoma serology, and stool for ova and parasite microscopy. Three real-time PCR assays targeting Schistosoma mansoni and S. haematobium were performed. Primary outcome measures were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), where both microscopy and serology were the composite reference standard against serum PCR. Of 8168 serum specimens submitted for Schistosoma serology, 638 (7.8%) were reactive and 6705 (82.1%) were non-reactive. Of 156,771 stool specimens submitted for ova and parasite testing, 46 (0.03%) were positive for eggs of S. mansoni. Four (0.5%) urine specimens were positive for eggs of S. haematobium. Combined serum PCRs targeting S. mansoni had a sensitivity and specificity of 27.8% (95% CI = 18.3-39.1%) and 100% (95% CI = 83.9-100%), respectively, with PPV of 100% (95% CI = 100%) and NPV of 26.9% (95% CI = 24.3-29.7%). The one serum sample positive for S. haematobium was also detectable by our S. haematobium PCR. No cross-reactivity was observed for all three PCR assays. Although serology is highly sensitive, parasitologic tests signify active infection, but are limited by low population-level sensitivity, particularly in non-endemic settings. Although serum PCR offered no performance advantage over stool microscopy, its role in diagnostic parasitology should be pursued due to its high-throughput and operator-independent nature.

ЧУВСТВИТЕЛЬНОСТЬ И СПЕЦИФИЧНОСТЬ ТЕСТ-СИСТЕМЫ В РЕАКЦИИ ИММУНОФЛЮОРЕСЦЕНЦИИ ПРИ ДИАГНОСТИКЕ TRYPANOSOMA EVANSI

The purpose of the research is to determine the sensitivity and specificity of the developed test system in the immunofluorescence reaction as one of the methods for diagnosing Trypanosoma evansi. The novelty of the test system is the use of a local trypanosome isolate, which will increase the diagnostic value and specificity, with further production in accordance with international GMP standards, which will guarantee the quality and safety of the studies. The work used parasitological research methods and serological methods of setting the reaction of immunofluorescence in various concentrations. As a result of the studies, the high sensitivity and specificity of the fluorescence reaction in relation to the Trypanosoma evansi strain was confirmed. Diagnostic immunoglobulin is highly sensitive to trypanosomes at concentrations of 1:1 and 1:10 in green-yellow and blue-violet spectra. The specificity of the diagnostic immunoglobulin was confirmed by the absence of reaction with the blood cells of horses, as well as with the T. equiperdum strain, in which luminescence is visible only in the shell and undulating membrane. The use of the developed test system shows the potential of the immunofluorescence reaction in modern practical veterinary serology and parasitology, and will also contribute to the fight against equine trypanosomiasis in the Republic of Kazakhstan.

ВИДОВОЕ РАЗНООБРАЗИЕ ГЕЛЬМИНТОВ ПИЩЕВАРИТЕЛЬНОЙ СИСТЕМЫ ЛЕЩА И ПЛОТВЫ САРАТОВСКОГО ВОДОХРАНИЛИЩА

The data on helminths of the digestive system of the bream Abramis brama Linnaeus, 1758 and the common roach Rutilus rutilus Linnaeus, 1758 (Osteichthyes, Cyprinidae) from the middle part of the Saratov reservoir (Koltsovo-Mordovinskaya fl oodplain) are given. In 2013–2016, 40 specimens of A. brama and 47 specimens of R. rutilus were studied by standard parasitological methods. Bream has 8 species of worms of different systematic groups, including Cestoda – 2, Trematoda – 1, Chromadorea – 3, Acanthocephala – 2. The community of intestinal and liver parasites of roach includes 5 species: Cestoda – 1, Aspidogastrea – 1, Chromadorea – 2, Acanthocephala – 1. The degree of invasion of fi sh by helminths circulating through food webs is low. The bream is dominated by the carp-specifi c nematode Rhabdochona denudata (Dujardin, 1845) Railliet, 1916 (Spirurida, Rhabdochonidae) (invasion intensity 27.5 %, invasion intensity 1–126 ind., abundance index 5.8 ind.). No dominant parasites were found in the common roach, three species (Contracaecum microcephalum (Rudolphi, 1819), Aspidogaster limacoides Diesing, 1835 and Raphidascaris acus (Bloch, 1779)) have the status of subdominants. During the research period, there were signifi cant fl uctuations in the qualitative and quantitative indicators of fi sh invasion (species composition of parasites, their occurrence and average number), which is determined by the feeding characteristics of bream and roach in each specifi c year.

Effects of sulphadoxine-pyrimethamine on maternal, neonatal and placental malaria in Port Harcourt, Rivers State, Nigeria.

The utilization of Intermittent Preventive Treatment (sulphadoxine-pyrimethamine) in pregnancy (IPTp-SP) for combating malaria has indicated control over adverse birth outcomes and has been recommended for use by pregnant women. The aim of this study was to determine the effectiveness of IPTp-SP on maternal, neonatal and placental malaria in Port Harcourt, Nigeria. 316 samples of maternal peripheral blood (MPB), placental blood (PLB), neonatal cord blood (NCB) and placental tissue (PT) were collected each from consenting mothers. Blood samples were processed and stained by the Giemsa method. Placental tissues were processed and stained in haematoxylin. Examination of samples for malaria parasitaemia was carried out using standard parasitological methods. Demography of participants was collected through questionnaires and from ante natal care (ANC) records. Overall prevalence of 74 (23.42%) was recorded. Age-related prevalence indicated that ≤ 20 years, 9 (56.25%) had the highest prevalence followed by 21-30 years (23.48%), and ≥41 years (16.67%) (p <0.05). Malaria in MPB showed that SP-users had 26 (13.20%) while non-users had 48 (40.33%) (p <0.05). In NCB, SP-users recorded 20 (10.15%) while non-users had 13 (10.92) (p>0.05). The prevalence in PLB and PT revealed that SP-users had a lower prevalence in PLB, 31 (15.73%) and PT, 12 (6.09%) while non-users recorded a higher prevalence 48 (40.33%) in PLB and 21 (17.65%) in PT (P<0.05). The utilization of IPTp-SP is seen to significantly reduce the occurrence of malaria in pregnancy, placental tissue and in neonates thereby helping in improving birth outcomes.

Frequency, subtypes distribution, and risk factors of Blastocystis spp. in COVID-19 patients in Tehran, capital of Iran: A case-control study

BackgroundRecent theories on the possible interactions between the intestinal parasites and COVID-19 have stated that these co-infections may cause immune imbalance and further complications in the affected patients. Until now, there is no data about Blastocystis subtypes as an intestinal parasite in COVID-19 patients. Therefore, the present work was done to evaluate the molecular prevalence of Blastocystis spp. and related risk factors in Iranian patients with COVID-19. MethodStool samples were gathered from 200 COVID-19 patients and 200 control, being matched regarding age, gender and residence. Then, stool samples were surveyed by parasitological methods, including direct slide smear and formalin-ether concentration. In the following, PCR and sequencing were used to detect Blastocystis spp. and their subtypes. ResultsThe frequency of Blastocystis spp. in patients with COVID-19 (7.5%; 15/200 by molecular method vs. 6%; 12/200 by microscopy method) was slightly higher than in individuals without COVID-19 (4.5%; 9/200 by molecular method vs. 4%; 8/200 by microscopy method), this difference was not statistically significant (P value = 0.57 for molecular method vs. P value = 0.81 for microscopy method). Regarding associated factors for Blastocystis spp., we found significant differences regarding the residence (rural), loose and watery stool with diarrhea, and duration of treatment (6 weeks <) in the COVID-19 group. Blastocystis ST3 was the most common subtype in the patients with COVID-19 and control group. ConclusionsBased on this results, health education, improved sanitation and good personal hygiene are highly recommended to prevent Blastocystis in COVID-19 patients.

Integrated survey of helminthic neglected tropical diseases and comparison of two mosquito sampling methods for lymphatic filariasis molecular xenomonitoring in the River Galana area, Kilifi County, coastal Kenya.

A lymphatic filariasis (LF) endemic focus along the River Galana/ Sabaki in Kilifi County, coastal Kenya, provided a platform to conduct an integrated survey for three helminthic neglected tropical diseases (NTDs), namely soil-transmitted helminthiasis (STH), schistosomiasis (SCH) and LF. Additionally, the study compared the performance of two mosquito trapping methods for LF molecular xenomonitoring (MX). Cross-sectional surveys measuring STH, SCH and LF prevalence were conducted in four villages. Mosquitoes were trapped using the CDC light trap (CDC-LT) and the Ifakara A tent trap (Ifakara-TT) methods and stored in pools which were tested for Wuchereria bancrofti DNA using the real-time polymerase chain reaction assay. A total of 907 people (436 adults; 471 children) participated in the parasitological testing. Among the STH infections, Trichuris trichiura and hookworms were most prevalent among the children and adult populations, respectively. The schistosome worm eggs detected belonged to the species Schistosoma haematobium and the prevalence of the infection was generally higher among the children compared with the adult population. The prevalence of LF infection among the adult population ranged from 1.8% to 7.6% across all 4 villages (P < 0.05). A total of 3,652 mosquitoes, including Anopheles, Culex, Mansonia, and Aedes species were collected. One mosquito pool consisting of Anopheles mosquitoes tested positive for filarial DNA out of 1,055 pools that were tested. The CDC-LT caught significantly more mosquitoes compared with the Ifakara-TT (P < 0.001). This study demonstrated that integrated epidemiological surveys using standard parasitological and entomological methods can provide useful information on co-endemic parasitic diseases which could help direct interventions and surveillance activities.

Study of Secondary Bacterial Infections Among the Patients with Cutaneous and Mucocutaneous Leishmaniasis in Thamar City- Yemen.

The main objective of this study was to determine the how common secondary bacterial infections that are isolated from patients with cutaneous and mucocutaneous leishmaniasis in Thamar City- Yemen. This study included patients admitted to the Skin Disease Unit and the Thamar General Hospital lab between August 2020 and January 2021. Clinical samples were examined using standard parasitological and bacteriological methods. The study included 81 patients, and leishmania was found in 60 (74 percent) of them. Bacterial infections had also infected 60 cases. The most common secondary infection bacterial causative agents found in this study were 30 isolates (38%) of kocuria spp out of the total number 79 bacterial isolates, 28 (35.4%) isolates of Bacillus spp., 12 (15.2%) isolates of P. aeruginosa and 9 (11.4%) isolates of S. aureus. Sensitivity testing revealed that 16% of S. aureus was resistant to Azithromycin, 35% of Kocuria spp. were resistant to Cefotaxime and 30% were resistant to Ceftriaxone, 36% of Bacillus spp. were resistant to Azithromycin, and 20% were resistant to Ceftriaxone, 8.3% of P. aeruginosa were resistant to Azithromycin and 10% were resistant to Ceftriaxone. The majority of isolates (35–50%) were sensitive to Ofloxacin and Ciprofloxacin. The recent article's results indicate that bacterial infections are still a significant problem in patients with cutaneous leishmaniasis in Yemen, and should be considered when treating these patients.

Prevalence and Risk Factors of Schistosoma haematobium Infection Among Schoolchildren in the North of Khuzestan Province, Iran: Evidence for the End of Endemism

Background: Schistosomiasis is a chronic parasitic and often weakening disease, which was endemic in Khuzestan Province, Southwest Iran. Objectives: This study aimed to determine the prevalence of Schistosomiasis in the north of Khuzestan province. Methods: The current study was carried out based on the parasitological method to observe the ova of the parasite in urine by light microscopy and serology method based on titer the volume of Immunoglobulin G (IGg) in the blood serum of school children. Results: The observation of parasite eggs in urine samples and the prevalence of infection based on examining antibodies against Schistosoma haematobium infection in the blood serum of students was zero. Conclusions: Based on the findings, the cessation of transmission and elimination of the disease in the investigated area in the north of Khuzestan province have appeared.

Environmental DNA in human and veterinary parasitology - Current applications and future prospects for monitoring and control

Parasites are important pathogens with significant global economic, public and animal health impacts. Successful control or elimination of many parasitic diseases, not least neglected tropical parasites, will require scalable, sensitive and cost-effective monitoring tools. Environmental DNA (eDNA) methods, used extensively in ecology for biomonitoring in natural ecosystems, offer promising advantages such reduced costs and labor requirements for species monitoring. Yet, the use of eDNA-based methods in parasitology and disease surveillance, has only recently begun to be explored. With this review, we wish to give an up-to-date overview of current uses and limitations of eDNA in human and veterinary parasitology, and how existing challenges can be overcome to fully utilize the potential of eDNA for monitoring and control of parasitic diseases. We begin by systematically searching published literature to identify studies that apply eDNA methods in parasitology and synthesize the main findings from these studies. We find that eDNA applications in parasitology only account for a small proportion (73/1960) of all eDNA publications up to now, and even fewer (27/73) studies, that apply eDNA methods specifically for parasites of human or veterinary importance. The majority of studies concern snail-borne trematodes and their intermediate host snails, while a few apply eDNA for mosquito vector species detection. A strong geographical bias, with only very few studies undertaken on the African continent, where parasites are of the biggest public health concern, is also noted. Current obstacles hindering further advances of eDNA methods in parasitology include incomplete reference databases, and challenges related to real-time monitoring in remote areas, and in certain LMIC settings. Finally, we point to future opportunities for eDNA-based research in parasitology and highlight recent innovations in eDNA research, which could further develop its application for monitoring and control of parasitic diseases and vectors in the future.

Wheat Germ Oil and Propolis Decrease Parasite Burden and Restore Marked Histopathological Changes in Liver and Lung in Mice with Chronic Toxoplasmosis.

Toxoplasmosis is a parasitic zoonotic disease with a worldwide distribution. Its effects can be critical in immunocompromised patients. However, there is a limited availability of effective, low-toxicity drugs against this disease, particularly in its chronic form. The present study evaluated the effect of propolis and wheat germ oil (WGO) as safe, natural products to reduce Toxoplasma cysts in experimentally infected mice. For the experiment, five groups (10 mice per group) were examined: Group 1: negative control (noninfected, nontreated); Group 2: positive control (infected, nontreated); Group 3: infected and treated with WGO at a dose of 0.2 mg/1.5 mL per kg body weight/day; Group 4: infected and treated with 0.1 mL propolis extract/day; and Group 5: infected and treated with a combination of WGO and propolis at the same doses as Group 3 and 4. After the mice were sacrificed, liver and lung specimens underwent histopathological examination, and the parasite burden was investigated by parasitological methods and quantified using real-time polymerase chain reaction. Notably, the results showed a substantial decrease in parasitic burden in Group 5 compared to the control group. These results were further confirmed by molecular analysis and quantification of the DNA concentration of the Toxoplasma P29 gene after treatment in all tested samples. Furthermore, the combination of propolis and WGO restored all histopathological changes in the liver and lungs. Taken together, these findings provide remarkably promising evidence of the effects of the combination of WGO and propolis against chronic toxoplasmosis in mice.

Ecological and faunal characteristics of helminths of wetland birds in North-Western Uzbekistan

The article studies some faunistic features of helminths of wetland birds in the ecological crisis zone in North-Western Uzbekistan. Parasites of wetland birds collected in the basin of the lower reaches of the Amu Darya during the hunting seasons of 2018–2022 were used as material for this work. 500 individuals of birds representing 39 species from 24 genera, 13 families and 9 orders were examined using the parasitological dissection method. The article discusses the distribution and ecology of the helminths in the studied groups of birds. A total of 112 helminth species were identified in the region – 28 species belong to the class Cestoda, the class Trematoda is represented by 57 species, Nematoda – 23 and Acanthocephala – 4 species. 92 species of heminths were identified in the study area for the first time. The work provides original data on the structure of the helminth fauna from 9 orders of wetland birds. Various paths of helminth transmission in the circulation of infection were specified. The researchers identified four ways parasites use to enter their definitive host: helminths penetrate the host’s body when the latter eats other organisms, intermediate or reservoir hosts of helminths, which are part of the definitive’s host’s diet; helminths enter the host as mechanical impurity to food or water; they make active efforts to penetrate their host; helminths are transmitted by the intermediate host when the latter forages on the definitive host. Most of intermediate hosts for representatives of the class Cestoda were crustaceans – inhabitants of different types of water bodies; Oligochaeta are also included here as second or reservoir hosts. The development of trematodes occurs with the participation of aquatic molluscs acting as the first intermediate host. Various species of insects, fish, amphibians, reptiles and small mammals were identified as the second hosts. Nematoda use a wider range of intermediate hosts (Oligochaeta, crustaceans, insects – aquatic and terrestrial). Fish and amphibians were registered as the second and reservoir hosts of nematodes. The food chains of individual orders of wetland birds largely determine the composition of helminths parasitising them and are the main factor in the formation of their helminth fauna. These processes undoubtedly occur in time and space under the strict control of environmental factors.

Prevalence of Coccidiosis among local and exotic breeds of reared chickens in Azare Metropolis, Bauchi State Nigeria

Coccidiosis is a deadly disease that hampers chickens’ productivity and welfare. The disease is a major menace to the global poultry industry. Coccidiosis is caused by the apicomplexan parasite of the genus Eimeria. This study investigated the prevalence of Coccidiasis amongst the chickens reared in Azare metropolis. A total number of four hundred 400 feacal samples of live and slaughtered chickens were collected and examined using standard parasitological methods between March and June, 2022. The samples comprised of 246 female and 154 male; 300 exotic species and 100 local species. Chi-square test and simple percentage were used to analyze the generated data and p&lt;0.05 was adopted to determine the level of significance. An overall prevalence rate of 173 (43.3%) was recorded. The prevalence rate was higher in female 138 (56.1%) than male with 35 (22.7%). There was statistically significant difference in infection rate between the sexes of the Chickens. The lower age (1-4weeks) had the highest rate of prevalence rate of 64(59.8%) followed by age (5-16weeks) with 97(38.1%) and Adult (16weeks&gt;above) with lowest infection rate of 12(17.6%). Similarly, high infection rates were observed among local breed 64(64%) than exotic breed 109(36.3%). The difference between the age groups and the two breeds in prevalence rate was statistically significant (p&lt;0.05).Coccidiosis is endemic in both local and exotic chickens in the study area due to poor management system. It is therefore, recommended that good management system of rearing the chickens, regular vaccination against Coccidiosis, maintaining good hygienic practice as well as creating awareness among farmers on effect of this disease on their productivity and welfare should be ensured.

Studying the Effect of Human–Lactobacillus Ruteri on the Viability of Cryptosporidium Parvum

The goal of this study was to see how well Lactobacillus ruteri probiotic bacteria might infect Cryptosporidium parvum viability at various bacterial dilutions and statuses. Cryptosporidium was obtained from stool samples of diarrheal patients and diagnosed using parasitological methods, followed by molecular methods and cell line cultivation (Human adenocarcinoma cell). The treatment of Cryptosporidium Parvum by adding Lactobacillus ruteri dilutions and status were then incubated at incubation periods (24, 48, and 72 hr), detection of Cryptosporidium count was carried out microscopically, and the parasite's viability was determined by 3-(4,5 dimethylthiazol-2-yl)-2- Diphenyltetrazolium bromide(Mtt) technique. Lactobacillus ruteri can treat Cryptosporidium parvum by suppressing activity and lowering the count with extremely significant differences, according to the findings.

Prevalence of fish Parasites IN Bagrus bayad and Protopterus annectens from Upper River Benue in Mutum Biu, Taraba State, Nigeria

The prevalence of fish parasites in Bagrus bayad and Protopterus annectens from Upper River Benue in Mutum Biu, Taraba State, Nigeria was carried out using standard parasitological method. The objectives of this study were to investigate the parasite species spectrum, prevalence of each parasites species, overall prevalence, prevalence of parasite infection in relation to the size and sexes of Bagrus bayad and Protopterus annectens and the infection induced by parasites on/in their body parts, with the view of providing relevant information that can be used in combating the rate of economic losses that could be experienced as a result of parasitic infestations. 4 parasite taxa comprising of Twelve (12) parasite species (Trichodina spp., Henneguya spp. Cryptobia iubilans, Diphillobothrium latum, Hymenolepis nanna and Bothriocephalus aengypticus, Capillaria philipinensis, Caenorhabditis briggsae, Camallanus spp., Eustrongylides spp., Contracaecum spp. and Clinostomum spp. were recovered from the samples of fish species used during the study period. Twelve (12) of the parasite species were recovered from Bagrus bayad while seven (7) were recovered from Protopterus annectens. Among the body parts of Bagrus bayad, while intestine had the highest percentage parasite load (56.37%), highest percentage parasite load (46.22%) in Protopterus annectens was recorded for stomach (46.22%). Out of the 400 fish samples comprising of 200 each of Bagrus bayad and Protopterus annectens, 234 (58.50%) comprising of 99 (49.50%) and 135 (67.50%) samples each of Bagrus bayad and Protopterus annectens were infested with 369 and 119 parasites, respectively. However, there was no significant difference in the prevalence of infection and parasite load between the fish species, respectively (P&gt;0.05). Generally, larger sized samples of Bagrus bayad and Protopterus annectens had higher percentage of infection compared to the smaller sized of both samples although, the prevalence of infection in relation to sizes of both fish species was not statistically significant (P&gt;0.05). Female Bagrus bayad and Protopterus annectens had higher prevalence of infection (21.61%) and (16.63%) than the males (6.37%) and (6.24%), respectively. There was no significant difference in the prevalence of infection in relation to the sexes of both fish species (P&gt;0.05). Epidermal necrosis of the caudal fin and skin, thickened intestinal epithelium at the site of parasite attachment, hyperplasia of the intestinal villi and lamina propria, hyperplasia of infected gills at the secondary lamellae, ulceration of the stomach, abdominal distention, eroded and flattened intestinal folds and lymphocytic infiltration of intestinal mucosa and edema of the lamina propria of the intestine were observed with the infected samples of B. bayad. The infected samples of P. annectens exhibited epidermal necrosis of the skin.

Novel CRISPR-based detection of Leishmania species.

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods leads to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with Leishmania (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10−2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

Comparison of Diagnostic Methods for Detection of Trichomonas vaginalis in Prediagnosed Vaginitis Cases and Its Association with Various Pathogens

Parasitological diagnostic methods such as direct microscopy, staining examination and culturemethodsare frequently used in the diagnosis ofTrichomonasvaginalis(T. vaginalis).Though, nowadays, new diagnostic methods, especially DNA-based methods, are developing, enabling the simultaneous recognition of different pathogens.In our study,weevaluatedwhetherthe choice of multiplex polymerase chain reaction (PCR), in whichT. vaginalisand different pathogens can be detected,is be an alternativeto classical methods and to evaluate the possible coexistence of pathogens. In our study, swab samples taken during routine examination of 100 female patients who presented to Manisa Celal Bayar University and Manisa City Hospital Outpatient Clinics Obstetrics and Gynecology were evaluated.The presence ofT. vaginaliswas investigated in these samples by direct microscopy, Giemsa stain and culture.BesidesT. vaginalis, other possible agents were also investigated by real-time multiplex PCR method. At least one agent was detected in 85 (85%) of the 100 patient samples included in our study.T. vaginalis positivity was detected in 6 (6%)of thesamples by parasitological diagnosis methods and in 10 (10%)of thesamples by multiplex PCR.Additionally,with real-time multiplex PCR,Chlamydia trachomatisin 4 (4%),Neisseria gonorrhoeaein 3 (3%),Ureaplasma urealyticum/parvumin 68 (68%),Gardnerella vaginalisin 68 (68%) andHerpes simplex virus1/2 in 1 (1%) of thesamplepositivity was found.Mycoplasma genitalium, another agent examinedbymultiplex PCR, was not found positive inany sample.The Kappa value of theculture thatis a parasitological test and multiplex PCR forT. vaginalisshowed moderate agreement with 59.5%. It has been concluded that usingreal-time multiplex PCR method, which hashigh specificity and sensitivity, in addition to microscopy and culture methods in the diagnosis ofT. vaginalis, could contribute to the correct and effective treatment by detecting multiple infections.

Application of Ziehl Neelsen Staining Method for Taenia spp. Eggs Differentiation.

Three of Taenia species, named Taenia saginata, T. solium and T. asiatica can be found in Indonesia, but only T. solium can lead to neurocysticercosis. The morphology of those 3 Taenia spp. egg is indistinguishable by standard parasitology procedure. We aimed to use Ziehl Neelsen staining for differentiation of eggs of T. saginata and T. solium. As many as 40 containers of stool samples from the positive helminthiasis patients in Jakarta, Indonesia were collected during the year 2018. From each container, 10 slides prepared for staining with Kato-Katz technique as the preliminary examination. From those stool samples with positive taeniasis, we then once again made 10 slides/container for Ziehl Neelsen staining. The first 400 slides stained with Kato-Katz technique as preliminary test were all positive for Taenia spp. The second 400 slides, we got 244 slides that gave good results as we could distinguished between the eggs of T. saginata and T. solium, meanwhile the remainder 156 slides gave unconfirmed results. From those 244 slides, 154 slides showed T. saginata eggs with magenta colored and 90 slides showed T. solium eggs with blue/purple colored. The eggs of T. solium slightly smaller if compared to Taenia saginata and had round shape, meanwhile T. saginata eggs were oval in shape. Ziehl Neelsen staining method can be used as an alternative parasitological method to differentiate the eggs of T. saginata and T. solium.

Effects of negative air ions (NAIs) on Leishmania major: A novel tool for treatment of zoonotic cutaneous leishmaniasis (ZCL).

BackgroundCutaneous leishmaniasis (CL) is a Neglected Tropical Disease (NTD) that causes high morbidity in the tropics and sub-tropics. Despite the remarkable advancements in the treatment of CL, the available therapeutics are far from ideal and also cause serious adverse side effects. Negative air ions (NAIs) generators are widely available for domestic and industrial uses. Several studies have reported on positive effects of NAIs therapy on human health as a non-pharmaceutical treatment for respiratory disease, allergy, or stress-related health conditions, including infectious diseases. To our knowledge, no studies have examined the effectiveness of the NAIs therapy against Leishmania parasites. The aims of this study were to investigate the effect of NAIs therapy on Leishmania major (L. major) the causative agent of CL in in vitro and in a murine model.Methodology/Principal findingsIn vitro anti-leishmanial effects of NAIs therapy were measured by parasitological methods. NAIs therapy was assessed in vivo in L. major infected BALB/c mice by measuring the footpad (FP) lesion size and parasite load using metric caliper tool and qPCR, respectively. Immune responses in treated and non-treated mice were assessed by measuring the levels of IFN-γ, IL-4, NO and arginase activity. In vitro NAIs therapy significantly decreased the viability of Leishmania promastigotes and of amastigotes cultured in macrophages, but did not affect the host cells. NAIs therapy of L. major infected BALB/c mice resulted in reduced FP lesion size, diminished parasite burden, and importantly decreased induction of IL-4 and arginase activity in the presence of NAIs. In contrast IFN-γ and NO levels were significantly enhanced. NAIs therapy significantly diminished the progression of disease compared to the control group, but was less effective than amphotericin B treatment.ConclusionsOur study shows that NAIs treatment was effective in vitro and in Leishmania-infected mice, elicited a T-helper 1 (Th1) response and increased efficient cellular immunity, resulting in a diminished parasite load. Therefore, NAIs therapy can be considered as a useful and safe tool that can contribute to clearing L. major infections without inducing toxicity in host cells. The applications and mechanisms of NAIs therapy warrant further investigation especially in humans suffering from CL.

Endoparasites in wild populations of Cyprus mouflon (Ovis gmelini ophion).

The Cyprus mouflon (Ovis gmelini ophion) is a subspecies of wild sheep, endemic to Cyprus and strictly protected by national and international legislation. In the present study, 107 Cyprus mouflon faecal samples were collected from locations within the species geographical range and examined by standard parasitological methods, i.e. flotation, sedimentation and Ziehl-Neelsen stained smears. Parasites were found in 104 (97.39%) samples. First stage larvae of the lungworms Muellerius capillaris and Cystocaulus ocreatus were found in 93 (86.9%) and in 65 (60.7%) of the samples, respectively, strongylid eggs in 46 (43%), Eimeria spp. oocysts in 44 (41.1%) and Nematodirus spp. Trichuris spp. and Moniezia spp. eggs in 10 (9.3%), 7 (6.5%) and 1 (0.9%) sample, respectively. To the best of the authors' knowledge, this is the first survey on the endoparasites of Cyprus mouflon. The possible role of the parasites found on the health status of the animals and the risk of endoparasite transmission from domestic ruminants to Cyprus mouflon are discussed.