Genetic Identification of Trichomonas vaginalis by Using the Actin Gene and Molecular Based Methods

Background

Trichomonas vaginalis is the agent of urogenital tract infection that causes human trichomoniasis with some serious health complications. More understanding about genetic features of the parasite can be helpful in the study of the pathogenesis, drug susceptibility and epidemiology of the infection. For this end, we conducted analysis of the actin gene of T. vaginalis by applying the PCR-SSCP (PCR-Single Stranded Conformational Polymorphism) and nucleotide sequencing method.

Methods

Fifty T. vaginalis samples were collected from 950 women attending gynecology clinics in two cities of Iran, Hamadan and Tehran, from November 2010 to July 2011. After axenisation of isolates, all samples subjected to PCR-SSCP and nucleotide sequencing.

Results

According to the SSCP banding patterns and nucleotide sequencing, seven sequence types were detected among the isolates. Alignment of the nucleotide sequences showed five polymorphic sites in the different strain types. Amino acid substitution was not observed in the nucleotide sequence translation of the all sequences.

Conclusion

The actin gene analysis represents genetic diversity of T. vaginalis and it suggests that various strains can be responsible for clinically different trichomoniasis in infected individuals. It is expected that further studies will be conducted to increase our knowledge about relationship between the actin gene polymorphism and different biological behavior of the parasite.     

Study on ITS1 Gene of Iranian Trichomonas vaginalis by Molecular Methods

Background

Trichomoniasis is a worldwide protozoan parasitic disease and metronidazole is a choice drug for its treatment. Because of disease importance in public health and its controversial ideas about the prevalence of drug resistance, this study was carried out.

Methods

Fifty-two suspected vaginal samples were collected from 2006 to 2007 in Gynecology Maryam Hospital, Tehran, Iran. All isolates were examined by microscopic, culture and PCR techniques. The PCR products were analyzed by RFLP and CSGE methods and two suspected samples were sequenced.

Results

Trichomonas vaginalis was identified from all 52 samples. Of 52 isolates, 45 samples were successfully cultured and amplified by PCR except one. Seven were positive only by PCR. Finally, ITS1 fragment was successfully amplified in 51 of 52. CSGE analysis and PCR products digestion by MspI followed by sequencing showed nucleotide mutation at position 209 (C209T) of the ITS1 fragment in two (3.9%) of them.

Conclusion

The results showed mutation in ITS1 fragment of T. vaginalis in two (3.9%) of Iranian isolates which may be related to metronidazole resistance.     

Comparison of Resistant and Susceptible Strains of Trichomons vaginalis to Metronidazole Using PCR Method

Background

Metronidazole is drug of choice recommended by WHO for treatment of trichomoniasis, however, some reports claims drug resistance in Trichomonas vaginalis isolates recently. The objective of this study was to determine the minimum lethal concentration (MLC) of metronidazole in resistant and sensitive strains, as well as genetic patterns of these stains by PCR method.

Methods

From February 2006 to March 2007, in a cross sectional study, clinical and wet mount examination of vaginal smear along with culture were performed on 683 women attending to public and private outpatient clinics in Hamadan. Trichomoniasis marked based on major clinical symptoms. Diagnosis confirmed using wet mount microscopically and culture in Diamond medium. A serial concentration of metronidazole was provided and all isolated Trichomonas strains (resistant and sensitive) tested by standard method. Finally, all sensitive and resistant strains examined by PCR technique.

Results

Only 15/683, (2.2%) of patients clinically diagnosed trichomonal vaginitis were positive for T. vaginalis by wet smear and culture. The minimum lethal concentration (MLC) for clinically sensitive isolates was 25 µg/ml; however, this concentration for resistant isolates was 200 µg/ml after 24 h and 100 µg/ml after 50 h. The results of PCR examination of DNA from sensitive and resistant isolates had same pattern. The lanes appeared by two primers were 98 bp and 261 bp for both clinically sensitive and resistant strains.

Conclusion

Resistance to metronidazole in T. vaginalis has not relation to genetic variations and might be related to some physiologic pathways of organism.     

Molecular Identification of Leishmania Species Using Samples Obtained from Negative Stained Smears

Background

Cutaneous Leishmaniasis (CL) is a parasitic skin disease. Diagnosis primarily is based on clinical signs and microscopic observation of parasite on direct stained smears or tissue sections. Sensitivity of direct smear is not as high as molecular methods. The aim of this study was to identify and characterize Leishmania species among the negative direct smears obtained from skin ulcers suspected to CL by PCR method.

Methods

Among 81 patients with suspicious skin lesions to CL referred to the Parasitology lab, negative Giemsa stained smears were collected. DNA extraction performed by scraping stained smears, then PCR was performed.

Results

Among the DNA extracted from smears, L. tropica was isolated from 9 (11.1%) of the smears and L.major was not isolated from any samples.

Conclusion

Direct microscopy on stained smears for diagnosis of leishmaniasis is not enough accurate. PCR is recommended for clinically suspected lesions with negative result of direct smear.     

Detection of Acanthamoeba and Toxoplasma in River Water Samples by Molecular Methods in Iran

Background:

Free-living amoebae such as Acanthamoeba species may act as carriers of Cryptosporidium and Toxoplasma oocysts, thus, may play an important role in the water-borne transmission of these parasites. In the present study, a loop mediated isothermal amplification (LAMP) method for detection of Toxoplasma and a PCR assay were developed for investigation of Acanthamoeba in environmental water samples.

Methods:

A total of 34 samples were collected from the surface water in Guilan Province. Water samples were filtrated with membrane filters and followed by DNA extraction. PCR and LAMP methods used for detection of the protozoan parasites Acanthamoeba and Toxoplasma respectively.

Results:

Totally 30 and 2 of 34 samples were positive for Acanthamoeba and Toxoplasma oocysts respectively. Two samples were positive for both investigated parasites.

Conclusion:

The investigated water supplies, are contaminated by Toxoplasma and Acanthamoeba (oo)cystes. Acanthamoeba may play an important role in water-borne transmission of Toxoplasma in the study area. For the first time in Iran, protocol of LAMP method was used effectively for the detection of Toxoplasma in surface water samples in Iran.     

Ferredoxin Gene Mutation in Iranian Trichomonas vaginalis Isolates

Background

Trichomonas vaginalis causes trichomoniasis and metronidazole is its chosen drug for treatment. Ferredoxin has role in electron transport and carbohydrate metabolism and the conversion of an inactive form of metronidazole (CO) to its active form (CPR). Ferredoxin gene mutations reduce gene expression and increase its resistance to metronidazole. In this study, the frequency of ferredoxin gene mutations in clinical isolates of T.vaginalis in Tehran has been studied.

Methods

Forty six clinical T. vaginalis isolates of vaginal secretions and urine sediment were collected from Tehran Province since 2011 till 2012. DNA was extracted and ferredoxin gene was amplified by PCR technique. The ferredoxin gene PCR products were sequenced to determine gene mutations.

Results

In four isolates (8.69%) point mutation at nucleotide position -239 (the translation start codon) of the ferredoxin gene were detected in which adenosine were converted to thymine.

Conclusion

Mutation at nucleotide -239 ferredoxin gene reduces translational regulatory protein''s binding affinity which concludes reduction of ferredoxin expression. For this reduction, decrease in activity and decrease in metronidazole drug delivery into the cells occur. Mutations in these four isolates may lead to resistance of them to metronidazole.     

Molecular Diagnosis of Strongyloides stercoralis Infection by PCR Detection of Specific DNA in Human Stool Samples

Background

Strongyloidiasis is mostly an asymptomatic infection and diagnosis of latent infections is difficult due to limitations of current parasitological and serological methods. This study was conducted to set up a PCR-based method for molecular diagnosis of Strongyloides stercoralis infection by detection of copro-DNA in stool samples.

Methods

A total of 782 fresh stool samples were collected and examined by agar plate culture. Among those sixteen stool samples, which confirmed to be infected with S. stercoralis were examined as positive control to set up each single and nested PCR, using two primer sets designing to amplify partial ribosomal DNA of S. stercoralis genome. Since, single PCR method yielded higher efficacy in detecting positive samples, in the second step, 30 stool samples, which found negative for S. stercoralis by agar plate culture of single stool sample, were examined by single PCR. Data analysis was performed using McNemar''s χ² test, with consideration of a P-value of <0.05 as indication of significant difference.

Results

In amplification of DNA extracted from stool samples, single PCR detected S. stercoralis DNA target in all 16 positive samples, while nested PCR amplified DNA in only 75% of samples. In the second step, single PCR amplified S. stercoralis extracted DNA in 5 out of 30 samples which were negative by coproculture.

Conclusion

Single PCR method amplifying a short (100bp) target represented more efficacies for detection of S. stercoralis in faecal examination compared to agar plate culture and nested PCR, which amplified longer target.     

A preliminary investigation of microsatellite-based genotyping in Trichomonas vaginalis

The genetic epidemiology of Trichomonas vaginalis is poorly understood at present. The recent release of the organism's genome sequence opens the way to investigation of polymorphic markers allowing strain identification. We here report a preliminary analysis of microsatellite loci in T. vaginalis and show that this approach holds promise for future studies of infection transmission and organism diversity.     

Genetic characterization of three Cuban Trichomonas vaginalis virus. Phylogeny of Totiviridae family

Trichomonas vaginalis can be infected with double stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVV). This viral infection may have important implications for trichomonal virulence and disease pathogenesis. In this study we identified and genetic characterized three strains of TVVs isolated from T. vaginalis in Cuba. The three new predicted sequences of capsid protein and RNA-dependent RNA polymerase amounted to the previously determined 20 TVV sequences and other 21 viruses of Totiviridae family were used for a phylogenetic analysis. Four distinct monophyletic clades are shown in a phylogenetic tree. One corresponds with TVVs, other with Victorivirus, Leishmaniavirus and Eimeria brunetti virus and, other with viruses of the genus Totivirus and the last with Giardiavirus. The E. brunetti virus is identified in the phylogenetic tree as independent taxon between Leishmaniavirus and Victorivirus isolates, most closely related to Victorivirus. TVV constitute a monophyletic cluster distinguishable from all other viruses in Totiviridae family. This result suggested that TVV may be grouped in a separated genus and not inside of Giardiavirus. TVVs appear to be more closely related to protozoan viruses in the genus Leishmaniavirus and to fungal viruses in the genus Victorivirus than to other protozoan and fungal viruses in Giardiavirus and Totivirus. Among TVVs, four main groups can be recognized within Trichomonasvirus cluster, which correspond with the previous species classification proposed. Further studies, with more TVV strains, especially TVV3 and 4 strains, are needed in order to determine the phylogenetic relationship among Trichomonasvirus genus and specifically if TVV2 and 3 each also constitute a well-delimited group.     

Molecular Detection of Malaria in South Punjab with Higher Proportion of Mixed Infections

Background

Malaria is well known for its fatalities worldwide, Plasmodium vivax and the Plasmodium falciparum are the two important species of malaria reported from Pakistan and creating lots of morbidities across the country.

Method

Study was conducted to determine the Surveillance of malaria in South Punjab by microscopy and Polymerase chain reaction (PCR).

Result

samples out of 100 patients were found positive for malarial parasites. One patient was found with mixed infection, whereas P. falciparum and P. vivax infections were detected in 17 and 22 patients, respectively. In nested PCR, genus-specific primers for Plasmodium species. in round 1 and species-specific primers for P. falciparum and P. vivax in round 2 were used. By the application of PCR 41% were found to be infected by Plasmodium spp. Among Plasmodium positive patients: mixed, P. falciparum and P. vivax infection were detected in 10, 15 and 16 patients, respectively. Thirty nine microscopically positive patients confirmed to have Plasmodium spp. One negative by PCR, 2 microscopically negative patients had shown Plasmodium spp. infection (P. falciparum and P. vivax) by PCR. In total samples, P. falciparum, P. vivax and mixed infection accounted for 36.6%, 39.0% and 24.3%, respectively.

Conclusion

Microscopy was found deficient for interpretation of mixed infections, low parasitaemia, and species specific diagnosis. The sensitivity, specificity and efficacy of nested PCR was calculated 95%, 98% and 97%, respectively, showing PCR as a more effective and efficient diagnostic tool for malaria.     

Prevalence and Molecular Diagnosis of Babesia ovis and Theileria ovis in Lohi Sheep at Livestock Experiment Station (LES), Bahadurnagar,Okara, Pakistan

Background

Babesia ovis and Theileria ovis are among the important and main etiological agents causing ovine babesiosis and ovine theileriosis, causing severe economic losses among sheep and goats. The aim of the present study was to determine the prevalence and molecular diagnosis of B. ovis and T. ovis in Lohi sheep at Livestock Experiment Station Bahadurnagar, Okara, Pakistan.

Methods

The prevalence of B. ovis and T. ovis was investigated in 200 Lohi sheep of mixed age and sex by PCR during 2011. The assay was employed using primers Bbo-F & Bbo-R, specific for a 549-bp fragment in B. ovis genomic DNA and primers TSsr 170F & TSsr 670R, specific for a 520-bp fragment in T. ovis genomic DNA. The animals were also screened for both haemoparasites through stained thin blood smears.

Results

Thirty two (16%), 48 (24%) and 26 (13%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites, respectively, through microscopy. Sixty eight (34%), 73 (37%) and 42 (21%) were the number of animals found positive for B. ovis, T. ovis and for mixed infection with both parasites, respectively, through PCR test.

Conclusion

The results indicate the high sensitivity of PCR for surveying babesiosis and theileriosis and there is noteworthy prevalence of these diseases in sheep at an experimental station where environmental conditions are relatively controlled as compared to field conditions.     

Molecular Diagnosis of Cause of Anisakiasis in Humans,South Korea

Anisakiasis in humans in South Korea has been considered to be caused exclusively by the larvae of Anisakis simplex sensu stricto and Pseudoterranova decipiens. Recently, however, DNA sequencing of larvae from 15 of 16 anisakiasis patients confirmed the cause to be Anisakis pegreffii infection. Molecular analysis should be performed for all extracted larvae.     

Molecular and Biomorphometrical Identification of Ovine Babesiosis in Iran

Background

Ovine babesiosis is the most important haemoparasitic tick-borne disease of small ruminants in Iran caused by Babesia ovis, B. motasi, and B. crassa. The aim of this study was to characterize the species of ovine Babesia species isolated from different geographical region of Iran.

Methods

One hundred fifty four blood samples collected from animals, which demonstrated the pale mucous membranes or hyperthermia. The specimens were transferred to the laboratory and the blood smears stained with Geimsa, the morphological and biometrical data of parasite in any infected erythrocyte have been considered. Extracted DNA from each blood samples were used in PCR and semi nested- PCR in order to confirm the presence of the species.

Results

Microscopical observation on 154 blood smears determined 38 (24.67%) and 40 (26%) samples were infected by Babesia and Theileria respectively. The mixed infections occurred in four (2.6%) samples. The results of the PCR assays showed nine (5.85%), 81 (53%) and 18 (11.7%) were distinguished as Babesia, Theileria and mixed infection, respectively. Semi nested- PCR did not confirm the presence of B. motasi.

Conclusion

The causative organism of many cases of haemoprotozoal diseases, which recorded in previous studies, could be B. ovis or Theileria lestoquardi. The result confirmed that B. ovis was only species which causes babesiosis in the study areas. It seems that the biometrical polymorphisms could exist in B. ovis in Iran. This polymorphism could be a main problem in differentiation between B. ovis and B. motasi and it could be dissolved by specific PCR analysis.     

Current status and prospects for development of a vaccine against Trichomonas vaginalis infections

Trichomonas vaginalis is a sexually transmitted pathogen with an annual worldwide incidence of over 276 million infections, the highest of all curable and non-viral STI. A large proportion of cases are asymptomatic and under-diagnosed with conventional diagnostic tools. Infection has important maternal and fetal health consequences and can lead to a higher probability of HIV transmission and susceptibility. Lack of affordable accurate diagnostic tests globally and metronidazole resistance hinder T. vaginalis control efforts. Based on data from current vaccination studies in animal models, a human vaccine is achievable to intervene on the substantial incidence of infection.     

Phase II clinical trial with Praneem polyherbal tablets for assessment of their efficacy in symptomatic women with abnormal vaginal discharge (an ICMR task force study)

Abnormal vaginal discharge syndrome (AVDS) is a commonly observed gynaecological complaint for which women seek medical attention. The present study was conducted in six Indian Council of Medical Research centres with Praneem polyherbal tablets (PPT), to determine their efficacy in the treatment of symptomatic women with AVDS. Data are given on 141 subjects investigated. In total, 137 women (97%) reported complete (n=62, 44%) and partial (n=75, 53%) relief from symptoms after use of PPT for seven consecutive days. On speculum examination, 71 (74%) women were confirmed to be cured of AVDS. Microbiological tests could only be conducted microscopically for Trichomonas vaginalis, Candida albicans and bacterial vaginosis. It was observed that all women with T. vaginalis had this infection cured by PPT, and the cure rate was 77% for C. albicans and 68% for bacterial vaginosis. Seventy-eight women (55%) reported a transient burning sensation, mostly on the first 2 d of intake of PPT; however, they continued to use the tablets for the prescribed 7 d. This study lays the basis for an extended Phase II/III clinical trial, preferably randomized and comparing a larger number of women to confirm the safety and efficacy of PPT.     

Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) of some benzimidazole derivatives with trichomonicidal activity

Trichomonosis is a common sexually transmitted infectious disease linked to reproductive health complications. Recently, the benzimidazole nucleus has emerged as a promising scaffold to develop new trichomonicidal agents. Despite the fact that large amounts of experimental data have been accumulated over the past eight years, no quantitative studies have yet been reported on this class of compounds. In our effort to develop new antiparasitic benzimidazole derivatives, we report in this paper CoMFA and CoMSIA studies with an initial set of 70 benzimidazole derivatives with trichomonicidal activity. Four CoMFA models and eight CoMSIA models were generated; ten of these models had values of r² > 0.6 and q² > 0.5. The best CoMFA model had r² = 0.936 and q² = 0.634, and the best CoMSIA model had r² = 0.858 and q² = 0.642. These models were generated by using two conformer selection methodologies (minimum energy conformations and 3D similarity), and three charge types (Mulliken, Gasteiger-Hükel and electrostatic potential atomic charges). The putative active tautomers of 1H-benzimidazole derivatives were selected using 3D-QSAR calculations. All models were validated via an external test set with 13 molecules. The best models satisfied additional validation criteria. The contour maps generated show the most important features that a benzimidazole derivative should have for trichomonicidal activity; they also, suggest that substituents at the 2- and 6-positions are important in the generation of derivatives with strong activity.     

Molecular Study of Sheep Malignant Theileriosis at Barka Region in the Sultanate of Oman

Background

We used the PCR technique based on the abovementioned primer pair and sequencing to demonstrate the Theileria infection in the sheep samples collected from Sultanate of Oman.

Methods

According to the frame work of “integrated control of ticks and tick borne diseases in globalized world managed by EU-ICTTD-3 project, the samples from blood, liver, spleen, lymph node and lung were sent to the laboratory of Iranian Research Center for Ticks and Tick-borne Diseases (IRCTTD). Samples from blood smear and impression smears from liver, spleen, lymph node, and lung were analyzed by Geimsa staining. The DNA was extracted from the abovementioned samples and analyzed by PCR technique using specific primers derived from the nucleotide sequences of 18S rRNA gene of T. lestoquardi, which can amplify the common region in other Theileria and Babesia spp. Subsequently the amplified DNA was sequenced.

Results

The analysis of blood smears of the sheep was negative for piroplasmosis performed through the Giemsa staining. The impression smears prepared from liver, spleen, lymph node, and lung showed suspicious structures mimicking Theileria schizonts in some cells. The results showed an expected PCR product of 428 bp in length, which is specific for Theileria spp. The PCR products were subsequently sequenced. The corresponding nucleotide sequence is registered under accession number {"type":"entrez-nucleotide","attrs":{"text":"JF309152","term_id":"323695911","term_text":"JF309152"}}JF309152 in GenBank. The sequence alignment in GenBank showed that the PCR products had 99% homology to the known T. lestoquardi registered under accession number {"type":"entrez-nucleotide","attrs":{"text":"AF081135","term_id":"7576455","term_text":"AF081135"}}AF081135 in the GenBank.

Conclusion

Oman sheep are highly susceptible for Theileria infection and the infected sheep mostly die before the microschizonts or erythrocytic form of Theileria appears in the nucleated or erytrocytic cells respectively.     

Molecular Characterization of Eimeria Species Naturally Infecting Egyptian Baldi Chickens

Background:

Coccidiosis is a serious protozoal disease of poultry. The identification of Eimeria species has important implications for diagnosis and control as well as for epidemiology. The molecular characterization of Eimeria species infecting Egyptian baladi chickens was investigated.

Methods:

Eimeria species oocysts were harvested from intestines of naturally infected Egyptian baldi chickens. The morphometry characterization of oocysts along with COCCIMORPH software was done. The DNA was extracted initially by freezing and thawing then the prepared samples was subjected to commercial DNA kits. The DNA products were analyzed through conventional polymerase chain reaction by using amplified region (SCAR) marker.

Results:

The PCR results confirmed the presence of 7 Eimeria species in the examined fecal samples of Egyptian baldi breed with their specific ampilicon sizes being E. acervulina (811bp), E. brunette (626bp), E. tenella (539bp), E. maxima (272bp), E. necatrix (200bp), E. mitis (327bp) and E. praecopx (354bp). A sequencing of the two most predominant species of Eimeria was done, on E. tenella and E. máxima. Analysis of the obtained sequences revealed high identities 99% between Egyptian isolates and the reference one. Similarly, E. maxima isolated from Egyptian baldi chickens showed 98% nucleotide identities with the reference strain. Only single nucleotide substitution was observed among the Egyptian E. tenella isolates (A181G) when compared to the reference one. The Egyptian isolates acquired 4 unique mutations (A68T, C164T, G190A and C227G) in compared with the reference sequence.

Conclusion:

This is the first time to identify the 7 species of Eimeria from Egyptian baladi chickens.     

Detection of Coproantigens by Sandwich ELISA in Rabbits Experimentally Infected with Fasciola gigantica

Background

The study was targeted to report the appearance of coproantigens in feces and circulating antibodies in the serum of Fasciola gigantica experimentally infected rabbits.

Methods

Copro Hyper Immune Serum (HIS) and Excretory-Secretory Hyper Immune Serum (ES HIS) antigens were used in a sandwich ELISA for the detection of F. gigantica antigens in feces of 12 rabbits experimentally infected with different doses of F. gigantica encysted metacercariae (EMC) (10, 25 and 30 EMC). The relation between time of appearance of coproantigens in feces and anti-Fasciola antibodies in serum was evaluated.

Results

The earliest diagnostic coproantigen was recorded at 21^st, 25^th and 28^th day post-infection (p.i.) in groups of rabbits infected with 30, 25 and 10 F. gigantica EMC respectively. Both HIS and ES HIS were able to detect coproantigens in feces of rabbits infected with 30 EMC at day 21 p.i. The appearance of F. gigantica coproantigens in feces of infected rabbits was concurrent to the appearance of anti-Fasciola antibodies in blood (3^rd week p.i.). However, coproantigen has specific ability for direct assessment of active infection with minimal cross-reaction with other heterologous parasitic infections.

Conclusion

The findings hold promise for a more accurate diagnostic technique in the near future for suspected Fasciola infection.