ITS1 Polymerase Chain Reaction Research Articles

A PCR and RFLP-based molecular diagnostic algorithm for visceral leishmaniasis

Objective: To determine an algorithm for molecular diagnosis of visceral leishmaniasis (VL) by kinetoplast DNA (kDNA) (RV1/ RV2) and internal transcriber spacer (ITS1) (LITSR/L5.8S) polymerase chain reaction (PCR), complemented by ITS1 PCR restriction fragment length polymorphism (RFLP), using peripheral blood or bone marrow aspirate from patients with suspected VL.Methods: Biological samples were submitted to the gold standard for the diagnosis of VL and molecular diagnosis represented by ITS1 PCR, kDNA PCR, and ITS1 PCR RFLP. The samples were obtained from seven groups: group I, 82 samples from patients with confirmed VL; group H , 16 samples from patients under treatment for VL; groupII, 14 samples from dogs with canine visceral leishmaniasis (CVL); group II, a pool of six experimentally infected sandflies (Lutzomya longipalpis); group IV, 18 samples from patients with confirmed tegumentary leishmaniasis (TL) and groups Ή and VI were from control groups without VII.Results: The following gold standard and molecular examination results were obtained for each of the seven groups: group I : parasitologic and immunochromatographic tests showed a sensitivity of 76.3% (61 of 80) and 68.8% (55 of 80), respectively, and a sensitivity of 97.6% (80 of 82) and 92.7% (76 of 82) by ITS1 and kDNA PCR, respectively. After ITS1 PCR RFLP (Hae III) analysis of the 80 positive samples, 52.5% (42 of 80) generated three fragments of 180, 70, and 50 bp, corresponding to the pattern of Leishmania infantum infantum; group Π : negative for the parasitologic methods and positive for IrK39 (100%, 16 of 16), presented 12.5% (2 of 16) of positivity by ITS1 PCR and 25.0% (4 of 16) by kDNA PCR; group III: positive in the parasitologic and serologic tests (100%, 14 of 14), presented 85.7%(12 of 14) of positivity by ITS1 PCR and kDNA PCR. ITS1 PCR RFLP showed that 83.3% (10 of 12) of the canine samples contained parasites with profiles similar to L. infantum; groupIVpresented amplifications by ITS1 PCR and kDNA PCR. ITS1 PCR products were analyzed by RFLP, generating a profile similar to that of L. infantum; group V: positive in the parasitologic examination (100%, 18 of 18), presented 72.2% (13 of 18) of the samples by ITS1 PCR positive. A total of 69.2% (9 of 13) showed profiles corresponding to a Viannia complex by ITS1 PCR RFLP; and group Ή and group W were negative by ITS1 and kDNA molecular tests. Comparing the molecular results with the parasitologic and serologic diagnosis from group I, almost perfect agreement was found ( κ both>0.80, P<0.001). ITS1 and RV1/RV2 PCR detected 90.2% (74 of 82) of the samples. Two samples positive by RV1/RV2 were negative by LITSR/L5.8S, and six samples positive by LITSR/L5.8S were negative by RV1/RV2. Therefore, these two systems complemented each other; they diagnosed 100% of the samples as belonging to the Leishmania genus.Conclusions: We suggest an algorithm for the molecular diagnosis of VL, which must consider previous parasitologic and serologic (immunochromatographic) diagnoses, and should combine kDNA and ITS1 to determine the Leishmania subgenus using RFLP as a complement method to define the L. infantum species.

The use of molecular technology to investigate trypanosome infections in tsetse flies at Liwonde Wild Life Reserve.

Trypanosomes are protozoan flagellates that cause human African trypanosomiasis (HAT) and African animal trypanosomiasis (AAT). HAT is caused by Trypanosoma brucei rhodesiense in East and Central Africa and T.b. gambiense in West Africa, whereas AAT is caused by a number of trypanosome species, including T. brucei brucei, T. evansi, T. vivax, T. congolense, T. godfreyi and T. simiae. The aim of this study was to establish if tsetse flies at Liwonde Wild Life Reserve (LWLR) are infected with these trypanosomes and thus pose a risk to both humans and animals within and surrounding the LWLR. A total of 150 tsetse flies were caught. Of these, 82 remained alive after capture and were dissected such that the mid-gut could be examined microscopically for trypanosomes. DNA extractions were performed from both mid-guts and the 68 dead flies using a Qiagen Kit. Amplification techniques involved the Internal Transcriber Spacer 1 (ITS 1) conventional polymerase chain reaction (PCR) with primers designed to identify trypanosome species, and Repetitive Insertion Mobile Element - Loop Mediated Isothermal Amplification (RIME LAMP), a sequence specific to T. brucei. Analysis showed that 79/82 (96.3%) of the mid-guts examined microscopically were positive for trypanosomes and that 75/150 (50%) of the DNA extracts (from the mid-gut, and tsetse fly carcasses) were positive for T. brucei, as determined by the RIME LAMP method. ITS1 PCR further showed that 87/150 (58.0%) flies were positive for trypanosomes, of which 56/87 (64.4%) were T. brucei, 9/87 (10.3%) were T. vivax; 7/87 (8.1%) were T. simiae; 6/87 (6.9%) were T. congolense, and 6/87 (6.9%) were T. godfreyi. Ten samples had a mixture of infections. Our analysis demonstrated a mixture of infections from trypanosome species in tsetse flies at LWLR, and that T. brucei, the species that causes HAT, was the most common. Our study successfully used molecular techniques to demonstrate the presence of T. b. rhodesiense at LWLR, a species that causes HAT in both East and Central Africa.

MOLECULAR EPIDEMIOLOGICAL STUDIES ON TRYPANOSOMA EVANSI TYPE A AND TYPE B IN CAMELS (CAMELUS DROMEDARIES) FROM FIVE DIFFERENT REGIONS OF SAUDI ARABIA USING THE ITS1 RDNA AND ROTAT 1.2 VSG GENE

Trypanosoma evansi is the most widespread of the pathogenic salivarian trypanosomes and cause a serious disease called (surra) that is affect the domestic animals such camels and horses in Tropical and subtropical countries and often leads to reduced productivity and economic losses. Therefore, the objectives of the present study were to determine the prevalence rates of trypanosomiasis using polymerase chain reaction (PCR) among camels from five different regions of Saudi Arabia and to sequence and characterized the T. evansi from these animals. In the current study, 832 camel blood samples collected from five different regions of Saudi Arabia for detecting T. evansi. A generic ITS1-PCR and RoTat 1.2 VSG gene were applied in this study to analyze camels’ blood samples. Molecular analysis was performed using ITS1-PCRwhich showed that the highest prevalence of trypanosomes was observed in Al-Qaseem province (50.1%) followed Riyadh province (49%), whereas in Hail and the Northern Borders, there were fewer infections with trypanosomes (28.4% &17.6%), respectively. PCR amplification was carried out targeting RoTat 1.2 VSG gene on TS1-positive samples and some of them were negative for RoTat1.2. The test negative in RoTat 1.2 PCR but ITS1 PCR positive could suggest T. evansi type B. Presence of T. evansi type B is interest to the international community, as this has a message to redesign the existing molecular and serological diagnostic markers. However, to our knowledge this the first study demonstrating T. evansi type B out of Africa.

Prevalence and molecular characterization of typanosomes in dogs in Warri, Nigeria

Animal African Trypanosomosis (AAT) is a devastating protozoan disease of animal and humans caused by Trypanosoma spp. resulting in a great economic loss in the livestock industries, in the sub-Saharan Africa. A survey for canine trypanosomosis was carried out in Warri, Uvwie and Okpe Local Government Areas of Delta State, using molecular technique. A total of 110 dogs of different ages and sexes were randomly screened for trypanosome in Okpe (19), Uvwie (45) and Warri South (41) Local Government Areas of Delta State. Extracted DNA samples were subjected to polymerase chain reaction (PCR) test using generic primers set that target the internal transcribes spacer 1 (ITS 1) (CF:5’-CCGGAAGTTCACCGATATTG-3’ and BR: 5’-TGCTGCGTTCTTCAACGAA- 3’) of ribosomal DNA (rDNA). The gel electrophoresis of the ITS1 PCR products of 5 samples revealed band sizes of 700 bp which corresponded with the expected band size of Trypanosoma congolense. One (5%) dog from Okpe was positive while Uvwie and Warri south recorded 2 (4.3%) and 2 (4.7%) positives, respectively. The prevalence was significantly higher in Nigerian indigenous dogs than exotic breeds of dogs. Overall prevalence was 5 (4.5%). We concluded that canine trypanosomosis is prevalent in Warri, Uvwie and Okpe Local Government Areas of Delta State.Keywords: Prevalence; molecular chacterization; trypanosomes; dogs; Warri

A tsetse Glossina pallidipes harbors the pathogenic trypanosomes circulating in Liwale district, Tanzania.

African Animal Trypanosomiasis (AAT) is among several constraints hindering development of the livestock sector in Tanzania. A survey was conducted in Liwale district located in southern Tanzania in 2013 to determine the population density of Glossina species, distribution pattern and Trypanosome species infection rate in tsetse flies. A total of 200 flies were collected from the study area and three Glossina species were identified. The proportional abundance of all trapped flies was 90% (180) for Glossina pallidipes, 6% (12) for G. brevipalpis and 4% (8) for G. m. morsitans with apparent densities (fly/trap/day - FTD) of 0.44. Higher density of Glossina pallidipes was observed in villages closer to than those far from the Selous game reserve. Trypanosomes were detected and identified by microscopy and ITS1 polymerase chain reaction (PCR) assay on DNA purified from 200 flies. Glossina pallidipes was the only fly found infected by three Trypanosoma species, namely T. vivax (60%), T. simiae (10%) and T. brucei (30%) with an overall infection rate of 10% (20/200). A higher proportion of trypanosome infections were observed in female tsetse flies than in males. Results of this study show that G pallidipes is the major Glossina species harboring pathogenic trypanosomes in Liwale district and that the Selous game reserve is a potential reservoir of trypanosomes in terms of parasite abundance and species diversity.

Molecular characterization of Dirofilaria spp. circulating in Portugal

BackgroundDirofilariosis is a potentially zoonotic parasitic disease, mainly transmitted by mosquito vectors in many parts of the world. Data concerning the canine Dirofilaria species currently circulating in Portugal is scarce. Thereby, a large-scale study was conducted to determine the Dirofilaria spp. present in Portugal, based on a molecular approach, and also to optimize a reliable and highly sensitive species-specific polymerase chain reaction (PCR) assay that could be used for the simultaneous detection and differentiation of Dirofilaria immitis, Dirofilaria repens, and other concurrent filarial species in animal reservoirs.MethodsBlood samples were collected from three districts of Portugal (Coimbra, Santarém and Setúbal) between 2011 and 2013. Samples were tested using rapid immunomigration tests (Witness® Dirofilaria), modified Knott’s technique and acid phosphatase histochemical staining. In addition, molecular analysis was performed by amplification of the internal transcribed spacer (ITS) region using two different PCR protocols, specific for molecular screening of canine filarial species.ResultsOf the 878 dogs sampled, 8.8% (n = 77) were positive for D. immitis circulating antigen and 13.1% (n = 115) positive for microfilariae by the modified Knott’s technique. Of the 134 samples tested by acid phosphatase histochemical staining, 100 (74.6%) were positive for D. immitis. Overall, 13.7% (n = 120) were positive by PCR for D. immitis by ITS2, of which 9.3% (67/720) were also positive by ITS1. ITS2 PCR was the most sensitive and specific method, capable of detecting mixed D. immitis and A. reconditum infections. Heterozygosity, in the form of double peaks, was detected by sequencing of both ITS regions. No D. repens was detected by any of the diagnostic methods.ConclusionsThe present study confirmed D. immitis as the dominant species of the genus Dirofilaria infecting Portuguese dogs, based on sequencing of ITS1 and ITS2 PCR fragments. Additionally, ITS2 PCR was the most adequate method for diagnosis and prevalence estimation.

Molecular Diagnosis of Clinical Isolates of Cutaneous Leishmaniasis Using ITS1 and KDNA Genes and Genetic Polymorphism of Leishmania in Kashan, Iran.

Cutaneous leishmaniasis is a common skin disease caused by leishmania parasite. An accurate diagnosis of parasites species is possible using molecular techniques. This study was carried out to compare internal transcribed spacer (ITS1) and kinetoplast deoxyribonucleic acid (KDNA) genes for identifying Leishmania species by Polymerase Chain Reaction (PCR), furthermore, genetic diversity of isolates was studied. This research examined 130 patients who were suspected of cutaneous leishmaniasis and referred to Kashan's health centers from 2011-2014. After DNA extraction from serosity, PCR were performed using ITS1 and KDNA primers. Cutaneous Leishmaniasis was diagnosed by the observation of 320 bp band in the ITS1-PCR. The PCR products were digested with restriction enzyme HaeIII and then leishmania species were identified by patterns of enzymatic digestion. The diagnostic criteria of Cutaneous Leishmaniasis (CL) in KDNA-PCR were based on the observation of 760 and 650 bp for Leishmaniasis tropica and Leishmaniasis major, respectively. Twelve isolates of leishmania were sequenced and the phylogenetic tree was traced using the results of sequencing by Mega 4 software. Out of 130 suspected patients to CL, 70 (53.8%) and 98 (75.4%) isolates were positive by Restriction Fragment Length Polymorphism (RFLP) of ITS1 and KDNA, respectively. Using ITS1 PCR, 60 samples (85.7%) and 10 samples (14.3%) were identified as L. tropica and L. major, respectively, ITS1-PCR had 25.3% false negative, compare to microscopy. While, microscopy had false negative in 13 cases compare to KDNA-PCR. Due to the lower sensitivity of the PCR-RFLP of ITS1, KDNA-PCR is recommended for diagnosis of CL. The L. tropica and L. major are the causative agents of CL.

The Jordanian Mid Jordan Valley is a classic focus of Leishmania major as revealed by RFLP of 56 isolates and 173 ITS-1-PCR-positive clinical samples

The identity of the causative species of cutaneous leishmaniasis (CL) in the endemic Jordanian Mid Jordan Valley (JMidJV) was investigated using the polymerase chain reaction (PCR) amplifying the ribosomal internal transcribed spacer 1 (ITS-1) followed by the restriction fragment length polymorphism (RFLP). The geographical distribution of CL and the usefulness of ITS1 PCR in diagnosis of suspected CL in the study area were also addressed. Over the period from 2004 to 2009, 56 clinical isolates of Leishmania promastigotes and 185 lesion scrapings spotted on filter papers were obtained from suspected CL patients living in the JMidJV, which is divided into northern and southern districts. The majority (67.1%) of patients occurred in the populated eastern part of the southern district. Of the 185 suspected CL patients, 173 (93.5%) were confirmed positive using PCR. Leishmanial DNA was detected in 27 (90%) of 30 patients having clinically atypical lesions of CL and in 60 (92%) of 65 smear- and culture-negative cases having typical lesions of CL. The parasites in all of the 56 isolates and the 173 PCR-positive scrapings were classified as Leishmania major. In conclusion, PCR is useful in diagnosis of CL especially when smear and culture are negative. It is also recommended as a differential diagnostic tool of atypical lesions when CL is endemic. The identification of L. major as the causative species in such a considerable number of CL cases, representative of all mini foci of CL in the study area, shows that the JMidJV is a classic focus of L. major.

Comparison of PCR-based diagnoses for visceral leishmaniasis in Bangladesh

The diagnosis of visceral leishmaniasis (VL) is performed using multiple methods encompassing parasitological, serological and nucleic acid-based diagnostic tools, each method with its own unique advantages and disadvantages. Conventional parasitological methods are risky for the patient and require skilled personnel to collect specimens from spleen or bone marrow, and hence they are not generally available in impoverished areas. Polymerase chain reaction (PCR) has been validated as an excellent alternative to microscopy in terms of sensitivity and specificity. Here, we evaluate four different PCR assays targeting ITS1, ITS2, mini-exon and small subunit-rRNA (SSUrRNA) using DNA extracted from peripheral blood buffy coat in order to avoid more invasive processes. A total of 61 VL patients and 75 non-VL infected control individuals were enrolled. The VL patients were confirmed to be positive for Leishmania amastigotes in splenic smears by microscopy. Sensitivities of the PCR targeting ITS1, ITS2, SSUrRNA and mini-exon were 96.7%, 91.8%, 88.5% and 34.4%, respectively, while the specificity was 98.7% for all methods. Nested PCR for ITS1 resulted in 100% sensitivity. The efficacy of each PCR was evaluated with various Leishmania amastigote parasite loads in each spleen smear, graded from 1+ to 5+. The PCR targeting ITS1 showed 100% sensitivity for the detection of Leishmania donovani in all samples from grades ≥3, ≥4, and ≥5, respectively. The restriction fragment length polymorphism observed in ITS1 amplicons digested by HaeIII classified the parasite into L. donovani complex. The ITS1 PCR was found to be equal to conventional, but very invasive and risky parasitological diagnoses and superior to other PCR based methods in sensitivity and examination of genetic heterogeneity. We recommend the PCR targeting ITS1 using peripheral blood buffy coat DNA as an alternate, less invasive diagnostic choice for the confirmation of L. donovani infection.

Methods incorporating a polymerase chain reaction and restriction fragment length polymorphism and their use as a ‘gold standard’ in diagnosing Old World cutaneous leishmaniasis

Three polymerase chain reaction (PCR) assays — kinetoplast DNA (kDNA) PCR, 7SL PCR, ITS1 PCR — were compared for their ability to detect leishmanial parasites in the skin tissue aspirates of 212 Palestinian suspect cases of cutaneous leishmaniasis (CL). The kDNA PCR was the most sensitive, detecting 156/170 (91.8%) of positive samples confirmed by a set ‘gold standard’ but of poor specificity in identifying the species of Leishmania. The 7SL PCR detected 154/170 (90.5%) and the ITS1 PCR only 108/170 (63.5%) of the true positives, and both were 100% specific. The highest Kappa coefficient agreement (95% CI) was obtained with the 7SL PCR (0.792 ± 0.098). Restriction analysis of the products from the ITS1 PCR and 7SL PCR enabled identification of species of Leishmania. L. tropica was the predominant cause of CL, with L. major being less frequent.

Toxoplasma gondii in Capybara (Hydrochaeris hydrochaeris) antibodies and DNA detected by IFAT and PCR

Toxoplasmosis is considered nowadays as one of the most important foodborne diseases in the world. One of the emerging risks in acquiring infection with Toxoplasma gondii is the increasing popularity of wild animals and game meat. Capybara (Hydrochaeris hydrochaeris) is the world's largest extant rodent and is used for human consumption in many areas of South America, and in case it carries T. gondii cysts, it may act as a source of infection. In the present study, we detected infection with T. gondii in capybaras from the south of Brazil. Antibodies to T. gondii were assayed in the serum of capybaras using the indirect fluorescent antibody test (IFAT > or = 1:16). Blood, liver, heart, lymph nodes, and spleen tissues were collected and tested by polymerase chain reaction (PCR) for B1 gene and ITS1 region. The results showed that 61.5% (16/26) capybaras were seropositive to T. gondii. Titers of specific antibodies to T. gondii ranged from 1:16 to 1:512. Among the feral rodents studied, 7.7% (2/26) were PCR positive for B1 gene assay and 11.5% (3/26) were positive for ITS1 PCR assay; for both test, the prevalence was 15.4%. Liver, heart, and blood tissues were those which tested positive for the apicomplexan. Our findings show a high percentage of infection with T. gondii in asymptomatic capybaras. Based on those data, we hypothesize that the consumption of raw or undercooked capybara meat could be a source of infection for humans.

A Method for Extracting Genomic DNA from Individual Elaphostrongyline (Nematoda: Protostrongylidae) Larvae and Differentiation of Elaphostrongylus Spp. from Parelaphostrongylus Spp. by PCR Assay

This article reports a rapid and effective method for the extraction and purification of genomic DNA (gDNA) from individual first-stage larvae (L1) of elaphostrongyline nematodes that had been stored frozen or fixed in 95% ethanol for 1 to 5 years. The method was highly effective for L1s of all 6 species of elaphostrongylines, based on polymerase chain reaction (PCR) amplification of a partial fragment of the first internal transcribed spacer (ITS-1) of the ribosomal DNA. Differences were detected in the sizes of partial ITS-1 amplicons between the 2 elaphostrongyline genera, Elaphostrongylus and Parelaphostrongylus. The reliability of the ITS-1 PCR assay was tested by using L1s of unknown identity from Newfoundland and Labrador, Canada. The ability to consistently isolate gDNA from individual L1s, together with a simple PCR-based method to distinguish between Parelaphostrongylus and Elaphostrongylus, have important implications for diagnostic testing and for conducting epizootiological studies on these parasites of veterinary importance.

Genetic variation in North American black flies in the subgenus Psilopelmia (Simulium: Diptera: Simuliidae)

Resolution of the genetic heterogeneity of closely related insect species depends on the selection of reliable genetic markers derived from representative specimens. We report the results of a survey of genetic variability in nine species of black flies in the subgenusPsilopelmia Enderlein. Three regions of the mitochondrial genome and an amplicon including the internal transcribed spacer 1 of the nuclear ribosomal RNA gene cluster (ITS1) were amplified using the polymerase chain reaction (PCR), and the amplicons were examined for intraspecific and interspecific polymorphisms. Six of the seven Psilopelmia species that yielded PCR products in the ITS1 PCR reaction were found to generate products that were indistinguishable on the basis of size. Similarly, little interspecific variation was noted in the 16S rRNA amplicon among nine species of Psilopelmia assayed by heteroduplex analysis. In contrast, the remaining regions of the mitochondrial genome exhibited both intra- and inter-specific variation when analyzed by heteroduplex analysis. Information collected from the five amplicons could be employed to develop a classification scheme capable of distinguishing the nine species of Psilopelmia examined.