Article

Isolation and molecular characterization of Leishmania infantum in urine from patients with visceral leishmaniasis in Brazil

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  • Fundação Oswaldo Cruz (Fiocruz) - Instituto Aggeu Magalhães (IAM)
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... In the case of the follow-up samples, both the kinetoplast DNA-PCR and the ssrRNA-gene-PCR showed 1 positive sample among the 11 samples assessed (9.1%), while the gpi-PCR remained negative in all 11 instances (0.0%). Regarding the recorded cycle threshold (Ct) values, median Ct value (minimum, maximum) showed a significant difference in Kruskal Wallis testing (p = 0.0038) between the assays with 23 (10, 39) for the kinetoplast DNA-PCR, 27 (17,32) for the ssrRNA-gene PCR, and 38 (36,39) for the gpi-PCR, respectively. Thereby, Dunn's multiple comparisons tests indicated statistically significant differences between the gpi-PCR and both the kinetoplast DNA PCR (p < 0.01) and the ssrRNA-gene-PCR (p < 0.05), while no significance was detectable for differences of the Ct-values when comparing the latter two PCRs. ...
... Of note, diagnostic Leishmania-PCR from urine samples [32][33][34][35] as well as various associated nucleic acid extraction strategies [36] have been introduced and recently also summarized in reviews [37,38]. Published interpretations regarding the diagnostic reliability of these approaches vary [37,38]. ...
Article
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To perform PCR from serum for the diagnosis of visceral leishmaniasis is convenient and much less invasive than the examination of deeper compartments such as bone marrow. We compared three Leishmania-specific real-time PCRs with three different molecular targets (kinetoplast DNA, the small subunit-ribosomal RNA-(ssrRNA-)gene, the glucose-6-phosphate isomerase-(gpi-)gene) regarding their sensitivity and specificity in human serum. Residual sera from previous diagnostic assessments at the German National Reference Center for Tropical Pathogens Bernhard Nocht Institute for Tropical Medicine Hamburg and the Swiss Tropical and Public Health Institute were used. The sensitivities of kinetoplast DNA-PCR, ssrRNA-gene PCR, and gpi-PCR were 93.3%, 73.3%, and 33.3%, respectively, with 15 initial serum samples from visceral leishmaniasis patients, as well as 9.1%, 9.1%, and 0.0%, respectively, with 11 follow-up serum samples taken at various time points following anti-leishmanial therapy. Specificity was 100.0% in all assays as recorded with 1.137 serum samples from deployed soldiers and migrants without clinical suspicion of visceral leishmaniasis. Kinetoplast-DNA PCR from serum was confirmed as a sensitive and specific approach for the diagnosis of visceral leishmaniasis. The results also indicate the suitability of serum PCR for diagnostic follow-up after therapy, in particular regarding therapeutic failure in case of persisting positive PCR results.
... The first evidence of the presence of Leishmania in the urine of patients infected by L. donovani came in the 1930s through the detection of Leishman-Donovan bodies in the urine of infected patients [119]. The presence of viable Leishmania parasites in the urine of infected individuals is documented [120][121][122]. The crossing of the glomerular barrier by Leishmania is thought to be a consequence of VL renal lesions and renal failure [123,124]. ...
... In infected individuals, urine represents a fluid from which parasite DNA is easily extracted for detection and species identification [128], which has been probed in the urine of patients [113,121,129,130] and in animal reservoirs [131,132]. These searches were performed in VL caused by L. infantum [113,120,121,133]; in CL and VL-HIV+ patients infected by L. martiniquensis [134]; in CL due to L. major or L. tropica [113]; in South American cutaneous and mucocutaneous leishmaniasis caused by L. braziliensis, L. guyanensis, or L. peruviana [130]; and in canine visceral leishmaniasis [131,132,135]. The presence, in urine, of precipitin activities directed against several microorganisms has been known since 1948 [136]. ...
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Unicellular eukaryotes of the Trypanosomatidae family include human and animal pathogens that belong to the Trypanosoma and Leishmania genera. Diagnosis of the diseases they cause requires the sampling of body fluids (e.g., blood, lymph, peritoneal fluid, cerebrospinal fluid) or organ biopsies (e.g., bone marrow, spleen), which are mostly obtained through invasive methods. Body fluids or appendages can be alternatives to these invasive biopsies but appropriateness remains poorly studied. To further address this question, we perform a systematic review on clues evidencing the presence of parasites, genetic material, antibodies, and antigens in body secretions, appendages, or the organs or proximal tissues that produce these materials. Paper selection was based on searches in PubMed, Web of Science, WorldWideScience, SciELO, Embase, and Google. The information of each selected article (n = 333) was classified into different sections and data were extracted from 77 papers. The presence of Trypanosomatidae parasites has been tracked in most of organs or proximal tissues that produce body secretions or appendages, in naturally or experimentally infected hosts. The meta-analysis highlights the paucity of studies on human African trypanosomiasis and an absence on animal trypanosomiasis. Among the collected data high heterogeneity in terms of the I 2 statistic (100%) is recorded. A high positivity is recorded for antibody and genetic material detection in urine of patients and dogs suffering leishmaniasis, and of antigens for leishmaniasis and Chagas disease. Data on conjunctival swabs can be analyzed with molecular methods solely for dogs suffering canine visceral leishmaniasis. Saliva and hair/bristles showed a pretty good positivity that support their potential to be used for leishmaniasis diagnosis. In conclusion, our study pinpoints significant gaps that need to be filled in order to properly address the interest of body secretion and hair or bristles for the diagnosis of infections caused by Leishmania and by other Trypanosomatidae parasites.
... The first evidence of the presence of Leishmania in the urine of patients infected by L. donovani came in the 1930s through the detection of Leishman-Donovan bodies in the urine of infected patients [109]. The presence of viable Leishmania parasites in the urine of infected individuals is documented [110][111][112]. The crossing of the glomerular barrier by Leishmania is thought to be a consequence of VL renal lesions and renal failure [113,114]. ...
... In infected individuals, urine represents a fluid from which parasite DNA is easily extracted for detection and species identification [118], and has been probed in the urine of patients [103,111,119,120] and in animal reservoirs [121,122]. These searches were performed in VL caused by L. infantum [103,110,111,123]; in CL and VL-HIV+ patients infected by L. martiniquensis [124]; in CL due to L. major or L. tropica [103]; in South American cutaneous and mucocutaneous leishmaniasis caused by L. braziliensis, L. guyanensis or L. peruviana [120]; and in canine visceral leishmaniasis [121,122,125]. The presence, in urine, of precipitin activities directed against several microorganisms has been known since 1948 [126]. ...
Preprint
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Preprint of the paper published in International Journal for Molecular Sciences. Doi: 10.3390/ijms21051684 Unicellular eukaryotes of the Trypanosomatidae family include human and animal pathogens that belong to the Trypanosoma and Leishmania genera. Diagnosis of the diseases they caused requires the sampling of body fluids (blood, lymph, peritoneal fluid, cerebrospinal fluid, etc.) or organ biopsies (bone marrow, spleen, etc.), which are mostly obtained through invasive methods. Body fluids or appendages can be alternatives to these invasive biopsies but appropriateness remains poorly studied. To further address this question, we perform a systematic review on clues evidencing the presence of parasites, genetic material, antibodies, and antigens in body secretions, appendages, or the organs or proximal tissues that produce these materials. Paper selection was based on searches in PubMed, Web of Science, WorldWideScience, SciELO, Embase, Google. The information of each selected article (n=333) was classified into different sections and data were extracted from 77 papers. The presence of Trypanosomatidae parasites has been tracked in most of organs or proximal tissues that produce body secretions or appendages, in naturally or experimentally infected hosts. The meta-analysis highlights the paucity of studies on Human African Trypanosomiasis and a the absence on animal Trypanosomiasis. Among the collected data high heterogeneity in terms of the I 2 statistic (100%) is recorded. A high positivity is recorded for antibody and genetic material detection in urine of patients and dogs suffering leishmaniasis, and of antigen for leishmaniasis and Chagas disease. Data on conjunctival swab can be analyzed with molecular methods solely for dogs suffering canine visceral leishmaniasis. Saliva and hair/bristle showed a pretty good positivity that support their potential to be used for leishmaniasis diagnosis. In conclusion, our study pinpoints significant gaps that need to be Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 23 February 2020
... It is believed that crossing the glomerular barrier is a consequence of renal lesions and/or of renal insufficiency. Additionally, tubulointerstitial involvement and glomerulonephritis are two of the major causative agents of proteinuria, common in most patients with clinical episodes of visceral leishmaniasis [9,14,30,31]. ...
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The clinical-immunological spectrum of human Leishmania (L.) infantum chagasi-infections in the Brazilian Amazon has been defined using DTH/IFAT-IgG immune assays and the clinical statuses of infected individuals, revealing five profiles: three asymptomatic [Asymptomatic Infection (AI), Subclinical Resistant Infection (SRI), and Indeterminate Initial Infection (III)], and two symptomatic profiles [Subclinical Oligosymptomatic Infection (SOI) and Symptomatic Infection (SI = American visceral leishmaniasis/AVL)]. We evaluated the diagnostic potential of urine qPCR over the entire spectrum of infection. Resine Instagene Matrix® was used for DNA extraction from urinary sediment, with amplification carried out using SYBR® Green Taq with the RV1 and RV2 primers. We examined urine samples from 151 individuals from an endemic area of AVL in Par´a State in the Brazilian Amazon, including: 91 (60.3%) with diagnoses of previous infections [13 (14.3%) sharing the AI profile, 13 (14.3%) with the SRI profile, 43 (47.2%) with III, 12 (13.2%) with SI (treated AVL), and 10 (11%) with SI (untreated AVL)]; sixty (39.7%) were DTH () /IFAT-IgG () (the uninfected group). The urine qPCR was positive in 61.5% of both the AI and SRI profiles, 65% of the III profile, 50% of treated AVL, 100% of untreated AVL, and 6.7% of the uninfected group. Those results confirmed the urine qPCR diagnosis in 100% of untreated AVL cases as well as in more than 60% of the cases with asymptomatic AI, SRI, and III profiles – indicating it as a promising tool for monitoring the evolution of human L. (L.) infantum chagasi-infections in endemic areas.
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Background Liquid biopsy refers to the sampling and molecular analysis of body fluids such as blood, saliva, and urine in contrast to conventional tissue biopsies. Liquid biopsy approach can offer powerful non-invasive biomarkers (circulating markers) for diagnosis and monitoring treatment response of a variety of diseases, including parasitic infections.Methods In this review, we concentrate on cell-free DNA (cfDNA), microRNA (miRNA), and exosomes in the published literature.ResultsConsidering the high prevalence and severity of parasitic infections worldwide, circulating biomarkers can provide a new insight into the diagnosis and prognosis of parasites in the near future. Moreover, identifying and characterizing parasite- or host-derived circulating markers are important for a better understanding of the pathogenesis of parasite infection and host–parasite relationship at the molecular level. Profiling of biomarkers for parasitic diseases is a promising potential field, though further studies and optimization strategies are required, both in vitro and in vivo.Conclusion In this review, we discuss three approaches in the liquid biopsy including circulating cfDNA, miRNAs, and exosomes for diagnosis and evaluation of parasites and summarize circulating biomarkers in non-invasive samples during parasitic infections.
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Visceral leishmaniasis (VL) is an acute and deadly form of leishmaniasis, caused by Leishmania infantum parasite. Due to the toxicity and side effects of conventional treatment options, such as glucantime and other pentavalent drugs, finding novel drugs with fewer adverse effects is required. Artemether (ART), is one of the derivatives of artemisinin, which was shown to be effective in treating malaria and more recently, leishmaniasis. In this fundamental-applied research, we compared the effect of ART and nanostructure loaded with artemether (NLC-ART) on Leishmania infantum promastigotes and amastigotes, at different concentrations (2.5–5-10–25-50–100 μg/ml) using the MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay method after 24 and 48 h of treatment. Inhibitory concentration (IC50) values (μg/ml) of promastigote and amastigote of L. infantum to ART/ NLC-ART, after 48 h of treatment, were found to be 37.12 / 32.1 and 16.43 / 15.42, respectively. Moreover, we found that (NLC-ART), had the lowest cytotoxicity against the J774 macrophage cell line. Conclusion: The NLC-ART can be a good candidate for the treatment of visceral leishmaniasis.
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Leishmaniasis is a neglected tropical disease affecting humans and domesticated animals with high mortality in endemic countries. The pleiotropy of symptoms and the complicated gold-standard methods make the need for non-invasive, highly sensitive diagnostic tests imperative. Individual studies on molecular-based Leishmania diagnosis in urine show high discrepancy; thus, a data-evidenced comparison of various techniques is necessary. We performed a systematic review and meta-analysis using the bivariate method of diagnostic methods to pool sensitivities and specificities. We investigated the impact of DNA-extraction method, PCR type, amplified locus, host species, leishmaniasis form, and geographical region. The pooled sensitivity was 69.2%. Tests performed with the kit-based DNA extraction method and qPCR outweighed in sensitivity the phenol-chloroform-based and PCR methods, while their combination showed a sensitivity of 79.3%. Amplified locus, human or canine as host and cutaneous or visceral leishmaniasis revealed similar sensitivities. Tests in European and Middle Eastern countries performed better than tests in other regions (sensitivity 81.7% vs. 43.7%). A combination of kit-based DNA extraction and qPCR could be a safer choice for molecular diagnosis for Leishmania infection in urine samples in European–Middle Eastern countries. For the rest of the world, more studies are needed to better characterize the endemic parasite species.
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Visceral leishmaniasis is a serious and debilitating infection with high fatality rate in tropical and subtropical countries. As clinical symptoms of visceral leishmaniasis are not so specific, confirmatory diagnostic methods with high sensitivity and specificity are needed. Noninvasive methods have been developed using urine as a clinical sample for visceral leishmaniasis diagnosis. In fact, there is a clear correlation between kidney impairment and Leishmania DNA in urine. However, it has been proved that Leishmania nucleic acid may also be isolated from patients without any sign of renal involvement. Even though urine has become a promissing biological sample, it is still not widely used due to several issues, such as (i) incomprehension of the whole renal pathophysiology process in visceral leishmaniasis, (ii) presence of many amplification inhibitors in urine, and (iii) lack of an efficient urinary DNA extraction method. In this article, we performed a literature review to bring a new perspective for Leishmania DNA isolation in urine.
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Introduction: Polymerase chain reaction (PCR) may offer an alternative diagnostic option when clinical signs and symptoms suggest visceral leishmaniasis (VL) but microscopic scanning and serological tests provide negative results. PCR using urine is sensitive enough to diagnose human visceral leishmaniasis (VL). However, DNA quality is a crucial factor for successful amplification. Methods: A comparative performance evaluation of DNA extraction methods from the urine of patients with VL using two commercially available extraction kits and two phenol-chloroform protocols was conducted to determine which method produces the highest quality DNA suitable for PCR amplification, as well as the most sensitive, fast and inexpensive method. All commercially available kits were able to shorten the duration of DNA extraction. Results: With regard to detection limits, both phenol: chloroform extraction and the QIAamp DNA Mini Kit provided good results (0.1 pg of DNA) for the extraction of DNA from a parasite smaller than Leishmania (Leishmania) infantum (< 100fg of DNA). However, among 11 urine samples from subjects with VL, better performance was achieved with the phenol:chloroform method (8/11) relative to the QIAamp DNA Mini Kit (4/11), with a greater number of positive samples detected at a lower cost using PCR. Conclusion: Our results demonstrate that phenol:chloroform with an ethanol precipitation prior to extraction is the most efficient method in terms of yield and cost, using urine as a non-invasive source of DNA and providing an alternative diagnostic method at a low cost.
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A urine-polymerase chain reaction (PCR) assay was validated for diagnosis of human visceral leishmaniasis (VL), taking advantage of the accessibility of urine samples. Leishmania infantum DNA presence was examined in 17 urine samples from 17 patients with VL during a clinical episode and in 55 urine samples from 17 patients with VL monitored after treatment at different intervals. Fifty-nine urine samples from 59 controls with no history of VL were also studied. The urine-PCR test was positive in 15/17 samples obtained during the episode (sensitivity, 88%). None of the controls tested were urine-PCR positive (specificity, 100%). During the monitoring period, 25% of the samples gave a positive urine-PCR. Results were compared with other diagnostic methods, such as urine antigen detection and peripheral blood-PCR and culture, with good concordance during the clinical episode and differences in the follow-up period. This study suggests that urine-PCR is sensitive for diagnosis and may be useful to monitor treatment efficacy.
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Visceral leishmaniasis (VL) is a systemic protozoan disease that is transmitted by phlebotomine sandflies. Poor and neglected populations in East Africa and the Indian sub-continent are particularly affected. Early and accurate diagnosis and treatment remain key components of VL control. In addition to improved diagnostic tests, accurate and simple tests are needed to identify treatment failures. Miltefosine, paromomycin and liposomal amphotericin B are gradually replacing pentavalent antimonials and conventional amphotericin B as the preferred treatments in some regions, but in other areas these drugs are still being evaluated in both mono- and combination therapies. New diagnostic tools and new treatment strategies will only have an impact if they are made widely available to patients.
Article
The leishmaniases are parasitic diseases caused by different species of Leishmania, protozoa transmitted by sandflies, haematophagous biting insects. The reservoir hosts are man (anthroponotic cycle) and domestic or wild animals (zoonotic cycle). In man the disease takes four main clinical forms: visceral, cutaneous, mucocutaneous and diffuse cutaneous. Morbidity and mortality due to leishmaniasis are on the increase. Leishmaniasis, which is now found on four continents, is endemic in 82 countries (21 in the New World and 61 in the Old). The large number of endemic countries shows the global scale of the problem, though it is particularly severe in certain countries (90% of cases of visceral leishmaniasis come from 4 countries). Annual incidence is estimated at some 600,000 new clinical cases, officially reported, with a global prevalence of 12 million cases and a population at risk of approximately 350 million. It is very difficult to provide realistic estimates given the frequency of subclinical forms of visceral leishmaniasis, the large number of undiagnosed or unreported cases, the frequent absence of active screening and the fact that the leishmaniases are notifiable diseases only in a few countries (30 out of 82); nevertheless, it seems clear that official reporting of cases considerably underestimates the problem. Over the last two decades, it has become clear that leishmaniasis is a growing public health problem in terms of geographical extent and incidence with the occasional severe epidemic, such as that which occurred in Sudan.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
In a comparative study 88 patients were diagnosed as suffering from kala-azar (visceral leishmaniasis) using 3 parasitological methods simultaneously. Splenomegaly was absent in 4 cases. In 84 patients with splenomegaly, splenic aspiration appeared to be the most sensitive method (96.4%), followed by bone marrow aspiration (70.2%) and lymph node aspiration (58.3%). There was no relation between titres in the direct agglutination test and parasite load as determined by the number of parasitological methods which were positive or parasite density in splenic aspirates. Splenic aspiration and bone marrow aspiration were compared as an assessment of cure in kala-azar. In 6 (13%) of 46 patients tested, parasites were found, all by splenic aspiration. Bone marrow showed parasites in one of these. The literature with regard to parasitological investigations before and after treatment is reviewed.
Article
Fifty one patients in a series of 1000 parasitologically confirmed patients of kala-azar showing proteinuria were studied from the point of view of renal involvement. Forty two out of these patients showed blood urea above 40 mg/dl and 12 had decreased glomerular filtration rate as well. Serum creatinine level was normal in all patients including those with blood urea of more than 100 mg/dl. It is therefore suggested that although there may be glomerular involvement in some patients of kala-azar, the changes are reversible.
Article
We describe the case of a 33-year-old male patient with an acute visceral leishmaniasis (Leishmania donovani) associated with an acute renal failure. The clinical manifestations were dominated by fever, oliguric renal failure and hepatic alterations. Serum C3 and C4 fractions of complement were decreased, and a renal biopsy demonstrated an interstitial nephritis with no glomerular involvement. The clinical course was favorable with recuperation of renal function without sequels.
Article
Long-term storage of DNA is required for a number of genetic studies; prior to extraction, blood samples may be subject to elevated temperatures for variable intervals. We have studied the effect of temperatures ranging from -70 degrees C to +65 degrees C on human blood and on DNA extracted from it. DNA in solution stored at ambient temperatures up to 37 degrees C for 6 months was digestible by three different restriction endonucleases, whereas storage at 45 degrees C is deleterious after 6-7 weeks. DNA can be extracted from blood samples stored at -70 degrees C for at least 2 months or at 23 degrees C for a week or more, but blood stored at these temperatures may yield less high-molecular-weight DNA. Cell pellets from which plasma has been removed also can serve as a source of DNA. Isolated DNA stored dry for years (up to 30) is difficult to dissolve and may appear degraded, but a sample stored dry for 13 years and then in solution at -20 degrees C for 7 years appeared to be intact.
Article
PCR is being used increasingly, not just to establish the presence of specific nucleotide sequences, but also to ascertain their quantity. The technique of competitive PCR involves the coamplification of a quantified competitive sequence with the target mRNA. Relative abundance after amplification of the target and competitor is then analyzed using a predefined and exploitable difference between the target sequence and that of the competitor. ~1'2) Differences that have been reported previously involve restriction site variations induced by site-directed mutagenesis and incorporation of an intron within the competitor. (3) In this paper we report a simplified competitive PCR system based on interspecies sequences differences and similarities. The technique has been applied to study quantitative variations in [3-actin mRNA within serum-deprived human hepatocellular carcinoma cells (Hep 3B) in response to serum addition. By comparing the [3-actin gene nucleotide sequence of the rat with that of the human (4's) and obtaining a range of consensus sequences, an appropriate set of nucleotide sequences that best fit the criteria for primers and were identical in both species was selected (Fig. 1). By exploiting existing nucleotide differences
Article
In the early 1930s, investigators of visceral leishmaniasis stated that Leishman-Donovan bodies are found in body fluids of kala-azar patients, for example, in urine, feces, semen, and nasal and pharyngeal secretions. Based on this finding, we investigated the diagnostic potential of nasal secretions, tonsillopharyngeal mucosal swabs, and urine centrifugates inoculated into Schneider's Drosophila Medium (containing antibiotics and antifungal agents) as well as with Giemsa-stained smears. Consequently, 64 randomly selected patients with visceral leishmaniasis from Kenya (59 who were splenic culture or Giemsa stain positive and five who were culture negative but Giemsa stain positive) were tested by three noninvasive methods. These tests were all performed before the patients were treated with Pentostam. Cultures of nasal and tonsillopharyngeal swabs and urine centrifugates produced 28 positive samples representing 24 patients (37.5%). Moreover, a set of 25 Giemsa-stained slide smears made from the nasal and tonsillopharyngeal mucosa of 25 patients with visceral leishmaniasis who had not tested positive in cultures produced nine positives. Therefore, the overall total of patients who tested positive by all of the above methods was 33 or 51.6%. The cryopreserved Leishmania isolates were characterized by cellulose acetate electrophoresis using 20 enzyme systems. The isoenzyme profiles produced by the parasites were represented in five different L. donovani s.l. zymodemes. Representatives of these isolates were also characterized by DNA Southern blotting analysis, which corroborated the isoenzyme results.
Article
Report of a case of renal and urinary tract leishmaniasis in a 61-year old male patient. The ureteral lesion responded favourably to treatment, unlike the renal one which required conservative surgery. Review of the literature stressing the fact that no other case has been found of kidney and urinary tract involvement. It is concluded that in the future, at least in those areas with a high prevalence of leishmaniasis, this condition should be taken into account as differential diagnosis when facing a toxic picture with renal involvement of the calyceal ecstasies type.
Article
Age-related changes in nicotinic acetylcholine receptor (nAChR) subunit alpha4 and beta2 messenger RNA (mRNA) expression in the postmortem human frontal cortex and hippocampus was investigated using the reverse transcription-polymerase chain reaction (RT-PCR). In the frontal cortex, both alpha4 and beta2 subunit mRNA expression decreased with age. In the hippocampus, alpha4 subunit mRNA expression was unaltered, while beta2 subunit mRNA expression significantly decreased with age. These findings suggest that nAChR transcription decreases during aging with differing vulnerability between subunits and brain regions, which could in part contribute to the reduction in cognitive functions seen in the elderly.
Article
The effect of different EDTA concentrations on the DNA content of urine samples was examined and compared to untreated urine at various storage temperatures and times. The results indicate that adding EDTA increases the DNA stability for long time storage especially at low temperatures.
Article
The aim of this study was to evaluate the sensitivity and acceptability of self-taken vulval-introital (VI) samples, first-catch urine (FCU) samples and clinician-obtained cervical samples for the presence of genital Chlamydia trachomatis infections in women using the ligase chain reaction (LCR) assay. One hundred and four patients were enrolled, of whom 54 patients had chlamydial DNA in at least one of the samples tested. The sensitivity of the cervical sample was 96.3%, vulval-introital sample in LCR buffer 92.6%, vulval-introital swab collected dry 88.9%, FCU stored at +2–8°C 81.5%, FCU stored at room temperature 77.8% and FCU stored with 2% w/v boric acid at room temperature 87.0%. Self-taken vulval-introital LCR samples were shown to be an acceptable alternative to a clinician-obtained LCR sample. The addition of boric acid may overcome the need for a continuous cold chain for FCU samples.
Article
Four polymerase chain reaction (PCR)-based approaches were used to analyze diversity within 23 Sudanese isolates of Leishmania donovani. Methods compared were fingerprinting with single nonspecific primers, restriction analysis of the amplified ribosomal internal transcribed spacer (ITS) locus, single-stranded conformation polymorphism (SSCP), and sequencing of the ITS region. When PCR fingerprinting and restriction analysis of ITS were applied, highly similar fragment patterns were observed for all strains of L. donovani studied. The ITS1 locus gave five different SSCP profiles among the 23 Sudanese isolates, whereas the ITS2 locus was highly conserved with the exception of 1 isolate. Strains of L. donovani derived from other geographical areas were found to have different ITS2 patterns. SSCP analysis correlated well with results of DNA sequencing and confirmed that SSCP was able to detect genetic diversity at the level of a single nucleotide. SSCP had advantages over the other methods employed for investigation of sequence variation within the species L. donovani. There was no correlation between the form of clinical manifestation of the disease and the PCR fingerprinting, ITS-RFLP, or ITS-SSCP characteristics.
Article
Visceral leishmaniasis is common in less developed countries, with an estimated 500000 new cases each year. Because of the diversity of epidemiological situations, no single diagnosis, treatment, or control will be suitable for all. Control measures through case finding, treatment, and vector control are seldom used, even where they could be useful. There is a place for a vaccine, and new imaginative approaches are needed. HIV co-infection is changing the epidemiology and presents problems for diagnosis and case management. Field diagnosis is difficult; simpler, less invasive tests are needed. Current treatments require long courses and parenteral administration, and most are expensive. Resistance is making the mainstay of treatment, agents based on pentavalent antimony, useless in northeastern India, where disease incidence is highest. Second-line drugs (pentamidine and amphotericin B) are limited by toxicity and availability, and newer formulations of amphotericin B are not affordable. The first effective oral drug, miltefosine, has been licensed in India, but the development of other drugs in clinical phases (paromomycin and sitamaquine) is slow. No novel compound is in the pipeline. Drug combinations must be developed to prevent drug resistance. Despite these urgent needs, research and development has been neglected, because a disease that mainly affects the poor ranks as a low priority in the private sector, and the public sector currently struggles to undertake the development of drugs and diagnostics in the absence of adequate funds and infrastructure. This article reviews the current situation and perspectives for diagnosis, treatment, and control of visceral leishmaniasis, and lists some priorities for research and development.
Article
The aim of this study was to detect Leishmania infantum DNA by real-time PCR in urine from different groups of dogs with clinical leishmaniosis. Urine from 10 clinically healthy dogs and 43 dogs with clinical leishmaniosis diagnosed by positive serology and/or bone marrow PCR were studied. The group of 43 dogs with clinical leishmaniosis was divided into three subgroups: 13 dogs with renal insufficiency and proteinuria (urine protein-creatinine ratio greater than one), 13 dogs with only proteinuria, and 17 dogs with neither renal insufficiency nor proteinuria. The detection of Leishmania DNA was performed by light cycler real-time PCR using hybridization probes in each urine sample. Leishmania positive PCR was found in 47% (20/43) of the urine from leishmaniotic dogs, while all urine from clinically healthy dogs were negative. The percentages of positive Leishmania PCR were 85% (11/13) in dogs with renal insufficiency and proteinuria, 23% (3/13) in dogs with proteinuria and 35% (6/17) in dogs with neither renal insufficiency nor proteinuria. Dogs with renal insufficiency and proteinuria presented a statistical significant greater percentage of positive Leishmania PCR in urine when compared with the other subgroups (P<0.02). This study demonstrates the presence of Leishmania DNA in urine of dogs with leishmaniosis. Those dogs with severe renal damage present a greater number of Leishmania parasites in urine.
Article
In the Mediterranean basin and Middle East, including Iran, visceral leishmaniasis (VL), also known as kala-azar, is caused by Leishmania donovani infantum. For the first time, the use of urine samples for the diagnosis of VL in immunocompetent patients has been used in this study. Based on its high sensitivity and specificity, as well as simplicity, this approach can serve as a valuable tool in the diagnosis of VL. We studied 60 urine samples from 60 individuals, 30 of which were patients with VL confirmed by parasitology, serology, or molecular methods, 5 were from healthy individuals, and 25 were from patients with cutaneous leishmaniasis, malaria, brucellosis, and hydatid cyst. Out of 30 samples from confirmed VL immunocompetent patients, 29 were positive (sensitivity, 96.8%) by polymerase chain reaction (RV1 and RV2 primers), and all the remaining 30 samples either from healthy individuals or patients with other diseases were negative (specificity, 100%). High sensitivity, specificity, and simplicity of the test can serve as a valuable tool in the diagnosis of VL.
Diagnosis of visceral leishmaniasis by the polymerase chain reaction using blood, bone marrow and lymph node samples from patients from the Sudan
  • Andresen
Andresen, K., Gasim, S., Elhassan, A.M., Khalil, E.A.G., Barker, D.C., Theander, T.G., Kharazmi, A., 1997. Diagnosis of visceral leishmaniasis by the polymerase chain reaction using blood, bone marrow and lymph node samples from patients from the Sudan. Trop. Med. Int. Heal. 2, 440-444. http://dx.doi.org/10.1111/j.1365-3156. 1997.tb00166.x.
Leishmaniose viscérale autochtone avec insuffisance rénale aiguë par glomérulonéphrite infectieuse 25
  • V Chaigne
  • Y Knefati
  • R Lafarge
  • J Bronner
  • B Mc Gregor
  • D Fouque
  • J.-C Sabatier
Chaigne, V., Knefati, Y., Lafarge, R., Bronner, J., Mc Gregor, B., Fouque, D., Sabatier, J.-C., 2004. Leishmaniose viscérale autochtone avec insuffisance rénale aiguë par glomérulonéphrite infectieuse 25. pp. 179-183.