Diagnostic PCR with <i>Leishmania donovani</i> specificity using sequences from the variable region of kinetoplast minicircle DNA

Singh, Neeloo; Curran, Martin D.; Rastogil, Anil K.; Middleton, Derek; Sundar, Shyam

doi:10.1046/j.1365-3156.1999.00416.x

Cited by 23 publications

(24 citation statements)

References 11 publications

(9 reference statements)

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“…Use of peripheral blood is advantageous because the collection procedure is less invasive and safer than the splenic or bone marrow biopsy specimen collection. In earlier studies for diagnosis of VL due to L. donovani, the sensitivity of PCR for blood samples has been found to be in the range of 45 to 94% based on smaller sample sizes ranging from 17 to 42 (1,4,15,21,22,32,35). For detection of VL due to L. donovani infantum, which may have a different pathogenesis, sensitivities between 64 to 97% have been reported with blood samples (16,18,21).…”

Section: Discussionmentioning

confidence: 99%

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Development of a Species-Specific PCR Assay for Detection of Leishmania donovani in Clinical Samples from Patients with Kala-Azar and Post-Kala-Azar Dermal Leishmaniasis

et al. 2001

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We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.

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Section: Discussionmentioning

confidence: 99%

“…PCR products were analyzed in 1% agarose gel, and Southern blot analysis was done as described (14). Southern blots were hybridized with 32 P-labeled cloned L. donovani kDNA fragments using the conditions described (14).…”

Section: Methodsmentioning

confidence: 99%

Development of a Species-Specific PCR Assay for Detection of Leishmania donovani in Clinical Samples from Patients with Kala-Azar and Post-Kala-Azar Dermal Leishmaniasis

et al. 2001

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“…There are different target sequences used, which include ribosomal RNA genes, kinetoplast DNA (kDNA), miniexon-derived RNA (medRNA), and the β-tubulina gene region [103].…”

Section: Molecular Methods: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

“…The ideal sample is peripheral blood due to its non-invasive character. Using peripheral blood, the sensitivity ranges described vary from 70% to 100% [93,102,103].…”

Section: Molecular Methods: Polymerase Chain Reaction (Pcr)mentioning

confidence: 99%

Diagnosis of leishmaniasis

Elmahallawy

Martínez

Rodríguez‐Grangér

et al. 2014

J Infect Dev Ctries

151

119

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Leishmaniasis is a clinically heterogeneous syndrome caused by intracellular protozoan parasites of the genus Leishmania. The clinical spectrum of leishmaniasis encompasses subclinical ( not apparent), localized (skin lesion), and disseminated (cutaneous, mucocutaneous, and visceral) infection. This spectrum of manifestations depends on the immune status of the host, on the parasite, and on immunoinflammatory responses. Visceral leishmaniasis causes high morbidity and mortality in the developing world. Reliable laboratory methods become mandatory for accurate diagnosis, especially in immunocompromised patients such as those infected with HIV. In this article, we review the current state of the diagnostic tools for leishmaniasis, especially the serological test.

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“…Several studies have reported that qPCR has virtually no false positive results . Cases where qPCR had specificity lower than 1.0 were attributed to contamination of the respective biological specimens or reagents, or/and the absence of good gold standard for reference . This is therefore one of the diagnostic tests for which it is appropriate to use the method we have presented.…”

Section: Discussionmentioning

confidence: 86%

Estimation of infection prevalence and sensitivity in a stratified two-stage sampling design employing highly specific diagnostic tests when there is no gold standard

et al. 2015

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In this work, we describe a two-stage sampling design to estimate the infection prevalence in a population. In the first stage, an imperfect diagnostic test was performed on a random sample of the population. In the second stage, a different imperfect test was performed in a stratified random sample of the first sample. To estimate infection prevalence, we assumed conditional independence between the diagnostic tests and develop method of moments estimators based on expectations of the proportions of people with positive and negative results on both tests that are functions of the tests' sensitivity, specificity, and the infection prevalence. A closed-form solution of the estimating equations was obtained assuming a specificity of 100% for both tests. We applied our method to estimate the infection prevalence of visceral leishmaniasis according to two quantitative polymerase chain reaction tests performed on blood samples taken from 4756 patients in northern Ethiopia. The sensitivities of the tests were also estimated, as well as the standard errors of all estimates, using a parametric bootstrap. We also examined the impact of departures from our assumptions of 100% specificity and conditional independence on the estimated prevalence.

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Diagnostic PCR with Leishmania donovani specificity using sequences from the variable region of kinetoplast minicircle DNA

Cited by 23 publications

References 11 publications

Development of a Species-Specific PCR Assay for Detection of Leishmania donovani in Clinical Samples from Patients with Kala-Azar and Post-Kala-Azar Dermal Leishmaniasis

Development of a Species-Specific PCR Assay for Detection of Leishmania donovani in Clinical Samples from Patients with Kala-Azar and Post-Kala-Azar Dermal Leishmaniasis

Diagnosis of leishmaniasis

Estimation of infection prevalence and sensitivity in a stratified two-stage sampling design employing highly specific diagnostic tests when there is no gold standard

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