Diagnosis of visceral leishmaniasis by polymerase chain reaction of DNA extracted from Giemsa's solution-stained slides

Abstract

Visceral leishmaniasis (VL) is caused by the protozoan parasite Leishmania donovani and is a potentially fatal disease in endemic areas of the world. Nepal is an endemic area in which VL causes major public health problems in the lowland areas of the southeast regions. The aim of the present study was to evaluate the sensitivity of polymerase chain reaction (PCR) amplification for the detection of Leishmania DNA from Giemsa's solution-stained bone marrow slides. Bone-marrow samples were aspirated from a total of 115 VL suspected patients and used to prepare smears on glass slides and for the initiation of in vitro culture.Bone-marrow slides were used for microscopic observation and for DNA extraction, and subsequent PCR amplification. PCR analysis showed that all the positive samples were of Leishmania parasites. The PCR assay also showed a higher sensitivity (69%) than microscopic examination (57%) and culture (21%). In addition, PCR was able to detect VL in 12% of samples which were negative by microscopy. PCR of DNA extracted from Giemsa's solution-stained bone-marrow slides is a suitable tool for confirming diagnosis in patients with VL and may also be useful in the diagnosis of difficult cases. Bone-marrow smears are easily stored, and can be easily sent to research centers where PCR is available. This makes PCR is good option for diagnosis in the field.

Rapid identification of Leishmania spp. in formalin‐fixed, paraffin‐embedded tissue samples by fluorescence in situ hybridization

“…Although successful amplification of Leishmania DNA from formalin-fixed, paraffin-embedded tissue specimens, even on Giemsa-stained slides, to confirm the histological detection of Leishmania spp. has been described (Volpini et al 2006;Pandey et al 2010), we failed to confirm positive FISH or Giemsa results with long-fragment PCRs and consecutive sequencing (El Tai et al 2000) even from thick cuts of the paraffin-embedded, formalin-fixed materials (data not shown). This failure may be attributed to cross-linking between the DNA strands because of fixation with formalin as described for longer DNA segments that are required for reliable sequencing results (Hagen et al 2002).…”

“…has been described (Volpini et al. 2006; Pandey et al. 2010), we failed to confirm positive FISH or Giemsa results with long‐fragment PCRs and consecutive sequencing (El Tai et al.…”

“…Therefore, preliminary microscopic analysis by Giemsa staining is confirmed by PCR in some laboratories. Positive PCR results may be achieved from Giemsa‐stained slides (Pandey et al. 2010) even up to several decades after the sample was taken (Volpini et al.…”

“…Therefore, preliminary microscopic analysis by Giemsa staining is confirmed by PCR in some laboratories. Positive PCR results may be achieved from Giemsa-stained slides (Pandey et al 2010) even up to several decades after the sample was taken (Volpini et al 2006). Nevertheless, the reliability of histological examination is reduced if pathologists have limited experience with leishmaniasis because of its rare occurrence, for example, in European or US laboratories.…”

Comparative Study of Microscopy and Polymerase Chain Reaction for the Diagnosis of Suspected Visceral Leishmaniasis Patients in Nepal

“…9 Correct diagnosis and identification of Leishmania species is essential for choosing the correct and effective treatment and for the production of effective vaccines. 10 With the increasing incidence of drug resistance in endemic areas, early case-finding and accurate diagnosis for effective treatment is an essential component of VL control and is necessary in order to interrupt the cycle.…”