Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples

Mohammadiha, Anita; Mohebali, Mehdi; Haghighi, Ali; Mahdian, Reza; Abadi, Ashraf H.; Zarei, Zabihollah; Yeganeh, Farshid; Kazemi, Bahram; Taghipour, Niloofar; Akhoundi, Behnaz

doi:10.1016/j.exppara.2012.10.017

Cited by 59 publications

(51 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…For example, Mohammadiha and others reported that 3.6% (1/28) and 10.7% (3/28) of dogs from Leishmania nonendemic areas were positive by real-time PCR and ITS-based PCR, respectively. 30 The specificity of rK39 ICT with sera of dogs from nonendemic regions ranged from 94% to 100% according to some previous studies and a few false-positive reactions were also reported in dogs infected with Ehrlichia canis, Trypanosoma cruzi, or Neospora caninum. [35][36][37][38] It is important to isolate viable Leishmania from naturally exposed animals to clarify their role in the maintenance and …”

Section: Discussionmentioning

confidence: 99%

“…We obtained the highest positive rate (20%) using kDNA-based real-time PCR, consistent with several previous studies showing that kDNAbased PCR is more sensitive than serological and ITS1-based PCR. 29,30 kDNA is considered the most sensitive target for leishmaniasis diagnosis, because it contains ∼10,000 minicircles per parasite. 31 Samples that were positive based on PCR and/ or real-time PCR, but negative based on rK39 ICT, might have a low infection burden and, therefore, lower levels of anti-Leishmania antibodies, consistent with previous studies 32,33 in which some seronegative dogs were PCR positive.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Molecular and Serological Evidence of Leishmania Infection in Stray Dogs from Visceral Leishmaniasis–Endemic Areas of Bangladesh

Akter

Alam

Yasin

et al. 2016

The American Society of Tropical Medicine and Hygiene

View full text Add to dashboard Cite

Abstract. Visceral leishmaniasis (VL), or kala-azar, is mainly caused by two closely related Leishmania species, Leishmania infantum and Leishmania donovani. Leishmania infantum is responsible for zoonotic VL, with dogs as the main reservoir host in the Mediterranean, the Middle East, Asia, and South America. In the Indian subcontinent, VL is caused by L. donovani and is considered anthroponotic, although the only known vector, the sand fly, is zoophilic in nature. The role of domestic and stray dogs in VL transmission is still unclear in this area. We screened 50 stray dogs from VL-endemic areas of Bangladesh for serological and molecular evidence of Leishmania infection. We detected anti-Leishmania antibodies in six (12%) dog serum samples using rK39 immunochromatographic tests. We observed Leishmania kinetoplast DNA in 10 (20%) buffy coat DNA samples by real-time polymerase chain reaction (PCR), five of which were positive based on internal transcribed spacer 1-PCR. A sequencing analysis of the amplified products confirmed that the parasitic DNA was derived from L. donovani. Our findings support the hypothesis that stray dogs are an animal reservoir for L. donovani in this endemic region. Further studies are required to determine the precise role of dogs in the epidemiology of VL in Bangladesh.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Molecular and Serological Evidence of Leishmania Infection in Stray Dogs from Visceral Leishmaniasis–Endemic Areas of Bangladesh

Akter

Alam

Yasin

et al. 2016

The American Society of Tropical Medicine and Hygiene

View full text Add to dashboard Cite

show abstract

“…simultaneously, many of the described qPCR methods detect only some of the pertinent Leishmania spp. or strains (24,27,28,30). Although assays that target minicircle kinetoplast DNA (kDNA) are the most sensitive PCR methods for detecting Leishmania parasites because of the abundance of minicircles in each kinetoplast (13,29,31,32), the high-level sequence polymorphism among minicircles is a substantial limitation for systematic species identification solely on the basis of this marker.…”

mentioning

confidence: 99%

“…The advantages of real-time quantitative PCR (qPCR) approaches in comparison with conventional PCR assays include the potential for increased sensitivity and specificity and for a decreased risk of contamination of the laboratory environment (14,(18)(19)(20)(21)(22)(23)(24)(25)(26). The efficiency of real-time platforms for Leishmania species identification has been discussed (24,(27)(28)(29)(30).…”

mentioning

confidence: 99%

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

et al. 2017

View full text Add to dashboard Cite

Leishmaniasis in humans is caused by Leishmania spp. in the subgenera Leishmania and Viannia. Species identification often has clinical relevance. Until recently, our laboratory relied on conventional PCR amplification of the internal transcribed spacer 2 (ITS2) region (ITS2-PCR) followed by sequencing analysis of the PCR product to differentiate Leishmania spp. Here we describe a novel real-time quantitative PCR (qPCR) approach based on the SYBR green technology (LSG-qPCR), which uses genus-specific primers that target the ITS1 region and amplify DNA from at least 10 Leishmania spp., followed by analysis of the melting temperature (T m ) of the amplicons on qPCR platforms (the Mx3000P qPCR system [Stratagene-Agilent] and the 7500 real-time PCR system [ABI Life Technologies]). We initially evaluated the assay by testing reference Leishmania isolates and comparing the results with those from the conventional ITS2-PCR approach. Then we compared the results from the real-time and conventional molecular approaches for clinical specimens from 1,051 patients submitted to the reference laboratory of the Centers for Disease Control and Prevention for Leishmania diagnostic testing. Specimens from 477 patients tested positive for Leishmania spp. with the LSG-qPCR assay, specimens from 465 of these 477 patients also tested positive with the conventional ITS2-PCR approach, and specimens from 10 of these 465 patients had positive results because of retesting prompted by LSG-qPCR positivity. On the basis of the T m values of the LSG-qPCR amplicons from reference and clinical specimens, we were able to differentiate four groups of Leishmania parasites: the Viannia subgenus in aggregate; the Leishmania

show abstract

“…Apesar de o PCR kDNA ser potencialmente mais sensível, devido à presença de um alto número de cópias por parasito (Rodgers et al, 1990;De Paiva-Cavalcanti et al, 2015), e deste alvo ter se mostrado mais sensível em outros estudos que compararam kDNA com ITS-1 (Bensoussan et al, 2006;Mohammadiha et al, 2013), não existem trabalhos comparando diretamente esses dois alvos em pacientes com HIV. Além disso, no presente estudo, não houve concordância entre os métodos moleculares e nenhum paciente teve as duas PCR positivas.…”

Section: Discussionunclassified

Frequência da infecção por Leishmania spp. em pacientes com HIV/aids vivendo em área urbana

Cunha¹

View full text Add to dashboard Cite

Comparison of real-time PCR and conventional PCR with two DNA targets for detection of Leishmania (Leishmania) infantum infection in human and dog blood samples

Cited by 59 publications

References 33 publications

Molecular and Serological Evidence of Leishmania Infection in Stray Dogs from Visceral Leishmaniasis–Endemic Areas of Bangladesh

Molecular and Serological Evidence of Leishmania Infection in Stray Dogs from Visceral Leishmaniasis–Endemic Areas of Bangladesh

Detection and Differentiation of Leishmania spp. in Clinical Specimens by Use of a SYBR Green-Based Real-Time PCR Assay

Frequência da infecção por Leishmania spp. em pacientes com HIV/aids vivendo em área urbana

Contact Info

Product

Resources

About