A highly sensitive modified nested PCR to enhance case detection in leishmaniasis

Abstract

Background Human leishmaniasis is one of the major parasitic diseases with worldwide distribution. Sri Lanka is a recently established focus of leishmaniasis caused by a variant Leishmania donovani . Early case detection and management is a main approach identified for L. donovani control in the regional leishmaniasis elimination drive. Usefulness of light microscopy and in-vitro culture are limited in chronic, atypical or treated lesions though timely and accurate detection of all light microscopy/in-vitro culture negative cases of all forms of leishmaniasis is necessary for treatment. Timely treatment is important to minimize risk for death in visceral disease and undesired sequelae of long standing infection and illness on both patients and community. We described a 100% sensitive, Leishmania spp. specific modified version of a nested PCR (Mo-STNPCR) that also minimizes carry over and cross contaminations while facilitate investigation of light microscopy and in-vitro culture negative clinically suggestive cases of leishmaniasis. Methods Leishmania DNA was amplified using previously published P221: 5′-GGTTCCTTTCCTGATTTACG-3′ and P332: 5′-GGCCGGTAAAGGCCGAATAG-3’outer primers followed by a nested reaction using P223: 5′-TCCCATCGCAACCTCGGTT-3′ and P333: 5′-AAGCGGGCGCGGTGCTG-3′ inner primers that by passes the requirement of tube handling between the two steps of the conventional nested PCR. Leishmania DNA was detected in a range of infected tissue material. Infected material from patients with cutaneous leishmaniasis ( n = 30), visceral leishmaniasis ( n = 10) and from a control group including patients with non-leishmanial skin diseases ( n = 10), other systemic diseases ( n = 10) and healthy individuals ( n = 10) were examined with Mo-STNPCR. Results were further compared with those of light microscopy and in-vitro culture. Results Mo-STNPCR method was 100% sensitive and 100% specific for diagnosis of leishmaniasis. Light microscopy and in-vitro culture were positive in 75.0% ( n = 30/40) and 72.5% ( n = 29/40) samples respectively where combined results of them gave 87.5% ( n = 35/40) sensitivity. Mo-STNPCR did not cross react with control samples. Furthermore, Mo-STNPCR reduces the risk of cross-contaminations and carry over contaminations since the full reaction is carried out without opening the tubes. Per patient cost was calculated as 22 USD while the same was 3 and 6 USD for light microscopy and in-vitro culture respectively. Conclusion Mo-STNPCR method is a useful tool in detecting leishmaniasis in minority of cases that go undetected by first line investigations. ...

Leishmania donovaniInduced Cutaneous Leishmaniasis: An Insight into Atypical Clinical Variants in Sri Lanka

“…Atypical lesions seem to demonstrate delayed response to the first line antileishmanial treatment as shown in some other studies as well [40, 45]. This provides further supportive evidence for the possible strain differences between the Leishmania sp.…”

“…Curative sector needs measures to encourage early referrals, better professional awareness, high degree of clinical suspicion, and adopting more sensitive diagnostic tools before arriving at a negative conclusion especially in less leishmanial prevalent areas [41]. Recently developed modified single tube nested PCR method may be used for disease confirmation in microscopically negative ACL [45]. Unknown sequalae associated with ACL can further complicate disease combatting efforts in an endemic setting.…”

First Serological Study Revealing High Humoral Response and Evidence for Antigenic Heterogeneity in Leishmania donovani Induced CL in Sri Lanka

“…Group I : Cases of CL ( n = 200) which were confirmed for CL by LM and/or IVC and/or PCR [ 22 – 24 ]. Parasite materials of a small group of CL from selected cases ( n = 15/200) were confirmed as L. donovani in previous genetic studies carried out in home laboratory [ 3 – 5 ], assuming all were caused by L. donovani.…”

“…Furthermore, the availability of such a tool is important for the detection of microscopy negative cases. Currently, IVC and polymerase chain reaction (PCR) are used with limited availability for the diagnosis of such cases in Sri Lanka [ 21 , 22 ].…”

A Comprehensive Review of Cutaneous Leishmaniasis in Sri Lanka and Identification of Existing Knowledge Gaps

“…specific modified version of a nested PCR (Mo-STNPCR). This method can be used to confirm the diagnosis of microscopy and in vitro culture-negative clinically suggestive cases [66]. However, the cost of the Mo-STNPCR is higher than that of light microscopy and in vitro culture [66].…”