Quantitative PCR in Epidemiology for Early Detection of Visceral Leishmaniasis Cases in India

<div><p>Introduction</p><p>Studies employing serological, DTH or conventional PCR techniques suggest a vast proportion of Leishmania infected individuals living in regions endemic for Visceral Leishmaniasis (VL) remain asymptomatic. This study was designed to assess whether quantitative PCR (qPCR) can be used for detection of asymptomatic or early <i>Leishmania donovani</i> infection and as a predictor of progression to symptomatic disease.</p><p>Methods</p><p>The study included 1469 healthy individuals living in endemic region (EHC) including both serology-positive and -negative subjects. TaqMan based qPCR assay was done on peripheral blood of each subject using kDNA specific primers and probes.</p><p>Results</p><p>A large proportion of EHC 511/1469 (34.78%) showed qPCR positivity and 56 (3.81% of 1469 subjects) had more than 1 calculated parasite genome/ml of blood. However, the number of individuals with parasite load above 5 genomes/ml was only 20 (1.36% of 1469). There was poor agreement between serological testing and qPCR (k = 0.1303), and 42.89% and 31.83% EHC were qPCR positive in seropositive and seronegative groups, respectively. Ten subjects had developed to symptomatic VL after 12 month of their follow up examination, of which eight were initially positive according to qPCR and among these, five had high parasite load.</p><p>Discussion</p><p>Thus, qPCR can help us to detect significant early parasitaemia, thereby assisting us in recognition of potential progressors to clinical disease. This test could facilitate early intervention, decreased morbidity and mortality, and possibly interruption of disease transmission.</p></div

Bar graph to show Leishmania detected by qPCR in different group of EHC on the basis of their serological status (QP-qPCR positive, QN-qPCR negative, SP-sero positive, SN-sero negative).

<p>Bar graph to show Leishmania detected by qPCR in different group of EHC on the basis of their serological status (QP-qPCR positive, QN-qPCR negative, SP-sero positive, SN-sero negative).</p

Pogressors (disease conversion, VL cases) details: Parasite load and serostatus of EHC who were healthy at the first survey, but developed symptomatic VL at the time of the 12-month follow up.

<p>(pos - positive on either serologic test; neg – negative on both serologic tests).</p><p>Pogressors (disease conversion, VL cases) details: Parasite load and serostatus of EHC who were healthy at the first survey, but developed symptomatic VL at the time of the 12-month follow up.</p

Parasitemia range in blood buffy coat samples from healthy controls from endemic or nonendemic regions.

<p>(*converted into VL).</p><p>Parasitemia range in blood buffy coat samples from healthy controls from endemic or nonendemic regions.</p

Flow chart of study of serology in endemic healthy control individuals (EHC) from baseline serosurvey (year 1) to identification of progressors.

<p>SP – seropositive; SN – seronegative; QP – qPCR positive (CT cutoff 39); QN – qPCR negative; VL – progressors to symptomatic visceral leishmaniasis.</p

Taqman primers and probe for detection of <i>Leishmania</i> DNA in endemic healthy controls.

<p>Sequences correspond to the kDNA4 minicircle DNA primers and probe <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003366#pntd.0003366-Hasker2" target="_blank">[21]</a>.</p><p>Taqman primers and probe for detection of <i>Leishmania</i> DNA in endemic healthy controls.</p

Determinants for progression from asymptomatic infection to symptomatic visceral leishmaniasis: A cohort study.

BACKGROUND:Asymptomatic Leishmania donovani infections outnumber clinical presentations, however the predictors for development of active disease are not well known. We aimed to identify serological, immunological and genetic markers for progression from L. donovani infection to clinical Visceral Leishmaniasis (VL). METHODS:We enrolled all residents >2 years of age in 27 VL endemic villages in Bihar (India). Blood samples collected on filter paper on two occasions 6-12 months apart, were tested for antibodies against L. donovani with rK39-ELISA and DAT. Sero converters, (negative for both tests in the first round but positive on either of the two during the second round) and controls (negative on both tests on both occasions) were followed for three years. At the start of follow-up venous blood was collected for the following tests: DAT, rK39- ELISA, Quantiferon assay, SNP/HLA genotyping and L.donovani specific quantitative PCR. RESULTS:Among 1,606 subjects enrolled,17 (8/476 seroconverters and 9/1,130 controls) developed VL (OR 3.1; 95% CI 1.1-8.3). High DAT and rK39 ELISA antibody titers as well as positive qPCR were strongly and significantly associated with progression from seroconversion to VL with odds ratios of 19.1, 30.3 and 20.9 respectively. Most VL cases arose early (median 5 months) during follow-up. CONCLUSION:We confirmed the strong association between high DAT and/or rK39 titers and progression to disease among asymptomatic subjects and identified qPCR as an additional predictor. Low predictive values do not warrant prophylactic treatment but as most progressed to VL early during follow-up, careful oberservation of these subjects for at least 6 months is indicated

Determinants for progression from asymptomatic infection to symptomatic visceral leishmaniasis: A cohort study

Background: Asymptomatic Leishmania donovani infections outnumber clinical presentations, however the predictors for development of active disease are not well known. We aimed to identify serological, immunological and genetic markers for progression from L. donovani infection to clinical Visceral Leishmaniasis (VL). Methods: We enrolled all residents >2 years of age in 27 VL endemic villages in Bihar (India). Blood samples collected on filter paper on two occasions 6–12 months apart, were tested for antibodies against L. donovani with rK39-ELISA and DAT. Sero converters, (negative for both tests in the first round but positive on either of the two during the second round) and controls (negative on both tests on both occasions) were followed for three years. At the start of follow-up venous blood was collected for the following tests: DAT, rK39- ELISA, Quantiferon assay, SNP/HLA genotyping and L.donovani specific quantitative PCR. Results: Among 1,606 subjects enrolled,17 (8/476 seroconverters and 9/1,130 controls) developed VL (OR 3.1; 95% CI 1.1–8.3). High DAT and rK39 ELISA antibody titers as well as positive qPCR were strongly and significantly associated with progression from seroconversion to VL with odds ratios of 19.1, 30.3 and 20.9 respectively. Most VL cases arose early (median 5 months) during follow-up. Conclusion: We confirmed the strong association between high DAT and/or rK39 titers and progression to disease among asymptomatic subjects and identified qPCR as an additional predictor. Low predictive values do not warrant prophylactic treatment but as most progressed to VL early during follow-up, careful oberservation of these subjects for at least 6 months is indicated