A Correlative Study of Splenic Parasite Score and Peripheral Blood Parasite Load Estimation by Quantitative PCR in Visceral Leishmaniasis

Abstract

Parasitological diagnosis of visceral leishmaniasis (VL) by splenic smear is highly sensitive, but it is associated with the risk of severe hemorrhage. In this study, the diagnosis of VL using quantitative PCR (qPCR) in peripheral blood was evaluated in 100 patients with VL. Blood parasitemia ranged from 5 to 93,688 leishmania parasite genomes/ml of blood and positively correlated with splenic score (P < 0.0001; r 2 ‫؍‬ 0.58). Therefore, quantification of parasite genomes by qPCR can replace invasive procedures for diagnostic and prognostic evaluations. Visceral leishmaniasis (VL), also known as kala-azar, is a systemic protozoan disease that is caused by the parasite Leishmania donovani complex and is transmitted through the bite of an infected phlebotomine sand fly. VL is characterized by prolonged fever, splenomegaly, weight loss, and pancytopenia, and it is complicated by serious infection (1). These clinical features can easily be mistaken for other common febrile illnesses, such as malaria, enteric fever, tuberculosis, etc. If untreated, VL is fatal (2). There are an estimated 200,000 to 400,000 new cases of VL each year worldwide, and over 60% of these occur in the Northern Indian state of Bihar and the bordering regions of Nepal and Bangladesh (3). Therefore, it is necessary to use a diagnostic test that is highly sensitive and specific. At present, diagnosis is either confirmed parasitologically by the demonstration of amastigotes in bone marrow or spleen smears or by the demonstration of antibodies using the rapid immunochromatographic test, the direct agglutination test (DAT), or molecular tests (4-10). The sensitivity of amastigote detection in splenic smear is 96% to 98% (11, 12); however, splenic aspiration is associated with the risk of lifethreatening hemorrhage. Additionally, serological tests cannot discriminate between subclinical, current, and past infections (13,14). Various DNA-based molecular methods (PCR) have been used for the diagnosis of VL using blood, buccal swabs, or urine samples; however, their inability to discriminate between symptomatic or asymptomatic VL is a major drawback (7,15,16). Hence, more appropriate tools are urgently needed. Real-time quantitative PCR (qPCR) is a promising tool that can be employed in the diagnosis of VL (17, 18). qPCR distinguishes symptomatic from asymptomatic VL and has the ability to diagnose VL early (19,20). We recently reported the usefulness of real-time PCR (RT-PCR) for estimation of parasite load during the treatment of VL (21). In the present study, we looked for a correlation between the splenic smear score (5) and the number of circulating parasites in patients with active VL.The study was carried out at the Infectious Diseases Research Laboratory (Department of Medicine, Banaras Hindu University, Varanasi, India) and at its field site Kala-Azar Medical Research Centre (Muzaffarpur, Bihar, India). The study was approved by the Ethics Committee of the Institute of Medical Sciences, Banaras Hindu University, and all subjects provided writt...

Real-time PCR in detection and quantitation of Leishmania donovani for the diagnosis of Visceral Leishmaniasis patients and the monitoring of their response to treatment

“…A different SYBR green based reaction demonstrated only 79% sensitivity targeting another kDNA minicircle sequence [ 21 ], rising to 94.7% sensitivity when targeting a genomic single copy polymerase gene by Taqman chemistry [ 33 ]. 95.7% sensitivity and 100% sensitivity are also reported with parasitologically confirmed patients in two recent SYBR green reaction based studies targeting kinetoplast DNA [ 34 , 35 ].…”

Combining epidemiology with basic biology of sand flies, parasites, and hosts to inform leishmaniasis transmission dynamics and control

“…Recent Indian data show that the median blood parasite load by qPCR is 500-fold higher in kala-azar patients than in asymptomatically infected individuals [ 48 ]. Data from the same group confirm that parasite loads in peripheral blood correlate well with those in splenic aspirates [ 49 ]. High parasite loads were rare among asymptomatic infections and, when present, indicated individuals with high risk of subsequent development of kala-azar [ 50 ].…”

Causes and consequences of higher Leishmania infantum burden in patients with kala‐azar: a study of 625 patients

“…However, in the present study, which included a larger sample population, patients with HIV had a median blood parasite burden that was approximately three times higher than that in patients without HIV coinfection. While bone marrow parasitism may be organ specific, DNA detected in the blood reflects the level of parasitism in the body overall, as previously described in both animals and humans . Unfortunately, CD4+ counts were not registered in the database, and its influence on the parasite burden could not be ascertained.…”