Serological and Molecular Analysis of Leishmania Infection in Healthy Individuals from Two Districts of West Bengal, India, Endemic for Visceral Leishmaniasis

Abstract

Several epidemiological studies have indicated the presence of asymptomatic infections with in the Indian subcontinent, where parasite transmission is considered anthroponotic. In India, such asymptomatic cases have been identified in the state of Bihar. We explored here, the presence of asymptomatic infection among healthy individuals living in two districts in the state of West Bengal, India, using serological and molecular tests. Blood samples of 246 healthy individuals were collected from nine villages of Malda and Murshidabad districts in West Bengal, considered endemic for visceral leishmaniasis (VL). Real-time quantitative polymerase chain reaction (qPCR) was performed for the quantification of parasite load in the blood. In addition, two serological tests were carried out to demonstrate anti- antibodies: rK39 strip test and anti-total soluble antigen IgG using enzyme-linked immunosorbent assay method. Nearly one-fifth (53/246) of the screened population was positive in qPCR as against 10.97% (27/246) positive in rK39 strip test. A range of parasite load was observed in the blood of identified asymptomatic cases with a median value of 7.7 parasites/mL (range = 1-65). There was poor agreement between qPCR and serological tests (κ = 0.089, = 0.13), and 29.62% and 20.54% of the population were qPCR positive in seropositive and seronegative groups, respectively. Combined molecular and serological tests enhanced the capacity to detect asymptomatic infection in healthy individuals residing in the endemic areas of VL. A significant proportion of asymptomatic individuals was detected in the examined endemic regions of West Bengal that might play a role in promoting VL transmission.

Age trends in asymptomatic and symptomatic Leishmania donovani infection in the Indian subcontinent: A review and analysis of data from diagnostic and epidemiological studies

“…different individuals were seropositive according to the two tests. Poor agreement was also observed between rK39 RDT and qPCR results in the Kaushal et al [64] study ( κ = 0.089). This may be due to a number of factors–differences in the sensitivities and specificities of the tests, differences in what the tests measure (as described above), and how well the tests were standardised and performed.…”

“…Age-stratified data on prevalence of PCR positivity was available from only two studies [64,70] (Fig 3C). In the Topno et al study there was a decrease in the proportion PCR positive with age, although the trend was not significant, while in the Kaushal et al study the proportion qPCR positive was highest in the 19–44 age group (OR 2.87 (95% CI 1.28–6.43, p = 0.010) compared to the 0–18 age group).…”

Identification of Leishmania infantum in blood donors from endemic regions for visceral leishmaniasis

“…The molecular method of qPCR was selected because it has displayed better sensitivity and specificity than serological tests and other techniques (Reithinger and Dujardin, 2007; Ruiter et al ., 2014; Sudarshan et al, 2014). This has been demonstrated in a wide variety of peripheral blood samples in clinical and subclinical forms (Galluzzi et al ., 2018), including the identification of asymptomatic infections (Sudarshan and Sundar, 2014; Kaushal et al ., 2017). As for the selected system in our study, although the literature has considered TaqMan™ to have high specificity, we opted for SYBR Green, a relatively more economical and frequently used system, as it is accessible and viable for use in the public health field (Pereira et al ., 2014).…”

“…Even if the qPCR with the SYBR Green system is considered promising for the diagnosis of asymptomatic infection by L. infantum , no approach can, at the moment, be regarded as a gold standard (Kaushal et al ., 2017; Varani et al ., 2017; Galluzzi et al ., 2018). The ‘best approach’ depends on factors such as assay design, the selected target region, origin of the clinical sample, cycle temperature and the DNA extraction method (Gomes et al ., 2007; Medeiros et al ., 2017).…”

“…Several studies have shown that various serological techniques have low sensitivity and specificity, in addition to low agreement (Romero et al ., 2009; Silva et al ., 2011, 2013; Ferreira-Silva et al ., 2018). Recent studies have presented molecular techniques as viable alternatives with better indices of sensitivity and specificity in asymptomatic individuals (Sudarshan and Sundar, 2014; Kaushal et al ., 2017; Galluzzi et al ., 2018).…”