Publications by authors named "Kamlesh Gidwani"

31 Publications

Primary breast cancer biomarkers based on glycosylation and extracellular vesicles detected from human serum.

Cancer Rep (Hoboken) 2021 Aug 22:e1540. Epub 2021 Aug 22.

Department of Biochemistry, University of Turku, Turku, Finland.

Background: Breast cancer is a very common cancer that can be severe if not discovered early. The current tools to detect breast cancer need improvement. Cancer has a universal tendency to affect glycosylation. The glycosylation of circulating extracellular vesicle-associated glycoproteins, and mucins may offer targets for detection methods and have been only explored in a limited capacity.

Aim: Our aim was to develop an approach to detect the aberrant glycosylation of mucins and extracellular vesicle-associated glycoproteins from human sera using fluorescent nanoparticles, and preliminarily evaluate this approach for the differential diagnosis of breast cancer.

Methods And Results: The assay involved immobilizing glycosylated antigens using monoclonal antibodies and then probing their glycosylation by using lectins and glycan-specific antibodies coated on Eu -doped nanoparticles. Detection of mucin 1 and mucin 16 glycosylation with wheat germ agglutinin, and detection of the extracellular vesicle-associated CD63 were found to have better diagnostic ability for localized breast cancer than the conventional assays for mucin 1 and mucin 16 based tumor markers when the receiver operating characteristics were compared.

Conclusions: These results indicate that successful differential diagnosis of primary breast cancer may be aided by detecting cancer-associated glycosylation of mucin 1 and mucin 16, and total concentration of CD63, in human serum.
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http://dx.doi.org/10.1002/cnr2.1540DOI Listing
August 2021

Detection of bladder cancer with aberrantly fucosylated ITGA3.

Anal Biochem 2021 Sep 5;628:114283. Epub 2021 Jun 5.

Department of Life Technologies, Division of Biotechnology, University of Turku, Turku, Finland.

We describe a simple, non-invasive assay to identify fucosylated-glycoisoform of integrin alpha-3 (ITGA3) directly from unprocessed urine. ITGA3 was detected directly from the urine of bladder cancer (BlCa) (n = 13) and benign prostatic hyperplasia (BPH) (n = 9) patients with the use of lectins coated on europium-doped-nanoparticles (Eu-NPs). Lectin Ulex europaeus agglutinin-I (UEA) showed enhanced binding with BlCa-derived ITGA3. The evaluation with individual samples showed that a glycovariant ITGA3-UEA assay could significantly discriminate BlCa from BPH patients (p = 0.007). The detection of aberrantly fucosylated-isoform of ITGA3 from urine can be used to distinguish BlCa from age-matched benign controls in a simple sandwich assay.
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http://dx.doi.org/10.1016/j.ab.2021.114283DOI Listing
September 2021

HE4 in the evaluation of tumor load and prognostic stratification of high grade serous ovarian carcinoma.

Acta Oncol 2020 Dec 8;59(12):1461-1468. Epub 2020 Oct 8.

Department of Obstetrics and Gynecology, Turku University Hospital and University of Turku, Turku, Finland.

Objective: Human epididymis protein 4 (HE4) is a validated, complementary biomarker to cancer antigen 125 (CA125) for high grade serous ovarian carcinoma (HGSC). Currently, there are insufficient data on the utility of longitudinal HE4 measurement during HGSC treatment and follow up. We set to provide a comprehensive analysis on the kinetics and prognostic performance of HE4 with serial measurements during HGSC treatment and follow up.

Methods: This prospective study included 143 patients with advanced HGSC (ClinicalTrials.gov identifier: NCT01276574). Serum CA125 and HE4 were measured at baseline, before each cycle of chemotherapy and during follow up until first progression. Baseline biomarker values were compared to the tumor load assessed during surgery and to residual disease. Biomarker nadir values and concentrations at progression were correlated to survival.

Results: The baseline HE4 concentration distinguished patients with a high tumor load from patients with a low tumor load assessed during surgery (<.0001). The baseline CA125 level was not associated with tumor load to a similar extent (=.067). At progression, the HE4 level was an independent predictor of worse survival in the multivariate analysis (=.002). All patients that were alive 3 years post-progression had a serum HE4 concentration below 199.20 pmol/l at the 1st recurrence.

Conclusion: HE4 is a feasible biomarker in the treatment monitoring and prognostic stratification of patients with HGSC. Specifically, the serum level of HE4 at first relapse was associated with the survival of patients and it may be a useful complementary tool in the selection of second line treatments. This is to the best of our knowledge the first time this finding has been reported.
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http://dx.doi.org/10.1080/0284186X.2020.1827157DOI Listing
December 2020

Glycovariant-based lateral flow immunoassay to detect ovarian cancer-associated serum CA125.

Commun Biol 2020 08 21;3(1):460. Epub 2020 Aug 21.

Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland.

Cancer antigen 125 (CA125) is a widely used biomarker in monitoring of epithelial ovarian cancer (EOC). Due to insufficient cancer specificity of CA125, its diagnostic use is severely compromised. Abnormal glycosylation of CA125 is a unique feature of ovarian cancer cells and could improve differential diagnosis of the disease. Here we describe the development of a quantitative lateral flow immunoassay (LFIA) of aberrantly glycosylated CA125 which is widely superior to the conventional CA125 immunoassay (CA125IA). With a 30 min read-out time, the LFIA showed 72% sensitivity, at 98% specificity using diagnostically challenging samples with marginally elevated CA125 (35-200 U/mL), in comparison to 16% sensitivity with the CA125IA. We envision the clinical use of the developed LFIA to be based on the substantially enhanced disease specificity against the many benign conditions confounding the diagnostic evaluation and against other cancers.
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http://dx.doi.org/10.1038/s42003-020-01191-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442799PMC
August 2020

Exploratory Analysis of CA125-MGL and -STn Glycoforms in the Differential Diagnostics of Pelvic Masses.

J Appl Lab Med 2020 03;5(2):263-272

Institute of Biomedicine, Research Center for Cancer, Infections and Immunity, Department of Pathology, University of Turku and Turku University Hospital, Turku, Finland.

Background: The cancer antigen 125 (CA125) immunoassay (IA) does not distinguish epithelial ovarian cancer (EOC) from benign disease with the sensitivity needed in clinical practice. In recent studies, glycoforms of CA125 have shown potential as biomarkers in EOC. Here, we assessed the diagnostic abilities of two recently developed CA125 glycoform assays for patients with a pelvic mass. Detailed analysis was further conducted for postmenopausal patients with marginally elevated conventionally measured CA125 levels, as this subgroup presents a diagnostic challenge in the clinical setting.

Methods: Our study population contained 549 patients diagnosed with EOC, benign ovarian tumors, and endometriosis. Of these, 288 patients were postmenopausal, and 98 of them presented with marginally elevated serum levels of conventionally measured CA125 at diagnosis. Preoperative serum levels of conventionally measured CA125 and its glycoforms (CA125-MGL and CA125-STn) were determined.

Results: The CA125-STn assay identified EOC significantly better than the conventional CA125-IA in postmenopausal patients (85% vs. 74% sensitivity at a fixed specificity of 90%, P = 0.0009). Further, both glycoform assays had superior AUCs compared to the conventional CA125-IA in postmenopausal patients with marginally elevated CA125. Importantly, the glycoform assays reduced the false positive rate of the conventional CA125-IA.

Conclusions: The results indicate that the CA125 glycoform assays markedly improve the performance of the conventional CA125-IA in the differential diagnosis of pelvic masses. This result is especially valuable when CA125 is marginally elevated.
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http://dx.doi.org/10.1093/jalm/jfz012DOI Listing
March 2020

A longitudinal analysis of CA125 glycoforms in the monitoring and follow up of high grade serous ovarian cancer.

Gynecol Oncol 2020 03 27;156(3):689-694. Epub 2019 Dec 27.

Department of Obstetrics and Gynecology, Turku University Hospital and University of Turku, Turku, Finland. Electronic address:

Objective: Cancer antigen 125 (CA125) is generally considered the gold standard of biomarkers in the diagnosis and monitoring of high grade serous ovarian carcinoma (HGSC). We recently reported, that two CA125 glycoforms (CA125-STn and CA125-MGL) have a high specificity to HGSC and further hypothesized, that these cancer specific glycoforms are feasible candidates as biomarkers in HGSC treatment and follow up.

Methods: Our cohort consisted of 122 patients diagnosed with HGSC. Serum samples were collected longitudinally at the time of diagnosis, during treatment and follow up. Serum levels of CA125, CA125-STn and CA125-MGL were determined and compared or correlated with different end points (tumor load assessed intraoperatively, residual disease, treatment response, progression free survival).

Results: Serum CA125-STn levels at diagnosis differentiated patients with low tumor load and high tumor load (p = 0,030), indicating a favorable detection of tumor volume. Similarly, the CA125-STn levels at diagnosis were significantly lower in patients with subsequent complete cytoreduction than in patients with suboptimal cytoreduction (p = 0,025). Conventional CA125 did not differentiate these patients (p = 0,363 and p = 0,154). The CA125-STn nadir value predicted the progression free survival of patients. The detection of disease relapse was improved with CA125-STn, which presented higher fold increase in 80,0% of patients and earlier increase in 37,0% of patients.

Conclusions: CA125-STn showed promise as a useful biomarker in the monitoring and follow up of patients with HGSC utilizing a robust and affordable technique. Our findings are topical as a suitable indicator of tumor load facilitates patient selection in an era of new targeted therapies.
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http://dx.doi.org/10.1016/j.ygyno.2019.12.025DOI Listing
March 2020

Nanoparticle-aided glycovariant assays to bridge biomarker performance and ctDNA results.

Mol Aspects Med 2020 04 29;72:100831. Epub 2019 Nov 29.

Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland. Electronic address:

Numerous immunoassay based cancer biomarkers established in the 1970 and 1980'ies are widely used in clinical routine. Initial expectations of biomarkers such as CEA, CA125, CA19-9, AFP to provide decisive help in the diagnosis of early stage, pre-symptomatic cancers have not been realized. Thus, they are primarily used for monitoring disease progression and occasionally being useful as prognostic indicators. This limitation is due to the marker also being measurable in healthy individuals and frequently at elevated concentrations in common benign conditions. Most conventional tumor markers are glycosylated and interestingly specific alterations of the glycostructure part can often be seen early in the cancerous process. Conventional double monoclonal immunoassays are however blind to such changes as they are based on peptide epitope recognition. Wide selections of carbohydrate recognizing macromolecules, lectins, but also glycan structure recognizing antibodies are potentially useful for detecting such changes. Despite numerous attempts generating proof-of-principle evidence for this, such assays have generally not been successfully introduced into clinical routine. The affinity constants of lectin and glycan specific antibodies for their corresponding carbohydrate structures may be up to several orders too low to provide the detection limits and robustness expected from routine tumor markers. In this review, we describe an approach based on the use of highly fluorescent Eu-chelate dyed nanoparticles onto which lectins or glycan specific antibodies are coated to provide the necessary binding strength and signal amplification to provide low detection limits, while maintaining the original glycan-structure specificity. This concept applied to three markers, PSA, CA125 and CA15-3 provide glycoform assays of greatly enhanced cancer specificity using sample volumes similar or lower than corresponding traditional ELISAs. For ovarian cancer, we show that this new approach when applied to ovarian cyst fluid samples provide results similar to the performance obtained with ctDNA determinations of a set of 17 driver mutations and greatly superior compared to corresponding conventional immunoassays. Based on our results, we predict that the nanoparticle-lectin concept will enable a new generation of simple, low-cost biomarker assays of highly improved cancer specificity. Such tools should ideally be evaluated together with determination of ctDNA to establish early detection schemes for cancers e.g. ovarian, pancreas, lung where the detection rate of early stage disease is presently unacceptably low.
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http://dx.doi.org/10.1016/j.mam.2019.11.001DOI Listing
April 2020

Europium Nanoparticle-Based Sialyl-Tn Monoclonal Antibody Discriminates Epithelial Ovarian Cancer-Associated CA125 from Benign Sources.

J Appl Lab Med 2019 11 23;4(3):299-310. Epub 2019 Aug 23.

Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland.

Background: The Sialyl-Thomsen-nouveau antigen (STn) is abundantly produced on many types of human epithelial cancers including epithelial ovarian cancer (EOC). We previously developed an EOC-specific lectin sandwich immunoassay (CA125) using a human macrophage galactose-binding lectin coated on fluorescent europium nanoparticles (Eu-NPs) as a tracer and an anti-CA125-specific mAb for capture. Here we have identified a novel STn-mAb that efficiently recognizes the EOC-associated STn antigen on CA125 when coated on Eu-NPs.

Method: CA125 from the ovarian cancer cell line OVCAR-3, placental homogenate, and ascites fluid from patients with liver cirrhosis was captured by anti-CA125 antibody immobilized on microtitration wells and traced with anti-STn-mAb-Eu-NPs. Samples from EOC or patients with endometriosis with marginally increased conventional CA125 immunoassay (CA125; 35-200 U/mL) and healthy controls were analyzed.

Results: An analytically sensitive CA125 assay that specifically recognized the CA125 isoform produced by OVCAR-3 was achieved. Serum CA125 concentration was significantly higher in EOC patients than in those with endometriosis ( < 0.001). Furthermore, the sensitivity for detection of EOC with CA125 assay was 73.3% when 95% of endometriosis cases were undetectable.

Conclusion: Our findings suggest that Eu-NPs-based CA125 assay could help reduce the false-positive rates of CA125 to improve differential diagnosis. The results encourage studying further the potential use of CA125 to detect EOC at earlier clinical stages. This approach warrants further investigation in other cancers as well.
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http://dx.doi.org/10.1373/jalm.2018.028266DOI Listing
November 2019

Lectin nanoparticle assays for detecting breast cancer-associated glycovariants of cancer antigen 15-3 (CA15-3) in human plasma.

PLoS One 2019 25;14(7):e0219480. Epub 2019 Jul 25.

Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland.

Cancer antigen 15-3 (CA15-3) is widely utilized for monitoring metastatic breast cancer (BC). However, its utility for early detection of breast cancer is severely limited due to poor clinical sensitivity and specificity. The glycosylation of CA15-3 is known to be affected by BC, and therefore it might offer a way to construct CA15-3 glycovariant assays with improved cancer specificity. To this end, we performed lectin-based glycoprofiling of BC-associated CA15-3. CA15-3 expressed by a BC cell line was immobilized on microtitration wells using an anti-CA15-3 antibody. The glycosylation of the immobilized CA15-3 was then detected by using lectins coated onto europium (III)-doped nanoparticles (Eu+3-NPs) and measuring the time-resolved fluorescence of Eu. Out of multiple lectin-Eu+3-NP preparations, wheat germ agglutinin (WGA) and macrophage galactose-type lectin (MGL) -Eu3+-NPs bound to the BC cell line-dericed CA15-3 glycovariants (CA15-3Lectin). To evaluate the clinical performance of these two lectin-based assays, plasma samples from metastatic BC patients (n = 53) and healthy age-matched women (n = 20).Plasma CA15-3Lectin measurements better distinguished metastatic BC patients from healthy controls than the conventional CA15-3 immunoassay. At 90% specificity, the clinical sensitivity of the assays was 66.0, 67.9 and 81.1% for the conventional CA15-3, CA15-3MGL and CA15-3WGA assays, respectively. Baseline CA15-3MGL and CA15-3WGA were correlated to conventional baseline CA15-3 levels (r = 0.68, p<0.001, r = 0.90, p>0.001, respectively). However, very low baseline CA15-3MGL levels ≤ 5 U/mL were common in this metastatic breast cancer patient population.In conclusion, the new CA15-3Lectin concept could considerably improve the clinical sensitivity of BC detection compared to the conventional CA15-3 immunoassays and should be validated further on a larger series of subjects with different cancer subtypes and stages.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0219480PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658058PMC
February 2020

A Nanoparticle-Based Approach for the Detection of Extracellular Vesicles.

Sci Rep 2019 07 11;9(1):10038. Epub 2019 Jul 11.

Department of Biochemistry, Division of Biotechnology, University of Turku, Turku, Finland.

The analysis of extracellular vesicles (EVs) typically requires tedious and time-consuming isolation process from bio-fluids. We developed a nanoparticle-based time resolved fluorescence immunoassay (NP-TRFIA) that uses biotinylated antibodies against the proteins of tetraspanin family and tumor-associated antigens for capturing EVs from urine samples and cell culture supernatants without the need for isolation. The captured-EVs were detected either with Eu-chelate or Eu-doped nanoparticle-based labels conjugated either to antibodies against the tetraspanins or lectins targeting the glycan moieties on EVs surface. The NP-TRFIA demonstrated specific capturing and detection of EVs by antibodies and lectins. Lectin-nanoparticle based assays showed 2-10 fold higher signal-to-background ratio compared with lectin-chelate assays. The nanoparticle assay concept allowed surface glycosylation profiling of the urine derived-EVs with lectins. It was also applied to establish an assay showing differential expression of tumor-associated proteins on more aggressive (higher ITGA3 on DU145- and PC3-EVs) compared to less aggressive (higher EpCAM on LNCaP-EVs) PCa- cell lines derived-EVs. This NP-TRFIA can be used as a simple tool for analysis and characterization of EVs in urine and cell culture supernatants. Such approach could be useful in identification of disease-specific markers on the surface of patient-derived urinary EVs.
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http://dx.doi.org/10.1038/s41598-019-46395-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6624270PMC
July 2019

A Nanoparticle-Lectin Immunoassay Improves Discrimination of Serum CA125 from Malignant and Benign Sources.

Clin Chem 2016 10 18;62(10):1390-400. Epub 2016 Aug 18.

Department of Biochemistry/Biotechnology, University of Turku, Turku, Finland;

Background: Measurement of serum cancer antigen 125 (CA125) is the standard approach for epithelial ovarian cancer (EOC) diagnostics and follow-up. However, the clinical specificity is not optimal because increased values are also detected in healthy controls and in benign diseases. CA125 is known to be differentially glycosylated in EOC, potentially offering a way to construct CA125 assays with improved cancer specificity. Our goal was to identify carbohydrate-reactive lectins for discriminating between CA125 originating from EOC and noncancerous sources.

Methods: CA125 from the OVCAR-3 cancer cell line, placental homogenate, and ascites fluid from patients with cirrhosis were captured on anti-CA125 antibody immobilized on microtitration wells. A panel of lectins, each coated onto fluorescent europium-chelate-doped 97-nm nanoparticles (Eu(+3)-NPs), was tested for detection of the immobilized CA125. Serum samples from high-grade serous EOC or patients with endometriosis and healthy controls were analyzed.

Results: By using macrophage galactose-type lectin (MGL)-coated Eu(+3)-NPs, an analytically sensitive CA125 assay (CA125(MGL)) was achieved that specifically recognized the CA125 isoform produced by EOC, whereas the recognition of CA125 from nonmalignant conditions was reduced. Serum CA125(MGL) measurement better discriminated patients with EOC from endometriosis compared to conventional immunoassay. The discrimination was particularly improved for marginally increased CA125 values and for earlier detection of EOC progression.

Conclusions: The new CA125(MGL) assay concept could help reduce the false-positive rates of conventional CA125 immunoassays. The improved analytical specificity of this test approach is dependent on a discriminating lectin immobilized in large numbers on Eu(+3)-NPs, providing both an avidity effect and signal amplification.
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http://dx.doi.org/10.1373/clinchem.2016.257691DOI Listing
October 2016

Role of lectin microarrays in cancer diagnosis.

Proteomics 2016 Apr 29;16(8):1257-65. Epub 2016 Mar 29.

Department of Biochemistry/Biotechnology, University of Turku, Turun yliopisto, Finland.

The majority of cell differentiation associated tumor markers reported to date are either glycoproteins or glycolipids. Despite there being a large number of glycoproteins reported as candidate markers for various cancers, only a handful are approved by the US Food and Drug Administration. Lectins, which bind to the glycan part of the glycoproteins, can be exploited to identify aberrant glycosylation patterns, which in turn would help in enhancing the specificity of cancer diagnosis. Although conventional techniques such as HPLC and MS have been instrumental in performing the glycomic analyses, these techniques lack multiplexity. Lectin microarrays have proved to be useful in studying multiple lectin-glycan interactions in a single experiment and, with the advances made in the field, hold a promise of enabling glycomic profiling of cancers in a fast and efficient manner.
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http://dx.doi.org/10.1002/pmic.201500404DOI Listing
April 2016

Leishmania specific CD4 T cells release IFNγ that limits parasite replication in patients with visceral leishmaniasis.

PLoS Negl Trop Dis 2014 Oct 2;8(10):e3198. Epub 2014 Oct 2.

Karolinska Institutet, Department of Microbiology Tumor and Cell Biology, Stockholm, Sweden.

Visceral leishmaniasis (VL) is associated with increased circulating levels of multiple pro-inflammatory cytokines and chemokines, including IL-12, IFNγ, and TNFα, and elevated expression of IFNγ mRNA in lesional tissue such as the spleen and bone marrow. However, an immunological feature of VL patients is that their peripheral blood mononuclear cells (PBMCs) typically fail to respond to stimulation with leishmanial antigen. Unexpectedly, it was recently shown that Leishmania specific IFNγ, can readily be detected when a whole blood stimulation assay (WBA) is used. We sought to define the conditions that permit whole blood cells to respond to antigen stimulation, and clarify the biological role of the IFNγ found to be released by cells from VL patients. CD4+ T cells were found to be crucial for and the main source of the IFNγ production in Leishmania stimulated whole blood (WB) cultures. Complement, antibodies and red blood cells present in whole blood do not play a significant role in the IFNγ response. The IFNγ production was reduced by blockade of human leukocyte antigen (HLA)-DR, indicating that the response to leishmanial antigens observed in WB of active VL patients is a classical HLA- T cell receptor (TCR) driven reaction. Most importantly, blockade of IFNγ in ex-vivo splenic aspirate cultures demonstrated that despite the progressive nature of their disease, the endogenous IFNγ produced in patients with active VL serves to limit parasite growth.
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http://dx.doi.org/10.1371/journal.pntd.0003198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4183461PMC
October 2014

Strong association between serological status and probability of progression to clinical visceral leishmaniasis in prospective cohort studies in India and Nepal.

PLoS Negl Trop Dis 2014 23;8(1):e2657. Epub 2014 Jan 23.

Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.

Introduction: Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4-10 to 1. We assessed the risk of progression from infection to disease as a function of DAT and rK39 serological titers.

Methods: We used available data on four cohorts from villages in India and Nepal that are highly endemic for Leishmania donovani. In each cohort two serosurveys had been conducted. Based on results of initial surveys, subjects were classified as seronegative, moderately seropositive or strongly seropositive using both DAT and rK39. Based on the combination of first and second survey results we identified seroconvertors for both markers. Seroconvertors were subdivided in high and low titer convertors. Subjects were followed up for at least one year following the second survey. Incident VL cases were recorded and verified.

Results: We assessed a total of 32,529 enrolled subjects, for a total follow-up time of 72,169 person years. Altogether 235 incident VL cases were documented. The probability of progression to disease was strongly associated with initial serostatus and with seroconversion; this was particularly the case for those with high titers and most prominently among seroconvertors. For high titer DAT convertors the hazard ratio reached as high as 97.4 when compared to non-convertors. The strengths of the associations varied between cohorts and between markers but similar trends were observed between the four cohorts and the two markers.

Discussion: There is a strongly increased risk of progressing to disease among DAT and/or rK39 seropositives with high titers. The options for prophylactic treatment for this group merit further investigation, as it could be of clinical benefit if it prevents progression to disease. Prophylactic treatment might also have a public health benefit if it can be corroborated that these asymptomatically infected individuals are infectious for sand flies.
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http://dx.doi.org/10.1371/journal.pntd.0002657DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3900391PMC
September 2014

Molecular and serological markers of Leishmania donovani infection in healthy individuals from endemic areas of Bihar, India.

Trop Med Int Health 2013 May 7;18(5):548-54. Epub 2013 Mar 7.

Infectious Disease Research Laboratory, Banaras Hindu University, Varanasi, India.

Objectives: Recent epidemiological reports indicate that asymptomatic human infections with Leishmania donovani, the causative agent of visceral leishmaniasis or Kala-azar (KA), occur frequently in India. We explored markers of infection.

Methods: Blood samples were collected from 286 healthy subjects from 16 villages in the Muzaffarpur district of Bihar. These individuals were classified into three groups: (i) persons with no history of KA and living in a house where no KA cases were previously reported, (ii) persons with no history of KA but living in a house where KA cases were diagnosed at the time of sampling or in the past, and (iii) successfully treated KA patients. Each sample was tested using a Leishmania-specific PCR to detect Leishmania DNA, and two serological tests to demonstrate anti-Leishmania antibodies: the Direct Agglutination Test and rK39 ELISA.

Results: PCR positivity was similar among the three groups (20-25%). In contrast, among treated patients, the percentage of serologically positive individuals was roughly five times that of healthy individuals with no KA history, as measured with either test. Living in a house where KA had been reported did not affect seropositivity.

Conclusion: A significant proportion of asymptomatic infections of Leishmania exist in endemic regions. Using a combination of molecular and serological tests increases the capacity to detect infections at different stages. Further work is required to understand the kinetics of the markers.
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http://dx.doi.org/10.1111/tmi.12085DOI Listing
May 2013

Latent infection with Leishmania donovani in highly endemic villages in Bihar, India.

PLoS Negl Trop Dis 2013 14;7(2):e2053. Epub 2013 Feb 14.

Institute of Tropical Medicine, Antwerp, Belgium.

Introduction: Asymptomatic persons infected with the parasites causing visceral leishmaniasis (VL) usually outnumber clinically apparent cases by a ratio of 4-10 to 1. We describe patterns of markers of Leishmania donovani infection and clinical VL in relation to age in Bihar, India.

Methods: We selected eleven villages highly endemic for Leishmania donovani. During a 1-year interval we conducted two house to house surveys during which we collected blood samples on filter paper from all consenting individuals aged 2 years and above. Samples were tested for anti-leishmania serology by Direct Agglutination Test (DAT) and rK39 ELISA. Data collected during the surveys included information on episodes of clinical VL among study participants.

Results: We enrolled 13,163 persons; 6.2% were reactive to DAT and 5.9% to rK39. Agreement between the tests was weak (kappa = 0.30). Among those who were negative on both tests at baseline, 3.6% had converted to sero-positive on either of the two tests one year later. Proportions of sero-positives and sero-converters increased steadily with age. Clinical VL occurred mainly among children and young adults (median age 19 years).

Discussion: Although infection with L. donovani is assumed to be permanent, serological markers revert to negative. Most VL cases occur at younger ages, yet we observed a steady increase with age in the frequency of sero-positivity and sero-conversion. Our findings can be explained by a boosting effect upon repeated exposure to the parasite or by intermittent release of parasites in infected subjects from safe target cells. A certain proportion of sero-negative subjects could have been infected but below the threshold of antibody abundance for our serologic testing.
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http://dx.doi.org/10.1371/journal.pntd.0002053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3573094PMC
July 2013

Leishmaniasis direct agglutination test: using pictorials as training materials to reduce inter-reader variability and improve accuracy.

PLoS Negl Trop Dis 2012 13;6(12):e1946. Epub 2012 Dec 13.

Royal Tropical Institute Koninklijk Instituut voor de Tropen, Amsterdam, The Netherlands.

Background: The Direct Agglutination Test (DAT) has a high diagnostic accuracy and remains, in some geographical areas, part of the diagnostic algorithm for Visceral Leishmaniasis (VL). However, subjective interpretation of results introduces potential for inter-reader variation. We report an assessment of inter-laboratory agreement and propose a pictorial-based approach to standardize reading of the DAT.

Methodology: In preparation for a comparative evaluation of immunochromatographic diagnostics for VL, a proficiency panel of 15 well-characterized sera, DAT-antigen from a single batch and common protocol was sent to nine laboratories in Latin-America, East-Africa and Asia. Agreement (i.e., equal titre or within 1 titer) with the reading by the reference laboratory was computed. Due to significant inter-laboratory disagreement on-site refresher training was provided to all technicians performing DAT. Photos of training plates were made, and end-titres agreed upon by experienced users of DAT within the Visceral-Leishmaniasis Laboratory-Network (VL-LN).

Results: Pre-training, concordance in DAT results with reference laboratories was only 50%, although agreement on negative sera was high (94%). After refresher training concordance increased to 84%; agreement on negative controls increased to 98%. Variance in readings significantly decreased after training from 3.3 titres to an average of 1.0 titre (two-sample Wilcoxon rank-sum (Mann-Whitney) test (z = -3,624 and p = 0.0003)).

Conclusion: The most probable explanation for disagreement was subjective endpoint reading. Using pictorials as training materials may be a useful tool to reduce disparity in results and promote more standardized reading of DAT, without compromising diagnostic sensitivity.
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http://dx.doi.org/10.1371/journal.pntd.0001946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3521667PMC
May 2013

Visceral leishmaniasis in rural bihar, India.

Emerg Infect Dis 2012 Oct;18(10):1662-4

Institute of Tropical Medicine, Antwerp, Belgium.

To identify factors associated with incidence of visceral leishmaniasis (VL), we surveyed 13,416 households in Bihar State, India. VL was associated with socioeconomic status, type of housing, and belonging to the Musahar caste. Annual coverage of indoor residual insecticide spraying was 12%. Increasing such spraying can greatly contribute to VL control.
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http://dx.doi.org/10.3201/eid1810.111083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471608PMC
October 2012

Reassessment of immune correlates in human visceral leishmaniasis as defined by cytokine release in whole blood.

Clin Vaccine Immunol 2012 Jun 25;19(6):961-6. Epub 2012 Apr 25.

Infectious Disease Research Laboratory, Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.

Depressed cell-mediated immunity in human visceral leishmaniasis (VL) (also known as kala-azar), revealed as the inability of peripheral blood mononuclear cells (PBMCs) to respond to Leishmania antigen, remains a hallmark of and is thought to underlie the progressive nature of this disease. We recently reported the ability of a whole-blood, gamma interferon (IFN-γ) release assay to detect subclinical infections among healthy individuals living in an area where kala-azar is endemic (Bihar, India) and the surprising result that patients with active VL also secreted significant levels of antigen-specific IFN-γ in this assay. We were interested in ascertaining whether these findings would be true for a larger cohort of subjects and in employing the whole-blood assay to detect additional cytokines that might better correlate with the disease status of infected individuals. We evaluated IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin-10 (IL-10) release in 35 patients with active VL, 54 patients with VL who were cured, 27 patients with other diseases, 52 healthy controls who lived in regions where VL or kala-azar is not endemic (NEHCs [for nonendemic healthy controls]), and 147 healthy controls who lived in regions where kala-azar is endemic (EHCs [for endemic healthy controls]). The cellular responses of the EHCs were correlated with their serological antibody titers against Leishmania donovani and Phlebotomus argentipes saliva. The whole-blood cells from the majority of both active (80%) and cured (85%) VL patients, as well as 24% of EHCs with presumed subclinical infections, produced significantly elevated levels of IFN-γ. The findings do not support a severe Th1 response defect in kala-azar. Importantly, only the patients with active VL also produced IL-10, which in conjunction with IFN-γ better reflects the immune responses that distinguish individuals with active disease from cured or subclinically infected, immune individuals.
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http://dx.doi.org/10.1128/CVI.00143-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3370446PMC
June 2012

Serological markers for leishmania donovani infection in Nepal: Agreement between direct agglutination test and rK39 ELISA.

Trop Med Int Health 2010 Nov;15(11):1390-4

B.P. Koirala Institute of Health Sciences, Dharan, Nepal.

Visceral leishmaniasis (VL) is an important vector-borne disease caused by Leishmania donovani in the Indian subcontinent. The actual incidence and role of asymptomatic infections in the region are not wellknown. We used the direct agglutination test (DAT) and the rK39 ELISA as L. donovani infection markers in 10 VL endemic villages in Nepal. DAT titre distribution showed two subgroups in the population (infected and non-infected individuals), while rK39 did not. The agreement between both tests was moderate (j = 0.53; 95% CI 0.49–0.57). More research is needed to develop validated markers for Leishmania infection.
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http://dx.doi.org/10.1111/j.1365-3156.2010.02631.xDOI Listing
November 2010

Incidence of symptomatic and asymptomatic Leishmania donovani infections in high-endemic foci in India and Nepal: a prospective study.

PLoS Negl Trop Dis 2011 Oct 4;5(10):e1284. Epub 2011 Oct 4.

Institute of Tropical Medicine, Antwerp, Belgium.

Incidence of Leishmania donovani infection and Visceral Leishmaniasis (VL) was assessed in a prospective study in Indian and Nepalese high-endemic villages. DAT-seroconversion was used as marker of incident infection in 3 yearly surveys. The study population was followed up to month 30 to identify incident clinical cases. In a cohort of 9034 DAT-negative individuals with neither active signs nor history of VL at baseline, 42 VL cases and 375 asymptomatic seroconversions were recorded in the first year, giving an infection:disease ratio of 8.9 to 1. In the 18 months' follow-up, 7 extra cases of VL were observed in the seroconverters group (N=375), against 14 VL cases among the individuals who had not seroconverted in the first year (N=8570) (RR=11.5(4.5
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http://dx.doi.org/10.1371/journal.pntd.0001284DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3186756PMC
October 2011

Serological markers of sand fly exposure to evaluate insecticidal nets against visceral leishmaniasis in India and Nepal: a cluster-randomized trial.

PLoS Negl Trop Dis 2011 Sep 13;5(9):e1296. Epub 2011 Sep 13.

Banaras Hindu University, Varanasi, India.

Background: Visceral leishmaniasis is the world' second largest vector-borne parasitic killer and a neglected tropical disease, prevalent in poor communities. Long-lasting insecticidal nets (LNs) are a low cost proven vector intervention method for malaria control; however, their effectiveness against visceral leishmaniasis (VL) is unknown. This study quantified the effect of LNs on exposure to the sand fly vector of VL in India and Nepal during a two year community intervention trial.

Methods: As part of a paired-cluster randomized controlled clinical trial in VL-endemic regions of India and Nepal we tested the effect of LNs on sand fly biting by measuring the antibody response of subjects to the saliva of Leishmania donovani vector Phlebotomus argentipes and the sympatric (non-vector) Phlebotomus papatasi. Fifteen to 20 individuals above 15 years of age from 26 VL endemic clusters were asked to provide a blood sample at baseline, 12 and 24 months post-intervention.

Results: A total of 305 individuals were included in the study, 68 participants provided two blood samples and 237 gave three samples. A random effect linear regression model showed that cluster-wide distribution of LNs reduced exposure to P. argentipes by 12% at 12 months (effect 0.88; 95% CI 0.83-0.94) and 9% at 24 months (effect 0.91; 95% CI 0.80-1.02) in the intervention group compared to control adjusting for baseline values and pair. Similar results were obtained for P. papatasi.

Conclusions: This trial provides evidence that LNs have a limited effect on sand fly exposure in VL endemic communities in India and Nepal and supports the use of sand fly saliva antibodies as a marker to evaluate vector control interventions.
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http://dx.doi.org/10.1371/journal.pntd.0001296DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3172194PMC
September 2011

Interferon-gamma release assay (modified QuantiFERON) as a potential marker of infection for Leishmania donovani, a proof of concept study.

PLoS Negl Trop Dis 2011 Apr 19;5(4):e1042. Epub 2011 Apr 19.

Banaras Hindu University, Varanasi, India.

Background: In areas endemic for visceral leishmaniasis (VL), a large number of infected individuals mount a protective cellular immune response and remain asymptomatic carriers. We propose an interferon-gamma release assay (IFN-γRA) as a novel marker for latent L. donovani infection.

Methods And Findings: We modified a commercial kit (QuantiFERON) evaluating five different leishmania-specific antigens; H2B, H2B-PSA2, H2B-Lepp12, crude soluble antigen (CSA) and soluble leishmania antigen (SLA) from L. donovani with the aim to detect the cell-mediated immune response in VL. We evaluated the assay on venous blood samples of active VL patients (n = 13), cured VL patients (n = 15), non-endemic healthy controls (n = 11) and healthy endemic controls (n = 19). The assay based on SLA had a sensitivity of 80% (95% CI = 54.81-92.95) and specificity of 100% (95% CI = 74.12-100).

Conclusion: Our findings suggest that a whole-blood SLA-based QuantiFERON assay can be used to measure the cell-mediated immune response in L. donovani infection. The positive IFN-γ response to stimulation with leishmania antigen in patients with active VL was contradictory to the conventional finding of a non-proliferative antigen-specific response of peripheral blood mononuclear cells and needs further research.
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http://dx.doi.org/10.1371/journal.pntd.0001042DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3079582PMC
April 2011

Longlasting insecticidal nets for prevention of Leishmania donovani infection in India and Nepal: paired cluster randomised trial.

BMJ 2010 Dec 29;341:c6760. Epub 2010 Dec 29.

London School of Hygiene and Tropical Medicine, London WC1E 7HT, UK.

Objective: To test the effectiveness of large scale distribution of longlasting nets treated with insecticide in reducing the incidence of visceral leishmaniasis in India and Nepal.

Design: Paired cluster randomised controlled trial designed to detect a 50% reduction in incidence of Leishmania donovani infection.

Setting: Villages in Muzaffarpur district in India and Saptari, Sunsari, and Morang districts in Nepal.

Participants: 13 intervention and 13 control clusters. 12 691 people were included in the analysis of the main outcome (infection), and 19 810 were enrolled for the secondary (disease) end point.

Intervention: Longlasting insecticidal nets (treated with deltamethrin) were distributed in the intervention clusters in December 2006.

Main Outcome Measures: Infection was determined by direct agglutination test at 12 and 24 months after the intervention in those who had negative results (titre <1:1600) at baseline. The effect estimate was computed as the geometric mean of the risk ratios for seroconversion for each cluster pair (net/no net), with its 95% confidence interval. Formal tests of effect of no intervention were obtained with a paired t test.

Results: There was no significant difference in the risk of seroconversion over 24 months in intervention (5.4%; 347/6372) compared with control (5.5%; 345/6319 people) clusters (risk ratio 0.90, 95% confidence interval 0.49 to 1.65) nor in the risk of clinical visceral leishmaniasis (0.99, 0.46 to 1.40). Adjustment for covariates did not alter these conclusions.

Conclusions: There is no evidence that large scale distribution of longlasting insecticidal nets provides additional protection against visceral leishmaniasis compared with existing control practice in the Indian subcontinent. The observed effect was small and not significant, though the confidence intervals did not exclude a 50% change in either direction.

Trial Registration: Clinical Trials NCT 2005-015374.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3011370PMC
http://dx.doi.org/10.1136/bmj.c6760DOI Listing
December 2010

Persistence of Leishmania donovani antibodies in past visceral leishmaniasis cases in India.

Clin Vaccine Immunol 2011 Feb 15;18(2):346-8. Epub 2010 Dec 15.

Banaras Hindu University, Varanasi, India.

The persistence of anti-Leishmania donovani antibodies in past visceral leishmaniasis (VL) cases was retrospectively assessed by means of the direct agglutination test (DAT) and the rK39 enzyme-linked immunosorbent assay (ELISA). Antibody titers remained high for an extended period of time in past cases of VL. These results highlight the need to carefully elicit the history of patients with VL symptoms.
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http://dx.doi.org/10.1128/CVI.00473-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3067357PMC
February 2011

The epidemiology of Leishmania donovani infection in high transmission foci in India.

Trop Med Int Health 2010 Jul 6;15 Suppl 2:12-20. Epub 2010 May 6.

Banaras Hindu University, Varanasi, India.

Objective: Visceral Leishmaniasis (VL) is highly prevalent in Bihar, India. India and its neighbours aim at eliminating VL, but several knowledge gaps in the epidemiology of VL may hamper that effort. The prevalence of asymptomatic infections with Leishmania donovani and their role in transmission dynamics are not well understood. We report data from a sero-survey in Bihar.

Methods: Demographic and immunological surveys were carried out in July and November 2006, respectively in 16 highly VL endemic foci in Muzaffarpur district in Bihar. Household and individual information was gathered and capillary blood samples were collected on filter papers. Direct agglutination test (DAT) was used to determine infected individuals (cut-off titre 1:1600). DAT results were tabulated against individual and household variables. A multivariate generalized estimating equation (GEE) model was used to study the prevalence of serologically positive individuals taking into account the clustering at household and cluster levels.

Results: Of study subjects 18% were DAT positive, and this proportion increased with age. Women had a significantly lower prevalence than men >14 years old. Owning domestic animals (cows, buffaloes or goats) was associated with a higher risk of being DAT positive [OR 1.16 (95% CI 1.01-1.32)], but socio-economic status was not.

Conclusions: Prevalence of leishmanial antibodies was high in these communities, but variable. Demographic factors (i.e. marriage) may explain the lower DAT positivity in women >14 years of age. Within these homogeneously poor communities, socio-economic status was not linked to L. donovani infection risk at the individual level, but ownership of domestic animals was.
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http://dx.doi.org/10.1111/j.1365-3156.2010.02519.xDOI Listing
July 2010

Evaluation of ex vivo human immune response against candidate antigens for a visceral leishmaniasis vaccine.

Am J Trop Med Hyg 2010 May;82(5):808-13

Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.

People cured from visceral leishmaniasis (VL) develop protection mediated by Th1-type cellular responses against new infections. We evaluated cytokine responses against 6 defined candidate vaccine antigens in 15 cured VL subjects and 5 healthy endemic controls with no evidence of previous exposure to Leishmania parasites. Of the 6 cytokines examined, only interferon-gamma (IFN-gamma) differentiated cured VL patients from non-exposed individuals, with cured patients mounting a significantly higher IFN-gamma response to a crude parasite antigen preparation. Among candidate vaccine antigens tested, the largest number of cured subjects recognized cysteine proteinase B, leading to heightened IFN-gamma responses, followed by sterol 24-c-methyltransferase. These two antigens were the most immunogenic and protective antigens in a murine VL model, indicating a relationship between T cell recall responses of humans cured from VL and protective efficacy in an experimental model. Further studies may help prioritize antigens for clinical development of a subunit vaccine against VL.
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http://dx.doi.org/10.4269/ajtmh.2010.09-0341DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2861380PMC
May 2010

Measurement of recent exposure to Phlebotomus argentipes, the vector of Indian visceral Leishmaniasis, by using human antibody responses to sand fly saliva.

Am J Trop Med Hyg 2010 May;82(5):801-7

London School of Hygiene and Tropical Medicine, London, United Kingdom.

Antibody (IgG) responses to the saliva of Phlebotomus argentipes were investigated using serum samples from regions of India endemic and non-endemic for visceral leishmaniasis (VL). By pre-adsorbing the sera against the saliva of the competing human-biting but non-VL vector P. papatasi, we significantly improved the specificity of a P. argentipes saliva enzyme-linked immunosorbent assay. Using this method, we observed a statistically significant correlation between antibodies to P. argenitpes saliva and the average indoor density of female sand flies. Additionally, the method was able to detect recent changes in vector exposure when sera from VL patients were assayed before, during, and after hospitalization and protected from sand fly bites under untreated bed nets. Collectively, these results highlight the utility of antibodies to P. argentipes saliva as an important tool to evaluate VL vector control programs.
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http://dx.doi.org/10.4269/ajtmh.2010.09-0336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2861389PMC
May 2010

Evaluation of leishmanin skin test in Indian visceral leishmaniasis.

Am J Trop Med Hyg 2009 Apr;80(4):566-7

Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.

We evaluated the performance of the leishmanin skin test (LST) in 50 patients with visceral leishmaniasis (VL), 124 cured VL patients at different time intervals, 125 healthy controls from an endemic region (HEC), and 14 healthy controls from a non-endemic region (NEHC). The leishmanin antigen used was based on Leishmania major and was obtained from the Pasteur Institute (Iran). A positive LST was found in 14.0% of patients with active VL, 40.3% of cured patients, 21.6% of HECs, and 0% in NEHCs. The 14% positivity in patients with active VL conflicts with the widely held opinion that such patients should be negative because of anergy. Also, a lack of sensitivity of the LST was observed in cured VL patients. An LST antigen produced from L. donovani strains from India should be explored.
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April 2009
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