Risk-free point-of-care visceral leishmaniasis diagnostics: combining buffy coat microscopy and immunoassay in tertiary rural hospitals in Sudan

Background Visceral leishmaniasis (VL) is a zoonotic protozoal vector-borne disease that is a major public health challenge. In Argentina, canine (CVL) and human visceral leishmaniasis (HVL) have recently emerged. There is a lack of standardised diagnostic tests for CVL, which hinders control of CVL and HVL. Methodology/Principal findings Sampling was carried out in Puerto Iguazú, Argentina, comprising 190 asymptomatic, oligosymptomatic and polysymptomatic dogs. The following diagnostics were applied: microscopy of lymph node aspirate (LNA); three immunochromatographic rapid diagnostic tests (RDTs), prototype rK28-ICT, rK39-ICT (both Coris BioConcept), commercial rK39 (InBios); ELISA for IgG, IgG1 and IgG2, against rK28, rK39 or crude lysate antigen. DNA detection and analysis, with 30 dogs, was of the ITS1 region using skin samples, and loop-mediated isothermal amplification (LAMP; Eiken Loopamp) of buffy coat, skin scrape or LNA. 15.4% of dogs were positive by LNA microscopy. The rK28 RDT had higher seropositivity rate (61%) than either a prototype rK39 RDT (31.4%) or commercial rK39 RDT (18.8%), without cross-reactivity with six other pathogens. IgG anti-rK39 ELISA antibody titres, but not IgG2, were positively correlated with number of clinical signs. LAMP with LNA had a higher positivity rate than PCR; buffy coat sampling was more sensitive than skin scrape. ITS1 confirmed Leishmania (Leishmania) infantum as the agent of CVL. Leishmania (Viannia) spp. was detected in skin samples from two dogs, compatible with Leishmania (Viannia) braziliensis. Conclusions/Significance Seroprevalence confirmed rapid increase in CVL in Puerto Iguazú. The rK28 RDT test potentially has great value for improved point-of-care diagnosis. Given cost reduction and accessibility, commercial LAMP may be applicable to buffy coat. RDT biomarkers of CVL clinical status are required to combat spread of CVL and HVL. The presence of Viannia, perhaps as an agent of human mucocutaneous leishmaniasis (MCL), highlights the need for vigilance and surveillance.

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Evaluation of qPCR on blood and skin microbiopsies, peripheral blood buffy coat smear, and urine antigen ELISA for diagnosis and test of cure for visceral leishmaniasis in HIV-coinfected patients in India: a prospective cohort study

BMJ Open ◽

10.1136/bmjopen-2020-042519 ◽

2021 ◽

Vol 11 (4) ◽

pp. e042519

Author(s):

Sophie I Owen ◽

Sakib Burza ◽

Shiril Kumar ◽

Neena Verma ◽

Raman Mahajan ◽

...

Keyword(s):

Bone Marrow ◽

Visceral Leishmaniasis ◽

Hiv Infection ◽

Peripheral Blood ◽

Medical Science ◽

Bone Marrow Aspiration ◽

Buffy Coat ◽

Tropical Medicine ◽

Urine Antigen ◽

Antigen Elisa

IntroductionHIV coinfection presents a challenge for diagnosis of visceral leishmaniasis (VL). Invasive splenic or bone marrow aspiration with microscopic visualisation of Leishmania parasites remains the gold standard for diagnosis of VL in HIV-coinfected patients. Furthermore, a test of cure by splenic or bone marrow aspiration is required as patients with VL-HIV infection are at a high risk of treatment failure. However, there remain financial, implementation and safety costs to these invasive techniques which severely limit their use under field conditions.Methods and analysisWe aim to evaluate blood and skin qPCR, peripheral blood buffy coat smear microscopy and urine antigen ELISA as non-invasive or minimally invasive alternatives for diagnosis and post-treatment test of cure for VL in HIV-coinfected patients in India, using a sample of 91 patients with parasitologically confirmed symptomatic VL-HIV infection.Ethics and disseminationEthical approval for this study has been granted by The Liverpool School of Tropical Medicine, The Institute of Tropical Medicine in Antwerp, the University of Antwerp and the Rajendra Memorial Research Institute of Medical Science in Patna. Any future publications will be published in open access journals.Trial registration numberCTRI/2019/03/017908.

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QBC® for the diagnosis of human and canine american visceral leishmaniasis: preliminary data

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822001000600013 ◽

2001 ◽

Vol 34 (6) ◽

pp. 577-581 ◽

Cited By ~ 16

Author(s):

Daniel B. Liarte ◽

Ivete L. Mendonça ◽

Francisco C.O. Luz ◽

Elza A.S. de Abreu ◽

Gustavo W.S. Mello ◽

...

Keyword(s):

Bone Marrow ◽

Visceral Leishmaniasis ◽

Preliminary Data ◽

Peripheral Blood ◽

Buffy Coat ◽

Promising Method ◽

Blood Parasites ◽

Leishmania Chagasi ◽

Asymptomatic Carriers ◽

Fluorescent Method

"Quantitative Buffy Coat" (QBC®) is a direct and fast fluorescent method used for the identification of blood parasites. Since Leishmania chagasi circulates in blood, we decided to test it in American visceral leishmaniasis (AVL). Bone marrow (BM) and peripheral blood (PB) of 49 persons and PB of 31 dogs were analyzed. QBC® was positive in BM of 11/11 patients with AVL and in 1/6 patients with other diseases. Amastigotes were identified in PB of 18/22 patients with AVL and in none without AVL. The test was positive in 30 out of the 31 seropositive dogs and in 28/28 dogs with Leishmania identified in other tissues. QBC® is a promising method for diagnosis of human AVL, and possibly for the exam of PB of patients with AVL/AIDS, for the control of the cure and for the identification of asymptomatic carriers. Because it is fast and easy to collect and execute, QBC® should be evaluated for programs of reservoir control.

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Diagnostic accuracy of loop-mediated isothermal amplification (LAMP) for detection of Leishmania DNA in buffy coat from visceral leishmaniasis patients

Parasites & Vectors ◽

10.1186/1756-3305-5-280 ◽

2012 ◽

Vol 5 (1) ◽

pp. 280 ◽

Cited By ~ 47

Author(s):

Md Gulam Musawwir Khan ◽

Khondaker Rifat Hasan Bhaskar ◽

Md Abdus Salam ◽

Tania Akther ◽

Gerd Pluschke ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Diagnostic Accuracy ◽

Buffy Coat ◽

Isothermal Amplification ◽

Loop Mediated Isothermal Amplification

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Peripheral Blood Buffy Coat Smear: a Promising Tool for Diagnosis of Visceral Leishmaniasis

Journal of Clinical Microbiology ◽

10.1128/jcm.05067-11 ◽

2011 ◽

Vol 50 (3) ◽

pp. 837-840 ◽

Cited By ~ 18

Author(s):

M. A. Salam ◽

M. G. M. Khan ◽

K. R. H. Bhaskar ◽

M. H. Afrad ◽

M. M. Huda ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Peripheral Blood ◽

Buffy Coat ◽

Promising Tool

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The assessment of acute chest pain in New Zealand rural hospitals utilising point-of-care troponin

Journal of Primary Health Care ◽

10.1071/hc18007 ◽

2018 ◽

Vol 10 (1) ◽

pp. 90 ◽

Cited By ~ 1

Author(s):

Rory Miller ◽

Garry Nixon

Keyword(s):

New Zealand ◽

Chest Pain ◽

Point Of Care ◽

Acute Chest Pain ◽

Rural Hospitals

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Autochthonous canine visceral leishmaniasis cases occur in Paraná state since 2012: isolation and identification of Leishmania infantum

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-29612019083 ◽

2020 ◽

Vol 29 (1) ◽

Author(s):

Renata Cristina Ferreira Dias ◽

Aline Kuhn Sbruzzi Pasquali ◽

Vanete Thomaz-Soccol ◽

Eliane Maria Pozzolo ◽

Luciana Chiyo ◽

...

Keyword(s):

Visceral Leishmaniasis ◽

Leishmania Infantum ◽

Buffy Coat ◽

Control Measures ◽

Canine Visceral Leishmaniasis ◽

Sample Collection ◽

Isolation And Identification ◽

Collaborative Control ◽

Stage 4 ◽

The City

Abstract The aim of this study was to confirm the emergence of canine visceral leishmaniasis among dogs in Foz do Iguaçu. The disease was diagnosed through the isolation and molecular identification of Leishmania infantum. In the first sample collection stage (2012), three lymph node aspirates and 46 buffy coat samples were obtained mostly from the dogs that were seroreagents for leishmaniasis. In the second sample collection stage (2013), the buffy coat samples were collected from 376 dogs located close to Paraguay, Paraná river, center and peripheral parts of the city. The DNA from the six isolates, four from the first sampling stage (4/49) and two from the second sampling stage (2/376), was subjected to polymerase chain reaction using the K26F/R primers. The isolate was confirmed as L. infantum by sequencing. As none of the dogs had ever left the city, the isolates were confirmed as autochthonous. Further, the study confirmed the emergence of canine visceral leishmaniasis in Paraná through the identification of L. infantum among dogs in Foz do Iguaçu city. Hence, collaborative control measures should be designed and implemented by the public agencies and research institutions of Brazil, Argentina, and Paraguay to control the spread of visceral leishmaniasis.

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Diagnosis of Kala-Azar by Nested PCR Based on Amplification of the Leishmania Mini-Exon Gene

Journal of Clinical Microbiology ◽

10.1128/jcm.36.8.2173-2177.1998 ◽

1998 ◽

Vol 36 (8) ◽

pp. 2173-2177 ◽

Cited By ~ 18

Author(s):

Ken Katakura ◽

Shin-Ichiro Kawazu ◽

Toshimitsu Naya ◽

Koichi Nagakura ◽

Mamoru Ito ◽

...

Keyword(s):

Bone Marrow ◽

Visceral Leishmaniasis ◽

Nested Pcr ◽

Northwest China ◽

Buffy Coat ◽

Invasive Procedures ◽

Blood Samples ◽

Kala Azar ◽

Pcr Method ◽

Exon Gene

To diagnose visceral leishmaniasis (kala-azar), we have developed a nested PCR method based on amplification of the mini-exon gene, which is unique and tandomly repeated in the Leishmania genome. Nested PCR was sufficiently sensitive for the detection of DNA in an amount equivalent to a single Leishmania parasite or less. We examined the usefulness of this PCR method using bone marrow aspirates and buffy coat cells collected from kala-azar patients who had or had not received chemotherapy in northwest China. We obtained PCR positivity for all of the parasitologically positive bone marrow samples from the patients. Some ambiguities with the primary PCR results were eliminated by the subsequent nested PCR. The buffy coat samples from 7 of 12 patients with splenomegaly were positive by the nested PCR, although only 2 of them were positive for parasites by culture. However, buffy coat samples from nine children, whose splenomegaly has been reduced and clinically cured by antimony treatment, were all negative. Thus, this nested PCR method represents a new tool for the diagnosis of kala-azar with patient blood samples instead of bone marrow or spleen aspirates obtained by more invasive procedures.

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Could kDNA-PCR in Peripheral Blood Replace the Examination of Bone Marrow for the Diagnosis of Visceral Leishmaniasis?

Journal of Parasitology Research ◽

10.1155/2016/1084353 ◽

2016 ◽

Vol 2016 ◽

pp. 1-7 ◽

Cited By ~ 4

Author(s):

Natalia Souza de Godoy ◽

Marcos Luiz Alves Andrino ◽

Regina Maia de Souza ◽

Erika Gakiya ◽

Valdir Sabbaga Amato ◽

...

Keyword(s):

Bone Marrow ◽

Visceral Leishmaniasis ◽

Peripheral Blood ◽

Gold Standard ◽

Buffy Coat ◽

Molecular Techniques ◽

Parasitological Examination ◽

Parasitological Diagnosis ◽

Group 2 ◽

Group 1

The aim of this study was to evaluate whether the molecular (kDNA-PCR) and parasitological diagnosis in peripheral blood (PB) could replace the invasive and painful bone marrow collection (BM) in the diagnosis of visceral leishmaniasis (VL). PB from suspected VL patients was evaluated by parasitological and molecular techniques using as the gold standard (GS) a combination of clinical, epidemiological, and immunochromatographic test (PB-rK39) results and parasitological examination of BM. Based on the GS, 38 samples from 32 patients were grouped: Group 1, 20 samples of VL cases, and Group 2, 18 samples of non-VL cases. In order to evaluate the parasitological and molecular techniques in PB, the samples were examined. From Group 1, PB kDNA-PCR was positive in 20 samples and in 19 of 20 in BM kDNA-PCR examination. However, the parasitological examination of buffy coat was insensitive, being able to detect only 4 cases from Group 1. All samples from Group 2 were negative. We concluded that, for the diagnosis of visceral leishmaniasis, the parasitological examination of peripheral blood was not useful; however, molecular diagnosis by kDNA-PCR, performed in peripheral blood, could be useful to replace the parasitological examination of bone marrow.

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