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Abstract
Introduction HIV 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 analysis We 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 dissemination Ethical 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 number CTRI/2019/03/017908.
- HIV & AIDS
- molecular diagnostics
- parasitology
- thoracic medicine
- tropical medicine
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Strengths and limitations of this study
This study will evaluate noninvasive and minimally invasive alternatives to splenic or bone marrow aspiration in HIV-infected patients for diagnosis of visceral leishmaniasis (VL) in India.
If an effective alternative diagnostic(s) method is identified as a result of this study, a reduction in the use of invasive sampling methods for diagnosis and test of cure of VL in HIV-infected patients could be made.
The study addresses both issues of initial diagnosis and test of cure.
This study is limited to HIV-infected patients presenting at hospital who are likely to be presenting with more advanced disease.
The use of the minimally invasive techniques do not have standardised approach methods.
Introduction
In the absence of treatment, visceral leishmaniasis (VL) caused in India by a parasitic infection of Leishmania donovani (LD), also known as kala-azar, is usually fatal.1 2 The state of Bihar in India remains VL endemic; having failed to meet previous elimination targets, new targets were set for sustained elimination in India by 2020.3 Infection with HIV leads to a loss of immune cells and a susceptibility to comorbidities. VL is recognised as an opportunistic infection in HIV.4 5 In 2017, between 83 806 (0.12%) and 158 675 (0.23%) individuals were estimated to be infected with HIV in Bihar.6 Of 2077 VL patients, aged ≥14 years, screened in Bihar between 2011 and 2013, 5.6% were found to be HIV-positive, while up to 20% of reported VL patients from highly endemic districts in Bihar are coinfected with HIV.7 8 HIV-VL-coinfected patients have much higher rates of treatment failure and relapse than those without HIV.5
Current diagnostics for VL are invasive or do not distinguish between past and current infections.9 VL in India is currently diagnosed by a combination of clinical presentation, rK39 rapid diagnostic test (RDT) and parasitological confirmation of tissue aspirates in those presenting with relapse. Splenic aspirates are the gold standard for diagnosis of VL, with a sensitivity of 93%–98%. The procedure is invasive, requires a significant skill set and carries a small risk of fatal haemorrhage (1 in 1000). When splenic aspiration is not possible (ie, unpalpable spleen, low platelet or haemoglobin (Hb)), bone marrow aspirates (BMAs) have a reasonable sensitivity between 50% and 78%.10 Although invasive and painful, BMA does not carry the haemorrhage risk associated with splenic aspiration.11 Additionally, both require the capacity to conduct microscopic confirmation of LD bodies in macrophages.11
Test of cure (ToC) is a practice whereby following completion of treatment, a repeat comparative diagnostic test is conducted to ensure effectiveness of treatment. It is necessary in VL-HIV due to the relatively high incidence of treatment failure in this cohort of patients, and the high mortality risk associated with incomplete or ineffective treatment of VL-HIV. ToC for VL in HIV-infected patients is currently carried out by parasitological confirmation at day 29 in splenic aspirates when possible, or BMAs when splenic aspiration is not possible and remain the only established way to determine treatment success. Tests which detect antibodies cannot be used due to the persistent circulation of anti-Leishmania antibodies following infection, whether or not the patient has symptoms of disease.
Diagnostics such as the rK39 ELISA and the rK39 RDT detect presence of anti-Leishmania antibodies to rK39 Leishmania antigen. rK39 is a routinely used diagnostic method; however, data on the sensitivity of the rK39 RDT in HIV-coinfected individuals in India do not exist. Kalon Biological (Guilford, UK) has developed an ELISA which detects Leishmania antigen excreted in urine, enabling noninvasive detection of current infection.9 Case–control evaluation of the urine antigen ELISA carried out by Kalon Biological found a sensitivity of 95.2% (n=105, Bangladesh) and 100% (n=18, Kenya). 48/48 and 17/17 healthy negative control samples from Bangladesh and Kenya, respectively, were found to be negative (Kalon Biological). Leishmania antigen excreted in the urine was previously demonstrated to be effective for measurement of treatment effect in non-HIV-infected patients with VL in Ethiopia and may provide a noninvasive alternative to tissue aspiration for both diagnosis and ToC.9
qPCR is a highly sensitive technique to detect current infection, allowing parasite DNA present in blood or other tissue to be detected and quantified. In this study, we will use the qPCR assay targeting Leishmania kinetoplast DNA as previously described by Adams et al.12 Animal studies have also shown that the skin harvests parasites long after the infectious sand fly bite took place13 14 and the same is assumed to be the case in humans. Direct comparisons between blood parasite load and skin parasite load in humans, however, are scarce, since skin biopsies are painful and not suitable for large clinical studies. Recently, however, a novel device was developed to take virtually painless microbiopsies from the skin. Finally, the use of peripheral blood buffy coat smear microscopy has been shown to be of value in immunocompetent patients with VL in Bangladesh, where 92% were found to be positive for LD bodies in buffy coat smear microscopy, against splenic aspiration as the gold standard.5 We can therefore consider a priori that in VL-HIV-coinfected patients, this may be similar if not higher.
The diagnostic potential of the urine antigen ELISA, blood and qPCR, and peripheral blood buffy coat smear microscopy for diagnosis of VL in HIV-infected patients in India is yet to be established, therefore we aim to evaluate the sensitivity and specificity of these tests in this population. Additionally, we aim to evaluate the urine antigen ELISA, qPCR and peripheral blood buffy coat smear microscopy as a diagnostic tool for active VL infection and ToC for patients coinfected with HIV on the Indian subcontinent (ISC) to potentially eliminate the need for repeated and invasive splenic and bone marrow aspirations.
Study objective(s)
Primary objective(s)
To evaluate the sensitivity and specificity of the urine antigen ELISA, qPCR (blood and skin microbiopsies) and peripheral blood buffy coat smear microscopy, singularly or in combination, as a diagnostic and ToC at day 29 for symptomatic VL in HIV-positive patients compared with the gold standard parasitological visualisation by splenic aspirate at days 0 and 29, respectively.
Secondary objective(s)
To evaluate the sensitivity and specificity of the urine antigen ELISA, qPCR (blood and skin microbiopsies) and peripheral blood buffy coat smear microscopy, singularly or in combination, as a ToC at day 15 for symptomatic VL in HIV-positive patients compared with the gold standard of parasitological confirmation of splenic aspirate at day 29.
To conduct a pilot study to evaluate the sensitivity and specificity of the urine antigen ELISA, qPCR (blood and skin microbiopsies) and peripheral blood buffy coat smear microscopy, singularly or in combination, as a diagnostic and ToC at day 29 for symptomatic VL in patientsHIV-positive patients compared with BMA in a subset of patients contraindicated for splenic aspiration at days 0 and 29, respectively.
To investigate the kinetics of LD infection during treatment (days 0, 3, 8, 15 and 29) using urine antigen ELISA, qPCR (blood and skin microbiopsies) and peripheral blood buffy coat smear microscopy.
Methods and analysis
Participants of the study will be patients admitted at the Rajendra Memorial Research Institute of Medical Sciences (RMRIMS), Patna, India. Blood, urine and skin microbiopsy samples will be collected from HIV-positive patients with suspected VL (meeting the WHO definition of an rK39 RDT-positive test with clinical case definition15) at baseline who have undergone parasitological confirmation of VL (splenic aspiration or bone marrow aspiration when splenic aspiration is contraindicated) as per the standard of care (figure 1).
Study workflow.
Approximately 15% of patients are contraindicated for splenic aspiration and require bone marrow aspiration for diagnosis. These patients form a subset of patients with atypical presentation or more severe disease who would benefit from less invasive and highly sensitive diagnostics. Patients who are confirmed positive by splenic aspiration will be recruited to the main cohort of the study. Patients who are confirmed positive by bone marrow aspiration will be recruited to the pilot component. All parasitologically confirmed patients will undergo treatment for VL as per the standard of care (figure 1).
Furthermore, blood, urine and skin microbiopsy samples will be collected during treatment and at the end of treatment. Patients who are negative by splenic or bone marrow aspiration will not receive treatment as per the standard of care and will not undergo further sampling. qPCR will be conducted on blood and skin samples, buffy coat smear microscopy on blood samples and urine antigen ELISA on urine samples. Measurements of CD4 counts, full blood counts and screening of tuberculosis (TB) (GeneXpert and chest X-ray) will be conducted as standard for these patients.
Main entry criteria
Inclusion criteria
WHO definition of a VL suspect: rK39 RDT-positive + clinical symptoms.
Has undergone parasitological confirmation (splenic or bone marrow aspiration) as per standard of care.
Diagnosis of HIV as per National AIDS Control Organization guidelines.
Patients found positive by parasitological confirmation (splenic aspiration) in the diagnostic section will be continued to follow-up.
18 years of age or above.
Given written consent.
Exclusion criteria
Medical emergency or any other severe chronic medical condition which makes participation in the study medically inadvisable.
Participant refusal.
Splenic and bone marrow aspiration contraindicated.
Sample size
The primary objective is to determine the diagnostic accuracy of the urine antigen ELISA, qPCR (blood, spleen and skin microbiopsy samples) and peripheral blood buffy coat smear microscopy for diagnosis of VL in HIV-positive patients compared with the reference standard (clinical examination and parasitological confirmation). Splenic aspirates are the current gold standard for diagnosis of VL and have a sensitivity between 93% and 98%. Case–control evaluation of the urine antigen ELISA carried out by Kalon Biological found a sensitivity of 95.2% in a cohort of 105 patients in Bangladesh. We expect the sensitivity of the urine ELISA, qPCR and buffy coat smear microscopy singularly or in combination to be 95% sensitive. Therefore, we calculated sample size based on an expected proportion of positive patients using a sensitivity of 95%. The same number of patients will be used to determine the diagnostic accuracy of the urine antigen ELISA, qPCR (blood, spleen and skin microbiopsy samples) and peripheral blood buffy coat smear microscopy as a ToC for VL in HIV-positive patients compared with the reference standards (clinical examination and parasitological confirmation tests).
Sample size to estimate a proportion
Precision=5%
95% confidence (z statistic=1.96)
n=73
An additional four patients (5%) were added to the sample size to account for patients who may default.
n=77
Of the total patients recruited to the study, approximately 15% are expected to be contraindicated for splenic aspiration and will undergo bone marrow aspiration as per standard practice. To meet the required precision for the analysis of the main cohort, an additional 15 patients contraindicated for spleen will be recruited to a pilot study.
n = 91
Selection of patients
Patients to be recruited will be screened at the RMRIMS, Patna, India. Consecutive patients with a suspected diagnosis of HIV-VL as per the inclusion criteria will be invited to participate. All laboratory tests are to be conducted at the RMRIMS, Patna, India or an appropriate quality-assured laboratory. Informed consent will be taken by study staff who have passed the National Institutes of Health (NIH) Protecting Human Research Participants Ethics course (https://phrp.nihtraining.com) or equivalent. A screening and recruitment log will be maintained.
Schedule of events
Suspects will be screened with an rK39 RDT and a clinical examination as per standard practice (WHO definition of a VL suspect). Clinical assessment includes temperature (axillary), spleen size (left costal margin on the anterior axillary line to the tip of the spleen medially), liver size (the mid-clavicular line for its total span), body weight and height. At this point, consent will be taken. Patients who are RDT-positive are confirmed through routine parasitological confirmation by splenic aspiration, and where splenic aspiration is not contraindicated. Patients are excluded from splenic aspiration based on an unpalpable spleen, platelet count <40x109/L or Hb <50 g/L, or with significantly prolonged prothrombin time (PT). Patients contraindicated for splenic aspiration will undergo a bone marrow aspiration as per standard practice and will be recruited to the pilot study. Patients will participate in the diagnostic section only, or the diagnostic section and the follow-up section, depending on the result of the splenic or bone marrow aspiration.
Study clinical information will be obtained by a study nurse or doctor on enrolment case record forms (CRFs):
Sociodemographic information (eg, sex).
HIV-related information (eg, diagnosis, WHO staging and opportunistic infections).
Antiretroviral therapy (ART)-related information (eg, CD4 counts, ART regimen, opportunistic infection (OI) treatment and ART adherence).
VL/post-kala-azar dermal leishmaniasis (PKDL)-related information (eg, VL/PKDL history and VL/PKDL symptoms).
Past and current medical conditions (eg, malaria, chronic comorbidities and concomitant medication).
VL-focused examination (eg, vital signs, VL signs and symptoms).
The schedule of sampling is detailed in table 1. The schedule of tests is detailed in table 2. Patients with confirmed VL-HIV will be given a course of combination treatment for VL and initiated on ART, where not already on ART, as per standard practice. Further sampling will then be conducted as per the schedule detailed in table 1, day 0 being day of diagnosis prior to treatment starting. The standard course of treatment for VL in HIV ends on day 14.
Schedule of sampling
Schedule of tests
A blood sample will be taken for CD4 count, full blood count, qPCR and peripheral blood buffy coat smear microscopy as per the schedule of events (tables 1 and 2). Patients on the ward undergo routine sampling for tests, where possible sampling will be matched with routine sampling to avoid repeated venepuncture. A urine sample will be taken for urine antigen ELISA. The skin microbiopsy device takes minimally invasive and virtually painless skin samples, and samples will be taken from the nape of the neck as well as on the lower arm for qPCR. The same sample taken for standard microscopic diagnosis of the splenic aspirate will be used for qPCR.
To measure parasitic load by skin qPCR, DNA will be isolated from the microbiopsy device and kinetoplast DNA will be looked for. This will provide a semiquantitative result in relation to a standard curve of known concentration of cultured parasites.
Formal ToC will be carried out by parasitological confirmation and clinical examination at day 29 as per standard practise. Information regarding treatment failure will be noted.
Laboratory testing and sample storage
Testing procedures
All samples will be stored at −80°C until the study ends. This will allow samples to be tested in batch to reduce costs. Testing in batch will also allow blinding of laboratory staff to results of previous time points.
Data analysis and statistical methods
91 consecutive patients meeting the inclusion criteria will be screened for LD infection by qPCR (blood and skin), peripheral blood buffy coat smear microscopy, and urine antigen ELISA at baseline and at times previously shown in the schedule of sampling (table 1) and schedule of tests (table 2).
Baseline
Results of the qPCR, peripheral blood buffy coat smear microscopy and urine antigen ELISA at baseline will be compared with the gold standard diagnosis (parasitological confirmation).
Sensitivity and specificity with 95% confidence intervals will be calculated as follows:
where the above values are shown in table 3.
Contingency table to calculate sensitivity and specificity
A Kappa coefficient will be used to determine the level of agreement between the evaluation tests and the gold standard.
The continuous variables of the baseline and demographic characteristics will be summarised using number of patients (n), mean, SD, median, minimum, maximum and confidence intervals. The categorical variable gender will be summarised using number of patients (n) and percentage (%).
Test of cure
Patients who test positive for the gold standard at baseline will continue to be screened for infection by qPCR (blood and skin), peripheral blood buffy coat smear microscopy and urine antigen ELISA at times previously shown in the schedule of sampling (table 1) and schedule of tests (table 2).
Results of the qPCR (blood and skin), peripheral blood buffy coat smear microscopy and urine antigen ELISA at days 15 and 29 will be compared with the gold standard ToC (parasitological confirmation) at day 29. Sensitivity and specificity with 95% confidence intervals will be calculated as above.
The continuous variables will be summarised using number of patients (n), mean, SD, median, minimum, maximum and confidence intervals.
Further analysis
Wherever possible, analysis will also be stratified by markers of disease severity and other subgroups such as HIV viral load, CD4+ T cell counts and the use of highly active antiretroviral therapy (HAART).
Risk/benefit assessment
Potential risks related to this study are minimal. Invasive procedures such as splenic aspirate and BMA will only be done as per routine clinical workup; no additional aspirates will be done for the purpose of this study. Urine sampling does not pose any physical risks. Risks during blood or skin sampling are minimal when adhering to standard hygienic rules and include vasovagal reaction, bleeding or infection. Some discomfort may be felt during the skin microbiopsy; however, the device has been designed to be virtually painless. Risk of breach in confidentiality will be minimised using unique personal codes on the case report forms, with the subject’s enrolment list linking unique personal codes to the names of the participants in a locked and secured office.
The potential benefit of this study mainly lies in the possibility to identify a diagnostic tool and/or ToC that makes the current invasive splenic or BMAs no longer necessary in the workup of HIV-VL coinfection. This would limit the risks associated to these tests (fatal haemorrhage in case of splenic aspirates of 1/1000 and painful procedure in case of BMA) and significantly increase the comfort of these patients during their treatment process. Additionally, due to the complexities of these invasive tests, they are only available in specialist centres. If a ToC at day 15 is equally accurate as the current (parasitological) ToC at day 29, this would decrease the duration of hospitalisation by half for many patients and eliminate the need to return to the hospital after being released for others, improving access to care for this vulnerable group of patients.
Patient and public involvement
There was no patient or public involvement in the development of research questions and the study design.
Discussion
Considering the discomfort, iatrogenic risk and level of technical skill involved in parasitological confirmation, development of tests which can diagnose current infection and determine ToC that are safer, better tolerated and less technically demanding are required. HIV-infected patients diagnosed with VL in India have recently been shown to have better treatment outcomes with a combination therapy of liposomal amphotericin B and miltefosine over 14 days (CTRI/2015/05/005807).10 The choice of day 29 for ToC is based on older treatment regimens which were traditionally a month long. As such, evidence for the diagnostic accuracy of a ToC at the end of a shorter treatment (eg, day 15 in this case) may allow patients to be discharged without the need to return on day 29 for ToC; however, it is also possible that the extended time to the day 29 ToC is required to counter ‘slow response’ in the viscera to treatment.
The sensitivity of the rK39 RDT is well established in immunocompetent patients with sensitivities identified by systematic review of approximately 97% on the ISC and 85% in east Africa.15 16The rK39 RDT was found to have a sensitivity of 77% in HIV-coinfected individuals compared with 87% in HIV-negative patients with VL in Ethiopia.17 As these antibodies remain present even after successful treatment, they cannot be used either as a diagnostic tool in suspected relapse nor as a ToC following treatment and therefore need to be used in combination with a clinical history or parasitological proof to distinguish between past or current infection.11
qPCR is considered a proxy for parasite load in an individual but is currently restricted to use for research purposes. The potential for qPCR on blood for use in monitoring of treatment effect has been demonstrated in a cohort in Bangladesh.18 However, measuring parasite load using qPCR on blood samples might not be the best proxy for measuring parasite load in an individual. A first study in Ethiopia suggested that qPCR in skin was more sensitive in detecting parasite DNA than qPCR in blood. However, more data are needed to validate this skin microbiopsy device.19
Once complete, the results of this study have the potential to inform alternative minimally invasive and noninvasive tools for diagnosis and ToC in VL patients coinfected with HIV. This would allow clinicians to move away from tissue aspirations, methods which carry a risk of discomfort to the patient and a risk of fatal haemorrhage in the case of splenic aspiration. These interventions may also allow diagnosis within less specialised healthcare facilities.
Ethics and dissemination
This study has been approved by the ethics boards of The Liverpool School of Tropical Medicine, The Institute of Tropical Medicine in Antwerp, the University of Antwerp and the RMRIMS in Patna. The results of this study will be published in an open source, peer-reviewed journal. Results will also be presented to policy makers at national and international level. In particular, the WHO Guidelines Review Committee (GRC) who are due to provide global updated management guidelines for VL-HIV coinfection in 2021. Reporting of results will follow Standards for Reporting of Diagnostic Accuracy Studies (STARD) guidelines. Data will be made available on request.
Acknowledgments
We would like to thank the field teams and the patients.
References
Footnotes
Twitter @SophieOwen3
Contributors SIO, SB, ERA: conceptualised the study; SIO, SB, SK, NV, RM, AH, KP, KC: methodology of the study; SIO, SB:
writing of the original draft; SIO, SB, SK, NV, RM, AH, KP, KC, ERA, PD: writing, review and editing of the manuscript; ERA:
funding acquisition.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.