by Professor Paul Kaye
Leishmaniasis is classified as a neglected tropical disease. It is the cause of a huge health burden and is common in Asia, Africa, South and Central America, and even southern Europe. This article discusses how flow cytometry can help to evaluate diagnosis, monitor the effects of therapy and help in the creation of a vaccine.

Background

The leishmaniases are a family of devastating diseases, affecting a great many people across the globe and presenting a significant risk to both public health and socioeconomic development. The leishmaniases are vector-borne diseases, caused by infection with one of 20 species of the parasitic protozoan Leishmania (Fig. 1), transmitted through the bite of the infected female phlebotomine sand fly.
They can be broadly classified as tegumentary leishmaniases (TLs), affecting the skin and mucosa, and visceral leishmaniasis (VL), affecting internal organs. Whereas VL is responsible for over 20¦000 deaths per year, TL are non-life-threatening, chronic and potentially disfiguring, and account for around two-thirds of the global disease burden.
Within TL, there are three subtypes: self-healing lesions at the location of sand fly bite (cutaneous leishmaniasis; CL), lesions that spread from the original skin lesion to the mucosae (mucosal leishmaniasis; ML), and those which spread uncontrolled across the body (disseminated or diffuse cutaneous leishmaniasis; DCL). VL, also known as kala azar, involves major organs including the spleen, liver and bone marrow. In addition, patients recovering from VL after drug treatment often develop post kala-azar dermal leishmaniasis (PKDL), a chronic skin condition, characterized by nodular or macular lesions beginning on the face and spreading to the trunk and arms. As it may develop in up to half of patients previously treated and apparently cured from VL, it is thought that PKDL plays a central role in community transmission of VL.
The World Health Organization designates leishmaniasis as a neglected tropical disease (NTD), which together affect more than one|billion people across 149 countries worldwide; true prevalence may be even higher. Disproportionately, NTDs affect the poorest, malnourished individuals, and contribute to a vicious circle of poverty and disease. The significant physical marks, including ulcers, often left in the wake of the TLs may have an impact on mental health and perpetuate social stigma associated with the diseases [5]. There are over 1|million new cases of TL and 0.5|million new cases of VL each year, which together account for the loss of approximately 2.4|million disability-adjusted life years.

Treatment challenges

Leishmaniasis treatment can be quite difficult since at-risk populations may lack access to healthcare, and the limited battery of drugs has been increasingly compromised by resistance. Additionally, because the parasites in question are eukaryotic, they are not dissimilar from human cells, so the medication is also liable to be harmful – even fatal – to host as well as to pathogen.
Although the burden of VL in South Asia has been reduced with single-dose liposomal amphotericin B, the drug is less effective in other geographic locations, namely East Africa. Various drug combinations have been tested, unsuccessfully, and new chemical entities and immune-modulators are in early stages of development and as yet untested in the field. Unfortunately, little has changed in the treatment for CL for the past 50|years.
No vaccines are currently approved for any form of human leishmaniasis, although vaccines for canine VL have reached the market. Barriers to vaccine development include the limited investment in leishmaniases R&D and the high costs involved in bringing new products to those that need them.

Current work

My work on leishmaniasis has taken a holistic view, rooted in the immunology of the host-parasite interaction, but employing tools and approaches that span many disciplines: mathematics, ecology, vector biology and most recently neuroscience. Thirty years of discovery science has led to the development of a candidate for a therapeutic vaccine for PKDL, the mysterious sequela to VL [6]. ‘Therapeutic’ vaccines are given after an individual is infected with a pathogen and are designed to enhance our immune system and help eliminate the infection.
With colleagues from Sudan, we are in the midst of a phase IIb clinical trial funded by the Wellcome Trust, evaluating the efficacy of this therapeutic vaccine in Sudanese patients with persistent PKDL.
However, the research has been a long time in the making and has a long way to go. To continue to make progress, we linked with colleagues in Ethiopia, Kenya and Uganda and at the European Vaccine Initiative (http://www.euvaccine.eu/) in Germany, to develop a new research consortium to evaluate the immune status of people suffering from leishmaniasis. For example, using flow cytometry for blood and multiplexed immunohistochemistry for tissue biopsies, we can enumerate the proportions of lymphocytes, monocytes and neutrophils based on surface marker expression (e.g. CD3, CD19, CD14, CD16), and characterize their function, for instance by expression of cytokines (e.g. interferon-gamma) or other cell surface proteins that define function state. To support this endeavour, we recently received a grant from the European & Developing Countries Clinical Trials Partnership (EDCTP) that will allow us to not only extend our vaccine programme in Sudan [9] but also to address other important research challenges.
To develop vaccines and indeed new drugs, we often need tools capable of performing in-depth comparisons of how the body’s immune system is coping with the infection when a patient is first admitted to hospital and how it changes as the patient undergoes treatment and is hopefully cured. For example, recent evidence suggests that during infection, T lymphocytes may become ‘exhausted’ and unable to fight infection and the exhausted state can be identified by expression of surface molecules such as programmed cell death protein|1 (PD-1) and lymphocyte activation gene 3 protein (LAG-3). It is important to know if exhaustion can be reversed following treatment or whether we need to stimulate new populations of T lymphocytes. By understanding these nuanced changes in immune cells in our blood, we can design ways to improve how vaccines and drugs work in concert with immune cells, and understand why some patients might relapse from their disease or develop PKDL. Flow cytometry is a central tool for immunologists and plays a critical role in uncovering mechanisms of immunity and in assessing how well vaccines work and biomarkers of drug response. It uses antibodies that recognize specific molecules or markers on the surface or inside immune cells, such as those mentioned above, that help us predict their function. These antibodies are fluorescently labelled and the fluorescent signal can be detected by exposing each cell individually to laser light as they pass through a small aperture, the essence of flow cytometry.
For flow cytometry to be beneficial in this project, we needed to purchase five new flow cytometers that could meet exacting standards. They needed to be sufficiently sensitive to identify rare cell populations, often with low levels of surface marker expression. For multicentre research projects, reproducibility of data between sites is essential. Hence, we needed excellent inter-machine reproducibility and the Figure 2. Initial training course with recently appointed flow managers (Credit: Dr Karen Hogg, University of York) | 10 manufacturer had to be able to provide service support across the region. In our search for the right flow cytometer to support the consortium, we settled upon the CytoFLEX, Beckman Coulter Life Sciences’ research flow cytometer, which uses avalanche photodiode detection to arrive at the required level of sensitivity. With assistance from Beckman Coulter, we devised and have run initial training courses with a group of recently appointed flow managers from each partner country, to share standard operating procedures, develop high-level data analysis strategies as well as to provide instruction in routine instrument maintenance.
Beckman Coulter also provides another important aid to reducing errors in flow cytometry for multisite projects such as this, namely freeze-dried antibody cocktails (DURAClone panels) [10], that allow highly multiplexed phenotyping of small volumes of blood added directly to a single tube. Particularly for investigators in remote locations, the use of dry, preformulated reagents, rather than liquid (‘wet’) antibodies, removes the need for a cold chain. Equally importantly, staining of cells when manual mixing of 15 or 16 antibodies is required can introduce data inconsistencies when conducted by different individuals and at different locations.
Together, these innovations have allowed us to establish a new network for flow cytometry in East Africa that will allow us to identify and functionally characterize and identify the types of immune cells present in the blood during these devastating diseases. We will match this data with similar multiplexed techniques in pathology to compare blood immune cell profiles with those of cells found in the skin, to give a more complete picture of the host response to infection before and after treatment or vaccination.

Future Directions

As mentioned, we are currently in the midst of an efficacy trial of our therapeutic vaccine, ChAd63-KH. The technology we are using is similar to that being used by researchers at the university of Oxford to develop a coronavirus vaccine. In short, we introduce two genes from Leishmania parasites (KMP-11 and HASPB1) into a well-studied chimpanzee adenovirus (ChAd63 viral vector). After vaccination with this vaccine, host cells become infected with the virus and express the Leishmania proteins in a way that can be recognized efficiently by the immune system. We are particularly interested in how well this vaccine can generate T|cells to fight the infection.
With the first of our clinical objectives now well underway – the ongoing therapeutic clinical trial in patients with PKDL will be completed in mid-2021 – we have two additional goals. The next, funded by EDCTP, is to start a new clinical trial to determine whether the vaccine can prevent progression from VL to PKDL. And finally, we hope to develop a human challenge model of leishmaniasis to test the vaccine for its ability to protect against infection by different forms of parasite. This would open the way to the development of a cost-effective prophylactic vaccine to prevent these diseases occurring in vulnerable populations across the world.
Our research also has larger ambitions for the long term. Our East African partners are also linked together through their work on leishmaniasis in drug development, as members of the Leishmaniasis East Africa Platform group, established to help coordinate drug development activities in the region by the Drugs for Neglected Diseases Partnership. Central questions about why the disease varies between countries are being addressed, and the increased capacity for flow cytometry will additionally support patient monitoring during drug trials conducted by DNDi or other groups. Indeed, through the capacity building this project provides, we hope this project will extend its reach beyond leishmaniasis, providing muchneeded support for research on other neglected diseases of poverty that affect people in the region, including bacterial, fungal, other parasitic and viral diseases. By continuing to demonstrate the analytical power of flow cytometry and its role in helping design much-needed therapies, we hope to open up additional discovery research possibilities for colleagues in Africa and around the world.
The research described in this article is part of the EDCTP2 programme supported by the European Union (grant number RIA2016V-1640; PREV_PKDL; https://www.prevpkdl.eu).
The author
Paul Kaye PhD, FRCPath, FMedSci
Hull York Medical School, University of York, York, UK
E-mail: paul.kaye@york.ac.uk

Elevated hormone flags liver problems in mice with methylmalonic acidemia. Researchers have discovered that a hormone, fibroblast growth factor 21 (FGF21), is extremely elevated in mice with liver disease that mimics the same condition in patients with methylmalonic acidemia (MMA), a serious genomic disorder. Based on this finding, medical teams treating patients with MMA will be able to measure FGF21 levels to predict how severely patients’ livers are affected and when to refer patients for liver transplants.

The findings also might shed light on more common disorders such as fatty liver disease, obesity and diabetes by uncovering similarities in how MMA and these disorders affect energy metabolism and, more specifically, the function of mitochondria, the cells’ energy powerhouses. The study was conducted by researchers at the National Human Genome Research Institute (NHGRI), part of the National Institutes of Health.

“Findings from mouse studies usually take years to translate into health care treatment, but not in this case,” said Charles P. Venditti, M.D., Ph.D., senior author and senior investigator in the NHGRI Medical Genomics and Metabolic Genetics Branch. “We can use this information today to ensure that patients with MMA are treated before they develop severe complications.”

MMA is a genomic disease that impairs a person’s ability to break down food proteins and certain fatty acids. The condition affects roughly 1 in 50,000 children born in the United States and can be detected through newborn screening. Children with MMA suffer from frequent life-threatening metabolic crises when they encounter a minor viral illness or other stressors like trauma, dietary imbalance or surgery. They must adhere to a special low-protein diet and take various supplements their entire lives.

The NHGRI team created a new mouse model and used it to discover key pathways that were affected during a fasting challenge to model a metabolic crisis in a patient with MMA. It enabled them to identify markers that they could then measure in MMA patients to assess the severity of the dysfunction in their mitochondria, specifically in the liver.
The MMA mice also allowed them to study the response to liver-directed gene therapy and to compare the findings in patients after liver transplant surgery. Liver transplants give patients with MMA a missing enzyme and ease some of the symptoms, but do not cure the disease. Kidney transplantation, on the other hand, is necessary when these patients reach terminal stages of renal failure, an expected chronic complication of MMA. Selecting which patients would benefit from a liver or combined liver/kidney transplant as opposed to just a kidney transplant is an important clinical decision for families and their clinicians.

“We found that having MMA, whether in a mouse or person, causes stress pathways to be chronically activated and can impair their ability to respond to acute stress,” said Irini Manoli, M.D., Ph.D., lead author and associate investigator in NHGRI’s Medical Genomics and Metabolic Genetics Branch. “Our new markers can accurately predict how effective a therapy, whether cellular or genomic, might be for the patients.”
National Human Genome Research Institutewww.genome.gov/news/news-release/Elevated-hormone-flags-liver-problems-in-mice-with-methylmalonic-acidemia-MMA

by Dr Jacqueline Gosink
Gastrointestinal complaints are very common and can be difficult to diagnose. Among the many causes are genetic deficiencies in digestive enzymes. Molecular genetic analysis of polymorphisms in the patient’s DNA can determine if inborn enzyme deficits are behind the digestive problems, aiding differential diagnostics. Primary lactose intolerance, for example, is associated with polymorphisms in the regulatory region of the lactase gene (LCT), whereas hereditary fructose intolerance (HFI) is caused by mutations in the aldolase B gene (ALDOB). A PCR-based DNA microarray provides parallel determination of the two main lactose intolerance-associated polymorphisms (LCT‑13910C/T and LCT‑22018G/A ) as well as the four HFI-associated mutations (A149P, A174D, N334K and del4E4). The fast and simple determination includes fully automated data evaluation, ensuring highly standardized results.

Lactose intolerance

Primary lactose intolerance is a genetically caused deficiency of lactase, the enzyme responsible for splitting lactose into its constituent sugars glucose and galactose. In affected patients, undigested lactose is fermented in the ileum and large intestine, producing by-products such as short-chain fatty acids, methane and hydrogen, which cause the typical symptoms of abdominal pain, nausea, meteorism and diarrhea. Secondary manifestations include deficiencies, for example of vitamins, and as a result unspecific symptoms such as fatigue, chronic tiredness and depression.
Lactose intolerance represents the natural state in mammals. Lactase activity decreases after weaning and in adulthood is often only a fraction of the activity in infancy. Some humans, however, retain the ability to metabolize lactose into adulthood due to specific genetic variants. The frequency of lactase persistence is around 35% worldwide, although it varies greatly between different population groups. It is prevalent in regions with a long tradition of pastoralism and dairy farming, for example in Europe and in populations of European descent. In large parts of eastern Asia, on the other hand, almost 100% of the population is lactose intolerant.
In addition to the primary genetically caused form of lactose intolerance there is also the secondary acquired form. This develops as a result of damage to the intestine, for example from other gastrointestinal diseases such as Crohn’s disease, coeliac disease, infectious enteritis or injury from abdominal surgery. The two forms need to be distinguished diagnostically because of the need for different treatment regimes. Whereas individuals with primary lactose intolerance must adhere to a lactose-free or low-lactose diet for life or alternatively take lactase supplements, those with secondary lactose intolerance need only restrict their dairy intake until the intestinal epithelium has regenerated through treatment of the underlying cause.
Diagnostics of lactose intolerance
Classic diagnostic tests for lactose intolerance are the hydrogen breath test and blood glucose tests, with which the patient’s ability to metabolize lactose is examined. However, these tests have a low specificity and sensitivity and are influenced by individual factors such as the composition of intestinal flora, colonic pH, gastrointestinal motility and sensitivity to lactose fermentation products. Moreover, they cannot distinguish between the primary and secondary forms of lactose intolerance. Molecular genetic testing complements these methods, enabling verification or exclusion of primary lactose intolerance with high probability, as well as differentiation of the primary and secondary forms. Genetic testing is, moreover, a non-invasive and more comfortable examination, which does not carry the risk of provoking symptoms of lactose intolerance in non-lactase-persistent individuals.
LCT polymorphisms
The main mutations associated with lactase persistence are LCT‑13910C>T and LCT‑22018G>A, which are located in the regulatory region of the lactase gene. According to current knowledge, homozygous carriers of the wild-type variants LCT‑13910CC and LCT‑22018GG develop lactose intolerance, while heterozygous carriers of the variants LCT‑13910CT and LCT‑22018GA only show corresponding symptoms in stress situations or with intestinal infections. Homozygous carriers of the mutant variants LCT‑13910TT and LCT‑22018AA are lactose tolerant as adults. These two polymorphisms are strongly coupled.

Hereditary fructose intolerance

HFI is caused by mutations in the gene for aldolase B, an enzyme essential for fructose metabolism. The mutations result in a reduction or loss in activity or stability of aldolase B, which is responsible for catalysing the breakdown of fructose-1-phosphate (F-1-P) to dihydroxyacetone phosphate and glyceraldehyde. The toxic intermediate F-1-P then accumulates in the body, causing symptoms such as nausea, vomiting and digestive disorders and in the longer term liver damage. HFI is a rare disease, occurring, for example, with a prevalence of 1 in 20¦000 in Europe. It manifests already in childhood, but may remain undiagnosed due to patients’ natural dislike of sweets, fruits and vegetables.
In addition to HFI, intolerance to fructose can also be caused by deficits in the transport of fructose into the enterocytes. This form is known as intestinal fructose intolerance or fructose malabsorption. It is much more common than HFI, occurring with a prevalence of about 30%. It is important to distinguish HFI from fructose malabsorption, because of the resulting difference in dietary requirements. Patients with HFI must completely eliminate fructose and its precursors (e.g. sucrose, sorbitol) from their diet to prevent damage to their organs. Patients with fructose malabsorption, however, should follow a fructose-restricted diet.
Diagnostics of HFI
Intolerance to fructose is usually diagnosed by means of the hydrogen breath test, in which a defined amount of fructose is ingested and then the amount of hydrogen in the exhaled air is measured. In patients with HFI, however, the intake of fructose carries the risk of a severe hypoglycaemic reaction. Therefore, a molecular genetic test for HFI should always be performed before a fructose load test. Early diagnosis of HFI is particularly important to avoid permanent damage to the liver, kidney and small intestine.
ALDOB mutations
In Europe the most frequent mutants associated with HFI are the amino acid substitutions A149P, A174D, N334K (in Human Gene Mutation Database nomenclature) and the deletion del4E4 in the aldolase B gene. For HFI to manifest, both alleles of an individual’s DNA must be affected by a mutation. In homozygous genotypes, the two alleles contain the same mutation (paternal and maternal inheritance). If the two alleles exhibit different mutations, this is referred to as a compound heterozygous HFI genotype.

Parallel genetic analysis

Molecular genetic determination of the polymorphisms associated with lactose intolerance and HFI enable diagnosis of these genetic conditions with high certainty. The EUROArray Lactose/Fructose Intolerance Direct enables simultaneous detection of the lactose-intolerance-associated polymorphisms ‑13910C/T and ‑22018G/A and the HFI-associated mutations A149P, A174D, N334K and del4E4. Thus, the two genetically caused metabolic disorders can be assessed with a single test.
he test can be performed on whole blood samples, eliminating the need for costly and time-consuming DNA isolation. In the test procedure (Fig. 1), the sections of DNA containing the alleles are first amplified by multiplex PCR using highly specific primers. During this process the PCR products are labelled with a fluorescent dye. The PCR mixture is then incubated with a microarray slide containing immobilized DNA probes. The PCR products hybridize with their complementary probes and are subsequently detected via the emission of fluorescence signals. The data is evaluated fully automatically using EUROArrayScan software (Fig. 2), and in the case of positive results, homozygous and heterozygous states are differentiated. Numerous integrated controls ensure high reliability of results, for example, by verifying that there are no other rare mutations in direct proximity to the tested positions which could interfere with the analysis.

Studies on blood donors

The performance of the EUROArray was investigated using 116 precharacterized samples from blood donors in Germany and from quality assessment schemes. The EUROArray revealed a sensitivity of 100% and a specificity of 100% with respect to the reference molecular genetic method.

Conclusions

Diagnosis of gastrointestinal disorders often involves a long and challenging process of diagnostic tests and restrictive diets. Since lactose and fructose are widely consumed in many diets, it is important to consider intolerance to these sugars during the diagnostic work-up. Simple genetic analysis enables primary lactose intolerance and HFI to be confirmed or excluded as the cause of gut problems. The parallel analysis offered by the EUROArray enables especially fast and effective diagnostics. Patients diagnosed with these genetic conditions can promptly adapt their diets to ease their symptoms. If the analysis is negative, the physician can focus on searching for other causes of the digestive complaints. The molecular genetic analysis thus provides valuable support for the gastroenterology clinic.
The author
Jacqueline Gosink PhD
EUROIMMUN AG, 23560 Lubeck, Germany