Visceral leishmaniasis (Kala-azar)- Causes, Lab diagnosis, Treatments

Visceral leishmaniasis (VL), also known as kala-azar, is a serious and potentially fatal disease caused by a group of protozoan parasites belonging to the genus Leishmania. These parasites are transmitted to humans by the bite of infected female phlebotomine sandflies, which are small blood-sucking insects that are active at night. VL is the most severe form of leishmaniasis, affecting the internal organs such as the spleen, liver, and bone marrow. It is characterized by irregular bouts of fever, weight loss, enlargement of the spleen and liver, and anemia. If left untreated, VL can be fatal in over 95% of cases.

VL is a neglected tropical disease that affects some of the poorest and most vulnerable populations in the world. It is associated with malnutrition, population displacement, poor housing, a weak immune system, and a lack of financial resources. An estimated 700 000 to 1 million new cases occur annually worldwide, with only a small fraction reported to the World Health Organization (WHO). Most cases occur in Brazil, east Africa, and India, where VL is endemic. VL can also occur in other regions, such as the Mediterranean basin, the Middle East, and Central Asia. VL has outbreak and mortality potential, especially in areas where HIV co-infection is prevalent.

VL can be diagnosed by various methods, such as microscopy, culture, animal inoculation, antigen detection, antibody detection, and molecular techniques. The diagnosis depends on the availability of laboratory facilities, the sensitivity and specificity of the tests, and the clinical presentation of the patient. The treatment of VL depends on the infecting Leishmania species, the geographical location, the drug availability and cost, and the patient`s condition. The standard treatment consists of pentavalent antimonial compounds, which are effective in most regions except in Bihar (India), where resistance has emerged. Other drugs include amphotericin B, liposomal amphotericin B, paromomycin, and miltefosine.

The prevention and control of VL require a combination of strategies, such as vector control, case detection and treatment, surveillance and monitoring, health education, and research. The WHO has set a target to eliminate VL as a public health problem in selected countries by 2020. This means reducing the annual incidence to less than 1 case per 10 000 people at the district level. To achieve this goal, WHO supports national programs and partners to implement effective interventions and strengthen health systems.

VL is a serious but preventable and treatable disease that affects millions of people worldwide. It is important to raise awareness about its causes, symptoms, diagnosis, and treatment among health workers and the general public. It is also essential to improve access to quality health care and diagnostic services for those who need them. Together, we can fight against this neglected tropical disease and save lives.

Visceral leishmaniasis (VL), also known as kala-azar, is caused by protozoan parasites of the genus Leishmania. There are several species of Leishmania that can cause VL, depending on the geographical region. The main ones are:

• Leishmania donovani, which is found in India and Eastern Africa.

• Leishmania infantum, which is found in the Mediterranean area, Central Asia and South America.

• Leishmania chagasi, which is considered synonymous with L. infantum and is found in Central and South America.

These parasites are transmitted by the bite of infected female phlebotomine sandflies, which are small blood-sucking insects that are active at night. The sandflies breed in moist and shady places, such as animal burrows, cracks in walls, piles of rubbish, and vegetation. Different species of sandflies can transmit different species of Leishmania. The main vectors are:

• Phlebotomus spp., which is common in the Old World (Africa, Asia, and Europe).

• Lutzomyia spp., which is common in the New World (the Americas).

The sandflies become infected when they feed on animals or humans that carry the parasites in their blood or tissues. The parasites multiply inside the sandflies and transform into a form that can infect a new host. When the sandflies bite a susceptible host, they inject the parasites along with their saliva.

The parasites then invade the cells of the immune system, such as macrophages, and multiply inside them. The infected cells migrate to various organs, especially the spleen, liver, and bone marrow, where they cause inflammation and damage. The parasites can also spread through blood transfusions or contaminated needles.

The risk of getting VL depends on several factors, such as:

• The presence and density of sandflies in the area.

• The presence and abundance of animal reservoirs that can maintain the parasite cycle, such as dogs, rodents, foxes, and marsupials.

• The genetic susceptibility and immune status of the host, which can influence the outcome of infection. People with HIV/AIDS or other conditions that weaken their immune system are more likely to develop severe disease.

• The environmental and social conditions that affect exposure to sandfly bites, such as poverty, malnutrition, deforestation, urbanization, and lack of vector control.

Visceral leishmaniasis (VL), also known as kala-azar, is the most severe form of leishmaniasis. It affects several internal organs, mainly the spleen, liver, and bone marrow. If left untreated, it can be fatal.

The symptoms of VL may vary depending on the type and strain of the parasite, the immune status of the host, and the duration of the infection. Some people may have no symptoms at all or only mild signs that are easily overlooked. Others may develop a chronic or progressive disease that can last for months or years.

The most common symptoms of VL are:

• Fever: This is usually the first and most persistent symptom. It may last for weeks or months and may be accompanied by chills, night sweats, and fatigue.

• Weight loss: This is due to loss of appetite, malabsorption of nutrients, and increased metabolic demands of the infection.

• Splenomegaly: This is the enlargement of the spleen, which is responsible for filtering blood and producing antibodies. The spleen may become so large that it can be felt or seen in the abdomen. Splenomegaly can cause abdominal pain, discomfort, and fullness. It can also reduce the number of blood cells and platelets, leading to anemia and bleeding problems.

• Hepatomegaly: This is the enlargement of the liver, which is involved in detoxifying substances and producing bile. The liver may also become inflamed (hepatitis) or scarred (cirrhosis). Hepatomegaly can cause jaundice (yellowing of the skin and eyes), dark urine, pale stools, and itching.

• Anemia: This is a condition in which the red blood cells are reduced in number or function. Red blood cells carry oxygen to the tissues and organs. Anemia can cause pallor (pale skin), weakness, dizziness, shortness of breath, and chest pain.

• Leukopenia: This is a condition in which the white blood cells are reduced in number or function. White blood cells fight infections and inflammation. Leukopenia can increase the risk of secondary infections by bacteria, fungi, or viruses.

• Thrombocytopenia: This is a condition in which the platelets are reduced in number or function. Platelets help with blood clotting and prevent bleeding. Thrombocytopenia can cause bruising, bleeding gums, nosebleeds, and internal bleeding.

• Hypergammaglobulinemia: This is a condition in which the level of gamma globulins (a type of antibody) is abnormally high in the blood. Gamma globulins are produced by plasma cells in response to antigens (foreign substances). Hypergammaglobulinemia can cause a reversal of the albumin-globulin ratio (a measure of protein balance) and hyperviscosity syndrome (a disorder of blood flow due to the thickening of the blood).

• Skin changes: Some people with VL may develop skin lesions or changes in skin color. The skin lesions may be nodules, ulcers, or plaques that resemble those of cutaneous leishmaniasis (CL), but they are usually fewer and smaller. The skin color changes may be hyperpigmentation (darkening) or depigmentation (lightening) of certain areas, especially on the face, hands, feet, and abdomen. These changes are more common in people with dark skin and are sometimes called kala-azar dermal leishmaniasis (KDL) or post-kala-azar dermal leishmaniasis (PKDL).

The symptoms of VL may worsen over time and lead to serious complications such as:

• Hemorrhage: This is excessive bleeding that can occur internally or externally. It can be caused by low platelet count, impaired clotting factors, or damage to blood vessels. Hemorrhage can result in shock (a life-threatening condition in which blood pressure drops and organs fail) or death.

• Infections: These are illnesses caused by microorganisms that invade the body. People with VL have a weakened immune system that makes them more susceptible to infections by bacteria, fungi, or viruses. Some common infections that affect people with VL are pneumonia (lung infection), tuberculosis (TB), diarrhea (intestinal infection), malaria (parasitic infection), HIV/AIDS (viral infection), and leishmaniasis co-infection (infection by another type of Leishmania parasite).

• Organ failure: This is a condition in which one or more organs stop working properly or completely. It can be caused by direct damage from the parasite or its toxins, inflammation, ischemia (lack of blood supply), or secondary infections. The most affected organs are the spleen, liver, kidneys, heart, lungs, and brain.

The diagnosis of visceral leishmaniasis (VL) or kala-azar requires direct or indirect evidence of the infection by Leishmania parasites. Direct evidence involves the detection of the parasite or its components in tissue samples, while indirect evidence involves the detection of antibodies or antigens in blood or other body fluids.

Direct evidence

Microscopy

The gold standard for the diagnosis of VL is the demonstration of amastigotes (the intracellular form of the parasite) in smears of tissue aspirates. The most common samples are:

• Peripheral blood sample

• Bone marrow biopsy: obtained by sternal or iliac crest puncture

• Splenic aspirate: obtained by a splenic puncture

• Enlarged lymph node

The smears of aspirates are stained with Leishman, Giemsa, or Wright`s stain and examined under oil immersion microscopy. Amastigotes can be seen within macrophages or occasionally extracellularly. They appear as small oval bodies with a dark nucleus and a rod-shaped kinetoplast.

The sensitivity and specificity of microscopy vary depending on the sample type and the parasite load. Splenic aspiration is the most sensitive method (95% to 99%) but also carries a risk of hemorrhage. Bone marrow biopsy shows amastigotes in approximately two-thirds of patients. Lymph node aspirates are less useful, especially in Indian VL. Peripheral blood smears have a low sensitivity (less than 10%) but may show a diurnal periodicity, with more positive smears during the day than at night.

Culture

Another method for direct detection of the parasite is culture. Different samples of blood, splenic, bone marrow, or lymph node aspirates can be inoculated into a culture medium and incubated at 22 to 24°C for one to four weeks. The most commonly used medium is NNN (Novy-MacNeal-Nicolle), which consists of defibrinated rabbit blood on a slant of Lockes solution agar. Another medium that can be used is Schneiders insect medium with 30% fetal calf serum.

The culture fluid is examined weekly for the presence of promastigotes (the extracellular flagellated form of the parasite) by microscopy. Promastigotes can be seen as motile slender bodies with long flagellum.

Culture is more sensitive than microscopy but less sensitive than splenic aspiration. It also takes longer and requires special laboratory facilities and skills. The advantage of culture is that it allows for species identification and drug susceptibility testing.

Animal inoculation

A very sensitive but rarely used method for direct detection of the parasite is animal inoculation. In this method, the sample material is inoculated intraperitoneally or intradermally into susceptible animals, such as hamsters, mice, or gerbils. The animals are kept at 23 to 26°C and examined periodically for signs of infection. Amastigotes can be demonstrated in smears taken from ulcers or nodules at the inoculation sites or from the spleen.

Animal inoculation is not suitable for routine diagnosis because it is time-consuming, expensive, and ethically questionable. It may be useful for research purposes or for isolating parasites that are difficult to grow in culture.

Molecular techniques

Molecular techniques based on polymerase chain reaction (PCR) have been developed for the detection of Leishmania DNA in various samples, such as blood, bone marrow, spleen, lymph node, skin lesions, urine, saliva, or buffy coat. PCR can amplify specific regions of Leishmania DNA and detect them by gel electrophoresis or hybridization probes.

PCR has several advantages over conventional methods: it is highly sensitive (up to 100%), specific (can differentiate between species and strains), fast (results within hours), and non-invasive (can use urine or saliva samples). However, PCR also has some limitations: it requires sophisticated equipment and trained personnel, it is prone to contamination and false positives, it is not standardized or validated across different laboratories, and it is expensive.

Indirect evidence

Detection of antigen

The detection of Leishmania antigen in serum or other body fluids can provide indirect evidence of infection. However, the concentration of antigen is usually very low and difficult to measure. Several methods have been developed for antigen detection, such as enzyme-linked immunosorbent assay (ELISA), latex agglutination test (LAT), immunochromatographic test (ICT), and PCR.

These methods have shown variable sensitivity and specificity depending on the antigen used and the sample type. Some antigens that have been used are rk39 (a recombinant kinesin-related protein), K9 (a recombinant protein derived from L. chagasi), K26 (a recombinant protein derived from L. infantum), and rK28 (a fusion protein composed of rk39, K9, and K26).

Two non-invasive antigen detection tests in urine for VL are under evaluation: ICT-KIM (based on rk39) and ICT-LF (based on K26). They have shown good specificity but only low to moderate sensitivity (48% to 87%).

Detection of antibodies

The detection of antibodies against Leishmania antigens in serum or other body fluids can also provide indirect evidence of infection. However, antibodies may persist after the cure or cross-react with other infections. Several methods have been developed for antibody detection, such as complement fixation test (CFT), indirect immunofluorescent antibody test (IFAT), counterimmunoelectrophoresis (CIEP), ELISA, direct agglutination test (DAT), immunochromatographic test (ICT), and Western blot.

These methods have shown variable sensitivity and specificity depending on the antigen used and the sample type. Some antigens that have been used are whole parasite lysate, rk39, K39/K26 fusion protein, rK28 fusion protein, rKRP42 protein derived from L. donovani, rKE16 protein derived from L. infantum, rLcKin protein derived from L. chagasi, rLdF12 protein derived from L. donovani, rLdF18 protein derived from L. donovani, rLdF29 protein derived from L. donovani, rLdF32 protein derived from L. donovani, rLdF38 protein derived from L. donovani, rLdF48 protein derived from L. donovani, rLdF51 protein derived from L. donovani, rLdF54 protein derived from L. donovani, rLdF56 protein derived from L. donovani, rLdF58 protein derived from L. donovani, rLdF59 protein derived from L. donovani, rLdF60 protein derived from L

Visceral leishmaniasis (VL) is a serious and potentially fatal disease that requires prompt and adequate treatment. The choice of treatment depends on several factors, such as the Leishmania species that caused the infection, the geographic location of the patient, the availability and cost of the drugs, the patient`s immune status, and the presence of any co-infections.

The main drugs available for the treatment of VL are:

• Pentavalent antimonials: These are the mainstays of therapy for most forms of VL. They are given by intravenous or intramuscular injection for 20 to 30 days. The most commonly used preparations are sodium stibogluconate and meglumine antimoniate. They have a cure rate of over 90% in most regions, except in Bihar (India), where there is widespread resistance to antimony.

• Amphotericin B: This is an antifungal drug that also has activity against Leishmania parasites. It is given by intravenous infusion for 15 to 20 days. It is effective against pentavalent-resistant VL and has a cure rate of over 90%. However, it has serious side effects, such as kidney toxicity, fever, and chills.

• Liposomal amphotericin B: This is a modified form of amphotericin B that is encapsulated in liposomes (tiny fat particles). This reduces its toxicity and enhances its efficacy. It is given by intravenous infusion for 5 to 10 days. It is the preferred treatment for VL in India and Nepal, where it has a cure rate of over 95%. It is also recommended for VL patients co-infected with HIV.

• Paromomycin: This is an aminoglycoside antibiotic that also has activity against Leishmania parasites. It is given by intramuscular injection for 21 days. It is effective against VL caused by L. donovani and L. infantum. It has a cure rate of over 90% and has fewer side effects than antimonials or amphotericin B.

• Miltefosine: This is an oral drug that was originally developed as an anticancer agent. It is effective against VL caused by L. donovani and L. infantum. It is given for 28 days and has a cure rate of over 90%. However, it has some drawbacks, such as gastrointestinal side effects, teratogenicity (causing birth defects), and the potential emergence of resistance.

Some of these drugs can be used in combination to reduce the duration, toxicity, and cost of treatment, as well as to prevent or overcome resistance. For example, a combination of miltefosine and liposomal amphotericin B has been shown to be highly effective for treating VL in patients co-infected with HIV.

The treatment of VL should be accompanied by supportive care, such as blood transfusion, nutritional supplementation, and management of any co-infections or complications. The patient should also be monitored for clinical response, adverse reactions, and relapse.

There is no vaccine or drug to prevent visceral leishmaniasis (VL) infection. The best way to prevent infection is to avoid or reduce exposure to sand fly bites, which transmit the Leishmania parasite. The following preventive measures are recommended for travelers and residents in endemic areas:

• Avoid outdoor activities, especially from dusk to dawn, when sand flies are most active.

• Wear long-sleeved shirts, long pants, socks, and shoes, and tuck the shirt into the pants to minimize the amount of exposed skin.

• Apply insect repellent to exposed skin and under the ends of sleeves and pant legs. The most effective repellents are those that contain DEET (N, N-diethylmetatoluamide).

• Stay in well-screened or air-conditioned areas. Sand flies are much smaller than mosquitoes and can get through smaller holes.

• Spray living and sleeping areas with an insecticide to kill insects.

• Use a bed net and tuck it under the mattress. If possible, use a bed net that has been treated with a pyrethroid-containing insecticide. The same treatment can be applied to screens, curtains, sheets, and clothing.

For people who have had VL and are coinfected with human immunodeficiency virus (HIV), secondary prophylaxis is recommended to prevent relapse of VL. Secondary prophylaxis consists of regular administration of antileishmanial drugs after the initial treatment of VL until the immune system is restored by antiretroviral therapy. The choice of antileishmanial drug depends on the availability, cost, and resistance patterns in different regions. Some of the drugs that have been used for secondary prophylaxis are:

• Pentavalent antimonials (sodium stibogluconate or meglumine antimoniate): These are the mainstays of therapy for all forms of leishmaniasis, but resistance is a problem in some areas, especially in Bihar, India. The usual dose is 20 mg/kg per day by intravenous or intramuscular injection for 28 days.

• Amphotericin B: This is an antifungal drug that has activity against Leishmania parasites. It can be given as conventional amphotericin B deoxycholate or as liposomal amphotericin B, which has fewer side effects and higher efficacy. The dose and duration vary depending on the formulation and the severity of infection.

• Miltefosine: This is an oral drug that has been shown to be effective against VL in India and other regions. It has a long half-life and can cause teratogenicity, so it should be used with caution in women of childbearing age. The usual dose is 50 to 100 mg per day for 28 days.

• Paromomycin: This is an aminoglycoside antibiotic that has activity against Leishmania parasites. It can be given as an intramuscular injection or as a topical cream for cutaneous leishmaniasis. The usual dose is 15 mg/kg per day for 21 days.

• Allopurinol: This is a drug that inhibits the synthesis of uric acid and has some antileishmanial activity. It can be used as an adjunctive therapy or as a prophylactic agent in HIV-VL co-infection. The usual dose is 10 to 20 mg/kg per day for 6 to 12 months.

Secondary prophylaxis should be continued until the CD4 cell count is above 200 cells/microliter for at least 6 months after starting antiretroviral therapy. Regular monitoring of clinical signs, laboratory tests, and parasitological markers is advised to detect any relapse or toxicity of the drugs.

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