Table of Contents
- Introduction to Visceral leishmaniasis (Kala-azar)
- Post-Kala-Azar Dermal Leishmaniasis (PKDL)
- Causes of Visceral leishmaniasis (Kala-azar)
- Symptoms of Visceral leishmaniasis (Kala-azar)
- Direct evidence
- Indirect evidence
- 1. Detection of antigen
- 2. Detection of antibodies
- 3. molecular diagnosis
- 4. Nonspecific serum test
- 5. Lelshmanin skin test (Montenegro test)
- 6. Blood count
- Treatments of Visceral leishmaniasis (Kala-azar)
Introduction to Visceral leishmaniasis (Kala-azar)
- Visceral leishmaniasis, also known as systemic leishmaniasis or kala-azar, is sometimes referred to as black fever or Dumdum fever in Asia.
- The disease usually appears 3 to 6 months after being bitten by a sandfly.
- It primarily infects the reticuloendothelial system, damaging internal organs such as the spleen and liver.
- It affects bone marrow and the immune system due to damage to these organs.
- Kala-azar is the most severe form of leishmaniasis and is usually fatal if left untreated.
- Annually, an estimated 50,000 to 90,000 new cases of visceral leishmaniasis occur worldwide, with only 25 to 45% reported to the WHO.
- It remains one of the top parasitic diseases with significant outbreak and mortality potential.
- This disease is the second-largest parasitic killer in the world after malaria, responsible for an estimated 20,000 to 30,000 deaths each year worldwide.
Post-Kala-Azar Dermal Leishmaniasis (PKDL)
- Post-kala-azar dermal leishmaniasis (PKDL) typically arises as a consequence of visceral leishmaniasis, appearing anywhere from one month to several years after treatment.
- In PKDL, parasites infiltrate the skin, leading to macular, papular, or nodular rashes, commonly on the face, upper arms, trunk, and other areas of the body.
- Approximately 5-10% of kala-azar patients develop PKDL.
- This condition mainly occurs in East Africa and the Indian subcontinent.
- While PKDL is not life-threatening, it is still considered a potential source of Leishmania infection.
Causes of Visceral leishmaniasis (Kala-azar)
- Visceral leishmaniasis (Kala-azar) is caused by:
- Leishmania donovani in India and Eastern Africa
- L. infantum in the Mediterranean area
- L. chagasi
- The disease is transmitted through the bite of small blood-sucking sandflies, primarily of the Phlebotomus and Sergentomyia genera in the Old World.
- In the New World, the primary vector is the Lutzomyia sandfly species.
- Sandflies are very small insects, less than 3.5 mm in length, about one-third the size of a small mosquito.
- They thrive in moist climates as they cannot withstand dehydration.
- These flies are nocturnal and are typically found in burrows, under rocks, or other shelters during the day.
- Both male and female sandflies obtain carbohydrates from plant juices, but females require a blood meal to reproduce.
- It is during this blood meal that the vector fly transmits the protozoa to the human host.
Symptoms of Visceral leishmaniasis (Kala-azar)
- The incubation period for visceral leishmaniasis (Kala-azar) ranges from 3 to 6 months, with symptoms potentially appearing up to 2 years after infection.
- At-risk populations include preschool children, immunocompromised individuals, and those who are undernourished.
- The frequency of visceral leishmaniasis has increased in patients with AIDS, intravenous drug users, and those who share contaminated syringes.
Early symptoms:
- Weight loss
- Weakness
- Fever lasting for weeks or months
- Enlarged spleen
- Anemia due to decreased blood cell production
- Bleeding
- Peculiar darkening of the skin
- Swollen lymph nodes
- Dry, rough, and dark or pigmented skin
- Brittle hair that falls out
- The disease is usually fatal if left untreated
Lab diagnosis of Visceral leishmaniasis (Kala-azar)
Diagnosing visceral leishmaniasis (Kala-azar) in the laboratory relies on both direct and indirect evidence.
Direct evidence
1. Microscopy
Samples:
- Samples for diagnosis include:
- Peripheral blood samples
- Biopsy from bone marrow (obtained via sternal or iliac crest puncture)
- Splenic aspirates (obtained via splenic puncture)
- Enlarged lymph nodes
- Smears of these aspirates are stained with Leishman, Giemsa, or Wright’s stain for detecting amastigote forms under light microscopy.
- Amastigotes should be visualized within macrophages under oil immersion for accurate diagnosis.
- Buffy coat of peripheral blood can be used for microscopic examination, showing diurnal periodicity in positivity.
- Splenic aspiration, despite the risk of hemorrhage, is the most sensitive method (95%) for diagnosing leishmaniasis.
- Bone marrow biopsy detects amastigotes in about two-thirds of patients.
- Lymph node aspirates are less useful in diagnosing Indian kala-azar but may be employed elsewhere.
- Microscopy specificity is high, but sensitivity varies: spleen (93% to 99%), bone marrow (53-86%), lymph node aspirates (53-65%).
- Microscopic examination is the classic confirmatory test for visceral leishmaniasis.
2. Culture
- Inoculating different samples (blood, splenic or bone marrow aspirates, tissues) onto NNN medium.
- Samples are placed in the water of condensation and incubated at 22-24°C for 1-4 weeks.
- Weekly, culture fluid drops are examined under high power objective or phase contrast illumination for motile promastigotes.
- Promastigotes can be microscopically demonstrated within a few days to 4 weeks in positive cultures.
- Another biphasic medium, such as Schneider’s drosophila tissue culture medium with 30% fetal calf serum added, can also be utilized.
3. Animal inoculation
- This method is highly sensitive but not employed for routine diagnosis.
- It involves inoculating the material intraperitoneally or intradermally into the skin of the nose and feet of Chinese golden hamsters, kept at 23-26°C.
- In positive cases, amastigotes can be demonstrated in smears taken from ulcers or nodules that develop at the sites of inoculation or from the spleen.
Indirect evidence
1. Detection of antigen
- The concentration of antigen in serum or other body fluids is typically low.
- ELISA and PCR assays have been developed to detect leishmanial antigens.
- Two non-invasive antigen detection tests for VL in urine are currently undergoing evaluation.
- These tests demonstrate good specificity but exhibit low to moderate sensitivity (48-87%).
2. Detection of antibodies
a. The complement fixation test was initially used as the first serological method to detect serum antibodies in visceral leishmaniasis (VL).
b. Various specific leishmanial antigens prepared from cultures have been employed in several antibody detection tests, including:
c. Indirect immunofluorescent antibody test (IFAT)
d. Counter immunoelectrophoresis (CIEP)
e. ELISA or Western Blot assays using whole parasite lysate have shown high diagnostic accuracy in many studies but are challenging to implement in field settings. ELISA is highly sensitive (80-100%), but its specificity varies (80-94%) depending on the antigen used.
Two validated tests commonly used in field settings are:
f. Direct agglutination test (DAT)
- Widely used for kala-azar diagnosis, based on antigen-antibody reaction with trypsin-treated, stained, and formalin-preserved promastigotes.
- Positive reaction results in agglutination with specific antibodies from VL-positive patients.
- Operates at room temperature but requires electricity, refrigeration, or a well-equipped laboratory, limiting its utility.
- Sensitivity ranges from 91% to 100%, and specificity ranges from 72% to 100% in various studies.
g. rk 39 immunochromatographic test
- Specific, rapid, and non-invasive test method.
- Developed using a recombinant leishmanial antigen (rk 39) consisting of 39 conserved amino acids in the kinesin region of L. infantum.
- Test sensitivity is 98%, and specificity is 90%.
- Recommended by the National Vector Borne Disease Control Programme (NVBDCP) in India.
- Results obtainable within 5 minutes using either LCT or dipstick formats, making them suitable for field use.
3. molecular diagnosis
a. DNA Probe:
- A DNA probe is a molecular diagnostic method used for detecting specific sequences of DNA within a sample.
- It involves hybridizing a labeled DNA probe to its complementary sequence in the target DNA.
- This method is sensitive and specific, capable of identifying the presence of Leishmania DNA in clinical samples.
- DNA probes are useful for identifying the parasite's genetic material directly, aiding in rapid diagnosis.
b. PCR (Polymerase Chain Reaction):
- PCR is a highly sensitive molecular technique used to amplify specific segments of DNA.
- It involves repeated cycles of DNA denaturation, primer annealing, and DNA synthesis using a DNA polymerase enzyme.
- PCR can detect even small amounts of Leishmania DNA in clinical samples, making it highly sensitive for diagnosis.
- This method is widely used in research and clinical laboratories due to its ability to amplify and detect DNA rapidly and accurately.
4. Nonspecific serum test
a. Napier’s Aldehyde test
- In this method, 1 mL of clear serum from the patient is taken in a small test tube.
- A drop of 40% formaldehyde (formalin) is added, shaken, and left at room temperature.
- A control tube with normal serum is also prepared.
- A positive reaction is indicated by jellification and opacification of the test serum, resembling coagulated egg white, occurring within 3-30 minutes.
- About 85% of patients with a disease duration of 4 months or more show a positive reaction.
- The Aldehyde test is consistently negative in cutaneous leishmaniasis (CL).
- The sensitivity of this test is poor, as low as 34%, indicating it is nonspecific as it primarily reflects increased serum gamma-globulin levels.
b. Chopra’s antimony test
- This test involves taking 0.2 mL of serum diluted 1:10 with distilled water in a Dreyer’s tube.
- A few drops of a 4% solution of urea stilbamine are overlaid on the serum.
- A positive test is indicated by the formation of a white flocculant precipitate.
- This test is considered more sensitive than the Aldehyde test.
- Both tests may yield false-positive reactions in various other diseases such as multiple myeloma, liver cirrhosis, tuberculosis, leprosy, schistosomiasis, and African trypanosomiasis.
5. Lelshmanin skin test (Montenegro test)
- It is a Delayed Hypersensitivity Test (DHT) named after its discoverer, Montenegro, in South America.
- Intradermal injection of 0.1 mL of killed promastigote suspension (containing 10^6 washed promastigotes/mL) is administered on the dorsoventral aspect of the forearm.
- A positive test result is characterized by induration and erythema of 5 mm or more, typically appearing 48-72 hours after injection.
- A positive result indicates previous exposure to the leishmanial parasite.
- The test shows positive results in African kala-azar but not in Indian and Mediterranean kala-azar.
- In active kala-azar cases, the test is negative but can turn positive approximately 6-8 weeks after successful treatment of the disease.
- DHT testing is valuable for diagnosing cutaneous leishmaniasis and serves mainly as an epidemiological tool, with limited utility in diagnosing acute visceral leishmaniasis (VL).
6. Blood count
- Complete blood count reveals normocytic normochromic anemia and thrombocytopenia.
- Leukocyte count shows leukopenia with a relative increase in lymphocytes and monocytes.
- Eosinophil granulocytes are absent throughout the disease progression.
- As the disease advances, leukocyte counts progressively decrease to 1,000/mm³ of blood or lower.
- The leukocyte to erythrocyte ratio is significantly altered, potentially ranging from 1:200 to 1:100 (normal ratio is 1:750).
- Serum analysis indicates hypergammaglobulinemia and a reversal of the albumin
- ratio.
- Liver function tests show mild elevations.
Treatments of Visceral leishmaniasis (Kala-azar)
- The decision to initiate treatment for leishmaniasis depends on the clinical syndrome, the infecting Leishmania species, the immune status of the host, and the availability and cost of drugs.
- Kala-azar generally responds better to treatment compared to other forms of visceral leishmaniasis (VL).
- The standard treatment primarily involves pentavalent antimonial compounds, which are the drugs of choice in most endemic regions worldwide.
- Resistance to antimony has been reported in Bihar, India, where alternatives such as amphotericin-B deoxycholate or miltefosine are preferred.
- Two main pentavalent antimonial preparations are available: Sodium stibogluconate and meglumine antimoniate.
- Dosage typically involves a daily administration of 20 mg/kg via rapid intravenous (IV) infusion or intramuscular (IM) injection for 20-30 days.
- Cure rates exceed 90% in most regions except Bihar, where resistance has reduced cure rates to 36%.
- Other drugs used for treatment include Amphotericin-B, Liposomal amphotericin-B, Paromomycin, and Miltefosine.
