Emergence of Drug Resistance

Development of drug resistance has magnified the already grim Kala azar situation in the country. Almost a quarter of cases of Kala azar from Bihar in 1992 were resistant to sodium stibogluconate, with children. accounting for 6 thousand of the resistant cases(2). Resistance to various anti Kala azar drugs is reported to be primary (unresponsive from. the beginning) as well as secondary (resistance acquired after a variable degree of. initial response to the drug)(3). Drug resistance is presently a definite therapeutic challenge and likely to assume alarming proportions in the near future.

Emergence of drug resistance in Kala azar is to a great extent related to delay in diagnosis and treatment. An early diagnosis and appro­priate therapy is critical for ,preventing, drug resistance. However, clinical suspicion. and laboratory diagnosis may be delayed on account of several factors. 

Late Clinical Suspicion/Diagnosis

The classical clinical presentation of Kala azar, namely intermittent fever, splenomegaly, hepatomegaly, failure to thrive in intants, brittle hair, scaly skin arid bleeding, manifestations may not be observed in all the cases. Subclinical infection may manifest with transient fever and hepato-splenomegaly which may progress to frank Kala azar or may even spontaneously get cured itself(4). Such cases may easily be missed unless investigated during the acute phase of their illness. The diagnosis may also be delayed because of failure to appreciate its rare presentations like cirrhosis of liver, glomerulonephritis, secondary amyloidosis, papilledema, nodules on eyelids, polymorphic waxy non-ulcerating papules on skin, progressive tremors and even meningitis(3;5). Lymphatic leishmaniasis, characterized by fever and generalized lymph node enlargement without hepatosplenomegaly, is mostly confused with glandular tuberculosis(4).

 Over-reliance on epidemiological demar­cations may be misleading because of recent conversion of non-endemic regions to en­demic areas as a consequence of population migration or importing of sandflies from neighboring endemic areas. Lack of aware­ness regarding rare but possible modes of acquisition like vertical transmission, trans­fusion induced, direct contact and inocula­tion(3) may further delay the screening of suspected cases of Kaia azar.

Limitations of Conventional Diagnostic Modalities

 Early Diagnosis

 Complement fixation test (CFT) and counterimmune electrophoresis (CIEP) are useful serological tests for. diagnosing early cases of Kala azar. CFT has a sensitivity of 96% but it is also positive in tuberculosis, leprosy and Chagas disease(6) and becomes positive within 21 days. In a non endemic area, CIEP .has a sensitivity of 100% in late cases but has a sensitivity of only 80% in early cases(3). It gives false positive results even in normal population of an endemic area. Enzyme immunosorbant assay (ELISA) has a sensitivity of 90% and specificity of 100% and can be a tool for epidemiological studies using dot ELISA(7). Direct agglutination test (DA T) has a sensitivity and specificity of 100% if a titer of I: 1600 is considered sugges­tive of Kala azar. Latex agglutination test (LA T) has a relatively lower sensitivity (80%) and specificity (96%).

 Indirect fluorescent antibody test (IFAT), a group specific test, detects. antibodies against Leishmania that appear early in the course of the disease and persists as long as six months after cure. This test, therefore, can also be used for monitoring treatment. A titer of 1:20 is significant and 1:28 is strongly sug­gestive of Kala azar(3).

Newer Diagnostic Techniques/Tests

Modifications in the conventional diag­nostic tests can enhance the sensitivity of these tests. Sensitivity of spleniclbone mar­row aspirate can be increased to 100% by cul­turing the aspirate in NNN media, modified USMARO media, M-199+20% FCS or RPMI-I6400+20% FCS media(8) to demon­strate the promastigote form. Use of buffy coat preparation, particularly with few doses of steroid cari enhance the sensitivity to about 90%(9).

Some highly promising tests, but not cur­rently available in India, include use of fucose mannose ligand (FML) and polymerase chain reaction (PCR)(lO). Use of FML, a complex glycoprotein found on the surface of parasite throughout its life cycle, has been found to be as specific and sensitive as recombinant anti­gen. It has the added advantage of identifying sub-clinical cases from endemic areas besides being capable of distinguishing cutaneous manifestations of visceral leishmaniasis from other closely. related cutaneous infections. PCR-Leishmania DNA called KDNA comprises of maxicircles and minicircles. The minicircles PLURKE 3 of leishmania strain UR6 contains two primers,' namely, LSUC and LSUL( I 0). Identification and character­ization of these sequences via PCR can theoretically detect even a single parasite in the body with a sensitivity and specificity unmatched by any other test(10).

Therapeutic Options

Pentavalent antimonials have stood. the test of time to remain the drug of first choice. However, with the development of resistance, the dose and duration of treatment has been increased now to 20 mg/kg/day intramuscu­larly for 20-40 days or even longer in late responders(1l). Therapy can be rendered more effective if the total daily dose is divided equally and given at an interval of 8 to 12 hours. But even with this regimen a primary unresponsiveness of 50% in hospitalized patients(12) and about 20% in peripherally treated patients is being reported. This is mainly due to constant transformation of Leishmania donovani and depressed levels of gamma interferon and interleukin-II(13). Therefore, even though antimonials have been in the field of chemotherapy for Kala azar in India for about 3 decades and have saved millions of lives, the alarming propor­tion of drug resistant cases makes it impera­tive to look for alternative second line drugs.

Treatment of Resistant Kala azar

Pentamidine, an effective second line drug for sodium stibogluconate (SSG) resistant cases, probably acts by damaging the kineto­plast-DNA mitochondrial complex. It is rec­ommended in a dose of 3 mg/kg in children under 12 years and 4 mg/kg in older children to be given on alternate days for a total of 15 injections. However, some workers believe that the duration of treatment should be guided by parasitological cure (confirmed by splenic aspiration) and not by fixed number of injections. Apart from its limited efficacy (77-81.5%), its toxicity which includes hypoglycemia, hyperglycemia, hypotension, tachycardia, nephrotoxicity, hepatotoxicity, arrythmias and sudden death is a limiting fac­tor for its wide spread use. Supervised setting is a must for using this drug.

  Amphotericin B, a polyene antibiotic, acts by binding to ergosterol in the plasma mem­brane of a parasite creating holes in it through which ions leak out and the parasite is killed(14). It should be used in resistant Kala azar in a dose of 0.5 to 1 mg/kg/day till a cumulative dose of 7-20 mg/kg is achieved. It is administered intravenously in 5% dextrose solution at a concentration of 0.1 mg/ml over 6-8 hours with close vigil on febrile and aller­gic reactions. Amphotericin B is a potentially toxic drug which can cause anaphylaxis, thrombocytopenia, convulsions, fever, hypo­kalemia, nephrotoxicity, unpredictable drug induced myocarditis and hepatic damage. Liposomal amphotericin B, compared to conventional amphotericin B, is preferentially taken by reticuloendothelial system and' pro­vides targeted delivery of the drug to organs like liver and the spleen with lower circulating half life and much higher peak plasma level at a dose as low as 2-3 mg/kg/day thereby resulting in lesser toxicity without necessarily com­promising the efficacy. Satisfactory results have been achieved with mere 3 injections of this drug in a dose of 2 mg/kg/day(15). Another antifungal drug which appears prom­ising is ketoconazole in children over 5 years(4) to be given orally in a dose of 2-15 mg/kg/day. Adverse effects include rashes, anorexia, nausea, vomiting, raised SGPT levels and hepatitis.

   Allopurinol, a xanthine oxidase inhibitor, can also be used with or without sodium stibogluconale (SSG) in a oral dose of 5-8 mg/kg/dose for 1-3 weeks. Adverse effects in­clude leucopenia, eosinopenia, headache and drowsiness.

Newer Antileishmania Drugs

Aminosidine, an amino glycoside anti­biotic, has been effectively used alone or in combination with SSG in adults. The results of a recently conducted randomized con­trolled trial in Bihar in patients of visceral leishmaniasis aged 6-50 years recommend it as a first line drug in a dose of 16-20 mg/kg/ day intramuscularly for 21 days(l7). Monotherapy, using aminosidine alone, may cause future problems .because when used alone for controlling any intracellular organism it may lead to drug resistance. Therefore a combination of aminosidine and SSG is considered to be more effective than the latter alone(l8) and may be preferred. However, the therapeutic preference of aminosidine for its .use in children is not well established because of limited trials in pediatric age group and the drug not being marketed at present.                      

Miltefosine, an alkaly 1 phospholipid developed as an oral anti neoplastic agent, has been found to be active against Leishmania donovani infection with minimal side effects. This appears to be a promising drug when given in a 28 days course even in antimony resistant cases(l9). The search for an effective oral drug still remains a priority even though some reports claim ca: response rate of 70% with rifampicin plus cptrimoxazole(20). Other newer antileishmania drugs under trial are inosine analogue, Difluoromethyl­ornithine, WR60Z6 (Primaquine derivative), methyl benzyl esters of leucine and ATPase proton inhibitors.

Monitoring of Therapy

Evaluation of effectiveness of therapy of Kala azar is as much important as early diag­nosis and initiation of appropriate therapy. Therefore, monitoring of patients should be regarded as mandatory since resistant cases need to be identified early. Response to therapy can be assessed by clinical response arid repeat bone marrow examination. How­ever, during the course of therapy other in­direct indicators can also be used for monitor­ing the therapeutic response of the patients. Evaluation of acute phase reactants like C-reactive protein (CRP), serum amyloid A protein and alpha 1 acid glycoprotein are less invasive tests for monitoring diseae activity, response to therapy and relapses in Kala azar(21). Our own results suggest that persistence of CRP levels greater than 12 mg/dl by day 10 of therapy predicts possible development of resistance in the course of treatment with SSG(22). Role of indirect fluorescent antibody test (IF AT) in the monitoring of disease activity is also being evaIuated with. encouraging results.

Splenectomy

Splenectomy should be reserved for cases. that are unresponsive to both first and second line drugs or those who have huge splenomegaly(3). Splenectomy helps by reducing. parasite load and improving the efficacy of drugs to kill the residual parasites. Therefore, splenectomy should be followed by SSG in a dose of 20 mg/kg intramuscularly for 20-40 days.

Prospects of a Vaccine

Vaccine trials using a combination of leishmania antigen and BCG vaccine are underway. Development of a safe and effective vaccine against Kala azar may go a long way to control this multifaceted disease with ever increasing epidemiological spectrum and therapeutic concerns. Till that time it is important to optimally utilize our limited resources, make an early diagnosis and follow an appropriate protocol.

Utpal Kant Singh,
Assistant Professor,
Upgraded Department of Pediatrics,
Patna Medical College,
 Patna - 800016, India.

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