Conrol of visceral leishmaniasis
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Preliminary diagnosis is based on the symptoms
and clinical signs of visceral leishmaniasis such as splenomagaly, hepatomegally
and high undulating fever. However these alone are not enough to differentiate
VL from other similar conditions such as malaria, relapsing fever, liver
abscess and trypanosomiasis.
Spleen and liver biopsy
Looking for parasites in the spleen and liver is one of the most accurate methods available to determine leishmanial infections. Ninty percent of the active cases show parasites in splenic and liver aspirates (Manson-Bahr, 1987). The smallest needle possible, preferably, 21-gauge (0.8 mm) should be used to minimise the risk of complications such as haemorrhage of the spleen (Williams, 1995). Part of the splenic aspirate can be used to make smears for direst microscopic examination and the rest should be cultured. L. donovani grows well on Novy- MacNeal- Nicolle (NNN) or Schneider's insect medium supplemented 10% v/v foetal calf serum, although other suitable growth media can be used just as well. Liver biopsy material is less likely to demonstrate parasites on direct examination or on culture, however histological examination will show amasatigotes in Kupffer cells in the portal system.
Marrow and lymph gland puncture
Marrow obtained from sternal or iliac crest puncture
is a much safer but a painful method. It is less likely to demonstrate
parasites in direct stained films (Williams, 1995), however, on culture
it can give positive results in up to 80% of the cases. Lymph gland puncture
gives positive results in 60% of the cases. Juice is extracted from any
enlarged lymph gland and subjected to both direct examination and culture
to give the best chance of diagnosis.
Blood buffy coat
Finding the leishmania parasites in blood in sometimes possible in patients with Kala-azar from Kenya and India. Blood in anticoagulant is centrifuged at 2000g for 10 min and the cells from the buffy coat removed and used to prepare smears and inoculate cultures. Amastigotes can be found in and around Macrophage cells. The volume used in culture inoculation is important, 1-3 drops on NNN or Schneider's medium has given successful results (Williams, 1995).
Serological diagnosis is based on the presence of specific humoral antibodies in cases of visceral leishmaniasis. There are a range of serological methods available for the diagnosis of VL varying in accuracy and specificity. With on-going research newer better methods are continually becoming available.
Indirect fluorescent antibody test (IFAT)
The IFAT is one of the most sensitive tests available. The test is based on detecting antibodies which are demonstrated in the very early stages of infection and undetectable six to nine months after cure. If the antibodies persist in low litres it is good indication of a probable relapse. Titres above 1/20 are significant and above 1/128 are diagnostic (Williams, 1995). There is a possibility of a cross reaction with trypanosomal sera, however, this can be overcome by using leishmania amastigotes as the antigen instead of the promastigotes (Gari-Toussaint et al, 1995).
Direct agglutination test
The DAT is a highly specific and sensitive test. It is cheap and simple to perform making it ideal for both field and laboratory use. The antigen is prepared from promastigotes of L. donovani and test can be carried out on plasma, serum, blood spots and whole blood. Serum titres of 1:3200 are considered positive (Al-Masum et al, 1995).
Enzyme linked immunosorbent assay (ELISA)
The ELISA.test is 100% specific with sensitivity above 98%. The antigen is prepared from promastigotes of L. donovani and the test can be performed on serum, plasma or blood spots collected on filter paper. It is useful in the field owing to its simplicity, however, false positives are not uncommon. Hence it is less preferential than the IFAT and DAT for laboratory diagnosis (Williams, 1995).
Formol gel test
The formol gel test has the advantage of being cheap and simple to perform. Serum obtained from about 5 ml of blood is mixed with one drop of 30% formaldehyde. A positive reaction is shown if the mixture solidifies and forms a white opaque precipitate within 20 minutes. A positive test can not be detected until 3 months after infection and becomes negative 6 months after cure. The test is non-specific since it is based on detecting raised levels of IgG and IgM which also result from other infections such as African trypanosomiasis, malaria and schistosomiasis (WHO expert committee report, 1991).
Diagnosis of visceral leishmaniasis based on serology is amongst other draw-backs, made less reliable due to cross-reaction with other microorganisms. Competition ELISA's making use of monoclonal antibodies are more specific but can not distinguish between past and present infection (Williams, 1995). Recently molecular probes, using kinetoplast DNA (kDNA), ribosomal RNA (rRNA), mini exon derived RNA (medRNA), and genomic repeats have been evaluated and used to a much higher sensitivity and specificity. The disticnt advantages of DNA hybridisation are that large numbers of samples can analysed quickly without compromising efficiency and many different types of samples can be processed including blood spots, tissue samples, splenic and bone marrow aspirates (Wilson, 1995). The probes are more specific since they are made to target regions on the kDNA or rRNA which are unique to a particular leishmania species, complex or isolate (Williams, 1995). A probe has been developed to recognise the genomic DNA repeat Lmet2 which is specific for L.donvani. This probe has been used to diagnose infection in human VL patients and in P. martini, the sandfly vector of L.donovani (Wilson, 1995).The early DNA probes were labelled with radioactive 32P, which were easy to use and gave high signals with low back-ground interference. However, these were for laboratory use only and were not suitable as diagnostic kits, since 32P has a short half-life of about 14 days and being radioactive they presented safety problems. Recently, a chemiluminscent hapten digoxigenin labelled kDNA probes have been developed. These probes are as sensitive as the 32P labelled probes (can detect as low as 100 parasites) and are stable enough to use in diagnostic kits which are more practical in the field. However, high back-ground signals make result interpretation difficult (Wilson, 1995; Williams, 1995). The latest developments in molecular diagnostic technology have come about as a direct result of the advent of the polymerase chain reaction (PCR). The PCR is able to amplify small amounts of DNA or RNA to larger usable quantities (Nuzum et al, 1994; Wilson, 1995; Williams, 1995). Although the PCR is able to detect a single copy of target DNA, repeat sequences are used to improve sensitivity. The early PCR assays rquired gel electrophoresis to interpret results, which was time consuming and not suitable for feild use. An improved PCR- solution hybridisation enzyme linked assay (PCR-SHELA) was developed and has been used to diagnose infection of L.donovani in patients in India, Kenya and Brazil with 90% sensitivity and 100% specificity (Nuzum et al, 1994). It has also been used as an epidemiological survey tool in central America to detect presence of L.chagasi (Carreira et al, 1995). The PCR-SHELA makes use of a biotin lablled probe, and the product can be detected using a spectrophotometer and the assay can be carried out in microtitre plates, hence useful in the field and large numbers of samples can be analysed simultaneously (Wilson, 1995).
The two pentavalent antimonial compounds, sodium
stibogluconate and meglumine antimoniate were first introduced in the 1940's
and have since been used as first-line chemotherapeutic agents against
all forms of leishmaniasis including visceral leishmaniasis. The two compounds
are derivatives of stibonic acid in which antimony is joined through oxygen
atoms to the carbon chain(s) of glucose. Sodium stibogluconate (sodium
antimony gluconate) is available commercially as Pentostam, Solustibosan,
Solyusurmin, Stibanate, and Dibanate to name but a few. Meglumine antimoniate
(N-methyl glucamine antimoniate) is marketed as Glucatime and Prostib
(Hashim et al, 1995). The drugs are administered parenterally and
are the safest currently available since they are rapidly excreted by the
kidneys and there is virtually no accumulation in the body. Potential side
effects include nausea, vomiting, diarrhoea, ECG changes and convulsions.
However, some of the side effects reported in the treatment of visceral
leishmaniasis are identical with features of the disease and have not always
been attributed clearly to the treatment (Bryceson, 1987). Sodium stibogluconate
is usually administered at a dose of 20 mg Kg-1 body weight
for 20-40 days, however due to wide-spread antimony resistant cases of
Indian PKDL and kala azar, treatment over four months is recomended. This
expensive and potentially toxic (Hashim et al, 1995)
If treatment with pentavalent antimonials
is unsuccessful a preparation of an aromatic diamidine, pentamidine isethionate
(Pentamidine) or pentamidine dimethane sulphonate (Lomidine) is used. Both
of these compounds are very effective in the treatment of Kala-Azar but
because of their toxicity and potential side effects they are used as drugs
of second choice. Pentamidine is administered parenterally. The drug binds
to the liver and the kidneys and is excreted slowly in the urine and faeces.
Only 50% of the injected dose is excreted in the urine over 5 days. Traces
can be detected in the urine up to 217 days and in the kidneys up to 240
days after a single injection. The toxic side effects of Pentamidine include
a sensation of burning, headache, tightness of the chest, dizziness, nausea,
vomiting and hypotension (WHO expert committee report, 1991). The more
serious side effects of Pentamidine are hypoglycaemia occurring probably
due to irreversible liver damage. Hyperglycaemia and acute pacreatitis
can also occur leading to diabetes. Pentamidine is generally effective
against all forms of leishmaniasis. However, a relatively high proportion
of patients with visceral leishmaniasis fail to respond in Kenya, India
and China due to increased resistance (Dietze et al, 1995; Thakur
et al,1995). Single doses of 2-4 mg Kg-1 body weight
are usually recommended 1-3 times a week. Doses of 2 mg Kg-1
body weight may be effective and better tolerated. In cases of visceral
leishmaniasis in Kenya thrice-weekly injections of 4 mg Kg-1
body weight are necessary and avoid serious toxicity. A short course of
10-20 days treatment followed by a second course after an interval of 10-15
days helps reduce the relapse reate in VL cases ( WHO expert committee
Amphotericin B is another drug of second choice used in the treatment of leishmaniasis. It is a polyene microlide antibiotic and acts on cell membrane sterols and phospholipids of leishmania spp (steck, 1974). It is very active, in vitro, killing extracellular and intracellular forms of leishmania at concentrations of 1mg per millilitre of medium. In hamsters and monkeys infected with L.donovani, Amphotericin B was up to 400 times as potent as sodium stibogluconate (Olliaro; Bryceson, 1993). Amphotericin B is unstable at other than neutral pH and when exposed to light and air (Bryceson,1987). It is formulated as a colloidal suspension which is administered as a slow intravenous infusion of 2 mg Kg-1 body weight on alternate days for 20-40 doses (Olliaro; Bryceson, 1993). Common side effects include anaphylaxis, thrombocytopenia, flushing, generalised pain, chills, fever, phlebitis, anaemia, convulsions, amorexia, decreased renal tubular and glomerular function and hypokalaemia in about one third of the patients treated (Olliaro, Bryceson, 1993). Amphotericin B binds preferentially to 24 subsuituted sterols. Such as ergosterol, which is the major cell membrane sterol of leishmanial but not mammalian cell membranes. To a lesser extent it binds to human cholesterol in membranes, this activities underlies its toxicity. (Olliario; Bryceson, 1993). In India Mishra et al, 1992, used lower doses 0.5 mg Kg-1 body weight on alternate days for a shorter duration (14 days) and achieved 100% cure with very little toxicity in patients suffering from VL. (Olliaro; Bryceson, 1993). Amphotericin B remains a valuable drug because of its specific made of action on leishmanial cell membrane sterols. Recent research has been orientated towards reducing its toxicity.
of old drugs.
Lipid associated Amphotericin B
There are three commercial preparations of lipid associated Amphotericin B (LAAMB) currently under trial for treatment of leishmaniasis, AmBisome (Vestar, USA) Amphocil (Liposome technology inc, USA) and Amphotericin B lipid complex (Bristol Meyers Squibb, USA). The idea behind LAAMB is that the lipid component will recognise receptors on specific cells and so the drug can be targeted to particular sites of infection, hence more drug will be available to interact with the parasite ergosterol, and less to react with the human cholesterol and so reducing its toxic effects (Hashim et al, 1995). Also these lipid molecules are phagocytosed by macrophages hence the drug is delivered exactly where the parasites live (Dietze et al, 1995).
Ambisome, is amphotericin B incorporated into
liposomes made of phosphatidyl choline, cholesterol and disteroyl phosphatidylglycerol.
It has been used successfully to treat VL patients unresponsive to standard
drugs (Olliaro; Bryceson, 1993). Amphocil, is a colloidal suspension of
Amphotericin B cholesterol sulphate. In hamsters infected with L.donovani,
Amphocil was 4-15 times as effective as conventional Amphotericin B (Olliora;
Bryceson, 1993). Trials on VL patient have shown promising results. The
Amphotericin B lipid complex, consists of the lipids dimirystoyl phosphatidylglycerol
and dimirystoyl phosphatidyl choline.
Hashim et al, 1995, reported of two PKDL patients successfuly treated with Ambisome without toxic side-effects. The Ambisome was administered at 3 mg Kg-1 body weight daily for one month intravenously in a 5% glucose solution as a slow drip. Amphocil was used to treat kala azar in Brazil at a dose of 2 mg Kg-1 body weight daily for 7 days. Side effects included chills, fever, increased respiratory rate and acute febrile reaction in 80% of the patients during infusion. This was overcome by administration of an anti-immflamatory agent, diclofenac potassium every day before each infusion of Amphocil. Because of these potentially dangerous adverse reactions, Amphocil is only recomended for adult use while Ambisome can be used for all ages (Dietze et al, 1995).
The emergence of drug resistance in leishmania
parasites is a major obstacle to their control. The lack of response to
pentavalent antimonials in visceral leishmaniasis has been a problem for
many years and is increasing world wide, occurring in about 5-70% of the
patients in some endemic areas (Ouellette; Papadopoulou, 1993). With no
real promising prospects of an effective vaccine in the near future, there
is an urgent need to develop new and better drugs. Amongst the new drugs
currently under clinical evaluation are aminosidine (Paromomycin), imidazoles
and triazoles and purine analogues (allopurinol). The latter works on the
principle that leishmania are unable to synthesise purines, allopurinol
is hydrolysed to allopurinol riboside, an analogue of inosine. This nucleoside
is incorporated instead of ATP into leishmanial RNA, where it interferes
with normal protein synthesis (Olliaro; Bryceson, 1993). Allopurinol alone
has not been very successful in the treatment of leishmaniasis, however
the synergistic effect of allopurinol with sodium stibogluconate has proved
useful in treating five antimony resistant cases of VL in Kenya (Olliaro;
Bryceson, 1993). One other drug that is showing optomistic results is aminosidine.
This is an antibiotic of the aminoglycoside family. Clinical trials in
India and Kenya have demonstrated that when aminosidine is used in combination
with sodium stibogluconate it is effective in treating unresponsive visceral
leishmaniasis (Chun et al, 1990; Thakur et al, 1992; Olliaro;
Immunotherapy is used to improve antileishmanial drug action. One of the methods employed is the use of interferon gamma (IFN-gamma) to treat visceral leishmanasis. The cell mediated immune system fails to recognise the leishmanial antigens, INF-gamma enhances the intracellular killing of the leishmania parasites and reduces the dose of antimony required for inhibition or killing of leishmania. In Brazil, a combination of sodium stibogluconate (20 mg/day for 10-28 days) and recombinant INF-gamma (35-500 microgrammes) has been used to successfuly to treat 6 out of 8 patients with refractory VL (Chance, 1995). An earlier study in Brazil, reported a 46% success rate and another one in Indian Kala azar patients produced a 69% cure rate (Sundar et al, 1994). Treatment with INF-gamma is known to produce only minor side effects which are usuall for cytokine therapy. Fever and general Flu-like symptoms were common side effects and were easily aleviated by treatment with antipyretic drugs (Sundar et al, 1995). Treatment with INF-gamma alone has been shown to produce a reduction in the numbers of L.donovani parasites in splenic aspirates, however INF-gamma is best used in combination with antimonial drugs (Sundar et al, 1995).
Elimination of sandfly vectors
In endemic areas such as Bangladesh and India sandfly control is often combined with malaria control and Brazil with malaria and chagas disease control programmes (WHO WWW site, 1997; Al-Masum et al, 1995). This is a cost effective method given the expense of mass spraying with chemicals such as DDT, Malathion, Fenitrothion, Propoxur and Diazinon. In endemic areas this has to be an on-going process and continued surveillance is required to keep the vector populations low (Al-Masum et al, 1995). In epidemics large scale spraying is required. Spraying should particularly include animal shelters, inside of buildings and the immediate surrounding areas. Self protection is important, several methods are available to avoid being bitten by sandflies including repellents such as diethyltoluamide (DEET) applied to the exposed areas of the body and clothing. Fine mesh screens (less than 16-mesh) can be applied to doors and windows and bed nets should be used impregnated with insecticides such as permethrin and deltamethrin. Mosquito nets are not effective since sandflies can get through the holes.
Elimination of reservoir hosts
In areas of anthroponotic foci of VL active case detection and treatment is imperative, especially asymptomatic seropositive patients. A recent study in Israel showed that the asymptomatic or sub-clinical cases are 4-30 times greater than those with the VL symptoms (Ephros et al, 1994). The immunofluoresent antibody test (IFAT), enzyme linked immunosorbent assay (ELISA), direct agglutination test (DAT), the western blott and the leishmanin skin test have all been useful in detecting seropositive cases of VL (Marty et al, 1994; Al-Masum et al, 1994; Rab et al, 1995; Shiddo et al, 1995). In endemic areas of zoonotic VL, elimination of wild animals and stray dogs can be carried out by shooting and by using poisoned baits impregnated with strychnine. Screening and treatment of domestic animals especially dogs is recomended using the various available techniques including the IFAT, DAT and ELISA (Marty et al, 1994).
Culture media and staining
NNN (Novy-MacNeal-Nicolle) medium (Liquid phase)
Place 90 ml of distilled water in a conical flask, place on heat and add 1.4 g of non-nutrient purified agar and 0.6 g of NaCl while continuously stirring with a glass rod to prevent the agar from burning. Once the agar has melted, pour the required amount in to culture vessels. Sterilise the agar by autoclaving at 121oC for 15 minutes then allow to cool to about 50oC. To each culture vessel add 15% v/v sterile defibrinated or citrated rabbit blood or fetal calf serum, mix and then let the agar set at a sloped angle. Once the agar has set, place the culture vessels in an up-right position in a fridge or an ice-bath to form condensation water at the bottom of the slope. The medium can be directly inoculated with biopsy samples such as marrow, blood and splenic aspirates and parasite growth can be observed within 5-7 days (Williams, 1995; WHO expert committee report, 1991).
RPMI-1640 medium (Liquid phase)
This general purpose medium is particularly useful for maintaining parasite stocks and is available from Sigma chemical, UK, in the form of a powder with or with-out phenol red pH indicator. Dissolve the recomended amount of powder in one litre of de-ionised water and stir with a magnetic stirrer until all of the powder has dissolved. Adjust the pH to 7.4 using 0.1 M HCl and filter sterilise using a 0.2 micrometer membrane filter. Then pour in to pre-autoclaved sterile bottles and add 10% v/v sterile fetal calf serum and check the medium for sterility by incubating at 37oC for 24 hours. If no bacterial growth is observed then the medium is safe to use. Store the medium at 4oC until required. For growing promastigotes, inoculate the medium with 1x 105 cells/ml. Regularly check for parasite growth by observing under x40 objective lens. To maintain growth sub-cultures will be required when parasites reach stationary phase (Sigma chemicals, UK, catalogue, 1996).
Staining biopsy smears
The Giemsa solution is ideal for staining smears of cultured parasites, blood samples, cerebrospinal fluid, gland fluid and splenic aspirates. Smear the biopsy material on to a glass microscope slide and allow it to dry. Flood the slide with methanol for 1-3 minutes to fix the smear. Shake off the excess methanol and flood the slide with Giemsa (1:10 dilution in buffered saline, pH 7.2) for 20 minutes for cultured parasites and 30 minutes for raw biopsy material. After this wash the slide with distilled water and when dry examine under oil immersion x100 objective lens (Williams, 1995).
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