Somali Federal Government Ministry of Health. Guidelines for diagnosis, treatment and prevention of visceral leishmaniasis in Somalia

Transcription

1 Somali Federal Government Ministry of Health Guidelines for diagnosis, treatment and prevention of visceral leishmaniasis in Somalia 2012

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3 Contents Acronyms... 4 Acknowledgements Introduction Background information Life-cycle and transmission patterns Human infection and disease Diagnosis Clinical diagnosis Laboratorydiagnosis Diagnosis of primary kala-azar Diagnosis of relapse Diagnosis of PKDL Treatment Treatment of primary kala-azar (new cases) Treatment of relapse of kala-azar Treatment of PKDL Other treatment related issues and special situations Treatment of concurrent infection and malnutrition Information system Prevention and control Annexes Annex 1. rk39 rapid diagnostic test procedure Annex 3. Lymph node aspirate procedure Annex 4. Bone marrow aspiration procedures Annex 5. Procedures for splenic aspiration Annex 6. Preparation and examination of aspirates. Grading of parasites Annex 7. Kala-azar laboratory register book (left page) Annex 8. Kala-azar treatment register book (left page) Annex 9. Kala-azar patient treatment card (front) Annex 10. Kala-azar patient discharge card Annex 11. Dosage and precautions for the use of sodium stibogluconate (SSG) Annex 12. Dosage and precautions for the use of paromomycin (aminosidine) Annex 13. Dosage, administration and precautions for meglumine antimoniate Annex 14. Anthropometry and nutrition therapy look-up tables Annex 15. Overview of treatment for concurrent illnesses in kala-azar Annex 16. Drug guidelines for kala-azar Annex 17. Kala-azar monthly reporting forms Annex 18. Kala-azar weekly reporting forms Annex 19. Kala-azar line listing file (to be sent to central database on regular basis)... 82

4 Acronyms DAT FDA IM IV KA ME ORS PKDL RBC RDT RR SSG TFC TOC VL WBC WHO Direct agglutination test Freeze dried antigen Intramuscular Intravenous Kala-azar Mercapto-ethanol Oral rehydration salt Post kala-azar dermal leishmaniasis Red blood cells Rapid diagnostic test Respiratory rate Sodium stibogluconate Therapeutic feeding centre Test of cure Visceral leishmaniasis White blood cells World Health Organization

5 Acknowledgements The Visceral Leishmaniasis (VL) guideline for Somalia has been updated through a highly participatory process involving officials from the Ministry of Health, Non-governmental Organizations supporting the various endemic kala-azar treatment centres, World Health Organization representatives from Somalia and EMRO, and national partners. I sincerely appreciate and commend the role of the World Health Organization in supporting the Ministry of Health technically and logistically without which this document would have not been materialized. I would like to thank health workers in kala-azar treatment facilities and the various INGOs supporting those facilities. I am fully cognizant that without their commitments and continuous daily efforts in diagnosing, treating and monitoring patients, no progress on kalaazar guidelines would have been possible. A special thank you is extended to Dr Marthe Everard, WR for Somalia, for her support, to Dr José Postigo of WHO EMRO, and Dr Mohamed M Fuje and Godela von Döhren of WHO Somalia for valuable comments, guidance and effective assistance during the guidelines preparation and printing process. The guidance provided in this document has been drawn from vast experience and lessons learnt from global, regional and local level. We hope the guideline will standardize and unify kala-azar management in the endemic regions and will be able to significantly reduce the high burden of kala-azar in the endemic states. Mr. Duale Adam Mohamed Director General Ministry of Health, TFG Somalia Mogadishu 23 rd July, 2012.

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7 1. Introduction 1.1 Background information Leishmaniases are caused by over 20 species of parasitic protozoa of the genus Leishmania. The disease, transmitted to humans by sandflies (Phlebotomus and Lutzomyia species), is endemic in 98 countries or territories, affecting around two million people each year. Depending on the species of the parasite and the immune response of the host the disease spectrum of leishmaniasis ranges from self-healing skin lesions to a fatal systemic disease called visceral leishmaniasis (VL) which is also known as kala-azar (KA), a Hindi term meaning black fever. Human leishmanial infections can result in 3 main forms of disease: Cutaneous leishmaniasis Muco-cutaneous leishmaniasis Visceral leishmaniasis (kala-azar) Visceral leishmaniasis (kala-azar) is a deadly disease caused by the protozoan Leishmania parasite, transmitted through the bite of Phlebotomus sandflies. The World Health Organization (WHO) estimates that globally about 500,000 new cases and over 50,000 deaths of kala-azar occur every year, over 90 % of these cases are from six countries: Bangladesh, Brazil, Ethiopia, India, Nepal and the Sudan. In Africa, there are five countries endemic for VL, namely Ethiopia, Kenya, Somalia, Uganda and the Sudan. Kalaazar generally affects poor and neglected populations living in remote rural areas. If not treated more than 95% of kala-azar cases will eventually die. Eastern Africa is one of the world s main kala-azar endemic areas, with the majority of the burden being concentrated in focal areas in the east and south-east of Sudan. In Somalia, kala-azar is caused by Leishmania donovani. Phlebotomus martinii is the predominant vector in Somalia, but also P. vansomerenae was reported as possible vector (Trans R Soc Trop Med Hyg Nov Dec;97(6):667 71). Man is believed to be the only reservoir and transmission is believed to be anthroponotic. Anecdotal cases were described as early as 1935, but VL was first officially reported in 1943, with the first outbreak reported in 1952 from Daarbuluk, Hargeisa. An endemic focus was described in 1965, with 12 cases diagnosed in Middle Shabele region, the majority of which occurred in young age groups and originated from the province capital Jowhar. Further VL cases were reported in from the Lower Juba region by MSF B (Belgian Section) and a case was identified in 1995 in Baidoa, Bay region. In 2001, an outbreak on the borders of Kenya, Ethiopia and Somalia began, with the majority of cases occurring in ethnic Somalis from nomadic tribes grazing cattle in the border area. Cases first occurred in Lower Juba, Bakool and Gedo regions, although VL in Gedo may have been introduced by the arrival of people displaced from Bakool in Cases continued to occur in Bakool, with approximately 140 cases reported annually from 2002 to A marked increase to 1,002 patients was observed in 2006, 80% of which came from two districts, Hoddur and Tiyeglow, in Bakool region and this outbreak continued up to

8 1.2 Life-cycle and transmission patterns Kala-azar is transmitted to human through the inoculation, during a blood meal, of the promastigotes by an infected female Phlebotomus sandfly. The form of the parasite that infects man is called a promastigote. The promastigotes are injected into the skin of a healthy person through the bite of an infected female sandfly. Following inoculation, the promastigotes are taken up by the phagocytic cells and develop into amastigotes. The amastigotes spread in the blood and multiply in the macrophages of the spleen, liver, bone marrow, lymph nodes, and the mucosa of the small intestines. Intracellular & free forms of amastigotes are ingested by a female sandfly while taking a blood meal. After about 72 hours, the amastigotes become flagellated promastigotes in the mid-gut of the sandfly. The promastigotes continue to multiply and fill up the sandfly gut. Subsequent migration to the mouthparts follows in 4 to 6 days time making promastigotes ready for inoculation when the vector takes the next blood meal (Figure 1). Different species of sandflies need different habitats to survive and have different biting patterns (in and outdoors, forest or village, day or night preferences). This has important implications for the transmission and possible control measures. Termite hills and acacia trees are assumed to be breeding and resting sites for P martinae. The sandfly is active throughout the dry season and it bites at night (from dusk to dawn). Figure 1. Life cycle of the leishmania parasite Human infection and disease Most individuals infected by Leishmania donovani will not develop the disease (90% of asymptomatic or sub-clinical infections). When the host immune system is not able to suppress the parasite, VL will develop. After an incubation period of 2 to 6 months, sometimes longer, patients will present with fever, anorexia, headache, sometimes with cough, abdominal pain, diarrhoea, vomiting, epistaxis (nose bleeding) and symptoms of anaemia. After several weeks of illness, splenomegally develops and weight loss becomes prominent, sometimes leading to severe malnutrition. If left untreated, the disease leads to death in more than 95% of the cases often from superimposed bacterial infection, severe anaemia or bleeding.

9 2. Diagnosis 2.1 Clinical diagnosis A patient will be considered as a clinical suspect of VL if she/he presents with a history of prolonged fever (2 weeks or more) associated with clinical splenomegaly or wasting (weight loss). Case definition of a clinical suspicion of VL History of prolonged fever (more than 2 weeks) AND splenomegaly or wasting (weight loss) NO ONE SHOULD BE TESTED FOR KALA-AZAR UNLESS THEY HAVE A FEVER. CHECK THE TEMPERATURE you may need to check in the afternoon. EXCEPTION: Occasionally you will clinically diagnose a severely malnourished patient with kala-azar without finding a fever because they are too sick. As only % of patients meeting this clinical case definition have kala-azar, the diagnosis needs to be confirmed serologically or parasitologically. The main differential diagnoses for KA patients are: Malaria Hyperactive malarial splenomegaly (HMS): formerly called Tropical Splenomegaly Syndrome (TSS). This condition results from multiple partially treated malaria episodes Schistosomiasis: the splenomegaly is caused by portal hypertension and the fever is usually caused by another condition (e.g. pneumonia) Brucellosis: the splenomegaly is usually not massive; hepatomegaly; joint, bone and occasionally neurological involvement Typhoid fever: high grade fever, bradycardia, duration of illness less than one month, impaired mental status, constipation Tuberculosis: usually no splenomegaly, but possible in case of milliary tuberculosis; respiratory symptoms are present. Splenic abscess Myeloproliferative diseases Malignancies of lymphoid origin (leukemias and lymphomas) Chronic haemolytic anaemia A kala-azar patient can have primary kala-azar, or relapse or PKDL. The definitions for each are as follows: Primary kala-azar: This refers to a patient who is diagnosed with KA for the first time. The patient had not been treated for KA before. Relapse: This refers to a patient who has completed full treatment course and then returns with proven kala-azar. If a patient was treated for at least 15 days with appropriate amount of antimonial as single therapy more than one month ago and then returns with proven KA 9

10 she/he is also considered as a relapse. Relapses are diagnosed parasitologically and are mainly observed within 6 months after treatment. Post kala-azar dermal leishmaniasis (PKDL): PKDL is a recognized complication, occurring in a very mild form in about 50% of kala-azar cases, and in severe forms in few cases. Usually, skin lesions develop months after the clinical cure of kala-azar, but sometimes PKDL occurs during treatment or even before kala-azar. In most of these cases however a previous infection occurred sub-clinically. In some cases no history of previous KA is known. The lesions of PKDL start on the face as small scattered hyper-pigmented macules and papules. The rash can become nodular and spread to the trunk and limbs. It is symmetrical and non itching. A grading system is used to describe the spread of the skin lesions: Grade 1: Scattered macular, rash on the face around the mouth with some lesions on the upper chest and upper arms. Grade 2: Dense macular, or nodular rash covering most of the face and extending to the chest, back and upper arms and legs. If extensive or black nodules, it is severe grade 2. Grade 3: Dense macular, rash, covering most of the body, including hands and feet. In grade 3 crusting ulcers, scaling and spreading to the mucosa of the lips and the palate occurs. PKDL might persist for years (up to 10 years have been reported). It is speculated that PKDL patients could form a reservoir of the parasite in the community. Bed nets should be given to PKDL patients to prevent transmission. Majority of PKDL cases are self limiting so treatment is not needed, only severe grade 2 and grade 3 are treated with specific medicines. PKDL is diagnosed clinically therefore none of the kala-azar laboratory tests (rapid tests, DAT and aspirates) should be done. 2.2 Laboratory diagnosis Before proceeding to any blood testing for primary visceral leishmaniasis (DAT or rapid diagnostic test) first exclude malaria by doing a blood test and treating if positive, or if no testing is possible simply treat and evaluate after 4 days. If the patient still has a clinical diagnosis of kala-azar do a blood test. A patient who is severely ill can be tested for kala-azar at the same time the person gets treatment for malaria. If the patient has been treated for kala-azar before with SSG for 15 days or more and is sick again they do NOT need a DAT test. That may have a relapse. Diagnostic techniques involve serological and parasitological tests: Serological tests: Rapid diagnostic test (RDT) (rk39 dipsticks- Inbios and DiaMed IT LEISH also known as Opti-Leish Direct agglutination test (DAT) Parasitological tests: Lymph node aspirate Splenic aspirate Bone marrow aspirate 10

11 Serological tests Several tests have been developed to detect antibodies against Leishmania in the blood or serum of VL patients. The most common serological tests used in diagnosis of kala-azar are the DAT and the rk39 dipstick tests. These tests indicate the presence of antibodies against Leishmania and so confirming the parasite (antigen) is or was present in the body. Inbios and DiaMed IT LEISH test are the commonly used and recommended rapid diagnostic tests in a dipstick format. The rapid tests allow diagnostic confirmation of kalaazar at the peripheral health facilities leading to early treatment and better prognosis. DAT is a robust and well-validated test that requires more material and training. The procedure is a bit complex, thus requires health facilities with a laboratory, cold chain and well trained staff. Serological tests can only be used for the diagnosis of primary kala-azar (patients with no prior history of kala-azar, the ones who had not been treated for kala-azar before). For patients with a prior history of KA who present with a suspicion of relapse one cannot rely on a serological test for diagnostic confirmation, as specific anti-leishmania antibodies can persist for several years. Rapid diagnostic (rk39 antigen-based) tests The rapid diagnostic rk39-based tests (RDT) are easy to perform, quick, cheap and give reproducible results. They can therefore be used for early diagnosis of visceral leishmaniasis at both peripheral and central levels. The RDTs detect specific antibodies against the kinesinrelated antigen that is present in Leishmania donovani. The procedure of the rk39 dipstick is simple with results available within minutes (annex 1). They do not require extra-material and results are stable overtime, allowing for quality control. The rk39 rapid tests are therefore the recommended tests for use in Somalia for confirmation of the diagnosis of kala-azar among clinical suspects with no prior history of the disease. Interpretation of Dipstick Results POSITIVE: This patient needs admission (if they were never treated before) and treatment. NEGATIVE: This patient must await the result of the DAT test. Do not let that person leave. Treat for all other illnesses while they wait. Give ferrous and folic acid while waiting. Direct agglutination test The DAT can be performed using either blood (dried blood on filter paper) or serum. The DAT antigen is prepared from formalin-killed promastigote stages of L. donovani cultures and stained blue for visibility. The test is semi-quantitative and gives antibody titres ranging from 1:100 up to 1: It is a highly sensitive (>95 %) and specific (>85 %) test when performed according to standardized procedures (annex 2). It requires a well-trained laboratory technician to undertake the process over a period of 2 to 3 days to obtain results. The DAT should be performed in a health facility with a laboratory, cold chain and well trained staff. 11

12 The clinical indications for parasitological diagnosis (spleen, bone marrow or lymph node aspiration) are the following: Clinical suspect with a prior history of kala-azar (suspicion of relapse) VL patient not responding to anti-leishmania treatment (test of cure) Clinical suspect with a borderline DAT result (1:400 1:1600) Clinical suspect with a negative rk39 dipstick or DAT results but with strong clinical suspicion of kala-azar AND absence of alternative diagnosis or no response to treatment of alternative diagnosis The DAT cut-off points vary between the types of antigen used either freeze dried antigen (FDA) or liquid antigen. The freeze dried antigen is more preferred in the field due to its stability during transportation and is therefore recommended for Somalia. The positive cutoff titre for freeze dried antigen is 1:3200 (well 7). Other cut-off points for FDA are as follows: DAT negative (< 1: 400): VL is very unlikely. Alternative diagnoses (e.g. malaria, disseminated tuberculosis, brucellosis, typhoid fever, etc.) should be looked for and treated. If there is no response to treatment for a proven or suspected alternative diagnosis and if clinical suspicion of kala-azar is high (i.e. much enlarged spleen), the tests can be repeated within a month or a parasitological test (lymph node/bone marrow/spleen aspiration) is performed to search for the Leishmania parasites. If the DAT is positive ( 1:3200): Kala-azar is very likely and appropriate treatment should be initiated. If the DAT is borderline (1: 400, 1:800 and 1:1600), the test should be repeated within one month or alternatively a parasitological test (lymph node/bone marrow/spleen aspiration) should be performed in the absence of contra-indication (s). Parasitological diagnosis Visceral leishmaniasis can be confirmed by microscopical examination of stained slides of lymph node, bone marrow, or spleen aspirates (annexes 3 6). Specificity of these tests is near 100% provided that slide staining is done properly and that the laboratory technicians are well trained. Spleen aspirate is more sensitive (96 %) than bone marrow (70 %) or lymph nodes (58%) aspirates. Bone marrow aspirate is a very painful and invasive medical procedure that needs expertise and optimal sterilization of the puncture material. The procedure of lymph node aspiration is also quite painful. Spleen aspiration should be limited to hospital settings or health facilities where there is adequate equipment and trained staff to manage complications appropriately. Transfusion facilities should be present. Provided that the test is performed properly, the rate of life threatening bleeding after a spleen puncture is very low. The patient must strictly rest in bed for at least eight hours after the procedure and remain under close nursing observation. Spleen aspiration is contra-indicated in the following situations: Spleen barely or not palpable Jaundice (a sign of possible liver dysfunction) Signs of active bleeding (nose, skin, digestive, etc ). A history of recent nose bleeding without active bleeding is not a contra-indication for spleen aspiration Severe anaemia (Haemoglobin < 5.5 mg/dl) Pregnancy Patient in very poor general condition 12

13 Low blood pressure Uncooperative patient or caretaker Lack of informed consent from patient or caretaker In patients with contra-indication(s) to spleen puncture, bone marrow or lymph node aspirates can be done, provided that enlarged lymph nodes are present. Figure 2 (below) shows the diagnostic algorithm to guide you when to use each of the tests mentioned above and how to interpret the result in terms of treatment decisions. All diagnostic tests must be properly recorded on the laboratory register book (annex 7). KA testing is done to clinically VL suspect patients since most people infected by Leishmania do not develop the disease (KA). It is crucial to enquire about any previous treatment for KA because serological tests will test positive even several years after a successful treatment. The first test to be used in a clinically VL suspect never treated before is the RDT and treatment should start when the RDT is positive. In case of a negative RDT result, then blood should be tested for DAT. If DAT result is positive the patient should be treated. If DAT result is borderline, the test should be repeated not earlier than one week later or to perform a parasitological test. If DAT result is negative another disease has to be considered. If a person shows a borderline DAT result and the parasitological test is positive the person has to be treated for KA. If the parasitological test is negative then re-test for DAT or search another diagnosis. In health facilities where either RDTs or DAT is not available the following is recommended: 2.3 Diagnosis of primary kala-azar RDT not available: Take a blood sample using filter paper to perform DAT either in your health facility or at the referral laboratory. At the same time you can do a parasitological test. If the parasitological test shows Leishmania parasites then treatment can be started. If parasites are not observed wait for the DAT results. DAT not available and RDT is negative or not available: Take a blood sample using filter paper to perform DAT at the referral laboratory. At the same time you can do a parasitological test. If the parasitological test shows Leishmania parasites then treatment can be started. If parasites are not observed wait for the DAT results. 13

14 Figure 2. Diagnostic algorithm of primary visceral leishmaniasis (VL) in Somalia Clinical VL suspect Fever > 2 weeks with splenomegaly or wasting (malaria and previous VL ruled out). Rapid diagnostic test Negative Positive Send blood for DAT VL treatment Positive Borderline Negative VL treatment Re-test in 1 week Search for other diagnosis and treat OR refer (1) DAT Freeze Dried Positive: 1:3200 Borderline (BL): 1:1600; 1:800; Lymph node, bone marrow or spleen aspirates Negative LN: lymph node aspirate BM: Bone marrow aspirate Search for other diagnosis and treat OR refer OR Re-test DAT in 1 week (1) Rarely DAT negative patients would need LN/SP/BM Positive VL treatment 14

15 2.4 Diagnosis of relapse A relapse of kala-azar means that a person has kala-azar but has already been treated before. Relapses usually occur within 6 months after treatment. If a patient comes with fever for more than 2 weeks and a palpable spleen ask if they have been treated for kala-azar before. A serological test cannot diagnose a relapse because it can still be positive for months to 2 3 years after treatment even if a person is feeling well. If the serological test was done and was negative, there is usually no relapse! 2.5 Diagnosis of PKDL Post kala-azar dermal leishmaniasis is usually known as PKDL. This is a rash that starts on the face. This rash may sometimes spread to the whole body but it always starts on the face. This rash usually starts within 6 months of having kala-azar. Sometimes it starts at the end of kala-azar treatment. Rarely the person was not previously treated for kala-azar. PKDL usually heals by itself. Sometimes it comes and goes for years. Sometimes it gets worse and worse. Sometimes it affects the mucous membranes. The diagnosis of PKDL is easier if you study the pictures. Note that people can die with severe PKDL and that PKDL itself may be a reservoir for kala-azar infection. NB: In Somalia PKDL cases have not been detected and it seems that it is very rare. 3. Treatment The treatment regimens should follow these national guidelines in all treatment centers in Somalia. All patients treated for KA must be registered in the designated KA treatment register book (Annex 8). On admission all patients should be screened for acute malnutrition and referred as necessary. On admission the KA treatment card will be also filled in so the data can be entered in the national data base of the Ministry of Health (annex 9). At discharge the patient will be given a card which will be useful for his/her reference, especially when the person visits another health facility or health personnel (annex 10). Patients must be encouraged to return for a follow-up visit six months after discharge to make sure the treatment was successful. The date of that post-treatment visit is the final cure date to be recorded in the KA treatment card. 3.1 Treatment of primary kala-azar (new cases) Treatment should normally be given only after confirmation of disease based on the clinical examination and the laboratory tests. At the same time, the presence and extent of concomitant infection should be ascertained, as this may influence the choice of therapy or supportive treatment. In many cases, supportive treatment, for example rehydration or nutritional supplementation, may be required before the start of therapy. Treatment should be given under the supervision of medical personnel. The objectives of kala-azar treatment are: to reduce parasites to below the level of detection ; to support the patient s nutrition and hydration; to treat complications; to prevent development of drug resistance, and 15

16 to reduce or interrupt transmission of infection in the community Hence, daily monitoring of signs and symptoms of each VL patient is necessary. Obtaining important baseline information such as, spleen size, hemoglobin, and body weight is also crucial. Weight should be taken weekly and drug dosage adjusted accordingly. Treatment on the spot is important since some patients need treatment for other illnesses. Treat all the illnesses while waiting for the DAT result. Give ferrous and folic acid tablets while waiting for the results (except if patient is undergoing inpatient treatment of severe acute malnutrition, please refer to IMAM guidelines) Treatment regimens (options) for primary kala-azar First line regimens for primary kala-azar Sodium stibogluconate (SSG) and paromomycin (combination therapy) In this combination therapy sodium stibogluconate, 20mg/kg body weight /day, and paromomycin injection, 15mg/kg body weight /day are given intramuscularly for 17 days (annex 11 12). This combination treatment is the first choice and line of therapy as the safety and efficacy is similar to the pentavalent antimonials monotherapy and considerably shortens the treatment duration. Sodium stibogluconate (monotherapy) SSG in monotherapy is administered as intramuscular injections 20 mg/ kg/day for 30 days. In the absence or stock ruptures of paramomycin the pentavalent antimonials sodium stibogluconate and meglumine antimoniate can be used in monotherapy (Annex 13). HOW TO CALCULATE THE DOSE OF SSG SSG vials contain 100 mg / ml so it is calculated as follows: Dose in ml = Body Weight in kg x 0.2 EXAMPLES: If your patient weighs 50 kg 50 x 0.2 =10 - so give 10 ml If your patient weighs 9 kg 9 x 0.2 = but give 2 ml (the lowest dose) Remember that: There is no upper limit for SSG, but doses above 10 ml should be given in 2 separate injections. The smallest dose is 2 ml for everyone over 5 kg in weight. For severe vomiting the SSG should be stopped for 2 to 5 days until the vomiting stops. If a child weighs 5 kg or less she/he is usually severely ill. Give 1 ml daily and follow closely. Patients with severe ascites may need a lower dose. If the patient has severe ascites: Subtract 5 kg from the weight of an adult Subtract 2 kg from the weight of a patient weighing between kg Subtract 1 kg from the weight of a patient weighting between kg Then calculate the dose of SSG. Weigh the patient weekly and recalculate the dose accordingly. 16

17 Conditions for withdrawal of SSG: Acute pancreatitis Aberrations of creatinine Jaundice developing during treatment Excessively high LFT values, i.e. > 5x normal values of SGPT/SGOT Any evidence of cardiotoxicity (prolonged QT interval, cardiac arrhythmia) Declining hematological measurements (HCT, total WBT counts) Uninterrupted vomiting Failure to respond favorably during the first 2 weeks of treatment If SSG needs to be withdrawn, then Ambisome can be used as second line option. Contraindications: Patients with known cardiac diseases. Co-administration of quinine or any other drug known to cause cardiac toxicity is contraindicated. How to calculate the dose of PAROMOMYCIN Paromomycin is 500 mg/ml in a 2 ml vial so it is calculated as follows:. Dose of paromomycin in ml = (weight in kg x 15) divided by 500 EXAMPLES: If your patient is 7 kg: (7 x 15) / 500 = 105 / 500 = 0.21 ml If your patient is 15 kg: (15 x 15) / 500 = 225 / 500 = 0.45 ml If your patient is 50 kg (50x 15) / 500 = 705 / 500 = 1.41 ml Remember that: To administer paromomycin: You must have 1 ml and 2 ml syringes to give this drug. The recommended dose is 15 mg/kg sulfate (equivalent to 11 mg/kg base); no maximum dose; monotherapy should not be used. Patients must remain well hydrated because paromomycin can affect the kidneys. Tell patients to drink enough that they pass urine 4 times a day. If patients have too much vomiting and diarrhea, do not give the injections. Patients over 60 years may have problems with their kidneys so should not get this. This medicine may NOT be given intravenously Weigh the patient weekly and recalculate the dose. Do not give during pregnancy unless there is no choice Liposomal amphotericin B (Ambisome ) Liposomal amphotericin B is the safest antileishmanial drug and needs to be available at the referral sites. It is presented as 50 mg per vial. Liposomal amphotericin B: 3 5 mg/kg per day intravenous by infusion over 6 10 days up to a total dose of 30 mg/kg. Ambisome requires cold chain. 17

18 Ambisome in VL is the first line treatment for pregnancy, severe patients and HIV coinfected patients Treatment of relapse of kala-azar First relapse A definitive or final cure is defined as an absence of signs and symptoms 6 months after initial cure (which was at the time of discharge). Therefore, to establish definite cure, active follow up should be done as part of the treatment centre activities. For definitive cure one looks at the clinical picture. No aspirate is necessary at follow-up unless relapse is clinically suspected. Patients should be instructed to return for follow-up 6 months after discharge or earlier in case they feel sick. If a person returns with a clinical suspicion of kala-azar, after having received full treatment and discharged with a negative test of cure (TOC), the patient has a relapse. It is impossible to differentiate a relapse from a new infection. Therefore, all are considered relapses and treated as such under this scenario. Relapses can occur in up to 5% of the treated patients. In HIV+ patients the relapse can go up to 50%. Most relapses occur within 6 months of initial discharge and these are called first relapses. A patient who had a first relapse, re-treated for that, and got cured again, can get a second relapse. Relapses tend to have a higher parasite load (but not necessarily so) and are difficult to treat. There must be two negative TOC s before discharging relapsed patients, because any further relapses will be more difficult to cure. The likelihood for a positive TOC after treating a relapse is higher than 10%. So, in every relapse case TOC s have to be done. To ensure that there is at least a week of treatment after the first negative TOC, two consecutive TOC s are performed one week apart. Relapse cases need close monitoring. Longer SSG therapy is associated with more side effects: kidney toxicity, pancreatitis, and arrhythmias are known complications. In-patient treatment (if feasible) is always preferred for relapse treatments. Usually, it is better to refer a relapsed patient for treatment in a hospital. The clinician should check each week or even more frequently whether the relapsed patient is clinically responding. Good signs of response are the clearance of fever, well-being of the patient (e.g. able to walk, appetite improving), spleen size reducing, hemoglobin increasing, weight stable or increasing. First line regimens for first relapse In case of first relapses, a new treatment schedule is started, and the following are acceptable: SSG 20 mg/kg/day IM PLUS paromomycin 15 mg/kg/day IM for days If parasitology is available, end of treatment should be guided by clinical assessment and test of cure aspirates. TOC aspirates can start on day 17 for two drug (SSG plus paromomycin) combination therapy. TOC aspirates should be done weekly. When TOC is available, the minimum days of SSG is 30 for combination therapy. Paromomycin is given for only 17 days (more than 21 days may be toxic). Sodium stibogluconate 20 mg/kg/day IM for 40 to 60 days If parasitology is available, end of treatment should be guided by clinical assessment and test of cure aspirates. TOC aspirates can start on day 30 for single drug (SSG) therapy. Then TOC aspirates should be done weekly. When TOC is available, the minimum number of days of 18

19 SSG is 40 for monotherapy. After 60 days of SSG, it is clear that the parasite is unresponsive to SSG and 2 nd line treatment should be instituted as stated below. Second line regimens for first relapse Liposomal amphotericin B (Ambisome ) Liposomal amphotericin B is the safest antileishmanial drug and needs to be available at the referral sites. It is presented as 50 mg per vial. Liposomal amphotericin B: 3 5 mg/kg per day intravenous by infusion over 6 10 days up to a total dose of 30 mg/kg. Ambisome requires cold chain. Ambisome in VL is mainly used for relapses, pregnancy, severe patients, HIV-co-infected patients and when SSG toxicity is not tolerated by the patient. Amphotericin B deoxycholate Dose: mg/kg/d intravenous for 30 alternate days (15 mg/kg total dose)- infused with 1 liter of 5% dextrose in 2-12 hrs. Before the full treatment course, the patient is given an initial test dose, which is the first 1 mg of the first dose to be infused over 20 to 30 minutes; and the patient observed for 1 hour. The remaining doses are given as 1 mg/kg body weight IV once per day every other day for 30 days (total of 15 treatment doses). Administration Amphotericin B is infused in 1 litre of dextrose 5% infusion running over 2 12 hours. The slower infusions decrease infusion related side-effects (chills, fever). Before starting therapy, hydrate the patient and maintain hydration with ORS and, if needed, IV fluids. This is important to decrease the risk of renal toxicity. Give potassium supplementation (1 tablet 3 /day for adults); if potassium tablets are not available, give one banana every eight hours (1 banana = 8 mmol KCl = 1 tablet KCl). Management of fever: give paracetamol before infusion or at the onset of symptoms. Avoid gentamycin, streptomycin, paromomycin or other drugs that can cause renal toxicity. Second relapses Second relapses are not very common but occur especially in immune suppressed patients. The treatment option for those cases is to administer amphotericin formulations at the highest dose Treatment of PKDL When to treat PKDL The rash shows blackening especially around the nose The rash is dense the skin is almost covered with bumps that are almost touching The rash includes mucous membranes especially the nose, eyes or mouth The rash is pealing or scaling The patient with this rash is also ill with fever (after treatment for possible concurrent illnesses) and has a big spleen Any patient who develops PKDL during kala-azar treatment: This patient should be continued on treatment until the PKDL is cured. 19

20 The rare patient who has a new decrease in vision and eye pain with the rash. Iritis is rarely part of PKDL. End of treatment of PKDL Treatment of PKDL can be completed when the rash is no longer palpable. Feel the rash with your finger. If the skin is smooth you can stop treatment even if you can still see the rash. Everyone must have at least 30 days of SSG. The treatment regimen consists of SSG 20 mg/kg/day IM for days OR SSG 20 mg/kg/day IM for 30 to 60 days plus paromomycin 15 mg/kg/day IM for 17 days. 3.4 Other treatment related issues and special situations Interruption of SSG treatment Interruption during treatment can occur in two conditions: medically initiated interruption or severe vomiting and defaulting by the patient: If interruption is less than 5 days, continue the treatment from where the patient stopped taking the treatment and continue until the full course of treatment is given. If interruption is 5 14 days, the patient should continue from where they stopped but must have an adequate test of cure parasitologically at the end of treatment. If interruption is 15 days or more, irrespective of the number of days of previous treatment, the patient needs readmission for parasitology testing; if positive, restart treatment as day one and do a test of cure before discharge. If negative, follow up is necessary and parasitological test if need be. Evaluating cure At the end of treatment with combination or monotherapy, the patient should be re-assessed. This usually includes clinical and laboratory examinations as described below: Clinical response Many patients get worse during the first few days of treatment. Patients, at the extremes of age, with severe anaemia, severely malnourished, in a state of collapse, and presenting with vomiting, pneumonia, or bleeding are at high risk during the first 10 days of treatment. After 7 to 10 days the patients become afebrile, and begin to look stronger, become more mobile, with increased alertness and appetite. By day 14 the spleen size regresses, hemoglobin rises and there is weight gain (note: loss of oedema may mask weight gain during treatment). By the end of successful treatment, patients are afebrile, usually have a smaller spleen than on admission, and have an increased hemoglobin level (though most remain anaemic). All the patient conditions get improved. Initial parasitological cure the role of TOC Splenic aspirate is more sensitive for diagnosis and for TOC. Lymph node aspirate (LNA) has been used for TOC when splenic aspirate is not possible. There is no clinical sign that correlates with a positive test-of-cure aspirate (TOC) or that predicts increased risk of relapse. The exceptions are signs of co-existing TB or HIV, both of which will increase the risk of treatment failure. TOC is done for assurance that discharge is appropriate for patients who may have difficulty returning for care/follow-up. When done systematically it is also a way to monitor the emergence of drug resistance. 20

21 Non-response is defined as no decrease in the grade of parasitology from before treatment to after adequate treatment. For those patients who are not aspirated on admission, use 4+ at TOC as defining primary unresponsiveness. Anyone with any TOC of 4+ (or a TOC that does not become negative by 60 days of SSG) should get second line treatment. Clinical judgment will always play the final role in any decision-making process given the variability of individual VL cases. Kala-azar treatment in pregnancy Kala-azar treatment during pregnancy cannot be differed as untreated VL during pregnancy may lead to severe disease, characterized by high grade anaemia, spontaneous loss of the fetus, and congenital VL because of transplacental transfer of parasites, even in apparently low symptomatic cases. Unfortunately, no drug of VL is proven to be safe in pregnancy however, none are known to be harmful. Pregnant women should therefore be treated with the safest available anti-leishmanial drugs. Amphotericin B deoxycholate or its liposomal formulation is safe and effective for pregnant women and their fetuses, and is therefore recommended as first-line treatment for these patients. The dosage and administration is the same as other patients. Pentavalent antimonials are less safe in pregnancy, as they can result in spontaneous abortion, preterm deliveries and hepatic encephalopathy in the mother and vertical transmission. Paromomycin: Ototoxicity in the fetus is the main concern and hence should be avoided as much as possible. Leishmania HIV coinfection Visceral leishmaniasis is an AIDS-defining condition and a valid entry point for starting antiretroviral treatment, irrespective of CD4+ count. The baseline CD4+ count is lower in visceral leishmaniasis HIV co-infected patients, as visceral leishmaniasis itself causes a reduction in CD4+ cells. The impact of antiretroviral treatment on visceral leishmaniasis in co-infected patients includes reduction of incidence of VL, higher survival rates, a reduction in relapse rate and possible immune reconstitution inflammatory syndrome. HIV and Leishmania infection reinforce each other. HIV patients are more likely to develop visceral leishmaniasis (due to reactivation of a dormant infection or clinical manifestation after primary infection). Patients characteristically have high disseminated parasite loads. Visceral leishmaniasis negatively affects the response to antiretroviral treatment and is difficult to cure in co-infected patients. The prognosis of co-infected patients is characterized by a high mortality rate during the first episode, increased anti-leishmanial drug toxicity (predominantly with antimonials), poor long-term clinical response, parasitological cure and a high relapse rate over a lifetime. The risk factors for relapse are: no antiretroviral treatment, low CD4+ cell count, previous visceral leishmaniasis episode, failure to achieve clinical or parasitological cure during the first episode and no secondary prophylaxis. Antimonials are more toxic in HIV patients, necessitating careful monitoring for pancreatitis and cardiotoxicity. Treatment of VL in HIV co-infected patient Amphotericin B deoxycholate or lipid formulations should be considered first and pentavalent antimonials only in areas of no significant resistance and when lipid formulations of amphotericin B are unavailable. 21

22 Lipid formulations (e.g., Liposomal Amphotericin B, Ambisome) infused at a dose of 3 5 mg/kg daily or intermittently for 10 doses (days 1 5, 10, 17, 24, 31 and 38) up to a total dose of 40 mg/kg are recommended Treatment of concurrent infection and malnutrition General kala-azar patient management Because infection with Leishmania depresses the immune system, patients with kalaazar are at increased risk for other infections. Additionally, the severity of such infections in kala-azar patients may be greater than in patients without kala-azar. All concurrent illnesses should be treated immediately and aggressively. Many kala-azar patients are severely malnourished. Because of this and their immune depression, these guidelines include routine treatments similar to those given in therapeutic feeding centers. Kala-azar patients can die during treatment. Those at most risk are those who are very young (< 3 years), those who are old, those severely malnourished, those severely anemic, those with prolonged disease (more than 2 month history), and those with vomiting. Please give special care for these patients! All kala-azar patients should be screened for malaria and managed promptly following the national treatment guidelines: If treated with Artemisinin Plus Fansidar in the previous 7 days no more treatment is needed. If not treated and not tested, the patient must be tested for malaria or treated. If the test is positive give Artemisinine plus Fansidar. NB: Remember not to use Fansidar in the first 2 months of pregnancy, use Quinine instead. On admission give Vitamin A to all VL patients except pregnant women: Vitamin A - 200,000 IU if 6 months to 1 year use 100,000 IU; if < 6 months, use 50,000 IU do not give to pregnant women until after delivery. Amoxicillin for 5 days Tinidazole for 3 days or metronidazole for 7 days Remember to also treat all other illnesses and complaints on admission to send children for measles vaccination if not previously vaccinated Daily tablets if not being treated for acute malnutrition Vit C and multivitamins not needed if there is good food available Ferrous plus folic acid 22

23 * If severely malnourished, give folic acid alone for first 7 days. If the patient is pregnant give 2 ferrous each day Discharge tablets Ferrous plus folic acid 30 tablets for daily use Fansidar* to all patients with a palpable or visible pregnancy that is less than 9 months. Albendazole* for non-pregnant patients if your area has round worms. Note that most severely malnourished patients have iron stored in their body, but kalaazar patients may have none. For this reason we add the ferrous to the folic acid on day 7 rather than waiting for day 14 as in many stabilization centre (SC) protocols. Treatment of malnutrition Promoting good nutritional status of patients is key in the management of kala-azar. Experience has shown that 70 75% of the diagnosed cases are severely or moderately malnourished and in desperate need of nutritional supplements. The latest round of nutrition and mortality surveys conducted in October 2011 indicate extremely high levels of global acute malnutrition and mortality compared to normal levels for this time of year, yet show some improvements from the round of surveys from August In most of the regions the rates of acute malnutrition remain near or above 30%, and depict a Very Critical nutrition phase. Therefore, these guidelines include the treatment of acute malnutrition in line with the National Guidelines for Integrated Management of Acute Malnutrition (IM-SAM). 23

24 New admissions Guidelines for diagnosis, treatment and prevention of visceral leishmaniasis in Somalia Admission criteria for moderately and severely malnourished Kala-azar cases: Admission criteria for children from 6 to 59 months of age SC OTP TSFP SAM with complications SAM no-complications MAM W/H (Z-score) < - 3 < - 3 < - 2 MUAC (mm) < 115 < 115 < 125 Oedema Bilateral oedema +++ any oedema Marasmus with No oedema, or Bilateral oedema +, ++ No oedema Appetite NO APPETITE Pass appetite test N/A Uncontrollable vomiting Fever > 39 ºC Hypothermia < 35 ºC Complications Lower respiratory tract infection Severe anaemia No complications No complications Extensive skin infection Very weak, apathetic unconscious, convulsions Admission criteria for other categories Indicator SAM MAM Infants less than 6 months W/H (W/L Z-scores) < - 3 Static weight or loosing weight at home Oedema Present Absent Clinical presentation To weak to suckle breast Poor feeding Older Children and Adolescents (5 18 years) W/H (Z-scores) < - 3 < - 2 MUAC (mm) <145 for children 5 10 years and < 160 for children years N/A Oedema Present Absent Adults (oder than 18 years) BMI and clinical signs < 16 with weight loss in the last 4 weeks < 16 but no weight loss Oedema Present Absent Pregnant and lactating women MUAC (mm) < 210 with weight loss in the last 4 weeks < 210 with no weight loss in the last 4 weeks SAM-Severe Acute Malnutrition; MAM-Moderate Acute Malnutrition See annex 14 for Anthropometry look-up tables Nutritional treatment for severely malnourished kala-azar cases Severely malnourished patients can be treated as inpatients (in Stabilization Centres) or outpatients (in Outpatient Therapeutic Feeding Centres) depending on whether or not they present with medical complications. It is likely that most patients with kala-azar will need to be treated as inpatients due to the daily injections that they must receive. However, once the 24

25 need for injections is over than they can be transferred to outpatient treatment to complete their nutrition rehabilitation. Infants should always be treated as inpatients. Infants 0-5 months with SAM who are not breastfed are particularly at risk and require protection and support to reduce the risks of artificial feeding. For these infants and their caregivers, the potential for restoring or establishing breastfeeding should always be explored to the maximum. Further, it is not appropriate to provide infants 0-5 months with RUTF because the reflex of swallowing semisolid foods is not yet present. Nutritional rehabilitation of severely malnourished patients uses several specialized products. F-75 Therapeutic milk is used in the first phase of inpatient care to stabilize the patient. Once the patient is stabilized they can be moved to transition phase which uses either F-100 therapeutic milk or Ready-to-Use Therapeutic Food (RUTF). From their they move to a rapid weight gain phase which almost always uses RUTF unless the patient is an infant or has other complications. F-75, F-100, and RUTF are all given based on body weight and look-up tables for quantities can be seen in Annex 14. Nutritional treatment for moderate malnourished Kala-azar cases The treatment of moderately malnourished patients should use Ready-to-Use Supplementary Food (RUSF) or fortified flour porridge. RUSF is the preferred treatment. Target group Children 6 59 months Above 5 years (older children 5-9 yrs, adolescents 10 18yrs and Adults) Treatment Provide 3 sachets of RUTF per day for the duration of the treatment Provide 2 sachets of RUTF per day for the duration of the treatments Basic food rations Food rations should be provided to all kala-azar patients for the duration of treatment except for the initial inpatient treatment of severely malnourished patients who should only consume the specialized nutritional products. 25

26 Daily food ration (per person/grams) BENEFICIARY CATEGORY Eligible beneficiaries) Cereal Pulses Oil Salt Sugar CSB Total Duration 1) IFP inpatient HIV, TB, Kalazar, Leprosis inpatient During hospitalization 2) IFP outpatient HIV, TB, Kalazar, Leprosis outpatient 3) IFP caretaker One caretaker for one HIV, TB, Kalazar, Leprosis inpatient During ambulatory treatment period (ARV, DOTS) When accompanying inpatients for treatment in hospitals 4) IFP family ration Two family members for one HIV, TB, Kalazar, Leprosis outpatient During ambulatory treatment period (ARV, DOTS) 5) TFP caretaker One caretaker for one SAM child 6-59 months During therapeutic treatment at TF center Systematic medicine for severe malnutrition Name of Product When Age / Weight Prescription Dose 6 months to < 1 year IU VITAMIN A* AT ADMISSION 1 year IU Single dose on admission DO NOT GIVE TO CHILDREN WITH OEDEMA AMOXICILLIN AT ADMISSION All beneficiaries See protocol 3 times a day for 7 days MEASLES VACCINATION AT ADMISSION From 9 months (standard) Single dose on admission < 1 year Do not give None ALBENDAZOLE SECOND VISIT months 2 years 200 mg (1/2 tablet) 400 mg (1 tablet) Single dose on day 7 * VITAMIN A: do not give if child has already received within last 3 months. DO NOT provide to patients with oedema. Wait for oedema to fully subside. 26

27 Treatment of diarrhea ORS, made with boiled water, left to cool, or with borehole water. Zinc supplements, 10mg/day for infants below 6 months and 20mg/day for patients 6 months and over. Tinidazole for 3 days, then continue tablets to 5 days if diarrhea continues (no more than 10 days total) OR Albendazole single dose. ask if they have nausea or vomiting and treat if they do Diarrhea 4 times or more in 24 hours OR any diarrhea with blood OR any diarrhea with fever: ORS, made with boiled water, left to cool or with borehole water. Ciprofloxacin AND Tinidazole for 3 days to 5 days if diarrhea persists.. (Tindazole for persistent diarrhea may be given for a total of 10 days.) ask if they have nausea or vomiting and treat if they do Second line treatment: substitute metronidazole for 7-10 days for the tinidazole Unresponsive diarrhea: Consider sending a stool specimen to the laboratory. Consider potassium supplement (slow K) for patients with severe diarrhea and vomiting. This protocol is for use with kala-azar patients only. Viruses cause most diarrhea in the world and there is no medicine other than oral rehydration salt (ORS) and zinc supplementation. Kala-azar patients are different; they get more dysentery and they are too weak. Note that zinc supplementation and ORS is not recommended if the patient is undergoing treatment for severe acute. Diarrhea with associated weight loss in a patient with severe acute malnutrition should be treated with ReSoMal as per IMAM guidelines. If the diarrhea is not causing weight loss than rehydration should be done with potable water alone. ORS is contraindicated in the treatment of diarrhea or vomiting when there is severe acute malnutrition. Any diarrhea: People do not usually die of the germ causing diarrhea, they die because they lost too much water or they are starving. Anyone with diarrhea must eat and drink more than normal or they may die. ORS, local made with safe water, and high energy biscuits are good choices. Causes of diarrhea Syndromic diagnosis (diagnosis based on history and physical) is good. The syndromic diagnosis is based on evidence from well-equipped hospitals and on knowing what diseases are in your area. Sometimes there are confusing cases. If you have a lab this can sometimes help sort out these confusions. Remember though, one stool specimen is not sensitive it is simply a help. You may need multiple specimens. Remember too that patients can have more than one germ. Use the below table as a quick reference for treatment of various pathogens causing diarrhoea: 27

28 Pathogen Fever common Stool exam Other symptoms Treatment Viral No Normal ORS ORS for all! Giardia No Giardia Nausea, vomiting, burping, bloating, abdominal cramp, May or may not have diarrhea Albendazole or Tinidazole or Metronidazole Ameba No RBC <5 WBC Ameba Trophs Bacterial Yes WBC RBC abdominal pain, tenesmus, sometimes with blood mixed with mucus Many times a day diarrhea, may be bloody, may be very ill Tinidazole or Metronidazole Ciprofloxin or cotrimoxazole Treatment of vomiting Patients who vomit only with cough do not need medication for vomiting., Give paracetamol if they have a fever Give promethazine tablets for 2 days but patient has difficulty of taking orally, give IM. Tell the patient to take the tablet and then wait 1 hour before taking any other tablets or eating. Tell the caretaker that the patient will get a little sleepy from promethazine, but they should be awakened to drink. Give ORS and asses hydration status frequently. Continue breastfeeding Consider stopping the SSG for 2 to 5 days. Any patient who has nausea or vomiting should continue eating and drinking but in small amounts 5 to 7 or more times a day. Any patient taking tinidazole or metronidazole should be asked about vomiting. Both these drugs are common causes of vomiting. You may treat with promethazine. Severe vomiting Be careful of these patients who cannot stop vomiting, patients who vomit too much to keep down water, ORS or milk. Stop the SSG for 2 to 5 days until the vomiting is gone. If the vomiting stops within 5 days, continue the SSG from the dose you stopped. SSG can cause nausea and vomiting. Vomiting is associated with death in kala-azar patients. Please give close attention to these patients. Remember that malaria and meningitis cause also cause vomiting; check your patients carefully! Give promethazine injection. 28

29 Always start promethazine tablets following the injection. Start the promethazine tablet 1/2 hour after the injection so they do not vomit it; the effect of the injection will stop in one or two hours. The effect of the tablets lasts 3 to 6 hours. Always give ORS, give small amounts every 15 minutes. They may need an IV of Saline or Ringers Lactate if they are dehydrated. Please refer them to help! Special notes for children < 12kg who are vomiting Take the temperature and make sure the child has no other disease such as malaria or meningitis. Try giving small amounts of ORS every 5-10 minutes. Give paracetamol if needed. You may give promethazine syrup after you have checked for other illnesses. Patients who vomit only when coughing do not need promethazine. Treatment of malaria Every one with a documented fever after the first 7 days of kala-azar treatment with no obvious source of the fever should repeat a blood film or RDT for malaria if possible. Consider malaria treatment if there is no other source of fever and no laboratory test is available. Remember that pregnant mothers and young children get malaria easily. Treat with ACT and paracetamol if more than 2 weeks from the last malaria treatment for uncomplicated malaria. Treat with Quinine for severe malaria (severe vomiting, a seizure in an adult, repeated seizures in a child, talking crazy, hypoglycemia, jaundice, pulmonary edema, Hb < 5, or any altered mental status). Note that SSG should not be used with quinine (due to prolonged QT interval and cardiotoxicity). If treatment with quinine is inevitable, stop SSG for VL while quinine is being given. After quinine treatment is finished, resume SSG injections, starting 24hrs after the last dose of quinine. Consider shortening quinine usage for 3-5 days and finish with other antimalaria medicines. This helps to resume SSG within short period. NB: refer to the national Malaria guideline for detailed dosage and duration of treatment. Treatment of pneumonia The diagnosis follows the health worker guidelines so check the last version of the integrated management childhood illness (IMCI) guidelines in use in Somalia: kala-azar patients can get a dry cough that does not need antibiotics kala-azar patients commonly get a pneumonia that does need antibiotics adults as well as children get pneumonia fever, productive cough, chest pain with breathing, and fast breathing are all part of a diagnosis of pneumonia WHO-IMCI guidelines for diagnosing pneumonia in children based on respiratory rate (RR): Diagnose pneumonia if a child 2 12 months has RR > 50; NOTE: normal RR is up to 60 for 0-2mo, 40 for infants, 30 for 12 months 5 years 29

30 Treatment of mild to moderate pneumonia amoxicillin for 7 days. Amoxicillin is preferred over cotrimoxazole. Amoxicillin but not cotrimoxazole will treat most pneumonia from aspiration that occurs with vomiting or seizures or a concurrent ginigivitis. Erythromycin is a reasonable first line drug for older children and adults who do not have the aspiration risks. Erythromycin can cause vomiting which is already a problem for kala-azar patients. Patients treated successfully for pneumonia will usually have their fever subside in 3 days and their cough and crepitations usually subside in 8 days. Treatment of pneumonia not improving by day 3 or 4 ceftriaxone injection, chloramphenicol capsules Initial treatment of all serious pneumonia ceftriaxone once daily injection second line treatment, chloramphenicol oral or by injection 3 or 4 times a day Signs of serious pneumonia infants breathing too fast to breast feed pneumonia patients with significant vomiting, unable to keep down medicines patients with respiratory distress (retractions, flaring nose, grunting) unexplained abnormal mental status Please refer these seriously ill patients to the senior medical staff. They may be acidotic from dehydration or sepsis, they may have severe malaria or meningitis with the pneumonia. Patients who have pneumonia all through their kala-azar treatment and/or who are unresponsive to antibiotics may have tuberculosis (TB). Document any fever. Take the temperature in the evening. Adults may need a sputum test. Ask children with continuing pneumonia if there is a family history of pulmonary TB. Treatment of tooth or gum pain - with or without bleeding clean teeth twice a day Procain penicillin or amoxicillin for 5 days paracetamol as needed (never aspirin) Treatment of bleeding Nose bleeding: Pinch the nose at the end of the bony part for 10 minutes. 30

31 If the nose bleeding will not stop with pinching: pack the nose with petroleum jelly on gauze and leave the pack in for at least 2 days. If you pack the nose, treat with amoxicillin (for prevention of sinusitis). Gum bleeding: treat with penicillin or amoxicillin for 5 days. Give vitamin C Occasionally you will need epinephrine on gauze compresses to stop the bleeding If the patient has jaundice: give vitamin K IM for 5 days. Treatment of conjunctivitis Tetracycline eye ointment is used for red conjunctiva especially if there is drainage. Any sign of trachoma should be treated with tetracycline eye ointment. Gentamicin or chloramphenicol eye drops for abundant pus discharge or for failure of tetracycline ointment. Use until 2 days after pus discharge is gone. Make sure this patient got vitamin A on admission! Patients who are severely malnourished can have a second dose of vitamin A. Iritis is a rare disease that can accompany PKDL. There is eye pain and the vision is blurry like looking through a cloud. There is usually little or no pus. Please notify a doctor. The patient needs to continue the SSG. They may also need steroid eye drops Treatment of infected wounds Clean and treat with penicillin for 5 days, they may need gentian violet. If the wound is not improving consider cloxacillin. If they have an injection abscess it may need draining and cloxacillin. Treatment of herpes zoster This is an extremely painful rash that can occur at the end of, or just after kala-azar treatment. It is only on one side of the body and usually covers only a small area. The skin is red and develops blisters. This is NOT associated with HIV when it occurs at the end or after kala-azar treatment (or if it occurs in the elderly). Treat with 5 to 10 days with paracetamol or ibuprophen (ibuprophen is OK to use ONLY at the end of kala-azar treatment). If the wounds are open you can use gentian violet Treatment of pain Treat with paracetamol (for severe pain consider tramadol). Never use aspirin for kala-azar patients, this can cause bleeding * Ibuprophen and indometacin may cause bleeding so should be avoided See treatment and medicines for concomitant diseases in annexes

32 4. Information system This is a crucial aspect to allow data collection and analysis for monitoring and evaluation of the activities. In addition to the register books and patient s forms, data should be summarized in the monthly report forms and then submitted to MOH. When an outbreak is declared a weekly report form is also to be filled in (annex 17 18). 5. Prevention and control Leishmaniasis control in general is primarily based on finding and treating cases, combined where feasible with vector control and, in some zoonotic foci, control of animal reservoirs. In practice, providing access to sensitive diagnostics and quality treatment, and prevention of sandfly bites are currently the only feasible options in Somalia. Active case finding is may be opted for as it becomes essential if patients have difficulties and delays in reaching treatment. Prevention should aim at reducing the number of bites by wearing appropriate clothes (long sleeved) and repellents (ash, neem oil, commercial) during the evenings, especially during the dry season when kala-azar is transmitted. The use of long lasting insecticide- treated nets (LLINs) provides personal protection in areas of transmission. Fine mesh (jersey fabric) bed nets with or without impregnation will also protect, but are hot. Impregnated bed nets should be distributed to all Kala-azar patients at treatment centre. Health education should focus on; Appropriate usage of LLINs, including the mode of transmission of kala-azar The danger of sleeping outside without being protected by a mosquito net The signs and symptoms of kala-azar, and the availability and location of treatment centres, aiming at improving early reporting of symptomatic cases. 32

33 6. Annexes Annex 1. rk39 rapid diagnostic test procedure The utility of a rapid diagnostic test for visceral leishmaniasis lies in its simplicity. Several brands of test with rk39 antigen are available. Operators should always read the package insert carefully, and follow the manufacturer s instructions. This is especially important with regard to the type of specimen used: serum or whole blood. Some brands can be used only with serum, while others can be used with whole blood collected by finger prick. Test procedure In general, the test procedure is as follows (figure 3): 1. Remove the test strip from the pouch and place it on a flat surface. 2. Place a specified amount of patient specimen (serum or finger-prick blood) on the absorbent pad on the bottom of the strip. 3. Add the specified amount of buffer provided. 4. Read the result after min, according to the manufacturer s instructions. Some brands require a slightly different procedure, for example: 1. Take a test tube or a U-bottom microtitre plate. 2. Add a specified amount of buffer to the tube or well. 3. Add a specified amount of specimen (blood or serum) to the tube or well and mix. 4. Immerse the test strip into the buffer specimen mixture. 5. Read the result after min, according to the manufacturer s instructions. Points to consider for optimizing use of rapid diagnostic tests Have a clear management plan to deal with positive and negative results. Follow biosafety standards and precautions for handling blood and other body fluids. Ensure proper storage conditions. Do not use damaged or expired tests. Adhere strictly to the manufacturer s instructions. Use test kits within 1 h of removal from pouch. Read the results within the time specified by the manufacturer. Do not reuse a test. Interpretation of the test Positive result: When both control and test lines appear, the sample tested has antibodies against recombinant K39 antigen of Leishmania. Even a faint line should be considered positive. Negative result: When only the control line appears, there are no antibodies against recombinant K39 antigen of Leishmania present in the patient s sample. Invalid result: When no control line appears, a fresh patient sample should be tested with a new strip. 33

34 Advantages and disadvantages of the rk39 test Advantages Simple to perform with minimal training. Does not require a laboratory. Can be performed with finger-prick whole blood, serum or plasma. Kits can be transported and stored at ambient temperature (up to 30 C). Results are available within min. Disadvantages Cannot distinguish between active cases and relapse in previously treated cases. Therefore, interpretation must always be accompanied by clinical case definition. In patients with advanced HIV infection, a negative result does not rule out a diagnosis of visceral leishmaniasis. A. diamed-it leish This is a ready-made kit, and so follow strictly the instructions provided by the manufacturers. The test can be used either on finger prick blood or plasma/serum. The kit is provided with a device containing 2 wells: that of conjugate well (red line) and wash well. The device should be used within 15 minutes of opening the packet. Figure 3. Procedures for the DiaMed-IT LEISH test 34

35 Test procedure Take out the device from its package and place it horizontally on a flat surface. Write the name and an identifying number of the patient on the space provided. Tear open the ampoule of buffer, add 1 drop to the conjugate well and 4 drops to the wash well, and allow to stand for 1 minute. Add 8-12 l blood/serum/plasma to the conjugate well by squeezing the pipette gently. Stir gently with the upper end of the pipette and allow to stand for 1 minute Pull the device apart by holding the device with wells between thumb and forefinger, and with the other hand pulling out the dipstick holder (with label). Then place the wells on a flat surface, and insert the legs of the dipstick holder into the holes besides the conjugate well (with red line) so that the dipstick end reaches the bottom of the conjugate well. Allow to stand 10 minutes (5-10 minutes for serum/plasma). Transfer the dipstick to the second well (wash well) and allow to stand 10 minutes (5 minutes for serum/plasma). N.B. the reaction field should then be completely cleared of blood or serum/plasma, and the control band should be clearly visible. Remove the dipstick from the wash well and click it back into the clear plastic piece. Close the wells with the well cover, break them off, and break the two legs off from the clear plastic piece (to be discarded). Read the reaction and interpret the results. Keep the dipstick slide for future reference. The appearance of dark purple bands on the dipstick demonstrates a positive reaction in the presence of a control band. The test is invalid if the control band is not visible. A very faint line/band must be considered as positive reaction. DiaMed -IT LEISH Set. 35

36 B. Inbios kalazar detect test This is also a ready-made kit, and so follow strictly the instructions provided by the manufacturers. The test can be used either on finger prick blood or plasma/serum. The kit is provided in pouches containing strips and appropriate buffers. Test procedure Take out the test strips from its package and allow reach room temperature. Add l blood/serum/plasma to the area beneath the arrows shown in the strips. Place the test strip into a test tube or well of a 96 well tissue culture plate so that the end of the strip is facing downward. Add two to three drops (150 l) of the chase buffer provided with the test kit Read the results in 10 minutes 36

37 Annex 2. Direct agglutination test procedure Principle of DAT Infection with L. donovani results in production of antibodies against the parasite. These antibodies can be demonstrated in blood or serum by agglutination test. The DAT antigen is a whole, killed, promastigotes from cultures of L. donovani which have been stained blue for visibility. These are suspended in solution, and when left in a V-shaped well, will slowly fall to the tip of the V, giving a dense blue dot. If anti-leishmania antibodies are present from the blood or serum, the blue-stained parasites (DAT antigen), will become cross-linked by the antibodies (directly agglutinated) and will settle to the bottom of the well as a hazy blue mat or a cloud, NOT a dot. By diluting the serum 2-fold in each well starting at 100x dilution, the titer (quantity of antibody) can be measured. These procedures are for freeze-dried presentation of DAT. Collection of blood specimen for DAT Collect capillary blood from the finger or the toe or heel in infants. Method Requirements DAT request form DAT registration book Whatman No.3 filter paper Sterile lancet Disinfectant e.g. iodine, alcohol etc. Cotton wool Scissors Plastic bag Pen (ball point) Paper clips Procedure Cut the circular filter paper into 8-16 segments depending on the size. Each segment can be used for one patient s test only. Draw a circle of approximately 1cm in radius on the segment of the filter paper. Record the patient s details in the laboratory registration book (annex 7). Write the DAT number on the patient s request form and also write this number on the segment. Soak a piece cotton wool in iodine or alcohol and disinfect finger, toe or heel thoroughly. Allow the skin to dry. Using the sterile lancet, prick the finger firmly so that blood flows freely without excessive squeezing. Wipe the first drop of blood with a plug of dry cotton wool. The first drop of blood is normally contaminated with dirt and tissue fluid. 37

38 Squeeze the finger gently and collect the next drop of blood into the circle on the segment of the filter paper. Check the DAT number on the filter paper before collecting the blood. Make sure the blood soaks through both sides of the filter paper and fills the circle Allow the filter paper to dry. Apply pressure to the finger prick with a dry cotton wool. Discard the used cotton wool into the waste bucket and the lancet into the sharps container. When dry, very dry, clip the filter paper with the request form and put into a plastic bag. Store in a fridge or cool box until ready to send for testing or until ready for performing the test. Elution of samples Day 1 Requirements Laboratory register Microtitre plates (V-shaped) Sample (dry in filter paper) Micropipette to measure 125 µl Normal saline Paper punch (5mm) Scissors Marker (permanent) Forceps Refrigerator Procedure Write the number of the samples in order in the laboratory register. The list should be labeled to show which row of which microtitre plate corresponds to which sample. e.g. The list can be labeled A, B, C, D, E, F, G, and H in order. The samples need to be grouped by 8 samples in each group or less. Group one is called Plate I, group two is called Plate II and so on. Label the microtitre plates to correspond with the laboratory register. i.e. Get out the microtiter plates and write on the plate itself the plate number. The first one is plate I. Using the paper punch, punch out a sample of filter-paper blood. Using the forceps, put the punched sample of filter-paper blood into the well of the first column of the microtitre plates corresponding to the position recorded on the patient laboratory register. Ensure that the punched filter paper blood is properly inserted in the well. e.g. The sample listed as A should go in the first row labeled A. The sample listed as B should go in the second row labeled B, and so on. Take the micropipette and adjust it to measure 125ul, fix the pipette tip firmly and pipette 125 ml of normal saline. Add the 125ul of normal saline to each well with a sample paper. Make sure that the punched filter paper blood is completely immersed in the saline. 38

39 Cover the plates with another microtire plate incubate in the fridge (at 40C) overnight for at least 8 hours. Dilution and titration Day 2 Requirements Measuring cylinder 50ml container (plastic or glass bottle or conical tubes) Research (Repetitive) pipette l, adjustable; brand Handy-Step or Eppendorf with volume display, Combitip ejection. Multipipette μl Eppendorf or Handy-Step adjustable with Volume display and tip ejection. Multichannel pipette, 8 channels, 5 50 l Eppendorf, adjustable volume and tip ejection. Micropipette tips (yellow tips for 100ul Multipette and the Multichannel pipette, blue tips for up to 1000ul Research pipette, combitips standard 1.25ml and combitips plus 2.5 ml) 5 10 ml syringe 2-mercaptoethanol (2-ME) Normal saline DAT antigen (Freeze dried antigen OR Liquid antigen) Freeze dried control sera Preparation of diluent 1. To be used with Freeze dried antigen (FDA) Measure 50ml of normal saline and put in the container Adjust the multipipette to measure 390ul, pipette 390ul of 2-ME and add to the normal saline then mix gently. Reconstitution of the Freeze dried antigen (FDA) Add 5ml of fresh normal saline to the vial of the antigen Mix gently by rotating and tilting the vial. Do not shake Let it stand for about 10 minutes before use. Freeze dried antigen is kept at room temperature. Reconstitution of the freeze dried control Use a new set of control sera with every new batch of DAT antigen. Make sure all the freeze dried powder is on the bottom of the vial. (The control kits made in Amsterdam contain 2ul of serum each) Strictly follow the manufacturers instructions on the procedure for reconstitution especially the amount of normal saline or diluent to be added to the vial. Either add 100ul or 200ul of normal saline or diluent to the vial of the control sera depending on the manufacturers instructions. Mix gently by rotating. Let it stand for at least 10 minutes before use. 39

40 Dilution of samples Filter paper blood Take the microtitre plates with the eluted blood out of the fridge and allow it to come to room temperature (Serum dilution in column 1 is 1:50). Reconstituted control sera Fill the control well(s) in column 1 with 100ul of reconstituted control serum. This is a 1:50 dilution Adjust the pipette to measure 50ul. Fill the wells from columns 2 to 12 with 50ul diluent. Adjust the multichannel pipette to measure 50ul. Place 8 standard tips (Yellow tips) on the multichannel pipette, make sure they are firmly fixed to avoid pipetting errors. If the multichannel pipette is not available, you may use a single channel pipette and pipette each row separately. Mix the contents in column 1 by pipetting in and out at least 5 times. Avoid forming bubbles by expelling air prior to inserting the pipette tips into the wells and using a slow action. Pipette 50ul from column 1 and transfer to column 2. Continue this mixing and transferring until column 11. Discard the last 50ul from column 11. Do not add to column 12. (Serial ilution). Column 12 is the negative control. Adding antigen Gently rotate the antigen bottle to mix it. Do not shake the bottle as this may destroy the antigen Adjust the pipette to measure 50ml and fit the pipette tips. Add 50ul antigen to every well except wells in column 1. It is advisable that, to avoid contamination, start with wells in column 12 (negative control) and add row by row, and also change the pipette tip every time antigen is taken out of the bottle. Rotate the plates gently, both clockwise and anticlockwise. Cover the plates with a spare microtitre plate, leave them on a level surface at room temperature for about 12 to 18 hours. 40

41 Reading the DAT results Day 3 Put the plates against a white background. Estimate the titre by comparing the dots in column 12 (negative control) to those of the samples in the other columns. The titre is expressed as the last dilution that shows a difference compared to the negative control. A dark blue dot indicates that the result is negative and no reaction took place whereas a hazy blue mat or cloudy appearance indicates that reaction took place. The highest titre will be last dilution that still shows a hazy blue mat or cloud. Record the result in the lab book by titer and meaning. For example record well 9, positive. If you are not sure of the meaning (positive, negative or borderline) simply record the titer. Currently the positive titer is well 7 or 3200 with the FD antigen. Report the results in the patients request form. The titres are as follows. Column: Dilution: 1:50 1:100 1:200 1:400 1:800 1:1600 1:3200 1:6400 1: : :51200 See below an example of positive result: sample E: well 11, titre 1:51200, positive 41

42 Annex 3. Lymph node aspirate procedure Collection of lymph node aspirate The most common site for collection of the lymph node aspirate for diagnosis of kala-azar is the inguinal glands. Infected glands will be swollen. Method Items required: Sterile needle (21G ) Syringe (10ml) Clean glass slide Iodine (disinfectant) or sterile cotton swabs Cotton wool Procedure Allow the patient to lie comfortably. Prepare the syringe by pulling the piston back as far as possible. Feel both sides of the inguinal area to locate a swollen gland. Disinfect the chosen site with swollen glands using a piece of cotton wool soaked in iodine or suitable disinfectant. Take the gland between the thumb and index finger of the left hand. Hold it steady, at the same time making it stand out. Introduce the needle at right angle into the centre of the gland in two stages:- a) First pierce the skin b) Second penetrate the gland With your left hand, gently knead the gland. With your right hand, revolve the needle in both directions. The glandular fluid will ooze into the needle. Withdraw the needle in one rapid movement, holding the thumb over the hub. Then apply a swab dipped in iodine to the point of entry. Attach the syringe (piston pulled back) to the needle. Place the needle on the slide. Push the piston gently down the barrel to discharge the glandular fluid contained in the needle onto the slide Make a thin film using the fluid on the slide. The fluid can be discharged in more than one slide. 42

43 Annex 4. Bone marrow aspiration procedures Materials needed Sterile BM needle 10 ml syringe Clean microscope slides NNN or any other suitable culture medium Wooden applicator or tooth picks Spirit lamp with sufficient flame Drapes Sterile gloves Sterile cotton and gauze Plaster Labels Pen and pencil/marker Biopsy aspiration needles (recommended sizes): Regular/Adults: Adults: Orthopaedic: Paediatric: Infant: Pre-operative procedures 4-inch, 11-gauge 4-inch, 8-gauge 6-inch, 10/11-gauge 3 ½ -inch, 13-gauge 2-inch, 13-gauge No specific procedures are needed. Aspiration procedures 1. Place the patient in a right or left lateral decubitus position with the back comfortably flexed and the top knee drawn toward the chest. 2. Locate the posterior iliac spine and mark with ink or thumb nail pressure 3. Using sterile technique, prepare the skin with anti-septics and drape 4. Using sterile syringe, apply/infiltrate the marked area with local anaesthetic especially the periosteum 5. Make a 3-mm skin incision with a scalpel blade over the marked area. 6. Hold the needle with the proximal end in the palm and the index finger against the shaft near the tip. 7. With the stylet locked in place, introduce the needle through the incision pointing toward the anterior superior iliac spine and bring it into contact with the posterior iliac spine. 8. Using gentle but firm pressure, advance the needle to bore through the iliac spine. 9. Rotate the needle in an alternating clock-wise and counter-clockwise motion. Entrance into the marrow cavity is generally detected by decreased resistance. 43

44 10. Remove the stylet, and check for presence/absence of marrow material. If not, proceed to bore until marrow is found in the tips of the stylet. 11. With a syringe locked into the proximal portion, apply a negative pressure. 12. The material can then be expelled on to clean slides and also inoculated into appropriate culture medium (preferably NNN medium) N.B. For cultures, insert needle into a tube containing culture medium and push the plunger into the barrel to expel contents of the needle on to the side walls of the tube or directly into the liquid phase of the medium. You may repeat this once or twice until the LN material is visible in the tube. For safety purposes, inoculate 2 culture tubes. For smears, expel any remaining material gently on clean glass slides holding tip of needle on the surface of slide, and spread evenly into a smear immediately using a linear motion. More material can be obtained at the end of the plunger or the needle (or tip of syringe) after removing the plunger and needle. Tooth picks or wooden applicators may be used for this purpose. It is important that culture tubes are not over loaded by large amounts of inoculum, which is not uncommon with BM aspirates. 13. Slides can be stained with Leishman, Giemsa or Wright s stain (See Annex 4). NNN cultures should be incubated at 25 0 C for up to 2 weeks. Post-operative procedures No specific procedures are needed 44

45 Annex 5. Procedures for splenic aspiration Splenic aspiration should be performed only if the following conditions are met: absence of clinical contraindication(s): - signs of active bleeding (e.g. epistaxis, rectal bleeding, skin bruises) - jaundice (a potential marker of liver dysfunction) - pregnancy - spleen barely palpable - bad general condition (e.g. cardiovascular shock, altered consciousness) absence of biological contraindication(s): - severe anaemia (haemoglobin count 5 g/l) - difference in prothrombin time between patient and control > 5 s - platelet count < /ml rapid access to blood transfusion in case of bleeding The two important prerequisites for the safety of the procedure are rapidity, so that the needle remains within the spleen for less than l s; and precision, so that the entry and exit axes of the aspirating needle are identical to avoid tearing the splenic capsule. The procedure is as follows: 1. Clean three glass slides and label them with patient s name, date and the words splenic aspirate. Have culture medium ready (if available) and labelled in the same way as the slides. Attach a 1 1/4 -inch 21-gauge ( mm) needle to a 5-ml syringe. Place all items on a table at the bedside. 2. Inform the patient about the procedure. Check all clinical and biological contraindications again. Palpate the spleen and outline its margins on the patient s abdomen with a pen. For safety, the spleen should be palpable at least 3 cm below the costal margin on expiration. Use an alcohol swab to clean the skin at the site of aspiration, and allow the skin to dry. 3. With the 21-gauge (0.8-mm) needle attached to the 5-ml syringe, just penetrate the skin, midway between the edges of the spleen, 2 4 cm below the costal margin. Aim the needle cranially at an angle of 45 to the abdominal wall. The actual aspiration is done as follows: pull the syringe plunger back to approximately the 1-ml mark to apply suction, and with a quick in-and-out movement push the needle into the spleen to the full needle depth and then withdraw it completely, maintaining suction throughout. 4. For young, restless children, have two assistants hold the child (arms folded across the chest, with shirt raised to obstruct the line of vision, and pelvis held firmly). Carry out the aspiration as a single-stage procedure, using the same landmarks, angles and suction as in step 3, all in one quick motion. The insertion should be timed with the patient s breathing so that the diaphragm is not moving; this should be done during fixed expiration if the child is crying. Only a minute amount of splenic material is obtained, but this is sufficient for culture and smear. 5. If culture is available: slowly pull the plunger back to the 2 3-ml mark, and, using sterile techniques, insert the needle into a tube containing culture medium and briskly push the plunger into the barrel to expel the contents of the needle onto the side walls of the tube. lf necessary, repeat once or twice until splenic material is visible in the tube. Replace the cap on the tube and invert to wash splenic material on the side of the tube. Repeat the procedure for the second tube of culture medium. Sterile techniques are essential throughout. 6. Expel material (or additional material if culture is available) gently onto glass slides, holding the needle tip on the surface of the slide. Immediately spread evenly with the needle, using a linear (not circular) motion. The smear should be slightly thinner than a 45

46 thick blood film for malaria. Remove the needle, and use the end of it to obtain additional material from the tip of the syringe and spread it on slides. Further material found on the end of the plunger may be dabbed directly onto a slide and spread. Allow the slides to dry. 7. Write the time of aspiration on the patient s chart, with the instructions: "Record pulse and blood pressure every half hour for 4 h, then every hour for 6 h. Patient must remain in bed for 12 h." Ensure that the patient understands the instructions. Enter the procedure in the notes, and sign. 8. Take the slides (and medium) to the laboratory for preparation and microscopic examination. 46

47 Annex 6. Preparation and examination of aspirates. Grading of parasites Preparation of the aspirates Prepare thin films of the splenic aspirate material or lymph gland fluid. Spread the films on a clean glass slide immediately after collection, before the material clots. Slides are stained with Giemsa as for a thin malaria film and examined under oil immersion Items required: Methanol Glass slide Slide rack Giemsa Buffer solution ph 7.2 Microscope Thin films Collect a drop of the aspirate/fluid on one end of the slide; about 1-2 cm from one end. Place the slide horizontally on a flat surface. Hold the sides of second slide (spreader) or coverslip on to the center of the specimen slide and move it backwards until it touches the drop of the fluid. Let the fluid spread along its base. At an angle of between , move the spreader firmly and steadily across the specimen slide to make a film. A good film should have a conical tail and should cover about two-thirds of the slide. Let the film dry. Fix the dried smear by dipping in absolute methanol for a few seconds and allow to airdry on a drying/draining rack. Fixation Place the slides horizontally on the slides rack and leave to air dry. Fix the slides by dipping them in 100% methanol for 1minute. The methanol must be stored in a tightly closed bottle to prevent absorption of water. Staining: Stain the slides with Giemsa stain 1: 10 concentration; 1ml of stock Giemsa stain to 9ml buffer solution ph 7.2. The slides can either be stained in a staining trough or on a staining rack. Staining in trough Place the slides in a staining trough Pour the stain gently into the trough until the slides are totally covered. Avoid pouring the stain directly onto the film Leave the slides to stain for minutes. 47

48 Pour clean water into the trough to float off the scum on the surface of the stain. The water should be poured into the end of the trough. Gently pour off the stain and rinse again in clean water. Then pour off the water. Remove the slides one by one and place them in vertical position on a slides rack to dry Staining on a rack Use a test tube or a small container to hold the prepared stain Gently pour the stain onto the slide or use a pipette to drop the stain on to the slide Leave the slides to stain for minutes Gently flush the stain off the slide by adding drops of clean water. Never pour the stain off the slides, otherwise the surface scum will stick to the film and spoil if for microscopic examination Place the slides in vertical position on a slides rack to dry Microscopic examination of stained aspirates Method Place the microscope on a firm bench, free from vibration. Switch on the light source. If there is no inbuilt light source, adjust the flat side of the mirror to reflect the light up through the condenser. Adjust the eyepieces by sliding them horizontally until they fit both eyes comfortably and the two fields merge. Centre the condenser if applicable. Prepare the slide for examination by putting a drop of oil immersion on the smear. Clean and dry underneath of the slide by wiping with cotton gauze/wool or tissue paper. Rotate the nosepiece until the low power objective is in position (x10). A slight resistance and a click are felt as the objective moves to the correct position. Place the slide carefully on the stage. Never place the slide on the stage when the x40 or x100 (oil immersion) objectives are in position as this may scratch the lenses. Adjust the illumination. Focus the specimen by racking the stage up to the top and then while observing through the eyepiece, rack the stage down slowly using the coarse adjustment knob, until the image comes into view. Use the fine adjustment knob to focus the image sharply. Scan the film and select a part that is well stained, free of staining debris and well populated with white blood cells. Swing the required objective into position i.e. oil immersion (x100) objective. Focus using the fine adjustment knob. Never use the coarse adjustment knob because the objectives are perfocal. Adjust the illumination by opening the iris diaphragm as required, for oil immersion objective open the iris diaphragm fully. Examine the specimen systematically, moving from field to field using the knobs that control the mechanical stage. For example, start at the selected site and move horizontally to the top right hand corner. Move the slide down by one field and then horizontally in the other direction to the end of the smear. Continue until the whole specimen has been examined. 48

49 After examination, lower the stage or swing the lowest power objective into position before removing the slide from the stage. Never remove the slide from the stage when x40 or x100 objectives are in position, as this may scratch the lenses. Wipe the oil immersion objective using lens cleaning tissue. Switch off the microscope. Identification of the Leishmania parasite Leishmania amastigotes are oval or round and are usually seen in the cytoplasm of monocytes. Free parasites may be seen if the host cells are ruptured during preparation of the film. In stained preparations, Leishmania amastigotes contain two visible structures: Nucleus Kinetoplast The nucleus and kinetoplast stain dark reddish. The cytoplasm stains pale pink. The nucleus of the host cell may be pushed to one side by the multiplying parasites. Leishmania amastigotes are sometimes referred to as L.D. bodies. Free amastigotes Intra-cellular amastigotes If Leishmania amastigotes are seen, report the slide as: Lymphnode or splenic aspirate: Leishmania amastigotes seen and indicate the grade. Examine at least 1000 fields before declaring a film to be negative. Grading of slides Parasite grading has several uses. It increases the sensitivity of parasite detection, provides an objective measure of the speed of response to treatment, distinguishes quickly between slow responders and nonresponders, and provides an indication of parasite load that is useful in research. 49