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1. MALARIA DIAGNOSIS PRESENTED BY: LABORATORY TEAM (ZIA ANGELINA H/C)

2. Introduction Human malaria is caused by one of the protozoan parasites Plasmodium falciparum, vivax, ovale , malariae , knowlesi -for forested regions of South East Asia. Malaria is transmitted through the bite of an infected female Anopheles mosquito. Malaria still remains the leading cause of morbidity and mortality globally with an estimated 228 million cases (WHO, 2018) and 438,000 deaths in sub-Saharan Africa alone ( Zgambo et al., 2017). In 2022, Uganda accounted for approximately 5.1% of global malaria cases, ranked third in terms of malaria burden and eighth in malaria-attributed death worldwide (WHO, 2023).

3. Life cycle of malaria parasite

4. Sample views of four different malaria parasite species and their life-stages

5. Diagnostic tools for human infections with malaria Parasitological diagnosis of malaria requires examination of blood smear for the presence of malaria parasites which is the ‘gold standard’ of malaria diagnosis. 1. Blood film examination (Microscopy) Thick blood film: detection and quantification of parasites Thin blood film: species identification and parasite density Note: Blood films may be negative due to sequestration of the parasitized erythrocyte in peripheral capillary in severe malaria, as well as in placental vessels in pregnant women

6. Thick and thin smear

7. Microscopy Thick smear Thin smear

8. Reporting of malaria results If the result is negative, report as “no malaria parasites seen (No mps seen)”. If there are malaria parasites, report the approximate numbers of parasites as below; 1-10 parasites per 100 high power fields………………………..+ 11-20 parasites per 100 high power fields ……………………. ++ 1-10 parasites in every high power field………………………. +++ >10 parasites in every high power field.………………………..++++

9. 2. Rapid diagnostic test (RDTs) Rapid test detects parasite antigens. They give only a qualitative result (pos or neg) in 2-15 minutes and may remain positive several days or weeks following effective treatment. Note: Smear still required to confirm RDT result Tests which detect histidine-rich protein 2 (HRP2) are specific for P. falciparum while those that detect parasite lactate dehydrogenase (pLDH) –optimal or aldose have the ability to differentiate between P. falciparum and non falciparum malaria

10. Principle for Malaria Ag P.f test device The SD BIOLINE Malaria Ag P.f test device contains a membrane strip, which is precoated with mouse monoclonal antibodies specific to HRP-II of P. falciparum at the test line region ( P.f ). The mouse monoclonal antibodies specific to HRP-II of P.f -colloid gold conjugate reacts with the Malaria Plasmodium falciparum antigen in the specimen. They move along the membrane chromatographically to the test region “ P.f ” and form a visible line as the antibody-antigen-antibody gold particle complex with high degree of sensitivity and specificity.

11. Interpretation of test results The Test result is non-reactive if one coloured line appears in the control line region (C) and no coloured line appears in the test line region ( P.f ). The Test result is reactive if coloured line appears in the control region (C) and another coloured line appears in test region ( P.f ). The Test result is invalid if there is no control line in the control line region (C) and even if a coloured line appears in the test line region ( P.f ), the test should be repeated with a new test cassette. Do not report invalid results.

12. Principle for Malaria Ag P.f /Pan test device The SD BIOLINE Malaria Ag P.f /Pan test device contains a membrane strip, which is precoated with mouse monoclonal antibodies specific to HRP-II of P. falciparum on test line P.f region and with mouse monoclonal antibodies specific to lactate dehydrogenase of Plasmodium species Pan (P. falciparum, P. vivax, P. ovale and P. malariae ) on test tine Pan region respectively. The mixture of mouse monoclonal antibodies specific to HRP-II of P.f and mouse monoclonal antibodies specific to pLDH of pan-colloid gold conjugate reacts with the malaria antigen in the specimen. They move along the membrane chromatographically to the test region ( P.f and Pan) and form a visible line as the antibody-antigen-antibody gold particle complex with high degree of sensitivity and specificity.

13. Interpretation of test results The Test result is non-reactive if one coloured line appears in the control line (C) region and no coloured line appears in the test line ( P.f and Pan) regions. The Test result is P.f reactive if coloured line appears in the control (C) region and a coloured line appears in P.f region. The Test result is Pan ( P.v , P.m or P.o ) reactive if coloured line appears in the control (C) region and a coloured line appears in Pan region. The Test result is invalid if there is no control line in the control line (C) region and even if a coloured line appears in the test line ( P.f and Pan) regions, the test should be repeated with a new test cassette. Do not report invalid results.

14. Importance of MRDTs Rapid diagnosis. Can differentiate falciparum and other species. Suitable for microscopists less experienced in examining blood films. Can detect malaria antigens up to 14 days.

15. 3. Molecular Diagnosis Parasite nucleic acids are detected using polymerase chain reaction(PCR).This technique is more accurate than microscopy. However, it is expensive and requires a specialized laboratory. Used to identify or confirm species Lengthy turnaround time

16. Additional examinations Complete blood count: Hemoglobin level and thrombocytopenia Blood glucose level-to be measured routinely to detect hypoglycemia (<3 mmol/l or 55mg/dl) in patients with severe malaria and those with malnutrition.

17. Blood examination for malaria parasites must be done for the following groups of patients: Patients who present with clinical features of severe malaria Patients who have taken antimalarial treatment for 2 days and symptoms persist. Children aged less than 4 months with symptoms of uncomplicated malaria Pregnant women with symptoms of uncomplicated malaria

18. Key messages Suspect malaria among individuals with fever and a recent history of travel, or fever without an alternative etiology A malaria blood smear is needed for all suspected malaria cases, but a rapid diagnostic test can shorten time to treatment initiation Species determination in all cases is necessary to determine when to treat dormant liver parasites (hypnozoites). Early malaria diagnosis and prompt treatment can prevent severe disease and death and reduce the risk of malaria transmission Expanding use of malaria prophylaxis to all travelers to malaria endemic countries is a key prevention strategy.

19. Prevention Avoid mosquito bites by: Wearing long sleeves or trousers Insecticide treated mosquito bed nets Repellent creams or sprays Cover doors and windows with screens Close doors and windows ( atleast from 6pm), etc Malaria prophylaxis for travelers travelling to endemic areas Vaccination

20. A case study: Reported monthly malaria infections at Zia Angelina H/C, Wakiso district, Central Uganda in 2024

21. Positive By Gender

22. References (WHO. World malaria report 2023. Geneva: World Health Organization; 2023).

23. Thank you