Tackling problems in malaria & syphilis screening. Are we doing enough?

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1 Tackling problems in malaria & syphilis screening. Are we doing enough? Dr Prashant Agarwal Additional Professor Department of Transfusion Medicine SGPGIMS, Lucknow

2 Malaria Protozoan disease :genus plasmodium Most imp parasitic disease of humans Transmission in 103 countries; 2.4 billion of world s population at risk Incidence : million clinical cases/year million deaths/year

3 Problem statement in India Consistently declining trend in annual malaria incidence. Major endemic areas are NE states (Assam), AP, Chhattisgarh, Gujarat, Jharkhand, MP, Maharashtra, Rajasthan and Orissa. Infections are mostly caused by P.vivax and P.falciparum. P.malariae has restricted distribution in Tumkur and Hassan districts of Karnataka. The current epidemiology of malaria in the country is described according to WHO guidelines by API. In the year 2000, million blood smears were examined with an API of 2.04/1000 population.

4 Agent for malaria Caused by 4 species of genus plasmodium: P.falciparum P.vivax (widest geog. distribution) P.malariae P.ovale Modes of transmission Vector transmission: bite of infected female Anopheline mosquito Direct transmission: by blood transfusion, i/v inj in drug addicts Congenital malaria: from mother to new born (rare)

5 Malaria transmission cycle

6 Transfusion Transmitted Malaria Accurate data on incidence of TTM are confounded due to underreporting esp in endemic areas. Transfusion transmitted malaria (TTM) first reported in 1911 by woolsey, More than 3000 cases have been described so far Annual incidence of TTM varies cases/ million units in the USA and Canada 50 or more cases per million units in endemic countries

7 Infectious dose is estimated to be 1-10 parasites per unit of blood. Donors implicated are invariably asymptomatic plasmodial carriers, semi immune individuals with parasite densities below the limit of detection of currently available assays. WB and PRBC represent the most common source of TTM Cases involving platelets, leucocytes, FFP, frozen RBCs have all been reported. TTM often manifests with non specific clinical features Caused by transmission of asexual forms of ery. schizogony. Also c/d as trophozoite induced malaria

8 Difference between TTM and vector borne malaria SPOROZOITE INDUCED MALARIA TROPHOZOITE INDUCED MALARIA Transmission By vector By blood transfusion Pre-ery schizogony Incubation period Exo-ery schizogony Present Long i.e days May be present Absent Short i.e days Absent Relapses May occur Do not occur

9 Malaria Risk Reduction Strategies NON ENDEMIC POPULATION Rely upon the appropriate donor deferral criteria and upon preciseness of information given by donor regarding their travel and malaria risk. Problems: Correct identification of malaria risk individual is difficult. Semi immune donors may carry parasite for long duration (>10 years) risk of TTM as they are not picked up by donor questionaire affect donor pool and many donors do not return To address above problems, many countries have combined travel based risk exclusion with serological testing to the restriction period.

10 Screening of Donors to prevent TTM Focus on minimizing the risk of introducing MP into blood supply without unnecessarily excluding any potential donor. Deferral guidelines are based on: LOCATION- Countries are identified as malaria risk or not but these distinctions not absolute. DURATION OF RESIDENCE IN MALARIAL AREA- Longer the period of residence, more the risk of becoming semiimmune PERIOD SINCE LAST VISIT IN MALARIAL AREA- at least 3 month deferral in case of endemic areas like India.

11 AABB guidelines for donor deferral to prevent TTM Donor information Had diagnosis of malaria/traveled or lived in malaria endemic area and had unexplained symptoms of malaria Lived for atleast 5 consecutive years in areas where malaria is endemic Traveled to an area where malaria is endemic Deferral period 3 years after becoming asymptomatic 3 years after departure from that area 12 months after departing from that area

12 Malaria Risk Reduction Strategies ENDEMIC POPULATION Travel based restriction and serological testing ineffective Strategies: Screening by microscopy for MP and Ag detection in India and many countries of Africa. PCR to screen donated blood in Veitnam. Segregation of donations from low and high risk localities with specific targeting of blood from low risk donors to low risk/vulnerable recipients. Provision of routine antimalarial treatment to transfusion recipients Addition of antimalarial drugs into blood units: efficacy not assessed. Judicious use of blood so that patients are not exposed unnecessarily.

13 Concerns : Difficult to differentiate TTM and conventional malaria. Most donors/pts already infected with malaria or are at high risk. Sensitivities of currently available assays between parasites/µl, thus low parasitemia is missed TTM. Most donors immune so Ab based assays not applicable (would result in high discarding of collected blood)

14 Screening of blood Donations 4 specific targets for donation screening. Intracellular parasite Microscopy Plasmodial antibody Enzyme immunoassay Plasmodial antigen Immunochromatographic tests based on LDH, HRP-2 Plasmodial nucleic acid

15 Direct Parasite Detection by microscopy Examination of Giemsa or Wright stained thick and thin blood film Sensitivity of 5-50 parasite/µl in experienced hands, routinely 500 parasites/µl. Problems In Screening By Microscopy Time consuming. Labor intensive. Interpretation requires considerable expertise. Lysis of RBCs during staining of thick film. Prolonged positivity post treatment due to observation of large no. of circulating dead parasites

16 Direct Parasite Detection by Fluorescence Microscopy Based on dyes with affinity to parasite nucleic acid. Acridine orange and BCP excited at 490 nm and exhibit apple green or yellow fluorescence. Sensitivity for parasites <100/µl ranges from 41-93%. Excellent specificity for P. falciparum (>93%). Lower specificity for non P.falciparum infections (52%). Limitations in screening blood donors as needs specialized equipment and low sensitivity

17 Antibody Detection Antibodies produced 1-14 days after infection and detectable for months to years after parasite clearance. Positive result indicate current clinical/subclinical malaria or immunity in parasitemic individuals. In endemic areas like India, Ab positivity is 20-90% Positive results compromise the efficacy of test for screening purpose. Increased discard rate In non endemic pop. Ab +ve donors 1-2% :Ab screening has found favour.

Antigen Detection Immunochromatographic tests based on the capture of parasite Ag from peripheral blood using conjugated monoclonal Ab against a malaria Ag

18 Antigen Detection Immunochromatographic tests based on the capture of parasite Ag from peripheral blood using conjugated monoclonal Ab against a malaria Ag target. Assay sensitivities range from 100 to 1000 parasites/µl. Give rapid results (15-20 min). Available in dipstick format and use whole blood as sample of choice

19 Malaria antigens currently targeted by RDT are: 1. HRP-2:- a water soluble protein produced by asexual stages and young gametocytes of P.falciparum. It is expressed on RBC membrane and 1 st Ag used in RDT 2. pldh:- an enzyme found in glycolytic pathway. Different isomers are produced by sexual and asexual stages of all 4 plasmodium species. 3. Aldolase:- another enzyme in the glycolytic pathway of malaria parasite. Ab produced are panspecific in reaction.

20 Expensive Limitations of antigen tests Relatively insensitive False negative results: In first few days of malarial infection when Ab is not detectable. In individuals with gene deletion for production of HRP-2 Monoclonal IgG Ab used may cross react with serum RF, causing false positive results.

21 Nucleic Acid Testing for screening of malaria High sensitivity with ability to detect 5 parasites or less/µl of blood. Methods using nested PCR and reverse transcription PCR enable all 4 species to be identified. Not suitable for universal donor screening due to High cost Complex and time consuming Transient parasitemia not detected parasite load not detected

22 Hemozoin Detection for malaria screening in blood donors Basis: detection of the hemozoin, a heme malarial breakdown product ingested by monocytes. The property of hemozoin to depolarize laser light discriminate normal monocytes from those having ingested hemozoin, thereby providing indication of potential infection. Unsuitable for donor screening due to lack of sensitivity (62%).

23 Comparison of various methods for malaria screening Parameter M/E PCR QBC HRP-2 pldh Sensitivity >100 >100 Specificity All spp All spp P.f. good P.f. only All spp. Parasite density Yes No No Crude estimat. Crude estimat. Time min 24 h min 20 min 20 min Skill req high high mod low low Equipment microscop PCR app Fluores. Microsc. Kit only Kit only Cost low high mod mod mod

24 Which is best method???? 100 parasites/unit (25 X 10-5 /µl) 10 parasites/unit (2.5 X 10-5 /µl) PCR (0.05 to 0.1parasites/µl) Microscopy (5 parasites/µl) Antigen detection (10 to 100 parasites/µ l) Parasite densities (parasites/µl) parasites/unit correspond to 2.5 X 10-5 /µl which is 4000 times lower than the PCR threshold

25 Indian scenario Guidelines for prevention of TTM given by Drug and cosmetic act of India are: Mandatory to screen every unit for MP though the method has not been specified. Deferral period for malaria: 3 months, duly treated. DGHS Technical Manual recommends antimalarial prophylaxis to all recipients of blood. (not followed anywhere in the country) Most of the donated blood across the country is Inadequately screened for malaria. Malaria immunoprophylaxis to all blood recipients is also not feasible practically.

26 Dilemma for appropriate testing method Microscopy cumbersome and labor intensive for high volume screening Malaria antibody screening not indicative of active infection and results in unnecessary high discarding of collected blood units as the antibody may persist up to several years after infection. PCR and antigen detection tests have limited availability.

27 Data from blood donors and recipients scanty. No reports of TTM from India. Studies indicate: - Only 67% of blood centers do screening for MP, that too by slide method. - No reports on units positive or discarded. - Only serological studies indicating Ag prevalence of 0.35% and Ab prevalence of 12.39% (IFAT) and 19.37% (ELISA). Author Suhailur Rehman et al(2015) Tulika Chandra et al (2013) Prevalance 0.12% 0.01% Gita Negi et al 0.08% Bahadur et al..(2010) 0.03% Chattoraj et al (2008) Chaudhary and Tetali (2007) None None Malaria prevalence in indian blood donors

28 In a nutshell The critical lack of evidence about the clinical impact of TTM and the absence of an effective and feasible screening method are impediments to rational decision making about when and how to screen blood for malaria. There are no screening tools for malaria that are practical, affordable and suitably sensitive for use by blood banks. Cost benefit studies should be conducted to point out the most suitable method to be used for the prevention of TTM. One should however bear in mind the complete prevention of TTM may not be attainable, so malaria must be always considered in any patient with a febrile illness post operatively.

29 TRANSFUSION TRANSMITTED SYPHILIS

30 Epidemiology WHO estimates that the annual global incidence of syphilis is approx 12 million cases, most of which occur in developing countries Transfusion transmitted syphilis first described in 1915 By 1941,138 cases had been described Transfusion transmitted syphilis has become non-existent in US, no reported case in more than 30 yrs. From 1965 all over the world 3 cases reported from developing countries. Prevalence of syphilis among blood donors in India reported to be 0.7%.

31 Transfusion Transmitted Syphilis In recent years there is no documented case of transfusion transmitted syphilis : Low incidence rate of disease. Practice of refrigerated blood storage Improved donor selection process. Uniform application of screening test Transfusion recipients were often under antibiotic therapy

32 Serologic Testing For Syphilis Standard nontreponemal (reaginic) tests The serum of a person with syphilis contains a non-specific anti-lipid antibody (termed reagin), which is not found in normal serum. IgM and IgG Ab to lipoidal material released from damaged host cells and to lipoidal like antigens of T.pallidium Qualitative and semiquantitative Reactive 1-4 wks after chancre appears. Prozone phenomenon occurs in 2% of sera.

33 Specific treponemal antibody tests Detect antibodies to antigenic determinants of treponemes Qualitative methods Once positive,remain positive for life Differentiate true positives from false positives Methods include MHA-TP FTA-ABS FTA-ABS double staining

34 Other Serological Tests For Syphilis Enzyme immunoassay equal to or better than FTA-ABS and TP-PA tests in overall sensitivity and specificity amenable to automation, removing the variation and subjectivity of the human reader ID-PaGIA Syphilis Antibody Test Column agglutination based test simple, rapid (20 min), and useful test with sensitivity, specificity, and positive and negative predictive values of 91.9, 99.8, 99.2, and 98%, respectively

35 Rapid Tests Solid Phase Immunochromatographic assay Membrane strip pre-coated with recombinant T.pallidium antigens (MW 47,42,17&15KDa based on western blot analysis) on test band region. Visible line in the test region indicates positive result as the antigen-antibody-antigen gold particle complex forms. Sensitivity 99% & specificity 99.5%

36 NAT testing for syphilis Molecular biological expertise required Special precautions to prevent cross-contamination Commercially available NAT assays require more than 12 hours to perform The cost of each commercial NAT test is approximately 10 times that of the most expensive EIA test.

37 WHO Recommendations 2009 To minimize the risk of syphilis infection through the route of transfusion: 1.Screening should be performed using a highly sensitive and specific test for treponemal antibodies: either TPHA or enzyme immunoassay. 2.In populations where there is a high incidence of syphilis, screening should be performed using a non-treponemal assay: VDRL or RPR

38 Routine Blood Donor Testing For Syphilis RPR or an automated treponemal tests ( PK TM TP hemagglutination) for donor screening If reactive, it is confirmed by testing FTA-ABS or an enzyme immunoassay Blood centres that use the automated treponemal test for screening may further test the donor using RPR as a measure of recent exposure for donor counseling

39 Concerns regarding syphilis transmission Platelet concentrates stored at room temperature (22 C) or transfused within a few hours of collection carry a higher risk of transmitting syphilis. The risk high in developing countries with limited blood supplies where the blood is collected from family donors and transfused within hours. Screening test is essential to prevent Blood donor did not have a history of venereal disease or the presence of sores at the time of donation. Thus, syphilis can be transmitted from donors who clinically and biologically do not show any signs of their disease.

40 Rationale for screening syphilis Closely related to risk factors in the blood donor, in particular sexual behavior since the disease is primarily transmitted by the sexual route. The rates of infection are high among homosexual men bisexuality (men having sex with both men and women), two or more sexual partners, intravenous drug use a past history of syphilis treatment HIV sero positivity are closely related to transfusion-transmitted syphilis skin scarification (tattooing, blood rituals)

41 Donor Screening for Syphilis CONS Syphilis testing costs money Non specificity of tests results in a significant discard rate Sero positive donors with adequate treatment are often embarrassed and inappropriately deferred Syphilis rarely is transmitted by blood transfusion Current testing is not perfect--window period cases do occur

42 Donor Screening for Syphilis PROS The rarity of post transfusion syphilis may be caused, in part, by screening There may be public health value in screening High titer antibody can be passively transmitted There may be value as a surrogate test for HIV and/or hepatitis Testing is required by the Food and Drug Administration

43 Indian status Current syphilis tests may not be sensitive but it should be continued to exclude high-risk donors Gold standard method is still lacking because no test is ideal for all stages of syphilis Author Prevalance P. Pallavi et al % Sri Krishna et al % Singh et al % Arora % Bhattacharya % Khare V et al %

44 Conclusion Malaria and syphilis testing lacks ideal screening tests Malaria testing not being done universally in view of non availability of feasible tests Syphilis screening being done universally Both the testing need to done for a safe blood supply however better screening tests need to be investigated

45 Sensitivities and Specificities of Syphilis serology tests Standard Status Non-treponemal Tests Sensitivity (%) by Stage of Untreated Syphilis Specificity Test Primary Secondary Latent Late Nonsyphilis VDRL 78 (74-87) (88-100) 71 (34-94) 98 (96-99) RPR 86 (77-99) (95-100) (93-99) Standard Status Treponemal Tests Sensitivity (%) by Stage of Untreated Syphilis Specificity Test Primary Secondary Latent Late Nonsyphilis FTA-ABS 84 (70-100) (84-100) MHA-TP 76 (69-90) (97-100) 99 (98-100)

HUMASIS MALARIA ANTIGEN TEST HIGH SENSITIVE DIFFERENTIAL DIAGNOSIS OF MALARIA INFECTION

HUMASIS MALARIA ANTIGEN TEST HIGH SENSITIVE DIFFERENTIAL DIAGNOSIS OF MALARIA INFECTION References 1) World Malaria Report 2010, WHO 2) Rapid diagnostic tests for malaria parasites, Clin. Microbiol. Rev.