Sysmex Educational Enhancement and Development No

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1 SEED Malaria Sysmex Educational Enhancement and Development No Malaria diagnostics in the era of improved malaria control The purpose of this newsletter is to provide an overview of the role and limitations of RDTs and blood smear microscopy in the diagnosis of suspected malaria in malaria endemic regions. Key words: malaria, rapid diagnostic test (RDT), thin smear, thick smear, microscopy, parasitaemia, Plasmodium falciparum, Plasmodium vivax, WHO, Sysmex The global status of malaria Malaria continues to pose a major public health challenge. As of 2015, there were more than 200 million malaria cases and more than 400,000 malaria deaths worldwide. More than 3 billion people are still at risk of infection in 97 countries and territories. Depending on the intensity of transmission and the parasite species involved, the clinical and public health impact of malaria is geographically variable. However, in the last few years, extensive efforts to control malaria have remarkably resulted in a significant reduction in the mortality and incidence rates. In fact, nonmalarial febrile illnesses cause more deaths than malaria even in malaria-endemic countries and in the absence of accurate or available diagnostics, there is still widespread presumptive treatment of every fever with antimalarial drugs. Not only does this contribute to the generation of drug resistance but also a significant number of preventable deaths due delayed or missed diagnosis of bacterial sepsis or acute viral illnesses. There is no combination of signs and symptoms that will reliably distinguish malaria from other causes of acute febrile illness. This poor specificity leads to overtreatment of malaria. Other possible causes of fever should always be considered as alternate or additional treatment may be required. It is generally acknowledged that the widespread use of diagnostic testing for malaria will improve the outcome of non-malarial febrile illnesses. What also needs to be considered is that there are a large number of people living in high transmission areas that may test positive for malaria but remain asymptomatic as they have developed a degree of immunity against the malaria parasite. The role of diagnostics Diagnosis of malaria involves identification of malaria parasite or its antigens in the blood of the patient. Although this seems simple, the efficacy of the diagnosis is subject to many factors. The different forms of the five malaria species; the different intraerythrocytic stages of the parasite life cycle; the prevalence of different species in the area; population movements; the inter-relation between the levels of transmission, immunity, parasitaemia, and the symptoms; the problems of recurrent malaria, drug resistance, persisting viable or non-viable parasitaemia, and sequestration of the parasites in the deeper tissues; and the

2 2 use of chemoprophylaxis or even presumptive treatment on the basis of clinical diagnosis can all have a bearing on the identification and interpretation of malaria parasitaemia on a diagnostic test. The WHO 2015 treatment guidelines recommend that all cases of suspected malaria should have a parasitological test (microscopy or rapid diagnostic test (RDT)) to confirm the diagnosis and that such tests should be supported by a quality assurance programme. Treatment should only be initiated once the diagnosis is confirmed, unless the patient is acutely ill and has a high probability of malaria where any delay in confirming the diagnosis may compromise the clinical outcome. Wherever possible, malaria diagnosis should include the identification of the parasite species as well as quantitation of malaria burden (parasite count) as this provides prognostic and disease monitoring information, vital for proper clinical management of patients. Rapid diagnostic tests (RDTs) Malaria RDTs are relatively simple to perform and interpret, require limited training, and provide rapid results thus have the potential to greatly improve the quality of malaria management, especially in more remote areas where access to good quality microscopy services is limited. Malaria RDTs detect specific proteins produced by malaria parasites that are present in the blood of infected individuals (see table 1). Depending on the antigen target, RDTs may detect a single species (either P. falciparum or P.vivax), multiple species (P. falciparum, P. vivax, P. ovale and P.malariae) and some distinguish between P.falciparum and non-p.falciparum species. To date, there are no commercially available RDTs that are able to specifically identify P ovale and P malariae and none are sufficiently sensitive to detect the presence of P. knowlesi infection (sensitivity is <50% meaning that 1 in 2 cases would test negative and be missed). There are well over 200 different malaria RDTs commercially available however, the specificities, sensitivities, numbers of false positives, numbers of false negatives and temperature tolerances of these tests vary considerably. False positive results have been observed in patients with rheumatoid factor, hepatitis C, toxoplasmosis, human African trypanosomiasis, dengue, leishmaniasis, Chagas disease, and schistosomiasis. This is particularly problematic in regions where there is an overlap in the distribution of malaria and other infectious diseases with very similar clinical pictures, for example in South East Asia where both dengue and malaria are common. Furthermore, RDTs, most notably pfhrp2 based tests; tend to remain positive for several weeks after successful malaria treatment, making it impossible to distinguish between residual antigen from a previous infection and a new infection, especially in high transmission areas where residents are repeatedly exposed to malaria. Table 1 Histidine rich protein-2 (HRP2) Lactate Dehydrogenase (LDH) Aldolase P. falciparum pfhrp2 pldh or pfldh* paldo P. vivax pldh or pvldh paldo P. ovale pldh paldo P. malariae pldh paldo * 90% similarity with pan-plasmodium LDH (pldh)

3 3 The sensitivity of RDTs is also not universal, hence false negative tests also require consideration. Exposure to excessive heat and humidity during transport and storage may reduce the efficacy of the test and lead to reduced sensitivities. LDH based tests appear to be less stable which has been a contributing factor to the predominant use of HRP2 based RDTs. A new challenge has since emerged which is the absence of production of the HRP2 protein due to the occurrence of pfhrp2 gene deletions in P falciparum parasites. This was first reported in Peru in 2010 but has since become more widespread including several parts of Africa, although prevalence rates vary. This is a growing area of concern and will require close monitoring and possible switch to RDTs targeting plasmodial LDH or aldolase, as these enzymes are essential for parasite survival and consequently deletion would kill the parasite. As RDTs are single-use devices, quality assurance testing of the kits must entail batch testing, i.e. testing one RDT on a daily basis and/or each time a new box is opened against a known positive specimen. Another limitation of malaria RDTs is that they are qualitative and therefore cannot be used to monitor response to therapy, in addition to their persistent positivity post successful treatment and clearance of parasites. The main role of RDTs is to serve as a screening test where laboratory infrastructure that can provide microscopy and other tests is lacking. Consequently, regular training and stringent competency testing is essential to ensure the required test quality level is maintained. Smear preparation and staining Blood obtained by pricking a finger or earlobe is the ideal sample because the density of malaria parasites is greater in blood from this capillary-rich area. Blood obtained by venipuncture collected in EDTA anticoagulant tubes is acceptable if used shortly after being drawn. If there is a delay in smear preparation, then the morphology of the parasites and blood cells becomes distorted making it difficult to accurately identify the malaria species. Both thick and thin blood films should be prepared. This can be done on the same slide or separately. Post-drying and fixation, the smears are stained with a Romanowsky type stain. The choice of stain depends on local practices with Field s, Giemsa s, Wright s and Leishman s all being suitable. a) Thick smear: The correct thickness of the thick smear can be tested by reading a newspaper through the unstained film. The print should be barely visible. It is dried for 30 minutes and not fixed with methanol. Prior to staining the thick smear is flooded with water. This causes the red blood cells to be breakdown leaving only the white blood cells any malaria parasites present as detectable elements. Peripheral blood smear microscopy Both RDTs and blood films can be used to diagnose malaria, but information regarding parasitaemia level and confirmation of malaria parasite species can only be obtained from the microscopic analysis of blood films. Although microscopic analysis is potentially both sensitive and specific and remains the gold standard for laboratory confirmation of malaria against which other diagnostic methods have traditionally been measured, this depends on the quality of the reagents, of the microscope, and on the experience of the laboratory technologist. Maintaining skilled laboratory staff capable of efficiently reading blood films is extremely challenging in many countries. A good quality thick film should on average have about WBC per high power field (HPF), exhibit a pale blue-grey background (lysed RBC) free of stain precipitates, dust and other artefacts. WBC nuclei should be stained deep purple and platelets should be clearly visible and bright pink. a) Thin smear: This should be air dried for 10 minutes and then fixed in methanol. While fixing the thin smear, all care should be taken to avoid exposure of the thick smear to methanol (if the smears have both been prepared on a single slide).

4 4 A good quality thin film has the presence of a tail or feathered edge with evenly distributed RBCs and no or very few overlapping cells. The RBCs should be stained grey-pink. An example of a thick and thin smear is shown in figures 1 and 2 respectively. Smear assessment a) Thick smear A thick film is always used to search for malaria parasites. The film consists of many layers of red and white blood cells. During staining, the haemoglobin in the red cells dissolves so that large amounts of blood can be examined quickly and easily. Malaria parasites, when present, are more concentrated than in a thin film and are easier to see and identify. However, due to the haemolysis and slow drying, the parasite morphology can get distorted, making the thick smear unsuitable for the differentiation of species. Thick smears are therefore primarily used as a screening test to detect the presence of malaria. The thick smear also lends itself to look for gametocytes, as these are usually present in low numbers, if at all. Fig. 1 An example of a thick smear. Free-lying malaria parasites are easily visible and identified with an arrow. The smear should be examined using the 100 oil immersion objective lens. Select an area that is wellstained, free of stain precipitate and well-populated with white blood cells (10-20 WBCs/field). The WHO recommends that at least 100 fields, each containing approximately 20 WBCs, be screened before calling a thick smear negative. In the hands of an expert microscopist the sensitivity limit of the thick smear is about parasites/μl. Fig. 2 An example of a thin smear. Malaria forms are clearly visible within RBCs. The double chromatin dot (arrow) is typical of Plasmodium falciparum. b) Thin smear The thin smear preserves the integrity of cells therefore intraerythrocytic parasites are observed with their morphology intact. The thin smear is used as an adjunct to the thick smear to identify the malaria species, including the diagnosis of mixed infections, and to quantify parasitaemia. Because a much smaller volume of blood is used to make a thin smear, it is far less sensitive than a thick smear to detect the presence of parasites. It is therefore not suited as a screening tool for malaria, especially in non-immune individuals or pregnant women who may have very low parasitaemia levels. Additionally, one can assess for the presence of gametocytes, schizonts and malaria pigment (haemozoin) in neutrophils and monocytes. The presence of malaria pigment has been shown to correlate with severity of disease therefore it provides prognostic information.

5 5 c) Parasite density Parasitaemia estimations can be performed from the thick or thin films with each method having its limitations. Parasite counts from the thin film tend to be more accurate than those obtained from the thick film because there is little or no parasite loss during staining. However, the thin film method is mostly appropriate for higher parasite densities only. Should the thick or thin film be used? i. Thick film - if the number of parasites (excluding gametocytes) is less than or equal to twice that of WBCs in the HPFs counted, then the thick film can be used to estimate parasite density. ii. Thin film - if the number of parasites is greater than twice the number of WBCs, then the thin film will provide a more accurate estimation of parasite density. Estimating parasite density on thick films Detection and counting of parasites is performed against a standard number of WBCs or HPFs on the thick film. The number of fields to be counted will depend on the parasite density. If 100 parasites in 200 white blood cells are observed, stop counting, and record the results as the number of parasites per 200 white blood cells. If not, continue until 500 WBCs have been counted. Counting should begin from the first good quality field seen whether or not there are parasites in the field of view, as this takes into account the entire volume of blood examined for the parasite density calculations. According to the WHO malaria microscopy manual, a slide should only be declared negative for malaria if no parasites are seen after reading at least 2500 WBCs or 200 HPFs. This is clearly very timeconsuming and in routine practice smear examination is generally stopped sooner. Both counting methods have limitations. i. WBC parasite density calculations using WBC as a counting benchmark is commonly based on an arbitrary assumed WBC count of 8 x 109/L (8000/μL). If the actual WBC is significantly lower, then parasite counts will be overestimated and vice versa. Ideally, the actual WBC count should be obtained from the same sample but this is rarely done as smears are mostly made from blood collected directly onto the slide from the finger prick. ii. HPF the area of the film in view in a HPF, and therefore the volume of blood screened for parasites, depends on the FN of the ocular lens of the microscope. The FN or field number indicates the diaphragm size of the eyepiece which in turn defines the image area of the specimen in view. The higher the FN, the greater the volume of blood examined. Consequently, smears viewed with a microscope having an eye piece with a higher FN value will result in higher parasite counts. Estimating parasite density on thin films Parasite detection is performed against a standard number of RBCs on the thin film in case of high parasite density (i.e. If 100 parasites are present in each field of a thick film under the 100X objective, calculate the parasite count on the thin film). This helps to reduce inaccuracy and imprecision caused by the loss of parasites during staining and by counting large numbers of parasites in a field. Counting of RBCs must begin from the first good quality field to improve accuracy of the parasite density estimate. In the case of a mixed infection, all parasitized RBCs can be counted together for clinical case management. The presence of gametocytes should be noted, but not included in the count. Schizonts should be included but a note made for the clinical report as this is highly suggestive of severe disease in P falciparum infections. The ideal field for counting is one that has about 250 RBCs. Parasitised and other RBCs should be counted until at least 2000 RBCs have been counted. Cells containing more than 1 parasite should be counted as 1. Schizonts should be included. The calculation of parasite density is done as shown in table 1 on the next page.

6 6 Table 2 Formula for calculating parasite density from a thick and thin film examinations: Thick Film WBC Parasite density per µl = (Number of parasites counted (WBC count per µl Number of WBCs counted) (if WBC value is not known, 8,000/μL is commonly used) HPF Parasite density per µl = Number of parasites counted (Number of HPFs Volume of blood per HPF) (Volume of blood per HPF is variable based on thickness of blood drop and FN of eye piece so this method is not generally recommended) Thin Film RBC Parasite density per µl = Number of parasites counted RBC count per µl Number of RBCs counted (if RBC value is not known, 5,000,000 / μl is commonly used) b) Parasite speciation and documentation of parasite life cycle stage Identification of the malaria species is important for clinical management as well as epidemiological surveillance. Of the Plasmodium species that affect humans, only P. vivax and P. ovale form hypnozoites, which are dormant parasite stages in the liver that cause relapses of infection weeks to years after the primary infection. Thus, a single bite from an infected mosquito may result in repeated bouts of illness. Standard antimalarial drugs such as Artemisinin combination therapies (ACT) are only effective in killing the intraeyrthrocytic parasites, which are the cause of clinical illness. Consequently, patients with P. vivax and P. ovale malaria need to be treated with an additional drug (Primaquine) to eliminate the dormant hypnozoites and prevent future relapses. Targeting hypnozoites in the interest of the patient as well as being vital for malaria control programmes. Likewise, it is important to identify the presence of the different life stages of the malaria parasite. It is unusual to see later stage trophozoites or schizonts in P. falciparum malaria, as these are usually sequestered in the deeper tissues.). The presence of more mature parasite forms (>20% of parasites as late trophozoites and schizonts) and of more than 5% of neutrophils containing malarial pigment indicates more advanced disease and a worse prognosis. Hence it is important to notify the clinician. In P. vivax infection late stage trophozoites, schizonts and gametocytes are quite commonly observed in the peripheral blood smear. Gametocytes are occasionally observed in P. falciparum infection and are easily identifiable by their characteristic banana-shape. If present in larger numbers or if they persist after treatment has commenced this may be a sign of drug resistance. If the asexual forms are partially stressed by antimalarial drugs but are not killed quickly because a reduced susceptibility to the drugs, then a greater number of merozoites released from the mature schizonts are directed into the sexual lifecycle. Hence an increase in gametocytes is observed in the peripheral blood. With the current focus on malaria elimination possibilities, there is a renewed interest in gametocytes. Identifying the presence of gametocytes, especially in asymptomatic carriers that would otherwise not be treated for malaria, is increasingly being acknowledged as vital in the control and ultimate elimination of malaria. Targeting such individuals with specific gametocidal drugs is seen as an important factor in breaking the cycle of transmission back to the mosquito.

7 7 Advantages and disadvantages of current routine malaria diagnostics a) RDTs RDTs are simple to use with minimal training facilitating parasitological confirmation at community level. A formal laboratory infrastructure is generally not required. This is their greatest advantage and has aided major improvements in the quality of clinical management of malaria. The shelf life of test strips is however impacted by exposure to excessive heat and humidity so storage conditions need to be controlled. A drawback is that the identification of species is dependent on the choice of RDT with many being non-specific. Furthermore, RDTs are purely qualitative which does not allow for disease monitoring. They are prone to a significant number of false positives (cross-reactivity) as well as being unable to differentiate between a recent and new recurrent infection. False negatives are linked to poor storage conditions as well as the emergence of HRP2 mutations in P. falciparum. The sensitivity of RDTs is variable but in general the lower limit of detection is ~ 200 parasites/μl (~0.004%). b) Microscopy The advantage of microscopy is that it is an established, relatively simple technique that is familiar to most laboratory technologists. The benefit is that the microscope can be used for multiple diagnostic investigations and the stains are relatively inexpensive. Peripheral smear examination allows for the diagnosis of malaria, identification of species, quantification of parasite load, identification of different life cycle stages, including gametocytes, as well as looking for the presence of malaria pigment. Furthermore, reviewing the thin smear also caters for a general review of the patient s haematolological state. This benefit may well become more important as malaria prevalence declines and febrile illnesses will become more likely to be due to other infectious causes. In this case, a smear review would be invaluable. The disadvantage of microscopy is that it is entirely dependent on the experience and diligence of the microscopist, the quality of the smear, stain and microscope. Consequently, the sensitivity is highly variable. In the hands of experts, thick smears can detect as low as ~5 parasites/μl but the average routine lab worker with regular exposure to malaria microscopy will achieve a lower limit of detection more in the region of 50 parasites/μl. The thin smear by virtue of examining a much lower volume of blood is less sensitive with an average lower limit of detection of ~200 parasites/μl. How many HPFs are examined before a smear is declared negative is also not uniform from laboratory to laboratory and amongst laboratory technologists, but obviously the more time that is spent one examining more fields the greater the likelihood of picking up a very low level of parasitaemia. The sensitivity of microscopic malaria detection is therefore influenced by the workload. Excessive workload is the number one factor of poor performance. In recognition of this WHO has revised its earlier slides/person/day guidelines to a much lower figure which takes into account the slide positivity rate, the skill of the worker, the need to identify species and other factors. (Please see WHO Malaria Microscopy Quality Assurance Manual, version 1, 2009 for details) What are the alternatives? a) PCR Molecular techniques such as polymerase chain reaction (PCR) are well established research tools. PCR has a significantly higher sensitivity than other conventional malaria diagnostic tests and is best when it comes to species identification. PCR can be qualitative or quantitative with the latter being quite complex. Both require a good laboratory infrastructure and skilled personnel. PCR, whilst acknowledged to be superior, is currently not a reality for the malaria diagnostic mass market.

8 8 b) Sysmex technology Born from many decades of experience in routine blood cell analysis, Sysmex has developed new innovative diagnostic tools to differentiate between viral, bacterial and parasitic causes of infection, with confirmation in cases of malaria. Using this technology acute cases of malaria will be rapidly identified, confirmed and treated. The technology provides a parasite count, can distinguish between species as well as identify different life cycle stages. Furthermore, it provides full blood count values as part of the analysis. Non-malarial illness will be identified as either being suspected to be of bacterial or viral origin which in turn will support the intelligent use or withholding of antibiotics and direct further diagnostic investigation in selective cases. Furthermore, the technology also provides prognostic information about the severity of disease, irrespective of cause. This technology, still under development and currently for research use only, will be discussed in more detail in a subsequent edition of SEED. Take home message Parasitological confirmation of malaria prior to treatment is vital Distinction between malaria and non-malarial causes of fever is becoming increasingly important as malaria prevalence drops. RDTs and microscopy both have a very important role to play but have their limitations PCR addresses many of these shortcomings but it is too cumbersome and complex for routine use New innovative diagnostic tools based on Sysmex technology are under development to address these issues. References 1. WHO Malaria quality assurance manual, version 1, WHO Methods manual. Microscopy for the detection, identification and quantification of malaria parasites on stained thick and thin blood films in research settings Compiled by Dr Marion Münster Distributor of Sysmex, "Concern - Energomash" CJSC, Azatutyan avenue 26/8, Yerevan 0014, Armenia Tel.: , , , info@c-e.am,