Original articles

Plasmodium lactate dehydrogenase assay to detect malarial parasites [PDF]

SONIA MALIK, SHOEB KHAN, ANUPAM DAS, J C SAMANTARAY

INTRODUCTION

With 2 million cases reported in 2000,1 malaria has re-emerged as a major health problem. One of the most important problems in controlling malaria is limited access to effective diagnosis and treatment. In India, healthcare is provided by para-healthcare workers at the domiciliary or village level.1 In such situations, it usually takes 1 week for collection of the smear, transporting it to the primary health centre2 (PHC) and obtaining laboratory confirmation of the diagnosis. All cases with fever are presumptively treated as being due to malaria and given chloroquine. This may result in overtreatment and has a bearing on the malarial parasite developing resistance to chloroquine. The WHO now emphasizes full treatment of patients after identification of the malarial species.2 Identification of the malarial parasite requires microscopic observation of the parasite on a stained blood smear, which is labour-intensive and depends on the quality of the stained preparation as well as of the microscopes, besides requiring considerable skill for its interpretation.3 Decentralization of slide examination from regional or district-level laboratories to PHCs has resulted in deterioration of the quality of slide examination, mainly due to technical problems such as poor maintenance of microscopes, improper staining and inadequate supervision.4 All these limitations necessitate consideration of alternate strategies for the diagnosis of malaria, which are reliable, rapid and technically less demanding.
    One such method, the Plasmodium lactate dehydrogenase (pLDH) assay, an immunochromatography-based dipstick product, is based on the detection of a soluble parasitic enzyme3 and provides a means of non-microscopic detection of malarial parasites. pLDH is expressed at high levels in the asexual stages of all four malaria species>3 and enables rapid differentiation (within 10 minutes) between a P. falciparum and a non-P. falciparum infection. However, in India, where a substantial number of malaria infections (51%) are caused by P. vivax (Source: Directorate of National Vector-borne Diseases Control Programme, 22 Shamnath Marg, Delhi, 2002), there have been few studies on the efficacy of pLDH.
    This study was designed to assess the sensitivity and specificity of the pLDH assay compared with microscopy in detecting and differentiating P. falciparum and P. vivax malaria. Unlike most of the previously reported studies in which pLDH assay has been compared with only Giemsa-stained thick and thin smears, we compared the assay with 3 microscopic techniques, viz. Giemsa-stained films, acridine orange-stained films and quantitative buffy coat assay (QBC), which is a concentration technique. Worldwide, QBC examination of the blood has been demonstrated to be as good5 and at times a more sensitive6 method for the detection of malaria parasites than a thick film. In this study, thick film examination was substituted with QBC.
    This study was conducted at the All India Institute of Medical Sciences, New Delhi. The important features of malaria in this region are moderate to low endemicity, P. vivax predominance (accounting for roughly two-thirds of all cases of malaria7,8) and focal P. falciparum transmission. Exacerbation occurs during the monsoon and post-monsoon periods. However, our institution being a tertiary care centre, caters mainly to referred complicated cases from Delhi and the adjoining states. Hence, the number of P. falciparum cases (complicated malaria) reported is higher than P. vivax.

METHODS

Blood samples from patients attending the hospital with symptoms suggestive of malaria were examined for malarial parasites. Only samples submitted for routine diagnosis were used in this study. From each blood sample, two thin films and a QBC preparation were made. One of the thin films was stained with Giemsa and examined under a 100 x oil immersion lens. The other was stained with acridine orange and examined using a binocular microscope fitted with a paralens adapter. Two hundred oil immersion fields of the blood films were examined by three examiners before being reported as negative.9 The parasite count per microlitre of blood was obtained assuming a white cell count of 8000/ml. 9 For QBC, 60 ml of blood was collected in commercially supplied malaria detection tubes as per the manufacturer’s instructions (Becton Dickinson). Each QBC tube was examined until the parasites were detected or for a maximum of 5 minutes.
    The pLDH assay was performed using commercially supplied dipsticks (OptiMAL®, Diamed, Switzerland) according to the manufacturer’s instructions. The dipstick contains a set of two capture antibodies of which one is specific for P. falciparum pLDH while the other is a pan-specific pLDH antibody and recognizes all four species of malarial parasite. P. falciparum infection is indicated by colour development at the zones of the P. falciparum-specific and the pan-specific antibodies. A non-P. falciparum (P. vivax, P. malariae, P. ovale) malarial infection is indicated by colour development only at the pan-specific antibody band.10
    The sensitivity, specificity, positive predictive value and negative predictive value were calculated.

RESULTS
A total of 124 blood samples were examined by microscopy (Giemsa, acridine orange staining and QBC) and pLDH assay (Tables I and II). Among all the microscopic techniques, QBC was the most sensitive as it identified three P. falciparum-positive samples and one P. vivax, which were negative on thin films stained with acridine orange and Giemsa (Table II). However, the pLDH assay identified 2 blood samples with P. falciparum infection, which were negative by microscopy. The sensitivity and specificity of pLDH assay was 94.7% and 97.6%, respectively, for detection of P. falciparum, and 90.4% and 100%, respectively, for P. vivax. There was one case of mixed P. falciparum and P. vivax infection, which was identified as P. by the pLDH assay and has been considered with P. falciparum cases. Similarly, one case of P. malariae, identified as P. vivax by pLDH assay (as described earlier), has been considered with P. vivax cases for the purpose of calculating the sensitivity and specificity

DISCUSSION
Several trials worldwide have reported that pLDH assay is an effective diagnostic test for malaria with a sensitivity for P. falciparum and P. vivax detection ranging from 94% to 95% and from 88% to 96%, respectively.11,12 A hospital- and a field-based study from central India13 also reported that pLDH assay was a useful test for the diagnosis of malaria as well as for monitoring treatment. The results of the hospital-based study were encouraging (overall sensitivity of 100% and specificity of 97% for the diagnosis of malaria). However, in the field setting, the specificity of the test was not high (100% sensitivity and 67% specificity for the overall detection of malaria). In a field study from Viet Nam,14 the pLDH assay did not work well and its overall sensitivity (49.7%) was the lowest reported in the literature so far. Also, authors from Myanmar expressed concern over the sensitivity of the pLDH assay as 57% of P. falciparum cases were missed by this assay. The reasons for such poor sensitivity were not clear, but probably batch variations in the quality of the kit were the principal suspected cause as an earlier study by the same authors had shown excellent results with this assay.
    We found the pLDH assay to be a useful tool for the detection of P. falciparum malaria. The assay missed only 2 cases of P. falciparum, which had only gametocytes on microscopy. These two patients were known cases of P. falciparum infection and had completed their treatment. They were being treated for P. falciparum malaria-related complications. pLDH is produced only by live asexual forms and to a lesser amount by sexual forms of the parasite.12 It is likely that the gametocytes seen on microscopy were non-viable or fewer in number and hence were not detected in the pLDH assay. The pLDH assay has been shown to have a lower sensitivity for gametocyte carrier cases.12 Excluding these 2 cases, pLDH assay had a 100% sensitivity for P. falciparum detection even at parasite counts as low as <40/µl. This is the maximum limit of microscopic sensitivity, which can be reached only by the most experienced examiners.10 There were two blood samples identified as P. falciparum by the immunochromato-graphic test which were not detected by microscopy. These two patients had received inadequate doses of chloroquine in the past. Both these patients recovered clinically on administration of quinine. One of the patients became negative for pLDH antigen on subsequent testing (the other patient could not be followed up), giving credence to the fact that the patient indeed suffered from falciparum malaria. A P. falciparum infection may be easily missed when the parasites are sequestered11 in the deep capillaries (spleen, liver, bone marrow) and are present in insufficient numbers for detection in blood films10 as might happen in patients partially treated for malaria. pLDH assay may provide a more precise diagnosis of P. falciparum infection in such cases. This also highlights the importance of clinical history and follow up of patients for, in the absence of these, the above two P. falciparum-positive patients would have been dismissed as being false positive.
    The pLDH assay indicated mixed infections with P. falciparum and P. vivax as P. falciparum, which is acceptable given the configuration of the test,12 where P. falciparum could react with both monoclonal antibodies on the strip. Though this format is advantageous, given the potentially fatal nature of P. falciparum infection, this will inevitably miss infections due to non-P. falciparum species. The incorporation of a P. vivax-specific antibody in the dipstick which does not cross-react with P. falciparum would obviate this problem.
    The results of the pLDH assay for P. vivax detection were also encouraging. It had a 90.4% sensitivity for the detection of P. vivax, which is higher than that reported earlier for ICT Malaria Pf/Pv (70–75).16,17 This would help in differentiating P. vivax malaria from non-malarial fevers and prevent unnecessary expenditure on antibiotics. However, the failure of pLDH assay in this study to detect two P. vivax-positive samples is a matter for concern. This false-negative result could not be due to low parasitaemia as parasite counts in these 2 cases were 5000/ml and 389/ml, respectively. In this study, pLDH assay correctly identified P. vivax at counts as low as <80/ml (Table II). The chances of human error too are unlikely as all the operators in the study were well-trained and the tests were performed according to the manufacturer’s instructions. Besides, in discrepant cases, the assay was repeated.
    Plasmodial gene deletion isolates, which express little or no pLDH antigen and result in a negative pLDH assay, could be a possibility. Such a possibility for P. falciparum gene deletion isolates that do not express HRP-2 has been postulated, although the same evidence for pLDH has not been discovered.10 However, important variations in the sensitivity of the pLDH assay have been reported among different geographical locations14 within the same country. The probability of occurrence of such variations was also indicated in a recent study from Mumbai in western India,18 wherein pLDH assay misidentified 6 of 31 P. falciparum infections as P. vivax. It was postulated that reduced production of falciparum-specific pLDH antigen, due to a different geographic strain, probably accounted for non-appearance of the falciparum-specific band. India is a large country with a multitude of geographical locations and vast biodiversity. The possibility of these variations existing in other regions of India needs to be substantiated in multicentric trials.
    Our results indicate that the pLDH assay can be an effective tool in the fight against malaria, especially in countries where both species are co-endemic and laboratory support is limited. Besides, a pLDH assay can also be used to assess parasitological clearance and possibly monitor treatment.19 This would help in the early detection of drug-resistant cases and prevent transmission of drug resistance. However, the cost of the test is an important factor (US$ 3 per test13) and needs to be made cheaper if it were to play a vital role in the National Malaria Control Programme. With indigenous mass production, this assay could also become affordable, as happened in the case of HRP-2 based dipsticks. In view of a possibility of plasmodial gene deletions leading to variations in the assay’s sensitivity as suggested above, we feel it needs evaluation in larger multicentric trials before its wider deployment.