Field of the Invention
This invention relates to a detection kit for rapid malaria diagnosis from urine samples and in particular, this invention relates to rapid immunochromatographic test of antigens inurine indicative of malaria caused by both Plasmodium falciparum and Plasmodiun vivax species. The present invention relies on the fact that in clinical malaria, febrile patients shed elevated levels of rifin family of variant surface antigens proteins, including (PFNF135_04387) gene specific for P. falciparum and (PVIIG_02112) gene specific for P. vivax, in urine and the test detects those proteins shed in the urine of individuals infected with malaria using monoclonal antibodies against them.
Furthermore, the present invention refers to a method for the production of the kit and the use of this kit for the rapid detection of malaria from non-invasive sample urine. This invention also relates to the detection kit which is convenient and fast for operation, is suitable for on-site detection, and is high in specificity, detection sensitivity, accuracy, and reliability and application prospect is promising.
Background of the invention and the related Prior Art
It is estimated that the number of patients suffering from malaria is three to five hundred million per year throughout the world, with a death toll of 1.5 to 2.7 million per year. In addition, malaria parasites are indigenous to 100 or more countries predominantly in tropical and subtropical areas, and thus half the global population constantly faces the risk of malaria infection. Therefore, early detection and early treatment are important for

preventing increases in the seriousness of the symptoms, and also for avoiding spreading of the infection.
Malaria breeds poverty and underdevelopment in vast regions of the world, including the Americas, thus contributing to issues of global concern such as illegal migration and security. Nearly 142 million people or 16% of the population of the Americas is among those who are at risk for malaria and 40 million of them are at moderate to high risk. Rolling back malaria is possible. In the Americas, miners, loggers, banana and sugarcane plantation workers, indigenous groups, populations in areas of armed and/or social conflict, and people along areas of common epidemiologic interest / border areas are also susceptible to the disease. Large numbers of Rapid diagnostic kits (RDTs) have been developed for early diagnosis of malaria by different researchers (Binaxnow malaria test kits, Moody et al 2005, Endeshaw et al 2008). However, all the current methods are invasive as they require blood for diagnosis. It has recently been found that it is possible to detect malaria parasite antigens/DNA in samples of urine and saliva from malaria patients (Mharakurwa et al, 2006, Buppan et al, 2010). But the existing lacunae is that till now, no work has been carried out or reported for development of antibody based diagnostics to detect these antigens from urine of malaria patient.

Malaria continues to pose major public health problem in India mainly in eastern states. Among them Odisha has topped the list of malaria deaths in the country with 77 deaths followed by West Bengal and Meghalaya, which have recorded 59 and 44 deaths respectively in 2016. There is geographical variation in the distribution of malaria cases within the districts, blocks and sub-centres. The 10 southern districts having only 27 % of state's population with more than 50% tribal population, contribute around 64% of deaths due to malaria in the state and these communities bear a disproportionately heavy burden of disease. Out of total of 6688 sub-centers in the state, 2880 (43%) with a population of 14.3 million (36%) have an Annual Parasite Incidence (API) of more than five. Around 85% of the cases reported from the state are due to Plasmodium falciparum malaria (Pf). Due to operational difficulties, the risk of malaria continues to be high in remote, rural, tribal, inaccessible, forested and forest fringed areas. "The major setback associated with the disease is late diagnosis since symptoms go unnoticed in the first few hours especially in children, blood taboo in tribal community, dependency on health practitioner for blood test, absence of required number of health workers, awareness and negligence on the part of patients have given rise to the alarming situation. Thus Progress in diagnostic coverage requires wider access to safe, non-invasive, rapid, effective, low-cost, and easy-to-use tests at primary-level points of care, including community health workers, private clinics, medicine shops, and front-line government health facilities. Rapid and non-invasive diagnosis of malaria can avert most deaths in tribal community where blood taboo is prevailed. And there has been no study carried out that has assessed the diagnosis of malaria rapidly and in non-invasive way.

In India currently all malaria diagnostic methods (microscopy and RDTs) are invasive as they depend on blood samples for diagnosis. These detection methods are time-consuming, usually of 2-3 days culture and phenotyping. There are few kits imported from foreign countries like BinaxNow malaria test kit, ACCU-TELL MALARIA P.F/P.V WHOLE BLOOD TEST (CE) is available, but they are not cost effective in India and also none of them are invasive i.e diagnose by using urine and saliva of the patients. Hence there is an immense significance in developing such non invasive, rapid, highly sensitive, cost effective malaria diagnostics in India that serves the needs of scientist of the bioresearch, hospitals and health care markets.
According to the document US20100279319, The detection of PfHRP II in saliva offers a practical alternative to PfHRP II detection in blood for malaria diagnosis and offers some distinct advantages over blood. Collection of saliva is non-invasive, simple, safe, stress free, painless, and can be done by individuals with limited training, including patients. It does not require blood cell lysis that diminishes HRPII antigen availability and detection. No special equipment is needed for collection and it allows for multiple or serial collections outside of the hospital. Detecting parasite antigens in saliva to determine presence or absence of parasites could be valuable for communities with blood taboos and reduce compliance problems associated with collection of blood.

The patent document CA2672357 states that a rapid immunochromatographic test device suitable to detect an antibody and/or antigen in at least one sample, uses of said device for detecting diseases in a sample, a method for the production of said device as well as a kit comprising the device.The other document WO2008071336 discloses an immuno¬chromatographic detection cup and its use for simultaneously detecting at least one antigen, e.g. tuberculosis antigen, malaria antigen and pneumonia antigen, and at least one antibody, e.g. HIV antibody, HCV antibody and H. pylori antibody, in a urine sample. The urinary detection cup comprises (a) a sample-collecting container; (b) a conjugate releasing pad comprising a gold labelled antibody X and an oligonucleotide linked antibody X', wherein both antibodies are directed against the same antigen, and a gold labelled antigen Y and an oligo-nucleotide linked antigen Y', wherein both antigens are recognized by the same antibody; (c) a test means inside the container separated from the conjugate releasing pad, which test means comprises a region comprising an oligo¬nucleotide complementary to the oligo-nucleotide linked to antibody X', a region comprising an oligo-nucleotide complementary to the oligo-nucleotide linked antigen Y', and at least one region comprising a control antibody and/or a control antigen; and (d) at least two sample absorbent pads linked to the test device at different positions.

The document US8034551 describes a method for malaria testing which enables testing for the presence of malaria infection and the extent of the infection in a convenient manner; and a reagent or kit which can be used in the method. The method for malaria testing according to the present invention includes the step of detecting a liver-type fatty acid binding protein present in urine collected from a subject animal. The extent of infection is determined to be higher when a larger amount of the liver-type fatty acid binding protein is present in a test sample.
According to the document, PCT/KR2007/006962 the invention has been conceived to solve the problems of the conventional techniques as described above, and the inventors of the present invention have developed a kit for diagnosis of malaria, which exhibits excellent specificity and sensitivity and is capable of discriminating infection of Plasmodium vivax, distinguishing the kinds of Plasmodia, and identifying mixed malaria infection with ease and economically. Further, the inventors developed a diagnosis kit including monoclonal and polyclonal antibodies to lactate dehydrogenase of P vivax and monoclonal antibodies to lactate dehydrogenase of Plasmodium falciparum.
To solve the foregoing problems, the present invention provides a method of detecting dual infection of malaria and a kit for diagnosing the same by preparing a specific monoclonal antibody to P. vivax lactate dehydrogenase (LDH) and simultaneously using a specific monoclonal antibody to P. falciparum LDH.

An aspect of the present invention provides a kit for diagnosing malaria, including specific monoclonal and polyclonal antibodies to P. vivax lactate dehydrogenase, a specific monoclonal antibody to P. falciparum lactate dehydrogenase, and a simultaneously specific monoclonal antibody to P. vivax lactate dehydrogenase and P. falciparum lactate dehydrogenase.
The document US4466917 states the antisera and monoclonal antibodies directed against the sporozoite stage of the malaria parasite capable of providing protection against infection in both animals and humans. The invention further provides a purified antigen derived from sporozoites of the malaria parasite, the antigen being suitable for use as a vaccine against malarial infections in both animals and humans. The invention further provides means for preparing said antigen and a vaccine comprising said antigen.
It may be understood that others skilled in the art, while adapting this invention by applying current and future knowledge, for its use in various conditions of service, may go for some changes in the details without, however, departing from its original scope and spirit or sacrificing any of its advantages.
Summary of the invention
This invention relates to a detection kit for rapid malaria diagnosis and in particular, this invention relates to a detection kit for rapid malaria diagnosis in which the specific antibodies against urine specific antigens of Plasmodium falciparum and Plasmodium

vivax is used. The technology relies on the fact that in clinical malaria, febrile patients shed elevated levels of rifin family of variant surface antigens proteins, including (PFNF135_04387) gene specific for P. falciparum and (PVIIG_02112) gene specific for P. vivax, in urine and the test detects those proteins shed in the urine of individuals infected with malaria using monoclonal antibodies against them.
More particularly, this present invention relates to a method of detecting the malaria in which kit is detecting antigens in urine indicative of malaria caused by both Plasmodium falciparum and Plasmodiun vivax species. Furthermore, this invention also relates to the detection kit which is convenient and fast for operation, is suitable for on-site detection, and is high in specificity, detection sensitivity, accuracy, and reliability and application prospect is promising.
Detailed description of the invention with accompanying drawings
For the purpose of facilitating an understanding of the invention, there is illustrated in the accompanying drawings a preferred embodiment thereof, from an inspection of which, when considered in connection with the following description, the invention, its construction and operation, and many of its advantages should be readily understood and appreciated.
The principal object of the invention is to provide a detection kit for rapid malaria diagnosis.

The other embodiment of the invention is to provide a detection kit for rapid malaria diagnosis in which the specific antibodies against urine specific antigens/proteins of Plasmodium falciparum and Plasmodium vivax and is used.
The other embodiment of the invention is to provide a method of detecting the malaria in which kit is detecting specific antigens in urine indicative of malaria caused by both Plasmodium falciparum and Plasmodiun vivax species.
The other object of the invention is to provide the detection kit which is convenient and fast for operation, is suitable for on-site detection, and is high in specificity, detection sensitivity, accuracy, and reliability and application prospect is promising.
The other embodiment of the invention is to provide a detection kit for rapid malaria diagnosis which utilizes the principle of immunochromatography. As the test sample urine flows through the membrane assembly of the device after addition of the clearing buffer, the colored monoclonal antibody complexes the antigen in the lysed sample. This complex moves further on the nitrocellulose membrane to the test region where it is immobilised by the ami- vivax specific monoclonal antibody and/ or the anti- falciparum specific antibody coated on the membrane leading to formation of pink-purple colored band/s which confirms a positive test result. A band will appear under Pf at the test region in falciparum malaria positive samples, while a band will appear under Pv in vivax malaria positive samples. Appearance of band under Pf as well as Pv in the test region suggests a mixed infection. Absence of colored band/s in the test region indicates a

negative test result. The unreacted conjugate and unbound complex if any move further on the membrane and are subsequently immobilised by anti rabbit antibodies coated on the membrane at the control region, forming a pink-purple band. This control band serves to validate the test performance.
The other object of the invention is to provide the detection kit which will provide a cost-effective approach for the screening of large populations in epidemiological surveys while being affordable, rapid, non-invasive, and safe for patients and technicians in resource-poorenvironments.
The novel features that are considered characteristic of the present invention are set forth with particularity in the appended claims. The invention itself, however, both as to its organization and its method of operation, together with additional objects and advantages thereof, will best be understood from the foDowing description of certain specific embodiments,
The invention relates to early diagnosis of malaria and its essential appropriate treatment. The impugned invention lies in the rapid immunochromatographic detection kit and for detecting antigens in urine indicative of malaria caused by both Plasmodium falciparum and Plasmodiun vivax species. This kit detects malaria parasite antigens in samples of urine from malaria patients. Furthermore this kit, thus, need to be simple, practical, and applicable, especially in malaria-endemic and resource-poor regions of India. Apart from

that this non-invasive needles otherwise leads to the risk of accidental infection such as
diseases like tuberculosis, AIDS etc.
Having described the invention in detail with particular reference to the illustrative
drawings, it will now be more particularly defined by means of claims appended
hereinafter.
While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.
The present invention has been described in detail, including the preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of the present disclosure, may make modifications and/or improvements on this invention and still be within the scope and spirit of this invention as set forth in the following claims.

We claim:
1) A non-invasive rapid malaria detection kit which comprises:
Individually pouched devices with
• Membrane assembly predispensed antibodies at the respective regions.
• Desiccant pouch.
• 5 μl sample loop.

2) The detection kit as claimed in claim 1 utilizes non-invasive sample urine for the detection of specific antigens from P. falciparum and P. vivax malaria.
3) The detection kit as claimed in claim 1 which utilizes anti-falciparum specific monoclonal antibody against urine specific (PFNF135_04387) protein/gene of P. falciparum species.
4) The detection kit as claimed in claim 1 which utilizes anti-vivax specific monoclonal antibody against urine specific (PVTIG_02112) protein/gene of P. vivax species.
5) The detection kit as claimed in claim 1 can detect the P. falciparum infected malaria, P. vivax infected malaria as well as mixed infection with both the species simultaneously.
6) A method of detecting the malaria in which the specific antibodies against urine specific antigens of Plasmodium falciparum and Plasmodium vivax is used.
7) The method for detection kit as claimed in claim 9 utilizes the principle of immunochromatography. A band will appear under Pf at the test region in falciparum

malaria positive samples, while a band will appear under Pv in vivax malaria positive
samples.
8) Appearance of band under Pf as well as Pv in the test region suggests a mixed
infection. Absence of colored band/s in the test region indicates a negative test result.