DIFFERENTIAL DIAGNOSIS OF MALARIA BY PERIPHERAL BLOOD SMEAR, ANTIGEN DETECTION AND CENTRIFUGED BUFFY COAT SMEAR EXAMINATION

Rahul Prabhas, Dr. Ritu Garg, Dr. Varsha A Singh, Arun Kumar, Nitin Goel, Ashish Kothari, Shyam Sundar Bera ,Md Mustafa Sofiur Rahman, Dr Ashwani Mehra, and Harit Chauhan , Department of Microbiology, MMIMSR, Mullana and Ambala. Haryana. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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Malaria imposes great socio-economic burden on humanity, and with six other diseases (diarrhea, HIV/AIDS, tuberculosis, measles, hepatitis B, and pneumonia), accounts for 85% of global infectious disease burden. 3 A clinical diagnosis of malaria is traditional among medical doctors. Clinical diagnosis is based on the patients 'signs and symptoms, and on physical findings on examination. The earliest symptoms of malaria are very nonspecific and variable, and include fever, headache, weakness, myalgia, chills, dizziness, abdominal pain, diarrhea, nausea, vomiting, anorexia, and pruritus. 4 Rapid and effective malaria diagnosis not only alleviates suffering, but also decreases community transmission. The nonspecific nature of the clinical signs and symptoms of malaria may result in over-treatment of malaria or nontreatment of other diseases in malaria-endemic areas, and miss diagnosis in non-endemic areas. 5 In the laboratory, malaria is diagnosed using different techniques, e.g. conventional microscopic diagnosis by staining thin and thick peripheral blood smears, other concentration techniques, e.g. quantitative buffy coat (QBC) method, rapid diagnostic tests, and molecular diagnostic methods, such as polymerase chain reaction(PCR) . Some advantages and short comings of these methods have also been described, related to sensitivity, specificity, accuracy, precision, time consumed, cost-effectiveness, labor intensiveness, the need for skilled microbiologist, and the problem of inexperienced technicians. 6,7 New diagnostic methods and approaches are increasingly important in efforts to improve surveillance, the precision of transmission data, and the detection of cases of malaria infection. The ability to reliably diagnose malaria infection is fundamental to both the management of individual patients as well as public health efforts to control the disease. The working group on malaria and the UN Millennium Project took a number of measures into account in their proposed global plan, which involves the measurable target to reduce malaria morbidity and mortality by 75% by 2015 from the 2005 baseline level. 8 However, asymptomatic carriers and patients with mild clinical manifestations and low parasitic density in less endemic countries are more common, and detecting infection in these people is more important because these asymptomatic carriers will continue to cause onward transmission silently. 9,10 Moreover, quality assurance protocols for microscopy are difficult to implement in elimination areas. Although microscopic diagnosis is sensitive and specific and remains the standard method for diagnosing malaria, it is not universally available, not considered a rapid diagnostic method, and requires a high level of expertise. Fluorescence microscopy was shown to improve the sensitivity, but not the specificity, of habitual microscopy-based procedures. Because of concerns about the sensitivity of rapid diagnostic tests in infections with low parasite densities, and their uncertain specificity for species other than P. falciparum, standardized quality assurance protocols are needed to confirm the diagnosis in the large numbers of suspicious negative results. 11, 12, and 13 A clinical diagnosis of malaria is still challenging because of the non-specific nature of the signs and symptoms, which overlap considerably with other common, as well as potentially life-threatening diseases, e.g. common viral or bacterial infections, and other febrile illnesses. The overlapping of malaria symptoms with other tropical diseases impairs diagnostic specificity, which can promote the indiscriminate use of anti-malarial and compromise the quality of care for patients with non-malarial fevers in endemic areas. 14 In the laboratory, malaria is diagnosed using different techniques, e.g. conventional microscopic diagnosis by staining thin and thick peripheral blood smears, other concentration techniques, e.g. quantitative buffy coat (QBC) method, rapid diagnostic tests, and molecular diagnostic methods, such as polymerase chain reaction (PCR). The accurate diagnosis of malaria infection is important in order to reduce severe complications and mortality. Malaria infection cannot be diagnosed clinically as the presenting clinical signs and symptoms mimic other tropical infections and therefore must be confirmed by laboratory diagnosis. 15 A previous study from India had developed, standardized and reported on the feasibility of a modified centrifuged buffy coat smear (CBCS) examination for diagnosis of malaria in which a wintrobe's tube is used to obtain a buffy coat. This new technique combined most of the advantages of the existing techniques.
The main benefit of CBC is that they are more reliable and accurate to perform and interpret. Clinical diagnosis of malaria is extremely difficult even to an experienced medical practitioner. Reliable laboratory methods are needed to 1156 assist the clinical diagnosis of malaria. Considering this, We conducted a study for the diagnosis of malaria with the aim to find out an easy, feasible and reasonable technique from the modifications of commonly employed techniques. The present study is based on -comparative study of peripheral blood smear, antigen detection and centrifuged buffy coat smear examination for diagnosis of malaria" In the present study, we have used the antigen tests as the gold standard.
The present study demonstrated the performance of a modified technique for diagnosis of malaria by incorporating a centrifugation-enhanced step into the conventional method of smear preparation and examination for malaria. This helps to concentrate the parasites, which are then easily visualized by microscopy. Keeping an eye on these state of the art techniques in the diagnosis of malaria, the present investigation was planned with clearly laid down -Aims and Objectives‖ stated here after.

Material and Method:-
The present study was conducted on the suspected cases of Malaria whose samples were received from different wards and OPDs in the department of Microbiology MMIMSR, Mullana with a target of 50 positive cases by RDT. A relevant history has been taken from the patient and performa given in the Annexure I was filled. Clinical sample was blood. The study was carried out in the Department of Microbiology, Maharishi Markendeshwar Institute of Medical Science And Research for a period of one year from April 2015 to February 2016. A prospective study were carried out in the department of microbiology, MMIMSR, Mullana Study were conducted on 50 positive blood samples by RDT attending OPD and IPD of MMIMSR Mullana.
Sample Processing:-Peripheral blood smear:-Preparation of thick and thin smear:-Thick smear: A big drop of blood is spread over half inch square area on a clean glass slide. The Thickness of the film should be such that it allow newsprint to be read. The film was dried and kept in distilled water in a koplin jar for 5-10 minutes for dehemoglobinization. Thin smear: A small drop of blood is taken on a corner of a slide. It is spread by another slide at an angle of 45oC and then is lowered to an angle of 30oC and is pushed gently to the left, till the blood is exhausted.
Centrifuged buffy coat smears were prepared by using 2 ml blood collected in a wide bore 4 ml tube with EDTA which was centrifuged (2000-3000 rpm for 15 min). The supernatant plasma was separated and layer of buffy coat and equal thickness of RBC layer just below was picked up to prepare smears which were stained by leishman stain Microscopic examination of the stained film.
Examine the blood smear first:-Screen the smear near the feathery tail end. Screen 200-300 oil immersion fields examined before the smears are considered as negative then we screened thick smear. Interpretation of Results: Plasmodium vivax: detection of asexual forms i.e. ring form, late trophozoites and schizonts, gametocytes Plasmodium falciparum: gametocytes and ring forms.

Result:-
A total of 248 clinically suspected cases of malaria were enrolled in the study. Out of 248 samples 50 blood samples were positive for malaria either by PBS or by RDT. Then on these 50 samples we have done comparison of PBS and centrifuged buffy coat smear examination by taking antigen detection test as gold standard.
Graph 1:-Prevalence of malaria among clinically suspected cases. Table 1shows the prevalence of malaria among clinically suspected cases. A total of 129 cases were found to be positive using rapid diagnostic test. Thus prevalence of malaria among clinically suspected cases was 20.15 %. Total 248 100    [10] and mixed infection 2 out of total 50 blood samples.    Graph 5:-Month wise distribution of malarial species. Table 5 Shows the distribution of malaria positive patients according to change in season. A maximum of 11 and 13 were seen in the month of July and August and minimum 2 and 2 were seen in the month of November and December.  Res. 5(4), 1154-1163 1161 Graph 6:-Sensitivity, specificity, positive predictive value, negative predictive value of PBF and CBCS in comparison to antigen test. Table 6 shows the specificity, sensitivity, of PBS and CBCS in comparison to antigen test. It was observed that while both PBS (100%) and CBCS (100%) had excellent specificity, PBS had low sensitivity (94%) in detecting the malaria parasites as compared with CBCS (100%).

Discussion:-
Half of the world's population is at risk of malaria, with an estimated 243 million cases worldwide. Prompt parasitological confirmation by microscopy or alternatively by RDTs is recommended for all patients with suspected malaria before treatment is started. 16 The results of present study indicated that 50 (20.16%) were infected with malaria and and the rest 198 (79.20%) were malaria negative out of total 248 blood samples.  This matched the results of the study done by Iqbal et al 17 [2003] who obtained blood specimens from 930 suspected malaria patients attending BHU and found 231 [25%] to be positive.
From the above finding, it is observed that some results are closed to and some remarkably differ from the present study. The difference may be due to the fact that studied were conducted at different geographical areas and the disease prevalence differ from region to region. Aggarwal N et al 18 [1997] concluded that the event of positivity might depend on the relative prevalence of malaria in different regions. Although according to World Health Organization Malaria risk chart [2011], major part of India falls into malaria risk zone.
In the present study it shows the distribution of malaria positive cases using different methods.  [2007]. Most of the point prevalence studies in India have been carried out for outbreak/epidemic investigations. There is very limited information on age-and sex-specific seasonal prevalence of malaria in different paradigms in the country. In the available studies, age and sex classification used is arbitrary. The burden is generally higher in men than women in all age groups. Children in the states of Assam, Arunachal Pradesh, and Rajasthan had a higher incidence of malaria than adults, whereas in the indo gangatic plains, the situation was reversed.
The result of present study indicated that maximum of 36 (27.9%) and 28 (21.7%) were seen in the month of July and August and minimum 6 (4.6%) and 5 (3.8%) were seen in the month of November The three slides reported as negative out of fifty by peripheral blood smear, but on other hand these false negative slides were observed positive by centrifuged buffy coat smear and antigen detection kit .Thus performance of a modified technique for diagnosis of malaria by incorporating a centrifugation-enhanced step into the conventional method of smear preparation and examination for malaria which gives positive result even in the case of low parasitemia as compared with conventional peripheral blood smear examination which gives false negative result in low parasitemia. 1163

Conclusion:-
The present study was done on the comparison of PBS, antigen detection test and CBC. As we know microscopic examination of peripheral blood smears (PBSs) as stained thick and/or thin blood smears is the standard method for malaria diagnosis, which is easily available and has low cost but its reliability is questionable at low level of parasitaemia and RDTs which are capable to detect malaria cases with good sensitivity and specificity but they are prove to be costlier options in resource limited areas. So the development of easy, rapid and accurate tests for the reliable detection of plasmodia infection is highly desirable. The present study ultimately concluded the performance of a modified technique for diagnosis of malaria by incorporating a centrifugation-enhanced step into the conventional method of smear preparation and examination for malaria which gives positive result even in the case of low parasitemia as compared with conventional peripheral blood smear examination which gives false negative result in low parasitemia. This helps to concentrate the parasites, which are then easily visualized by microscopy. The CBCS technique fulfills most of these criteria and may be adopted for rapid and reliable diagnosis of malaria in resource-limited settings.