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Background & Objectives: The highly sensitive method for a true understanding of malaria prevalence is of utmost importance for India's elimination strategy. The PCR reaction type with rapid detection, cost-effectiveness, and less workforce should be preferable. Multiplex PCR type accomplishes the present requirement by saving time and resources to find true surveillance data for malaria, especially in low-parasitemia/asymptomatic groups or populations., Methods: The present study focuses on designing multiplex PCR (mPCR) to detect simultaneously Plasmodium genus (PAN) and two common Plasmodium species found in India. It is compared to standard nested PCR on 195 clinical samples to diagnose malaria. The mPCR was designed with a minimum number of primers, leading to less clogging and effective and enhanced detection. It contains one common reverse primer and three forward primers amplifying three targeted genes corresponding to P. falciparum, P. vivax, and Plasmodium genus., Results: The sensitivity and specificity for mPCR were 94.06 and 95.74, respectively. The limit of detection for mPCR was 0.1 parasites/µl. The study has shown a ROC curve area for the mPCR of 0.949 for Plasmodium genus and P. falciparum and 0.897 for P. vivax with standard nPCR., Interpretation & Conclusion: The mPCR is rapid in detecting species together, cost-effective, and requires fewer human resources than the standard nPCR. Therefore, the mPCR can be used as an alternative technique for the higher sensitive detection of the malaria parasite. It could also become a vital tool for determining malaria prevalence, facilitating the application of the most effective measures.

Background & Objectives: Microscopy is considered as the gold standard for malaria diagnosis, however sub-microscopic infections can only be detected by Polymerase chain reaction, which demands high cost and elaborate laboratory setup. The Micro-chip PCR based Truenat Malaria Pv-Pf and Pf assay is a portable solution for detection of sub-microscopic/asymptomatic cases of malaria in the field, three lots of which were evaluated for P. falciparum and P. vivax malaria., Methods: Three lots of Truenat® Malaria Pv-Pf and Pf assay (kits) were assessed using blood samples of P. vivax and P. falciparum as well as malaria negative blood samples. DNA was extracted from the blood samples using the Trueprep Auto v2 Universal Cartridge based sample prep device and real time qPCR was performed using Truelab DUO micro PCR Analyzer with three lots of Truenat® Malaria Pv-Pf and Pf Assays. Mean, Standard deviation and one-way analysis of variance (ANOVA) was used to assess the significance of inter-lot variability in Cycle threshold values., Results: The Truenat® Malaria Pv-Pf and Pf assays identified the malaria parasites with 100% accuracy. Based on the test for variance (ANOVA) the inter-lot variability in cycle threshold values were not significant, indicating a high degree of precision., Interpretation & Conclusion: Based on high accuracy and precision between different lots, the Truenat® Malaria Pv-Pf and Pf assays were found to be suitable for the diagnosis of sub-microscopic infections in field conditions to provide support in elimination of malaria., Competing Interests: None

Background & Objectives: Malaria continues to be a significant public health problem in tropical countries including India; however, there are limited tools to predict occurrence of severe disease due to malaria. This study was designed to evaluate the role of Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Vascular endothelial growth factor (VEGF) and Asymmetric Dimethylarginine (ADMA)as disease biomarkers in uncomplicated malaria (UM) and severe malaria (SM)., Methods: This is a prospective observational study carried out at All India Institute of Medical Sciences (AIIMS), tertiary referral hospital in New Delhi, India. The study population included patients diagnosed with malaria (Plasmodium falciparum or Plasmodium vivax) either by rapid diagnostic kit test or positive peripheral smear and age more than 12 years. Forty-nine patients (25 with SM, 24 with UM) and 22 controls were recruited. In addition to routine investigations, serum concentrations of Ang-1, Ang-2, VEGF and ADMA were measured using ELISA technique., Results: We observed Ang-1 serum levels to be significantly lower in patients with severe malaria (7775 pg/ml) compared to uncomplicated malaria (17629 pg/ml) and healthy controls (43472 pg/ml) [p <0.001]. Ang-2 levels were significantly higher in severe malaria (11100 pg/ml) compared to uncomplicated malaria (7315 pg/ml) and healthy controls (3679 pg/ml) (p <0.001). The ratio of Ang-2/Ang-1 was significantly higher in patients with severe malaria. VEGF serum levels was significantly lower in severe malaria (130.36 pg/ml) compared to uncomplicated malaria (317.3 pg/ml). The Ang-1, Ang-2 and VEGF levels were able to differentiate severe malaria from uncomplicated malaria caused by P. vivax but not with P. falciparum., Interpretation & Conclusion: We conclude that Ang-1, Ang-2 and VEGF are markers of disease severity in vivax malaria., Competing Interests: None

Background & Objectives: Loop-mediated isothermal amplification (LAMP) is an emerging nucleic acid based diag- nostic approach that is easily adaptable to the field settings with limited technical resources. This study was aimed to evaluate the LAMP assay for the detection and identification of Plasmodium falciparum and P. vivax infection in malaria suspected cases using genus and species-specific assay., Methods: The 18S rRNA-based LAMP assay was evaluated for diagnosis of genus Plasmodium, and species- specific diagnosis of P. falciparum and P. vivax, infection employing 317 malaria suspected cases, and the results were compared with those obtained by 18S nested PCR (n-PCR). All the samples were confirmed by microscopy for the presence of Plasmodium parasite., Results: The n-PCR was positive in all Plasmodium-infected cases (n=257; P. falciparum=133; P. vivax=124) and negative in microscopy negative cases (n=58) except for two cases which were positive for P. vivax, giving a sen- sitivity of 100% (95% CI: 97.04-100%) and a specificity of 100% (95% CI: 88.45-99.5%). Genus-specific LAMP assay missed 11 (3.2%) microscopy and n-PCR confirmed vivax malaria cases. Considering PCR results as a refer- ence, LAMP was 100% sensitive and specific for P. falciparum, whereas it exhibited 95.16% sensitivity and 96.7% specificity for P. vivax. The n-PCR assay detected 10 mixed infection cases while species-specific LAMP detected five mixed infection cases of P. vivax and P. falciparum, which were not detected by microscopy., Interpretation & Conclusion: Genus-specific LAMP assay displayed low sensitivity. Falciparum specific LAMP assay displayed high sensitivity whereas vivax specific LAMP assay displayed low sensitivity. Failed detection of vivax cases otherwise confirmed by the n-PCR assay indicates exploitation of new targets and improved detection methods to attain 100% results for P. vivax detection.

Background & Objectives: The search for effective polymorphic markers in Plasmodium vivax is highly demanding to understand its transmission in a population. Due to the limited knowledge existing for P. vivax, the search for polymorphic markers for population studies is ongoing. The MSP gene family of Plasmodia has been linked with immune evasion. To study the circulating parasite population P. vivax merozoite surface protein 3β (PvMSP3β) polymorphic marker was used to investigate the genetic diversity of P. vivax in natural infections., Methods: Polymorphism of PvMSP3β gene was determined in 46 P. vivax blood samples from six different regions of India by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) with PstI., Results: Two different parasite genotypes, viz. type-A and type-B were detected among 46 samples that were positive for PCR, based on the size of the amplification. RFLP analysis with PstI showed 22 allelic groups and 15.2% samples revealed mixed infections on analysis., Conclusion: PvMSP3β was found to be an effective molecular marker for P. vivax as it shows high diversity in India and multiple genotypes easily distinguishable without the need for sequencing.

Background & Objective: A cross - sectional study was conducted to determine the prevalence of Plasmodium infections among migrant workers in Thailand., Methods: A total of 241 migrants at Kanchanaburi, Pathumthani and Nakornpathom provinces of Thailand were recruited in our surveillance. Blood samples were examined for human malaria parasites by using microscopy and semi - nested multiplex PCR (SnM- PCR)., Results: Laboratory diagnosis revealed 6.2% total positive rate. As compared to microscopy (26.7%), SnM- PCR was more sensitive (93.3%) for malaria. Plasmodium falciparum was predominant than P. vivax (53% : 40%, respectively). The majority of positive cases were from Myanmar workers who had low parasitaemia and without symptoms. The highest prevalence (13.7%) was found among migrant workers from Kanchanaburi province in western Thailand., Conclusion: These findings indicate risk of malaria transmission from migrant workers. Malaria surveillance should be included in the health- screening program for migrants in Thailand to manage this health risk.

Background & Objectives: Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) are responsible for most of the global burden of malaria. With changing spectrum of clinical presentation in malaria, pulmonary system involvement has always been under diagnosed. The present study was planned to estimate the pulmonary system involvement in patients with malaria from north- western India (Bikaner). STUDY DESIGN & DESCRIPTION OF THE PATIENTS: Our study was conducted during 2007 to 2009 in 200 cases of severe malaria [Pf , Pv, and mixed (Pf + Pv)] with pulmonary involvement. It included adult patients of both sexes (145 males and 55 females) belonging to all age groups. The diagnosis of Pf and Pv was confirmed by demonstrating asexual form of parasites in peripheral blood smear and OptiMal test., Main Outcome Measures: Pulmonary involvement was observed in 30% (60/200) patients among which cough in 24% , dyspnea in 12%, acute respiratory distress syndrome (ARDS) in 7% , bronchitis in 3% and pneumonia in 1.5% were the major clinical manifestations of malaria. Metabolic acidosis and low oxygen saturation was observed in 7% patients. Chest X- ray abnormality in 11.5% patients, 7% had bilateral infiltrates, 1.5% had inflammatory patch and 3% had findings suggestive of bronchitis. Spirometry findings showed 17% patients had early small airway obstruction. All the patients with ARDS had poor disease outcome., Results & Conclusion: Our results suggest that pulmonary system involvement was observed in patients infected with Pf and Pv. If these clinical presentations are ignored, it may lead to delay in diagnosis and can alter the outcome and prognosis of the disease. Therefore, early diagnosis of malaria induced ARDS can significantly affect the outcome.

Objective: This retrospective study was conducted to determine the incidence of various complications of Plasmodium vivax malaria based on review of case records., Methods: The case records of all confirmed cases of malaria over the period of one year (September 2005-August 2006) were studied. Complete blood count, peripheral blood findings, liver and kidney functions were reviewed. The results of rapid diagnostic test for malaria (OptiMAL test, Diamed AG, Switzerland) were correlated with the peripheral blood smear findings in the patients in whom it was requested. All abnormal results like a positive direct Coomb's test were noted. Findings were clinically correlated., Results: There were 265 confirmed cases by peripheral blood examination. Of these 221 were due to Plasmodium vivax and 41 due to P. falciparum. Two cases had mixed infection and in one case the species could not be identified as it showed only malarial pigment. The peak incidence of malaria was seen in September 2005 and August 2006. The complications in P. vivax were thrombocytopenia, biochemical evidence of hepatic dysfunction, renal damage, positive DCT and death due to ARDS. Thrombocytopenia was seen in 213 patients with counts < 20 x 103/microl in 13 patients. Nine (4%) patients had serum bilirubin >3 mg/dl with normal liver enzymes. Liver enzymes were elevated in 60 patients with seven patients showing liver enzymes level, three times the normal. Renal dysfunction was seen in 17 patients with serum creatinine ranging from 1.3-10.65 mg/dl. One patient went into acute renal failure following quinine therapy and showed red cell fragments in the peripheral blood. In two children DCT was positive with the peripheral smear showing RBC agglutinates around the parasitised RBC. There were three maternal deaths at about 32 weeks gestation due to ARDS. The peripheral blood smear in these patients showed WBC agglutinates., Conclusion: This paper is presented to highlight that P. vivax malaria though considered to be a benign entity can also have a severe and complicated course which is usually associated with P. falciparum malaria.

Background & Objectives: Plasmodium vivax is geographically widespread and responsible for > 50% of malaria cases in India. Increased drug resistance of the parasite highlights the immediate requirement of early and accurate diagnosis as well as new therapeutics. In view of this, the present study was undertaken to amplify P. vivax (Indian strains) lactate dehydrogenase gene (PvLDH) which has been identified as a good target for antimalarials as well as diagnostics., Methods: P. vivax infected clinical blood samples were collected from southern part of India and were tested with established diagnostic parameters (ICT, Giemsa staining). Total DNA was extracted from blood samples and subjected to PCR using two sets of primers, one for the amplification of full PvLDH gene (951 bp) and the other for a partial PvLDH gene fragment (422bp), covering a variable antigenic region (140aa) as compared to other plasmodial species., Results & Conclusion: PCRs for both the full and partial gene targets were optimised and found to be consistent when tested on several P. vivax positive clinical samples. In addition, full gene PCR was found to specifically detect only P. vivax DNA and could be used as a specific molecular diagnostic tool. These amplified products can be cloned and expressed as a recombinant protein that might be useful for the development and screening of antimalarials as well as for diagnostic purposes.

Background and Objectives: Although polymerase chain reaction (PCR) is a new technique in the diagnosis of malaria with very high accuracy; light microscopy is still conventional diagnostic method in Iran. In this study we checked the accuracy of light microscopy using the results of PCR as gold standard in Iran., Methods: The blood samples were collected from 124 febrile cases in Kahnooj district. The blood slides were read by microscopists, and double checked by experts in provincial referral laboratory. DNA samples were processed by PCR to amplify species-specific sequences of 18s subunit ribosomal ribonucleic acid (18ssrRNA) genes of Plasmodium vivax and P. falciparum., Results: The sensitivity and specificity of microscopy in the detection of Plasmodium spp infection were 77% (95% CI: 46-94%) and 100% (95% CI: 95-100%), correspondingly. Also, the estimated positive and negative predictive values were 100% (95% CI: 66-100%) and 97% (95% CI: 91-99%), respectively., Interpretation and Conclusion: According to these results, we believe that the accuracy of light microscopy in the diagnosis of malaria in Kahnooj was acceptable. Expert micorscopists in endemic areas of Iran such as Kahnooj and available equipments in one hand and expensive PCR test on the other hand may convince that in current situation we do not have to change the diagnostic method.