Plasmodium DNA, in addition to being used for molecular diagnosis of malaria, find utility in monitoring patient responses to antimalarial drugs, drug resistance studies, genotyping and sequencing purposes. Over the years, numerous protocols have been proposed for extracting Plasmodium DNA from a variety of sources. Given that DNA isolation is fundamental to successful molecular studies, here we review the most commonly used methods for Plasmodium genomic DNA isolation, emphasizing their pros and cons. A comparison of these existing methods has been made, to evaluate their appropriateness for use in different applications and identify the method suitable for a particular laboratory based study. Selection of a suitable and accessible DNA extraction method for Plasmodium requires consideration of many factors, the most important being sensitivity, cost-effectiveness and, purity and stability of isolated DNA. Need of the hour is to accentuate on the development of a method that upholds well on all these parameters.

Background & objectives: Traditionally, Plasmodium falciparum has been attributed to cause severe malaria, whereas P. vivax is considered to cause "benign" tertian malaria. Recently, there has been an increasing body of evidence challenging this conviction. However, the spectrum and degree of severity of the disease caused by P. vivax, as per World Health Organization (2012) remains unclear. Thus, in this prospective study, we aimed at comparing the severity of malaria caused by P. vivax, P. falciparum and dual infection. Methods: Adult patients presenting to Christian Medical College, Vellore from October 2012 to September 2013 with microscopically confirmed malaria were included in the study. Their clinical and laboratory parameters were recorded and analyzed. Paired t-test and chi-square with 95% CI and post-hoc analyses using the Scheffι post-hoc criterion were used to assess the statistical significance at the level of α <0.05. Results: In total, 131 cases of malaria were identified during the study period, comprising 83 cases of P. vivax, 35 cases of P. falciparum and 13 cases of mixed vivax and falciparum infections. The spectrum and degree of hematological, hepatic, renal, metabolic, central nervous system complications of vivax malaria was not different from that of falciparum group. Thrombocytopenia and hyperbilirubinemia were the most common laboratory abnormalities identified in all the groups. Interpretation & conclusion: This cross-sectional comparative study clearly demonstrates that clinical features, complications and case-fatality rates in vivax malaria can be as severe as in falciparum malaria. Hence, vivax malaria could not be considered benign; and appropriate preventive strategies along with antimalarial therapies should be adopted for control and elimination of this disease.

Background & objectives: Control of vivax malaria is challenging due to persistence of hypnozoites causing relapses and safety concerns with primaquine in G6PD deficient individuals. We present the epidemiology of malaria with emphasis on recurrence of vivax malaria over a period of four years in southwest Delhi among patients reporting to malaria clinic. Methods: Microscopic examination of stained blood smears of fever patients attending malaria clinic was performed. Confirmed malaria cases were treated as per the national treatment guidelines. The epidemiological data of confirmed malaria cases including demographic characteristics, age, gender and past history of malaria were analysed. Patients were asked to report in case of occurence of fever. Results: From January 2011 to December 2014, 429 Plasmodium vivax, 24 P. falciparum and three mixed infection cases were reported to the Malaria Clinic at National Institute of Malaria Research, New Delhi. Malaria cases peaked in the months of August and September during all the four years. Recurrent episodes of vivax malaria were observed in 14.72% patients to whom primaquine was not dispensed, while the prevalence was 4.02% among those who received primaquine. The relapsing patterns observed were of both short as well as long latency P. vivax phenotypes. The entomological survey of area from where malaria patients reported, showed prevalence of Anopheles stephensi. Interpretation & conclusion: The study showed presence of persistent P. vivax malaria with strains causing both frequent and long latency recurrences (probable relapses) in southwest Delhi. This highlights the need to evaluate primaquine regimens against both these strains and formulate strategies to improve compliance to 14-days primaquine treatment.

Background & objectives: Dengue is one of the major public health problems worldwide, transmitted mainly by Aedes aegypti and Ae. albopictus mosquitoes. Rapid urbanisation and industrialisation have led to an increase in vector population in Northeastern states of India. In 2013, Guwahati, the capital city of Assam, India experienced an outbreak of dengue. This study was undertaken with an objective to determine infection rates of dengue viruses (DENV) in both the established vectors present in this region. Methods: During the outbreak (2013), adults and larvae of both the vector species were collected from different container habitats found in case reporting areas and container index was also recorded. The mosquitoes were first pooled, homogenised and processed for NS1-ELISA. This was followed by RT-PCR of the mosquito pools. Results: Both Ae. aegypti and Ae. albopictus were found breeding in containers with container index in the range of 29.41 to 80%. Six pools of Ae. aegypti were found to be positive for NS1 antigen. RT-PCR assay revealed positivity in only the NS1-ELISA positive pools, exhibiting circulation of serotype DENV-2. Minimum infection rate of female and male Ae. aegypti was recorded as 10.87 and 11.03 respectively. Interpretation & conclusion: This is the maiden report of detection of DENV in wild caught Ae. aegypti mosquitoes from Northeastern Region of India. The study also demonstrates the presence of transovarial transmission of dengue virus in this part of country. This information is useful in respect of both entomological as well as epidemiological point of view for taking appropriate vector control measures.

Background & objectives: Dengue is one of the most important vector-borne viral infection which may lead to haemorrhage or even shock. The present study was conducted with an objective to study the epidemiological and demographic characteristics of dengue disease during the year 2013 in Saurashtra region of Gujarat state, India. Methods: A longitudinal study was conducted at sentinel surveillance centre for dengue disease at microbiology department of tertiary care hospital in Rajkot, Gujarat from 1 January 2013 to 31 December 2013. A total of 4366 blood samples were collected during study period and serologically tested for dengue NS1 antigen and IgM antibody by capture ELISA testing from various districts of Saurashtra region. Patients with acute onset of illness, high grade fever, severe headache, backache, musculoskeletal pain or retro-bulbar pain with or without rashes were considered as clinically suspected case of dengue virus infection. Patient who presented with fever and found positive for Dengue NS1 Ag and/or IgM Ab was considered as a case. A pre-tested structured proforma was used as a tool for data collection. The data was entered into EpiInfo software and analyzed by using appropriate statistical test (chi-square test). Results: Out of 4366 tested samples, 41.3% samples were found positive for dengue. Majority of samples were tested in age group 16-25 yr (33.9%) and 26-45 yr (31.1%). The ratio of male cases (60.8%) was higher than female cases. Two-third (68.7%) suspected cases were residing in urban areas of Saurashtra region. More than half of the cases (57%) were diagnosed within five days of fever by NS1 antigen test. The reporting of number of cases increased from July 2013, which reached to peak during September 2013 followed by decrease till December 2013. Interpretation & conclusion: Dengue predominately affected males and urban population. A seasonal occurrence was reported mainly during monsoon season. A long term serosurveillance study may help to provide more information about the intensity, seasonal incidence and seasonal effect.

Background & objectives: The indications for platelet transfusion in dengue fever are clearly defined in World Health Organization (WHO) guidelines (2011) for dengue fever, but physicians face practical difficulty in its implementation in an epidemic setting. On one hand there is an intense social pressure created by the panic-struck parents to transfuse platelets in presence of bleeding and on the other hand there is a need for its judicious use as the requirement is more than its availability. The study was aimed to assess the clinico-hematological parameters, and the requirement and need for platelet transfusion in children with dengue fever. Material & Methods: All children (0-12 yr of age) diagnosed and confirmed with dengue fever at a tertiary care hospital in Puducherry between 1 August 2012 and 31 January 2015 were reviewed retrospectively from hospital case records as per the revised WHO guidelines for dengue fever. The diagnosis was confirmed by NS1 antigen- based ELISA test or dengue serology for IgM and IgG antibodies and the data were analyzed using SPSS 16.0 statistical software. Results: Out of 261 cases of dengue fever, hemorrhagic manifestations were observed in 52 children (19.9%), which mainly included petechiae (38.5%), gum bleeding (34.6%) and melena (26.9%). Thrombocytopenia was seen in 211 (80.8%) cases. Bleeding manifestations were present in 20(39.2%), 8(15.7%), 13(25.5%) and 11(21.6%) cases with platelet count <50,000/mm³, 50,000-100,000/mm³, 1-1.50,000/mm³, and >1.50,000/mm³ respectively. Bleeding manifestations did not always correlate with platelet count in non-severe dengue infection in comparison to severe dengue infection. The most common mode of presentation of severe dengue infection was shock with 102(39.1%) cases and among them only 22 children (21.6%) had bleeding. About 17 children (6.5%) with severe dengue infection required platelet transfusion and out of them, 12 children (70.6%) had a platelet count <20,000/ mm3 whereas five children (29.4%) had platelet count in the range of 20,000-50,000/mm³. Interpretation & conclusion: Platelet transfusion was required in children with severe dengue infection in the form of significant spontaneous bleed, shock and severe thrombocytopenia. Bleeding should not be considered only indicator to transfuse platelets as it occurred in children even with normal platelet counts. The community and treating physicians should be educated regarding the judicious transfusion of platelets. Unnecessary and empirical use of platelets should be completely avoided especially during an epidemic when there is scarcity in its availability.

Background & objectives: Oviposition is an important event in the life cycle of mosquito which is a decisive factor for the fate of its progeny. If oviposition is prevented, the life cycle of mosquito is disrupted and the population growth can be reduced. Diethyl-phenylacetamide (DEPA) is an effective topical repellent against various haematophagous insects. The study reports the egg retention effect and subsequent lethal effect of DEPA on the gravid females of Aedes aegypti and Ae. albopictus mosquitoes. Methods: DEPA was applied at 1000 mg/l onto water surface in oviposition bowls under no-choice condition. Experiments had three replicates with three control and treated cages separately, containing 100 gravid females of each test mosquito species. Every day freshly treated oviposition bowls were provided to the gravid mosquitoes till the last surviving female of the cohort was dead. On completion of one experimental day (24 h) the number of eggs laid in control and treatment bowls were counted manually to assess the oviposition response and longevity of test mosquito species. Results: DEPA at 1000 mg/l concentration reduced the egg deposition and affected the longevity of gravid females. In the DEPA treated cages >1100 eggs were retained by both the species. In the treated cages, about 50% of Ae. albopictus and 41% of Ae. aegypti females died, retaining eggs in their abdomen. The average life span of Ae. aegypti and Ae. albopictus females in control cages were 43 and 50 days respectively, while the females of both the species exposed to DEPA in treated cages survived for 16-22 days. Interpretation & conclusion: Presence of DEPA odor on water surface forced the gravid Aedes females to retain their eggs, affecting their fecundity and longevity. This indigenously developed topical repellent DEPA may be applied to container's water that act as breeding grounds for dengue vectors to deter gravid females from oviposition in the vicinity of a household.

Background & objectives: Lymphatic filariasis (LF) is endemic in the state of Assam and mass drug administration (MDA) programme for LF elimination is being implemented in the state since 2004. A study on prevalence of microfilaria (mf), disease endemicity and vector infection was carried out in a tea garden population of Dibrugarh, Assam (India) to assess the effect of ongoing MDA programme on elimination of LF. Methods: Finger prick thick blood smears (20 mm³) were made from individuals aged ≥2 yr old during night blood survey in between 2000-0000 hrs during the period of November 2012 to February 2013. Blood smears were dehaemoglobinised, stained with Giemsa and examined under microscope for presence of mf. Indoor resting mosquitoes were collected during 0600-1000 hrs and female Culex quinquefasciatus were dissected and examined under microscope for larval forms of the parasite. Results: A total of 634 blood smears were collected and screened for mf and 47 (7.41%) individuals were found microfilaraemic, with predominance of males (74.5%). Highest mf rate (20.0%) was seen in the males of 30-39 yr age group while in females, age group of 10-19 yr recorded maximum mf rate (5.48%). Entomological collection and dissection of Cx. quinquefasciatus revealed presence of larval stages of the parasite and infection and infectivity rates recorded were 13.20 and 3.70%, respectively. Chronic clinical manifestations in the form of elephantiasis and hydrocele were recorded in 33 (5.73%) subjects of the 575 examined. Interpretation & conclusion: Mass drug administration data showed six rounds of MDA with drug distribution coverage in between 63.42 and 95.93% in the study population. Out of 634 individuals examined 47 were found microfilaraemic giving an overall infection rate of 7.41%. Mosquito vector infection and infectivity rates were 13.20 and 3.70%, respectively. Presence of high mf rate, vector infectivity rate and clinical cases in the study population after six rounds of MDA warrants concerted efforts to be made for effective implementation and monitoring of MDA for success of LF elimination programme.

Background & objectives: Japanese encephalitis virus (JEV), a mosquito borne pathogen, is one of the major causes of viral encephalitis in eastern Uttar Pradesh, India. The objective of this work was to evaluate the entomological based virological surveillance of Japanese encephalitis (JE) in the highly endemic area of eastern Uttar Pradesh. Methods: The study was carried out during September 2010 to March 2013 in Gorakhpur district of Uttar Pradesh. A total of 251 adult mosquito pools and 64 water samples containing larvae were collected from the District of Gorakhpur. Water pH, turbidity, and oxygen level were analyzed for vector breeding index (BI). In addition, 393 serum/cerebrospinal fluid (CSF) samples of acute encephalitis syndrome (AES) suspected cases were collected from the district hospital. Results: The various Culex species found included, Cx. quinquefasciatus (26.83%), Cx. vishnui (22.29%), Cx. pseudovishnui (20.73%), Cx. tritaeniorhynchus (12.71%), Cx. whitmorei (9.04%), and Cx. gelidus (8.25%). Highest minimum infection rate (MIR) was calculated for Cx. tritaeniorhynchus (2.32), followed by Cx. vishnui (1.98) and Cx. pseudovishnui (0.71). All the larvae samples were negative for JEV. The mean number larvae of Cx. tritaeniorhynchus and Cx. pseudovishnui was negatively correlated with pH (r = - 0.45 and r = - 0.63) and turbidity (r = - 0.30 and r = - 0.37). In contrast, positive correlation was observed in case of Cx. quinquefasciatus. A total of 41 clinical samples were found positive for JEV by IgM ELISA. The rainfall was significantly associated with Japanese encephalitis incidence and showed positive correlation to disease transmission (p = 0.02, r = 0. 66). Interpretation & conclusion: The findings showed the rapid dissemination of JEV within a population, facilitated by different species of Culex in the region. As JE is a vaccine-preventable disease, an immunization programme, an effective vector control strategy and application of standard hygiene practices in these endemic areas could result in a considerable reduction in morbidity and mortality due to JE.

Background & objectives: The heartworm Dirofilaria immitis is an important mosquito-borne zoonotic nematode of domestic and wild mammals throughout the world, causing cardiopulmonary dirofilariasis. This parasite has been reported from carnivores in some provinces of Iran. However, in the present study, the occurrence of this filarial nematode is reported for the first time in wild canids of the North Khorasan Province, located in northeast Iran, based on morphological and molecular characteristics. Methods: The carcasses of 45 golden jackals (Canis aureus), 16 foxes (Vulpes vulpes), 15 dogs (Canis familiaris), and one wolf (Canis lupus) were necropsied between 2013 and 2014. Results: By gross examination, adult filarial nematodes were found in the cardiovascular system of four jackals (8.9%). The morphological characteristics of the recovered heartworms were compatible with D. immitis. DNA sequencing of the cytochrome c oxidase subunit I (cox1) gene of all four isolates was identical, showing 100% homology with several sequences registered in GenBank from other countries. No adult D. immitis was found in any of the other animals examined. Interpretation & conclusion: D. immitis is circulating in wildlife of the study area, suggesting the relevance of developing control programmes to prevent transmission of the disease to humans and domestic animals.