Rapid and inexpensive method of PCR ready DNA isolation from human peripheral blood and saliva.

BACKGROUND: The isolation of nucleic acids is a frequently performed procedure in the molecular biology area. Although several rapid DNA isolation techniques from human peripheral blood and saliva have been developed, there are still some disadvantages - volume, time, cost, and yield are a few notable ones. OBJECTIVE: We aim to develop a rapid and inexpensive method to isolate high-molecular-weight genomic DNA from human peripheral blood and saliva that can be used for molecular biology experiments. METHODS: Five DNA isolation methods with slightly varying protocols were used. High-quality DNA obtained from one specific method was further amplified by PCR and the template with good amplification was further used for performing RFLP and sequencing. RESULTS: Out of 5 different isolation methods (R1 to R5), DNA obtained from the R4 was of good quality (molecular weight is > 10 kb and 260/280 ratio is 1.89 ± 0.2), which allows successful PCR amplification and good separation in Restriction Fragment Length Polymorphism analysis. Sequencing by the Sanger Sequencing produced a good readable sequence of an amplified fragment from Method R4 DNA. CONCLUSION: In the present study we have developed a simple, rapid, and cost-effective DNA isolation method, which uses low sample volume and yields good quantity and high-quality product. The DNA obtained is highly fit for molecular genetics research applications.

The Effect of Socioeconomic Factors and Indoor Residual Spraying on Malaria in Mangaluru, India: A Case-Control Study.

India faces 0.5 million malaria cases annually, including half of all Plasmodium vivax malaria cases worldwide. This case-control study assessed socioeconomic determinants of urban malaria in coastal Mangaluru, Karnataka, southwestern India. Between June and December 2015, we recruited 859 malaria patients presenting at the governmental Wenlock Hospital and 2190 asymptomatic community controls. We assessed clinical, parasitological, and socioeconomic data. Among patients, p. vivax mono-infection (70.1%) predominated. Most patients were male (93%), adult (median, 27 years), had no or low-level education (70.3%), and 57.1% were daily labourers or construction workers. In controls (59.3% male; median age, 32 years; no/low-level education, 54.5%; daily labourers/construction workers, 41.3%), 4.1% showed asymptomatic Plasmodium infection. The odds of malaria was reduced among those who had completed 10th school grade (aOR, 0.3; 95% CI, 0.26-0.42), lived in a building with a tiled roof (aOR, 0.71; 95% CI, 0.53-0.95), and reported recent indoor residual spraying (aOR, 0.02; 95% CI, 0.01-0.04). In contrast, migrant status was a risk factor for malaria (aOR, 2.43; 95% CI, 1.60-3.67). Malaria in Mangaluru is influenced by education, housing condition, and migration. Indoor residual spraying greatly contributes to reducing malaria in this community and should be promoted, especially among its marginalised members.

MiRNA-146a Polymorphism Was Not Associated with Malaria in Southern India.

Micro-RNAs (miRNAs) play a crucial role in immune regulation, and a common miRNA-146a polymorphism (rs2910164) increased the odds of falciparum malaria in pregnant African women. Here, we examined whether this association holds true in a different population, that is, 449 mainly male and adult malaria patients and 666 community controls in southwestern India. Plasmodium vivax malaria (67%) predominated over falciparum malaria (11%) and mixed species infections (22%). Overall, 59% of the study participants carried the miRNA-146a polymorphism. However, it was not associated with the odds of malaria, irrespective of parasite species. This underlines the importance of considering the complexities of clinical manifestations of malaria, genetic background, and parasite species when disentangling the role of human genetic variation, including those of miRNAs in malaria.

Duffy antigen receptor for chemokines gene polymorphisms and malaria in Mangaluru, India.

BACKGROUND: Duffy blood group antigens serve as receptors for Plasmodium vivax invasion into erythrocytes, and they are determined by polymorphisms of the Duffy antigen receptor for chemokines (DARC), also known as Fy glycoprotein (FY). Duffy negativity, i.e., absence of the antigens, protects against P. vivax infection and is rare among non-African populations. However, data on DARC polymorphisms and their impact on Plasmodium infection in India are scarce. METHODS: In a case-control study among 909 malaria patients and 909 healthy community controls in Mangaluru, southwestern India, DARC polymorphisms T-33C (rs2814778), G125A (rs12075), C265T (rs34599082), and G298A (rs13962) were genotyped. Associations of the polymorphisms with the odds of malaria, parasite species and manifestation were assessed. RESULTS: Among patients, vivax malaria (70%) predominated over falciparum malaria (9%) and mixed species infections (21%). DARC T-33C was absent and C265T was rare (1%). FYB carriage (deduced from DARC G125A) was not associated with the risk of malaria per se but it protected against severe falciparum malaria (P = 0.03), and hospitalization (P = 0.006) due to falciparum malaria. Vice versa, carriage of DARC 298A was associated with increased odds of malaria (aOR, 1.46 (1.07-1.99), P = 0.015) and vivax malaria (aOR, 1.60 (1.14-2.22), P = 0.006) and with several reported symptoms and findings of the patients. CONCLUSION: This report from southern India is the first to show an independent effect of the DARC 298A polymorphism on the risk of malaria. Functional studies are required to understand the underlying mechanism. Moreover, FYB carriage appears to protect against severe falciparum malaria in southern India.

Characterization of Plasmodium vivax pvmdr1 Polymorphisms in Isolates from Mangaluru, India.

India accounts for approximately half of the global Plasmodium vivax cases, but information as to the presence of chloroquine (CQ) resistance is scarce. In an observational study in Mangaluru, south-western India, of 116 vivax malaria patients analyzed, 89.5% (102/114) had cleared parasitemia on days two or three of CQ treatment. Two remaining patients presented on days four and five without parasitemia. One hundred eight isolates of these 116 patients were successfully sequenced for pvmdr1 polymorphisms. Eight non-synonymous polymorphisms but no wild-type isolate were detected. Ten pvmdr1 haplotypes were observed with mutations T958M and F1076L occurring in all isolates, whereas the candidate CQ resistance marker Y976F was present in one isolate only. Pvmdr1 polymorphisms were not associated with early parasite clearance. The high proportion of early parasite clearance and the virtual absence of pvmdr1 Y976F and of sextuple pvmdr1 mutants suggest that CQ in the study area is still sufficiently effective. However, the abundance of pvmdr1 mutations in the local parasite population warrants monitoring.

MiRNA-146a polymorphism increases the odds of malaria in pregnancy.

BACKGROUND: Plasmodium falciparum infection during pregnancy is a major cause of poor maternal health, adverse foetal outcome and infant mortality in sub-Saharan Africa. Genetic disposition is involved in susceptibility to malaria in pregnancy and its manifestation. MicroRNAs (miRNAs) influence gene regulation including that of innate immune responses. A miRNA-146a rs2910164 G > C single nucleotide polymorphism (SNP) has been associated with increased risks of several diseases, but no data as to malaria are available. METHODS: The association between miRNA-146a rs2910164 and P. falciparum infection among 509 Ghanaian women attending antenatal care (ANC) and 296 delivering Ghanaian primiparae was investigated. Malaria parasites were diagnosed by microscopy and PCR. Leukocyte-associated hemozoin in placental samples was recorded as well. Proportions were compared between groups by Fisher's exact test, and logistic regression models were used to adjust for possible confounders. RESULTS: By PCR, P. falciparum infection was detected in 63% and 67% of ANC attendees and delivering primiparae, respectively. In both groups, two in three women were either heterozygous or homozygous for miRNA-146a rs2910164. Among ANC attendees, homozygosity conferred increased odds of infection (adjusted odds ratio (aOR), 2.3; 95% CI, 1.3-4.0), which was pronounced among primigravidae (aOR, 5.8; 95% CI, 1.6-26) but only marginal in multigravidae. Likewise, homozygosity for miRNA-146a rs2910164 in primiparae increased the odds of past or present placental P. falciparum infection almost six-fold (aOR, 5.9; 95% CI, 2.1-18). CONCLUSIONS: These results indicate that SNP rs2910164 G > C is associated with increased odds for P. falciparum infection in first-time pregnant women who are considered to lack sufficient acquired immune responses against pregnancy-specific strains of P. falciparum. These findings suggest that miRNA-146a is involved in protective malarial immunity, and specifically in the innate component.

Molecular Evidence for Plasmodium falciparum Resistance to Sulfadoxine-Pyrimethamine but Absence of K13 Mutations in Mangaluru, Southwestern India.

In most of India, sulfadoxine-pyrimethamine (SP) plus artesunate serves as first-line treatment for uncomplicated falciparum malaria. In 112 clinical Plasmodium falciparum isolates from Mangaluru, southwestern India, we sequenced molecular markers associated with resistance to SP, lumefantrine, and artemisinin (pfdhfr, pfdhps, pfmdr1, and K13). The pfdhfr double mutation 59R-108N combined with the dhps 437G mutation occurred in 39.3% and the pfdhfr double mutation plus the pfdhps double mutation 437G-540E in additional 24.1%. As for pfmdr1, the allele combination N86-184F-D1246 dominated (98.2%). K13 variants were absent. No evidence for artemisinin resistance was seen. However, the antifolate resistance alleles compromise the current first-line antimalarial sulfadoxine-pyrimethamine plus artesunate, which may facilitate the emergence of artemisinin resistance. Artemether-lumefantrine, introduced in northeastern parts of the country, in the study area faces the predominant pfmdr1 NFD genotype, known to impair lumefantrine efficacy. Further monitoring of resistance alleles and treatment trials on alternative artemisinin-based combination therapies are required.

Manifestation of malaria in Mangaluru, southern India.

BACKGROUND: Severe and fatal vivax malaria is increasingly reported from India. In Mangaluru, southern India, malaria is focused in urban areas and associated with importation by migrant workers. In Wenlock Hospital, the largest governmental hospital, the clinical, parasitological and biochemical characteristics of malaria patients were assessed. METHODS: During the peak malaria season in 2015 (June to December), outpatients were interviewed and clinically assessed. Malaria was ascertained by microscopy and PCR assays, concentrations of haemoglobin, creatinine and bilirubin, as well as thrombocyte count, were determined, and severe malaria was defined according to WHO criteria. RESULTS: Among 909 malaria patients, the vast majority was male (93%), adult (median, 26 years) and of low socio-economic status. Roughly half of them were migrants from beyond the local Karnataka state, mostly from northern and northeastern states. Vivax malaria (69.6%) predominated over mixed Plasmodium vivax-Plasmodium falciparum infection (21.3%) and falciparum malaria (9.0%). The geometric mean parasite density was 3412/µL. As compared to vivax malaria, patients with falciparum malaria had higher parasite density and more frequently showed impaired general condition, affected consciousness and splenomegaly. Also, they tended to more commonly have anaemia and increased creatinine levels, and to be hospitalized (7.3%). Mixed-species infections largely assumed an interim position. Severe malaria (3.5%) was not associated with parasite species. No fatality occurred. CONCLUSION: In this study, uncomplicated cases of malaria predominated, with P. falciparum causing slightly more intense manifestation. Severe malaria was infrequent and fatalities absent. This contrasts with the reported pattern of manifestation in other parts of India, which requires the analysis of underlying causes.

Artemisinin Resistance-Associated K13 Polymorphisms of Plasmodium falciparum in Southern Rwanda, 2010-2015.

Emerging artemisinin resistance is a threat to global malaria control. Mutations in the Plasmodium falciparum Kelch 13 (K13) propeller domain confer artemisinin resistance and constitute molecular markers for its detection and monitoring. We sequenced 222 P. falciparum isolates obtained from community children in the Huye District of southern Rwanda in 2010, 2014, and 2015 to investigate the presence of K13 polymorphisms. No polymorphisms were observed in 2010 but they were present in 2.5% and 4.5% in 2014 and 2015, respectively. In 2015, two isolates showed candidate K13 resistance mutations (P574L and A675V), which are common in southeast Asia and associated with delayed parasite clearance. K13 polymorphisms in southern Rwanda are infrequent but include variants associated with artemisinin resistance. Establishing correlations with local treatment response and in vitro resistance assays are needed in addition to further monitoring K13 polymorphisms in the study area.

Matrix metalloproteinase-9 polymorphism 1562 C > T (rs3918242) associated with protection against placental malaria.

Phagocytosis of malaria pigment (hemozoin) induces increased activity of matrix metalloproteinase (MMP)-9, an endopeptidase involved in cytokine regulation. In this study, we examined whether a common functional MMP-9 promoter polymorphism (rs3918242) affects Plasmodium falciparum infection in pregnancy. Eighteen percent of Ghanaian primiparae carried the minor T allele. It was associated with reduced odds of placental hemozoin and of placental as well as peripheral blood parasitemia. The results indicate that a common MMP-9 polymorphism protects against placental malaria indicating that this endopeptidase is involved in susceptibility to P. falciparum.

Reduced prevalence of placental malaria in primiparae with blood group O.

BACKGROUND: Blood group O protects African children against severe malaria and has reached high prevalence in malarious regions. However, its role in malaria in pregnancy is ambiguous. In 839 delivering Ghanaian women, associations of ABO blood groups with Plasmodium falciparum infection were examined. METHODS: Plasmodium falciparum infection was diagnosed in placental blood samples by microscopy and PCR assays. Present or past infection was defined as the detection of parasitaemia or haemozoin by microscopy, or a positive PCR result. Blood groups were inferred from genotyping rs8176719 (indicating the O allele) and rs8176746/rs8176747 (distinguishing the B allele from the A allele). RESULTS: The majority of women had blood group O (55.4%); present or past P. falciparum infection was seen in 62.3% of all women. Among multiparae, the blood groups had no influence on P. falciparum infection. In contrast, primiparae with blood group O had significantly less present or past infection than women with non-O blood groups (61.5 vs 76.2%, P = 0.007). In multivariate analysis, the odds of present or past placental P. falciparum infection were reduced by 45% in blood group O primiparae (aOR, 0.55 [95% CI, 0.33-0.94]). CONCLUSIONS: The present study shows a clear protective effect of blood group O against malaria in primiparae. This accords with findings in severe malaria and in vitro results. The data underline the relevance of host genetic protection among primiparae, i.e. the high-risk group for malaria in pregnancy, and contribute to the understanding of high O allele frequencies in Africa.

No association of the p53 codon 72 polymorphism with malaria in Ghanaian primiparae and Rwandan children.

The p53 protein is a key cell-signaling mediator integrating host responses to various types of stress. A common polymorphism of the encoding TP53 gene (codon 72, Pro > Arg, rs1042522) is associated with susceptibility to virus-related and other cancers. The p53 has also been shown to be central for successful Plasmodium liver stage infection. We examined whether the polymorphism is associated with P. falciparum infection in Ghanaian primiparae and Rwandan children. The allele frequency of TP53 codon 72 Arg was 0.30 among 314 Ghanaian primiparae and 0.31 among 545 Rwandan children, respectively, and it was not associated with infection prevalence or parasite density. This does not exclude p53 to be of pathophysiological relevance in malaria but argues against a major respective role of the TP53 codon 72 polymorphism.