Performance Evaluation of Biozentech Malaria Scanner in Plasmodium knowlesi and P. falciparum as a New Diagnostic Tool

The computer vision diagnostic approach currently generates several malaria diagnostic tools. It enhances the accessible and straightforward diagnostics that necessary for clinics and health centers in malaria-endemic areas. A new computer malaria diagnostics tool called the malaria scanner was used to investigate living malaria parasites with easy sample preparation, fast and user-friendly. The cultured Plasmodium parasites were used to confirm the sensitivity of this technique then compared to fluorescence-activated cell sorting (FACS) analysis and light microscopic examination. The measured percentage of parasitemia by the malaria scanner revealed higher precision than microscopy and was similar to FACS. The coefficients of variation of this technique were 1.2–6.7% for Plasmodium knowlesi and 0.3–4.8% for P. falciparum. It allowed determining parasitemia levels of 0.1% or higher, with coefficient of variation smaller than 10%. In terms of the precision range of parasitemia, both high and low ranges showed similar precision results. Pearson’s correlation test was used to evaluate the correlation data coming from all methods. A strong correlation of measured parasitemia (r²=0.99, P<0.05) was observed between each method. The parasitemia analysis using this new diagnostic tool needs technical improvement, particularly in the differentiation of malaria species.     

Performance of Microscopy for the Diagnosis of Malaria and Human African Trypanosomiasis by Diagnostic Laboratories in the Democratic Republic of the Congo: Results of a Nation-Wide External Quality Assessment

The present External Quality Assessment (EQA) assessed microscopy of blood parasites among diagnostic laboratories in the Democratic Republic of the Congo. The EQA addressed 445 participants in 10/11 provinces (October 2013–April 2014). Participants were sent a panel of five slides and asked to return a routinely stained slide which was assessed for quality of preparation and staining. Response rate was 89.9% (400/445). For slide 1 (no parasites), 30.6% participants reported malaria, mostly Plasmodium falciparum. Only 11.0% participants reported slide 2 (Plasmodium malariae) correctly, 71.0% reported “malaria” or “Plasmodium falciparum” (considered acceptable). Slide 3 contained Plasmodium falciparum (109/μl) and Trypanosoma brucei brucei trypomastigotes: they were each reported by 32.5% and 16.5% participants respectively, 6.0% reported both. Slide 4 (Trypanosoma) was recognised by 44.9% participants. Slide 5 (Plasmodium ovale) was correctly reported by 6.2% participants, another 68.8% replied “malaria” or “Plasmodium falciparum” (considered acceptable). Only 13.6% of routine slides returned were correctly prepared and stained. The proportion of correct/acceptable scores for at least 4/5 slides was higher among EQA-experienced participants compared to first time participants (40.9% versus 22.4%, p = 0.001) and higher among those being trained < 2 years ago compared to those who were not (42.9% versus 26.3%, p = 0.01). Among diagnostic laboratories in Democratic Republic of the Congo, performance of blood parasite microscopy including non-falciparum species and Trypanosoma was poor. Recent training and previous EQA participation were associated with a better performance.     

Circulating IL-6, IL-10, and TNF-alpha and IL-10/IL-6 and IL-10/TNF-alpha ratio profiles of polyparasitized individuals in rural and urban areas of gabon

Malaria, blood-borne filarial worms and intestinal parasites are all endemic in Gabon. This geographical co-distribution leads to polyparasitism and, consequently, the possibility of immune-mediated interactions among different parasite species. Intestinal protozoa and helminths could modulate antimalarial immunity, for example, thereby potentially increasing or reducing susceptibility to malaria. The aim of the study was to compare the cytokine levels and cytokine ratios according to parasitic profiles of the population to determine the potential role of co-endemic parasites in the malaria susceptibility of populations. Blood and stool samples were collected during cross-sectional surveys in five provinces of Gabon. Parasitological diagnosis was performed to detect plasmodial parasites, Loa loa, Mansonella perstans, intestinal helminths (STHs) and protozoan parasites. Nested PCR was used to detect submicroscopic plasmodial infection in individuals with negative blood smears. A cytometric bead array was used to quantify interleukin (IL)-6, IL-10 and tumour necrosis factor (TNF)-α in the plasma of subjects with different parasitological profiles. Median IL-6 and IL-10 levels and the median IL-10/TNF-α ratio were all significantly higher among individuals with Plasmodium (P.) falciparum infection than among other participants (p<0.0001). The median TNF-α level and IL-10/IL-6 ratio were higher in subjects with STHs (p = 0.09) and P. falciparum-intestinal protozoa co-infection (p = 0.04), respectively. IL-6 (r = -0.37; P<0.01) and IL-10 (r = -0.37; P<0.01) levels and the IL-10/TNF-α ratio (r = -0.36; P<0.01) correlated negatively with age. Among children under five years old, the IL-10/TNF-α and IL-10/IL-6 ratios were higher in those with intestinal protozoan infections than in uninfected children. The IL-10/TNF-α ratio was also higher in children aged 5–15 years and in adults harbouring blood-borne filariae than in their control counterparts, whereas the IL-10/IL-6 ratio was lower in those aged 5–15 years with filariae and intestinal parasites but higher in adults with intestinal parasitic infections. Asymptomatic malaria is associated with a strong polarization towards a regulatory immune response, presenting high circulating levels of IL-10. P. falciparum/intestinal protozoa co-infections were associated with an enhanced IL-10 response. Immunity against malaria could differ according to age and carriage of other parasites. Helminths and intestinal protozoa can play a role in the high susceptibility to malaria currently observed in some areas of Gabon, but further investigations are necessary.     

Multiplex qPCR for Detection and Absolute Quantification of Malaria

We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R² values greater than 0.99 and the STDEV of each replicate was <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria.     

Malaria Morbidity in High and Seasonal Malaria Transmission Area of Burkina Faso

Background

Malariometric parameters are often primary endpoints of efficacy trials of malaria vaccine candidates. This study aims to describe the epidemiology of malaria prior to the conduct of a series of drug and vaccine trials in a rural area of Burkina Faso.

Methods

Malaria incidence was prospectively evaluated over one year follow-up among two cohorts of children aged 0–5 years living in the Saponé health district. The parents of 1089 children comprising a passive case detection cohort were encouraged to seek care from the local health clinic at any time their child felt sick. Among this cohort, 555 children were randomly selected for inclusion in an active surveillance sub-cohort evaluated for clinical malaria during twice weekly home visits. Malaria prevalence was evaluated by cross-sectional survey during the low and high transmission seasons.

Results

Number of episodes per child ranged from 0 to 6 per year. Cumulative incidence was 67.4% in the passive and 86.2% in the active cohort and was highest among children 0–1 years. Clinical malaria prevalence was 9.8% in the low and 13.0% in the high season (p>0.05). Median days to first malaria episode ranged from 187 (95% CI 180–193) among children 0–1 years to 228 (95% CI 212, 242) among children 4–5 years. The alternative parasite thresholds for the malaria case definition that achieved optimal sensitivity and specificity (70–80%) were 3150 parasites/µl in the high and 1350 parasites/µl in the low season.

Conclusion

Clinical malaria burden was highest among the youngest age group children, who may represent the most appropriate target population for malaria vaccine candidate development. The pyrogenic threshold of parasitaemia varied markedly by season, suggesting a value for alternative parasitaemia levels in the malaria case defintion. Regional epidemiology of malaria described, Sapone area field centers are positioned for future conduct of malaria vaccine trials.     

IL-4 Haplotype -590T, -34T and Intron-3 VNTR R2 Is Associated with Reduced Malaria Risk among Ancestral Indian Tribal Populations

Background

Interleukin 4 (IL-4) is an anti-inflammatory cytokine, which regulates balance between T_H1 and T_H2 immune response, immunoglobulin class switching and humoral immunity. Polymorphisms in this gene have been reported to affect the risk of infectious and autoimmune diseases.

Methods

We have analyzed three regulatory IL-4 polymorphisms; -590C>T, -34C>T and 70 bp intron-3 VNTR, in 4216 individuals; including: (1) 430 ethnically matched case-control groups (173 severe malaria, 101 mild malaria and 156 asymptomatic); (2) 3452 individuals from 76 linguistically and geographically distinct endogamous populations of India, and (3) 334 individuals with different ancestry from outside India (84 Brazilian, 104 Syrian, and 146 Vietnamese).

Results

The -590T, -34T and intron-3 VNTR R2 alleles were found to be associated with reduced malaria risk (P<0.001 for -590C>T and -34C>T, and P = 0.003 for VNTR). These three alleles were in strong LD (r²>0.75) and the TTR2 (-590T, -34T and intron-3 VNTR R2) haplotype appeared to be a susceptibility factor for malaria (P = 0.009, OR = 0.552, 95% CI = 0.356 –0.854). Allele and genotype frequencies differ significantly between caste, nomadic, tribe and ancestral tribal populations (ATP). The distribution of protective haplotype TTR2 was found to be significant (χ² ₃ = 182.95, p-value <0.001), which is highest in ATP (40.5%); intermediate in tribes (33%); and lowest in caste (17.8%) and nomadic (21.6%).

Conclusions

Our study suggests that the IL-4 polymorphisms regulate host susceptibility to malaria and disease progression. TTR2 haplotype, which gives protection against malaria, is high among ATPs. Since they inhabited in isolation and mainly practice hunter-gatherer lifestyles and exposed to various parasites, IL-4 TTR2 haplotype might be under positive selection.     

CXCL10 Gene Promoter Polymorphism -1447A>G Correlates with Plasma CXCL10 Levels and is Associated with Male Susceptibility to Cerebral Malaria

The risk factors for cerebral malaria (CM) and the wide variation in clinical manifestations of malaria are poorly understood. Recent studies indicate that interferon gamma inducible chemokine, CXCL10, is a strong predictor of both human and experimental cerebral malaria. Increased plasma and cerebrospinal fluid levels of CXCL10 were tightly associated with fatal CM in Indian and Ghanaian patients. In the present study, we hypothesized that in a subset of malaria patients, CM susceptibility is associated with variation in CXCL10 expression. We determined whether polymorphisms in the CXCL10 gene promoter region played a role in the clinical status of malaria patients and addressed the genetic basis of CXCL10 expression during malaria infection. Following extensive bioinformatics analyses, two reported single nucleotide polymorphisms in the CXCL10 promoter (−135G>A [rs56061981] and −1447A>G [rs4508917]) were identified among 66 CM and 69 non-CM Indian patients using PCR-restriction fragment length polymorphism assay. Individuals with the −1447(A/G) genotype were susceptible to CM (adjusted odds ratio [AOR] = 2.60, 95% CI = 1.51–5.85, p = 0.021). In addition, individuals with the −1447(A/G) genotype had significantly higher plasma CXCL10 levels than individuals with the −1447(A/A) genotype. Stratifying patients according to gender, the observed association of CM with over expression of CXCL10 were more pronounced in males than in female patients (AOR = 5.47, 95% CI = 1.34–22.29, p = 0.018). Furthermore, −135G>A polymorphism conferred a decreased risk of CM among males (AOR = 0.19, 95% CI = 0.05–0.78, p = 0.021). Polymorphisms in the CXCL10 gene promoter sequence were associated with increased CXCL10 production, which is linked to severity of CM. These results suggest that the −1447A>G polymorphism in CXCL10 gene promoter could be partly responsible for the reported variation underlying severity of CM outcomes particularly in males.     

MR Scanner Systems Should Be Adequately Characterized in Diffusion-MRI of the Breast

Breast imaging represents a relatively recent and promising field of application of quantitative diffusion-MRI techniques. In view of the importance of guaranteeing and assessing its reliability in clinical as well as research settings, the aim of this study was to specifically characterize how the main MR scanner system-related factors affect quantitative measurements in diffusion-MRI of the breast. In particular, phantom acquisitions were performed on three 1.5 T MR scanner systems by different manufacturers, all equipped with a dedicated multi-channel breast coil as well as acquisition sequences for diffusion-MRI of the breast. We assessed the accuracy, inter-scan and inter-scanner reproducibility of the mean apparent diffusion coefficient measured along the main orthogonal directions (<ADC>) as well as of diffusion-tensor imaging (DTI)-derived mean diffusivity (MD) measurements. Additionally, we estimated spatial non-uniformity of <ADC> (NU_<ADC>) and MD (NU_MD) maps. We showed that the signal-to-noise ratio as well as overall calibration of high strength diffusion gradients system in typical acquisition sequences for diffusion-MRI of the breast varied across MR scanner systems, introducing systematic bias in the measurements of diffusion indices. While <ADC> and MD values were not appreciably different from each other, they substantially varied across MR scanner systems. The mean of the accuracies of measured <ADC> and MD was in the range [−2.3%,11.9%], and the mean of the coefficients of variation for <ADC> and MD measurements across MR scanner systems was 6.8%. The coefficient of variation for repeated measurements of both <ADC> and MD was < 1%, while NU_<ADC> and NU_MD values were <4%. Our results highlight that MR scanner system-related factors can substantially affect quantitative diffusion-MRI of the breast. Therefore, a specific quality control program for assessing and monitoring the performance of MR scanner systems for diffusion-MRI of the breast is highly recommended at every site, especially in multicenter and longitudinal studies.     

External Quality Assessment of Reading and Interpretation of Malaria Rapid Diagnostic Tests among 1849 End-Users in the Democratic Republic of the Congo through Short Message Service (SMS)

Background

Although malaria rapid diagnostic tests (RDT) are simple to perform, they remain subject to errors, mainly related to the post-analytical phase. We organized the first large scale SMS based external quality assessment (EQA) on correct reading and interpretation of photographs of a three-band malaria RDT among laboratory health workers in the Democratic Republic of the Congo (DR Congo).

Methods and Findings

High resolution EQA photographs of 10 RDT results together with a questionnaire were distributed to health facilities in 9 out of 11 provinces in DR Congo. Each laboratory health worker answered the EQA by Short Message Service (SMS). Filled-in questionnaires from each health facility were sent back to Kinshasa. A total of 1849 laboratory health workers in 1014 health facilities participated. Most frequent errors in RDT reading were i) failure to recognize invalid (13.2–32.5% ) or negative test results (9.8–12.8%), (ii) overlooking faint test lines (4.1–31.2%) and (iii) incorrect identification of the malaria species (12.1–17.4%). No uniform strategy for diagnosis of malaria at the health facility was present. Stock outs of RDTs occurred frequently. Half of the health facilities had not received an RDT training. Only two thirds used the RDT recommended by the National Malaria Control Program. Performance of RDT reading was positively associated with training and the technical level of health facility. Facilities with RDT positivity rates >50% and located in Eastern DR Congo performed worse.

Conclusions

Our study confirmed that errors in reading and interpretation of malaria RDTs are widespread and highlighted the problem of stock outs of RDTs. Adequate training of end-users in the application of malaria RDTs associated with regular EQAs is recommended.     

Malaria Transmission in Bissau,Guinea-Bissau between 1995 and 2012: Malaria Resurgence Did Not Negatively Affect Mortality

Introduction

As Plasmodium falciparum prevalence decreases in many parts of Sub-Saharan Africa, so does immunity resulting in larger at risk populations and increased risk of malaria resurgence. In Bissau, malaria prevalence decreased from ∼50% to 3% between 1995 and 2003. The epidemiological characteristics of P. falciparum malaria within Bandim health and demographic surveillance site (population ∼100000) between 1995 and 2012 are described.

Methods and Findings

The population was determined by census. 3603 children aged <15 years that were enrolled in clinical trials at the Bandim health centre (1995–2012) were considered incident cases. The mean annual malaria incidence per thousand children in 1995–1997, 1999–2003, 2007, 2011, 2012 were as follows; age <5 years 22→29→4→9→3, age 5–9 years 15→28→4→33→12, age 10–14 years 9→15→1→45→19. There were 4 campaigns (2003–2010) to increase use of insecticide treated bed nets (ITN) amongst children <5 years. An efficacious high-dose chloroquine treatment regime was routinely used until artemisinin based combination therapy (ACT) was introduced in 2008. Long lasting insecticide treated bed nets (LLIN) were distributed in 2011. By 2012 there was 1 net per 2 people and 97% usage. All-cause mortality decreased from post-war peaks in 1999 until 2012 in all age groups and was not negatively affected by malaria resurgence.

Conclusion

The cause of decreasing malaria incidence (1995–2007) was probably multifactorial and coincident with the use of an efficacious high-dose chloroquine treatment regime. Decreasing malaria prevalence created a susceptible group of older children in which malaria resurged, highlighting the need to include all age groups in malaria interventions. ACT did not hinder malaria resurgence. Mass distribution of LLINs probably curtailed malaria epidemics. All-cause mortality was not negatively affected by malaria resurgence.     

Commutability of Possible External Quality Assessment Materials for Cardiac Troponin Measurement

Background

The measurement of cardiac troponin is crucial in the diagnosis of myocardial infarction. The performance of troponin measurement is most conveniently monitored by external quality assessment (EQA) programs. The commutability of EQA samples is often unknown and the effectiveness of EQA programs is limited.

Methods

Commutability of possible EQA materials was evaluated. Commercial control materials used in an EQA program, human serum pools prepared from patient samples, purified analyte preparations, swine sera from model animals and a set of patient samples were measured for cTnI with 4 assays including Abbott Architect, Beckman Access, Ortho Vitros and Siemens Centaur. The measurement results were logarithm-transformed, and the transformed data for patient samples were pairwise analyzed with Deming regression and 95% prediction intervals were calculated for each pair of assays. The commutability of the materials was evaluated by comparing the logarithmic results of the materials with the limits of the intervals. Matrix-related biases were estimated for noncommutable materials. The impact of matrix-related bias on EQA was analyzed and a possible correction for the bias was proposed.

Results

Human serum pools were commutable for all assays; purified analyte preparations were commutable for 2 of the 6 assay pairs; commercial control materials and swine sera were all noncommutable; swine sera showed no reactivity to Vitros assay. The matrix-related biases for noncommutable materials ranged from −83% to 944%. Matrix-related biases of the EQA materials caused major abnormal between-assay variations in the EQA program and correction of the biases normalized the variations.

Conclusion

Commutability of materials has major impact on the effectiveness of EQA programs for cTnI measurement. Human serum pools prepared from patient samples are commutable and other materials are mostly noncommutable. EQA programs should include at least one human serum pool to allow proper interpretation of EQA results.     

A four-year surveillance program for detection of Plasmodium falciparum chloroquine resistance in Honduras

Countries could use the monitoring of drug resistance in malaria parasites as an effective early warning system to develop the timely response mechanisms that are required to avert the further spread of malaria. Drug resistance surveillance is essential in areas where no drug resistance has been reported, especially if neighbouring countries have previously reported resistance. Here, we present the results of a four-year surveillance program based on the sequencing of the pfcrt gene of Plasmodium falciparum populations from endemic areas of Honduras. All isolates were susceptible to chloroquine, as revealed by the pfcrt “CVMNK” genotype in codons 72-76.     

Abolishing Fees at Health Centers in the Context of Community Case Management of Malaria: What Effects on Treatment-Seeking Practices for Febrile Children in Rural Burkina Faso?

Introduction

Burkina Faso started nationwide community case management of malaria (CCMm) in 2010. In 2011, health center user fees for children under five were abolished in some districts.

Objective

To assess the effects of concurrent implementation of CCMm and user fees abolition on treatment-seeking practices for febrile children.

Methods

This is a natural experiment conducted in the districts of Kaya (CCMm plus user fees abolition) and Zorgho (CCMm only). Registry data from 2005 to 2014 on visits for malaria were collected from all eight rural health centers in the study area. Annual household surveys were administered during malaria transmission season in 2011 and 2012 in 1,035 randomly selected rural households. Interrupted time series models were fitted for registry data and Fine and Gray’s competing risks models for survey data.

Results

User fees abolition in Kaya significantly increased health center use by eligible children with malaria (incidence rate ratio for intercept change = 2.1, p <0.001). In 2011, in Kaya, likelihood of health center use for febrile children was three times higher and CHW use three times lower when caregivers knew services were free. Among the 421 children with fever in 2012, the delay before visiting a health center was significantly shorter in Kaya than in Zorgho (1.46 versus 1.79 days, p <0.05). Likelihood of visiting a health center on the first day of fever among households <2.5km or <5 km from a health center was two and three times higher in Kaya than in Zorgho, respectively (p <0.001).

Conclusions

User fees abolition reduced visit delay for febrile children living close to health centers. It also increased demand for and use of health center for children with malaria. Concurrently, demand for CHWs’ services diminished. User fees abolition and CCMm should be coordinated to maximize prompt access to treatment in rural areas.     

Cost Implications of Improving Malaria Diagnosis: Findings from North-Eastern Tanzania

Background

Over diagnosis of malaria contributes to improper treatment, wastage of drugs and resistance to the few available drugs. This paper attempts to estimate the rates of over diagnosis of malaria among children attending dispensaries in rural Tanzania and examines the potential cost implications of improving the quality of diagnosis.

Methodology/Principal Findings

The magnitude of over diagnosis of malaria was estimated by comparing the proportion of outpatient attendees of all ages clinically diagnosed as malaria to the proportion of attendees having a positive malaria rapid diagnostic test over a two month period. Pattern of causes of illness observed in a <2 year old cohort of children over one year was compared to the pattern of causes of illness in <5 year old children recorded in the routine health care system during the same period. Drug and diagnostic costs were modelled using local and international prices. Over diagnosis of malaria by the routine outpatient care system compared to RDT confirmed cases of malaria was highest among <5 year old children in the low transmission site (RR 17.9, 95% CI 5.8–55.3) followed by the ≥5 year age group in the lower transmission site (RR 14.0 95%CI 8.2–24.2). In the low transmission site the proportion of morbidity attributable to malaria was substantially lower in <2 year old cohort compared to children seen at routine care system. (0.08% vs 28.2%; p<0.001). A higher proportion of children were diagnosed with ARI in the <2 year old cohort compared to children seen at the routine care system ( 42% vs 26%; p<0.001). Using a RDT reduced overall drug and diagnostic costs by 10% in the high transmission site and by 15% in the low transmission site compared to total diagnostic and drug costs of treatment based on clinical judgment in routine health care system.

Implications

The introduction of RDTs is likely to lead to financial savings. However, improving diagnosis to one disease may lead to over diagnosis of another illness. Quality improvement is complex but introducing RDTs for the diagnosis of malaria is a good start.     

Optimized Pan-species and Speciation Duplex Real-time PCR Assays for Plasmodium Parasites Detection in Malaria Vectors

Background

An accurate method for detecting malaria parasites in the mosquito’s vector remains an essential component in the vector control. The Enzyme linked immunosorbent assay specific for circumsporozoite protein (ELISA-CSP) is the gold standard method for the detection of malaria parasites in the vector even if it presents some limitations. Here, we optimized multiplex real-time PCR assays to accurately detect minor populations in mixed infection with multiple Plasmodium species in the African malaria vectors Anopheles gambiae and Anopheles funestus.

Methods

Complementary TaqMan-based real-time PCR assays that detect Plasmodium species using specific primers and probes were first evaluated on artificial mixtures of different targets inserted in plasmid constructs. The assays were further validated in comparison with the ELISA-CSP on 200 field caught Anopheles gambiae and Anopheles funestus mosquitoes collected in two localities in southern Benin.

Results

The validation of the duplex real-time PCR assays on the plasmid mixtures demonstrated robust specificity and sensitivity for detecting distinct targets. Using a panel of mosquito specimen, the real-time PCR showed a relatively high sensitivity (88.6%) and specificity (98%), compared to ELISA-CSP as the referent standard. The agreement between both methods was “excellent” (κ = 0.8, P<0.05). The relative quantification of Plasmodium DNA between the two Anopheles species analyzed showed no significant difference (P = 0, 2). All infected mosquito samples contained Plasmodium falciparum DNA and mixed infections with P. malariae and/or P. ovale were observed in 18.6% and 13.6% of An. gambiae and An. funestus respectively. Plasmodium vivax was found in none of the mosquito samples analyzed.

Conclusion

This study presents an optimized method for detecting the four Plasmodium species in the African malaria vectors. The study highlights substantial discordance with traditional ELISA-CSP pointing out the utility of employing an accurate molecular diagnostic tool for detecting malaria parasites in field mosquito populations.     

Genetic Variants in the Fat and Obesity Associated (FTO) Gene and Risk of Alzheimer's Disease

Background

Recent studies showed that polymorphisms in the Fat and Obesity-Associated (FTO) gene have robust effects on obesity, obesity-related traits and endophenotypes associated with Alzheimer''s disease (AD).

Methods

We used 1,877 Caucasian cases and controls from the NIA-LOAD study and 1,093 Caribbean Hispanics to further explore the association of FTO with AD. Using logistic regression, we assessed 42 SNPs in introns 1 and 2, the region previously reported to be associated with AD endophenotypes, which had been derived by genome-wide screenings. In addition, we performed gene expression analyses of neuropathologically confirmed AD cases and controls of two independent datasets (19 AD cases, 10 controls; 176 AD cases, 188 controls) using within- and between-group factors ANOVA of log₁₀ transformed rank invariant normalized expression data.

Results

In the NIALOAD study, one SNP was significantly associated with AD and three additional markers were close to significance (rs6499640, rs10852521, rs16945088, rs8044769, FDR p-value: 0.05<p<0.09). Two of the SNPs are in strong LD (D′>0.9) with the previously reported SNPs. In the Caribbean Hispanic dataset, we identified three SNPs (rs17219084, rs11075996, rs11075997, FDR p-value: 0.009<p<0.01) that were associated with AD. These results were confirmed by haplotype analyses and in a metaanalysis in which we included the ADNI dataset. FTO had a significantly lower expresssion in AD cases compared to controls in two independent datasets derived from human cortex and amygdala tissue, respectively (p = 2.18×10−5 and p<0.0001).

Conclusions

Our data support the notion that genetic variation in Introns 1 and 2 of the FTO gene may contribute to AD risk.     

A comparison and optimization of methods and factors affecting the transformation of Escherichia coli

DNA manipulation routinely requires competent bacteria that can be made using one of numerous methods. To determine the best methods, we compared four commonly used chemical methods (DMSO, MgCl₂–CaCl₂, CaCl₂ and Hanahan''s methods) on frequently used Escherichia coli (E. coli) strains: DH5α, XL-1 Blue, SCS110, JM109, TOP10 and BL21-(DE3)-PLysS. Hanahan''s method was found to be most effective for DH5α, XL-1 Blue and JM109 strains (P<0.05), whilst the CaCl₂ method was best for SCS110, TOP10 and BL21 strains (P<0.05). The use of SOB (super optimal broth) over LB [Luria–Bertani (broth)] growth media was found to enhance the competency of XL-1 Blue (P<0.05), dampened JM109′s competency (P<0.05), and had no effect on the other strains (P>0.05). We found no significant differences between using 45 or 90 s heat shock across all the six strains (P>0.05). Through further optimization by means of concentrating the aliquots, we were able to get further increases in transformation efficiencies. Based on the optimized parameters and methods, these common laboratory E. coli strains attained high levels of TrE (transformation efficiency), thus facilitating the production of highly efficient and cost-effective competent bacteria.     

Submicroscopic malaria parasite carriage: how reproducible are polymerase chain reaction-based methods?

The polymerase chain reaction (PCR)-based methods for the diagnosis of malaria infection are expected to accurately identify submicroscopic parasite carriers. Although a significant number of PCR protocols have been described, few studies have addressed the performance of PCR amplification in cases of field samples with submicroscopic malaria infection. Here, the reproducibility of two well-established PCR protocols (nested-PCR and real-time PCR for the Plasmodium 18 small subunit rRNA gene) were evaluated in a panel of 34 blood field samples from individuals that are potential reservoirs of malaria infection, but were negative for malaria by optical microscopy. Regardless of the PCR protocol, a large variation between the PCR replicates was observed, leading to alternating positive and negative results in 38% (13 out of 34) of the samples. These findings were quite different from those obtained from the microscopy-positive patients or the unexposed individuals; the diagnosis of these individuals could be confirmed based on the high reproducibility and specificity of the PCR-based protocols. The limitation of PCR amplification was restricted to the field samples with very low levels of parasitaemia because titrations of the DNA templates were able to detect < 3 parasites/µL in the blood. In conclusion, conventional PCR protocols require careful interpretation in cases of submicroscopic malaria infection, as inconsistent and false-negative results can occur.