Distinction of Plasmodium falciparum recrudescence and re-infection by MSP2 genotyping: A caution about unstandardized classification criteria

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum </it>genotyping with molecular polymorphic markers is widely employed to distinguish recrudescence from re-infection in antimalarial drug efficacy monitoring programmes. However, limitations occur on agarose gel DNA measurements used to resolve the polymorphisms. Without empirical data, the current distinction of pre- and post-treatment bands, as persistent or new infection, is subjective and often varying by author. This study measures empirical tolerance limits for classifying different-sized bands as same or different alleles during MSP2 genotyping.</p> <p>Methods</p> <p><it>P. falciparum </it>field samples from 161 volunteers were genotyped by nested PCR using polymorphic MSP2 family-specific primers. Data were analysed to determine variability of band size measurements between identical MSP2 alleles randomized into different agarose lanes.</p> <p>Results</p> <p>The mean (95% CI) paired difference in band size between identical alleles was 9.8 bp (1.48 – 18.16 bp, p = 0.022) for 3D7/IC and 2.54 (-3.04 – 8.05 bp, p = 0.362) for FC27. Based on these findings, pre- and post-treatment samples with 3D7/IC alleles showing less than 18 bp difference corresponded to recrudescence, with 95% confidence, while greater difference indicated new infection. FC27 allele differences were much narrower. For both 3D7/IC and FC27 amplicon, allele detection sensitivity was significantly higher with 13 μl compared to 20 μl or 30 μl lane loading volumes.</p> <p>Conclusion</p> <p>During MSP genotyping, it is useful to standardize classifications against measurement of background variability on identical alleles, in order to obtain reliable findings. It is critical to use a fixed optimal lane loading volume for constant allele patency, to avoid the disappearance or false appearance of new infection.</p

PCR detection of Plasmodium falciparum in human urine and saliva samples

BACKGROUND: Current detection or screening for malaria infection necessitates drawing blood by fingerprick or venipuncture, which poses risks and limitations for repeated measurement. This study presents PCR detection of Plasmodium falciparum in human urine and saliva samples, and illustrates this potential application in genotyping malaria infections. METHODS: Urine and saliva were obtained from 47 thick film positive and 4 negative individuals one day after collection of blood slides and filter paper blood spots. P. falciparum DNA was extracted from blood, urine and saliva, in separate groups, using the Chelex method or Qiagen DNEasy(® )kit (urine and saliva only). Blood, urine and saliva extracts were subjected to PCR in separate batches. Amplicons from the various sample types were examined for MSP2 polymorphisms and restriction fragment patterns on DHFR amino acid codon 59. RESULTS AND DISCUSSION: Malaria infections exhibited primarily low-grade parasite densities, with a geometric mean of 775 asexual parasites/μl. Regularly matching polymorphic MSP2 genotypes were found between the corresponding urine, saliva and peripheral blood amplicons of each individual, with different inter-individual polymorphic genotypes. Amplicon yields were significantly dependent on DNA extraction method, parasite density and primer set (p < 0.001). A Qiagen(® )kit extraction had more than 2× higher amplicon yield than the Chelex method, for both urine and saliva. Amplicon yields were 1.6 fold higher from saliva than urine. For each unit increase in log parasite density, the probability of amplicon enhanced 1.8 fold. Highest amplicon yields were obtained from the primer set with the shortest PCR product. CONCLUSION: P. falciparum infection is detectable by PCR on human urine and saliva samples. Subject to further refinement of extraction technique and amplicon yields, large-scale malaria parasite screening and epidemiological surveys could be possible without the need to collect blood and use of needles or sharps

Escalating Plasmodium falciparum antifolate drug resistance mutations in Macha, rural Zambia

<p>Abstract</p> <p>Background</p> <p>In Zambia the first-line treatment for uncomplicated malaria is artemisinin combination therapy (ACT), with artemether-lumefantrine currently being used. However, the antifolate regimen, sulphadoxine-pyrimethamine (SP), remains the treatment of choice in children weighing less than 5 kg and also in expectant mothers. SP is also the choice drug for intermittent preventive therapy in pregnancy and serves as stand-by treatment during ACT stock outs. The current study assessed the status of <it>Plasmodium falciparum </it>point mutations associated with antifolate drug resistance in the area around Macha.</p> <p>Methods</p> <p>A representative sample of 2,780 residents from the vicinity of Macha was screened for malaria by microscopy. At the same time, blood was collected onto filter paper and dried for subsequent <it>P. falciparum </it>DNA analysis. From 188 (6.8%) individuals that were thick film-positive, a simple random sub-set of 95 <it>P. falciparum </it>infections were genotyped for DHFR and DHPS antifolate resistance mutations, using nested PCR and allele-specific restriction enzyme digestion.</p> <p>Results</p> <p><it>Plasmodium falciparum </it>field samples exhibited a high prevalence of antifolate resistance mutations, including the DHFR triple (Asn-108 + Arg-59 + Ile-51) mutant (41.3%) and DHPS double (Gly-437 + Glu-540) mutant (16%). The quintuple (DHFR triple + DHPS double) mutant was found in 4 (6.5%) of the samples. Levels of mutated parasites showed a dramatic escalation, relative to previous surveys since 1988. However, neither of the Val-16 and Thr-108 mutations, which jointly confer resistance to cycloguanil, was detectable among the human infections. The Leu-164 mutation, associated with high grade resistance to both pyrimethamine and cycloguanil, as a multiple mutant with Asn-108, Arg-59 and (or) Ile-51, was also absent.</p> <p>Conclusion</p> <p>This study points to escalating levels of <it>P. falciparum </it>antifolate resistance in the vicinity of Macha. Continued monitoring is recommended to ensure timely policy revisions before widespread resistance exacts a serious public health toll.</p

A Simple Chelex Protocol for DNA Extraction From Anopheles Spp

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens. We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km(2) vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 μl of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 μl was retained while the other 10 μl was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol(9,10). The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction(9). Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality, a PCR for identification of Anopheles gambiae sibling species(10) and a nested PCR for typing of Plasmodium falciparum infection. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min\u27 processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain

Skin, Thermal and Umbilical Cord Care Practices for Neonates in Southern, Rural Zambia: A Qualitative Study

Background: In Choma District, southern Zambia, the neonatal mortality rate is approximately 40 per 1000 live births and, although the rate is decreasing, many deliveries take place outside of formal facilities. Understanding local practices during the postnatal period is essential for optimizing newborn care programs. Methods: We conducted 36 in-depth interviews, five focus groups and eight observational sessions with recently-delivered women, traditional birth attendants, and clinic and hospital staff from three sites, focusing on skin, thermal and cord care practices for newborns in the home. Results: Newborns were generally kept warm by application of hats and layers of clothing. While thermal protection is provided for preterm and small newborns, the practice of nighttime bathing with cold water was common. The vernix was considered important for the preterm newborn but dangerous for HIV-exposed infants. Mothers applied various substances to the skin and umbilical cord, with special practices for preterm infants. Applied substances included petroleum jelly, commercial baby lotion, cooking oil and breastmilk. The most common substances applied to the umbilical cord were powders made of roots, burnt gourds or ash. To ward off malevolent spirits, similar powders were reportedly placed directly into dermal incisions, especially in ill children. Conclusions: Thermal care for newborns is commonly practiced but co-exists with harmful practices. Locally appropriate behavior change interventions should aim to promote chlorhexidine in place of commonly-reported application of harmful substances to the skin and umbilical cord, reduce bathing of newborns at night, and address the immediate bathing of HIV-infected newborns

Changes in the Burden of Malaria Following Scale up of Malaria Control Interventions in Mutasa District, Zimbabwe

Background: To better understand trends in the burden of malaria and their temporal relationship to control activities, a survey was conducted to assess reported cases of malaria and malaria control activities in Mutasa District, Zimbabwe. Methods: Data on reported malaria cases were abstracted from available records at all three district hospitals, three rural hospitals and 25 rural health clinics in Mutasa District from 2003 to 2011. Results: Malaria control interventions were scaled up through the support of the Roll Back Malaria Partnership, the Global Fund to Fight AIDS, Tuberculosis and Malaria, and The President\u27s Malaria Initiative. The recommended first-line treatment regimen changed from chloroquine or a combination of chloroquine plus sulphadoxine/ pyrimethamine to artemisinin-based combination therapy, the latter adopted by 70%, 95% and 100% of health clinics by 2008, 2009 and 2010, respectively. Diagnostic capacity improved, with rapid diagnostic tests (RDTs) available in all health clinics by 2008. Vector control consisted of indoor residual spraying and distribution of longlasting insecticidal nets. The number of reported malaria cases initially increased from levels in 2003 to a peak in 2008 but then declined 39% from 2008 to 2010. The proportion of suspected cases of malaria in older children and adults remained high, ranging from 75% to 80%. From 2008 to 2010, the number of RDT positive cases of malaria decreased 35% but the decrease was greater for children younger than five years of age (60%) compared to older children and adults (26%). Conclusions: The burden of malaria in Mutasa District decreased following the scale up of malaria control interventions. However, the persistent high number of cases in older children and adults highlights the need for strategies to identify locally effective control measures that target all age groups

Validation of oral fluid samples to monitor serological changes to Plasmodium falciparum: An observational study in southern Zambia

<p>Abstract</p> <p>Background</p> <p>In formerly endemic areas where malaria transmission has declined, levels of population immunity to <it>Plasmodium falciparum </it>provide information on continued malaria transmission and potentially susceptible populations. Traditional techniques for measuring serological responses to <it>P. falciparum </it>antigens use plasma or dried blood spots (DBS). These invasive procedures pose a biohazard and may be unacceptable to communities if performed frequently. The use of oral fluid (OF) samples to detect antibodies to <it>P. falciparum </it>antigens may be a more acceptable strategy to monitor changes in population immunity.</p> <p>Methods</p> <p>An enzyme immunoassay was optimized to detect antibodies to whole, asexual stage <it>P. falciparum </it>antigens. Optical density (OD) values from paired DBS and OF samples collected as part of a community-based survey of malaria parasitaemia were compared.</p> <p>Results</p> <p>Oral fluid and dried blood spot samples were collected from 53 participants in Southern Province, Zambia. Their ages ranged from 1 to 80 years and 45% were female. A statistically significant correlation (r = 0.79; P < 0.01) was observed between OD values from OF and DBS samples. The OF assay identified all DBS-confirmed positive and negative samples, resulting in 100% sensitivity and specificity.</p> <p>Conclusions</p> <p>Oral fluid is a valid alternative specimen for monitoring changes in antibodies to <it>P. falciparum </it>antigens. As OF collection is often more acceptable to communities, poses less of a biohazard than blood samples and can be performed by community volunteers, serological surveys using OF samples provide a strategy for monitoring population immunity in regions of declining malaria transmission.</p

Factors Associated With Sustained Use of Long-Lasting Insecticide-Treated Nets Following a Reduction in Malaria Transmission in Southern Zambia

Understanding factors influencing sustained use of long-lasting insecticide-treated nets (LLIN) in areas of declining malaria transmission is critical to sustaining control and may facilitate elimination. From 2008 to 2013, 655 households in Choma District, Zambia, were randomly selected and residents were administered a questionnaire and malaria rapid diagnostic test. Mosquitoes were collected concurrently by light trap. In a multilevel model, children and adolescents of 5-17 years of age were 55% less likely to sleep under LLIN than adults (odds ratio [OR] = 0.45; 95% confidence interval [CI] = 0.35, 0.58). LLIN use was 80% higher during the rainy season (OR = 1.8; CI = 1.5, 2.2) and residents of households with three or more nets were over twice as likely to use a LLIN (OR = 2.1; CI = 1.4, 3.1). For every increase in 0.5 km from the nearest health center, the odds of LLIN use decreased 9% (OR = 0.9; CI = 0.88, 0.98). In a second multilevel model, the odds of LLIN use were more than twice high if more than five mosquitoes (anopheline and culicine) were captured in the house compared with households with no mosquitoes captured (OR = 2.1; CI = 1.1, 3.9). LLIN use can be sustained in low-transmission settings with continued education and distributions, and may be partially driven by the presence of nuisance mosquitoes

Plasmodium Falciparum HRP2 Elisa for Analysis of Dried Blood Spot Samples in Rural Zambia

Background: Dried blood spots are commonly used for sample collection in clinical and non-clinical settings. This method is simple, and biomolecules in the samples remain stable for months at room temperature. In the field, blood samples for the study and diagnosis of malaria are often collected on dried blood spot cards, so development of a biomarker extraction and analysis method is needed. Methods: A simple extraction procedure for the malarial biomarker Plasmodium falciparum histidine-rich protein 2 (HRP2) from dried blood spots was optimized to achieve maximum extraction efficiency. This method was used to assess the stability of HRP2 in dried blood spots. Furthermore, 328 patient samples made available from rural Zambia were analysed for HRP2 using the developed method. These samples were collected at the initial administration of artemisinin-based combination therapy and at several points following treatment. Results: An average extraction efficiency of 70% HRP2 with a low picomolar detection limit was achieved. In specific storage conditions HRP2 was found to be stable in dried blood spots for at least 6 months. Analysis of patient samples showed the method to have a sensitivity of 94% and a specificity of 89% when compared with microscopy, and trends in HRP2 clearance after treatment were observed. Conclusions: The dried blood spot ELISA for HRP2 was found to be sensitive, specific and accurate. The method was effectively used to assess biomarker clearance characteristics in patient samples, which prove it to be ideal for gaining further insight into the disease and epidemiological applications

Submillimeter Continuum Properties of Cold Dust in the Inner Disk and Outflows of M82

Deep submillimeter (submm) continuum imaging observations of the starburst galaxy M82 are presented at 350, 450, 750 and 850 micron wavelengths, that were undertaken with the Submillimetre Common-User Bolometer Array (SCUBA) on the James Clerk Maxwell Telescope in Hawaii. The presented maps include a co-addition of submm data mined from the SCUBA Data Archive. The co-added data produce the deepest submm continuum maps yet of M82, in which low-level 850 micron continuum has been detected out to 1.5kpc, at least 10% farther in radius than any previously published submm detections of this galaxy. The overall submm morphology and spatial spectral energy distribution of M82 have a general north-south asymmetry consistent with H-alpha and X-ray winds, supporting the association of the extended continuum with outflows of dust grains from the disk into the halo. The new data raise interesting points about the origin and structure of the submm emission in the inner disk of M82. In particular, SCUBA short wavelength evidence of submm continuum peaks that are asymmetrically distributed along the galactic disk suggests the inner-disk emission is re-radiation from dust concentrations along a bar (or perhaps a spiral) rather than edges of a dust torus, as is commonly assumed. Higher resolution submm interferometery data from the Smithsonian Submillimeter Array and later Atacama Large Millimeter Array should spatially resolve and further constrain the reported dust emission structures in M82.Comment: Accepted by the Astronomical Journal -- 28 pages and 14 figure