Plasmodium knowlesi Infection in Humans, Cambodia, 2007–2010

Two cases of Plasmodium knowlesi infection in humans were identified in Cambodia by 3 molecular detection assays and sequencing. This finding confirms the widespread distribution of P. knowlesi malaria in humans in Southeast Asia. Further wide-scale studies are required to assess the public health relevance of this zoonotic malaria parasite

Rapid diagnostic tests for molecular surveillance of Plasmodium falciparum malaria -assessment of DNA extraction methods and field applicability

Background: The need for new malaria surveillance tools and strategies is critical, given improved global malaria control and regional elimination efforts. High quality Plasmodium falciparum DNA can reliably be extracted from malaria rapid diagnostic tests (RDTs). Together with highly sensitive molecular assays, wide scale collection of used RDTs may serve as a modern tool for improved malaria case detection and drug resistance surveillance. However, comparative studies of DNA extraction efficiency from RDTs and the field applicability are lacking. The aim of this study was to compare and evaluate different methods of DNA extraction from RDTs and to test the field applicability for the purpose of molecular epidemiological investigations. Methods: DNA was extracted from two RDT devices (Paracheck-PfW and SD Bioline Malaria Pf/Pan (R)), seeded in vitro with 10-fold dilutions of cultured 3D7 P. falciparum parasites diluted in malaria negative whole blood. The level of P. falciparum detection was determined for each extraction method and RDT device with multiple nested-PCR and real-time PCR assays. The field applicability was tested on 855 paired RDT (Paracheck-Pf) and filter paper (Whatman (R) 3MM) blood samples (734 RDT negative and 121 RDT positive samples) collected from febrile patients in Zanzibar 2010. RDT positive samples were genotyped at four key single nucleotide polymorphisms (SNPs) in pfmdr1 and pfcrt as well as for pfmdr1 copy number, all associated with anti-malarial drug resistance. Results: The P. falciparum DNA detection limit varied with RDT device and extraction method. Chelex-100 extraction performed best for all extraction matrixes. There was no statistically significant difference in PCR detection rates in DNA extracted from RDTs and filter paper field samples. Similarly there were no significant differences in the PCR success rates and genotyping outcomes for the respective SNPs in the 121 RDT positive samples. Conclusions: The results support RDTs as a valuable source of parasite DNA and provide evidence for RDT-DNA extraction for improved malaria case detection, molecular drug resistance surveillance, and RDT quality control.ACT Consortium through Bill and Melinda Gates Foundation; Swedish International Development Agency (SIDA) [SWE 2009-193]; Swedish Civil Contingencies Agency (MSB) [2010-7991]; Swedish Medical Research Council (VR) [2009-3785]; Goljes Foundationinfo:eu-repo/semantics/publishedVersio

Management of uncomplicated malaria in febrile under five-year-old children by community health workers in Madagascar: reliability of malaria rapid diagnostic tests

<p>Abstract</p> <p>Background</p> <p>Early diagnosis, as well as prompt and effective treatment of uncomplicated malaria, are essential components of the anti-malaria strategy in Madagascar to prevent severe malaria, reduce mortality and limit malaria transmission. The purpose of this study was to assess the performance of the malaria rapid diagnostic tests (RDTs) used by community health workers (CHWs) by comparing RDT results with two reference methods (microscopy and Polymerase Chain Reaction, PCR).</p> <p>Methods</p> <p>Eight CHWs in two districts, each with a different level of endemic malaria transmission, were trained to use RDTs in the management of febrile children under five years of age. RDTs were performed by CHWs in all febrile children who consulted for fever. In parallel, retrospective parasitological diagnoses were made by microscopy and PCR. The results of these different diagnostic methods were analysed to evaluate the diagnostic performance of the RDTs administered by the CHWs. The stability of the RDTs stored by CHWs was also evaluated.</p> <p>Results</p> <p>Among 190 febrile children with suspected malaria who visited CHWs between February 2009 and February 2010, 89.5% were found to be positive for malaria parasites by PCR, 51.6% were positive by microscopy and 55.8% were positive by RDT. The performance accuracy of the RDTs used by CHWs in terms of sensitivity, specificity, positive and negative predictive values was greater than 85%. Concordance between microscopy and RDT, estimated by the Kappa value was 0.83 (95% CI: 0.75-0.91). RDTs stored by CHWs for 24 months were capable of detecting <it>Plasmodium falciparum </it>in blood at a level of 200 parasites/μl.</p> <p>Conclusion</p> <p>Introduction of easy-to-use diagnostic tools, such as RDTs, at the community level appears to be an effective strategy for improving febrile patient management and for reducing excessive use of anti-malarial drugs.</p

Surveillance for Malaria Elimination in Swaziland: A National Cross-Sectional Study Using Pooled PCR and Serology

BACKGROUND: To guide malaria elimination efforts in Swaziland and other countries, accurate assessments of transmission are critical. Pooled-PCR has potential to efficiently improve sensitivity to detect infections; serology may clarify temporal and spatial trends in exposure. METHODOLOGY/PRINCIPAL FINDINGS: Using a stratified two-stage cluster, cross-sectional design, subjects were recruited from the malaria endemic region of Swaziland. Blood was collected for rapid diagnostic testing (RDT), pooled PCR, and ELISA detecting antibodies to Plasmodium falciparum surface antigens. Of 4330 participants tested, three were RDT-positive yet false positives by PCR. Pooled PCR led to the identification of one P. falciparum and one P. malariae infection among RDT-negative participants. The P. falciparum-infected participant reported recent travel to Mozambique. Compared to performing individual testing on thousands of samples, PCR pooling reduced labor and consumable costs by 95.5%. Seropositivity was associated with age ≥20 years (11·7% vs 1·9%, P<0.001), recent travel to Mozambique (OR 4.4 [95% CI 1.0-19.0]) and residence in southeast Swaziland (RR 3.78, P<0.001). CONCLUSIONS: The prevalence of malaria infection and recent exposure in Swaziland are extremely low, suggesting elimination is feasible. Future efforts should address imported malaria and target remaining foci of transmission. Pooled PCR and ELISA are valuable surveillance tools for guiding elimination efforts

Sub-microscopic malaria cases and mixed malaria infection in a remote area of high malaria endemicity in Rattanakiri province, Cambodia: implication for malaria elimination

BACKGROUND: Malaria microscopy and rapid diagnostic tests are insensitive for very low-density parasitaemia. This insensitivity may lead to missed asymptomatic sub-microscopic parasitaemia, a potential reservoir for infection. Similarly, mixed infections and interactions between Plasmodium species may be missed. The objectives were first to develop a rapid and sensitive PCR-based diagnostic method to detect low parasitaemia and mixed infections, and then to investigate the epidemiological importance of sub-microscopic and mixed infections in Rattanakiri Province, Cambodia. METHODS: A new malaria diagnostic method, using restriction fragment length polymorphism analysis of the cytochrome b genes of the four human Plasmodium species and denaturing high performance liquid chromatography, has been developed. The results of this RFLP-dHPLC method have been compared to 1) traditional nested PCR amplification of the 18S rRNA gene, 2) sequencing of the amplified fragments of the cytochrome b gene and 3) microscopy. Blood spots on filter paper and Giemsa-stained blood thick smears collected in 2001 from 1,356 inhabitants of eight villages of Rattanakiri Province have been analysed by the RFLP-dHPLC method and microscopy to assess the prevalence of sub-microscopic and mixed infections. RESULTS: The sensitivity and specificity of the new RFLP-dHPLC was similar to that of the other molecular methods. The RFLP-dHPLC method was more sensitive and specific than microscopy, particularly for detecting low-level parasitaemia and mixed infections. In Rattanakiri Province, the prevalences of Plasmodium falciparum and Plasmodium vivax were approximately two-fold and three-fold higher, respectively, by RFLP-dHPLC (59% and 15%, respectively) than by microscopy (28% and 5%, respectively). In addition, Plasmodium ovale and Plasmodium malariae were never detected by microscopy, while they were detected by RFLP-dHPLC, in 11.2% and 1.3% of the blood samples, respectively. Moreover, the proportion of mixed infections detected by RFLP-dHPLC was higher (23%) than with microscopy (8%). CONCLUSIONS: The rapid and sensitive molecular diagnosis method developed here could be considered for mass screening and ACT treatment of inhabitants of low-endemicity areas of Southeast Asia

HIV Viral Load Testing in Laos

The number of people living with HIV/AIDS in the People’s Democratic Republic of Laos (also known as Laos PDR) is estimated at 13,600 and the number of people in need of antiretroviral therapy at 8,000. Today, around 3,200 HIV infected individuals receive treatment in seven centres throughout the country. Until recently, antiretroviral treated patients were followed-up only on the basis of clinical and immunological criteria. In 2009 the Centre d'Infectiologie Christophe Mérieux in Laos PDR (CICML) signed a collaboration agreement with the national Centre of HIV/AIDS/STI (CHAS) for the implementation of HIV viral load testing (VLT) in the country, leading to the technological transfer of the ANRS generic assay (HIV Generic charge virale, Biocentric, Bandol, France). The introduction of HIV VLT has been accompanied through national HIV workshops every 6 months. From June 2009 to December 2011, HIV viral load has been measured in 1,782 antiretoviral-treated patients. Of these, 97% were on reverse-transcriptase inhibitor -based 1st line regimen. HIV viral load was undetectable (<250 copies/ml) for 1,491 out of 1,782 (84%) antiretroviral-treated patients. Four months after adherence strengthening, HIV viral load became undetectable for 179/247 patients (72.5%) while 68/247 patients (27.5%) remained viremic (median viral load: 4.1 Log10). This report demonstrates the feasibility of setting up an affordable HIV viral load generic test at a national level in Laos PDR. Interestingly, only 16% of antiretroviral-treated patients presented with detectable VL at their first viral load measurement. Importantly, almost two thirds of them have controlled their viral load after strengthening their adherence to treatment.La République démocratique populaire du Laos compterait 13 600 personnes atteintes du VIH/SIDA dont 8000 auraient besoin d’un traitement antirétroviral. Aujourd’hui, environ 3200 individus infectés par le VIH reçoivent un traitement dans sept centres répartis dans tout le pays. Jusqu’à récemment, le suivi des patients traités par antirétroviraux se faisait uniquement sur la base de critères cliniques et immunologiques. En 2009, le Centre d'Infectiologie Christophe Mérieux du Laos (CICML) a signé un accord de collaboration avec le Programme national SIDA du Laos (CHAS) pour la mise en œuvre de tests de détection de la charge virale de VIH (VLT) dans le pays, qui permettra le transfert technologique de la technique générique ANRS (HIV Generic charge virale, Biocentric, Bandol, France). L’introduction du VLT appliqué au VIH s’est faite au travers d’ateliers nationaux sur le VIH tous les 6 mois. De juin 2009 à décembre 2011, la charge virale de VIH a été mesurée chez 1782 patients traités par antirétroviraux. Parmi eux, 97 % prenaient un traitement de première intention à base d’inhibiteur de la transcriptase inverse. La charge virale de VIH était indétectable (<250 copies/ml) chez 1491 des 1782 (84 %) patients traités par antirétroviraux. Quatre mois après renforcement de l’administration du traitement, la charge virale de VIH est devenue indétectable chez 179/247 patients (72,5 %) tandis que 68/247 patients (27,5 %) sont demeurés virémiques (charge virale médiane : 4,1 Log10). Cet article montre la faisabilité de la mise en place d’un test de détection générique de la charge virale de VIH abordable au niveau national au Laos. Il est intéressant de noter que seuls 16 % des patients traités par antirétroviraux ont présenté une charge virale détectable lors de la première mesure. En outre, près des deux-tiers d’entre eux ont pu maîtriser leur charge virale par une meilleure administration du traitement.El número de personas que padecen VIH/SIDA en la República Democrática Popular de Laos (también conocida como RDP Lao) se estima en 13.600, y el número de personas que necesitan un tratamiento antirretroviral es de 8.000. En la actualidad, cerca de 3.200 personas infectadas por el VIH reciben tratamiento en siete centros repartidos por todo el país. Hasta hace poco, el seguimiento de los pacientes tratados con fármacos antirretrovirales dependía exclusivamente de criterios clínicos e inmunológicos. En el 2009, el Centro de Infectología Christophe Mérieux de PDR Lao (CICML) suscribió un acuerdo de colaboración con el Centro Nacional de VIH/SIDA/ITS (CHAS) para la implantación del estudio de la carga viral del VIH (VLT, por sus siglas en inglés) en el país, lo que comportaría la transferencia de la tecnología del ensayo genérico ANRS (carga viral genérica del VIH, Biocentric, Bandol, Francia). La introducción del estudio de la carga viral del VIH se ha visto respaldada por la realización de varios talleres nacionales sobre el VIH cada 6 meses. Desde junio de 2009 a diciembre de 2011 se ha medido la carga vírica del VIH de 1.782 pacientes tratados con antirretrovirales. El 97 % de ellos seguía un régimen antirretroviral de primera línea basado en inhibidores de la transcriptasa inversa. No se detectó carga viral del VIH (<250 copias/ml) en 1.491 de 1.782 (84 %) de los pacientes tratados con antirretrovirales. Cuatro meses después del fortalecimiento de la adherencia, no se detectó carga viral del VIH en 179 de 247 pacientes (72,5 %) mientras que 68 de 247 pacientes (27,5 %) seguían siendo virémicos (carga viral media: 4,1 Log10). Este informe demuestra la viabilidad de elaborar un estudio genérico de la carga viral del VIH económico a nivel nacional en la RDP Lao). Cabe destacar que solamente el 16 % de los pacientes tratados con antirretrovirales presentaron una carga viral detectable en su primera medición de la carga viral. Es significativo que casi dos terceras partes de ellos hayan conseguido controlar su carga viral después de fortalecer su adherencia al tratamiento

Self-assembled multifunctional core–shell highly porous metal–organic framework nanoparticles

Core-shell nanoparticles (NPs) are attracting increasing interest in nanomedicine as they exhibit unique properties arising from the combined assets of core and shell materials. Porous nanoscale metal–organic frameworks (nanoMOFs) are able to incorporate with high payloads a large variety of drugs. Like other types of NPs, nanoMOFs need to be functionalized with engineered coatings to ensure colloidal stability, control in vivo fate and drug release. To do so, a novel biodegradable cyclodextrin (CD)-based shell was designed in this study. Water soluble γ-CD-citrate oligomers grafted or not with fluorophores were successfully synthesized using citric acid as crosslinker and efficiently anchored onto the surface of porous nanoMOFs. As compared to monomeric CDs, the oligomeric CD coatings could offer higher interaction possibilities with the cores and better possibilities to graft functional moieties such as fluorescent molecules. The amounts of γ-CD-citrate oligomers onto the nanoMOFs were as high as 53 ± 8 wt%. The yield reached up to 86% in the optimized system. These core–shell nanocomposites were stable upon storage, in contrast to the naked nanoMOFs. In addition, the presence of the coating prevented the doxorubicin (DOX)-loaded nanoMOFs from aggregation. Moreover, due to the presence of fluorophores conjugated to the shell, fluorescence-lifetime microscopy enabled deciphering the coating mechanism. DOX loadings reached 48 ± 10 wt% after 24 h incubation with the drug solution. After coating for additional 24 h, DOX loadings reached 65 ± 8 wt%

A method of active case detection to target reservoirs of asymptomatic malaria and gametocyte carriers in a rural area in Southern Province, Zambia

<p>Abstract</p> <p>Background</p> <p>Asymptomatic reservoirs of malaria parasites are common yet are difficult to detect, posing a problem for malaria control. If control programmes focus on mosquito control and treatment of symptomatic individuals only, malaria can quickly resurge if interventions are scaled back. Foci of parasite populations must be identified and treated. Therefore, an active case detection system that facilitates detection of asymptomatic parasitaemia and gametocyte carriers was developed and tested in the Macha region in southern Zambia.</p> <p>Methods</p> <p>Each week, nurses at participating rural health centres (RHC) communicated the number of rapid diagnostic test (RDT) positive malaria cases to a central research team. During the dry season when malaria transmission was lowest, the research team followed up each positive case reported by the RHC by a visit to the homestead. The coordinates of the location were obtained by GPS and all consenting residents completed a questionnaire and were screened for malaria using thick blood film, RDT, nested-PCR, and RT-PCR for asexual and sexual stage parasites. Persons who tested positive by RDT were treated with artemether/lumefantrine (Coartem^®). Data were compared with a community-based study of randomly selected households to assess the prevalence of asymptomatic parasitaemia in the same localities in September 2009.</p> <p>Results</p> <p>In total, 186 and 141 participants residing in 23 case and 24 control homesteads, respectively, were screened. In the case homesteads for which a control population was available (10 of the 23), household members of clinically diagnosed cases had a 8.0% prevalence of malaria using PCR compared to 0.7% PCR positive individuals in the control group (p = 0.006). The case and control groups had a gametocyte prevalence of 2.3% and 0%, respectively but the difference was not significant (p = 0.145).</p> <p>Conclusions</p> <p>This pilot project showed that active case detection is feasible and can identify reservoirs of asymptomatic infection. A larger sample size, data over multiple low transmission seasons, and in areas with different transmission dynamics are needed to further validate this approach.</p

Optimising Strategies for Plasmodium falciparum Malaria Elimination in Cambodia: Primaquine, Mass Drug Administration and Artemisinin Resistance

Malaria elimination requires a variety of approaches individually optimized for different transmission settings. A recent field study in an area of low seasonal transmission in South West Cambodia demonstrated dramatic reductions in malaria parasite prevalence following both mass drug administration (MDA) and high treatment coverage of symptomatic patients with artemisinin-piperaquine plus primaquine. This study employed multiple combined strategies and it was unclear what contribution each made to the reductions in malaria.A mathematical model fitted to the trial results was used to assess the effects of the various components of these interventions, design optimal elimination strategies, and explore their interactions with artemisinin resistance, which has recently been discovered in Western Cambodia. The modelling indicated that most of the initial reduction of P. falciparum malaria resulted from MDA with artemisinin-piperaquine. The subsequent continued decline and near elimination resulted mainly from high coverage with artemisinin-piperaquine treatment. Both these strategies were more effective with the addition of primaquine. MDA with artemisinin combination therapy (ACT) increased the proportion of artemisinin resistant infections, although much less than treatment of symptomatic cases with ACT, and this increase was slowed by adding primaquine. Artemisinin resistance reduced the effectiveness of interventions using ACT when the prevalence of resistance was very high. The main results were robust to assumptions about primaquine action, and immunity.The key messages of these modelling results for policy makers were: high coverage with ACT treatment can produce a long-term reduction in malaria whereas the impact of MDA is generally only short-term; primaquine enhances the effect of ACT in eliminating malaria and reduces the increase in proportion of artemisinin resistant infections; parasite prevalence is a better surveillance measure for elimination programmes than numbers of symptomatic cases; combinations of interventions are most effective and sustained efforts are crucial for successful elimination

Field-based evidence of fast and global increase of Plasmodium falciparum drug-resistance by DNA-microarrays and PCR/RFLP in Niger

<p>Abstract</p> <p>Background</p> <p>Over the last years, significant progress has been made in the comprehension of the molecular mechanism of malaria resistance to drugs. Together with <it>in vivo </it>tests, the molecular monitoring is now part of the survey strategy of the <it>Plasmodium </it>sensitivity. Currently, DNA-microarray analysis allows the simultaneous study of many single nucleotide polymorphisms (SNP) of <it>Plasmodium </it>isolates. In December 2005, the International Federation of the Red Cross distributed two million three hundred thousand long-lasting insecticide nets to pregnant women and mothers of under five years children in the whole Niger. Then, Niger adopted artemisinin-based combination therapy as first-line treatment.</p> <p>Methods</p> <p>Thirty four SNPs of <it>pfcrt, pfdhfr, pfdhps, pfmdr </it>and <it>pfATPase </it>were analysed by DNA-microarray and PCR/RFLP in two villages – Zindarou and Banizoumbou – with different durations of malaria transmission. The main objective of the study was to measure the dynamics <it>of Plasmodium falciparum </it>resistant strains and associated factors.</p> <p>Results</p> <p>This study shows a global and clear increase of the drug-resistance associated molecular markers frequencies during a relatively short-time period of four years. Markers associated with resistance to chloroquine and sulphonamids were more frequently found in the short transmission zone than in the long transmission one. The <it>pfcrt76T </it>mutation is significantly more present at Banizoumbou than Zindarou (38.3% vs 25.2%, p = 0.013).</p> <p>This work allowed the screening of several field strains for five SNPs of <it>PfATPase6 </it>gene. The <it>pfATPase6S769N</it>, candidate mutation of resistance to artemisinin was not found. However the <it>pfATPsaeA623E </it>mutation was found in 4.7% of samples.</p> <p>Conclusion</p> <p>A significant increase of several SNPs frequencies was highlighted over a four-year period. The polymorphism of five <it>PfATPase6 </it>gene SNPs was described. The global, large and fast increase of the molecular resistance is discussed in the context of current changes of health policy and malaria control in Niger.</p