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

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

"Epidemiology and aetiology of influenza-like illness among households in metropolitan Vientiane, Lao PDR": A prospective, community-based cohort study.

Respiratory diseases are a major contributor to morbidity and mortality in many tropical countries, including Lao PDR. However, little has been published regarding viral or bacterial pathogens that can contribute to influenza-like illness (ILI) in a community setting. We report on the results of a community-based surveillance that prospectively monitored the incidence of ILI and its causative pathogens in Vientiane capital in Lao PDR. A cohort of 995 households, including 4885 study participants, were followed-up between May 2015 and May 2016. Nasopharyngeal swabs, throat swabs, and sputum specimens were collected from ILI cases identified through active case-finding. Real-Time PCR was used to test nasopharyngeal swabs for 21 respiratory pathogens, while throat and sputum samples were subjected to bacterial culture. Generalized linear mixed models were used to assess potential risk factors for associations with ILI. In total, 548 episodes of ILI were reported among 476 (9.7%) of the study participants and 330 (33.2%) of the study households. The adjusted estimated incidence of ILI within the study area was 10.7 (95%CI: 9.4-11.9) episodes per 100 person-years. ILI was significantly associated with age group (p<0.001), sex (p<0.001), and number of bedrooms (p = 0.04) in multivariate analysis. In 548 nasopharyngeal swabs, the most commonly detected potential pathogens were Streptococcus pneumoniae (17.0%), Staphylococcus aureus (11.3%), influenza A (11.1%; mostly subtype H3N2), rhinovirus (7.5%), and influenza B (8.0%). Streptococci were isolated from 42 (8.6%) of 536 throat swabs, most (27) of which were Lancefield Group G. Co-infections were observed in 132 (24.1%) of the 548 ILI episodes. Our study generated valuable data on respiratory disease burden and patterns of etiologies associated with community-acquired acute respiratory illness Laos. Establishment of a surveillance strategy in Laos to monitor trends in the epidemiology and burden of acute respiratory infections is required to minimize their impact on human health

Correction: "Epidemiology and aetiology of influenza-like illness among households in metropolitan Vientiane, Lao PDR": A prospective, community-based cohort study.

[This corrects the article DOI: 10.1371/journal.pone.0214207.]

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

Approche génétique de l'environnement de Plasmodium Falciparum dans sa phase érythrocytaire au Rattanakiri

Le Cambodge présente la particularité d être endémique pour les quatre espèces de Plasmodium infectant l Homme et d avoir de fortes prévalences de maladies héréditaires du globule rouge. Des mutations touchant l hémoglobine comme l hémoglobine E et d autres comme le déficit en G6PD (au travers notamment de la mutation G6PD Viangchan) y ont été identifiées. En 2001, la politique national de lutte contre le paludisme a été mise en place dans le Rattanakiri, province du Nord-est du Cambodge. Celle-ci repose sur l utilisation des tests rapides pour le diagnostic des accès palustres en formant du personnel local, les Village Malaria Worker , l utilisation d ACT et la distribution de moustiquaires imprégnées d insecticide. Nos travaux reposent sur deux études transversales menées dans huit villages avant et après la mise en place du programme de lutte (respectivement en 2001 et 2002) dans cette région de forte endémie palustre. Ce travail de thèse a permis de mettre au point une méthode moléculaire rapide de diagnostic des infections plasmodiales utilisable à large échelle et d établir les limites de la mise au point et de l utilisation de puces à ADN dans ce type d emploi. L utilisation de cette nouvelle technique a permis de mieux caractériser les infections plasmodiales dans une zone reculée du Cambodge où elles sont beaucoup plus fréquentes que ne le laissait penser la microscopie ou qu auraient pu l objectiver les tests rapides. Notre travail a donc souligné la difficulté du défi que constitue l élimination de ces infections plasmodiales par dépistage et traitement systématique, objectif actuellement affiché par les autorités sanitaires locales et internationales. Nos travaux de génotypage humain ont enfin permis de révéler des taux de prévalence élevés de portage de l hémoglobine E et, dans certaines communautés, de mutations du gène de la G6PD. Cette dernière anomalie génétique peut poser un problème de sécurité au déploiement de l utilisation de la primaquine sans dépistage des déficits en G6PD ou d élimination des infections à P. vivax faute d utilisation de la primaquine.Cambodia has the characteristic of being an endemic country for all four Plasmodium species pathogenic for human, as well as being characterised by high prevalence of red blood cell illnesses. Hemoglobin mutation, as hemoglobin E, and other G6PD deficiencies (Viangchan mutation) have been identified. In 2001, a national health policy against malaria has been set up in the North-East province of Cambodia, Rattanakiri. Such polity consists of: staff training (known as Village Malaria Worker) on the use of diagnostic rapid tests for malaria, the use of ACT, and the distribution of impregnated bednets. Our work consists of two transversal studies conducted in eight villages in such high endemic region, before and after the health policy took place (2001 and 2002 respectively). The present thesis work allowed us to set up a rapid molecular methodology for the diagnosis of Plasmodium infection on large scale, and the evaluation of DNA microarray use limits in such application. The use of this new molecular technique gave us the opportunity of better characterise Plasmodium infections in this isolated area of Cambodia, where they are much more frequent than shown by microscopy or rapid test. Moreover, our work highlighted the challenge of infection reduction by mass screening and treatment policy in Cambodia. Finally, the human genotyping study highlighted the high prevalence of hemoglobin E and, in some communities, G6PD Viangchan mutation. This last genetic abnormality could represent a problem for primaquine use without concurrent screening for G6PD deficiency.AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocSudocFranceF

2009 A(H1N1) seroconversion rates and risk factors among the general population in Vientiane Capital, Laos.

To assess 2009 A(H1N1) seroconversion rates and their determinants within an unvaccinated population in Vientiane Capital, Laos.CoPanFlu Laos, a general population cohort of 807 households and 4,072 participants was established in March 2010. Sociodemographic data, epidemiological data, and capillary blood samples were collected from all the household members in March, and again in October 2010, in order to assess the level of antibodies to 2009 A(H1N1) with the haemagglutination inhibition assay. 2009 A(H1N1) seroconversion was defined as a fourfold or greater increase in titre between inclusion and follow-up. Determinants for pandemic influenza infection were studied using the generalized estimating equations model, taking household clustering into account.Between March and November 2010, 3,524 paired sera were tested. Prior to the pandemic, our cohort was almost completely vaccine-naive for seasonal influenza. The overall seroconversion rate among nonvaccinated individuals (n = 2,810) was 14.3% (95%CI [13.0, 15.6]), with the highest rate for participants under 20 yo (19.8%, 95%CI [17.4, 22.4]) and the lowest rate for participants over 60 yo (6.5%, 95%CI [3.7, 10.4]). Participants with lower baseline titres had significantly higher infection rates, with a dose-effect relationship. Odds ratios (ORs) ranged from 76.5 (95%CI [27.1, 215.8]), for those with a titre at inclusion of 1∶10, to 8.1 (95%CI [3.3, 20.4]), for those with a titre of 1∶40. Having another household member with a titre ≥1∶80 was associated with a higher likelihood of immunity (OR = 3.3, 95%CI [2.8, 3.9]).The determinants and age distribution for seroconversion within a vaccine-naive population were similar to those found in developed countries. This pandemic was characterized by strong epidemiological determinants, regardless of geographical zone and level of development. Moreover, we detected pre-existing cross-reacting antibodies in participants over 60 yo, which could not have originated from former multiple vaccination as has been suggested elsewhere

Hepatitis C in Laos: A 7-Year Retrospective Study on 1765 Patients

International audienceHepatitis C virus (HCV) is a global health concern, notably in Southeast Asia, and in Laos the presentation of the HCV-induced liver disease is poorly known. Our objective was thus to describe a comprehensive HCV infection pattern in order to guide national health policies. A study on a group of 1765 patients formerly diagnosed by rapid test in health centres was conducted at the Centre of Infectiology Lao Christophe Merieux in Vientiane. The demographic information of patients, their infection status (viral load: VL), liver function (aminotransferases) and treatments were analysed. Results showed that gender distribution of infected people was balanced; with median ages of 53.8 for men and 51.6 years for women (13-86 years). The majority of patients (72%) were confirmed positive (VL > 50 IU/mL) and 28% of them had high VL (> 6log10). About 23% of patients had level of aminotransferases indicative of liver damage (> 40 IU/mL); but less than 20% of patients received treatment. Patients rarely received a second sampling or medical imaging. The survey also showed that cycloferon, pegylated interferon and ribavirin were the drugs prescribed preferentially by the medical staff, without following any international recommendations schemes. In conclusion, we recommend that a population screening policy and better management of patients should be urgently implemented in the country, respecting official guidelines. However, the cost of biological analysis and treatment are significant barriers that must be removed. Public health resolutions should be immediately enforced in the perspective of meeting the WHO HCV elimination deadline by 2030

Perinatal hepatitis B virus transmission in Lao PDR: A prospective cohort study.

BackgroundMother-to-child transmission of hepatitis B virus (HBV) is the main cause of new infections worldwide. We aimed at assessing the percentage of infants successfully immunized in two major hospitals in Vientiane, Lao PDR where HB immune globulin (HBIg) is not available.MethodsWe studied a prospective cohort of chronically HBV infected pregnant women and their infants until 6 months post-partum from January 2015 to March 2017. All infants received HB vaccine at birth and 6, 10 and 14 weeks thereafter, and HBV status was assessed at 6 months of age. HBV surface gene sequencing was performed in infected mother-infant pairs.ResultsOf 153 mothers with HB surface antigen (HBsAg), 60 (39%) had detectable serum HBe antigen (HBeAg). HBeAg positive pregnant women were younger than those negative (median age 26 versus 28 years; p = 0.02) and had a significantly higher HBV viral load at delivery (median 8.0 versus 4.0 log10 IU/mL, p 8.5 log10 IU/mL. However, only four (3.3%, 95% CI 0.5% to 7.0%) had a virus strain closely related to their mother's strain. HBV surface gene mutations were detected in 4 of the 5 infected infants. Anti-HBs antibody levels were below 10 IU/L in 10 (9%) uninfected infants at 6 months of age.ConclusionsMother-to-child transmission occurred less frequently than expected without the use of HBIg. Adding HBIg and/or maternal antiviral prophylaxis may have prevented some of these infections. The observation of unsatisfactory levels of anti-HBs antibodies in 9% of the uninfected infants at 6 months highlights the need for improvement of the universal immunization procedures