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Background The production and use of malaria rapid diagnostic tests (RDTs) has risen dramatically over the past 20 years. In view of weak or non-existing in vitro diagnostics (IVD) regulations and post-marketing surveillance (PMS) systems in malaria endemic countries, the World Health Organization, later joined by the Foundation for Innovative New Diagnostics, established an independent, centralized performance evaluation and Lot Testing (LT) programme to safeguard against poor quality of RDTs being distributed through the public health sector of malaria endemic countries. RDT performances and manufacturer quality management systems have evolved over the past decade raising questions about the future need for a centralized LT programme. Results Between 2007 and 2017, 6056 lots have been evaluated, representing approximately 1.6 Billion RDTs. A total of 69 lots (1.1%) failed the quality control. Of these failures, 26 were detected at receipt of the RDT lot in the LT laboratory, representing an estimated 7.9 million poor quality RDTs, and LT requesters were advised that RDTs were not of sufficient quality for use in patient management. Forty-three were detected after long-term storage in the laboratory, of which 24 (56%) were found to be due to a major issue with insufficient buffer volume in single use buffer vials, others predominantly showing loss of sensitivity. The annual cost of running the programme, based on expenses recorded in years 2014–2016, an estimated volume of 700 lots per year and including replenishment of quality control samples, was estimated at US$ 178,500 ($US 255 per lot tested). Conclusions Despite the clear benefits of the centralized LT programme and its low cost compared with the potential costs of each country establishing its own PMS system for RDTs, funding concerns have made its future beyond 2020 uncertain. In order to manage the risks of misdiagnosis due to low quality RDTs, and to ensure the continued safety and reliability of malaria case management, there is a need to ensure that an effective and implementable approach to RDT quality control continues to be available to programmes in endemic countries.

Malaria rapid diagnostic tests (RDTs) emerged in the early 1990s into largely unregulated markets, and uncertain field performance was a major concern for the acceptance of tests for malaria case management. This, combined with the need to guide procurement decisions of UN agencies and WHO Member States, led to the creation of an independent, internationally coordinated RDT evaluation programme aiming to provide comparative performance data of commercially available RDTs. Products were assessed against Plasmodium falciparum and Plasmodium vivax samples diluted to two densities, along with malaria-negative samples from healthy individuals, and from people with immunological abnormalities or non-malarial infections. Three measures were established as indicators of performance, (i) panel detection score (PDS) determined against low density panels prepared from P. falciparum and P. vivax wild-type samples, (ii) false positive rate, and (iii) invalid rate, and minimum criteria defined. Over eight rounds of the programme, 332 products were tested. Between Rounds 1 and 8, substantial improvements were seen in all performance measures. The number of products meeting all criteria increased from 26.8% (11/41) in Round 1, to 79.4% (27/34) in Round 8. While products submitted to further evaluation rounds under compulsory re-testing did not show improvement, those voluntarily resubmitted showed significant increases in P. falciparum (p = 0.002) and P. vivax PDS (p

(2004). The in-vitro antimalarial activity of ferrochloroquine, measured against Cambodian isolates of Plasmodium falciparum. Annals of Tropical Medicine & Parasitology: Vol. 98, No. 4, pp. 419-424.

Cambodia, especially the western border with Thailand, is known as a hot spot for multidrug-resistant malaria. Pyrimethamine resistance was first reported in that border area in the early 1950s (27), followed by chloroquine resistance in the late 1950s, sulfadoxine-pyrimethamine (SP) resistance in the late 1960s, and mefloquine resistance in the late 1980s (31). Since 1986 (5), the Cambodian National Malaria Control Program (NMCP) has set up a program to monitor the efficacy of SP and mefloquine monotherapy, specifically in the provinces bordering Thailand, Vietnam, and Laos. Between 1995 and 1999, failure rates based on 14-day follow-up periods were 7 to 30% for a low dose of mefloquine (15 mg/kg) and 7 to 10% for the higher doses (20 to 25 mg/kg of body weight) in provinces in northwest Cambodia bordering Thailand, while mefloquine efficacy remained 100% elsewhere (M. B. Denis, personal communication). These findings were consistent with reports from Thailand (21). In 2000, studies of the clinical efficacy of artesunate-mefloquine (AM) (12 mg/kg artesunate and 1,000 mg mefloquine given over 3 days) were conducted, again in northwestern Cambodia; they confirmed 100% efficacy based on a 14-day follow-up (M. B. Denis, personal communication). The NMCP decided to switch to AM combination therapy in the same year. During an informal consultation on the monitoring of antimalarial drug efficacy in the Mekong Subregion organized by the World Health Organization (WHO) in Phnom Penh, Cambodia, in October 2000, it was agreed that each country should strengthen its capacity to monitor the efficacy of the first-line antimalarial drug. Monitoring should be conducted by therapeutic efficacy studies (TES) at selected sentinel sites on a regular basis, at least every other year for each site, with a 28-day follow-up. In 2001, the Center for Parasitology, Entomology, and Malaria Control of Cambodia (CNM) conducted TES in two provinces: Sampovloun, in the northwestern part of the country near the Thai border, and Snoul, along the Vietnamese border. Both studies confirmed the high efficacy of AM, with cure rates of 96% and 100%, respectively (7). In 2002, the failure rates of artesunate-mefloquine increased to 14.3% in the northwest part of the country along the Thai-Cambodian border (7). In vitro monitoring conducted on isolates from Plasmodium falciparum-infected patients during the years 2001 and 2002 showed significantly higher geometric mean 50% inhibitory concentrations (GMIC50s) for mefloquine, chloroquine, and quinine in the western provinces than in the eastern provinces (13). The molecular marker of chloroquine resistance is also known to differ in its geographic distribution within Cambodia: the P. falciparum crt (pfcrt) CVIETIF//ISS haplotype was detected in 92% of isolates from the west but in only 11% of those from the east (14). Since 2001, regular monitoring of the efficacy of artemisinin-based combination therapy (ACT), coupled with in vitro assessment and molecular marker assays, has continued uninterruptedly. This article reports the temporal and geographic trends in the in vitro drug susceptibility assay results and the prevalence of single nucleotide polymorphisms (SNPs) in isolates from infected patients enrolled in the TES during the years 2001 to 2007. These findings were also analyzed in relation to the clinical outcomes of ACT.

Background In Cambodia, estimates of the malaria burden rely on a public health information system that does not record cases occurring among remote populations, neither malaria cases treated in the private sector nor asymptomatic carriers. A global estimate of the current malaria situation and associated risk factors is, therefore, still lacking. Methods A large cross-sectional survey was carried out in three areas of multidrug resistant malaria in Cambodia, enrolling 11,652 individuals. Fever and splenomegaly were recorded. Malaria prevalence, parasite densities and spatial distribution of infection were determined to identify parasitological profiles and the associated risk factors useful for improving malaria control programmes in the country. Results Malaria prevalence was 3.0%, 7.0% and 12.3% in Sampovloun, Koh Kong and Preah Vihear areas. Prevalences and Plasmodium species were heterogeneously distributed, with higher Plasmodium vivax rates in areas of low transmission. Malaria-attributable fevers accounted only for 10–33% of malaria cases, and 23–33% of parasite carriers were febrile. Multivariate multilevel regression analysis identified adults and males, mostly involved in forest activities, as high risk groups in Sampovloun, with additional risks for children in forest-fringe villages in the other areas along with an increased risk with distance from health facilities. Conclusion These observations point to a more complex malaria situation than suspected from official reports. A large asymptomatic reservoir was observed. The rates of P. vivax infections were higher than recorded in several areas. In remote areas, malaria prevalence was high. This indicates that additional health facilities should be implemented in areas at higher risk, such as remote rural and forested parts of the country, which are not adequately served by health services. Precise malaria risk mapping all over the country is needed to assess the extensive geographical heterogeneity of malaria endemicity and risk populations, so that current malaria control measures can be reinforced accordingly.

Plasmodium ovale malaria has been reported in various countries in southeast Asia, but never in Cambodia. Using a species-specific polymerase chain reaction (PCR) targeting the small subunit (SSU) ribosomal RNA (rRNA) gene, we detected P. ovale in nearly 4% of the inhabitants of a northeastern Cambodian village. Plasmodium ovale was associated with at least one other Plasmodium species, and two quadruple infections were detected. The diagnosis was confirmed by microscopy and by SSU rRNA PCR product sequencing. The sequences shared 96-99% identity with published sequences, and displayed a substantial heterogeneity with 2-4 different haplotypes per sample. Nine distinct SSU rRNA haplotypes were identified, including seven novel variants. Phylogenetic analysis showed two major genetic clusters, suggesting amplification of two distinct gene sets and/or P. ovale variants from each sample. Our data indicate that P. ovale was overlooked in Cambodia until now, and call for the implementation of larger prevalence surveys and accurate diagnosis methods in this country.

Background Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation.