Abstract
Background
Sri Lanka, an island nation, has eliminated endemic malaria transmission. Maintaining elimination in the continued presence of vectors requires vigilance in screening people ...travelling from high malaria-risk areas and a rapid response with focal screening for infections identified in the community. Such screening requires accurate and very rapid assays that enable an immediate response. Both microscopy and rapid diagnostic tests (RDTs) have limitations including sensitivity and speed in screening large numbers, while polymerase chain reaction (PCR) is practical only as laboratory confirmation. This study assessed the utility of ‘Gazelle’, a novel rapid malaria assay based on magneto-optical detection of haemozoin, a by-product of malaria parasite metabolism.
Methods
Between October 2020 and March 2021, two groups of individuals were screened for malaria by four methods, namely, microscopy, Rapid Diagnostic Test (RDT), Gazelle and PCR. Passive case detection was carried out for confirmation of diagnosis amongst individuals suspected of having malaria. Individuals at high-risk of acquiring malaria, namely persons returning from malaria endemic countries, were screened by active case detection.
Results
Of the 440 individuals screened for malaria, nine malaria positives were diagnosed by PCR, microscopy and the HRP2 band of RDT, which included five
Plasmodium falciparum
infections, two
Plasmodium ovale
, and one each of
Plasmodium vivax
and
Plasmodium malariae.
Gazelle correctly detected the
P. vivax, P. ovale
and
P. malariae
infections within the 2 min test time, but did not detect two
P. falciparum
infections giving a sensitivity of 77.8%. Specificity was 100%.
Discussion
The Gazelle, a portable bench top device proved useful to screen a large number of blood samples for non-falciparum parasites within 5 minutes of sample input. Species differentiation, and improvement in
P. falciparum
detection, will be important to broaden utility.
After eliminating local malaria transmission and being certified as a malaria-free country, Sri Lanka is facing the challenge of imported malaria. At the same time, the country has the unique ...opportunity to be a case study for other countries in a similar situation by approaching this issue systematically, guided by evidence. This study demonstrates the importance of developing a mechanism to detect imported malaria and adopting an evidence-based approach to study the resistance of imported malaria to anti-malarial medicines. This is a prospective study of patients diagnosed with imported malaria in Sri Lanka and treated according to the national treatment guidelines, over 24 months (2015/2016). The clinical features, time to diagnosis, origin of the infection, infecting species, parasite density and the treatment given were recorded. All patients were followed up for 28 days, and in the case of Plasmodium vivax and P. ovale infections, the follow up period was extended to 12 months to establish treatment failures and relapses. Fifty nine uncomplicated and 15 severe imported malaria cases were reported in Sri Lanka during the study period. Most of these infections originated in either Sub-Saharan Africa or South and Southeast Asia. Having a P. vivax infection and low parasitic counts were significantly associated with relative diagnostic delay. One of the 14 uncomplicated P. falciparum patients and two of the 12 severe P. falciparum malaria patients who were followed up till day 28 had a late clinical failure. The others responded adequately to treatment both clinically and parasitologically. There was no treatment failure reported amongst any other species. This study, which is the first to assess the therapeutic response of imported malaria in Sri Lanka after elimination, demonstrates that the current antimalarial treatment policies and strategies in Sri Lanka have been effective against infections acquired overseas up until the end of year 2016.
Sri Lanka after eliminating malaria in 2012, is in the prevention of re-establishment (POR) phase. Being a tropical country with high malariogenic potential, maintaining vigilance is important. All ...malaria cases are investigated epidemiologically and followed up by integrated drug efficacy surveillance (iDES). Occasionally, that alone is not adequate to differentiate Plasmodium falciparum reinfections from recrudescences. This study evaluated the World Health Organization and Medicines for Malaria Venture (MMV) recommended genotyping protocol for the merozoite surface proteins (msp1, msp2) and the glutamate-rich protein (glurp) to discriminate P. falciparum recrudescence from reinfection in POR phase.
All P. falciparum patients detected from April 2014 to December 2019 were included in this study. Patients were treated and followed up by iDES up to 28 days and were advised to get tested if they develop fever at any time over the following year. Basic socio-demographic information including history of travel was obtained. Details of the malariogenic potential and reactive entomological and parasitological surveillance carried out by the Anti Malaria Campaign to exclude the possibility of local transmission were also collected. The msp1, msp2, and glurp genotyping was performed for initial and any recurrent infections. Classification of recurrent infections as recrudescence or reinfection was done based on epidemiological findings and was compared with the genotyping outcome.
Among 106 P. falciparum patients, six had recurrent infections. All the initial infections were imported, with a history of travel to malaria endemic countries. In all instances, the reactive entomological and parasitological surveillance had no evidence for local transmission. Five recurrences occurred within 28 days of follow-up and were classified as recrudescence. They have not travelled to malaria endemic countries between the initial and recurrent infections. The other had a recurrent infection after 105 days. It was assumed a reinfection, as he had travelled to the same malaria endemic country in between the two malaria attacks. Genotyping confirmed the recrudescence and the reinfection.
The msp1, msp2 and glurp genotyping method accurately differentiated reinfections from recrudescence. Since reinfection without a history of travel to a malaria endemic country would mean local transmission, combining genotyping outcome with epidemiological findings will assist classifying malaria cases without any ambiguity.
Malaria was eliminated from Sri Lanka in 2012, and the country received WHO-certification in 2016. The objective of this paper is to describe the epidemiology of malaria elimination in Sri Lanka, and ...the key technical and operational features of the elimination effort, which may have been central to achieving the goal, even prior to schedule, and despite an ongoing war in parts of the country. Analysis of information and data from the Anti Malaria Campaign (AMC) of Sri Lanka during and before the elimination phase, and the experiences of the author(s) who directed and/or implemented the elimination programme or supported it form the basis of this paper. The key epidemiological features of malaria on the path to elimination included a steady reduction of case incidence from 1999 onwards, and the simultaneous elimination of both Plasmodium falciparum and Plasmodium vivax. Against the backdrop of a good health infrastructure the AMC, a specialized programme within the Ministry of Health operated through a decentralized provincial health system to implement accepted strategies for the elimination of malaria. Careful planning combined with expertise on malaria control at the Central level with dedicated staff at all levels at the Centre and on the ground in all districts, for several years, was the foundation of this success. The stringent implementation of anti-relapse treatment for P. vivax through a strong collaboration with the military in whose cadres most of the malaria cases were clustered in the last few years of transmission would have supported the relatively rapid elimination of P. vivax. A robust case and entomological surveillance and investigation system described here enabled a highly focused approach to delivering interventions leading to the interruption of transmission.
Application of flow cytometry (FCM) to microbial analysis of milk is hampered by the presence of milk proteins and lipid particles. Here we report on the development of a rapid (< or = 1-h) FCM assay ...based on enzymatic clearing of milk to determine total bacteria in milk. When bacteria were added to ultra-heat-treated milk, a good correlation (r > or = 0.98) between the FCM assay and the more conventional methods of plating and direct microscopic counting was achieved. Raw milk data showed a significant correlation (P < 0.01) and a good agreement (r = 0.91) between FCM and standard plate count methods. The detection limit of the FCM assay was < or = 10(4) bacteria ml of milk(-1). This limit is below the level of detection required to satisfy legislation in many countries and states.
The country received malaria-free certification from WHO in September 2016, becoming only the second country in the WHO South East Asia region to be declared malaria-free. Imported malaria cases ...continue to be reported, with 278 cases reported between 2013 and 2017. The diagnosis of a severe Plasmodium vivax patient co-infected with HIV and tuberculosis is discussed with an overview of the rapid response mounted by the Anti Malaria Campaign (AMC), Sri Lanka.
A Sri Lankan gem miner who returned from Madagascar on the 6th of April 2018 presented to a private hospital for a malaria diagnostic test on the 21st April, 2 days after the onset of fever. He came on his own for this test due to the awareness he had regarding the risk of imported malaria. As the patient was positive for P. vivax malaria, he was admitted to a government hospital for further management. The patient had features of severe malaria upon admission with a systolic BP < 80 mmHg and thrombocytopaenia (38,000 cells/mm
). Treatment with IV artesunate was initiated immediately and management was carried out rapidly and efficiently by the clinicians with guidance from the staff of the AMC headquarters, which resulted in a rapid recovery of the patient. IV artesunate was followed by a course of artemether plus lumefantrine and the blood smear was negative for malaria by the 2nd day. A 14-day course of primaquine was commenced after excluding a G6PD deficiency. Due to an accidental needle stick injury of a health care worker attending on the patient was tested for HIV and subsequently tuberculosis and was found to be positive for both infections. The patient was discharged on the 1st of May with instructions for follow up visits for malaria. Management of the HIV and tuberculosis infections was attended to by the clinicians and staff of the appropriate disease control programmes (i.e. the national STD/AIDS Control Programme in Sri Lanka and the National Programme for tuberculosis control and chest diseases).
It is important to consider comorbid conditions and immunosuppression when a patient with a benign form of malaria presents with severe manifestations. Measures should be strengthened to prevent importation of diseases, such as malaria and AIDS through migrant workers who return from high-risk countries.
Monitoring the quality and safety of milk requires careful analysis of microbial and somatic cell loading. Our aim was to demonstrate proof of the principle that flow cytometry (FCM), coupled with ...fluorescence techniques for distinguishing between cell types, could potentially be employed in a wide variety of biological assays relevant to the dairy industry. To this end, we studied raw milk samples and ultraheat-treated milk, into which known numbers of bacteria or mouse cells were inoculated. For bacterial analyses, protein and lipids were removed, whereas only centrifugal lipid clearing was needed for somatic cell analyses. Cleared samples were stained with fluorescent dyes or with bacterial-specific fluorescent-labeled oligonucleotides and analyzed by FCM. A fluoresceinated peptide nucleic acid probe enabled efficient enumeration of bacteria in milk. Dual staining of samples with fluorescent dyes that indicate live (5-cyanol-2,3-ditolyl tetrazolium chloride, CTC or SYTO 9) or damaged cells (oxonol or propidium iodide, PI) enabled determination of viable bacteria in milk. Gram-positive and -negative bacteria were distinguished using hexidium iodide and SYTO 13 in dual staining of cleared milk samples. An FCM-based method gave a good correlation (
r=0.88) with total microscopic counts of somatic cells in raw milk. The FCM method also correlated strongly (
r=0.98) with the standard Fossomatic method for somatic cell detection. We conclude that FCM, coupled with fluorescence staining techniques, offers potentially diverse and rapid approaches to biological safety and quality testing in the dairy industry. Potential application of flow cytometers to a broad range of assays for milk biological quality should make this instrumentation more attractive and cost effective to the dairy industry and indeed the broader food industry.
Sri Lanka has reached zero indigenous malaria cases in November 2012, two years before its targeted deadline for elimination. Currently, the biggest threat to the elimination efforts are the risk of ...resurgence of malaria due to imported cases. This paper describes two clusters of imported malaria infections reported in 2013 and 2014, one among a group of Pakistani asylum-seekers resident in Sri Lanka, and the other amongst local fishermen who returned from Sierra Leone. The two clusters studied reveal the potential impact of imported malaria on the risk of reintroducing the disease, as importation is the only source of malaria in the country at present. In the event of a case occurring, detection is a major challenge both amongst individuals returning from malaria endemic countries and the local population, as malaria is fast becoming a "forgotten" disease amongst health care providers. In spite of a very good coverage of diagnostic services (microscopy and rapid diagnostic tests) throughout the country, malaria is being repeatedly overlooked by health care providers even when individuals present with fever and a recent history of travel to a malaria endemic country. Given the high receptivity to malaria in previously endemic areas of the country due to the prevalence of the vector mosquito, such cases pose a significant threat for the reintroduction of malaria to Sri Lanka. The challenges faced by the Anti Malaria Campaign and measures taken to prevent the resurgence of malaria are discussed here.
Sri Lanka has achieved 'malaria-free' status and is now in the phase of prevention of re-introduction of malaria. Imported malaria remains a challenge to resurgence of the disease. The diagnostic ...challenges encountered and the rapid response initiated to manage a Plasmodium infection, which was later confirmed as Plasmodium knowlesi, the first reported case from Sri Lanka, is discussed.
An army officer who returned from Malaysia in October 2016 was found to be positive for Plasmodium both by microscopy and rapid diagnostic test (RDT) by the Anti Malaria Campaign Sri Lanka (AMC) during his third visit to a health care provider. Microscopy findings were suspicious of P. knowlesi infection as the smears showed parasite stages similar to both Plasmodium malariae and Plasmodium falciparum. Nested PCR at AMC confirmed Plasmodium genus, but not the species. In the absence of species confirmation, the patient was treated as a case of P. falciparum. The presence of P. knowlesi was later confirmed by a semi-nested PCR assay performed at the Environmental Health Institute, National Environmental Agency in Singapore. The parasite strain was also characterized by sequencing the circumsporozoite gene. Extensive case investigation including parasitological and entomological surveillance was carried out.
Plasmodium knowlesi should be suspected in patients returning from countries in the South Asian region where the parasite is prevalent and when blood smear results are inconclusive.
Sri Lanka sustained its malaria-free status by implementing, among other interventions, three core case detection strategies namely Passive Case Detection (PCD), Reactive Case Detection (RACD) and ...Proactive Case Detection (PACD). The outcomes of these strategies were analysed in terms of their effectiveness in detecting malaria infections for the period from 2017 to 2019.
Comparisons were made between the surveillance methods and between years, based on data obtained from the national malaria database and individual case reports of malaria patients. The number of blood smears examined microscopically was used as the measure of the volume of tests conducted. The yield from each case detection method was calculated as the proportion of blood smears which were positive for malaria. Within RACD and PACD, the yield of sub categories of travel cohorts and spatial cohorts was ascertained for 2019.
A total of 158 malaria cases were reported in 2017-2019. During this period between 666,325 and 725,149 blood smears were examined annually. PCD detected 95.6 %, with a yield of 16.1 cases per 100,000 blood smears examined. RACD and PACD produced a yield of 11.2 and 0.3, respectively. The yield of screening the sub category of travel cohorts was very high for RACD and PACD being 806.5 and 44.9 malaria cases per 100,000 smears, respectively. Despite over half of the blood smears examined being obtained by screening spatial cohorts within RACD and PACD, the yield of both was zero over all three years.
The PCD arm of case surveillance is the most effective and, therefore, has to continue and be further strengthened as the mainstay of malaria surveillance. Focus on travel cohorts within RACD and PACD should be even greater. Screening of spatial cohorts, on a routine basis and solely because people are resident in previously malarious areas, may be wasteful, except in situations where the risk of local transmission is very high, or is imminent. These findings may apply more broadly to most countries in the post-elimination phase.
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