Plasmodium vivax Latent Liver Stage Infection and Relapse: Biological Insights and New Experimental Tools.

Plasmodium vivax is the most widespread human malaria parasite, in part because it can form latent liver stages known as hypnozoites after transmission by female anopheline mosquitoes to human hosts. These persistent stages can activate weeks, months, or even years after the primary clinical infection; replicate; and initiate relapses of blood stage infection, which causes disease and recurring transmission. Eliminating hypnozoites is a substantial obstacle for malaria treatment and eradication since the hypnozoite reservoir is undetectable and unaffected by most antimalarial drugs. Importantly, in some parts of the globe where P. vivax malaria is endemic, as many as 90% of P. vivax blood stage infections are thought to be relapses rather than primary infections, rendering the hypnozoite a major driver of P. vivax epidemiology. Here, we review the biology of the hypnozoite and recent discoveries concerning this enigmatic parasite stage. We discuss treatment and prevention challenges, novel animal models to study hypnozoites and relapse, and hypotheses related to hypnozoite formation and activation.

Higher-Dose Primaquine to Prevent Relapse of Plasmodium vivax Malaria.

BACKGROUND: In most of the Americas, the recommended treatment to prevent relapse of Plasmodium vivax malaria is primaquine at a total dose of 3.5 mg per kilogram of body weight, despite evidence of only moderate efficacy. METHODS: In this trial conducted in Brazil, we evaluated three primaquine regimens to prevent relapse of P. vivax malaria in children at least 5 years of age and in adults with microscopy-confirmed P. vivax monoinfection. All the patients received directly observed chloroquine for 3 days (total dose, 25 mg per kilogram). Group 1 received a total primaquine dose of 3.5 mg per kilogram (0.5 mg per kilogram per day) over 7 days with unobserved administration; group 2 received the same regimen as group 1 but with observed administration; and group 3 received a total primaquine dose of 7.0 mg per kilogram over 14 days (also 0.5 mg per kilogram per day) with observed administration. We monitored the patients for 168 days. RESULTS: We enrolled 63 patients in group 1, 96 in group 2, and 95 in group 3. The median age of the patients was 22.4 years (range, 5.4 to 79.8). By day 28, three P. vivax recurrences were observed: 2 in group 1 and 1 in group 2. By day 168, a total of 70 recurrences had occurred: 24 in group 1, 34 in group 2, and 12 in group 3. No serious adverse events were noted. On day 168, the percentage of patients without recurrence was 58% (95% confidence interval [CI], 44 to 70) in group 1, 59% (95% CI, 47 to 69) in group 2, and 86% (95% CI, 76 to 92) in group 3. Survival analysis showed a difference in the day 168 recurrence-free percentage of 27 percentage points (97.5% CI, 10 to 44; P<0.001) between group 1 and group 3 and a difference of 27 percentage points (97.5% CI, 12 to 42; P<0.001) between group 2 and group 3. CONCLUSIONS: The administration of primaquine at a total dose of 7.0 mg per kilogram had higher efficacy in preventing relapse of P. vivax malaria than a total dose of 3.5 mg per kilogram through day 168. (Supported by the U.S. Agency for International Development; ClinicalTrials.gov number, NCT03610399.).

A Novel Ex Vivo Assay to Evaluate Functional Effectiveness of Plasmodium vivax Transmission-Blocking Vaccine Using Pvs25 Transgenic Plasmodium berghei.

BACKGROUND: Plasmodium falciparum and Plasmodium vivax account for >90% global malaria burden. Transmission intervention strategies encompassing transmission-blocking vaccines (TBV) and drugs represent ideal public health tools to eliminate malaria at the population level. The availability of mature P. falciparum gametocytes through in vitro culture has facilitated development of a standard membrane feeding assay to assess efficacy of transmission interventions against P. falciparum. The lack of in vitro culture for P. vivax has significantly hampered similar progress on P. vivax and limited studies have been possible using blood from infected patients in endemic areas. The ethical and logistical limitations of on-time access to blood from patients have impeded the development of P. vivax TBVs. METHODS: Transgenic murine malaria parasites (Plasmodium berghei) expressing TBV candidates offer a promising alternative for evaluation of P. vivax TBVs through in vivo studies in mice, and ex vivo membrane feeding assay (MFA). RESULTS: We describe the development of transmission-competent transgenic TgPbvs25 parasites and optimization of parameters to establish an ex vivo MFA to evaluate P. vivax TBV based on Pvs25 antigen. CONCLUSIONS: The MFA is expected to expedite Pvs25-based TBV development without dependence on blood from P. vivax-infected patients in endemic areas for evaluation.

Forest-goers as a heterogeneous population at high-risk for malaria: a case-control study in Aceh Province, Indonesia.

BACKGROUND: A major challenge to malaria elimination is identifying and targeting populations that are harbouring residual infections and contributing to persistent transmission. In many near-elimination settings in Southeast Asia, it is known that forest-goers are at higher risk for malaria infection, but detailed information on their behaviours and exposures is not available. METHODS: In Aceh Province, Indonesia, a near-elimination setting where a growing proportion of malaria is due to Plasmodium knowlesi, a case-control study was conducted to identify risk factors for symptomatic malaria, characteristics of forest-goers, and key intervention points. From April 2017 to September 2018, cases and controls were recruited and enrolled in a 1:3 ratio. Cases had confirmed malaria infection by rapid diagnostic test or microscopy detected at a health facility (HF). Gender-matched controls were recruited from passive case detection among individuals with suspected malaria who tested negative at a health facility (HF controls), and community-matched controls were recruited among those testing negative during active case detection. Multivariable logistic regression (unconditional for HF controls and conditional for community controls) was used to identify risk factors for symptomatic malaria infection. RESULTS: There were 45 cases, of which 27 were P. knowlesi, 17 were Plasmodium vivax, and one was not determined. For controls, 509 and 599 participants were recruited from health facilities and the community, respectively. Forest exposures were associated with high odds of malaria; in particular, working and sleeping in the forest (HF controls: adjusted odds ratio (aOR) 21.66, 95% CI 5.09-92.26; community controls: aOR 16.78, 95% CI 2.19-128.7) and having a second residence in the forest (aOR 6.29, 95% CI 2.29-17.31 and 13.53, 95% CI 2.10-87.12). Male forest-goers were a diverse population employed in a variety of occupations including logging, farming, and mining, sleeping in settings, such as huts, tents, and barracks, and working in a wide range of group sizes. Reported use of protective measures, such as nets, hammock nets, mosquito coils, and repellents was low among forest-goers and interventions at forest residences were absent. CONCLUSIONS: Second residences in the forest and gaps in use of protective measures point to key malaria interventions to improve coverage in forest-going populations at risk for P. knowlesi and P. vivax in Aceh, Indonesia. Intensified strategies tailored to specific sub-populations will be essential to achieve elimination.

Radical cure for Plasmodium vivax malaria after G6PD qualitative testing in four provinces in Cambodia, results from Phase I implementation.

BACKGROUND: Cambodia aims to eliminate all forms of malaria by 2025. In 2020, 90% of all malaria cases were Plasmodium vivax. Thus, preventing P. vivax and relapse malaria is a top priority for elimination. 14-day primaquine, a World Health Organization-recommended radical cure treatment regimen, specifically targets dormant hypnozoites in the liver to prevent relapse. Cambodia introduced P. vivax radical cure with primaquine after glucose-6-phosphate dehydrogenase (G6PD) qualitative testing in 2019. This paper presents Cambodia's radical cure Phase I implementation results and assesses the safety, effectiveness, and feasibility of the programme prior to nationwide scale up. METHODS: Phase I implementation was carried out in 88 select health facilities (HFs) across four provinces. Males over 20kgs with confirmed P. vivax or mixed (P. vivax and Plasmodium falciparum) infections were enrolled. A descriptive analysis evaluated the following: successful referral to health facilities, G6PD testing results, and self-reported 14-day treatment adherence. P. vivax incidence was compared before and after radical cure rollout and a controlled interrupted time series analysis compared the estimated relapse rate between implementation and non-implementation provinces before and after radical cure. RESULTS: In the 4 provinces from November 2019 to December 2020, 3,239 P. vivax/mixed infections were reported, 1,282 patients underwent G6PD deficiency testing, and 959 patients received radical cure, achieving 29.6% radical cure coverage among all P. vivax/mixed cases and 98.8% coverage among G6PD normal patients. Among those who initiated radical cure, 747 patients (78%) completed treatment. Six patients reported side effects. In implementation provinces, an average 31.8 relapse cases per month were estimated signaling a 90% (286 cases) reduction in relapse compared to what would be expected if radical cure was not implemented. CONCLUSIONS: Plasmodium vivax radical cure is a crucial tool for malaria elimination in Cambodia. The high coverage of radical cure initiation and adherence among G6PD normal patients demonstrated the high feasibility of providing radical cure at point of care in Cambodia. Incomplete referral from community to HFs and limited capacity of HF staff to conduct G6PD testing in high burden areas led to lower coverage of G6PD testing. Phase I implementation informed approaches to improve referral completion and patient adherence during the nationwide expansion of radical cure in 2021.

Malaria elimination in West Java, Indonesia: A descriptive-and-qualitative study.

BACKGROUND OBJECTIVES: Following World Health Organization (WHO) plans for thirty-five malaria-endemic countries, Indonesia will eliminate malaria by 2030. As one of the Indonesian provinces, West Java targeted subnational malaria elimination in 2022. This article aims to describe malaria surveillance data and elimination programs, including weaknesses in sustaining the program. METHODS: This study used secondary data from malaria surveillance information system regencies/cities' case reports for 2019-2022 and achievement data of sub-national malaria elimination certification from each regency/city from 2014-2022. The data was confirmed from the evaluation study document, analysis of reported cases, and interviews. RESULTS: Most cases were confirmed by microscopic examination (84.1% in 2021 and 94.4% in 2022) and rapid diagnostic tests (57% in 2019 and 58.1% in 2020). Malaria is more prevalent among men (93% in 2019, 95% in 2020, 96% in 2021, and 95.9% in 2022) and productive ages of 15-64 years (98.8% in 2019, 100% in 2020, 99.2% in 2021, and 98.8% in 2022), frequently occurs in the military (56.3% in 2019, 75.7% in 2020, 45.2% in 2021) and police (40.5% in 2022), often uses passive case detection for identifying cases (97.9% in 2019 and 2020, 95.2% in 2021, and 97.6% in 2022), and the majority undergo inpatient treatment (86.4% in 2019, 81.7% in 2021, and 82.6% in 2022). Most positive cases originated from imported cases, and last indigenous cases were still found in 2019. Plasmodium vivax dominated malaria cases and and relapses were high (55.0% in 2020, and 47.3% in 2022). INTERPRETATION CONCLUSION: All regencies/cities have obtained sub-national malaria elimination certification in 2022. West Java has the potential to be verified for Java-Bali sub-national malaria elimination targeted in 2023, albeit cases of imported malaria still occur. It is imperative to address the issue of imported cases transitioning into locally transmitted cases (introduced) by effective coordination across all regencies/cities and inter-provincial efforts.

Immunity against sexual stage Plasmodium falciparum and Plasmodium vivax parasites.

The efficient spread of malaria from infected humans to mosquitoes is a major challenge for malaria elimination initiatives. Gametocytes are the only Plasmodium life stage infectious to mosquitoes. Here, we summarize evidence for naturally acquired anti-gametocyte immunity and the current state of transmission blocking vaccines (TBV). Although gametocytes are intra-erythrocytic when present in infected humans, developing Plasmodium falciparum gametocytes may express proteins on the surface of red blood cells that elicit immune responses in naturally exposed individuals. This immune response may reduce the burden of circulating gametocytes. For both P. falciparum and Plasmodium vivax, there is a solid evidence that antibodies against antigens present on the gametocyte surface, when co-ingested with gametocytes, can influence transmission to mosquitoes. Transmission reducing immunity, reducing the burden of infection in mosquitoes, is a well-acknowledged but poorly quantified phenomenon that forms the basis for the development of TBV. Transmission enhancing immunity, increasing the likelihood or intensity of transmission to mosquitoes, is more speculative in nature but is convincingly demonstrated for P. vivax. With the increased interest in malaria elimination, TBV and monoclonal antibodies have moved to the center stage of malaria vaccine development. Methodologies to prioritize and evaluate products are urgently needed.

Implications of conformational flexibility, lipid binding, and regulatory domains in cell-traversal protein CelTOS for apicomplexan migration.

Malaria and other apicomplexan-caused diseases affect millions of humans, agricultural animals, and pets. Cell traversal is a common feature used by multiple apicomplexan parasites to migrate through host cells and can be exploited to develop therapeutics against these deadly parasites. Here, we provide insights into the mechanism of the Cell-traversal protein for ookinetes and sporozoites (CelTOS), a conserved cell-traversal protein in apicomplexan parasites and malaria vaccine candidate. CelTOS has previously been shown to form pores in cell membranes to enable traversal of parasites through cells. We establish roles for the distinct protein regions of Plasmodium vivax CelTOS and examine the mechanism of pore formation. We further demonstrate that CelTOS dimer dissociation is required for pore formation, as disulfide bridging between monomers inhibits pore formation, and this inhibition is rescued by disulfide-bridge reduction. We also show that a helix-destabilizing amino acid, Pro127, allows CelTOS to undergo significant conformational changes to assemble into pores. The flexible C terminus of CelTOS is a negative regulator that limits pore formation. Finally, we highlight that lipid binding is a prerequisite for pore assembly as mutation of a phospholipids-binding site in CelTOS resulted in loss of lipid binding and abrogated pore formation. These findings identify critical regions in CelTOS and will aid in understanding the egress mechanism of malaria and other apicomplexan parasites as well as have implications for studying the function of other essential pore-forming proteins.

Impact of Chemoprophylaxis on Plasmodium vivax and Plasmodium ovale Infection Among Civilian Travelers: A Nested Case-Control Study With a Counterfactual Approach on 862 Patients.

BACKGROUND: The impact of chemoprophylaxis targeting Plasmodium falciparum on Plasmodium vivax and Plasmodium ovale, which may remain quiescent as hypnozoites in the liver, is debated. METHODS: We conducted a nested case-control analysis of the outcomes of P. vivax and P. ovale infections in imported malaria cases in France among civilian travelers from 1 January 2006, to 31 December 2017. Using adjusted logistic regression, we assessed the effect of chemoprophylaxis on the incubation period, time from symptoms to diagnosis, management, blood results, symptoms, and hospitalization duration. We analyzed the effect of blood-stage drugs (doxycycline, mefloquine, chloroquine, chloroquine-proguanil) or atovaquone-proguanil on the incubation period. We used a counterfactual approach to ascertain the causal effect of chemoprophylaxis on postinfection characteristics. RESULTS: Among 247 P. vivax- and 615 P. ovale-infected travelers, 30% and 47%, respectively, used chemoprophylaxis, and 7 (3%) and 8 (1%) were severe cases. Chemoprophylaxis users had a greater risk of presenting symptoms >2 months after returning for both species (P. vivax odds ratio [OR], 2.91 [95% confidence interval {CI}, 1.22-6.95], P = .02; P. ovale OR, 2.28 [95% CI, 1.47-3.53], P < .001). Using drugs only acting on the blood stage was associated with delayed symptom onset after 60 days, while using atovaquone-proguanil was not. CONCLUSIONS: Civilian travelers infected with P. vivax or P. ovale reporting chemoprophylaxis use, especially of blood-stage agents, had a greater risk of delayed onset of illness. The impact of chemoprophylaxis on the outcomes of infection with relapse-causing species calls for new chemoprophylaxis acting against erythrocytic and liver stages.

Successful elimination of falciparum malaria following the introduction of community-based health workers in Eastern Myanmar: A retrospective analysis.

BACKGROUND: Myanmar has a large majority of all malaria in the Greater Mekong Subregion. In the past decade, substantial progress was made in malaria control. The residual burden of malaria is in remote areas where currently recommended malaria elimination approaches are generally not feasible. In such hard-to-reach communities in Mon state, East Myanmar, Medical Action Myanmar introduced community health workers (CHWs) to deliver early diagnosis and treatment for malaria. We conducted a retrospective analysis to assess the impact of this intervention. METHODS AND FINDINGS: This retrospective analysis involved data collected routinely from a CHW programme in Mon state conducted between 2011 and 2018. A network of 172 CHWs serving a population of 236,340 was deployed. These CHWs carried out 260,201 malaria rapid diagnostic tests (RDTs) to investigate patients with acute febrile illness. The median blood examination rate was 1.33%; interquartile range (IQR) (0.38 to 3.48%); 95% CI [1.28%, 1.36%] per month. The changes in malaria incidence and prevalence in patients presenting with fever were assessed using negative binomial regression mixed effects models fitted to the observed data. The incidence of Plasmodium falciparum malaria (including mixed infections) declined by 70%; 95% CI [65%, 75%]; p < 0.001 for each year of CHW operation. The incidence of P. vivax malaria declined by 56%; 95% CI [50%, 62%]; p < 0.001 per year. Malaria RDT positivity rates for P. falciparum and P. vivax declined by 69%; 95% CI [62%, 75%]; p < 0.001 and 53%; 95% CI [47%, 59%]; p < 0.001 per year, respectively. Between 2017 and 2018, only 1 imported P. falciparum case was detected in 54,961 RDTs. The main limitations of the study are use of retrospective data with possible unidentified confounders and uncharacterised population movement. CONCLUSIONS: The introduction of CHWs providing community-based malaria diagnosis and treatment and basic health care services in remote communities in Mon state was associated with a substantial reduction in malaria. Within 6 years, P. falciparum was eliminated and the incidence of P. vivax fell markedly.

Cytochrome P450 2D6 profiles and anti-relapse efficacy of tafenoquine against Plasmodium vivax in Australian Defence Force personnel.

Plasmodium vivax infections and relapses remain a major health problem for malaria-endemic countries, deployed military personnel, and travelers. Presumptive anti-relapse therapy and radical cure using the 8-aminoquinoline drugs primaquine and tafenoquine are necessary to prevent relapses. Although it has been demonstrated that the efficacy of primaquine is associated with Cytochrome P450 2D6 (CYP2D6) activity, there is insufficient data on the role of CYP2D6 in the anti-relapse efficacy of tafenoquine. We investigated the relationship between CYP2D6 activity status and tafenoquine efficacy in preventing P. vivax relapses retrospectively using plasma samples collected from Australian Defence Force personnel deployed to Papua New Guinea and Timor-Leste who participated in clinical trials of tafenoquine during 1999-2001. The CYP2D6 gene was amplified from plasma samples and fully sequenced from 92 participant samples, comprised of relapse (n = 31) and non-relapse (n = 61) samples, revealing 14 different alleles. CYP2D6 phenotypes deduced from combinations of CYP2D6 alleles predicted that among 92 participants 67, 15, and 10 were normal, intermediate, and poor metabolizers, respectively. The deduced CYP2D6 phenotype did not correlate with the corresponding participant's plasma tafenoquine concentrations that were determined in the early 2000s by high-performance liquid chromatography or liquid chromatography-mass spectrometry. Furthermore, the deduced CYP2D6 phenotype did not associate with P. vivax relapse outcomes. Our results indicate that CYP2D6 does not affect plasma tafenoquine concentrations and the efficacy of tafenoquine in preventing P. vivax relapses in the assessed Australian Defence Force personnel.

Using Plasmodium knowlesi as a model for screening Plasmodium vivax blood-stage malaria vaccine targets reveals new candidates.

Plasmodium vivax is responsible for the majority of malaria cases outside Africa. Unlike P. falciparum, the P. vivax life-cycle includes a dormant liver stage, the hypnozoite, which can cause infection in the absence of mosquito transmission. An effective vaccine against P. vivax blood stages would limit symptoms and pathology from such recurrent infections, and therefore could play a critical role in the control of this species. Vaccine development in P. vivax, however, lags considerably behind P. falciparum, which has many identified targets with several having transitioned to Phase II testing. By contrast only one P. vivax blood-stage vaccine candidate based on the Duffy Binding Protein (PvDBP), has reached Phase Ia, in large part because the lack of a continuous in vitro culture system for P. vivax limits systematic screening of new candidates. We used the close phylogenetic relationship between P. vivax and P. knowlesi, for which an in vitro culture system in human erythrocytes exists, to test the scalability of systematic reverse vaccinology to identify and prioritise P. vivax blood-stage targets. A panel of P. vivax proteins predicted to function in erythrocyte invasion were expressed as full-length recombinant ectodomains in a mammalian expression system. Eight of these antigens were used to generate polyclonal antibodies, which were screened for their ability to recognize orthologous proteins in P. knowlesi. These antibodies were then tested for inhibition of growth and invasion of both wild type P. knowlesi and chimeric P. knowlesi lines modified using CRISPR/Cas9 to exchange P. knowlesi genes with their P. vivax orthologues. Candidates that induced antibodies that inhibited invasion to a similar level as PvDBP were identified, confirming the utility of P. knowlesi as a model for P. vivax vaccine development and prioritizing antigens for further follow up.

Adherence to 14-day radical cure for Plasmodium vivax malaria in Papua, Indonesia: a mixed-methods study.

BACKGROUND: Reducing the risk of recurrent Plasmodium vivax malaria is critical for malaria control and elimination. Primaquine (PQ) is the only widely available drug against P. vivax dormant liver stages, but is recommended as a 14-day regimen, which can undermine adherence to a complete course of treatment. METHODS: This is a mixed-methods study to assess socio-cultural factors influencing adherence to a 14-day PQ regimen in a 3-arm, treatment effectiveness trial in Papua, Indonesia. The qualitative strand, consisting of interviews and participant observation was triangulated with a quantitative strand in which trial participants were surveyed using a questionnaire. RESULTS: Trial participants differentiated between two types of malaria: tersiana and tropika, equivalent to P. vivax and Plasmodium falciparum infection, respectively. The perceived severity of both types was similar with 44.0% (267/607) perceiving tersiana vs. 45.1% (274/607) perceiving tropika as more severe. There was no perceived differentiation whether malaria episodes were due to a new infection or relapse; and 71.3% (433/607) acknowledged the possibility of recurrence. Participants were familiar with malaria symptoms and delaying health facility visit by 1-2 days was perceived to increase the likelihood of a positive test. Prior to health facility visits, symptoms were treated with leftover drugs kept at home (40.4%; 245/607) or bought over the counter (17.0%; 103/607). Malaria was considered to be cured with 'blue drugs' (referring to dihydroartemisinin-piperaquine). Conversely, 'brown drugs,' referring to PQ, were not considered malaria medication and instead were perceived as supplements. Adherence to malaria treatment was 71.2% (131/184), in the supervised arm, 56.9% (91/160) in the unsupervised arm and 62.4% (164/263) in the control arm; p = 0.019. Adherence was 47.5% (47/99) among highland Papuans, 51.7% (76/147) among lowland Papuans, and 72.9% (263/361) among non-Papuans; p < 0.001. CONCLUSION: Adherence to malaria treatment was a socio-culturally embedded process during which patients (re-)evaluated the characteristics of the medicines in relation to the course of the illness, their past experiences with illness, and the perceived benefits of the treatment. Structural barriers that hinder the process of patient adherence are crucial to consider in the development and rollout of effective malaria treatment policies.

Treatment of uncomplicated Plasmodium vivax with chloroquine plus radical cure with primaquine without G6PDd testing is safe in Arba Minch, Ethiopia: assessment of clinical and parasitological response.

BACKGROUND: Ethiopia rolled out primaquine nationwide in 2018 for radical cure along with chloroquine for the treatment of uncomplicated Plasmodium vivax malaria in its bid for malaria elimination by 2030. The emergence of anti-malarial drug resistance would challenge the elimination goal. There is limited evidence on the emergence of chloroquine drug resistance. The clinical and parasitological outcomes of treatment of P. vivax with chloroquine plus radical cure using low dose 14 days primaquine were assessed in an endemic area of Ethiopia. METHODS: A semi-directly observed 42-days follow up in-vivo therapeutic efficacy study was conducted from October 2019 to February 2020. Plasmodium vivax mono-species infected patients (n = 102) treated with a 14 days low dose (0.25 mg/kg body weight per day) primaquine plus chloroquine (a total dose of 25 mg base/kg for 3 days) were followed for 42 days to examine clinical and parasitological outcomes. Samples collected at recruitment and days of recurrence were examined by 18 S based nested polymerase chain reaction (nPCR) and Pvmsp3α nPCR-restriction fragment length polymorphism. Asexual parasitaemia and the presence of gametocytes were assessed on the scheduled days using microscopy. Clinical symptoms, haemoglobin levels, and Hillmen urine test were also assessed. RESULTS: Of the 102 patients followed in this study, no early clinical and parasitological failure was observed. All patients had adequate clinical and parasitological responses within the 28 days of follow up. Late clinical (n = 3) and parasitological (n = 6) failures were observed only after day 28. The cumulative incidence of failure was 10.9% (95% confidence interval, 5.8-19.9%) on day 42. Among the paired recurrent samples, identical clones were detected only in two samples on day 0 and day of recurrences (day 30 and 42) using Pvmsp3α genotyping. No adverse effect was detected related to the low dose 14 days primaquine administrations. CONCLUSION: Co-administration of CQ with PQ in the study area is well tolerated and there was no recurrence of P. vivax before 28 days of follow up. Interpretation of CQ plus PQ efficacy should be done with caution especially when the recurrent parasitaemia occurs after day 28. Therapeutic efficacy studies with appropriate design might be informative to rule out chloroquine or primaquine drug resistance and/or metabolism in the study area.

Malaria misdiagnosis in the routine health system in Arba Minch area district in southwest Ethiopia: an implication for malaria control and elimination.

BACKGROUND: Plasmodium falciparum and Plasmodium vivax are coendemic in Ethiopia, with different proportion in different settings. Microscopy is the diagnostic tool in Ethiopian health centres. Accurate species-specific diagnosis is vital for appropriate treatment of cases to interrupt its transmission. Therefore, this study assessed the status of species-specific misdiagnosis by microscope compared with polymerase chain reaction (PCR). METHODS: A health facility based cross-sectional study was conducted from November 2019 to January 2020 in Kolla Shelle Health centre, Arba Minch Zuria district. The study population were suspected malaria cases, who visited the health centre for a diagnosis and treatment. Consecutive microscopy positive cases as well as a sample of microscopically negative cases were included for molecular analysis by polymerase chain reaction (PCR). RESULTS: 254 microscopically negative and 193 microscopically positive malaria suspects were included. Of the 193 malaria positive cases, 46.1% [95% confidence interval (CI) 38.9-53.4] (89/193) were P. falciparum infection, 52.3% (95% CI 45.0-59.5) (101/193) were P. vivax infection, and 1.6% (3/193) had mixed infection of P. falciparum and P. vivax. Of the microscopically positive cases of P. falciparum, 3.4% (3/89) were P. vivax and 11.2% (10/89) were mixed infections with P. falciparum and P. vivax and a single case was negative molecularly. Similarly, of the microscopically positive P. vivax cases, 5.9% (6/101) were P. falciparum and 1% (1/101) was mixed infection. Single case was negative by molecular technique. Of the 254 microscopically negative cases, 0.8% were tested positive for P. falciparum and 2% for P. vivax by PCR. Considering molecular technique as a reference, the sensitivity of microscopy for detecting P. falciparum was 89.2% and for P. vivax, it was 91.2%. The specificity of microscopy for detecting P. falciparum was 96.1% and for P. vivax, it was 97.7%. However, the sensitivity of microscopy in detecting mixed infection of P. falciparum and P. vivax was low (8.3%). CONCLUSION: There were cases left untreated or inappropriately treated due to the species misidentification. Therefore, to minimize this problem, the gaps in the microscopic-based malaria diagnosis should be identified. It is recommended to regularly monitor the competency of malaria microscopists in the study area to improve species identification and diagnosis accuracy.

Effect of adherence to primaquine on the risk of Plasmodium vivax recurrence: a WorldWide Antimalarial Resistance Network systematic review and individual patient data meta-analysis.

BACKGROUND: Imperfect adherence is a major barrier to effective primaquine radical cure of Plasmodium vivax. This study investigated the effect of reduced adherence on the risk of P. vivax recurrence. METHODS: Efficacy studies of patients with uncomplicated P. vivax malaria, including a treatment arm with daily primaquine, published between January 1999 and March 2020 were identified. Individual patient data from eligible studies were pooled using standardized methodology. Adherence to primaquine was inferred from i) the percentage of supervised doses and ii) the total mg/kg dose received compared to the target total mg/kg dose per protocol. The effect of adherence to primaquine on the incidence of P. vivax recurrence between days 7 and 90 was investigated by Cox regression analysis. RESULTS: Of 82 eligible studies, 32 were available including 6917 patients from 18 countries. For adherence assessed by percentage of supervised primaquine, 2790 patients (40.3%) had poor adherence (≤ 50%) and 4127 (59.7%) had complete adherence. The risk of recurrence by day 90 was 14.0% [95% confidence interval: 12.1-16.1] in patients with poor adherence compared to 5.8% [5.0-6.7] following full adherence; p = 0.014. After controlling for age, sex, baseline parasitaemia, and total primaquine dose per protocol, the rate of the first recurrence was higher following poor adherence compared to patients with full adherence (adjusted hazard ratio (AHR) = 2.3 [1.8-2.9]). When adherence was quantified by total mg/kg dose received among 3706 patients, 347 (9.4%) had poor adherence, 88 (2.4%) had moderate adherence, and 3271 (88.2%) had complete adherence to treatment. The risks of recurrence by day 90 were 8.2% [4.3-15.2] in patients with poor adherence and 4.9% [4.1-5.8] in patients with full adherence; p < 0.001. CONCLUSION: Reduced adherence, including less supervision, increases the risk of vivax recurrence.

Community acceptability, participation, and adherence to mass drug administration with primaquine for Plasmodium vivax elimination in Southern Thailand: a mixed methods approach.

BACKGROUND: Mass drug administration (MDA) with primaquine (PQ) is being considered for accelerating Plasmodium vivax elimination in remaining active foci. This study aimed to determine the acceptability of MDA with PQ in malaria endemic villages in a malarious setting in the South of Thailand undergoing MDA with PQ. METHODS: A cross-sectional mixed-methods approach was conducted in seven malaria endemic villages where MDA with PQ was implemented. The data were collected from community villagers and health workers using structured questionnaires, in-depth interviews, and focus group discussions. Descriptive statistics and logistic regression models were used for quantitative data analysis. Thematic analysis was applied for qualitative data. RESULTS: Among a total of 469 participants from the MDA villages, 293 participants were eligible for MDA with PQ and 79.86% (234) completed 14-days of PQ. The logistic regressions indicated that males (adjusted odds ratio: 2.52 [95% confidence interval: 1.33-4.81]) and those who are farmers (2.57 [1.12-5.90]) were most likely to participate in the MDA. Among 293 participants in the post-MDA study, 74.06% had originally agreed to participate in the MDA with PQ while 25.94% had originally reported not wanting to participate in the MDA. Of those who originally reported being willing to participate in the MDA, 71.23% followed through with participation in the first or second round. Conversely, 93.24% of those who originally reported not being willing to participate in the MDA did in fact participate in the MDA. Factors contributing to higher odds of agreeing to participate and following through with participation included being male (1.98 [1.06-3.69]) and correctly responding that malaria is preventable (2.32 [1.01-5.35]) with some differences by village. Five key themes emerged from the qualitative analyses: concern about side effects from taking PQ; disbelief that malaria could be eliminated in this setting; low overall concern about malaria infections; misunderstandings about malaria; and a general need to tailor public health efforts for this unique context. CONCLUSION: While the reported likelihood of participating in MDA was high in this setting, actual follow-through was relatively moderate, partially because of eligibility (roughly 71% of those in the follow-up survey who originally agreed to participate actually followed through with participation). One of the largest concerns among study participants was PQ-related side effects-and these concerns likely heavily influenced participant adherence to the MDA. The results of this study can be used to tailor future MDAs, or other public health interventions, in this and potentially other similar settings.

Implementing a novel capture and ligation probe-PCR method in mass screen and treatment to support malaria elimination efforts in the China-Myanmar border region.

BACKGROUND: Mass screening and treatment (MSAT) for malaria elimination lacks an ideal diagnostic tool to allow sensitive and affordable test of the target population in the field. This study evaluated whether Capture and Ligation Probe-PCR (CLIP-PCR) could be used in a field MSAT in Laiza City, Myanmar. METHODS: On day 0, two dried blood spots were collected from each participant. On day 1, all samples were screened for Plasmodium in a 20 m2 laboratory with workbench, a biosafety cabinet, a refrigerator, a benchtop shaking incubator and a qPCR machine, by four technicians using CLIP-PCR with sample pooling, at a health clinic of the Chinese bordering town of Nabang. On day 2, all positives were followed up and treated. RESULTS: Of 15,038 persons (65% of the total population) screened, 204 (1.36%) were CLIP-PCR positives. Among them, 188, 14, and 2 were infected with Plasmodium vivax, Plasmodium falciparum, and P. vivax/P. falciparum mix, respectively. The testing capacity was 538 persons/day, with a cost of US$0.92 /person. The proportion of submicroscopic infection was 64.7%. All positive individuals received treatment within 72 h after blood collection. CONCLUSION: Using CLIP-PCR in MSAT in low transmission settings can support the malaria elimination efforts in the China-Myanmar border region.

Interventions for promoting patients' adherence to 14-day primaquine treatment in a highly malaria-endemic township in Myanmar: a qualitative study among key stakeholders.

BACKGROUND: Plasmodium vivax malaria is considered a major threat to malaria eradication. The radical cure for P. vivax malaria normally requires a 14-day administration of primaquine (PQ) to clear hypnozoites. However, maintaining adherence to PQ treatment is a significant challenge, particularly in malaria-endemic rural areas. Hence, this study aimed to formulate interventions for promoting patients' commitment to PQ treatment in a highly malaria-endemic township in Myanmar. METHODS: A qualitative study was conducted in Waingmaw Township in northern Myanmar, where P. vivax malaria is highly endemic. Key stakeholders including public health officers and community members participated in focus group discussions (FGDs) and in-depth interviews (IDIs) in September 2022. Data were collected using validated guidelines, translated into English, and visualized through thematic analysis. RESULTS: Responsible individuals from different levels of the Myanmar National Malaria Control Programme participated in the IDIs. Most of them reported being aware of the markedly increasing trend of P. vivax and the possibility of relapse cases, especially among migrants who are lost to follow-up. Workload was a key concern surrounding intervention implementation. The respondents discussed possible interventions, such as implementing directly observed treatment (DOT) by family members, piloting a shorter PQ regimen, expanding the community's malaria volunteer network, and strengthening health education activities using local languages to promote reasonable drug adherence. FGDs among community members revealed that although people were knowledgeable about malaria symptoms, places to seek treatment, and the use of bed nets to prevent mosquito bites, most of them still preferred to be treated by quack doctors and rarely used insecticide-treated nets at worksites. Many often stopped taking the prescribed drugs once the symptoms disappeared. Nevertheless, some respondents requested more bed nets to be distributed and health promotion activities to be conducted. CONCLUSION: In rural areas where human resources are limited, interventions such as implementing family member DOT or shortening PQ regimens should be introduced to enhance the radical cure for the P. vivax infection. Disseminating information about the importance of taking the entire treatment course and emphasizing the burden of relapse is also essential.