Glucose-6-phosphate Dehydrogenase Testing Research Articles

Pregnancy Favism and Severe Hemolytic Anemia in a Patient with Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency: A Case Report

Introduction: Favism is a genetic disease-causing hemolytic anemia in Mexico, primarily due to glucose-6-phosphate dehydrogenase (G6PD) deficiency. Factors triggering favism include infections, pregnancy, certain drugs, and eating beans. The disease is inherited recessively and is more common in men. Diagnosing and managing favism during pregnancy is crucial, as it can lead to severe hemolytic anemia and neonatal jaundice. Case presentation: A female patient, aged 18, presented with severe anemia and thrombocytopenia during her 20-week pregnancy. During pregnancy, the patient experienced jaundice and anemia at 21.5 weeks of gestation. Despite not presenting evidence of bleeding, transfusions were performed due to critical hemoglobin levels. G6PD deficiency was confirmed at 9 days of hospital admission, indicating severe hemolytic anemia. Clinical discussion: The hemolytic crisis in a pregnant woman with favism was triggered by pregnancy and repeated infections of candidiasis. Clinical symptoms of hemolysis were confirmed through G6PD and Coombs tests, and blood transfusion was recommended to prevent complications. The patient's hemoglobin was monitored for signs of active intravascular hemolysis, and a folic acid tablet was recommended during the crisis. Post-crisis management involved periodic controls of hemoglobin, hematocrit, lactate dehydrogenase, and bilirubin. Conclusion: Favism, a prevalent condition in Mexico, should be considered in patients with a family history of hemolytic anemia or compatible symptoms. Early diagnosis and proper management are crucial to prevent complications in the mother and fetus. A multidisciplinary approach, genetic counseling, and medical education are essential for effective management.

Assessing tafenoquine implementation in Brazil: a qualitative evaluation of perceptions of healthcare providers and Plasmodium vivax patients (QualiTRuST Study).

To eliminate malaria by 2035, Brazil must address Plasmodium vivax. Previously, first-line treatment was chloroquine plus 7-day primaquine (PQ) without glucose-6-phosphate dehydrogenase (G6PD) deficiency testing. In 2021, point-of-care quantitative G6PD testing and single-dose tafenoquine (TQ) were piloted in two municipalities. This study evaluated healthcare professional (HCP) and patient perceptions of TQ implementation. This qualitative observational study in Manaus and Porto Velho municipalities evaluated the pilot implementation of the new P. vivax malaria treatment algorithm in high/medium-complexity healthcare units (phase one), then low-complexity units (phase two). Qualitative data collection began 30days after the first TQ treatment in each phase, i.e., October 2021 and March 2022. Perceptions of TQ were assessed using semi-structured in-depth interviews and field notes until saturation. Data were analysed through debriefing sessions, and systematic organization in Excel and MAXQDA, with themes derived by inductive and deductive analysis. The study included 55 patients who received TQ and 94 HCPs. HCPs viewed the TQ single-dose regimen as a significant advancement over 7-day PQ, enhancing adherence. Patients appreciated the shorter duration of treatment and perceived a rapid clinical recovery and fewer side effects. HCPs also noted that TQ resulted in fewer recurrences of P. vivax. The single-dose administration of TQ facilitated complete supervision of the treatment, reduced HCP workload and ensured that patients received the necessary care and did not share the medication with family members. TQ packaging instilled patient trust, though HCPs working in the community found the packaging too bulky. Prescription insecurities among HCPs after initial training prompted requests for additional training. While some patients initially doubted single-dose efficacy, confidence grew with experience. TQ implementation increased awareness of pharmacovigilance and enhanced patient communication, with HCPs adhering to protocols for monitoring haemolysis symptoms. Single-dose TQ for P. vivax malaria in Brazil's Amazon region was positively received by HCPs and patients. Positive perceptions of the medication may aid in improving patient adherence to malaria treatment, thereby reducing malaria recurrences. The findings underscore the importance of adaptive training to optimize P. vivax radical cure implementation.

Perspectives of healthcare professionals on training for quantitative G6PD testing during implementation of tafenoquine in Brazil (QualiTRuST Study).

Effective radical cure of Plasmodium vivax malaria is essential for malaria elimination in Brazil. P. vivax radical cure requires administration of a schizonticide, such as chloroquine, plus an 8-aminoquinoline. However, 8-aminoquinolines cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency, requiring prior screening to exclude those at risk. Brazil is pioneering the implementation of tafenoquine, a single-dose 8-aminoquinoline indicated for P. vivax patients with >70% of normal G6PD activity. Tafenoquine implementation in Manaus and Porto Velho, two municipalities located in the western Brazilian Amazon, included comprehensive training of healthcare professionals (HCPs) on point-of-care quantitative G6PD testing and a new treatment algorithm for P. vivax radical cure incorporating tafenoquine. Training was initially provided to higher-level facilities (phase one) and later adapted for primary care units (phase two). This study analyzed HCP experiences during training and implementation and identified barriers and facilitators. In-depth interviews and focus discussion groups were conducted 30 days after each training for a purposive random sample of 115 HCPs. Thematic analysis was employed using MAXQDA software, analyzing data through inductive and deductive coding. Analysis showed that following the initial training for higher-level facilities, some HCPs did not feel confident performing quantitative G6PD testing and prescribing the tafenoquine regimen. Modifications to the training in phase two resulted in an improvement in understanding the implementation process of the G6PD test and tafenoquine, as well as in the knowledge acquired by HCPs. Additionally, knowledge gaps were addressed through in situ training, peer communication via a messaging app, and educational materials. Training supported effective deployment of the new tools in Manaus and Porto Velho and increased awareness of the need for pharmacovigilance. A training approach for nationwide implementation of these tools was devised. Implementing quantitative G6PD testing and tafenoquine represents a significant shift in P. vivax malaria case management. Consistent engagement with HCPs is needed to overcome challenges in fully integrating these tools within the Brazilian health system.

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

BackgroundCambodia 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.MethodsPhase 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.ResultsIn 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.ConclusionsPlasmodium 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.

Tafenoquine following G6PD screening versus primaquine for the treatment of vivax malaria in Brazil: A cost-effectiveness analysis using a transmission model.

Malaria transmission modelling has demonstrated the potential impact of semiquantitative glucose-6-phosphate dehydrogenase (G6PD) testing and treatment with single-dose tafenoquine for Plasmodium vivax radical cure but has not investigated the associated costs. This study evaluated the cost-effectiveness of P. vivax treatment with tafenoquine after G6PD testing using a transmission model. We explored the cost-effectiveness of using tafenoquine after G6PD screening as compared to usual practice (7-day low-dose primaquine (0.5 mg/kg/day) without G6PD screening) in Brazil using a 10-year time horizon with 5% discounting considering 4 scenarios: (1) tafenoquine for adults only assuming 66.7% primaquine treatment adherence; (2) tafenoquine for adults and children aged >2 years assuming 66.7% primaquine adherence; (3) tafenoquine for adults only assuming 90% primaquine adherence; and (4) tafenoquine for adults only assuming 30% primaquine adherence. The incremental cost-effectiveness ratios (ICERs) were estimated by dividing the incremental costs by the disability-adjusted life years (DALYs) averted. These were compared to a willingness to pay (WTP) threshold of US$7,800 for Brazil, and one-way and probabilistic sensitivity analyses were performed. All 4 scenarios were cost-effective in the base case analysis using this WTP threshold with ICERs ranging from US$154 to US$1,836. One-way sensitivity analyses showed that the results were most sensitive to severity and mortality due to vivax malaria, the lifetime and number of semiquantitative G6PD analysers needed, cost per malaria episode and per G6PD test strips, and life expectancy. All scenarios had a 100% likelihood of being cost-effective at the WTP threshold. The main limitations of this study are due to parameter uncertainty around our cost estimates for low transmission settings, the costs of G6PD screening, and the severity of vivax malaria. In our modelling study that incorporated impact on transmission, tafenoquine prescribed after a semiquantitative G6PD testing was highly likely to be cost-effective in Brazil. These results demonstrate the potential health and economic importance of ensuring safe and effective radical cure.

Implementation of Glucose-6-Phosphate Dehydrogenase (G6PD) testing for Plasmodium vivax case management, a mixed method study from Cambodia.

Plasmodium vivax remains a challenge for malaria elimination since it forms dormant liver stages (hypnozoites) that can reactivate after initial infection. 8-aminoquinolone drugs kill hypnozoites but can cause severe hemolysis in individuals with Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency. The STANDARD G6PD test (Biosensor) is a novel point-of-care diagnostic capable of identifying G6PD deficiency prior to treatment. In 2021, Cambodia implemented the Biosensor to facilitate radical cure treatment for vivax malaria. To assess the Biosensor's implementation after its national rollout, a mixed-methods study was conducted in eight districts across three provinces in Cambodia. Interviews, focus group discussions, and observations explored stakeholders' experiences with G6PD testing and factors influencing its implementation. Quantitative data illustrative of test implementation were gathered from routine surveillance forms and key proportions derived. Qualitative data were analyzed thematically. The main challenge to implementing G6PD testing was that only 49.2% (437/888) of eligible patients reached health centers for G6PD testing following malaria diagnosis by community health workers. Factors influencing this included road conditions and long distances to the health center, compounded by the cost of seeking further care and patients' perceptions of vivax malaria and its treatment. 93.9% (790/841) of eligible vivax malaria patients who successfully completed referral (429/434) and directly presented to the health center (360/407) were G6PD tested. Key enabling factors included the test's acceptability among health workers and their understanding of the rationale for testing. Only 36.5% (443/1213) of eligible vivax episodes appropriately received primaquine. 70.5% (165/234) of female patients and all children under 20 kilograms never received primaquine. Our findings suggest that access to radical cure requires robust infrastructure and income security, which would likely improve referral rates to health centers enabling access. Bringing treatment closer to patients, through community health workers and nuanced community engagement, would improve access to curative treatment of vivax malaria.

Preliminary Investigation into the Prevalence of G6PD Deficiency in a Pediatric African American Population Using a Near-Patient Diagnostic Platform.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is prevalent in the African American population. We identified eighteen G6PD-deficient samples (9%) in a study of residual, de-identified whole blood specimens from 200 African American pediatric patients using a point-of-care instrument. This highlights the possibility of a rapid time to result for G6PD testing, which can be valuable in some clinical scenarios.

Severe Hemolysis during Primaquine Radical Cure of Plasmodium vivax Malaria: Two Systematic Reviews and Individual Patient Data Descriptive Analyses.

Primaquine (PQ) kills Plasmodium vivax hypnozoites but can cause severe hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. We conducted two systematic reviews. The first used data from clinical trials to determine the variety of definitions and frequency of hematological serious adverse events (SAEs) related to PQ treatment of vivax malaria. The second used data from prospective studies and case reports to describe the clinical presentation, management, and outcome of severe PQ-associated hemolysis necessitating hospitalization. In the first review, SAEs were reported in 70 of 249 clinical trials. There were 34 hematological SAEs among 9,824 patients with P. vivax malaria treated with PQ, nine of which necessitated hospitalization or blood transfusion. Criteria used to define SAEs were diverse. In the second review, 21 of 8,487 articles screened reported 163 patients hospitalized after PQ radical cure; 79.9% of whom (123 of 154) were prescribed PQ at ≥ 0.5 mg/kg/day. Overall, 101 patients were categorized as having probable or possible severe PQ-associated hemolysis, 96.8% of whom were G6PD deficient (< 30% activity). The first symptoms of hemolysis were reported primarily on day 2 or 3 (45.5%), and all patients were hospitalized within 7 days of PQ commencement. A total of 57.9% of patients (77 of 133) had blood transfusion. Seven patients (6.9%) with probable or possible hemolysis died. Even when G6PD testing is available, enhanced monitoring for hemolysis is warranted after PQ treatment. Clinical review within the first 5 days of treatment may facilitate early detection and management of hemolysis. More robust definitions of severe PQ-associated hemolysis are required.

Molecular Characterization of Erythrocyte Glucose-6-Phosphate Dehydrogenase Deficiency in Different Ethnic Groups of Blood Donors in Mauritania.

Glucose-6-phosphate-dehydrogenase (G6PD) deficiency is the most frequent enzymopathy worldwide; it is a genetic disorder that affects red blood cells and causes hemolysis. Here, we conducted a study on G6PD-deficient subjects in Mauritania to evaluate the molecular characteristics associated with a deficiency in this enzyme and the frequency of nucleotide polymorphisms in the glucose-6-phosphate dehydrogenase gene. A total of 943 blood samples were collected from blood donors (803 males and 140 females; 364 white Moors; 439 black Moors; 112 Pulaar; 18 Wolof; 10 Soninke). All blood samples were analyzed using a rapid screening test. G6PD status was analyzed quantitatively by the Randox G6PD test. Samples deficient in G6PD were extracted from the whole blood samples and subjected to DNA genotyping. The most frequent G6PD variants were determined by two molecular techniques: restriction fragment length polymorphism (RFLP) and multiplex PCR using the GENESPARK G6PD African kit. A total of six single nucleotide polymorphisms (SNPs) (G202A, A376G, A542T, G680T, C563T, and T968C) were identified. The prevalence of G6PD deficiency in this population sample was 8.1%. The most common mutation was A376G/202A and was characterized by the G6PD A-phenotype, which is more common in the G6PD-deficient black Moors population. The wilaya in Nouakchott was the most affected among the 13 wilayas studied. This study shows, for the first time, the presence of the G680T mutation.

Genetic variants causing G6PD deficiency: Clinical and biochemical data support new WHO classification.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency in erythrocytes causes acute haemolytic anaemia upon exposure to fava beans, drugs, or infection; and it predisposes to neonatal jaundice. The polymorphism of the X-linked G6PD gene has been studied extensively: allele frequencies of up to 25% of different G6PD deficient variants are known in many populations; variants that cause chronic non-spherocytic haemolytic anaemia (CNSHA) are instead all rare. WHO recommends G6PD testing to guide 8-aminoquinolines administration to prevent relapse of Plasmodium vivax infection. From a literature review focused on polymorphic G6PD variants we have retrieved G6PD activity values of 2291 males, and for the mean residual red cell G6PD activity of 16 common variants we have obtained reliable estimates, that range from 1.9% to 33%. There is variation in different datasets: for most variants most G6PD deficient males have a G6PD activity below 30% of normal. There is a direct relationship between residual G6PD activity and substrate affinity (Km G6P ), suggesting a mechanism whereby polymorphic G6PD deficient variants do not entail CNSHA. Extensive overlap in G6PD activity values of individuals with different variants, and no clustering of mean values above or below 10% support the merger of class II and class III variants.

Accelerating towards P. vivax elimination with a novel serological test-and-treat strategy: a modelling case study in Brazil

Plasmodium vivax malaria is challenging to control and eliminate. Treatment with radical cure drugs fails to target the hidden asymptomatic and hypnozoite reservoirs in populations. PvSeroTAT, a novel serological test-and-treat intervention using a serological diagnostic to screen hypnozoite carriers for radical cure eligibility and treatment, could accelerate P.vivax elimination. Using a previously developed mathematical model of P.vivax transmission adapted to the Brazilian context as a case study for implementation, we evaluate the public health impact of various deployment strategies of PvSeroTAT as a mass campaign. We compare relative reductions in prevalence, cases averted, glucose-6-phosphate dehydrogenase (G6PD) tests, and treatment doses of PvSeroTAT campaigns to strengthened case management alone or mass drug administration (MDA) campaigns across different settings. Deploying a single round of PvSeroTAT with 80% coverage to treat cases with a high efficacy radical cure regimen with primaquine is predicted to reduce point population prevalence by 22.5% [95% UI: 20.2%-24.8%] in a peri-urban setting with high transmission and by 25.2% [95% UI: 9.6%-42.2%] in an occupational setting with moderate transmission. In the latter example, while a single PvSeroTAT achieves 9.2% less impact on prevalence and averts 300 less cases per 100,000 than a single MDA (25.2% [95% UI: 9.6%-42.2%] point prevalence reduction versus 34.4% [95% UI: 24.9%-44%]), PvSeroTAT requires 4.6 times less radical cure treatments and G6PD tests. Layering strengthened case management and deploying four rounds of PvSeroTAT six months apart is predicted to reduce point prevalence by a mean of 74.1% [95% UI: 61.3%-86.3%] or more in low transmission settings with less than 10 cases per 1000 population. Modelling predicts that mass campaigns with PvSeroTAT are predicted to reduce P.vivax parasite prevalence across a range of transmission settings and require fewer resources than MDA. In combination with strengthened case management, mass campaigns of serological test-and-treat interventions can accelerate towards P.vivax elimination. This project was funded in part by the Bill and Melinda Gates Foundation and the National Health and Medical Research Council.

Assessing the Operational Feasibility of Integrating Point-of-Care G6PD Testing into Plasmodium vivax Malaria Management in Vietnam.

Plasmodium vivax cases represent more than 50% of a diminishing malaria case load in Vietnam. Safe and effective radical cure strategies could support malaria elimination by 2030. This study investigated the operational feasibility of introducing point-of-care quantitative glucose-6-phosphate dehydrogenase (G6PD) testing into malaria case management practices. A prospective interventional study was conducted at nine district hospitals and commune health stations in Binh Phuoc and Gia Lai provinces in Vietnam over the period of October 2020 to October 2021. The STANDARD™ G6PD Test (SD Biosensor, Seoul, Republic of Korea) was incorporated to inform P. vivax case management. Case management data and patient and health care provider (HCP) perspectives, as well as detailed cost data were collected. The G6PD test results were interpreted correctly by HCP and the treatment algorithm was adhered to for the majority of patients. One HCP consistently ran the test incorrectly, which was identified during the monitoring and resulted in provision of refresher training and updating of training materials and patient retesting. There was wide acceptability of the intervention among patients and HCP albeit with opportunities to improve the counseling materials. Increasing the number of facilities to which the test was deployed and decreases in the malaria cases resulted in higher per patient cost for incorporating G6PD testing into the system. Commodity costs can be reduced by using the 10-unit kits compared to the 25 unit kits, particularly when the case loads are low. These results demonstrate intervention feasibility while also highlighting specific challenges for a country approaching malaria elimination.

A Review of the Current Status of G6PD Deficiency Testing to Guide Radical Cure Treatment for Vivax Malaria.

Plasmodium vivax malaria continues to cause a significant burden of disease in the Asia-Pacific, the Horn of Africa, and the Americas. In addition to schizontocidal treatment, the 8-aminoquinoline drugs are crucial for the complete removal of the parasite from the human host (radical cure). While well tolerated in most recipients, 8-aminoquinolines can cause severe haemolysis in glucose-6-phosphate dehydrogenase (G6PD) deficient patients. G6PD deficiency is one of the most common enzymopathies worldwide; therefore, the WHO recommends routine testing to guide 8-aminoquinoline based treatment for vivax malaria whenever possible. In practice, this is not yet implemented in most malaria endemic countries. This review provides an update of the characteristics of the most used G6PD diagnostics. We describe the current state of policy and implementation of routine point-of-care G6PD testing in malaria endemic countries and highlight key knowledge gaps that hinder broader implementation. Identified challenges include optimal training of health facility staff on point-of-care diagnostics, quality control of novel G6PD diagnostics, and culturally appropriate information and communication with affected communities around G6PD deficiency and implications for treatment.

Glucose-6-Phosphate Dehydrogenase (G6PD) Measurement Using Biosensors by Community-Based Village Malaria Workers and Hospital Laboratory Staff in Cambodia: A Quantitative Study.

Vivax malaria can relapse after an initial infection due to dormant liver stages of the parasite. Radical cure can prevent relapses but requires the measurement of glucose-6-phosphate dehydrogenase enzyme (G6PD) activity to identify G6PD-deficient patients at risk of drug-induced haemolysis. In the absence of reliable G6PD testing, vivax patients are denied radical curative treatment in many places, including rural Cambodia. A novel Biosensor, 'G6PD Standard' (SD Biosensor, Republic of Korea; Biosensor), can measure G6PD activity at the point of care. The objectives of this study were to compare the G6PD activity readings using Biosensors by village malaria workers (VMWs) and hospital-based laboratory technicians (LTs), and to compare the G6PD deficiency categorization recommended by the Biosensor manufacturer with categories derived from a locally estimated adjusted male median (AMM) in Kravanh district, Cambodia. Participants were enrolled between 2021 and 2022 in western Cambodia. Each of the 28 VMWs and 5 LTs received a Biosensor and standardized training on its use. The G6PD activities of febrile patients identified in the community were measured by VMWs; in a subset, a second reading was done by LTs. All participants were tested for malaria by rapid diagnostic test (RDT). The adjusted male median (AMM) was calculated from all RDT-negative participants and defined as 100% G6PD activity. VMWs measured activities in 1344 participants. Of that total, 1327 (98.7%) readings were included in the analysis, and 68 of these had a positive RDT result. We calculated 100% activity as 6.4 U/gHb (interquartile range: 4.5 to 7.8); 9.9% (124/1259) of RDT-negative participants had G6PD activities below 30%, 15.2% (191/1259) had activities between 30% and 70%, and 75.0% (944/1259) had activities greater than 70%. Repeat measurements among 114 participants showed a significant correlation of G6PD readings (rs = 0.784, p < 0.001) between VMWs and LTs. Based on the manufacturer's recommendations, 285 participants (21.5%) had less than 30% activity; however, based on the AMM, 132 participants (10.0%) had less than 30% activity. The G6PD measurements by VMWs and LTs were similar. With the provisions of training, supervision, and monitoring, VMWs could play an important role in the management of vivax malaria, which is critical for the rapid elimination of malaria regionally. Definitions of deficiency based on the manufacturer's recommendations and the population-specific AMM differed significantly, which may warrant revision of these recommendations.

Peripheral blood smear diagnosis of G6PD deficiency in an 83-year-old man.

An 83-year-old male was admitted to the emergency department for abrupt onset of jaundice and asthenia. Vital signs were in range, and physical examination was unremarkable except for jaundice and hyperchromic urine. Full blood count on admission showed his hemoglobin concentration to be 81 g/L with no alteration in white blood cell (WBC) or platelets count. Biochemical tests were significant for high bilirubin level 417.07 μmol/L (indirect bilirubin 369.53 μmol/L) and abnormal lactate dehydrogenase level 1520 μmol/L. The hypothesis of hemolytic anemia was confirmed by further laboratory tests, which showed undetectable haptoglobin and increased reticulocyte count 196 × 109/L. Direct and indirect Coombs tests were both negative, suggesting a Coombs-negative hemolytic anemia. A first blood smear did not show schistocytes and the coagulation profile was in order. Medical history was negative for oxidant drugs assumption, prosthetic heart valves, and liver and kidney diseases. Chest radiography was normal, and a complete abdominal echography did not show hepatomegaly or splenomegaly. Meanwhile, the patient clinical course was complicated by pain and tightness in the chest and dizziness and an electrocardiogram was compatible with a non-ST-segment elevation myocardial infarction. A new set of lab tests showed severe anemia with 66 g/L of hemoglobin, and the patient undergone blood transfusion. In the suspicion of a hemolytic crisis in the context of glucose-6-phosphate dehydrogenase (G6PD) deficiency, an accurate anamnesis was collected and the patient confirmed the assumption of fava beans 3 days earlier after a long time. G6PD levels could not be assessed overnight, but a second blood smear was significant for bite, blister, and ghost cells (Figure 1 A and B), which are findings compatible with G6PD deficiency. Testing for G6PD was performed as soon as available and it revealed a significant G6PD deficiency (2.7 U/g Hb, normal range 8–14 U/g Hb, overestimated because of low Hb concentration). This is a case of G6PD deficiency diagnosed in 83-yer-old patient with no previous similar event in an unfavorable setting (emergency department by night). It highlights the necessity of high clinical suspicion of G6PD deficiency in the context of Coombs-negative hemolytic anemia, even in elderly patients. It is worth noting that different species of fava beans have a different capacity in inducing oxidative stress. Lastly, blood smear can still provide useful morphological data in supporting the diagnosis, regardless the immediate availability of G6PD test. Fabio Giglio conceived the study; D. Sannipoli, D. T. Clerici, and Fabio Giglio provided all clinical and hematological data; A. Boletini and P. Ronchi performed and analyzed morphology; D. Sannipoli and Fabio Giglio wrote the paper. All authors approved the paper. The authors declare no conflict of interest. Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Quantitative G6PD Deficiency Screening in Routine Malaria Diagnostic Units in the Brazilian Amazon (SAFEPRIM): An Operational Mixed-Methods Study.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency testing is not routinely performed before primaquine treatment in most Plasmodium vivax endemic areas, despite the risk of primaquine-associated hemolysis. This is due to the operational challenges associated with pragmatic G6PD testing and as such needs to be addressed. This mixed-methods operational study was aimed at implementing the quantitative point-of-care StandardTM G6PD (SD Biosensor, Korea) screening test in malaria treatment units (MTUs) in the municipalities of Rio Preto da Eva and Mâncio Lima, in the Brazilian Amazon, between mid-January 2020 and December 2020. In total, 1286 P. vivax cases were treated based on the Standard G6PD test: 1230 had activity equal to or greater than 4.0 U/g Hb, and 56 less than 4.0 U/g Hb. No G6PD deficient (G6PDd) genotypes were found in 96 samples from the 1230, and only 21 of the 56 G6PDd cases had confirmed G6PDd genotypes. Evaluations were conducted on the proficiency of health care professionals (HCPs) training to perform the test, the reliability of testing performed in the field, and the perceptions of HCPs and patients about the implementation. Post-training proficiency was 73.4% after a 4-hour training session. This study revealed that locations with lower malaria caseloads will need regular refresher training. The test was well accepted by both HCPs and patients. Signs and symptoms of hemolysis were not always associated with malaria treatment drugs by HCPs and patients. Point-of-care quantitative G6PD testing can be performed at MTUs in the Brazilian Amazon to inform treatment decisions with primaquine. Limitations related to technical and cultural aspects need to be addressed further when expanding screening to larger areas.

G6PD testing and radical cure for Plasmodium vivax in Cambodia: A mixed methods implementation study.

IntroductionCambodia aims to eliminate malaria by 2025, however tackling Plasmodium vivax (P.v) presents multiple challenges. The prevalence of glucose-6-phosphate dehydrogenase (G6PD) deficiency has prevented the deployment of 8-aminoquinolones for “radical cure”, due to the risk of severe haemolysis. Patients with P. vivax have therefore continued to experience recurrent relapses leading to cumulative health and socioeconomic burden. The recent advent of point of care testing for G6PD deficiency has made radical cure a possibility, however at the time of the study lack of operational experience and guidance meant that they had not been introduced. This study therefore aimed to design, implement and evaluate a new care pathway for the radical cure of P.vivax.MethodsThis implementation study took place in Pursat province, Western Cambodia. The interventions were co-developed with key stakeholders at the national, district, and local level, through a continuous process of consultations as well as formal meetings. Mixed methods were used to evaluate the feasibility of the intervention including its uptake (G6PD testing rate and the initiation of primaquine treatment according to G6PD status); adherence (self-reported); and acceptability, using quantitative analysis of primary and secondary data as well as focus group discussions and key informant interviews.ResultsThe co-development process resulted in the design of a new care pathway with supporting interventions, and a phased approach to their implementation. Patients diagnosed with P.v infection by Village Malaria Workers (VMWs) were referred to local health centres for point-of-care G6PD testing and initiation of radical cure treatment with 14-day or 8-week primaquine regimens depending on G6PD status. VMWs carried out follow-up in the community on days 3, 7 and 14. Supporting interventions included training, community sensitisation, and the development of a smartphone and tablet application to aid referral, follow-up and surveillance. The testing rate was low initially but increased rapidly over time, reflecting the deliberately cautious phased approach to implementation. In total 626 adults received G6PD testing, for a total of 675 episodes. Of these 555 occurred in patients with normal G6PD activity and nearly all (549/555, 98.8%) were initiated on PQ14. Of the 120 with deficient/intermediate G6PD activity 61 (50.8%) were initiated on PQ8W. Self-reported adherence was high (100% and 95.1% respectively). No severe adverse events were reported. The pathway was found to be highly acceptable by both staff and patients. The supporting interventions and gradual introduction were critical to success. Challenges included travel to remote areas and mobility of P.v patients.ConclusionThe new care pathway with supporting interventions was highly feasible with high levels of uptake, adherence and acceptability in this setting where high prevalence of G6PD deficiency is high and there is a well-established network of VMWs. Scaling up of the P.v radical cure programme is currently underway in Cambodia and a decline in reduction in the burden of malaria is being seen, bringing Cambodia a step closer to elimination.

Genotype-phenotype association and biochemical analyses of glucose-6-phosphate dehydrogenase variants: Implications for the hemolytic risk of using 8-aminoquinolines for radical cure.

Background: Plasmodium vivax remains the malaria species posing a major threat to human health worldwide owing to its relapse mechanism. Currently, the only drugs of choice for radical cure are the 8-aminoquinolines (primaquine and tafenoquine), which are capable of killing hypnozoites and thus preventing P. vivax relapse. However, the therapeutic use of primaquine and tafenoquine is restricted because these drugs can cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. This study aimed to assess and understand the hemolytic risk of using 8-aminoquinolines for radical treatment in a malaria endemic area of Thailand. Methods: The prevalence of G6PD deficiency was determined using a quantitative test in 1,125 individuals. Multiplexed high-resolution meltinging (HRM) assays were developed and applied to detect 12 G6PD mutations. Furthermore, biochemical and structural characterization of G6PD variants was carried out to understand the molecular basis of enzyme deficiency. Results: The prevalence of G6PD deficiency was 6.76% (76/1,125), as assessed by a phenotypic test. Multiplexed HRM assays revealed G6PD Mahidol in 15.04% (77/512) of males and 28.38% (174/613) of females, as well as G6PD Aures in one female. G6PD activity above the 30% cut-off was detected in those carrying G6PD Mahidol, even in hemizygous male individuals. Two variants, G6PD Murcia Oristano and G6PD Songklanagarind + Viangchan, were identified for the first time in Thailand. Biochemical characterization revealed that structural instability is the primary cause of enzyme deficiency in G6PD Aures, G6PD Murcia Oristano, G6PD Songklanagarind + Viangchan, and G6PD Chinese 4 + Viangchan, with double G6PD mutations causing more severe enzyme deficiency. Conclusion: In western Thailand, up to 22% of people may be ineligible for radical cure. Routine qualitative tests may be insufficient for G6PD testing, so quantitative tests should be implemented. G6PD genotyping should also be used to confirm G6PD status, especially in female individuals suspected of having G6PD deficiency. People with double G6PD mutations are more likely to have hemolysis than are those with single G6PD mutations because the double mutations significantly reduce the catalytic activity as well as the structural stability of the protein.

Glucose 6 Phosphate Dehydrogenase (G6PD) quantitation using biosensors at the point of first contact: a mixed method study in Cambodia

BackgroundQuantitative measurement of Glucose-6-Phosphate Dehydrogenase (G6PD) enzyme activity is critical to decide on appropriate treatment and provision of radical cure regimens for vivax malaria. Biosensors are point-of-care semi-quantitative analysers that measure G6PD enzyme activity. The main objective of this study was to evaluate the operational aspects of biosensor deployment in the hands of village malaria workers (VMWs) in Cambodia over a year.MethodsFollowing initial orientation and training at Kravanh Referral Hospital, each VMW (n = 28) and laboratory technician (n = 5) was provided a biosensor (STANDARD SD Biosensor, Republic of Korea) with supplies for routine use. Over the next 12 months VMWs convened every month for refresher training, to collect supplies, and to recalibrate and test their biosensors. A quantitative self-administered questionnaire was used to assess the skills necessary to use the biosensor after the initial training. Subsequently, VMWs were visited at their location of work for field observation and evaluation using an observer-administered questionnaire. All quantitative questionnaire-based data were analysed descriptively. Semi-structured interviews (SSIs) were conducted among all participants to explore their experience and practicalities of using the biosensor in the field. SSIs were transcribed and translated into English and underwent thematic analysis.ResultsA total of 33 participants completed the training and subsequently used the biosensor in the community. Quantitative assessments demonstrated progressive improvement in skills using the biosensor. VMWs expressed confidence and enthusiasm to use biosensors in their routine work. Providing G6PD testing at the point of first contact avoids a multitude of barriers patients have to overcome when travelling to health centres for G6PD testing and radical cure. Deploying biosensors in routine work of VMWs was also considered an opportunity to expand and strengthen the role of VMWs as health care providers in the community. VMWs reported practical concerns related to the use of biosensor such as difficulty in using two pipettes, difficulty in extracting the code chip from the machine, and the narrow base of buffer tube.ConclusionsVMWs considered the biosensor a practical and beneficial tool in their routine work. Providing VMWs with biosensors can be considered when followed by appropriate training and regular supervision. Providing community management of vivax malaria at the point of first contact could be key for elimination.

DIAGNOSTIC ACCURACY COMPARISON OF THE QUANTITATIVE POINT-OF-CARE ECO G6PD AND BREWERS TEST FOR ASSESSMENT OF G6PD DEFICIENCY IN INFECTIOUS DISEASES

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked, hereditary condition caused by mutations in the G6PD gene, that results in protein variants with different enzyme activity levels. G6PD provides reducing power to NADPH (reduced form of nicotinamide adenine dinucleotide phosphate). NADPH effect is critical in red blood cells (RBC) due to the absence of a nucleus. Clinical manifestations are usually mild and are induced by an exogenous agent, such as drugs, infections, or dietary (fava beans). The G6PD deficiency diagnosis is crucial in infectious diseases because both infections and treatment can induce acute hemolysis. Rarely, life-threatening cases can require transfusions of RBC and hemodialysis. The gold standard for the measurement of G6PD activity is a quantitative UV spectrophotometric assay, however, it is inaccessible in most cases. Methemoglobin reduction test (Brewer's test) is a qualitative spot test most used for G6PD deficiency screening, but is imprecise, requires a cold chain, some laboratory infrastructure, trained personnel, and is time-costing. Point-of-care (POC) is a simple and fast semi-quantitative test that is becoming increasingly important when a prompt diagnosis is required and in health care of out-of-reach communities. To compare the diagnostic accuracy of the quantitative POC ECO and Brewers test for assessment of G6PD deficiency. A cross-sectional diagnostic accuracy study was enrolled between May 2020 and December 2021 at the Instituto Nacional de Infectologia Evandro Chagas, Fundação Oswaldo Cruz. Individuals older than 17 years old with a suspicious diagnosis of an infectious disease were eligible. 125 convenience blood samples were tested to compare concordance analysis between Brewers test and POC STANDARD G6PD. The exclusion criteria were hemoglobin concentration < 7.0 g/dL due to technical device limitations. A total of 125 subjects (38 females and 87 males) were recruited. The median age was 42.0 years (range 18–76) in females and 45.0 years (range 19–78) in males. The median of G6PD activity was 7.1 U/g Hb (0.2-17.5). We observed a trend of concordance between the G6PD status (deficient or normal), with low frequencies of discordances in the screenings. The strength of agreement between G6PD tests was classified as almost perfect in all participants (k = 0.82, IC95% = 0.66,0.97) and in subgroups female (k = 0.83, IC95% = 0.59,1.00) and male (k = 0.81, IC95% = 0.59,1.00). The total concordance percentage (number of concordances/total) was 95% in females, 97% in males, and 96% in all participants. Here, we demonstrate POC ECO G6PD test could be performed during medical consultation, guiding the most appropriate therapy almost immediately. In addition to saving time and having comparable results to Brewer's test, it could be performed even in remote areas and with scarce resources, dispensing minimum laboratory requirements. Our findings will be of value to diagnose conditions that could have life-threatening consequences if not timely recognized, such as before initiating primaquine for the treatment of malaria. Therefore, it can improve healthcare in vulnerable isolated communities. We observed a high concordance result between Brewers test and POC STANDARD G6PD, which is becoming the reference test to diagnose G6PD deficiency worldwide.