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Background & Aims: In 2020, the World Health Organization (WHO) recommended peripartum antiviral prophylaxis (PAP) for pregnant women infected with hepatitis B virus (HBV) with high viremia (≥200,000 IU/ml). Hepatitis B e antigen (HBeAg) was also recommended as an alternative when HBV DNA is unavailable. To inform policymaking and guide the implementation of prevention of mother-to-child transmission strategies, we conducted a systematic review and meta-analysis to estimate the proportion of HBV-infected pregnant women eligible for PAP at global and regional levels. Methods: We searched PubMed, EMBASE, Scopus, and CENTRAL for studies involving HBV-infected pregnant women. We extracted proportions of women with high viremia (≥200,000 IU/ml), proportions of women with positive HBeAg, proportions of women cross-stratified based on HBV DNA and HBeAg, and the risk of child infection in these maternal groups. Proportions were pooled using random-effects meta-analysis. Results: Of 6,999 articles, 131 studies involving 71,712 HBV-infected pregnant women were included. The number of studies per WHO region was 66 (Western Pacific), 21 (Europe), 17 (Africa), 11 (Americas), nine (Eastern Mediterranean), and seven (South-East Asia). The overall pooled proportion of high viremia was 21.27% (95% CI 17.77-25.26%), with significant regional variation: Western Pacific (31.56%), Americas (23.06%), Southeast Asia (15.62%), Africa (12.45%), Europe (9.98%), and Eastern Mediterranean (7.81%). HBeAg positivity showed similar regional variation. After cross-stratification, the proportions of high viremia and positive HBeAg, high viremia and negative HBeAg, low viremia and positive HBeAg, and low viremia and negative HBeAg were 15.24% (95% CI 11.12-20.53%), 2.70% (95% CI 1.88-3.86%), 3.69% (95% CI 2.86-4.75%), and 75.59% (95% CI 69.15-81.05%), respectively. The corresponding risks of child infection following birth dose vaccination without immune globulin and PAP were 14.86% (95% CI 8.43-24.88%), 6.94% (95% CI 2.92-15.62%), 7.14% (95% CI 1.00-37.03%), and 0.14% (95% CI 0.02-1.00%). Conclusions: Approximately 20% of HBV-infected pregnant women are eligible for PAP. Given significant regional variations, each country should tailor strategies for HBsAg screening, risk stratification, and PAP in routine antenatal care. Impact and implications: In 2020, the WHO recommended that pregnant women who test positive for the hepatitis B surface antigen (HBsAg) undergo HBV DNA testing or HBeAg and those with high viremia (≥200,000 IU/ml) or positive HBeAg receive PAP. To effectively implement new HBV PMTCT interventions and integrate HBV screening, risk stratification, and antiviral prophylaxis into routine antenatal care services, estimating the proportion of HBV-infected pregnant women eligible for PAP is critical. In this systematic review and meta-analysis, we found that approximately one-fifth of HBV-infected pregnant women are eligible for PAP based on HBV DNA testing, and a similar proportion is eligible based on HBeAg testing. Owing to substantial regional variations in eligibility proportions and the availability and costs of different tests, it is vital for each country to optimize strategies that integrate HBV screening, risk stratification, and PAP into routine antenatal care services. Systematic review registration: This study was registered with PROSPERO (Protocol No: CRD42021266545).
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OBJECTIVE: Economic feasibility of eliminating mother-to-child transmission (MTCT) of hepatitis B virus (HBV) in highly endemic African countries remains uncertain. Prevention of MTCT (PMTCT) involves screening pregnant women for hepatitis B surface antigen (HBsAg), identifying those with high viral loads or hepatitis B e antigen (HBeAg), and administering tenofovir prophylaxis to high-risk women. We estimated the costs of integrating PMTCT services into antenatal care in Burkina Faso, based on four different strategies to select women for tenofovir prophylaxis: (1) HBV DNA (≥200 000 IU/mL), (2) HBeAg, (3) hepatitis B core-related antigen rapid diagnostic test (HBcrAg-RDT) and (4) all HBsAg-positive women. METHODS: Using a micro-costing approach, we estimated the incremental economic cost of integrating each strategy into routine antenatal care in 2024, compared to neonatal vaccination alone. Sensitivity analyses explored variations in prevalence, service coverage, test and tenofovir prices. RESULTS: HBcrAg-RDT strategy was the least expensive, with a total economic cost of US$3959689, compared to HBV DNA (US$6128875), HBeAg (US$4135233), and treat-all (US$4141206). The cost per pregnant woman receiving tenofovir prophylaxis varied from US$61.88 (Treat-all) to US$1071.05 (HBV DNA). The Treat-All strategy had the lowest marginal cost due to a higher number of women on tenofovir (66928) compared to HBV DNA (5722), HBeAg (10020), and HBcrAg-RDT (7234). In sensitivity analyses, the treat-all strategy became less expensive when the tenofovir price decreased. CONCLUSION: HBcrAg-RDT minimizes resource use and costs, representing 0.61% of Burkina Faso's 2022 health budget. This study highlights the potential economic feasibility of these strategies and provides valuable resources for conducting cost-effectiveness analyses.
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Antivirales , Hepatitis B , Transmisión Vertical de Enfermedad Infecciosa , Complicaciones Infecciosas del Embarazo , Atención Prenatal , Tenofovir , Humanos , Femenino , Burkina Faso , Embarazo , Atención Prenatal/economía , Atención Prenatal/métodos , Complicaciones Infecciosas del Embarazo/tratamiento farmacológico , Complicaciones Infecciosas del Embarazo/prevención & control , Tenofovir/uso terapéutico , Tenofovir/economía , Tenofovir/administración & dosificación , Hepatitis B/prevención & control , Hepatitis B/tratamiento farmacológico , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Antivirales/uso terapéutico , Antivirales/economía , Antivirales/administración & dosificación , Tamizaje Masivo/economía , Tamizaje Masivo/métodos , Antígenos e de la Hepatitis B/sangre , Análisis Costo-Beneficio , Antígenos de Superficie de la Hepatitis B/sangre , Adulto , ADN Viral , Virus de la Hepatitis B , Carga ViralRESUMEN
BACKGROUND: Elimination of mother-to-child transmission of hepatitis B virus (HBV) requires infant immunoprophylaxis and antiviral prophylaxis for pregnant women with high viral loads. Since real-time polymerase chain reaction (RT-PCR), a gold standard for assessing antiviral eligibility, is neither accessible nor affordable for women living in low-income and middle-income countries (LMICs), rapid diagnostic tests (RDTs) detecting alternative HBV markers may be needed. To inform future development of the target product profile (TPP) for RDTs to identify highly viremic women, we used a discrete choice experiment (DCE) and elicited preference and trade-off of healthcare workers (HCW) in Africa between the following four attributes of fictional RDTs: price, time-to-result, diagnostic sensitivity, and specificity. METHODS: Through an online questionnaire survey, we asked participants to indicate their preferred test from a set of two RDTs in seven choice tasks with varying levels of the four attributes. We used mixed multinomial logit models to quantify the utility gain or loss generated by each attribute. We attempted to define minimal and optimal criteria for test attributes that can satisfy ≥ 70% and ≥ 90% of HCWs, respectively, as an alternative to RT-PCR. RESULTS: A total of 555 HCWs from 41 African countries participated. Increases in sensitivity and specificity generated significant utility and increases in cost and time-to-result generated significant disutility. The size of the coefficients for the highest attribute levels relative to the reference levels were in the following order: sensitivity (ß = 3.749), cost (ß = -2.550), specificity (ß = 1.134), and time-to-result (ß = -0.284). Doctors cared most about test sensitivity, while public health practitioners cared about cost and midwives about time-to-result. For an RDT with 95% specificity, costing 1 US$, and yielding results in 20 min, the minimally acceptable test sensitivity would be 82.5% and the optimally acceptable sensitivity would be 87.5%. CONCLUSIONS: African HCWs would prefer an RDT with the following order of priority: higher sensitivity, lower cost, higher specificity, and shorter time-to-result. The development and optimization of RDTs that can meet the criteria are urgently needed to scale up the prevention of HBV mother-to-child transmission in LMICs.
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Virus de la Hepatitis B , Mujeres Embarazadas , Lactante , Femenino , Embarazo , Humanos , Virus de la Hepatitis B/genética , Carga Viral , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , Sensibilidad y Especificidad , Antivirales , Personal de SaludRESUMEN
BACKGROUND: Malaria continues to cause burden in various parts of the world. Haiti, a Caribbean country, is among those aiming to eliminate malaria within a few years. Two surveys were conducted in Haiti during which we aimed to evaluate the performance of the simple and rapid procedure for ultra-rapid extraction-loop-mediated isothermal amplification (PURE-LAMP) method with dried blood spots as an alternative diagnostic method for malaria in the context of low to very low rates of transmission. METHODS: Febrile and afebrile people were recruited from three administrative divisions within Haiti: Nippes, Sud and Grand'Anse, during the summers of 2017 (early August to early September) and 2018 (late July to late August). Their blood samples were tested by microscopy, rapid diagnostic tests (RDT), PURE-LAMP and nested PCR to detect Plasmodium infection. Sensitivity, specificity, positive and negative predictive values and kappa statistics were estimated with the nested PCR results as the gold standard. RESULTS: Among 1074 samples analyzed, a positive rate of 8.3% was calculated based on the nested PCR results. Among febrile participants, the rates in 2017 and 2018 were 14.6% and 1.4%, respectively. Three positives were detected among 172 afebrile participants in 2018 by PURE-LAMP and nested PCR, and all three were from the same locality. There was no afebrile participants recruited in 2017. The PURE-LAMP, RDT and microscopy had respective sensitivities of 100%, 85.4% and 49.4%. All of the testing methods had specificities over 99%. CONCLUSIONS: This study confirmed the high performance of the PURE-LAMP method to detect Plasmodium infection with dried blood spots and recommends its use in targeted mass screening and treatment activities in low endemic areas of malaria.
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Malaria Falciparum , Malaria , Humanos , Haití , Sensibilidad y Especificidad , Malaria/diagnóstico , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Diagnóstico Molecular/métodos , Malaria Falciparum/diagnóstico , Malaria Falciparum/epidemiología , Plasmodium falciparumRESUMEN
BACKGROUND: Sub-Saharan Africa is highly endemic for hepatitis B virus (HBV); historically, most people were exposed during childhood through vertical or horizontal transmission. Although all African countries now provide a three-dose infant hepatitis B vaccination starting at age 6-8 weeks, only a third of African countries have introduced birth dose (HepB-BD) vaccine. Adding HepB-BD is fundamental to prevent vertical transmission, but its effectiveness in preventing horizontal transmission, compared with the three-dose infant vaccination alone, is unknown. We aimed to estimate the risk of early horizontal transmission in children of hepatitis B surface antigen (HBsAg)-negative mothers in sub-Saharan Africa stratified according to the vaccination schedule. METHODS: In this systematic review and meta-analysis we searched MEDLINE, Global Health, Embase, African Index Medicus and African Journals Online from their inception to Oct 24, 2022, for studies reporting HBsAg or HBV DNA, or both, in children (aged 0-5 years) of HBsAg-negative mothers. We excluded studies if children were only tested at birth. Two reviewers independently screened the titles and abstracts of all articles and data were extracted using a standardised pre-piloted data extraction sheet, and authors were contacted if any important information was missing. The primary outcome was the risk of HBV infection in children of HBsAg-negative mothers, stratified by vaccination schedule (no vaccination, first dose at 6-8 weeks, or first dose at birth). We pooled the child risks of HBsAg or HBV DNA-positivity from the age of 0 years to 5 years via a random-effect meta-analysis using a generalised linear mixed model. The study was registered on PROSPERO, CRD42021236203. FINDINGS: Of 8856 articles identified, 27 studies evaluating 10â003 children of HBsAg-negative mothers were included. The pooled risks of infection were 6·16% (95% CI 3·05-12·04; 155/1407) in the no vaccination group, 0·21% (0·04-1·15; 10/3425) in children who received their first dose at 6-8 weeks, and 0·05% (0·00-1·32; 3/2902) in children who received their first dose at birth. The difference was not statistically significant in children who received their first dose at 6-8 weeks and children who received their first dose at birth after adjusting for the study period, region, and maternal HIV status (test of moderators p=0·37). INTERPRETATION: In children of HBsAg-negative mothers, the risk of infection might be minimal even with the vaccination starting at 6-8 weeks, without clear additional benefit from HepB-BD. When births take place at home and timely administration of HepB-BD is challenging, antenatal HBsAg screening and selective HepB-BD might allow efficient allocation of resources to mother and child pairs at high risk compared with universal HepB-BD. FUNDING: None. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.
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Virus de la Hepatitis B , Hepatitis B , Lactante , Recién Nacido , Niño , Femenino , Humanos , Embarazo , Madres , Antígenos de Superficie de la Hepatitis B , ADN Viral , Hepatitis B/epidemiología , Hepatitis B/prevención & control , Vacunas contra Hepatitis B , Transmisión Vertical de Enfermedad Infecciosa/prevención & control , África del Sur del Sahara/epidemiologíaRESUMEN
Background: To reduce mortality associated with hepatitis B virus (HBV) infection, timely detection of cirrhosis and early-stage hepatocellular carcinoma (HCC) is essential. In low-income countries, however, HBV-infected people have limited access to liver histopathology, a reference test. Recently, Asian studies have suggested the usefulness of an inexpensive serum biomarker called Mac-2 binding protein glycosylation isomer (M2BPGi) in staging liver fibrosis and predicting HCC in HBV-infected patients. Methods: We systematically searched PubMed for studies examining the performance of M2BPGi in staging liver fibrosis in HBV-infected people, published up to September 21, 2021, to elucidate the knowledge gap. We then conducted a cross-sectional study of 339 HBV-infected patients in The Gambia (cirrhosis = 65, HCC = 73, non-cirrhosis non-HCC = 201). We evaluated the association of M2BPGi with cirrhosis and HCC by computing odds ratios (ORs) derived from logistic regression. We also assessed the performance of M2BPGi to stage liver fibrosis in 49 patients who underwent liver biopsy (derivation set) and 217 patients with transient elastography (validation set). Using the derivation set we drew the receiver operating characteristics (ROC) curves to identify optimal M2BPGi thresholds to indicate significant fibrosis and cirrhosis using biopsy as a reference. We then applied these cut-offs to the validation set to obtain its sensitivity and specificity for indicating significant fibrosis and cirrhosis using transient elastography as a reference. Results: The systematic review identified 13 studies, all of which were conducted in East Asia and none in Africa. In The Gambia, positive M2BPGi was significantly associated with both cirrhosis (adjusted OR = 7.8, 95% CI = 3.1-19.7) and HCC (adjusted OR = 10.1, 2.6-40.2). The areas under the ROC curve (AUROC) in the derivation and validation set were 0.62 and 0.78, respectively, to diagnose significant fibrosis, and 0.80 and 0.89, respectively, to diagnose cirrhosis. By applying the optimal cut-offs, the sensitivity and specificity in the validation set were 61.5% and 93.4%, respectively, to diagnose significant fibrosis, and 72.5% and 92.2%, respectively, for cirrhosis. Conclusions: To the best of our knowledge, this is the first evaluation of M2BPGi in HBV-infected African population. The findings supported its accuracy in the diagnosis of cirrhosis in HBV-infected patients in West Africa.
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Carcinoma Hepatocelular , Hepatitis B , Neoplasias Hepáticas , Humanos , Virus de la Hepatitis B , Glicosilación , Estudios Transversales , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/complicaciones , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/complicaciones , Carcinoma Hepatocelular/metabolismo , Hepatitis B/complicaciones , Hepatitis B/diagnóstico , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/complicaciones , Neoplasias Hepáticas/metabolismoRESUMEN
Background: Viral hepatitis causes an important global health burden. In 2016, the World Health Assembly adopted an objective to globally eliminate this as a public health threat by 2030. However, significant gaps exist between countries in their progress. Haiti is the last country that has introduced infant hepatitis B vaccines into the routine immunization program in the Region of the Americas, and its schedule still does not incorporate birth dose vaccines. As the first step to raise awareness of viral hepatitis in this country, we conducted a systematic review and meta-analysis to estimate the prevalence of hepatitis B (HBV), C (HCV), and D (HDV) viruses in Haiti. Methods: We searched PubMed, EMBASE, Web of Science and Scopus for studies reporting the prevalence of HBV, HCV and HDV among Haitian, with no language restriction, published until November 30th, 2021. Prevalence was pooled via a random-effects meta-analysis using a generalized linear mixed model with the logit link. Results: Of 453 articles retrieved, 25 studies were included: 16 reported the prevalence of hepatitis B surface antigen (HBsAg), three for anti-HCV antibody, and six for both HBsAg and anti-HCV. No study was found for HDV prevalence. The pooled prevalence of HBsAg was 0.7% [95% confidence interval (CI): 0.3-1.4, I 2 = 77.7%] among children, 3.5% (95% CI: 2.8-4.4, I 2 = 93.2%) in the general adult population and 7.4% (95% CI: 4.0-13.3, I 2 = 83.9%) in high-risk adult population. The pooled prevalence of anti-HCV antibody was 0.9% (95% CI: 0.6-1.4, I 2 = 93.5%) among the general population and 1.4% (95% CI: 0.4-4.2, I 2 = 0.0%) in high-risk adult population. No study reported the prevalence of anti-HCV antibody exclusively in children. Interpretation: The prevalence of blood-borne hepatitis, particularly that of HBV, is substantial in Haiti. The introduction of birth dose hepatitis B vaccines and improving access to testing and treatment services should be urgently considered to meet the elimination goal. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022298081, identifier: PROSPERO (CRD42022298081).
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Hepatitis B , Hepatitis C , Hepatitis D , Hepatitis Viral Humana , Virosis , Adulto , Niño , Humanos , Lactante , Haití/epidemiología , Hepatitis B/epidemiología , Antígenos de Superficie de la Hepatitis B , Vacunas contra Hepatitis B , Prevalencia , Hepatitis C/epidemiología , Hepatitis D/epidemiología , Antígenos de la Hepatitis/inmunologíaRESUMEN
To achieve global hepatitis elimination by 2030, it is critical to prevent the mother-to-child transmission (MTCT) of hepatitis B virus (HBV). Since 2009, the WHO has recommended administering hepatitis B vaccine to all neonates within 24 h of birth to prevent MTCT. However, many countries in sub-Saharan Africa only provide hepatitis B immunization at the age of 6, 10, and 14 weeks or 8, 12, and 16 weeks using a combined vaccine. To accelerate the introduction of the hepatitis B birth dose vaccine (HepB-BD) into sub-Saharan Africa, it is critical to establish to what extent the addition of HepB-BD can further reduce HBV transmission in areas where three-dose infant vaccination has been implemented. We therefore designed a study to evaluate the impact, acceptability, and cost-effectiveness of incorporating the HepB-BD into the routine immunization program in a real-life field condition in Burkina Faso, where the hepatitis B vaccination is currently scheduled at 8-12-16 weeks. Through a multidisciplinary approach combining epidemiology, anthropology, and health economics, the Neonatal Vaccination against Hepatitis B in Africa (NéoVac) study conducts a pragmatic stepped wedge cluster randomized controlled trial in rural areas of the Hauts-Bassins Region. The study was registered in ClinicalTrials.gov (identifier: NCT04029454). A health center is designated as a cluster, and the introduction of HepB-BD will be rolled out sequentially in 24 centers. Following an initial period in which no health center administers HepB-BD, one center will be randomly allocated to incorporate HepB-BD. Then, at a regular interval, another center will be randomized to cross from the control to the intervention period, until all 24 centers integrate HepB-BD. Pregnant women attending antenatal care will be systematically invited to participate. Infants born during the control period will follow the conventional immunization schedule (8-12-16 weeks), while those born in the interventional period will receive HepB-BD in addition to the routine vaccines (0-8-12-16 weeks). The primary outcome, the proportion of hepatitis B surface antigen (HBsAg) positivity in infants aged at 9 months, will be compared between children born before and after HepB-BD introduction. The study will generate data that may assist governments and stakeholders in sub-Saharan Africa to make evidence-based decisions about whether to add HepB-BD into the national immunization programs.
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Limited access to nucleic acid testing (NAT) to quantify HBV DNA levels, an essential tool to determine anti-HBV treatment eligibility, represents a significant barrier to scale up HBV diagnostic services in resource-limited countries. Hepatitis B core-related antigen (HBcrAg) has the potential to become an affordable alternative because of its low cost (US$ <15/assay) and strong correlation with HBV DNA levels in treatment-naïve patients. However, the current assay requires plasma or serum. To further facilitate its application to decentralized settings, we developed and evaluated a standardized procedure to quantify HBcrAg using dried blood spots as a tool to diagnose HBV-infected people with high viraemia. We evaluated the following elution method optimized to quantify HBcrAg: suspension of a punched blood-soaked disc (11 mm) of Whatman 903 Protein Saver Card in 450 µL of PBS 0.05% Tween 20, followed by an incubation for 4 h at room temperature and a centrifugation at 10,000 g for 10 minutes. 150 µL of DBS eluate was used to quantify HBcrAg using chemiluminescent enzyme immunoassay (LUMIPULSE® G600II, Fujirebio). The limit of detection of dried blood spot HBcrAg in relation with HBV DNA levels was 19,115 IU/mL across the five major HBV genotypes (A/B/C/D/E). A strong linear correlation was confirmed between dried blood spot HBcrAg and HBV DNA levels (r = 0.94, p < 0.0001) in samples with high viral loads (range: 3.7-7.0 log IU/mL). The coefficient of variation ranged between 4.0-11.2% for repeatability and 3.9-12.2% for reproducibility. Analytical specificity was 100% (95% CI: 83.9-100%) in HBV-negative samples. Using our elution method, it may be possible to identify HBV-infected patients with high viraemia who need antiviral therapy using dried blood spot and HBcrAg. A large-scale clinical validation is warranted in resource-limited countries.
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Hepatitis B Crónica , Hepatitis B , ADN Viral , Hepatitis B/diagnóstico , Antígenos del Núcleo de la Hepatitis B , Virus de la Hepatitis B/genética , Hepatitis B Crónica/diagnóstico , Humanos , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: Malaria is one of the most important parasitic infectious diseases for which almost half of the world's population is at risk. Although several diagnostic methods are now available to detect the infection, more sensitive and applicable tests are still required in the field. The loop-mediated isothermal amplification (LAMP) method is a DNA amplification tool in which the DNA amplification can be achieved by incubation at a stable temperature. A malaria detection kit based on this methodology has already been commercialized and is being used in some countries. The kit includes two reaction tubes: one targeting the common Plasmodium genus (Pan tube) and the other specifically targeting Plasmodium falciparum (Pf tube). In parallel, a simple DNA extraction method, the procedure for ultra rapid extraction (PURE), which can produce a DNA solution suitable for the LAMP reaction without the use of a centrifuge, has also become available. In this study, the sensitivity of the combination of the PURE and LAMP methods (PURE-LAMP) was evaluated with archived dried clinical blood samples of imported malaria cases, including P. falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae. RESULTS: Using a nested PCR as the reference, 117 samples including 46 P. falciparum, 7 P. vivax, 9 P. ovale, 4 P. malariae, and 51 negative cases were tested. The PURE-LAMP Pan correctly identified 64 of the 66 positives and the 51 negatives. Among the Pan-positive samples 45 P. falciparum were also detected with the PURE-LAMP Pf. The PURE-LAMP Pan and PURE-LAMP Pf had respective sensitivities of 96.96% (95% CI 89.47-99.63) and 97.82% (95% CI 88.47-99.94) and common specificity of 1. CONCLUSION: The PURE-LAMP system is accurate when used with dried blood spots and extendable to the field.
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Pruebas Diagnósticas de Rutina/métodos , Malaria/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Plasmodium/aislamiento & purificación , Humanos , Malaria Falciparum/diagnóstico , Plasmodium falciparum/aislamiento & purificación , Sensibilidad y EspecificidadRESUMEN
A microscopy-based diagnosis is the gold standard for the detection and identification of malaria parasites in a patient's blood. However, the detection of cases involving a low number of parasites and the differentiation of species sometimes requires a skilled microscopist. Although PCR-based diagnostic methods are already known to be very powerful tools, the time required to apply such methods is still much longer in comparison to traditional microscopic observation. Thus, improvements to PCR systems are sought to facilitate the more rapid and accurate detection of human malaria parasites Plasmodium falciparum, P. vivax, P. ovale, and P. malariae, as well as P. knowlesi, which is a simian malaria parasite that is currently widely distributed in Southeast Asia. A nested PCR that targets the small subunit ribosomal RNA genes of malaria parasites was performed using a "fast PCR enzyme". In the first PCR, universal primers for all parasite species were used. In the second PCR, inner-specific primers, which targeted sequences from P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi, were used. The PCR reaction time was reduced with the use of the "fast PCR enzyme", with only 65 minutes required to perform the first and second PCRs. The specific primers only reacted with the sequences of their targeted parasite species and never cross-reacted with sequences from other species under the defined PCR conditions. The diagnoses of 36 clinical samples that were obtained using this new PCR system were highly consistent with the microscopic diagnoses.