Malaria cases sometimes go undetected using RDTs due to their inaccurate use, poor storage conditions and failure to detect low parasitaemia (<50parasites/μL). This could result in continuous ...transmission of malaria and sustenance of parasite reservoirs. Molecular diagnostic tools are more sensitive and specific than RDTs in the detection of plasmodium parasites. However, the Polymerase Chain Reaction (PCR) is not routinely used because equipment and reagents are expensive and requires highly skilled personnel. Loop-mediated isothermal amplification (LAMP) is a relatively new molecular diagnostic tool for malaria with all the advantages of PCR (sensitive and specific) without the mentioned disadvantages. However, it has not been evaluated extensively as a point of care diagnostic in the field. One hundred and fifteen used RDTs were collected from health facilities in Northern Namibia in a blind study and PCR and LAMP were used to determine the presence of Plasmodium DNA. The sensitivities and PPV were 40.91% and 90% respectively for RDTs, 72.73% and 100% respectively for PCR with LAMP as the golden standard. In low malaria transmission settings, LAMP can be also be considered for use as a surveillance tool to detect all sources of malaria and determine proportion of low parasitaemia infections in order to eliminate them.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
The growing prevalence of deadly microbes with resistance to previously life-saving drug therapies is a dire threat to human health. Detection of low abundance pathogen sequences remains a ...challenge for metagenomic Next Generation Sequencing (NGS). We introduce FLASH (Finding Low Abundance Sequences by Hybridization), a next-generation CRISPR/Cas9 diagnostic method that takes advantage of the efficiency, specificity and flexibility of Cas9 to enrich for a programmed set of sequences. FLASH-NGS achieves up to 5 orders of magnitude of enrichment and sub-attomolar gene detection with minimal background. We provide an open-source software tool (FLASHit) for guide RNA design. Here we applied it to detection of antimicrobial resistance genes in respiratory fluid and dried blood spots, but FLASH-NGS is applicable to all areas that rely on multiplex PCR.
Programmes naturally scale down IRS, but their approaches are unsystematic, and given the instability of hotspots in low endemic areas, how best to target IRS is unclear. Because Plasmodium ...falciparum transmission clusters around households,6,7 a reactive, mini-outbreak approach that targets a ring of households around index cases might be as effective as and more cost-effective than pre-season IRS. ...in settings with high outdoor transmission, alternative and novel vector control tools might be needed.11 Reactive focal mass drug administration could be used as a standalone or complementary intervention to target the parasite reservoir in humans.10 Third, targeted IRS can only be implemented with robust surveillance to detect, report, and investigate the origin and location of cases in a timely way. ...transitioning between two quite different interventions (pre-season campaigns and mini-outbreak response) will be challenging but necessary.
Local and cross-border importation remain major challenges to malaria elimination and are difficult to measure using traditional surveillance data. To address this challenge, we systematically ...collected parasite genetic data and travel history from thousands of malaria cases across northeastern Namibia and estimated human mobility from mobile phone data. We observed strong fine-scale spatial structure in local parasite populations, providing positive evidence that the majority of cases were due to local transmission. This result was largely consistent with estimates from mobile phone and travel history data. However, genetic data identified more detailed and extensive evidence of parasite connectivity over hundreds of kilometers than the other data, within Namibia and across the Angolan and Zambian borders. Our results provide a framework for incorporating genetic data into malaria surveillance and provide evidence that both strengthening of local interventions and regional coordination are likely necessary to eliminate malaria in this region of Southern Africa.
In areas of low and unstable transmission, malaria cases occur in populations with lower access to malaria services and interventions, and in groups with specific malaria risk exposures often away ...from the household. In support of the Namibian National Vector Borne Disease Program’s drive to better target interventions based upon risk, we implemented a health facility-based case control study aimed to identify risk factors for symptomatic malaria in Zambezi Region, northern Namibia. A total of 770 febrile individuals reporting to 6 health facilities and testing positive by rapid diagnostic test (RDT) between February 2015 and April 2016 were recruited as cases; 641 febrile individuals testing negative by RDT at the same health facilities through June 2016 were recruited as controls. Data on socio-demographics, housing construction, overnight travel, use of malaria prevention and outdoor behaviors at night were collected through interview and recorded on a tablet-based questionnaire. Remotely-sensed environmental data were extracted for geo-located village residence locations. Multivariable logistic regression was conducted to identify risk factors and latent class analyses (LCA) used to identify and characterize high-risk subgroups. The majority of participants (87% of cases and 69% of controls) were recruited during the 2016 transmission season, an outbreak year in Southern Africa. After adjustment, cases were more likely to be cattle herders (Adjusted Odds Ratio (aOR): 4.46 95%CI 1.05–18.96), members of the police or other security personnel (aOR: 4.60 95%CI: 1.16–18.16), and pensioners/unemployed persons (aOR: 2.25 95%CI 1.24–4.08), compared to agricultural workers (most common category). Children (aOR 2.28 95%CI 1.13–4.59) and self-identified students were at higher risk of malaria (aOR: 4.32 95%CI 2.31–8.10). Other actionable risk factors for malaria included housing and behavioral characteristics, including traditional home construction and sleeping in an open structure (versus modern structure: aOR: 2.01 95%CI 1.45–2.79 and aOR: 4.76 95%CI: 2.14–10.57); cross border travel in the prior 30 days (aOR: 10.55 95%CI 2.94–37.84); and outdoor agricultural work at night (aOR: 2.09 95%CI 1.12–3.87). Malaria preventive activities were all protective and included personal use of an insecticide treated net (ITN) (aOR: 0.61 95%CI 0.42–0.87), adequate household ITN coverage (aOR: 0.63 95%CI 0.42–0.94), and household indoor residual spraying (IRS) in the past year (versus never sprayed: (aOR: 0.63 95%CI 0.44–0.90). A number of environmental factors were associated with increased risk of malaria, including lower temperatures, higher rainfall and increased vegetation for the 30 days prior to diagnosis and residing more than 5 minutes from a health facility. LCA identified six classes of cases, with class membership strongly correlated with occupation, age and select behavioral risk factors. Use of ITNs and IRS coverage was similarly low across classes. For malaria elimination these high-risk groups will need targeted and tailored intervention strategies, for example, by implementing alternative delivery methods of interventions through schools and worksites, as well as the use of specific interventions that address outdoor transmission.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In low malaria-endemic settings, screening and treatment of individuals in close proximity to index cases, also known as reactive case detection (RACD), is practised for surveillance and response. ...However, other approaches could be more effective for reducing transmission. We aimed to evaluate the effectiveness of reactive focal mass drug administration (rfMDA) and reactive focal vector control (RAVC) in the low malaria-endemic setting of Zambezi (Namibia).
We did a cluster-randomised controlled, open-label trial using a two-by-two factorial design of 56 enumeration area clusters in the low malaria-endemic setting of Zambezi (Namibia). We randomly assigned these clusters using restricted randomisation to four groups: RACD only, rfMDA only, RAVC plus RACD, or rfMDA plus RAVC. RACD involved rapid diagnostic testing and treatment with artemether-lumefantrine and single-dose primaquine, rfMDA involved presumptive treatment with artemether-lumefantrine, and RAVC involved indoor residual spraying with pirimiphos-methyl. Interventions were administered within 500 m of index cases. To evaluate the effectiveness of interventions targeting the parasite reservoir in humans (rfMDA vs RACD), in mosquitoes (RAVC vs no RAVC), and in both humans and mosquitoes (rfMDA plus RAVC vs RACD only), an intention-to-treat analysis was done. For each of the three comparisons, the primary outcome was the cumulative incidence of locally acquired malaria cases. This trial is registered with ClinicalTrials.gov, number NCT02610400.
Between Jan 1, 2017, and Dec 31, 2017, 55 enumeration area clusters had 1118 eligible index cases that led to 342 interventions covering 8948 individuals. The cumulative incidence of locally acquired malaria was 30·8 per 1000 person-years (95% CI 12·8–48·7) in the clusters that received rfMDA versus 38·3 per 1000 person-years (23·0–53·6) in the clusters that received RACD; 30·2 per 1000 person-years (15·0–45·5) in the clusters that received RAVC versus 38·9 per 1000 person-years (20·7–57·1) in the clusters that did not receive RAVC; and 25·0 per 1000 person-years (5·2–44·7) in the clusters that received rfMDA plus RAVC versus 41·4 per 1000 person-years (21·5–61·2) in the clusters that received RACD only. After adjusting for imbalances in baseline and implementation factors, the incidence of malaria was lower in clusters receiving rfMDA than in those receiving RACD (adjusted incidence rate ratio 0·52 95% CI 0·16–0·88, p=0·009), lower in clusters receiving RAVC than in those that did not (0·48 0·16–0·80, p=0·002), and lower in clusters that received rfMDA plus RAVC than in those receiving RACD only (0·26 0·10–0·68, p=0·006). No serious adverse events were reported.
In a low malaria-endemic setting, rfMDA and RAVC, implemented alone and in combination, reduced malaria transmission and should be considered as alternatives to RACD for elimination of malaria.
Novartis Foundation, Bill & Melinda Gates Foundation, and Horchow Family Fund.
Pyrethroid-based indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) have been employed as key vector control measures against malaria in Namibia. However, pyrethroid resistance ...in Anopheles mosquitoes may compromise the efficacy of these interventions. To address this challenge, the World Health Organization (WHO) recommends the use of piperonyl butoxide (PBO) LLINs in areas where pyrethroid resistance is confirmed to be mediated by mixed function oxidase (MFO).
This study assessed the susceptibility of Anopheles gambiae sensu lato (s.l.) mosquitoes to WHO tube bioassays with 4% DDT and 0.05% deltamethrin insecticides. Additionally, the study explored the effect of piperonyl butoxide (PBO) synergist by sequentially exposing mosquitoes to deltamethrin (0.05%) alone, PBO (4%) + deltamethrin (0.05%), and PBO alone. The Anopheles mosquitoes were further identified morphologically and molecularly.
The findings revealed that An. gambiae sensu stricto (s.s.) (62%) was more prevalent than Anopheles arabiensis (38%). The WHO tube bioassays confirmed resistance to deltamethrin 0.05% in the Oshikoto, Kunene, and Kavango West regions, with mortality rates of 79, 86, and 67%, respectively. In contrast, An. arabiensis displayed resistance to deltamethrin 0.05% in Oshikoto (82% mortality) and reduced susceptibility in Kavango West (96% mortality). Notably, there was reduced susceptibility to DDT 4% in both An. gambiae s.s. and An. arabiensis from the Kavango West region. Subsequently, a subsample from PBO synergist assays in 2020 demonstrated a high proportion of An. arabiensis in Oshana (84.4%) and Oshikoto (73.6%), and 0.42% of Anopheles quadriannulatus in Oshana. Non-amplifiers were also present (15.2% in Oshana; 26.4% in Oshikoto). Deltamethrin resistance with less than 95% mortality, was consistently observed in An. gambiae s.l. populations across all sites in both 2020 and 2021. Following pre-exposure to the PBO synergist, susceptibility to deltamethrin was fully restored with 100.0% mortality at all sites in 2020 and 2021.
Pyrethroid resistance has been identified in An. gambiae s.s. and An. arabiensis in the Kavango West, Kunene, and Oshikoto regions, indicating potential challenges for pyrethroid-based IRS and LLINs. Consequently, the data highlights the promise of pyrethroid-PBO LLINs in addressing resistance issues in the region.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
Rapid diagnostic tests (RDTs) that rely on the detection of
Plasmodium falciparum
histidine-rich protein 2 (
Pf
HRP2) have become key tools for diagnosing
P. falciparum
infection. ...The utility of RDTs can be limited by
Pf
HRP2 persistence, however it can be a potential benefit in low transmission settings where detection of persistent
Pf
HRP2 using newer ultra-sensitive
Pf
HRP2 based RDTs can serve as a surveillance tool to identify recent exposure. Better understanding of the dynamics of
Pf
HRP2 over the course of a malaria infection can inform optimal use of RDTs.
Methods
A previously published mathematical model was refined to mimic the production and decay of
Pf
HRP2 during a malaria infection. Data from 15 individuals from volunteer infection studies were used to update the original model and estimate key model parameters. The refined model was applied to a cohort of patients from Namibia who received treatment for clinical malaria infection for whom longitudinal
Pf
HRP2 concentrations were measured.
Results
The refinement of the
Pf
HRP2 dynamic model indicated that in malaria naïve hosts,
P. falciparum
parasites of the 3D7 strain produce 33.6 × 10
−15
g (95% CI 25.0–42.1 × 10
−15
g) of
Pf
HRP2 in vivo per parasite replication cycle, with an elimination half-life of 1.67 days (95% CI 1.11–3.40 days). The refined model included these updated parameters and incorporated individualized body fluid volume calculations, which improved predictive accuracy when compared to the original model. The performance of the model in predicting clearance of
Pf
HRP2 post treatment in clinical samples from six adults with
P. falciparum
infection in Namibia improved when using a longer elimination half-life of 4.5 days, with 14% to 67% of observations for each individual within the predicted range.
Conclusions
The updated mathematical model can predict the growth and clearance of
Pf
HRP2 during the production and decay of a mono-infection with
P. falciparum
, increasing the understanding of
Pf
HRP2 antigen dynamics. This model can guide the optimal use of
Pf
HRP2-based RDTs for reliable diagnosis of
P. falciparum
infection and re-infection in endemic settings, but also for malaria surveillance and elimination programmes in low transmission areas.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
As malaria transmission decreases, the proportion of infections that are asymptomatic at any given time increases. This poses a challenge for diagnosis as routinely used rapid diagnostic tests (RDTs) ...miss asymptomatic malaria cases with low parasite densities due to poor sensitivity. Yet, asymptomatic infections can contribute to onward transmission of malaria and therefore act as infectious reservoirs and perpetuate malaria transmission. This study compared the performance of RDTs to loop-mediated isothermal amplification (LAMP) in the diagnosis of malaria during reactive active case detection surveillance.
All reported malaria cases in the Engela Health District of Namibia were traced back to their place of residence and persons living within the four closest neighbouring houses to the index case (neighbourhood) were tested for malaria infection with RDTs and dried blood spots (DBS) were collected. LAMP and nested PCR (nPCR) were carried out on all RDTs and DBS. The same procedure was followed in randomly selected control neighbourhoods.
Some 3151 individuals were tested by RDT, LAMP and nPCR. Sensitivity of RDTs and LAMP were 9.30 and 95.50%, respectively, and specificities were 99.27 and 99.92%, respectively, compared to nPCR. LAMP carried out on collected RDTs showed a sensitivity and specificity of 95.35 and 99.85% compared to nPCR carried out on DBS. There were 2 RDT samples that were negative by LAMP but the corresponding DBS samples were positive by PCR.
The study showed that LAMP had the equivalent performance as nPCR for the identification of Plasmodium falciparum infection. Given its relative simplicity to implement over more complex and time-consuming methods, such as PCR, LAMP is particularly useful in elimination settings where high sensitivity and ease of operation are important.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Reactive case detection (RACD) around passively detected malaria cases is a strategy to identify and treat hotspots of malaria transmission. This study investigated the unproven assumption on which ...this approach is based, that in low transmission settings, infections cluster over small scales.
A prospective case-control study was conducted between January 2013 and August 2014 in Ohangwena and Omusati regions in north central Namibia. Patients attending health facilities who tested positive by malaria rapid diagnostic test (RDT) (index cases) were traced back to their home. All occupants of index case households (n = 116 households) and surrounding households (n = 225) were screened for Plasmodium infection with a rapid diagnostic test (RDT) and loop mediated isothermal amplification (LAMP) and interviewed to identify risk factors. A comparison group of 286 randomly-selected control households was also screened, to compare infection levels of RACD and non-RACD households and their neighbours. Logistic regression was used to investigate spatial clustering of patent and sub-patent infections around index cases and to identify potential risk factors that would inform screening approaches and identify risk groups. Estimates of the impact of RACD on onward transmission to mosquitoes was made using previously published figures of infection rates.
Prevalence of Plasmodium falciparum infection by LAMP was 3.4%, 1.4% and 0.4% in index-case households, neighbors of index case households and control households respectively; adjusted odds ratio 6.1 95%CI 1.9-19.5 comparing case households versus control households. Using data from Engela, neighbors of cases had higher odds of infection adjusted OR 5.0 95%CI 1.3-18.9 compared to control households. All infections identified by RDTs were afebrile and RDTs identified only a small proportion of infections in case (n = 7; 17%) and control (0%) neighborhoods. Based on published estimates of patent and sub-patent infectiousness, these results suggest that infections missed by RDTs during RACD would allow 50-71% of infections to mosquitoes to occur in this setting.
Malaria infections cluster around passively detected cases. The majority of infections are asymptomatic and of densities below the limit of detection of current RDTs. RACD using standard RDTs are unlikely to detect enough malaria infections to dramatically reduce transmission. In low transmission settings such as Namibia more sensitive field diagnostics or forms of focal presumptive treatment should be tested as strategies to reduce malaria transmission.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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