A comparison between dissection techniques for the assessment of parity in Anopheles arabiensis and determination of sac stage in mosquitoes alive or dead on collection

BACKGROUND: The determination of parous rates in mosquitoes, despite numerous shortcomings, remains a tool to evaluate the effectiveness of control programs and to determine vectorial capacity in malaria vectors. Two dissection techniques are used for this. For one, the tracheoles of dried ovaries are examined with a compound microscope and in the other the follicular stalk of ovaries is examined, wet, with a stereomicroscope. The second method also enables the sac stage of parous insects (which provides information on the duration of the oviposition cycle) and the mated status of insects to be determined. Despite widespread use the two techniques have not previously been compared. METHODS: We compared the two dissection techniques using Anopheles arabiensis, collected with a tent-trap in Eritrea. The paired ovaries were removed in water and one was examined by each method. From a separate set of dissections from Tanzania, we also determined if the sac stages of Anopheles gambiae s.l. (83% of 183 identified by PCR being Anopheles arabiensis the remainder being A. gambiae) that were alive on collection were different to those that died on collection and what the implications for vectorial capacity estimation might be. RESULTS: Seven per cent of the dry ovaries could not be classified due to granulation (yolk) in the ovariole that obscured the tracheoles. The sensitivity of the dry dissection was 88.51% (CI [79.88-94.35%]) and the specificity was 93.55% (CI [87.68-97.17%]) among the 211 ovaries that could be classified by the dry technique and compared to the ovaries dissected wet. 1,823 live and 1,416 dead from Furvela tent-traps, CDC light-trap and window-trap collections were dissected 'wet' from Tanzania. In these collections parous insects were more likely to die compared to nulliparous ones. The proportion of parous mosquitoes with 'a' sacs (indicative of recent oviposition) was significantly greater in insects that were dead (0.36) on collection in the morning compared to those that were alive (0.12) (Chi square 138.93, p < 0.001). There was a preponderance of newly emerged virgin insects in the outdoor collection (Chi sq = 8.84, p = 0.003). CONCLUSIONS: In anophelines the examination of mosquito ovaries using transmitted light in a 'wet' dissection is a more useful and informative technique than examination of dry ovaries. In order to correctly estimate the duration of the oviposition cycle mosquitoes should be dissected as soon as possible after collection. Younger insects were more likely to attempt to feed outdoors rather than indoors.publishersversionpublishe

‘We like it wet’: a comparison between dissection techniques for the assessment of parity in Anopheles arabiensis and determination of sac stage in mosquitoes alive or dead on collection

Background The determination of parous rates in mosquitoes, despite numerous shortcomings, remains a tool to evaluate the effectiveness of control programs and to determine vectorial capacity in malaria vectors. Two dissection techniques are used for this. For one, the tracheoles of dried ovaries are examined with a compound microscope and in the other the follicular stalk of ovaries is examined, wet, with a stereomicroscope. The second method also enables the sac stage of parous insects (which provides information on the duration of the oviposition cycle) and the mated status of insects to be determined. Despite widespread use the two techniques have not previously been compared. Methods We compared the two dissection techniques using Anopheles arabiensis, collected with a tent-trap in Eritrea. The paired ovaries were removed in water and one was examined by each method. From a separate set of dissections from Tanzania, we also determined if the sac stages of Anopheles gambiae s.l. (83% of 183 identified by PCR being Anopheles arabiensis the remainder being A. gambiae) that were alive on collection were different to those that died on collection and what the implications for vectorial capacity estimation might be. Results Seven per cent of the dry ovaries could not be classified due to granulation (yolk) in the ovariole that obscured the tracheoles. The sensitivity of the dry dissection was 88.51% (CI [79.88–94.35%]) and the specificity was 93.55% (CI [87.68–97.17%]) among the 211 ovaries that could be classified by the dry technique and compared to the ovaries dissected wet. 1,823 live and 1,416 dead from Furvela tent-traps, CDC light-trap and window-trap collections were dissected ‘wet’ from Tanzania. In these collections parous insects were more likely to die compared to nulliparous ones. The proportion of parous mosquitoes with ‘a’ sacs (indicative of recent oviposition) was significantly greater in insects that were dead (0.36) on collection in the morning compared to those that were alive (0.12) (Chi square 138.93, p < 0.001). There was a preponderance of newly emerged virgin insects in the outdoor collection (Chi sq = 8.84, p = 0.003). Conclusions In anophelines the examination of mosquito ovaries using transmitted light in a ‘wet’ dissection is a more useful and informative technique than examination of dry ovaries. In order to correctly estimate the duration of the oviposition cycle mosquitoes should be dissected as soon as possible after collection. Younger insects were more likely to attempt to feed outdoors rather than indoors

Corrigendum to 'A novel multiplex qPCR assay for detection of Plasmodium falciparum with histidine-rich protein 2 and 3 (pfhrp2 and pfhrp3) deletions in polyclonal infections'.

The authors wish to correct two typographical errors in the manuscript. In the Methods (Section 5.3: Assay optimization), the concentration unit of dNTPs was wrongly written as 800 nM (nanomolar) and should be corrected to 800mM (millimolar). Furthermore, in Table S1 of the Supplementary material, the primers and probe sequences for Pfhrp3 are incorrect. They should be written: Pfhrp3_F2: 5’-ACGGATTTCATTTTAACCCTTCACGA-‘3, Pfhrp3_R2: 5’-TGAGAATCATCAAAACAAGCATTAGC-‘3 and Pfhrp3_probe: JOE’-ACAATTCCCATACTTTACATCATGCA-‘3 BHQ1. A revised Table S1 is included (below). The primers and probe sequences of Pfhrp3 in Figure 3S of the supplementary material are correct. The authors regret any confusion caused and appreciate the opportunity to correct these mistakes The authors would like to apologise for any inconvenience caused

Prevalence of CYP2C8*2 and *3 among Eritreans and its Potential Impact on Artesunate/Amodiaquine Treatment

Background: In Eritrea, artesunate–amodiaquine is the first-line treatment against uncomplicated malaria. Amodiaquine, which is mainly bio-transformed by CYP2C8, is known to be associated with adverse events of different severity. Extrapyramidal events are among the less common but have been reported with non-negligible frequency in Eritrea. This study was conducted to investigate the allele frequencies of CYP2C8*2 and *3, both associated with decreased amodiaquine metabolism, among the Eritrean population. Methods: During September–November 2018, dried blood samples from 380 participants and 17 patients who previously had experienced extrapyramidal symptoms following treatment of artesunate–amodiaquine were collected and PCR-RFLP genotyped for CYP2C8*2 and *3. Results: The allele frequencies of CYP2C8*2 and *3 were determined as 5.9% (95% CI: 4.4– 7.8) and 4.6% (95% CI: 3.2– 6.3), respectively. Four out of the 17 patients with extrapyramidal reactions showed to be carriers of the alleles. Conclusion: CYP2C8*2 and *3 frequencies among Eritreans were found to be intermediate between the documented for Caucasian and African populations. These findings, along with the alleles not being decisive for the occurrence of extrapyramidal events, might be of importance regarding the amodiaquine-containing malaria treatment in Eritrea. Furthermore, it suggests a significant proportion of slow amodiaquine metabolizers in the Sahel region, information of potential interest in the context of amodiaquine-involving seasonal malaria chemoprevention

Epidemiology of mutant Plasmodium falciparum parasites lacking histidine-rich protein 2/3 genes in Eritrea 2 years after switching from HRP2-based RDTs

Eritrea was the first African country to complete a nationwide switch in 2016 away from HRP2-based RDTs due to high rates of false-negative RDT results caused by Plasmodium falciparum parasites lacking hrp2/hrp3 genes. A cross-sectional survey was conducted during 2019 enrolling symptomatic malaria patients from nine health facilities across three zones consecutively to investigate the epidemiology of P. falciparum lacking hrp2/3 after the RDT switch. Molecular analyses of 715 samples revealed the overall prevalence of hrp2-, hrp3-, and dual hrp2/3-deleted parasites as 9.4% (95%CI 7.4–11.7%), 41.7% (95% CI 38.1–45.3%) and 7.6% (95% CI 5.8–9.7%), respectively. The prevalence of hrp2- and hrp3-deletion is heterogeneous within and between zones: highest in Anseba (27.1% and 57.9%), followed by Gash Barka (6.4% and 37.9%) and Debub zone (5.2% and 43.8%). hrp2/3-deleted parasites have multiple diverse haplotypes, with many shared or connected among parasites of different hrp2/3 status, indicating mutant parasites have likely evolved from multiple and local parasite genetic backgrounds. The findings show although prevalence of hrp2/3-deleted parasites is lower 2 years after RDT switching, HRP2-based RDTs remain unsuitable for malaria diagnosis in Eritrea. Continued surveillance of hrp2/3-deleted parasites in Eritrea and neighbouring countries is required to monitor the trend.</p

Novel sampling methods for monitoring Anopheles arabiensis from Eritrea

BACKGROUND: Studies comparing novel collection methods for host seeking and resting mosquitoes A. arabiensis were undertaken in a village in Eritrea. Techniques included an odor baited trap, a novel tent-trap, human landing collection and three methods of resting collection. A technique for the collection of mosquitoes exiting vegetation is also described. Pre-gravid rates were determined by dissection of host seeking insects and post-prandial egg development among insects collected resting. RESULTS: Overall 5,382 host-seeking, 2,296 resting and 357 A. arabiensis exiting vegetation were collected. The Furvela tent-trap was the most efficient, risk-free method for the collection of outdoor host-seeking insects, whilst the Suna trap was the least effective method. Mechanical aspirators (the CDC backpack or the Prokopack aspirator) were superior to manual aspiration in a dark shelter but there was no advantage over manual aspiration in a well-lit one. An estimated two-thirds of newly-emerged mosquitoes went through a pre-gravid phase, feeding twice before producing eggs. Mosquitoes completed gonotrophic development in a dark shelter but left a well-lit shelter soon after feeding. One blood-fed female marked in the village was recaptured 2 days after release exiting vegetation close to the oviposition site and another, shortly after oviposition, attempting to feed on a human host 3 days after release. Exit rates of males from vegetation peaked 3 min after the initial male had left. Unfed and gravid females exited approximately 6 min after the first males. CONCLUSIONS: Furvela tent-traps are suitable for the collection of outdoor biting A. arabiensis in Eritrea whilst the Prokopack sampler is the method of choice for the collection of resting insects. Constructing well-lit, rather than dark, animal shelters, may encourage otherwise endophilic mosquitoes to leave and so reduce their survival and hence their vectorial capacity.https://peerj.compm2022Zoology and Entomolog

Major threat to malaria control programs by Plasmodium falciparum lacking histidine-rich protein 2, Eritrea

False-negative results for Plasmodium falciparum histidine-rich protein (HRP) 2–based rapid diagnostic tests (RDTs) are increasing in Eritrea. We investigated HRP gene 2/3 (pfhrp2/pfhrp3) status in 50 infected patients at 2 hospitals. We showed that 80.8% (21/26) of patients at Ghindae Hospital and 41.7% (10/24) at Massawa Hospital were infected with pfhrp2-negative parasites and 92.3% (24/26) of patients at Ghindae Hospital and 70.8% (17/24) at Massawa Hospital were infected with pfhrp3-negative parasites. Parasite densities between pfhrp2-positive and pfhrp2-negative patients were comparable. All pfhrp2-negative samples had no detectable HRP2/3 antigen and showed negative results for HRP2-based RDTs. pfhrp2-negative parasites were genetically less diverse and formed 2 clusters with no close relationships to parasites from Peru. These parasites probably emerged independently by selection in Eritrea. High prevalence of pfhrp2-negative parasites caused a high rate of false-negative results for RDTs. Determining prevalence of pfhrp2-negative parasites is urgently needed in neighboring countries to assist case management policies

Increasing Prevalence of Artemisinin-Resistant HRP2-Negative Malaria in Eritrea: Increasing Prevalence of Artemisinin-Resistant HRP2-Negative Malaria in Eritrea

International audienceBackgroundAlthough the clinical efficacy of antimalarial artemisinin-based combination therapies in Africa remains high, the recent emergence of partial resistance to artemisinin in Plasmodium falciparum on the continent is troubling, given the lack of alternative treatments.MethodsIn this study, we used data from drug-efficacy studies conducted between 2016 and 2019 that evaluated 3-day courses of artemisinin-based combination therapy (artesunate–amodiaquine or artemether–lumefantrine) for uncomplicated malaria in Eritrea to estimate the percentage of patients with day-3 positivity (i.e., persistent P. falciparum parasitemia 3 days after the initiation of therapy). We also assayed parasites for mutations in Pfkelch13 as predictive markers of partial resistance to artemisinin and screened for deletions in hrp2 and hrp3 that result in variable performance of histidine rich protein 2 (HRP2)–based rapid diagnostic tests for malaria.ResultsWe noted an increase in the percentage of patients with day-3 positivity from 0.4% (1 of 273) in 2016 to 1.9% (4 of 209) in 2017 and 4.2% (15 of 359) in 2019. An increase was also noted in the prevalence of the Pfkelch13 R622I mutation, which was detected in 109 of 818 isolates before treatment, from 8.6% (24 of 278) in 2016 to 21.0% (69 of 329) in 2019. The odds of day-3 positivity increased by a factor of 6.2 (95% confidence interval, 2.5 to 15.5) among the patients with Pfkelch13 622I variant parasites. Partial resistance to artemisinin, as defined by the World Health Organization, was observed in Eritrea. More than 5% of the patients younger than 15 years of age with day-3 positivity also had parasites that carried Pfkelch13 R622I. In vitro, the R622I mutation conferred a low level of resistance to artemisinin when edited into NF54 and Dd2 parasite lines. Deletions in both hrp2 and hrp3 were identified in 16.9% of the parasites that carried the Pfkelch13 R622I mutation, which made them potentially undetectable by HRP2-based rapid diagnostic tests.ConclusionsThe emergence and spread of P. falciparum lineages with both Pfkelch13-mediated partial resistance to artemisinin and deletions in hrp2 and hrp3 in Eritrea threaten to compromise regional malaria control and elimination campaigns