Signs of multiple anthelmintic resistance in sheep gastrointestinal nematodes in Sweden.

Gastrointestinal nematodes in small ruminants are clinically and economically important parasites that often are controlled with anthelmintics. In this study, we compiled information on the anthelmintic efficacy collected on sheep farms according to routines established by Farm & Animal Health in Sweden. The efficacies of benzimidazoles (i.e. albendazole or fenbendazole, n = 30), ivermectin (n = 47), levamisole (n = 2) or moxidectin (n = 2) were examined between 2015 and 2021 in 81 treatment groups on 49 non-randomly selected farms in south-central Sweden. Drug efficacies were estimated with the faecal egg count reduction test. In addition, efficacy data were in most cases supplemented with data on the abundance of the three most common nematode genera in sheep by performing droplet digital (dd) PCR on coprocultures. Efficacies of <95% for benzimidazoles or ivermectin were identified in 37% and 77% of the tested groups, respectively. In addition, on 27 (55%) of the 49 farms where both benzimidazoles and ivermectin were tested, multiple resistance was found on 8 (30%). In contrast, on each of the two farms tested for levamisole and moxidectin both drugs proved to be 100% effective. However, because post-sampling was performed earlier than recommended in several susceptible groups (benzimidazoles = 15, and ivermectin = 10 groups), this could have underestimated the severity of the situation. Mainly larvae from the genus Haemonchus were detected in post-treatment coprocultures, in all groups with declared resistance, suggesting that this parasite was primarily associated with anthelmintic resistance. Unexpectedly, the DNA of larvae, which survived treatment, was also detected on farms declared as susceptible. Taken together, this indicates that the situation regarding the anthelmintic efficacy has deteriorated compared with the latest nationwide study on Swedish sheep farms conducted more than a decade ago. Unlike the previous study, the farm selection here was not strictly randomized but rather opportunistic i.e., only farms with a recognized parasite problem were included. Thus, there is a need for a truly randomized study to get an update on the extent of the situation of anthelmintic resistance at a national level, as well as to identify risk factors involved in the resistance selection. Research is also required to establish the optimal intervals for sampling post-treatment.

Assessment of three DNA extraction kits for the absolute quantification of strongyle nematode eggs in faecal samples.

BACKGROUND: Haemonchus contortus is one of the most pathogenic gastrointestinal nematodes of small ruminants. The current diagnostic approach for the detection of this species relies on coproscopic methods, which both have low sensitivity and are time consuming. Methods employing detection through DNA amplification, such as droplet digital polymerase chain reaction (ddPCR), offer an advantageous approach to the diagnosis of H. contortus. However, DNA extraction protocols need to be constantly updated for the optimal retrieval of diagnostically usable template. Here, we describe the evaluation of three genomic DNA extraction kits for the detection and quantification of H. contortus ITS2 amplicon DNA from faecal samples, using droplet digital PCR. RESULTS: DNA samples, extracted from faecal material with the Nucleospin DNA Stool kit, produced the highest amounts of ITS2 amplicon copies and had the lowest coefficient of variation across different dilutions and sample types (fresh or frozen) out of the tested kits (Nucleospin DNA Stool, E.Z.N.A.® Stool DNA Kit and QIAamp Fast DNA Stool Mini Kit). Furthermore, the protocol of this kit has the fewest number of steps and the price of DNA extraction per sample is reasonable (2.77 €). CONCLUSIONS: The Nucleospin DNA Stool kit is an attractive option for the detection and quantification of H. contortus DNA in faecal samples of small ruminants in a diagnostic setting.

Nematode parasitism affects lying time and overall activity patterns in lambs following pasture exposure around weaning.

We investigated the effects of gastrointestinal nematode (GIN) challenge on activity in first season grazing lambs naturally exposed to two different levels of multispecies GIN infections. Ewes and their twin-born lambs were turned-out to graze in two permanent pasture enclosures naturally contaminated with GIN the previous year, thereby exposing them to overwintering strongyle larvae. Animals in the low parasite exposure group (LP) were dewormed monthly with 0.2 mg ivermectin (Ivomec® vet, oral suspension) per kg body weight, whereas those in high parasite exposure group (HP) were left untreated. At weaning, lambs were allocated to one out of four groups based on weight and sex (HPE, n = 15; HPR, n = 15; LPE, n = 14; LPR, n = 14), in four nearby non-contaminated ley enclosures of similar size. Activity patterns were monitored from day -7, i.e. 7 days pre-weaning, until day 49, i.e. 49 days post-weaning, by fitting all lambs with IceQube sensors (IceRobotics). Body weight was monitored weekly from day -21, whereas faecal samples were investigated at days -21, 7, 35 and 49 for nematode faecal egg counts (EPG) using McMaster-technology and a validated Droplet Digital PCR protocol to determine nematode composition. All statistical analyses were performed in R studio, using mixed models with repeated measures. In the data analyses, weekly recordings was treated as a period, generating a total of eight periods. Average daily lying time had a significant interaction between parasite exposure and period (P = 0.0013), with animals in HP having a 101 ± 31 min shorter daily lying time compared to LP. Motion Index (MI; absolute value of the 3-D acceleration) had a significant interaction between parasite exposure and period (P = 0.0001) with lambs in group HP having a lower average daily MI compared with LP. Both body weight gain and EPG levels were significantly different (P<0.0001) between HP and LP groups during the course of the study. The molecular investigation showed that animals were predominantly infected with Teladorsagia spp., combined with low proportions of Haemonchus spp. In conclusion, this study shows that lying time and Motion Index of lambs around weaning was affected by moderate nematode infections. This indicates that there is a potential use of automated behaviour recordings as a diagnostic tool for detection of nematode parasites in lambs even at moderate infection levels.

First case of monepantel resistant nematodes of sheep in Sweden.

In this study, we describe for the first time monepantel (MOP) resistance in gastrointestinal nematodes (GIN) in a Swedish sheep flock. On the farm, which had recurrent problems with Haemonchus contortus infection, the efficacy of most available anthelmintics (AH) in Sweden (i.e. ivermectin, albendazole, levamisole and monepantel), was monitored. This was done with the faecal egg count reduction test (FECRT) on three different occasions between August 2017 and April 2020. Although, MOP was used in ewes for the first time in this herd in October 2018 and then demonstrated to be highly efficacious (100% reduction), MOP-resistant worms (52% reduction) appeared in lambs already in April 2020. Resistance was detected only after two further rounds of treatment of the lambs after weaning. It is assumed that a contributing factor to this extremely rapid development was related to the fact that ewes and lambs treated during the housing period were let out on clean pasture after treatment. The ewes were treated during the housing period 2018 and grazed a clean pasture the following spring. The same ewes were treated a second time after housing 2018. The lambs were grazed with these ewes in summer 2018 and after weaning they were treated and moved to another clean pasture during the fall 2018. Anthelmintic resistance was also confirmed on two occasions to different compounds of ivermectin and once to albendazole, but not to levamisole which was tested twice. In conclusion, this is the first description of triple resistance to AH drugs in GIN of sheep in Sweden.

Microscopic and molecular evidence of the presence of asymptomatic Plasmodium falciparum and Plasmodium vivax infections in an area with low, seasonal and unstable malaria transmission in Ethiopia.

BACKGROUND: The presence of asymptomatic infections has serious implications for malaria elimination campaigns. Since asymptomatic carriers do not seek treatment for their infection and may become gametocyte carriers, they undoubtedly contribute to the persistence of malaria transmission in a population. The presence of asymptomatic parasitemias was noted in areas with seasonal malaria transmission. In Ethiopia there is a paucity of data regarding the prevalence of asymptomatic malaria carriage. This study was undertaken to assess the presence and prevalence of asymptomatic Plasmodium falciparum and Plasmodium vivax infections in south-central Oromia, Ethiopia. METHODS: A total of 1094 apparently healthy individuals ≥ 2 years of age in south-central Oromia, Ethiopia, an area with seasonal and unstable malaria transmission, were screened for the presence of asymptomatic plasmodial infections. Finger-prick blood samples were taken from each participant for blood film preparation for microscopy and the rapid diagnostic test (RDT). Blood samples were also spotted on Whatman 3MM filter paper for parasite DNA extraction. RESULTS: The prevalence of asymptomatic Plasmodium carriage (P. falciparum, P. vivax and mixed species) was 5.0 % (55/1,094) as determined by microscopy, while the prevalence as determined using RDT was 8.2 % (90/1,094). PCR was done on 47 of 55 microscopy-confirmed and on 79 of 90 RDT-confirmed samples. PCR detected parasite DNA in 89.4 % (42/47) of the microscopy-positive samples and in 77.2 % (61/79) of the RDT-positive samples. No significant difference was observed in the prevalence of asymptomatic P. falciparum or P. vivax infections in the study area (P > 0.1). However, the prevalence of asymptomatic parasitaemia was significantly associated with gender (OR = 0.47, P = 0.015; being higher in males than females) and age (X(2) = 25, P < 0.001; being higher in younger than in older individuals). Age and parasite densities had an inverse relationship. CONCLUSIONS: This study confirms the presence of asymptomatic P. falciparum and P. vivax infections in south-central Oromia, an area with low, seasonal and unstable malaria transmission in Ethiopia. Of 55 microscopically confirmed asymptomatic infections, P. falciparum monoinfection accounted for 45.5 % and of 90 RDT positive asymptomatic infections, 66.7 % were P. falciparum. Although not statistically significant, P. falciparum accounted for a relatively large number of the asymptomatic infections as determined by both tests. The prevalence of asymptomatic parasitaemia was highest in the younger age group. HRP-2-based RDTs specific for P. falciparum showed high false positivity rate compared to Plasmodium lactate dehydrogenase (pLDH) specific to P. vivax. Although microscopy and RDT detected substantial numbers of asymptomatic infections in apparently healthy inhabitants, the use of a highly sensitive molecular diagnostics offers a more accurate assessment of the magnitude of asymptomatic infections.

Identification of large variation in pfcrt, pfmdr-1 and pfubp-1 markers in Plasmodium falciparum isolates from Ethiopia and Tanzania.

BACKGROUND: Plasmodium falciparum resistance to anti-malarials is a major drawback in effective malaria control and elimination globally. Artemisinin-combination therapy (ACT) is currently the key first-line treatment for uncomplicated falciparum malaria. Plasmodium falciparum genetic signatures at pfmdr-1, pfcrt, and pfubp-1 loci are known to modulate in vivo and in vitro parasite response to ACT. The objective of this study was to assess the distribution of these resistance gene markers in isolates collected from different malaria transmission intensity in Ethiopia and Tanzania. METHODS: Plasmodium falciparum clinical isolates were collected from different regions of Ethiopia and Tanzania. Genetic polymorphisms in the genes pfcrt, pfmdr-1 and pfubp-1 were analysed by PCR and sequencing. Frequencies of the different alleles in the three genes were compared within and between regions, and between the two countries. RESULTS: The majority of the isolates from Ethiopia were mutant for the pfcrt 76 and wild-type for pfmdr-1 86. In contrast, the majority of the Tanzanian samples were wild-type for both pfcrt and pfmdr-1 loci. Analysis of a variable linker region in pfmdr-1 showed substantial variation in isolates from Tanzania as compared to Ethiopian isolates that had minimal variation. Direct sequencing of the pfubp-1 region showed that 92.8% (26/28) of the Ethiopian isolates had identical genome sequence with the wild type reference P. falciparum strain 3D7. Of 42 isolates from Tanzania, only 13 (30.9%) had identical genome sequences with 3D7. In the Tanzanian samples, 10 variant haplotypes were identified. CONCLUSION: The majority of Ethiopian isolates carried the main marker for chloroquine (CQ) resistance, while the majority of the samples from Tanzania carried markers for CQ susceptibility. Polymorphic genes showed substantially more variation in Tanzanian isolates. The low variability in the polymorphic region of pfmdr-1 in Ethiopia may be a consequence of low transmission intensity as compared to high transmission intensity and large variations in Tanzania.

High prevalence of pfcrt-CVIET haplotype in isolates from asymptomatic and symptomatic patients in south-central Oromia, Ethiopia.

BACKGROUND: As a result of extensive chloroquine resistance (CQR) in Plasmodium falciparum in late 1990s, Ethiopia replaced CQ with sulphadoxine-pyrimethamine (SP) as first-line drug, which in turn was replaced by artemisinin combination therapy in 2004. Plasmodium falciparum resistance to CQ is determined by the mutation at K76T of the P. falciparum chloroquine resistance transporter (pfcrt) gene. Understanding diversity in the P. falciparum genome is crucial since it has the potential to influence important phenotypes of the parasite such as drug resistance. Limited data is available regarding the type of pfcrt mutant allelic type, the effect of CQ withdrawal and diversity of the parasite population in south-central Oromia, Ethiopia. METHODS: Finger-pricked blood spotted on Whatman 3MM filter papers were collected from falciparum malaria patients. Parasite DNA was extracted from individual blood spots on the filter papers. The presence of K76T mutations was determined using nested PCR for all isolates. Complete sequencing of mutations in pfcrt 72-76 was done for a set of randomly selected resistant isolates. Four microsatellite (MS) markers were analysed to determine the heterozygosity. RESULTS: Although CQ was withdrawn for more than a decade, 100% of the parasites still carried the pfcrt K76T mutation. All isolates were mutant at the K76T polymorphism. Based on combinations of MS markers, seven different Ethiopian CQR variants (E1-E7) were identified. Heterozygosity (H(e)) for MS flanking the pfcrt chloroquine resistance allele ranged from 0.00 (mscrt -29, -29.268 kb) to 0.21 (mscrt -2, -2.814 kb). H(e) ranged from 0.00 (msint 3, 0 kb) to 0.19 (msint 2, 0 kb) for MS within the pfcrt gene. Both intronic and MS flanking the pfcrt gene showed low levels of diversity. CONCLUSION: pfcrt CQR allele seems to be fixed in the study area. Of the different haplotypes associated with CQR, only the CVIET genotype was identified. No reversal to the wild-type has occurred in Ethiopia unlike in many Africa countries where CQR parasites declined after cessation of CQ use. Decreased diversity in CQR isolates surrounding pfcrt suggests CQ selection and homogenization among CQR parasite population. While mutation in msint 3 and mscrt -29 of the mutant pfcrt allele is being fixed, it seems that mutations in msint 2 and mscrt -2 are still evolving and may indicate the start of re-diversification of the population from a fixed 76 T population.

High prevalence of Plasmodium falciparum pfcrt K76T mutation in children with sickle cell disease at a tertiary hospital in north-western Tanzania.

The high prevalence of sickle cell disease (SCD) and trait in Sub-Saharan Africa coincides with the distribution of Plasmodiumfalciparum malaria. Due to prolonged heavy use of chloroquine (CQ) as an antimalarial, drug resistance has developed. Many countries including Tanzania abandoned the use of CQ for uncomplicated malaria, except its use as prophylaxis in patients with sickle cell disease. This study investigated the prevalence of malaria in SCD patients and mutations associated with CQ resistance. Children diagnosed with sickle cell disease attending both outpatient clinic and those admitted at Bugando Medical Centre in northwestern Tanzania were screened for malaria using thick blood smear. A dried blood spot on Whatman filter paper was also taken for polymerase chain reaction (PCR) and restriction fragment length polymorphism. Among 123 known patients with sickle cell disease, the prevalence of malaria by blood smear microscopy was 3.2% and by PCR was 13.8%. The prevalence of K76T mutation among the patients was 81.3%. The majority of the patients (72.4%) were using chloroquine prophylaxis. In conclusion, the prevalence of malaria parasitaemia among children with sickle cell disease attending BMC is low (3.2%) by microscopy but several children maintain sub patent infection detectable by PCR. The prevalence of chloroquine resistant P falciparum in these children was higher than that previously seen in normal population in Tanzania. We recommend special attention to be paid to patients with sickle cell disease while studying the dynamics of drug resistant parasites.

Detection of a substantial number of sub-microscopic Plasmodium falciparum infections by polymerase chain reaction: a potential threat to malaria control and diagnosis in Ethiopia.

BACKGROUND: Prompt and effective malaria diagnosis not only alleviates individual suffering, but also decreases malaria transmission at the community level. The commonly used diagnostic methods, microscopy and rapid diagnostic tests, are usually insensitive at very low-density parasitaemia. Molecular techniques, on the other hand, allow the detection of low-level, sub-microscopic parasitaemia. This study aimed to explore the presence of sub-microscopic Plasmodium falciparum infections using polymerase chain reaction (PCR). The PCR-based parasite prevalence was compared against microscopy and rapid diagnostic test (RDT). METHODS: This study used 1,453 blood samples collected from clinical patients and sub-clinical subjects to determine the prevalence of sub-microscopic P. falciparum carriages. Subsets of RDT and microscopy negative blood samples were tested by PCR while all RDT and microscopically confirmed P. falciparum-infected samples were subjected to PCR. Finger-prick blood samples spotted on filter paper were used for parasite genomic DNA extraction. RESULTS: The prevalence of sub-microscopic P. falciparum carriage was 19.2% (77/400) (95% CI = 15. 4-23.1). Microscopy-based prevalence of P. falciparum infection was 3.7% (54/1,453) while the prevalence was 6.9% (100/1,453) using RDT alone. Using microscopy and PCR, the estimated parasite prevalence was 20.6% if PCR were performed in 1,453 blood samples. The prevalence was estimated to be 22.7% if RDT and PCR were used. Of 54 microscopically confirmed P. falciparum-infected subjects, PCR detected 90.7% (49/54). Out of 100 RDT-confirmed P. falciparum infections; PCR detected 80.0% (80/100). The sensitivity of PCR relative to microscopy and RDT was, therefore, 90.7% and 80%, respectively. The sensitivity of microscopy and RDT relative to PCR was 16.5 (49/299) and 24.2% (80/330), respectively. The overall PCR-based prevalence of P. falciparum infection was 5.6- and 3.3 fold higher than that determined by microscopy and RDT, respectively. None of the sub-microscopic subjects had severe anaemia, though 29.4% had mild anaemia (10-11.9 g/dl). CONCLUSIONS: Asymptomatic, low-density malaria infection was common in the study area and PCR may be a better tool for measuring Plasmodium prevalence than microscopy and RDT. The inadequate sensitivity of the diagnostic methods to detect substantial number of sub-microscopic parasitaemia would undoubtedly affect malaria control efforts, making reduction of transmission more difficult. RDT and microscopy-based prevalence studies and subsequent reports of reduction in malaria incidence underestimate the true pictures of P. falciparum infections in the community. PCR, on the other hand, seems to have reasonable sensitivity to detect a higher number of infected subjects with low and sub-microscopic parasite densities than RDTs or microscopy.

Detecting adenosine triphosphatase 6 (pfATP6) point mutations that may be associated with Plasmodium falciparum resistance to artemisinin: prevalence at baseline, before policy change in Uganda.

The artemisinin based combination therapy (ACT) of artemether and lumefantrine (Co-artem) has recently replaced chloroquine and fansidar as the first line treatment policy drug in Uganda. It is necessary to develop practical procedures to monitor the likely emergence and spread of artemisinin resistant P. falciparum strains. We have analyzed the genotypes of PfATP6 in parasites from 300 stored filter paper samples from malaria patients who were diagnosed and treated in the years 1999 to 2004 at three field sites in Uganda. This is a period just prior to introduction of Co-artem. In order to develop a simple molecular procedure for mutation detection, regions of PfATP6 encoding protein domains important in artemisinin binding was amplified by nested PCR. Three DNA products, which together contain most of the coding region of amino acids located within the putative active site of pfATP6 were readily amplified. The amplified DNA was digested by restriction enzymes and the fragments sized by agarose gel electrophoresis. For the important codons 260, 263 and 769, methods using engineered restriction sites were employed. We did not find mutations at codons for the key residues Lys 260, Leu263, Gln266, Ser769 and Asn1039. Nucleotide sequencing of pfATPase6 gene DNA from at least 15 clinical isolates confirmed the above findings and suggested that mutations at these amino acid residues have not emerged in our study sites.