Improving gonorrhoea molecular diagnostics: Genome mining-based identification of identical multi-repeat sequences (IMRS) in Neisseria gonorrhoeae.

Background: Curable sexually transmitted infections (STIs), such as Neisseria gonorrhoeae (N. gonorrhoeae), are a major cause of poor pregnancy outcomes. The infection is often asymptomatic in pregnant women, and a syndrome-based approach of testing leads to a missed diagnosis. Culture followed by microscopy is inadequate and time-consuming. The gold standard nucleic acid amplification tests require advanced infrastructure settings, whereas point-of-care tests are limited to immunoassays with sensitivities and specificities insufficient to accurately diagnose asymptomatic cases. This necessitates the development and validation of assays that are fit for purpose. Methods: We identified new diagnostic target biomarker regions for N. gonorrhoeae using an algorithm for genome mining of identical multi-repeat sequences (IMRS). These were then developed as DNA amplification primers to design better diagnostic assays. To test the primer pair, genomic DNA was 10-fold serially diluted (100 pg/µL to 1 × 10-3 pg/µL) and used as DNA template for PCR reactions. The gold standard PCR using 16S rRNA primers was also run as a comparative test, and both assay products were resolved on 1% agarose gel. Results: Our newly developed N. gonorrhoeae IMRS-PCR assay had an analytical sensitivity of 6 fg/µL representing better sensitivity than the 16S rRNA PCR assay with an analytical sensitivity of 4.3096 pg/µL. The assay was also successfully validated using clinical urethral swab samples. We further advanced this technique by developing an isothermal IMRS, which was both reliable and sensitive for detecting cultured N. gonorrhoeae isolates at a concentration of 38 ng/µL. Combining isothermal IMRS with a low-cost lateral flow assay, we were able to detect N. gonorrhoeae amplicons at a starting concentration of 100 pg/µL. Conclusion: Therefore, there is a potential to implement this concept within miniaturized, isothermal, microfluidic platforms, and laboratory-on-a-chip diagnostic devices for highly reliable point-of-care testing.

Mapping brucellosis risk in Kenya and its implications for control strategies in sub-Saharan Africa.

In Sub-Saharan Africa (SSA), effective brucellosis control is limited, in part, by the lack of long-term commitments by governments to control the disease and the absence of reliable national human and livestock population-based data to inform policies. Therefore, we conducted a study to establish the national prevalence and develop a risk map for Brucella spp. in cattle to contribute to plans to eliminate the disease in Kenya by the year 2040. We randomly generated 268 geolocations and distributed them across Kenya, proportionate to the area of each of the five agroecological zones and the associated cattle population. Cattle herds closest to each selected geolocation were identified for sampling. Up to 25 cattle were sampled per geolocation and a semi-structured questionnaire was administered to their owners. We tested 6,593 cattle samples for Brucella immunoglobulin G (IgG) antibodies using an Enzyme-linked immunosorbent assay (ELISA). We assessed potential risk factors and performed spatial analyses and prevalence mapping using approximate Bayesian inference implemented via the integrated nested Laplace approximation (INLA) method. The national Brucella spp. prevalence was 6.8% (95% CI: 6.2-7.4%). Exposure levels varied significantly between agro-ecological zones, with a high of 8.5% in the very arid zone with the lowest agricultural potential relative to a low of 0.0% in the agro-alpine zone with the highest agricultural potential. Additionally, seroprevalence increased with herd size, and the odds of seropositivity were significantly higher for females and adult animals than for males or calves. Similarly, animals with a history of abortion, or with multiple reproductive syndromes had higher seropositivity than those without. At the herd level, the risk of Brucella spp. transmission was higher in larger herds, and herds with a history of reproductive problems such as abortion, giving birth to weak calves, or having swollen testes. Geographic localities with high Brucella seroprevalence occurred in northern, eastern, and southern regions of Kenya all primarily characterized by semi-arid or arid agro-ecological zones dominated by livestock pastoralism interspersed with vast areas with mixed livestock-wildlife systems. The large spatial extent of our survey provides compelling evidence for the widespread geographical distribution of brucellosis risk across Kenya in a manner easily understandable for policymakers. Our findings can provide a basis for risk-stratified pilot studies aiming to investigate the cost-effectiveness and efficacy of singular and combined preventive intervention strategies that seek to inform Kenya's Brucellosis Control Policy.

The African Swine Fever Isolate ASFV-Kenya-IX-1033 Is Highly Virulent and Stable after Propagation in the Wild Boar Cell Line WSL.

We describe the characterization of an African swine fever genotype IX virus (ASFV-Kenya-IX-1033), which was isolated from a domestic pig in western Kenya during a reported outbreak. This includes the efficiency of virus replication and in vivo virulence, together with genome stability and virulence, following passage in blood macrophages and in a wild boar lung cell line (WSL). The ASFV-Kenya-IX-1033 stock retained its ability to replicate in primary macrophages and retained virulence in vivo, following more than 20 passages in a WSL. At the whole genome level, a few single-nucleotide differences were observed between the macrophage and WSL-propagated viruses. Thus, we propose that the WSL is suitable for the production of live-attenuated ASFV vaccine candidates based on the modification of this wild-type isolate. The genome sequences for ASFV-Kenya-IX-1033 propagated in macrophages and in WSL cells were submitted to GenBank, and a challenge model based on the isolate was developed. This will aid the development of vaccines against the genotype IX ASFV circulating in eastern and central Africa.

Deletion of the CD2v Gene from the Genome of ASFV-Kenya-IX-1033 Partially Reduces Virulence and Induces Protection in Pigs.

Infection of pigs with the African swine fever virus (ASFV) leads to a devastating hemorrhagic disease with a high mortality of up to 100%. In this study, a CD2v gene deletion was introduced to a genotype IX virus from East Africa, ASFV-Kenya-IX-1033 (ASFV-Kenya-IX-1033-∆CD2v), to investigate whether this deletion led to reduced virulence in domestic pigs and to see if inoculation with this LA-ASFV could induce protective immunity against parental virus challenge. All pigs inoculated with ASFV-Kenya-IX-1033-ΔCD2v survived inoculation but presented with fever, reduced appetite and lethargy. ASFV genomic copies were detected in only one animal at one time point. Seven out of eight animals survived subsequent challenge with the pathogenic parental strain (87.5%) but had mild to moderate clinical symptoms and had a gross pathology compatible with chronic ASFV infection. All mock-immunised animals developed acute ASF upon challenge with ASFV-Kenya-IX-1033 and were euthanised upon meeting the humane endpoint criteria. ASFV genome copy numbers after challenge were similar in the two groups. ASFV-Kenya-IX-1033-∆CD2v is therefore a useful tool to investigate the development of immunity to ASFV genotype IX, but safety concerns preclude its use as a candidate vaccine without further attenuation.

Co-Deletion of A238L and EP402R Genes from a Genotype IX African Swine Fever Virus Results in Partial Attenuation and Protection in Swine.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), resulting in up to 100% mortality in pigs. Although endemic in most sub-Saharan African countries, where all known ASFV genotypes have been reported, the disease has caused pandemics of significant economic impact in Eurasia, and no vaccines or therapeutics are available to date. In endeavors to develop live-attenuated vaccines against ASF, deletions of several of the ~170 ASFV genes have shown contrasting results depending on the genotype of the investigated ASFV. Here, we report the in vivo outcome of a single deletion of the A238L (5EL) gene and double deletions of A238L (5EL) and EP402R (CD2v) genes from the genome of a highly virulent genotype IX ASFV isolate. Domestic pigs were intramuscularly inoculated with (i) ASFV-Ke-ΔA238L to assess the safety of A238L deletion and (ii) ASFV-Ke-ΔEP402RΔA238L to investigate protection against challenge with the virulent wildtype ASFV-Ke virus. While A238L (5EL) gene deletion did not yield complete attenuation, co-deletion of A238L (5EL) and EP402R (CD2v) improved the safety profile of the single deletions, eliciting both humoral and cellular immune responses and conferred partial protection against challenge with the virulent wildtype ASFV-Ke virus.

High prevalence of Plasmodium malariae and Plasmodium ovale in co-infections with Plasmodium falciparum in asymptomatic malaria parasite carriers in southwestern Nigeria.

Asymptomatic malaria parasite carriers do not seek anti-malarial treatment and may constitute a silent infectious reservoir. In order to assess the level of asymptomatic and symptomatic carriage amongst adolescents in a highly endemic area, and to identify the risk factors associated with such carriage, we conducted a cross-sectional survey of 1032 adolescents (ages 10-19 years) from eight schools located in Ibadan, southwestern Nigeria in 2016. Blood films and blood spot filter paper samples were prepared for microscopy and DNA analysis. The prevalence of asymptomatic malaria was determined using microscopy, rapid diagnostic tests and PCR for 658 randomly selected samples. Of these, we found that 80% of asymptomatic schoolchildren were positive for malaria parasites by PCR, compared with 47% and 9%, determined by rapid diagnostic tests and microscopy, respectively. Malaria parasite species typing was performed using PCR targeting the mitochondrial CoxIII gene, and revealed high rates of carriage of Plasmodium malariae (53%) and Plasmodium ovale (24%). Most asymptomatic infections were co-infections of two or more species (62%), with Plasmodium falciparum + P. malariae the most common (35%), followed by P. falciparum + P. malariae + P. ovale (21%) and P. falciparum + P. ovale (6%). Single infections of P. falciparum, P. malariae and P. ovale accounted for 24%, 10% and 4% of all asymptomatic infections, respectively. To compare the species composition of asymptomatic and symptomatic infections, further sample collection was carried out in 2017 at one of the previously sampled schools, and at a nearby hospital. Whilst the species composition of the asymptomatic infections was similar to that observed in 2016, the symptomatic infections were markedly different, with single infections of P. falciparum observed in 91% of patients, P. falciparum + P. malariae in 5% and P. falciparum + P. ovale in 4%.

Rapid CRISPR/Cas9 Editing of Genotype IX African Swine Fever Virus Circulating in Eastern and Central Africa.

African swine fever virus (ASFV) is the etiological agent of a contagious and fatal disease of domestic pigs that has significant economic consequences for the global swine industry. Due to the lack of effective treatment and vaccines against African swine fever, there is an urgent need to leverage cutting-edge technologies and cost-effective approaches for generating and purifying recombinant virus to fast-track the development of live-attenuated ASFV vaccines. Here, we describe the use of the CRISPR/Cas9 gene editing and a cost-effective cloning system to produce recombinant ASFVs. Combining these approaches, we developed a recombinant virus lacking the non-essential gene A238L (5EL) in the highly virulent genotype IX ASFV (ASFV-Kenya-IX-1033) genome in less than 2 months as opposed to the standard homologous recombination with conventional purification techniques which takes up to 6 months on average. Our approach could therefore be a method of choice for less resourced laboratories in developing nations.

The advancements, challenges, and future implications of the CRISPR/Cas9 system in swine research.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing technology has dramatically influenced swine research by enabling the production of high-quality disease-resistant pig breeds, thus improving yields. In addition, CRISPR/Cas9 has been used extensively in pigs as one of the tools in biomedical research. In this review, we present the advancements of the CRISPR/Cas9 system in swine research, such as animal breeding, vaccine development, xenotransplantation, and disease modeling. We also highlight the current challenges and some potential applications of the CRISPR/Cas9 technologies.

Malaria parasites regulate intra-erythrocytic development duration via serpentine receptor 10 to coordinate with host rhythms.

Malaria parasites complete their intra-erythrocytic developmental cycle (IDC) in multiples of 24 h suggesting a circadian basis, but the mechanism controlling this periodicity is unknown. Combining in vivo and in vitro approaches utilizing rodent and human malaria parasites, we reveal that: (i) 57% of Plasmodium chabaudi genes exhibit daily rhythms in transcription; (ii) 58% of these genes lose transcriptional rhythmicity when the IDC is out-of-synchrony with host rhythms; (iii) 6% of Plasmodium falciparum genes show 24 h rhythms in expression under free-running conditions; (iv) Serpentine receptor 10 (SR10) has a 24 h transcriptional rhythm and disrupting it in rodent malaria parasites shortens the IDC by 2-3 h; (v) Multiple processes including DNA replication, and the ubiquitin and proteasome pathways, are affected by loss of coordination with host rhythms and by disruption of SR10. Our results reveal malaria parasites are at least partly responsible for scheduling the IDC and coordinating their development with host daily rhythms.

The Toll-Like Receptor 2 agonist PEG-Pam2Cys as an immunochemoprophylactic and immunochemotherapeutic against the liver and transmission stages of malaria parasites.

Both vaccine and therapeutic approaches to malaria are based on conventional paradigms; whole organism or single antigen epitope-based vaccines administered with or without an adjuvant, and chemotherapeutics (anti-malaria drugs) that are toxic to the parasite. Two major problems that limit the effectiveness of these approaches are i) high levels of antigenic variation within parasite populations rendering vaccination efficacy against all variants difficult, and ii) the capacity of the parasite to quickly evolve resistance to drugs. We describe a new approach to both protection from and treatment of malaria parasites that involves the direct stimulation of the host innate immune response through the administration of a Toll-Like Receptor-2 (TLR2) agonist. The activity of PEG-Pam2Cys against the hepatocytic stages, erythrocytic stages and gametocytes of the rodent malaria parasite Plasmodium yoelii was investigated in laboratory mice. We show that administration of PEG-Pam2Cys, a soluble form of the TLR2 agonist S-[2,3-bis(palmitoyloxy)propyl] cysteine (Pam2Cys), significantly and dramatically reduces the numbers of malaria parasites that grow in the livers of mice following subsequent challenge with sporozoites. We also show that treatment can also clear parasites from the liver when administered subsequent to the establishment of infection. Finally, PEG-Pam2Cys can reduce the numbers of mosquitoes that are infected, and the intensity of their infection, following blood feeding on gametocytaemic mice. These results suggest that this compound could represent a novel liver stage anti-malarial that can be used both for the clearance of parasites following exposure and for the prevention of the establishment of infection.

Rapid identification of genes controlling virulence and immunity in malaria parasites.

Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites.

Within-host competition does not select for virulence in malaria parasites; studies with Plasmodium yoelii.

In endemic areas with high transmission intensities, malaria infections are very often composed of multiple genetically distinct strains of malaria parasites. It has been hypothesised that this leads to intra-host competition, in which parasite strains compete for resources such as space and nutrients. This competition may have repercussions for the host, the parasite, and the vector in terms of disease severity, vector fitness, and parasite transmission potential and fitness. It has also been argued that within-host competition could lead to selection for more virulent parasites. Here we use the rodent malaria parasite Plasmodium yoelii to assess the consequences of mixed strain infections on disease severity and parasite fitness. Three isogenic strains with dramatically different growth rates (and hence virulence) were maintained in mice in single infections or in mixed strain infections with a genetically distinct strain. We compared the virulence (defined as harm to the mammalian host) of mixed strain infections with that of single infections, and assessed whether competition impacted on parasite fitness, assessed by transmission potential. We found that mixed infections were associated with a higher degree of disease severity and a prolonged infection time. In the mixed infections, the strain with the slower growth rate was often responsible for the competitive exclusion of the faster growing strain, presumably through host immune-mediated mechanisms. Importantly, and in contrast to previous work conducted with Plasmodium chabaudi, we found no correlation between parasite virulence and transmission potential to mosquitoes, suggesting that within-host competition would not drive the evolution of parasite virulence in P. yoelii.

Malaria parasite genetics: doing something useful.

Genetics has informed almost every aspect of the study of malaria parasites, and remains a key component of much of the research that aims to reduce the burden of the disease they cause. We describe the history of genetic studies of malaria parasites and give an overview of the utility of the discipline to malariology.

DNA from pre-erythrocytic stage malaria parasites is detectable by PCR in the faeces and blood of hosts.

Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be "sub-microscopic" blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.