Polymorphisms in the human angiotensin converting enzyme gene (ACE) linked to susceptibility of COVID-19 and malaria infections in the Ghanaian population.

Genetic variations in the human angiotensin converting enzyme gene (ACE) influence ACE enzyme expression levels in humans and subsequently influence both communicable and non-communicable disease outcomes. More recently, polymorphisms in this gene have been linked to susceptibility and outcomes of infectious diseases such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and malaria infections. This study is the first to investigate the genetic diversity of ACE and ACE2 polymorphisms in the Ghanaian population. Archived filter blood blot samples from malaria patients aged ≤9 years were used. Molecular analysis for the detection of ACE rs4646994 (I/D), ACE2 rs2106809 (C/T) and rs2285666 (G/A) alleles as well as ACE2 exons 1-4 polymorphisms was conducted on 300 samples. The D allele (54%,162/300) was the most dominant polymorphism observed in the ACE rs4646994 gene whilst the G (68%, 204/300) and T alleles (59.3%,178/300) were the most frequent ACE2 rs2285666 and rs2106809 polymorphisms observed. For the 300 samples sequenced for ACE2 exons 1-4, analyses were done on 268, 282 and 137 quality sequences for exons 1, 2 and 3-4 respectively. For exon 1, the mutation D38N (2.2%; 6/268) was the most prevalent. The S19P and E37K mutations previously reported to influence COVID-19 infections were observed at low frequencies (0.4%, 1/268 each). No mutations were observed in exon 2. The N121K/T variants were the most seen in exons 3-4 at frequencies of 5.1% (K121, 7/137) and 2.9% (T121, 4/137) respectively. Most of the variants observed in the exons were novel compared to those reported in other populations in the world. This is the first study to investigate the genetic diversity of ACE and ACE2 genes in Ghanaians. The observation of novel mutations in the ACE2 gene is suggesting selection pressure. The importance of the mutations for communicable and non-communicable diseases (malaria and COVID-19) are further discussed.

Usefulness of a commercial LAMP assay for detection of malaria infection, including Plasmodium knowlesi cases, in returning travelers in Spain.

OBJECTIVE: Main malaria diagnosis is based on microscopic examination combined with rapid diagnostic tests. Both methods have low sensitivity and specificity. Loop-mediated isothermal amplification techniques have shown a sensitivity similar to PCR but with lower times of performance. This study aimed to assess a commercial LAMP for the diagnosis of malaria (Alethia® Malaria) against the Nested-Multiplex-Malaria PCR, including the analytical sensitivity and the operational characteristics. RESULTS: One hundred five samples out of 114 rendered valid results, obtaining 85 positive samples and 18 negative samples with an agreement of 98% compared to the reference method with a sensitivity, specificity and kappa coefficient of 98.84%, 94.74% and 0.94 respectively, with only two discrepant samples. The turnaround time was estimated in 1 h and 30 min, with a cost of 32.67€ per determination. The results showed several advantages of the Alethia® Malaria, as it was easy to perform, minimal training requirement and 40 min run. Moreover, it includes an internal control to avoid false negatives. However, it also showed some limitations such as the need for a specific amplification and detection device, the detection of only Plasmodium spp. and a very high price.

Molecular Detection and Characterization of Intestinal and Blood Parasites in Wild Chimpanzees (Pan troglodytes verus) in Senegal.

Wild chimpanzee populations in West Africa (Pan troglodytes verus) have dramatically decreased as a direct consequence of anthropogenic activities and infectious diseases. Little information is currently available on the epidemiology, pathogenic significance, and zoonotic potential of protist species in wild chimpanzees. This study investigates the occurrence and genetic diversity of intestinal and blood protists as well as filariae in faecal samples (n = 234) from wild chimpanzees in the Dindefelo Community Nature Reserve, Senegal. PCR-based results revealed the presence of intestinal potential pathogens (Sarcocystis spp.: 11.5%; Giardia duodenalis: 2.1%; Cryptosporidium hominis: 0.9%), protist of uncertain pathogenicity (Blastocystis sp.: 5.6%), and commensal species (Entamoeba dispar: 18.4%; Troglodytella abrassarti: 5.6%). Entamoeba histolytica, Enterocytozoon bieneusi, and Balantioides coli were undetected. Blood protists including Plasmodium malariae (0.4%), Trypanosoma brucei (1.3%), and Mansonella perstans (9.8%) were also identified. Sanger sequencing analyses revealed host-adapted genetic variants within Blastocystis, but other parasitic pathogens (C. hominis, P. malariae, T. brucei, M. perstans) have zoonotic potential, suggesting that cross-species transmission between wild chimpanzees and humans is possible in areas where both species overlap. Additionally, we explored potential interactions between intestinal/blood protist species and seasonality and climate variables. Chimpanzees seem to play a more complex role on the epidemiology of pathogenic and commensal protist and nematode species than initially anticipated.

KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum.

BACKGROUND: The emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum. METHODS: Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method. RESULTS: The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed. CONCLUSIONS: The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

Geographical distribution and species identification of human filariasis and onchocerciasis in Bioko Island, Equatorial Guinea.

Human filariae are vector-borne parasites and the causative agents of various diseases, including human onchocerciasis and lymphatic filariasis. Onchocerciasis causes a spectrum of cutaneous and ophthalmologic manifestations (including blindness) and has long been a major public health problem in Bioko Island (Equatorial Guinea). Bioko Island has been included in the WHO's Onchocerciasis Control Program since 1987. In Bioko Island, the specificity and sensitivity of clinical Onchocerca volvulus diagnosis is key. The objective of this work was to update onchocerciasis elimination progress in Bioko Island, after 18 years of mass ivermectin intervention, and the general filariasis situation through a rapid and accurate molecular method. A cross-sectional study was conducted in Bioko Island from mid-January to mid-February 2014. A total of 543 subjects were included in the study. Whole blood and one skin snip (from lumbar regions) were analysed with a real time PCR assay. Two other skin biopsies were analysed by an expert microscopist. All positive samples were confirmed by sequencing. Traditional microscopic examination of the skin biopsies failed to detect any microfilariae. However, 11 (2.03%) infections were detected using PCR assay, including one O. volvulus, two Mansonella streptocerca, seven Mansonella perstans and one Loa loa infections. PCR assays in blood detected 52 filariae-positive individuals (9.6%) which harboured M. perstans or L. loa. The low prevalence of O. volvulus confirms the success of the Onchocerciasis Control Programme and suggests that Mass Drug Administration in Bioko Island can be interrupted in the near future. The very high prevalence of M. perstans found in skin snips assays raises doubts about the reliability of microscope-based diagnosis of O. volvulus infections.

Nested multiplex PCR for identification and detection of human Plasmodium species including Plasmodium knowlesi.

OBJECTIVE: To develop a new technique for diagnosis of Plasmodium knowlesi and at the same time to be able to discriminate among the diverse species of Plasmodium causing human malaria. METHODS: In this study the nested multiplex malaria PCR was redesigned, targeting the 18S rRNA gene, to identify the fifth human Plasmodium species, Plasmodium knowlesi, together with the other human Plasmodium (Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) by amplified fragment size using only two amplification processes and including an internal reaction control to avoid false negatives. RESULTS: The technique was validated with 91 clinical samples obtained from patients with malaria compatible symptoms. The technique showed high sensitivity (100%) and specificity (96%) when it was compared to the reference method employed for malaria diagnosis in the Instituto de Salud Carlos Ⅲ and a published real-time PCR malaria assay. CONCLUSIONS: The technique designed is an economical, sensitive and specific alternative to current diagnosis methods. Furthermore, the method might be tested in knowlesi-malaria endemic areas with a higher number of samples to confirm the quality of the method.

Atypical Mansonella ozzardi Microfilariae from an Endemic Area of Brazilian Amazonia.

Mansonellosis is endemic in several regions of Africa, the Caribbean, and Latin America. Mansonella ozzardi and Mansonella perstans have been reported in Latin America, including the Amazon region. A morphological and molecular microfilariae study was performed in Pauini (Brazil). Blood samples were collected from 40 individuals, and were analyzed by Giemsa-stained blood film and by two different nested polymerase chain reactions which detect internal transcribed spacer-1 and the major sperm protein gene. By microscopy, 14 of 40 were positive: 11 as M. ozzardi and three as M. perstans-like infections. Both molecular methods detected 19 positive cases as M. ozzardi, including those 14 individuals detected by microscopy, without detectable genetic differences among any of the 19 positive samples. Molecular techniques showed an improvement of mansonellosis diagnosis and may become an effective tool to evaluate the present status of M. ozzardi and M. perstans in Latin America.

First case of a naturally acquired human infection with Plasmodium cynomolgi.

Since 1960, a total of seven species of monkey malaria have been reported as transmissible to man by mosquito bite: Plasmodium cynomolgi, Plasmodium brasilianum, Plasmodium eylesi, Plasmodium knowlesi, Plasmodium inui, Plasmodium schwetzi and Plasmodium simium. With the exception of P. knowlesi, none of the other species has been found to infect humans in nature. In this report, it is described the first known case of a naturally acquired P. cynomolgi malaria in humans.The patient was a 39-year-old woman from a malaria-free area with no previous history of malaria or travel to endemic areas. Initially, malaria was diagnosed and identified as Plasmodium malariae/P. knowlesi by microscopy in the Terengganu State Health Department. Thick and thin blood films stained with 10% Giemsa were performed for microscopy examination. Molecular species identification was performed at the Institute for Medical Research (IMR, Malaysia) and in the Malaria & Emerging Parasitic Diseases Laboratory (MAPELAB, Spain) using different nested PCR methods.Microscopic re-examination in the IMR showed characteristics of Plasmodium vivax and was confirmed by a nested PCR assay developed by Snounou et al. Instead, a different PCR assay plus sequencing performed at the MAPELAB confirmed that the patient was infected with P. cynomolgi and not with P. vivax.This is the first report of human P. cynomolgi infection acquired in a natural way, but there might be more undiagnosed or misdiagnosed cases, since P. cynomolgi is morphologically indistinguishable from P. vivax, and one of the most used PCR methods for malaria infection detection may identify a P. cynomolgi infection as P. vivax.Simian Plasmodium species may routinely infect humans in Southeast Asia. New diagnostic methods are necessary to distinguish between the human and monkey malaria species. Further epidemiological studies, incriminating also the mosquito vector(s), must be performed to know the relevance of cynomolgi malaria and its implication on human public health and in the control of human malaria.The zoonotic malaria cannot be ignored in view of increasing interactions between man and wild animals in the process of urbanization.

Nested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region.

We present filaria-nested polymerase chain reaction (PCR), which is based on amplification of first internal transcribed spacer rDNA to distinguish three parasitic filarial species (Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans) that can be found in the Amazon Region. Nested PCR-based identifications yielded the same results as those utilizing morphological characters. Nested PCR is highly sensitive and specific and it detects low-level infections in both humans and vectors. No cross-amplifications were observed with various other blood parasites and no false-positive results were obtained with the nested PCR. The method works efficiently with whole-blood, blood-spot and skin biopsy samples. Our method may thus be suitable for assessing the efficacy of filaria control programmes in Amazonia by recording parasite infections in both the human host and the vector. By specifically differentiating the major sympatric species of filaria, this technique could also enhance epidemiological research in the region.

Nested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region

We present filaria-nested polymerase chain reaction (PCR), which is based on amplification of first internal transcribed spacer rDNA to distinguish three parasitic filarial species (Onchocerca volvulus, Mansonella ozzardiand Mansonella perstans) that can be found in the Amazon Region. Nested PCR-based identifications yielded the same results as those utilizing morphological characters. Nested PCR is highly sensitive and specific and it detects low-level infections in both humans and vectors. No cross-amplifications were observed with various other blood parasites and no false-positive results were obtained with the nested PCR. The method works efficiently with whole-blood, blood-spot and skin biopsy samples. Our method may thus be suitable for assessing the efficacy of filaria control programmes in Amazonia by recording parasite infections in both the human host and the vector. By specifically differentiating the major sympatric species of filaria, this technique could also enhance epidemiological research in the region.

First case of detection of Plasmodium knowlesi in Spain by Real Time PCR in a traveller from Southeast Asia.

Previously, Plasmodium knowlesi was not considered as a species of Plasmodium that could cause malaria in human beings, as it is parasite of long-tailed (Macaca fascicularis) and pig-tailed (Macaca nemestrina) macaques found in Southeast Asia. A case of infection by P. knowlesi is described in a Spanish traveller, who came back to Spain with daily fever after his last overseas travel, which was a six-month holiday in forested areas of Southeast Asia between 2008 and 2009. His P. knowlesi infection was detected by multiplex Real time quantitative PCR and confirmed by sequencing the amplified fragment. Using nested multiplex malaria PCR (reference method in Spain) and a rapid diagnostic test, the P. knowlesi infection was negative. This patient was discharged and asymptomatic when the positive result to P. knowlesi was reported. Prior to this case, there have been two more reports of European travellers with malaria caused by P. knowlesi, a Finnish man who travelled to Peninsular Malaysia during four weeks in March 2007, and a Swedish man who did a short visit to Malaysian Borneo in October 2006. Taken together with this report of P. knowlesi infection in a Spanish traveller returning from Southeast Asia, this is the third case of P. knowlesi infection in Europe, indicating that this simian parasite can infect visitors to endemic areas in Southeast Asia. This last European case is quite surprising, given that it is an untreated-symptomatic P. knowlesi in human, in contrast to what is currently known about P. knowlesi infection. Most previous reports of human P. knowlesi malaria infections were in adults, often with symptoms and relatively high parasite densities, up to the recent report in Ninh Thuan province, located in the southern part of central Vietnam, inhabited mainly by the Ra-glai ethnic minority, in which all P. knowlesi infections were asymptomatic, co-infected with P. malariae, with low parasite densities and two of the three identified cases were very young children under five years old.