First report of Acanthamoebae spp. isolation from a volcanic mud spring in the Philippines.

Acanthamoebae spp. is considered highly adaptive. The present study aims to establish the occurrence of free-living amoebae, particularly Acanthamoebae, to exist in extreme environments such as volcanic mud springs. Fifty surface water samples were collected from mud springs (34 samples), and flat rocks (16 samples) were collected, processed, and cultured. After 14 days of incubation, 32 (64%) plates showed positive amoebic growth. Nineteen (55.8%) of these plates came from the mud spring collection site, while 13 (81.2%) samples are from flat rock sources. DNAs from positive samples were made to react to polymerase chain reaction (PCR) using primer sets JDP1 5'GGCCCAGATCGTTTACCGTGAA-3' and JDP2 5'TCTCACAAGCTGCTAGGGAGTCA-3' for cells that resemble Acanthamoebae. Sequencing and basic local alignment search tool (BLAST) revealed a 99% similarity of isolates to Acanthamoebae spp. Identification of Acanthamoebae spp that can survive in higher temperatures is important public health information. The existence of such isolates in the environment has dire health implications, which suggests revisitation of water treatment protocols. Detection of such organisms in environmental sources used for recreational purposes provides information to local and international tourists who frequent them. This will result in the mitigation of potential future infection.

Gastrointestinal parasites in Africa: A review.

Data on human gastrointestinal parasites (GIP) infections in the african sub-regions and countries are mainly lacking in terms of prevalence and population stratification by afflicted age group, symptomatology, multi-parasitism, and diagnostic methods. This study aims to describe the GIP reported in african countries and discuss the extent of the burden in the african context. Only 68.42% (39/57) of african countries reported human cases of GIP with helminths (45%, CI: 40-50%, I2: 99.79%) as the predominant parasitic group infecting the african population. On a regional scale, Central Africa had the highest pooled prevalence for GIP (43%, CI: 32-54%, I2: 99.74%), while the Central African Republic led all countries with a pooled prevalence of 90% (CI: 89-92%, I2: 99.96%). The vulnerable population (patients who are minorities, children, old, poor, underfunded, or have particular medical conditions) was the most affected (50%, CI: 37-62%, I2: 99.33%), with the predominance of GIP in the 6 to <20 years age group (48%, CI: 43-54%, I2: 99.68%). Reports on multi-parasitism (44%, CI: 40-48%, I2: 99.73%) were almost double the reports of single infections (43%, CI: 27-59%, I2: 99.77%) with combined molecular and non-molecular techniques demonstrating the best performance for GIP identification. The current review spans more than 40 years of GIP reports from the african continent. Geographical characteristics, environmental factors, habits of its inhabitants, and their health status play a crucial role in GIP modulation and behaviour in its captive hosts. Strategies for regular and enhanced surveillance, policy formation, and high-level community awareness are necessary to identify the true incidence in Africa and the transmission of the pathogens via water and food.

Epidemiology of free-living amoebae infections in Africa: a review.

FLA-related conditions are a rare medical occurrence. Despite their rarity, they are considered a public health concern for two reasons: the absence of a regular treatment regimen in the case of central nervous system infections and the fast progression of the symptoms leading to fatal outcomes. A total of 358 articles were retrieved from different databases (91 from PubMed, 26 from NCBI, 138 from Academia, 102 from Science Direct, and one from IJMED). 7 (46.6%) clinical cases came from Egypt, 2 (13.3%) cases of FLA infection came from Nigeria, 3 (20%) cases came from the Gambia, and 1 (6.6%) case was reported from African countries like Algeria, Tunisia, South Africa, and Zambia. Medical conditions caused by free-living amoeba are considered significant public health concerns. These ubiquitous organisms can cause both fatal and debilitating health conditions. Immediate diagnosis of cases and proper hygienic practices are necessary to provide direct medical intervention. They may be the key to reducing the morbidity and mortality rates from FLA-acquired infections. Although several government-led initiatives have been implemented to mitigate a plethora of parasitic diseases, the case of FLA-related conditions in African countries has yet to be realized.

Well water sources simultaneous contamination with Cryptosporidium and Acanthamoeba in East-Southeast Asia and Acanthamoeba spp. in biofilms in the Philippines.

Cryptosporidium is the leading agent of waterborne parasitic protozoan outbreaks and is the second leading cause of infant mortality due to diarrhoea worldwide. Acanthamoeba spp. causes Acanthamoeba keratitis (AK) and a life-threatening condition known as granulomatous amoebic encephalitis (GAE). The present study aimed to assess the water quality of an indigenous and a rural community for waterborne parasitic protozoan contamination. Aquatic samples (n = 22) were processed by filtration of 500 mL portion through a 1.2 µm pore size glass microfiber filter and eluted for light microscopy, culture in non-nutrient agar, and PCR analysis. Overall, 36% (8/22) of the investigated aquatic samples were positive for either Cryptosporidium spp. oocysts (13%; 3/22) or Acanthamoeba spp., (36%; 8/22) or both (13%; 3/22). Cryptosporidium spp. oocysts were detected in 27% (3/11) of wet season samples only while Acanthamoeba spp. were detected in 18% (2/11) and 55% (6/11) of wet and dry season samples, respectively. Subsequently, molecular detection for Acanthamoeba species identified A. lenticulata and A. hatchetti with 98-99% BLAST similarity. This is the first report on the simultaneous contamination of Cryptosporidium and Acanthamoeba in well water sources in East-Southeast Asia, the first detection of Acanthamoeba spp. in biofilms in the Philippines, and the longest viability demonstrated for A. lenticulata in two-year-old water samples stored at room temperature.

Epidemiology of free-living amoebae in the Philippines: a review and update.

Free-living amoebae (FLA) are considered environmental pathogens and thus pose a public health threat. Their ubiquity in natural sources may magnify the potential severity of health outcomes in the future. However, less attention was given despite several probable public health risks that arise from the presence of pathogenic strains in the environment. Here, we provide epidemiological data based on investigations involving the distribution and occurrence of free-living amoebae in the Republic of the Philippines. This aims to connect data of fragmented studies of these organisms and provide potential roadmaps in FLA research in the country. The majority of the reviewed articles (n = 19) focused on characterization studies (36.8%; 7/19) while environmental isolation and isolation from biological samples had an equal frequency of 31.6% (6/19) each. There is a great disparity between the established ubiquity in environmental sources and the number of cases of FLA infections in the country. FLA-related research in the Philippines is still in its inceptive stage with several gaps to fill, which can be used to formulate policy briefs in the future regarding its isolation, identification, diagnosis, therapeutic management, and control of FLA infections in the country.

Acanthamoeba species isolated from Philippine freshwater systems: epidemiological and molecular aspects.

Free-living amoeba (FLA) research in the Philippines is still in its infancy but has, by far, demonstrated the presence of potentially pathogenic species. Acanthamoeba may cause sight-threatening and central nervous system infections to humans, yet its epidemiologic distribution from local environmental sources is yet to be defined. The present study aimed to provide a baseline epidemiologic distribution of Acanthamoeba spp. in freshwater systems in the Philippines and establish potential pathogenicity of isolates through thermo-tolerance assay. A total of 63 water samples were collected from 13 freshwater systems all over the Philippine archipelago. The low-volume (50 ml) water samples were processed and cultured on non-nutrient agar lawned with Escherichia coli and observed for amoebic growth using light microscopy. Amoebic culture demonstrated 14.28% (9/63) positivity while further molecular testing of culture-positive plates using Acanthamoeba-specific primers demonstrated 100% (9/9) confirmation of Acanthamoeba species. Genotyping of Acanthamoeba isolates revealed T1, T3, T4, T5, T7, T11, and T15 genotypes. Thermo-tolerance assay demonstrated that T5 and T7 genotypes were potentially pathogenic strains. The evidence of environmental distribution of Acanthamoeba spp. in the freshwater systems in the Philippines and thermo-tolerance profile of isolates are significant aspects of amoeba study in public health and calls for initiatives in the dissemination of relevant information and the expansion of knowledge, awareness, and policies on pathogenic waterborne amoeba to mitigate, prevent, detect, and report cases of human infections.

Global prevalence and mortality of severe Plasmodium malariae infection: a systematic review and meta-analysis.

BACKGROUND: Severe complications among patients with Plasmodium malariae infection are rare. This is the first systematic review and meta-analysis demonstrating the global prevalence and mortality of severe P. malariae infection in humans. METHODS: The systematic review and meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. All research articles published on the severity and mortality of P. malariae infection cases in humans were retrieved from three public databases: PubMed, Scopus, and ISI Web of Science. The pooled prevalence estimate and 95% confidence interval (CI) of complications in patients with P. malariae malaria was analysed using the random-effects model provided in Stata software. The pooled odds ratio (OR) and 95% CI of severe malaria for P. malariae infection and Plasmodium falciparum infection were analysed using Review Manager software. RESULTS: Six studies were used to estimate the pooled prevalence of severe P. malariae malaria. Out of 10,520 patients infected with P. malariae, the pooled prevalence estimate of severe P. malariae infection was 3% (95% CI 2-5%), with high heterogeneity (I2: 90.7%). Severe anaemia (3.32%), pulmonary complications (0.46%), and renal impairments (0.24%) were the most common severe complications found in patients with P. malariae infection. The pooled proportion of severe anaemia for P. malariae infection and P. falciparum infection was comparable among the four included studies (OR: 0.74, 95% CI 0.22-2.45, I2 = 98%). The pooled proportion of pulmonary complications was comparable between patients with P. malariae infection and those with P. falciparum infection among the four included studies (OR: 1.44; 95% CI 0.17-12.31, I2: 92%). For renal complications, the funnel plot showed that the pooled proportion of renal complications for P. malariae infection and P. falciparum infection was comparable among the four included studies (OR: 0.94, 95% CI 0.18-4.93, I2: 91%). The mortality rate of patients with P. malariae infection was 0.17% (18/10,502 cases). CONCLUSIONS: This systematic review demonstrated that approximately two percent of patients with P. malariae infection developed severe complications, with a low mortality rate. Severe anaemia, pulmonary involvement, and renal impairment were the most common complications found in patients with P. malariae infection. Although a low prevalence and low mortality of P. malariae infection have been reported, patients with P. malariae infection need to be investigated for severe anaemia and, if present, treated aggressively to prevent anaemia-related death.

Prevalence of severe Plasmodium knowlesi infection and risk factors related to severe complications compared with non-severe P. knowlesi and severe P. falciparum malaria: a systematic review and meta-analysis.

BACKGROUND: Plasmodium knowlesi is a potential cause of severe and fatal malaria, but comprehensive studies of its pooled prevalence and risk factors are lacking. This study aimed to explore the prevalence and risk factors related to severe P. knowlesi infection. METHODS: A systematic review was conducted by retrieving all published articles on severe P. knowlesi available in Web of Science (ISI), Scopus, and PubMed (MEDLINE). Titles, abstracts, and full-text articles were screened, and any irrelevant studies were excluded. The random-effects model was used to compute the pooled prevalence estimate of severe P. knowlesi infection by a metaprop command provided in STATA software. Differences in demographic characteristics, clinical characteristics, and laboratory data were analysed using Review Manager Version 5.3 software for patients in the following groups: 1) patients with severe and non-severe P. knowlesi infection and 2) patients with severe P. knowlesi and severe P. falciparum infection. RESULTS: Out of the 2382 studies retrieved from the three databases, seven studies with a total enrolment of 1124 patients with P. knowlesi infections were eligible to be included in this systematic review and meta-analysis. The pooled prevalence estimate of severe P. knowlesi infection was 19% (95% CI: 11-27%, I2 = 93.7%). Severe acute kidney injuries (AKI) (77 cases, 45.6%), jaundice (71 cases, 42%), and hyperparasitaemia (55 cases, 32.5%) were the common clinical manifestations found among patients with severe complications. In comparison to non-severe P. knowlesi infections, patients with severe P. knowlesi infections had significantly higher age, leucocyte count, and parasitaemia levels (P < 0.05). In comparison to patients with severe P. falciparum infections, patients with severe P. knowlesi infections had significantly higher age, neutrophil count, and creatinine levels (P < 0.05). CONCLUSIONS: This systematic review and meta-analysis demonstrated a high proportion of severe P. knowlesi infections. Patients with severe P. knowlesi infections had higher age, leucocyte count, and parasitaemia levels than those with non-severe P. knowlesi infections. In addition, patients with severe P. knowlesi infections had higher age, neutrophil count, and creatinine levels than those with severe P. falciparum infections.

Severity and mortality of severe Plasmodium ovale infection: A systematic review and meta-analysis.

Plasmodium ovale can infect humans, causing malaria disease. We aimed to investigate the severity and mortality of severe P. ovale infection to increase the awareness of physicians regarding the prognosis of this severe disease and outcome-related deaths in countries in which this disease is endemic. Articles that were published in the PubMed, Scopus, and ISI Web of Science databases prior to January 5, 2020 and reported the prevalence of severe P. ovale infection were systematically searched and reviewed. Studies that mainly reported severe P. ovale infection according to the 2014 WHO criteria for the treatment of malaria were included. Two reviewers selected, identified, assessed, and extracted data from studies independently. The pooled prevalence of severe P. ovale mono-infections was estimated using the command "metaprop case population, random/fixed", which yielded the pooled estimate, 95% confidence interval (CI) and the I2 value, indicating the level of heterogeneity. Meta-analyses of the proportions were performed using a random-effects model to explore the different proportions of severity between patients with P. ovale and those with other Plasmodium species infections. Among the eight studies that were included and had a total of 1,365 ovale malaria cases, the pooled prevalence of severe P. ovale was 0.03 (95% CI = 0.03-0.05%, I2 = 54.4%). Jaundice (1.1%), severe anemia (0.88%), and pulmonary impairments (0.59%) were the most common severe complications found in patients infected with P. ovale. The meta-analysis demonstrated that a smaller proportion of patients with P. ovale than of patients with P. falciparum had severe infections (P-value = 0.01, OR = 0.36, 95% CI = 0.16-0.81, I2 = 72%). The mortality rate of severe P. ovale infections was 0.15% (2/1,365 cases). Although severe complications of P. ovale infections in patients are rare, it is very important to increase the awareness of physicians regarding the prognosis of severe P. ovale infections in patients, especially in a high-risk population.

Prevalence and proportion of Plasmodium spp. triple mixed infections compared with double mixed infections: a systematic review and meta-analysis.

BACKGROUND: Although mixed infection by two Plasmodium species has been recognized, mixed infection by three different Plasmodium species within one individual has not been clarified. This study sought to determine the pooled prevalence and proportion of triple mixed Plasmodium spp. infection compared with double mixed infection. METHODS: Articles from PubMed, Scopus, and Web of Science were searched for cross-sectional studies of triple mixed infection by Plasmodium species and then were retrieved and extracted. The pooled proportion and prevalence of triple mixed infection by Plasmodium species were subjected to random-effects analysis. The secondary outcomes were differences in the pooled proportion between triple mixed infection and double mixed infection by Plasmodium species reported in the included studies. RESULTS: Of 5621 identified studies, triple mixed infection data were available for 35 records, including 601 patients from 22 countries. The overall pooled prevalence of triple mixed infection was 4% (95% Confidence Interval (CI) 3-5%; I2 = 92.5%). The pooled proportion of triple mixed infection compared with double mixed infection was 12% (95% CI 9-18; I2 = 91%). Most of the included studies (29/35; 82.9%) presented a lower proportion of triple mixed infection than double mixed infection. Subgroup analysis demonstrated that the proportion of triple mixed infection was the highest in Oceania (23%; 95% CI 15-36%) and Europe (21%; 95% CI 5-86%), but the lowest in the USA (3%; 95% CI 2-4%). Moreover, the proportion of triple mixed infection was higher in residents (20%; 95% CI 14-29%) than in febrile patients (7%; 95% CI 4-13%), when compared with the proportion of double mixed infection. Subgroup analysis of the age groups demonstrated that, compared with the proportion of double mixed infection, triple mixed infection was lower in patients aged ≤ 5 years (OR = 0.27; 95% CI 0.13-0.56; I2 = 31%) and > 5 years (OR = 0.09; 95% CI 0.04-0.25, I2 = 78%). CONCLUSIONS: The present study suggested that, in areas where triple mixed infection were endemic, PCR or molecular diagnosis for all residents in communities where malaria is submicroscopic can provide prevalence data and intervention measures, as well as prevent disease transmission and enhance malaria elimination efforts.

Reduction in total leukocytes in malaria patients compared to febrile controls: A systematic review and meta-analysis.

BACKGROUND: Leukocyte alterations are a common hematological alteration among malaria patients. OBJECTIVES: This systematic review and meta-analysis aimed to provide data and evidence comparing alterations in total leukocyte counts in malaria patients compared to febrile/healthy subjects at baseline before treatment. A systematic review was conducted by following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement for reporting systematic reviews and meta-analyses. DATA SOURCES: Web of Science (ISI), Scopus, and Medline. STUDY ELIGIBILITY CRITERIA, PARTICIPANTS, AND INTERVENTIONS: All published articles reporting a total leukocyte count of patients infected with malaria, non-malaria (febrile or healthy group) at baseline before treatment before August 27, 2019, were retrieved, and data were extracted by two main reviewers independently. STUDY APPRAISAL AND SYNTHESIS METHODS: We used a forest plot, heterogeneity test (Cochran's Q), and the degree of heterogeneity (I2) to test whether the included studies were heterogeneous. The quality of the included studies was determined by a quality assessment guide based on the quality assessment tool developed by the Newcastle-Ottawa Scale (NOS). Cochran's Q (Chi-square) and Moran's I2 were used to evaluate heterogeneity. Meta-regression using STATA software was conducted to find the source of heterogeneity. A funnel plot with Egger's test was used to examine the significance of publication bias among the included studies. The mean differences were estimated using a random-effects model. RESULTS: Out of the 2,261 articles screened, 29 articles were included in this systematic review and meta-analysis. The heterogeneity test indicated that there was heterogeneity among the included studies with no publication bias. The meta-analysis demonstrated that the total leukocyte count was significantly lower in patients with malaria (n = 4,619) than in those without malaria (n = 10,056) (Z = 4.0, P-value < 0.00001, mean difference = -1.38, 95% CI = -2.06-(-0.71)). Leukocyte differential alterations, low lymphocyte counts (P-value <0.0001, mean difference = -1.03, 95% CI = -1.53-(-0.53)) and a high NL ratio were found in the malaria group (n = 1,579) compared to the non-malaria group (n = 4,991) (P-value <0.0001, mean difference = 0.6, 95% CI = 0.32-0.88). The subgroup analysis indicated that there was a significantly lower total leukocyte count in the malaria group (n = 3,545) than in the febrile group (n = 8,947) (Z = 1.33, P-value < 0.0001, mean difference = -1.76, 95% CI = -2.56-(-0.96)), but no significant difference was found between the malaria group (n = 1,232) and the healthy group (n = 1,679) (P-value > 0.05). LIMITATIONS: As the specific diagnoses in the febrile groups were not reported in the included studies so that the results of the present study need to be carefully interpreted. CONCLUSIONS AND IMPLICATIONS OF KEY FINDINGS: This systematic review demonstrated that the total leukocyte count was affected by malarial infection at baseline despite the heterogeneity of the included studies. Future work must aim to understand the treatment-related total leukocyte reduction during follow-up or post-treatment outcomes in malaria-endemic settings.

Waterborne protozoan pathogens in environmental aquatic biofilms: Implications for water quality assessment strategies.

Biofilms containing pathogenic organisms from the water supply are a potential source of protozoan parasite outbreaks and a significant public health concern. The aim of the present study was to demonstrate the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens (WBPP) in substrate-associated biofilms (SAB) and compare it to surface water (SW) and sediments with bottom water (BW) counterparts using manual filtration and elution from low-volume samples. For scenario purposes, simulated environmental biofilm contamination was created from in-situ grown one-month-old SAB (OM-SAB) that were spiked with Cryptosporidium parvum oocysts. Samples were collected from the largest freshwater reservoirs in Luzon, Philippines and a University Lake in Thailand. A total of 69 samples (23 SAB, 23 SW, and 23 BW) were evaluated using traditional staining techniques for Cryptosporidium, and Immunofluorescence staining for the simultaneous detection of Cryptosporidium and Giardia. WBPP were found in 43% SAB, 39% SW, and 39% BW of the samples tested in the present study with SAB results reflecting SW and BW results. Further highlights were demonstrated in the potential of using low-volume samples for the detection of parasites in source water. Scanning electron microscopy of OM-SAB samples revealed a naturally-associated testate amoeba shell, while Cryptosporidium oocysts spiked samples provided a visual profile of what can be expected from naturally contaminated biofilms. This study provides the first evidence for the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens in low-volume aquatic matrices and further warrants SAB testing along with SW and BW matrices for improved water quality assessment strategies (iWQAS).

First report of Cryptosporidium hominis in a freshwater sponge.

Identification of Cryptosporidium oocyst is essential in ensuring water quality fit for human use, consumption, and recreation. This communication proposes the supplemental analysis of substrate-associated biofilms, in particular, freshwater sponges in improving case finding of waterborne-protozoan pathogens (WBPP) in environmental aquatic samples. In this study, a small portion of a mature freshwater sponge under the Genus Spongilla was subjected to microscopic and molecular analysis to identify the presence of Cryptosporidium. Microscopic screening with modified Kinyoun's staining (MK) and microscopic confirmation using direct antibody fluorescent testing (IFT) returned with Cryptosporidium spp. positive findings. Molecular investigation resulted in the confirmation of Cryptosporidium hominis upon sequencing of PCR products and phylogenetic analysis. This is the first report of a pathogenic protozoan, C. hominis isolated from a freshwater sponge. The results of this study provide evidence of the value of expanding water quality assessment strategies to the analysis of substrate-associated biofilms and sponges in improving case finding of WBPP in natural aquatic environments.

Molecular identification of Vermamoeba vermiformis from freshwater fish in lake Taal, Philippines.

Free Living Amoebae (FLA) are considered ubiquitous. FLAs may infect various biological organisms which act as reservoir hosts. Infected freshwater fishes can pose a public health concern due to possible human consumption. This study aims to identify possible pathogenic FLAs present in freshwater fishes. Seventy five (75) Oreochromis niloticus were studied for the presence of FLAs. Fish organs were suspended in physiologic saline pelleted and cultured in non-nutrient agar (NNA) lawned with Escherichia coli and were incubated in 33 °C for 14 days. Eighteen (18) fish gills and nineteen (19) fish intestine samples presented with positive growth. Trophozoites and cystic stages of FLAs were subcultured until homogenous growth was achieved. Cells were harvested from cultured plates and DNA was extracted using Chelex resin. DNA was subjected to polymerase chain reaction using universal forward primer EukA and reverse primer EukB targeting the 18s RNA. Of the 37 plates that presented with positive amoebic growth, 9 samples showed the presence of DNAs and were sent for further purification and sequencing. Basic Local Alignment Search Tool (BLAST) results showed that protists isolated from fish organs in Lake Taal include: Eocercomonas (HM536152), Colpoda steinii (KJ607915) and Vermamoeba vermiformis (KC161965). The results showed that fresh-water fishes can harbour FLAs in the gut. It is proposed that freshwater reservoirs utilized for aquaculture be monitored for the presence of FLAs and extensive study be conducted on the pathogenicity of bacterial endosymbionts and infecting viruses to its mammalian and non-mammalian host.