Small Subunit Ribosomal Ribonucleic Acid Research Articles

Re-evaluation of the systematic position of the order Prostomatida (Protista: Ciliophora), with the establishment of two new genera and two new species

Abstract Prostomatean ciliates inhabit a wide range of aquatic environments and serve as a trophic link in food webs. However, the biodiversity and molecular phylogeny of the Prostomatea, especially the order Prostomatida, is poorly known owing to undersampling and the paucity of molecular data. Here, three prostomatid ciliates, Platina marina gen. nov., sp. nov., Parametacystis pulchra gen. nov., sp. nov. and Apsiktrata gracilis, were studied based on morphological data and small subunit ribosomal ribonucleic acid (SSU rRNA) gene sequences. The two new genera show a close relationship with the class Plagiopylea but a distant relationship with Metacystis, which bears a strong morphological similarity to the two new genera. The simplified oral ciliature can be regarded as a synapomorphy of the two new genera. The SSU rRNA gene of a representative of the family Apsiktratidae was sequenced for the first time. Morphological and phylogenetic analyses suggest that Apsiktratidae might be more closely related to Prorodontida than to Prostomatida, and the oral basket might be more phylogenetically informative than the brosse in distinguishing Prorodontida and Prostomatida. Supplementation with rare taxa reveals a close association between the Prostomatida and Trimyemidae. The putative secondary structure of the SSU rRNA V9 region suggests a sister relationship between the order Prostomatida and the class Plagiopylea.

Molecular phylogeny and taxonomy of three anaerobic plagiopyleans (Alveolata: Ciliophora), retrieved from two geographically distant localities in Asia and North America

Abstract Anaerobic ciliates are key organisms for studying adaptations to an anaerobic lifestyle, the evolution of eukaryotic cell organelles, mechanisms of energy metabolism, and symbiosis with prokaryotes; however, the biodiversity of their marine representatives remains poorly known. In this study, three species of ciliates belonging to the class Plagiopylea, i.e. Trimyema foissnerisp. nov.,Plagiopyla rarisetasp. nov., and Plagiopyla frontata, collected from various marine anoxic habitats from two geographically distant continents, Asia and North America, were investigated using morphological and molecular methods. Trimyema foissneri can be recognized by three complete somatic ciliary girdles, special arrangement of kinetosomes near the anterior end of the oral kineties, and the presence of a ventrolateral fragment. Plagiopyla rariseta is distinguished by sparse cilia on the frontal region and particular orientation of the buccal cavity. Plagiopyla frontata is newly recorded and is redescribed in detail and redefined based on new populations. Phylogenetic analyses based on the small subunit ribosomal ribonucleic acid (SSU rRNA) gene supported the generic assignment of the three species and corroborated the monophyly of the families Trimyemidae and Plagiopylidae. An improved key to the identification of the valid species of genus Trimyema is also supplied.

[Prevalence and genotyping of Cryptosporidium spp. and Giardia lamblia in dogs and cats from a pet hospital in Shanghai Municipality].

To investigate the prevalence and genotypes of Cryptosporidium spp. and Giardia lamblia in dogs and cats from a pet hospital in Shanghai Municipality. A total of 145 fresh fecal samples were collected from pet dogs and cats in a pet hospital in Shanghai Municipality during the period from November 2021 to June 2022, including 99 dog fecal samples and 46 cat fecal samples. The small subunit ribosomal ribonucleic acid (SSU rRNA) gene of Cryptosporidium and the triose phosphate isomerase (TPI) gene of G. lamblia were amplified using nested PCR assay, and the positive amplification products were sequenced from both directions. The sequence assembly was performed using the software Clustal X 2.1, and sequence alignment was conducted using BLAST. A phylogenetic tree was created with the Neighbor-Joining method using MEGA 11.0 to identify parasite species or genotype. The overall prevalence of Cryptosporidium and G. lamblia was 20.00% (29/145) in 145 pet dog and cat fecal samples, with the prevalence of 0.69% (1/145) and 19.31% (28/145) in Cryptosporidium and G. lamblia, respectively. G. lamblia was only detected in dog fecal samples, with prevalence of 18.18% (18/99), while the detection rates of Cryptosporidium and G. lamblia were 2.17% (1/46) and 21.74% (10/46) in cat fecal samples. Nucleotide sequence analysis showed that one Cryptosporidium positive sample was characterized as C. felis, and 28 G. lamblia positive samples were all characterized as Giardia assemblage A, which showed 100% sequence homology with human isolates of Giardia. Phylogenetic analysis revealed that the sequences obtained in this study belonged to the same branch with the reported Giardia assemblage A. Cryptosporidium and G. lamblia infection was prevalent in pet dogs and cats from the study pet hospital in Shanghai Municipality, and there is a zoonotic risk for the species and genotype. Intensified surveillance of Cryptosporidium and Giardia infection is recommended in pets and their owners, and improved management of pet keeping is required.

Molecular Epidemiology of Plasmodium species in Conflicted Federally Administered Tribal Area (FATA) Pakistan.

Military conflicts have been significant obstacles in detecting and treating infectious disease diseases due to the diminished public health infrastructure, resulting in malaria endemicity. A variety of violent and destructive incidents were experienced by FATA (Federally Administered Tribal Areas). It was a struggle to pursue an epidemiological analysis due to continuing conflict and Talibanization. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai agencies from May 2017 to May 2018. For Giemsa staining, full blood EDTA blood samples have been collected from symptomatic participants. Malaria-positive microscopy isolates were spotted on filter papers for future Plasmodial molecular detection by nested polymerase chain reaction (nPCR) of small subunit ribosomal ribonucleic acid (ssrRNA) genes specific primers. Since reconfirming the nPCR, a malariometric study of 762 patients found 679 positive malaria cases. Plasmodium vivax was 523 (77%), Plasmodium falciparum 121 (18%), 35 (5%) were with mixed-species infection (P. vivax plus P. falciparum), and 83 were declared negative by PCR. Among the five agencies of FATA, Khyber agency has the highest malaria incidence (19%) with followed by P. vivax (19%) and P. falciparum (4.1%). In contrast, Kurram has about (14%), including (10.8%) P. vivax and (2.7%) P. falciparum cases, the lowest malaria epidemiology. Surprisingly, no significant differences in the distribution of mixed-species infection among all five agencies. P. falciparum and P. vivax were two prevalent FATA malaria species in Pakistan's war-torn area. To overcome this rising incidence of malaria, this study recommends that initiating malaria awareness campaigns in school should be supported by public health agencies and malaria-related education locally, targeting children and parents alike.

Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan.

Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

The Biodiversity of Epilithic Microalgal Communities Colonising a Central Mediterranean Coastline

Schembri, S. and Zammit, G., 2022. The biodiversity of epilithic microalgal communities colonising a central Mediterranean coastline. Journal of Coastal Research, 38(2), 249–260. Coconut Creek (Florida), ISSN 0749-0208. Microbial communities colonise the coastal ecosystem around the Maltese islands. However, such communities are understudied, both in Malta and in the central Mediterranean. This research aims to increase current knowledge about the biodiversity of phototrophic communities growing along a central Mediterranean rocky shoreline and is the first such study to be performed on microorganisms growing in epilithic biofilms and microbial mats along the coastline in the Maltese islands. Samples were obtained using techniques that were noninvasive to the underlying substratum. These were studied by direct observation using light and electron microscopy, by culturing in vitro, by molecular analysis via sequencing of the small subunit ribosomal ribonucleic acid genes and phylogenetic analyses. Microscopic analyses revealed highly diverse communities made of both photosynthetic and heterotrophic organisms. The predominant microorganisms were simple filamentous cyanobacteria including species of Leptolyngbya, Phormidesmis, Nodosilinea, Toxifilum, Phormidium, and Lyngbya, as well as heterocytous Calothrix and Nunduva spp. The coccal cyanobacteria included species of Aphanocapsa and Chroococcus, whereas coccal microalgae belonged to Chlorella, Chlamydomonas, and Coelastrella spp., accompanied by diatoms of Navicula sp. These results include first records of cyanobacterial and microalgal barcodes that were genetically sequenced from a coastline in the central Mediterranean. Germlings of the filamentous macroalga Cladophora were embedded in the rocky substrate that was preconditioned by biofilm growth and ciliated protozoans, micronematodes, and microcrustaceans interacted with the microbial communities. The isolation of new cyanobacterial and microalgal strains from these phototrophic communities highlights the importance of employing a combined multiphasic approach to supplement current knowledge about the biodiversity of microbial communities colonising rocky shores.

Integrative taxonomy suggests that South American freshwater nematodes Echinocephalus and their host stingrays co-originated in late Oligocene to early Miocene

Abstract Nematoda of the genus Echinocephalus Molin, 1858 include species from the Gnathostomatidae family, the adult stages of which parasitize the stomach and spiral intestine of elasmobranchs as their final hosts. In the present study, we describe Echinocephalus spinosus n. sp. found parasitizing the spiral valve of the freshwater stingray Potamotrygon motoro from the Tapajós River, in the Amazon Basin, in the state of Pará, Brazil. In the study we performed morphological (light and scanning electron microscopy) and molecular (small subunit ribosomal ribonucleic acid (SSU rDNA) and mitochondrial cytochrome c oxidase I sequencing) analyses. E. spinosus n. sp. is only the second species of the Echinocephalus genus described from a strictly freshwater environment. The SSU rDNA based phylogenetic analyses showed Echinocephalus clade as a sister lineage of Gnathostoma, and that the new species arises as a sister to Echinocephalus cf. pseudouncinatus. Time-calibrated phylogenetic analysis revealed that the origin of the freshwater Echinocephalus coincides with the recently proposed origin of the freshwater host potamotrygonin stingray, namely the late Oligocene to early Miocene, when the western Amazon was dominated by the Pebas wetlands, an epicontinental marine/freshwater system.

Gut microbiota profile in patients with nonalcoholic fatty liver disease and presumed nonalcoholic steatohepatitis.

Background:The main composition of intestinal microbiota in nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) patients has not yet been elucidated. In this, case-control study, we identified differences of intestinal microbiota in male patients with NAFLD, presumed NASH, and healthy controls.Materials and Methods:We compared gut microbial composition of 25 patients with NAFLD, 13 patients with presumed NASH, and 12 healthy controls. Demographic information as well as clinical, nutritional, and physical activity data was gathered. Stool and blood samples were collected to perform the laboratory analysis. The taxonomic composition of gut microbiota was assessed using V4 regions of microbial small subunit ribosomal Ribonucleic acid genes sequencing of stool samples.Results:Firmicutes, Actinobacteria, and Bacteroidetes were the most frequently phyla in all groups. Our results revealed that Veillonella was the only genus with significantly different amounts in presumed NASH patients compared with patients with NAFLD (P = 2.76 × 10−6, q = 2.07 × 10−4, logFC = 5.52).Conclusion:This pilot study was the first study to compare gut microbial composition in patients with NAFLD and presumed NASH in the Middle East. Given the potential effects of gut microbiota on the management and prevention of NAFLD, larger, prospective studies are recommended to confirm this study's findings.

​Therapeutic Efficacy of Ethanolic Extract of Curcuma longa and its Component, Curcumin against Experimental Cryptosporidiosis in Mice

Background: Cryptosporidiosis caused by Cryptosporidium spp. is a zoonotic disease and is the most prevalent pathogens worldwide and leads to severe diarrhoeal diseases and affects the immunological status of the individual. Thus, the study was undertaken to examine the anti-cryptosporidial efficacy of curcumin in comparison with ethanolic extract of curcuma longa in immunocompromised mice infected with oocysts isolated from cattle calves of Jammu region and identified as Cryptosporidium parvum using nested PCR on small subunit ribosomal ribonucleic acid (SSU rRNA) gene. Methods: Two hundred female Swiss albino mice were equally divided into ten groups. Group I were kept as a healthy control, group II were immunocompromised, group III were immunocompromised and infected, group IV animals were immunocompromised, infected and treated orally with nitazoxanide. Animals in groups V to VII were immunocompromised, infected and treated with ethanolic extract of C. longa @ 4, 6 and 8 mg/kg/day/os respectively whereas groups VIII to X were immunocompromised, infected and treated with pure salt of curcumin @ 4, 6 and 8 mg/kg/day/os respectively for 5 successive days. Thus, mean oocysts per gram faeces, body weight gain and histopathological changes were measured in different groups. Result: Administration of curcumin as a therapeutic agent @ 8 mg/kg body weight for five days resulted in higher percent mean oocyst reduction of 74.03% and improved body weight gain in experimentally infected mice. Histopathological changes showed that treatment with oral curcumin (group X) in animals had minimal and improved intestinal lesions as compared to animals treated with C. longa (group VII). Altogether, curcumin showed promising anticryptosporidial effects under in vivo conditions and deserves further exploration.

Assessment of Microscopic Detection of Malaria with Nested Polymerase Chain Reaction in War-Torn Federally Administered Tribal Areas of Pakistan.

Diagnostic accuracy of malaria is critical for early treatment, control, and elimination of malaria, especially in war-affected malaria-endemic areas. Microscopic detection of Plasmodium species has been the gold standard in remote malaria-endemic regions. However, the diagnostic accuracy is still questioned, especially in discriminating mixed and submicroscopic parasitic levels. This study was designed to evaluate the diagnostic performance of microscopic examination against nested PCR analysis in war-torn malaria-endemic Federally Administered Tribal Areas (FATA) of Pakistan. Venous blood samples were collected from symptomatic patients for microscopic examination and nested PCR analysis from January 2016-December 2016 from five Agencies (Bajaur, Mohmand, Khyber, Orakzai and Kurram Agency) and four Frontier Regions (Peshawar, Kohat, Bannu, and Dera Ismail Khan Frontier Region) of FATA. Malaria-positive isolates were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes) for speciation. Among enrolled participants, 762 were found positive for malaria parasite on microscopic examination of the blood film. Plasmodium vivax was found in 623, Plasmodium falciparum in 132 and 7 were diagnosed with mixed infection (P. vivax and P. falciparum coinfection). Nested PCR detected Plasmodium infection in 679 samples (523 P. vivax, 121 P. falciparum, and 35 mixed infections). Compared with microscopy, the sensitivity of nested PCR was 98.94%, and specificity was 98.27%, while the sensitivity and specificity of slide microscopy 89.34% and 87.99% respectively. The conventional microscopy method has low sensitivity to detect the mixed infection as compared to nested PCR. High sensitivity and specificity observed in nested PCR make this molecular tool a useful technique for monitoring, controlling, and eliminating malaria-endemic regions.

Detection and molecular identification of Blastocystis isolates from humans and cattle in northern Egypt.

Blastocystis is one of the less well-understood waterborne protozoa of medical and veterinary importance. The present study aimed to detect, identify, and subtype Blastocystis in cattle and humans in northern Egypt and to investigate the potential for zoonotic transmission. Fecal samples were collected from 136 humans (68in contact with animals and 68 no animal contact) and 190 cattle from a rural area in Kafr El-Sheikh Province, Egypt. All samples were microscopically examined by direct wet mount technique and cultured in Jones' medium. Genomic DNA was extracted from positive cultures. A region of the Blastocystis small subunit ribosomal ribonucleic acid (SSU rRNA) gene was amplified using PCR. Blastocystis subtypes were identified by sequencing of the amplified SSU rRNA gene. The relatedness of sequence types obtained in this study and others submitted in GenBank was compared according to their genetic relatedness. Out of 136 human and 190 cattle samples, Blastocystis was detected in 53 (38.9%) and 37 (19.4%), respectively using either culture or direct wet mount. Blastocystis SSU rRNA gene was amplified in 55 samples (32 humans and 23 animals). Six subtypes were identified (STs 1-3in humans and ST4, ST10 and ST14 in cattle) and were distinguishable with inter (up to 0.075) and intra (up to 0.024) subtype variations. A close phylogenetic relationship between human and animal isolated STs 1-4 was demonstrated. To the best of our knowledge, this is the first study to investigate the the potentiality for zoonotic transmission of certain Blastocystis subtypes in northern Egypt.

New morphological and molecular perspectives about Macracanthorhynchus hirudinaceus (Acanthocephala: Oligacanthorhynchidae) from wild boar, Sus scrofa Linn., in Ukraine.

We make new morphological observations not previously reported for the old acanthocephalan Macracanthorhynchus hirudinaceus (Pallas, 1781) Travassos, 1917, described for the first time about 240 years ago. Our specimens were collected from the wild boar, Sus scrofa Linn., in Ukraine in 2005. We provide comparative morphometrics with other populations from Ukraine, Union of Soviet Socialist Republics, Europe, Brazil and the United States of America. Our specimens from the Ukraine had the smallest trunk (110-120 mm long), longest and thickest hooks, with the third hook being the largest, largest eggs, and a proboscis wider than long. We document the morphology of the proboscis, apical organ, hooks, hook roots, sensory pores, micropores, and eggs with scanning electron microscopy for the first time. We also provide chemical analysis of hooks and eggs using energy-dispersive X-ray analysis, discuss its micropores, and provide a new molecular profile based on 18S rDNA from a European population for the first time. Edge of hook tips feature high levels of calcium and phosphorous but the high level of sulphur is mostly found in the cortical layer of eggs. One new partial 18S rDNA sequence (482 nt length) was generated from an adult specimen of M. hirudinaceus. We present the first 18S rDNA published sequence for this cosmopolitan acanthocephalan obtained from Europe. The amplified region corresponded to the approximate middle region of the small subunit ribosomal ribonucleic acid gene, which is ~1800 nt in length. This molecular contribution is especially valuable in light of the extreme scarcity of genetic information about species of Macracanthorhynchus and of the family Oligacanthorhynchidae as a whole.

Evolutionary analyses of phylum Chaetognatha based on mitochondrial cytochrome oxidase I gene

Chaetognaths (arrow worms) are an enigmatic group of transparent planktonic invertebrates and play an important role in the marine food web. Their morphological and developmental features have raised extensive debates since the discovery of the phylum in the 18th century. Uncertainty in the phylogenetic placement of certain chaetognath species still exists and is puzzling many scientists who have tried to clarify this task. Studies using a portion of both small subunit ribosomal ribonucleic acid (SSU rRNA) and large subunit ribosomal ribonucleic acid (LSU rRNA) genes when integrated with conventional taxonomy were contributed to resolve taxonomical issues in this group. Here we present the first phylogenetic study of Chaetognatha based on a portion of mitochondrial cytochrome oxidase I (COI) gene and compare our results with the earlier morphological and molecular evolutionary hypotheses. This study includes 16 extant species, representing 8 genera and 6 of which are among the 9 extant families. We recommend the following clade structure for the phylum: Aphragmophora comprising Sagittidae with Pterosagittidae and Krohnittidae included in the Sagittidae and Phragmophora comprising Eukrohniidae, Spadellidae, and Heterokrohniidae. Phylogenetic analyses also supported the division of Phragmophora into two monophyletic groups: the Monophragmophora and Biphragmophora. Moreover, Ctenodontina/Flabellodontina and Syngonata/Chorismogonata suborders were not validated. Precise phylogenetic investigations using various molecular markers and specimens from diverse regions are definitely needed to provide an exact evolutionary concept on this phylum.

Stenamoeba dejonckheerei sp. nov., a Free-Living Amoeba Isolated from a Thermal Spring.

Two amoeboid organisms were obtained from water samples taken from a thermal spring, "Agua Caliente", in Northwestern Mexico. The isolates were obtained when samples were cultivated at 37 °C on non-nutrient agar coated with Escherichia coli. The initial identification of the isolates was performed morphologically using light microscopy. The samples were found to have trophozoite morphology consistent with members of the genus Stenamoeba, a genus derived in 2007 from within the abolished polyphyletic genus Platyamoeba. Further analysis was performed by sequencing PCR products obtained using universal eukaryotic primers for the small subunit ribosomal ribonucleic acid (SSU rRNA) gene. Sequencing primers were designed to allow the comparison of the 18S rRNA gene sequences of the new isolates with previous sequences reported for Stenamoeba. Phylogenetic relationships among sequences from Stenamoeba were determined using Maximum Likelihood analysis. The results showed the two "Agua Caliente" sequences to be closely related, while clearly separating them from those of other Stenamoeba taxa. The degrees of sequence differentiation from other taxa were considered sufficient to allow us to propose that the Mexican isolates represent a new species.

Molecular characterization of Giardia spp. and Cryptosporidium spp. from dogs and coyotes in an urban landscape suggests infrequent occurrence of zoonotic genotypes

Giardia spp. and Cryptosporidium spp. are common gastrointestinal parasites with the potential for zoonotic transmission. This study aimed to (1) determine the genotypes occurring in dogs and coyotes occupying a similar urban area; (2) determine if these hosts were infected with potentially zoonotic genotypes; (3) provide baseline molecular data. In August and September 2012, 860 dog owners living in neighborhoods bordering six urban parks in Calgary, Alberta, Canada, provided faecal samples from their dogs. From March 2012 through July 2013, 193 coyote faeces were also collected from five of six of the same parks. Direct immunofluorescence microscopy (DFA) indicated that Giardia spp. and Cryptosporidium spp. infected a total of 64 (7.4%) and 21 (2.4%) dogs, as well as 15 (7.8%) and three (1.6%) coyotes, respectively. Semi-nested, polymerase chain reactions targeting the 16S small-subunit ribosomal ribonucleic acid (SSU rRNA) and 18S SSU rRNA genes of Giardia spp. and Cryptosporidium spp., respectively, were conducted on samples that screened positive by DFA, and products were sequenced and genotyped. Dogs were infected with Giardia intestinalis canid-associated assemblages C (n = 14), D (n = 13), and Cryptosporidium canis (n = 3). Similarly, G. intestinalis assemblages C (n = 1), D (n = 1) and C. canis (n = 1), were detected in coyotes, as well as G. intestinalis assemblage A (n = 1) and Cryptosporidium vole genotype (n = 1). Dogs and coyotes were predominantly infected with host-specific genotypes and few potentially zoonotic genotypes, suggesting that they may not represent a significant risk for zoonotic transmission of these parasites in urban areas where these hosts are sympatric.

The first molecular detection of Blastocystis subtypes in human faecal samples from Ibague, Colombia

Blastocystis spp. has become one of the protozoans arousing the greatest scientific interest because of the controversy surrounding its biology; it is currently considered one of the most prevalent organisms in humans and animals worldwide. Such prevalence increases, especially in tropical countries where infection rates are high, highlighting the need to conduct studies focused on understanding this protozoan's biology. Interestingly, molecular tools are emerging as the best option for diagnosing this infection. This study was thus aimed at conventional PCR molecular detection and characterisation of Blastocystis spp. in human faecal samples from Ibagué, Colombia, using primers targeting the small subunit ribosomal ribonucleic acid (rRNA) gene. One hundred human faecal samples with confirmed Blastocystis spp. were studied, revealing the following subtype genetic diversity: ST1 50%, ST2 33% and ST3 17%. The results contributed to the limited information available regarding Blastocystis spp. in Colombia and created a reference point for further studies in the region.

Molecular identification of Leishmania tropica and L. infantum isolated from cutaneous human leishmaniasis samples in central Morocco

Cutaneous leishmaniasis (CL) in Marrakesh-Safi region located in the central-south part of Morocco is a public health problem. This study assessed the efficiency of a microscopic examination method in establishing the diagnosis of CL and PCR for the characterization and identification of the circulating Leishmania strains in different CL foci of the study area. A total of 297 smears obtained from cutaneous lesions of suspected patients with CL were stained with May-Grünwald Giemsa (MGG) for microscopic examination. For each positive smear, genomic DNA was extracted and PCR-analysed, targeting the small subunit ribosomal ribonucleic acid (ssu rRNA) gene to detect Leishmania DNA. Then, the internal transcribed spacer 1 (ITS1) was amplified and sequenced in order to identify the Leishmania species. The sensitivity and specificity of the conventional microscopy with ssu rRNA gene were compared by Leishmania nested PCR (LnPCR) and ITS1 gene by ITS-PCR. A total of 257 smears were positive in the microscopic examination, i.e. the detection rate of amastigotes by optical microscopy was 86.53% (257/297). The LnPCR was found to have a specificity and a sensitivity of 100%, each. Interestingly, the sequencing results showed that 99.61% (256/257) of the isolates had Leishmania tropica and 0.39% (1/257) had L. infantum infection. Though, classical microscopic examination is useful and economical, it is not sensitive enough, especially in endemic regions where several Leishmania species coexist. In such situations, PCR constitutes a complementary method for the identification of the causal species. The results indicate that both the L. tropica (dominant) and L. infantum are the causative agents of CL in the Marrakesh-Safi region. The rate of CL infection is high in Imintanout, and Chichaoua provinces. Hence, early diagnosis and prompt treatment of CL patients is necessary to prevent its extension to neighboring localities.

Biomarker Profiling of Microbial Mats in the Geothermal Band of Cerro Caliente, Deception Island (Antarctica): Life at the Edge of Heat and Cold.

Substrate–atmosphere interfaces in Antarctic geothermal environments are hot–cold regions that constitute thin habitable niches for microorganisms with possible counterparts in ancient Mars. Cerro Caliente hill in Deception Island (active volcano in the South Shetland Islands) is affected by ascending hydrothermal fluids that form a band of warm substrates buffered by low air temperatures. We investigated the influence of temperature on the community structure and metabolism of three microbial mats collected along the geothermal band of Cerro Caliente registering 88°C, 8°C, and 2°C at the time of collection. High-throughput sequencing of small subunit ribosomal ribonucleic acid (SSU rRNA) genes and Life Detector Chip (LDChip) microarray immunoassays revealed different bacterial, archaeal, and eukaryotic composition in the three mats. The mat at 88°C showed the less diverse microbial community and a higher proportion of thermophiles (e.g., Thermales). In contrast, microbial communities in the mats at 2°C and 8°C showed relatively higher diversity and higher proportion of psychrophiles (e.g., Flavobacteriales). Despite this overall association, similar microbial structures at the phylum level (particularly the presence of Cyanobacteria) and certain hot- and cold-tolerant microorganisms were identified in the three mats. Daily thermal oscillations recorded in the substrate over the year (4.5–76°C) may explain the coexistence of microbial fingerprints with different thermal tolerances. Stable isotope composition also revealed metabolic differences among the microbial mats. Carbon isotopic ratios suggested the Calvin–Benson–Bassham cycle as the major pathway for carbon dioxide fixation in the mats at 2°C and 8°C, and the reductive tricarboxylic acid cycle and/or the 3-hydroxypropionate bicycle for the mat at 88°C, indicating different metabolisms as a function of the prevailing temperature of each mat. The comprehensive biomarker profile on the three microbial mats from Cerro Caliente contributes to unravel the diversity, composition, and metabolism in geothermal polar sites and highlights the relevance of geothermal-cold environments to create habitable niches with interest in other planetary environments.

Occurrence of Cryptosporidium and Giardia in potable water sources in Chandigarh, Northern India

AbstractContamination with Cryptosporidium and Giardia from drinking water sources in a city in Northern India was assessed. A protocol modified from a standard ISO protocol, which includes filtration, concentration, separation and detection steps, was tested and showed comparable recovery efficiencies (Giardia mean = 77.4%, Cryptosporidium mean = 61.8% from the modified protocol, compared with Giardia mean = 61.6%, Cryptosporidium mean = 69% from the ISO protocol) at a substantial cost reduction. This protocol was used for analysing 71 samples of potable water from different areas of Chandigarh, where sampling locations were divided into groups according to the population density, which also partially equates with the level of infrastructure. Samples were collected during (n = 29) and outside the monsoon season (n = 42). Of all samples analysed, 16 (22.5%) were Cryptosporidium- and/or Giardia-positive. Parasites per sample were low (1–10 (oo)cysts per 10 L), although one sample contained large numbers of Giardia cysts (>1,000). Polymerase chain reaction analyses on the small subunit ribosomal ribonucleic acid (SSU rRNA), triose-phosphate isomerase (tpi), glutamate dehydrogenase (gdh) and beta-giardin (bg) gene sequences on Giardia-positive samples and SSU rRNA on Cryptosporidium-positive samples tended to be unsuccessful, although Giardia cysts of Assemblages B and C were identified. No association with the season was detected, but an association with the location of water supply was identified. Samples from areas with the lowest infrastructure were not associated with higher levels of contamination, but samples from the middle level were significantly more likely to be contaminated than those from the highest level of infrastructure. Results indicate that even in a city with a well-developed infrastructure, the contamination of potable water with protozoan parasites remains a public health risk.

Molecular characterization of Blastocystis in cattle in Turkey.

Blastocystis genus exist in a wide variety of hosts, including humans, birds, insects, annelids, amphibians, fish, and mammals. PCR-based molecular diagnostic methods have been successfully used to detect Blastocystis spp. in feces, and small subunit ribosomal ribonucleic acid (SSU rRNA) gene-based subtyping is the preferred method for diagnosis. There has been discussion about the subtypes of Blastocystis spp. which has been detected so far. To date, 26 different subtypes have been reported. The aim of this study was to determine the existence and diversity of Blastocystis spp. in cattle. In our study, a total of 80 stool samples were collected from cows and calves at 13 different farms in Burdur and one farm in Aydın. Using molecular method, a total of 9 samples out of 80 samples were found to be positive (11.25%) for Blastocystis. As a result of sequence analysis of Blastocystis positive samples, the subtype 14 was detected on seven samples, while in the other two samples, Blastocystis subtype 10 was identified. The ST10 and ST14 subtypes are commonly reported in animals but not isolated from human. Our analyses showed genetic differences among Blastocystis subtypes. Our study is the first Blastocystis subtyping study from cattle in Turkey.