Molecular Characterization and Genetic Diversity of Cryptosporidium spp. in Human Infections: A Review

IntroductionGlobally Cryptosporidium and Giardia species are the most common non-bacterial causes of diarrhoea in children and HIV infected individuals, yet data on their role in paediatric diarrhoea in Kenya remains scant. This study investigated the occurrence of Cryptosporidium species, genotypes and subtypes in children, both hospitalized and living in an informal settlement in Nairobi.MethodsThis was a prospective cross-sectional study in which faecal specimen positive for Cryptosporidium spp. by microscopy from HIV infected and uninfected children aged five years and below presenting with diarrhoea at selected outpatient clinics in Mukuru informal settlements, or admitted to the paediatric ward at the Mbagathi District Hospital were characterized. The analysis was done by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) of the 18srRNA gene for species identification and PCR-sequencing of the 60 kDa glycoprotein (GP60) gene for subtyping.Results C. hominis was the most common species of Cryptosporidium identified in125/151(82.8%) of the children. Other species identified were C. parvum 18/151(11.9%), while C. felis and C. meleagridis were identified in 4 and 2 children, respectively. Wide genetic variation was observed within C. hominis, with identification of 5 subtype families; Ia, Ib, Id, Ie and If and 21 subtypes. Only subtype family IIc was identified within C. parvum. There was no association between species and HIV status or patient type.Conclusion C. hominis is the most common species associated with diarrhoea in the study population. There was high genetic variability in the C. hominis isolates with 22 different subtypes identified, whereas genetic diversity was low within C. parvum with only one subtype family IIc identified.

Secondhand Smoke and CVD in Low- and Middle-Income Countries: A Case for Action

Despite the clinical and public health importance of Cryptosporidium parvum, little is known about its transmission dynamics in cattle and other farm animals, especially in Iran and other Mideast countries. Currently, the maintenance of the parasites on cattle farms and the role of herd-to-herd transmission in cryptosporidiosis epidemiology are not clear (1). Recent molecular epidemiologic studies of cryptosporidiosis have helped researchers to better understand the transmission of cryptosporidiosis in humans and the public health significance of Cryptosporidium spp. in animals and the environment (2, 3). Because of the ability of Cryptosporidium spp. to infect humans and a wide variety of animals, and because of the ubiquitous presence of Cryptosporidium oocysts in the environment, humans can acquire Cryptosporidium infections through several transmission routes, such as direct contact with infected persons (person-to-person transmission) or animals (zoonotic transmission), and ingestion of contaminated food (foodborne transmission) and water (waterborne transmission). The relative importance of these transmission routes in the epidemiology of cryptosporidiosis is not entirely clear, largely due to the fact that traditional diagnostic tools do not have the ability to differentiate sources of parasites (3). The use of molecular tools has been helpful in assessing the zoonotic potential of various Cryptosporidium species and the sources of human infection; it has begun to play a significant role in the characterization of transmission dynamics in different areas and in the determination of host specificity of various Cryptosporidium spp. The 60 kDa glycoprotein (gp60, also known as Cpgp15/45) gene encodes a precursor protein that is proteolytically cleaved to yield mature surface glycoproteins gp45 and gp15 (also known as Cp17), both of which are implicated in the attachment and invasion of enterocytes by sporozoites and merozoites. An important feature of this gene is its high degree of sequence polymorphism in C. hominis, C. parvum, and C. meleagridis isolates. Several subtype families have been identified in these species: 7 subtype families in C. hominis (Ia–Ig), 2 zoonotic (IIa, IId) and 10 non-zoonotic (IIb, IIc, IIe–IIl) subtype families in C. parvum, and 6 subtype families in C. meleagridis (4). Within each subtype family, there are multiple subtypes based primarily on the number of tri-nucleotide repeats coding for the amino acid serine, as suggested by Sulaiman et al. (2005) (5). The use of gp60 subtyping has allowed the identification of geographic and temporal differences in the transmission dynamics of cryptosporidiosis, the role of zoonotic infections in epidemiology, better appreciation of the public health significance of Cryptosporidium species/genotypes in ruminants and significance of parasite subtypes/strains in clinical manifestations and outbreak potentials, and the tracking of infection and contamination sources during outbreak and endemic investigations (1–5). To our knowledge, there are several molecular epidemiological studies that have documented the presence of C. parvum and C. hominis In Iran (Table 1) (6–12). However, the distribution of subtypes of the two species in humans, animals and environmental is unclear. In the first characterization of Cryptosporidium subtypes in humans and cattle in Iran by sequence analysis of the gp60 gene, 47 samples of C. parvum (22 from children and 25 from cattle) and three of C. hominis (all from children) were characterized. Nine subtypes (two of C. hominis and seven of C. parvum) belonging to four subtype families were found. Cattle were mainly infected with C. parvum IIa subtypes and humans mostly with C. parvum IIa and IId subtypes (Table 2). The predominance of IIa and IId subtypes underlines the importance of zoonotic Cryptosporidium transmission in Iran. Thus, cattle could be a source of human infection with C. parvum IIa in Iran (13–15). Although the source of IId subtypes in humans is not yet clear, IId subtypes are known to be common in sheep and goats in some countries such as Spain (16) and in dairy cattle in some other countries such as Egypt (17) and China (18). Further molecular study in humans and animals is needed in order to determine the extent and animal source of zoonotic transmission of cryptosporidiosis in Iran. Table 1 Distribution of Cryptosporidium spp. in humans in Mideast countries Table 2 Distribution of C. parvum subtypes in humans and cattle in Iran. The dominance of C. parvum and wide occurrence of IId C. parvum subtypes in humans in Iran is similar to the situation seen in other Mideast countries (9–12, 19–25) (Tables 1 and ​and3).3). Children in the Kuwait City are almost exclusively infected with IIa and IId subtypes, although they have little contact with farm animals. As the city uses desalinized sea water as drinking water, the C. parvum transmission appears to be anthroponotic in origin (5, 20). IId subtypes are also common in children in Saudi Arabia and Jordan (Table 3). In many industrialized nations in other areas, C. parvum infections are much less common in humans than C. hominis infections, with the exception of European countries and New Zealand, where both C. parvum and C. hominis are commonly seen in humans. In these industrialized nations, most C. parvum infections are caused by the IIa subtypes commonly found in cattle, indicating zoonotic transmission plays a significant role in cryptosporidiosis epidemiology. In contrast, humans in developing countries are much less commonly infected with C. parvum and most of the few C. parvum infections are caused by the anthroponotic IIc subtype family (2). Table 3 Distribution of C. parvum subtype families in humans in Mideast countries In conclusion, preliminary molecular epidemiological studies have revealed some unique features of cryptosporidiosis transmission in humans in Iran and other Mideast countries. As the C. parvum subtype family IId was the dominant family causing cryptosporidiosis in humans in Iran (13–15), zoonotic transmission could possibly be involved. However, more extensive sampling of both humans and farm animals, especially sheep and goats, and collection of epidemiological data in case-control and longitudinal studies are needed for a better understanding of the sources of C. parvum infections in humans in Iran and other Mideast countries.

Development of a multilocus sequence typing tool for high-resolution subtyping and genetic structure characterization of Cryptosporidium ubiquitum

Listeria monocytogenes (Lm) can persist in food processing environments (FPEs), surviving environmental stresses and disinfectants. We described an intensive environmental monitoring plan performed in Central Italy and involving food producing plants (FPPs) and retail grocery stores (RSs). The aim of the study was to provide a snapshot of the Lm circulation in different FPEs during a severe listeriosis outbreak, using whole genome sequencing (WGS) to investigate the genetic diversity of the Lm isolated, evaluating their virulence and stress resistance profiles. A total of 1217 samples were collected in 86 FPEs with 12.0% of positive surfaces at FPPs level and 7.5% at RSs level; 133 Lm isolates were typed by multilocus sequencing typing (MLST) and core genome MLST (cgMLST). Clonal complex (CC) 121 (25.6%), CC9 (22.6%), CC1 (11.3%), CC3 (10.5%), CC191 (4.5%), CC7 (4.5%) and CC31 (3.8%) were the most frequent MLST clones. Among the 26 cgMLST clusters obtained, 5 of them persisted after sanitization and were re-isolated during the follow-up sampling. All the CC121 harboured the Tn6188_qac gene for tolerance to benzalkonium chloride and the stress survival islet SSI-2. The CC3, CC7, CC9, CC31 and CC191 carried the SSI-1. All the CC9 and CC121 strains presented a premature stop codon in the inlA gene. In addition to the Lm Pathogenicity Island 1 (LIPI-1), CC1, CC3 and CC191 harboured the LIPI-3. The application of intensive environmental sampling plans for the detection and WGS analysis of Lm isolates could improve surveillance and early detection of outbreaks.

Cryptosporidiumfelis is an important cause of feline and human cryptosporidiosis. However, the transmission of this pathogen between humans and cats remains controversial, partially due to a lack of genetic characterization of isolates from cats. The present study was conducted to examine the genetic diversity of C. felis in cats in China and to assess their potential zoonotic transmission. A newly developed subtyping tool based on a sequence analysis of the 60-kDa glycoprotein (gp60) gene was employed to identify the subtypes of 30 cat-derived C. felis isolates from Guangdong and Shanghai. Altogether, 20 C. felis isolates were successfully subtyped. The results of the sequence alignment showed a high genetic diversity, with 13 novel subtypes and 2 known subtypes of the XIXa subtype family being identified. The known subtypes were previously detected in humans, while some of the subtypes formed well-supported subclusters with human-derived subtypes from other countries in a phylogenetic analysis of the gp60 sequences. The results of this study confirmed the high genetic diversity of the XIXa subtype family of C. felis. The common occurrence of this subtype family in both humans and cats suggests that there could be cross-species transmission of C. felis.

Dependency theory argues that due to unequal economic relationships, including exports, multinational corporations, and loans from multilateral lending institutions, high-income nations exploit the labor and resources of low- and middle-income nations. We extend this line of reasoning to the United States Export–Import Bank, as it has recently come under scrutiny for its lending in the forestry sector of low- and middle-income nations. Although this concern has been raised, we are not aware of any cross-national research that empirically evaluates if their investments adversely impact forests. Therefore, we examine the impact of the United States Export–Import Bank lending in the forestry sector on forest loss. Using a two-stage instrumental variable regression model to account for possible donor selection bias as well as ordinary least squares regression to analyze data for 78 low- and middle-income nations, we find that export credit agency financing is related to increased forest loss from 2001 to 2014. Our findings are consistent with dependency theory ideas that economic linkages with high-income nations increase forest loss in low- and middle-income nations.

Objective To understand the pathogenicity and multilocus sequence typing (MLST) characteristics of the pathogenic strain of Burkholderia pseudomallei (Bp) in Hainan, and provide a reference for studying the structural characteristics and phylogenetic characteristics of pathogenic Bp in Hainan. Methods The data of patients with melioidosis in Hainan, who were treated in Hainan General Hospital from 2011 to 2018, and Bp pathogenic strains isolated from patient infected specimens were collected. The Bp pathogenic strains were genotyped by MLST method, and the distribution characteristics of the sequence type (ST) were analyzed. The ST of all Bp pathogenic strains was analyzed by eBURST v3 software to establish the evolutionary relationship map. Results A total of 91 cases of melioidosis patients were studied, including 76 males and 15 females; the ages were mainly concentrated in 40 - 70 years old; clinical manifestations included sepsis, pulmonary infection and local abscess; and the most common diseases were diabetes. A total of 91 Bp pathogenic strains were observed, of which 85 Bp pathogenic strains were distributed in coastal areas of Hainan, accounting for 93.41%; identified by MLST method, 39 ST were discovered, the most common were ST46 (13 strains), ST55 (12 strains), ST50 (8 strains) and ST58 (7 strains), accounting for 14.29%, 13.19%, 8.79% and 7.69%, respectively; except ST46 was widely distributed in the coastal areas of Hainan, the ST55, ST50 and ST58 were concentrated in the southwest, northeast and southeast regions of Hainan. Compared with the MLST database, ST30 (3 strains) was currently found only in Hainan, ST562 (4 strains) had been reported in northern Australia, and the remaining ST models had been reported in southeast Asia. The eBURST v3 software divided the 39 ST into 3 subtypes and 18 individual types. Among them, the subtypes with ST300 as the original type had the most number of ST, including 17 ST, 57 Bp pathogenic strains; compared with the MLST database, the ST300 was mainly distributed in southeast Asian regions such as Thailand. Conclusions The ST of Bp pathogenic strains in Hainan has high regional diversity and genetic diversity, and is closely related to Bp in southeast Asian regions such as Thailand. Key words: Burkholderia pseudomallei; Multilocus sequence typing; Fungus structure

Cryptosporidiosis is one of the most important causes of moderate to severe diarrhea and diarrhea-related mortality in children under 2 years of age in low- and middle-income countries. In recent decades, genotyping and subtyping tools have been used in epidemiological studies of human cryptosporidiosis. Results of these studies suggest that higher genetic diversity of Cryptosporidium spp. is present in humans in these countries at both species and subtype levels and that anthroponotic transmission plays a major role in human cryptosporidiosis. Cryptosporidium hominis is the most common Cryptosporidium species in humans in almost all the low- and middle-income countries examined, with five subtype families (namely, Ia, Ib, Id, Ie, and If) being commonly found in most regions. In addition, most Cryptosporidium parvum infections in these areas are caused by the anthroponotic IIc subtype family rather than the zoonotic IIa subtype family. There is geographic segregation in Cryptosporidium hominis subtypes, as revealed by multilocus subtyping. Concurrent and sequential infections with different Cryptosporidium species and subtypes are common, as immunity against reinfection and cross protection against different Cryptosporidium species are partial. Differences in clinical presentations have been observed among Cryptosporidium species and C. hominis subtypes. These observations suggest that WASH (water, sanitation, and hygiene)-based interventions should be implemented to prevent and control human cryptosporidiosis in low- and middle-income countries.

BackgroundThe Chlamydia trachomatis incidence rate in Finnmark, the most northern and sparsely populated county in Norway, has been twice the national average. This population based cross-sectional study among Finnmark high school students had the following aims: i) to examine distribution of multilocus sequence types (STs) of C. trachomatis in a previously unmapped area, ii) to compare chlamydia genetic diversity in Finnmark with that of two urban regions, and iii) to compare discriminatory capacity of multilocus sequence typing (MLST) with conventional ompA sequencing in a large number of chlamydia specimens.MethodologyompA sequencing and a high-resolution MLST system based on PCR amplification and DNA sequencing of five highly variable genetic regions were used. Eighty chlamydia specimens from adolescents aged 15–20 years in Finnmark were collected in five high schools (n = 60) and from routine clinical samples in the laboratory (n = 20). These were compared to routine clinical samples from adolescents in Tromsø (n = 80) and Trondheim (n = 88), capitals of North and Central Norway, respectively.Principal FindingsompA sequencing detected 11 genotypes in 248 specimens from all three areas. MLST displayed 50 STs providing a five-fold higher resolution. Two-thirds of all STs were novel. The common ompA E/Bour genotype comprised 46% and resolved into 24 different STs. MLST identified the Swedish new variant of C. trachomatis not discriminated by ompA sequencing. Simpson's discriminatory index (D) was 0.93 for MLST, while a corrected Dc was 0.97. There were no statistically significant differences in ST genetic diversity between geographic areas. Finnmark had an atypical genovar distribution with G being predominant. This was mainly due to expansion of specific STs of which the novel ST161 was unique for Finnmark.Conclusions/SignificanceMLST revealed multiple new STs and a larger genetic diversity in comparison to ompA sequencing and proved to be a useful tool in molecular epidemiology of chlamydia infections.

Geographical and longitudinal analysis of Listeria monocytogenes genetic diversity reveals its correlation with virulence and unique evolution

Objective To analyze the genotypes of Neisseria gonorrhoeae (N.gonorrhoeae) epidemic strains in Wenzhou, eastern China, and to study the mechanism of tetracycline resistance in these strains. Methods A total of 77 N. gonorrhoeae strains were isolated from patients with gonorrhea. Antimicrobial susceptibility of these strains to penicillin, tetracycline, ciprofloxacin, spectinomycin, ceftriaxone and azithromycin was analyzed using E-test. PCR and DNA sequencing were used to detect the genes associated with tetracycline resistance, such as Tet-M, mtrR promoter region and mtrR coding region. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) were used to determine the molecular characteristics of all clinical isolates and tetracycline-resistant isolates, respectively. Results Among the 77 N. gonorrhoeae isolates, 74 (96.10%), 27 (35.06%) , 70 (90.91%) and 15 (19.48%) were resistant to penicillin, tetracycline, ciprofloxacin and azithromycin, respectively. All tested isolates were susceptible to spectinomycin and ceftriaxone. Nineteen isolates were resistant to tetracycline and all of them carried Tet-M gene. Among them, 17 had one deletion mutation of base A in mtrR promoter region and three had G45D mutation in mtrR coding region. NG-MAST classified the 19 tetracycline-resistant isolates into 11 different sequence types (ST). ST14781, ST1766 and ST1866 each accounted for 15.79% (three strains). Two ST (10.52%, 2/19) found in the present study had not been reported previously in the NG-MAST database. MLST showed the 19 tetracycline-resistant isolates belonged to 12 different STs, in which ST10899 accounted for 26.32% (five strains) and ST1600 accounted for 15.79% (three strains). Conclusions Mutations in mtrR promoter region and carrying Tet-M gene were associated with tetracycline resistance in N. gonorrhoeae. Clinical strains isolated in Wenzhou showed considerable molecular diversity. Measures should be implemented to monitor the spread of NG-MAST ST1766 and MLST ST1600 N. gonorrhoeae clones with high resistance to tetracycline in Wenzhou. Key words: Neisseria gonorrhoeae; Resistance rate; Tetracycline; NG-MAST; MLST

This study evaluates the effectiveness of artificial intelligence (AI) in mammography in a diverse population from a middle-income nation and compares it to traditional methods. A retrospective study was conducted on 543 mammograms of 467 Malays, 48 Chinese, and 28 Indians in a middle-income nation. Three breast radiologists interpreted the examinations independently in two reading sessions (with and without AI support). Breast density and BI-RADS categories were assessed, comparing the accuracy, sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) results. Of 543 mammograms, 69.2% had lesions detected. Biopsies were performed on 25%(n=136), with 66(48.5%) benign and 70(51.5%) malignant. Substantial agreement in density assessment between the radiologist and AI software (κ =0.606, p < 0.001) and the BI-RADS category with and without AI (κ =0.74, p < 0.001). The performance of the AI software was comparable to the traditional methods. The sensitivity, specificity, PPV, and NPV or radiologists alone, radiologist + AI, and AI alone were 81.9%,90.4%,56.0%, and 97.1%; 81.0%, 93.1%,55.5%, and 97.0%; and 90.0%,76.5%,36.2%, and 98.1%, respectively. AI software enhances the accuracy of lesion diagnosis and reduces unnecessary biopsies, particularly for BI-RADS 4 lesions. The AI software results for synthetic were almost similar to the original 2D mammography, with AUC of 0.925 and 0.871, respectively. AI software may assist in the accurate diagnosis of breast lesions, enhancing the efficiency of breast lesion diagnosis in a mixed population of opportunistic screening and diagnostic patients. • The use of artificial intelligence (AI) in mammography for population-based breast cancer screening has been validated in high-income nations, with reported improved diagnostic performance. Our study evaluated the usage of an AI tool in an opportunistic screening setting in a multi-ethnic and middle-income nation. • The application of AI in mammography enhances diagnostic accuracy, potentially leading to reduced unnecessary biopsies. • AI integration into the workflow did not disrupt the performance of trained breast radiologists, as there is a substantial inter-reader agreement for BI-RADS category assessment and breast density.

Molecular Characterization of Cryptosporidium spp. in HIV-infected Persons in Benin City, Edo State, Nigeria

Longitudinal multi-locus molecular characterisation of sporadic Australian human clinical cases of cryptosporidiosis from 2005 to 2008