Seroprevalence of Rickettsia Spp. and Orientia tsutsugamushi in Indigenous Populations from the Sierra Nevada de Santa Marta, Colombia.

Background: Rickettsia spp. are vector-borne zoonotic pathogens that cause febrile illness in humans. Rickettsioses is not included in the Colombian national surveillance system and is subsequently expected to be underreported. This cross-sectional study aimed to determine the seroprevalence of Rickettsia spp. and the closely related Orientia tsutsugamushi in two indigenous populations residing in the Sierra Nevada de Santa Marta, Colombia. Materials and Methods: Serum samples (n = 539) were collected from the Wiwa and Koguis people between 2021 and 2022. Serum samples were screened for spotted fever group (SFG) and typhus group (TG) Rickettsia spp. using the Fuller laboratories Rickettsia IgG IFA kit and for O. tsutsugamushi with the Scrub Typhus Detect™ IgG ELISA. Results: We observed an overall seroprevalence of 26.2% (95% confidence interval [CI] 22.5-30.1] for Rickettsia spp. of the SFG, 5.4% (95% CI 3.6-7.6) for Rickettsia spp. of the TG and 4.3% (95% CI 2.7-6.3) for O. tsutsugamushi. Common risk factors for zoonotic disease infections were assessed for 147 of the Wiwa participants. Increased odds of seropositivity for SFG Rickettsia spp. were observed for Wiwa participants who cared for livestock, including assisting with the birth of cattle (odds ratio [OR] = 8.85; 95% CI 1.54-50.90; p = 0.015) and goats (OR = 7.60; 95% CI 1.70-33.90; p = 0.008). Conclusions: These results highlight a notable exposure to Rickettsia spp., especially the SFG, in rural Colombia. Together with recent reports of high mortality for Rocky Mountain Spotted Fever in nearby regions of South America, more detailed investigations focusing on improving knowledge and awareness as well as "One Health" and "causes-of-fever" studies are needed. The characterization of Rickettsia spp. infections in humans, livestock, and tick vectors with their potential transmission routes could make a high impact on these easily treatable diseases.

ESBL-Type and AmpC-Type Beta-Lactamases in Third Generation Cephalosporin-Resistant Enterobacterales Isolated from Animal Feces in Madagascar.

Third generation cephalosporin-resistant (3GCR) Enterobacterales are known to be prevalent in Madagascar, with high colonization or infection rates in particular in Madagascan patients. Extended spectrum beta-lactamases (ESBLs) have been reported to be the predominant underlying resistance mechanism in human isolates. So far, little is known on antimicrobial resistance and its molecular determinants in Enterobacterales and other bacteria causing enteric colonization of Madagascan wild animals. To address this topic, swabs from 49 animal stool droppings were collected in the Madagascan Tsimanapesotsa National Park and assessed by cultural growth of bacterial microorganisms on elective media. In addition to 7 Acinetobacter spp., a total of 31 Enterobacterales growing on elective agar for Enterobacterales could be isolated and subjected to whole genome sequencing. Enterobacter spp. was the most frequently isolated genus, and AmpC-type beta-lactamases were the quantitatively dominating molecular resistance mechanism. In contrast, the blaCTX-M-15 gene, which has repeatedly been associated with 3GC-resistance in Madagascan Enterobacterales from humans, was detected in a single Escherichia coli isolate only. The identification of the fosfomycin-resistance gene fosA in a high proportion of isolates is concerning, as fosfomycin is increasingly used to treat infections caused by multidrug-resistant bacteria. In conclusion, the proof-of-principle assessment indicated a high colonization rate of resistant bacteria in stool droppings of Madagascan wild animals with a particular focus on 3GCR Enterobacterales. Future studies should confirm these preliminary results in a more systematic way and assess the molecular relationship of animal and human isolates to identify potential routes of transmission.

Seroprevalence of Coxiella burnetii in an Indigenous Population from the Sierra Nevada De Santa Marta, Colombia.

Coxiella burnetii is an underreported zoonotic pathogen in many rural regions globally. We investigated C. burnetii exposure in a remote indigenous tribe residing in the Sierra Nevada de Santa Marta, Colombia. The high seroprevalence of 35% (95% CI, 27-43%) demonstrates the need for One Health studies to identify risk factors, clinical impact, and potential medical, veterinary, and environmental interventions.

Evidence for Bartonella quintana in Lice Collected from the Clothes of Ethiopian Homeless Individuals.

Human lice, Pediculus humanus, can transmit various pathogens, including Bartonella quintana, Borrelia recurrentis, and Rickettsia prowazekii. Xenosurveillance is an epidemiological approach to assessing human infection risks performed by screening vectors of infectious disease agents. In the proof-of-principle study reported herein, the DNA of 23 human lice was collected from the clothes of 30 homeless Ethiopian individuals. These samples were assessed using 16S rRNA gene-specific pan-eubacterial PCR for screening, followed by Bartonella genus 16S-23S internal transcribed spacer (ITS) sequence-specific PCR, Bartonella genus gltA gene-specific PCR, and 16S rRNA gene PCR with specificity for relapsing-fever-associated Borrelia spp. with subsequent sequencing of the amplicons. In one sample, the pan-eubacterial 16S rRNA gene-specific screening PCR, the Bartonella genus 16S-23S ITS sequence-specific PCR, and the Bartonella genus gltA gene-specific PCR allowed for the sequencing of B. quintana-specific amplicons. In two additional samples, Bartonella genus gltA gene-specific PCR also provided sequences showing 100% sequence identity with B. quintana. In total, 3/23 (13.0%) of the assessed lice were found to be positive for B. quintana. Correlating clinical data were not available; however, the assessment confirmed the presence of B. quintana in the local louse population and thus an associated infection pressure. Larger-sized cross-sectional studies seem advisable to more reliably quantify the infection risk of lice-infested local individuals. The need for prevention by providing opportunities to maintain standard hygiene for Ethiopian homeless individuals is stressed by the reported findings, especially in light of the ongoing migration of refugees.

Parasitic, bacterial, viral, immune-mediated, metabolic and nutritional factors associated with nodding syndrome.

Nodding syndrome is a neglected, disabling and potentially fatal epileptic disorder of unknown aetiology affecting thousands of individuals mostly confined to Eastern sub-Saharan Africa. Previous studies have identified multiple associations-including Onchocerca volvulus, antileiomodin-1 antibodies, vitamin B6 deficiency and measles virus infection-yet, none is proven causal. We conducted a case-control study of children with early-stage nodding syndrome (symptom onset <1 year). Cases and controls were identified through a household survey in the Greater Mundri area in South Sudan. A wide range of parasitic, bacterial, viral, immune-mediated, metabolic and nutritional risk factors was investigated using conventional and state-of-the-art untargeted assays. Associations were examined by multiple logistic regression analysis, and a hypothetical causal model was constructed using structural equation modelling. Of 607 children with nodding syndrome, 72 with early-stage disease were included as cases and matched to 65 household- and 44 community controls. Mansonella perstans infection (odds ratio 7.04, 95% confidence interval 2.28-21.7), Necator americanus infection (odds ratio 2.33, 95% confidence interval 1.02-5.3), higher antimalarial seroreactivity (odds ratio 1.75, 95% confidence interval 1.20-2.57), higher vitamin E concentration (odds ratio 1.53 per standard deviation increase, 95% confidence interval 1.07-2.19) and lower vitamin B12 concentration (odds ratio 0.56 per standard deviation increase, 95% confidence interval 0.36-0.87) were associated with higher odds of nodding syndrome. In a structural equation model, we hypothesized that Mansonella perstans infection, higher vitamin E concentration and fewer viral exposures increased the risk of nodding syndrome while lower vitamin B12 concentration, Necator americanus and malaria infections resulted from having nodding syndrome. We found no evidence that Onchocerca volvulus, antileiomodin-1 antibodies, vitamin B6 and other factors were associated with nodding syndrome. Our results argue against several previous causal hypotheses including Onchocerca volvulus. Instead, nodding syndrome may be caused by a complex interplay between multiple pathogens and nutrient levels. Further studies need to confirm these associations and determine the direction of effect.

Should we treat Blastocystis sp.? A double-blind placebo-controlled randomized pilot trial.

BACKGROUND: Blastocystis sp. is a worldwide-distributed protist colonizing the guts of humans and a great variety of animals. It is unclear whether it is just a commensal or an infectious parasite that prompts eradication.The main objective of this study was to evaluate the usefulness of metronidazole in patients with gastrointestinal symptoms harbouring only Blastocystis sp. In addition, we explored whether Blastocystis subtype or concomitant parasitic infection detected by polymerase chain reaction (PCR) may influence treatment outcome. METHODS: We included adults with persistent gastrointestinal symptoms (>14 days) visiting a primary care physician and in whom stool microscopy revealed only Blastocystis sp. Eligible patients were randomized to receive 10 days of metronidazole or placebo, followed by a crossover if still symptomatic. The primary outcome was normal stool consistency. Secondary outcomes were the changes in other abdominal symptoms (bloating, flatulence, abdominal pain, number of daily bowel movements) and general wellbeing. After the clinical phase of the study, Blastocystis subtypes were determined by PCR sequencing and stool samples were tested for 11 other protozoa with an in-house PCR. RESULTS: We screened 581 outpatients for inclusion, of which 50 met the eligibility criteria. There was no difference in the primary outcome, nor any of the secondary outcomes between the subjects treated with metronidazole and placebo.The most frequent Blastocystis subtypes were ST4 (11/36) and ST2 (10/36). The in-house PCR was positive for other protozoa in 25% (10/40) of the patients. We identified Dientamoeba fragilis in 5, Entamoeba dispar in 3 and Cyclospora cayetanensis in 2 patients. Stratified analysis according to Blastocystis subtype or the presence of other protozoa showed no significant difference in treatment outcome with metronidazole or placebo. CONCLUSIONS: Among patients infected with Blastocystis sp., metronidazole, compared with placebo, was not better in improving gastrointestinal symptoms, irrespective of subtype or microscopically undetected coinfection with other protozoa.

SARS-CoV-2 from COVID-19 Patients in the Republic of Moldova: Whole-Genome Sequencing Results.

Since the onset of the COVID-19 pandemic, no viral genome sequences of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have been documented from the Republic of Moldova, a developing country geographically located in Eastern Europe between Romania and Ukraine. Here, we report the analysis of 96 SARS-CoV-2 sequences from Delta and Omicron variants of the SARS-CoV-2 cases in the Republic of Moldova obtained between August and November 2021 and between January and May 2022. Comparison to global viral sequences showed that among the Delta variant of the SARS-CoV-2, AY.122 (n = 25), followed by AY.4.2.3 (n = 6), AY.4 (n = 5), AY.43 (n = 3), AY.98.1 (n = 3), B.1.617.2 (n = 1), AY.125 (n = 1), AY.54 (n = 1), AY.9 (n = 1), AY.126 (n = 1), and AY.33 (n = 1) were the most frequently found lineages. Furthermore, 10 lineages of the Omicron variant, namely, BA.2 (n = 14), followed by BA.2.9 (n = 10), BA.1 (n = 5), BA.1.1 (n = 5), BA.1.18 (n = 4), BA.1.15.1 (n = 3), BA.1.17.2 (n = 2), BA.1.17 (n = 2), BA.1.15 (n = 1), and BA.2.1 (n = 1) were detected. In addition, we also identified the impact of the military crisis between Russia and Ukraine, when the COVID-19 epidemiological rules collapsed, on the distribution of Delta and Omicron variants in the Republic of Moldova. Additional studies are warranted to characterize further the impact of the war between Russia and Ukraine on the genomic epidemiology of the SARS-CoV-2 in the Republic of Moldova and Eastern Europe.

Metagenomic Sequencing for the Diagnosis of Plasmodium spp. with Different Levels of Parasitemia in EDTA Blood of Malaria Patients-A Proof-of-Principle Assessment.

Molecular diagnostic approaches are increasingly included in the diagnostic workup and even in the primary diagnosis of malaria in non-endemic settings, where it is difficult to maintain skillful microscopic malaria detection due to the rarity of the disease. Pathogen-specific nucleic acid amplification, however, bears the risk of overlooking other pathogens associated with febrile illness in returnees from the tropics. Here, we assessed the discriminatory potential of metagenomic sequencing for the identification of different Plasmodium species with various parasitemia in EDTA blood of malaria patients. Overall, the proportion of Plasmodium spp.-specific sequence reads in the assessed samples showed a robust positive correlation with parasitemia (Spearman r = 0.7307, p = 0.0001) and a robust negative correlation with cycle threshold (Ct) values of genus-specific real-time PCR (Spearman r = -0.8626, p ≤ 0.0001). Depending on the applied bioinformatic algorithm, discrimination on species level was successful in 50% (11/22) to 63.6% (14/22) instances. Limiting factors for the discrimination on species level were very low parasitemia, species-depending lacking availability of reliable reference genomes, and mixed infections with high variance of the proportion of the infecting species. In summary, metagenomic sequencing as performed in this study is suitable for the detection of malaria in human blood samples, but the diagnostic detection limit for a reliable discrimination on species level remains higher than for competing diagnostic approaches like microscopy and PCR.

Colitis caused by Entamoeba histolytica identified by real-time-PCR and fluorescence in situ hybridization from formalin-fixed, paraffin-embedded tissue.

Intestinal amoebiasis in a 35-year-old German patient with a 3 weeks travel history in Indonesia was initially misidentified as non-steroidal anti-inflammatory-drug associated colitis in colonoscopy and histopathological analysis. Furthermore, initial stool examination by microscopy and Entamoeba faecal antigen ELISA did not reveal any protozoan infection. When cessation of non-steroidal anti-inflammatory drug (NSAID) use and mesalazine treatment did not lead to clinical improvement, the patient presented to a specialist for tropical diseases. An intensive reinvestigation including a workup of formalin-fixed, paraffin-embedded colonic biopsies by molecular analysis with real-time PCR and fluorescence in situ hybridization (FISH) proofed the diagnosis of Entamoeba histolytica colitis. Molecular methods including real-time PCR and FISH for the diagnosis of amoebiasis from histopathological samples are rarely used for the diagnosis of E. histolytica infections. Bloody diarrhoea vanished after the onset of metronidazole treatment. In conclusion, the here-presented case demonstrates how modern molecular diagnostics may help to diagnose E. histolytica-associated colitis, even from difficult specimens like paraffin-embedded, formalin-fixed tissue.

Prevalence and Molecular Characterization of Mycobacterium bovis in Slaughtered Cattle Carcasses in Burkina Faso; West Africa.

This cross-sectional study was conducted at the slaughterhouses/slabs of Oudalan and Ouagadougou in Burkina Faso, between August and September 2013. It aimed at determining the prevalence of bovine tuberculosis (bTB) suggestive lesions in slaughtered cattle carcasses and to identify and characterize the mycobacteria isolated from these lesions. A thorough postmortem examination was conducted on carcasses of a total of 2165 randomly selected cattle. The overall prevalence of bTB suggestive lesions was 2.7% (58/2165; 95% CI 2.1-3.5%). Due to the low number of positive samples, data were descriptively presented. The lesions were either observed localized in one or a few organs or generalized (i.e., miliary bTB) in 96.6% (n = 57) and 3.4% (n = 2), respectively. The identified mycobacteria were M. bovis (44.4%, n = 20), M. fortuitum (8.9%, n = 4), M. elephantis (6.7%, n = 3), M. brumae (4.4%, n = 2), M. avium (2.2%, n = 1), M. asiaticum (2.2%, n = 1), M. terrae (2.2%, n = 1), and unknown non-tuberculous mycobacteria (NTM) (11.1%, n = 5). Moreover, eight mixed cultures with more than one Mycobacterium species growing were also observed, of which three were M. bovis and M. fortuitum and three were M. bovis and M. elephantis. In conclusion, M. bovis is the predominant causative agent of mycobacterial infections in the study area. Our study has identified a base to broaden the epidemiological knowledge on zoonotic transmission of mycobacteria in Burkina Faso by future studies investigating further samples from humans and animals, including wild animals employing molecular techniques.

Prevalence of Bacterial and Protozoan Pathogens in Ticks Collected from Birds in the Republic of Moldova.

Epidemiological knowledge on pathogens in ticks feeding on birds in Moldova is scarce. To reduce this gap of information, a total of 640 migrating and native birds of 40 species were caught from 2012 to 2015 and examined for the presence of ticks in the Republic of Moldova. Altogether, 262 ticks belonging to five tick species (Ixodes ricunus n = 245, Ixodes frontalis n = 12, Haemaphysalis punctata n = 2, Hyalomma marginatum n = 2 (only males), Dermacentor marginatus n = 1) were collected from 93 birds. Of these ticks, 250 (96%) were at the stage of a nymph and 9 at the stage of a larva (3%). One imago of I. frontalis and two imagoes of Hy. marginatum were found. Generally, ticks infested 14.1% of the assessed birds belonging to 12 species. DNA was extracted from individual ticks with subsequent PCR targeting Rickettsia spp., Borrelia spp. in general, as well as relapsing fever-associated Borrelia spp., in particular, Anaplasma phagocytophilum, Neoehrlichia mikurensis, Babesia spp. and Coxiella burnetii. The bird species Turdus merula showed the heaviest infestation with ticks and the highest incidence of infected ticks. Altogether, 32.8% of the assessed ticks (n = 86) were positive for one of the pathogens. DNA of Borrelia spp. was found in 15.2% (40/262) of the investigated ticks; in 7.6% of ticks (20/262), DNA of rickettsiae was detected; 6.9% (18/262) of the ticks were positive for A. phagocytophilum DNA; in 1.5% of the ticks (4/262), DNA of Neoehrlichia mikurensis was detected, followed by 1.5% (4/262) Babesia microti and 1.5% (4/262) Borrelia miyamotoi. Within the B. burgdorferi complex, B. garinii (n = 36) was largely predominant, followed by B. valaisiana (n = 2) and B. lusitaniae (n = 2). Among the detected Rickettsia spp., R. monacensis (n = 16), R. helvetica (n = 2) and R. slovaca (n = 1) were identified. In conclusion, the study provided some new information on the prevalence of ticks on birds in Moldova, as well as the presence of DNA of pathogens in the ticks. By doing so, it provided an additional piece in the puzzle of the global epidemiology of tick-transmitted infectious diseases from a geographic side from where respective surveillance data are scarce.

Comparison of the Serion IgM ELISA and Microscopic Agglutination Test for diagnosis of Leptospira spp. infections in sera from different geographical origins and estimation of Leptospira seroprevalence in the Wiwa indigenous population from Colombia.

Leptospirosis is among the most important zoonotic diseases in (sub-)tropical countries. The research objective was to evaluate the accuracy of the Serion IgM ELISA EST125M against the Microscopic Agglutination Test (MAT = imperfect reference test); to assess its ability to diagnose acute leptospirosis infections and to detect previous exposure to leptospires in an endemic setting. In addition, to estimate the overall Leptospira spp. seroprevalence in the Wiwa indigenous population in North-East Colombia. We analysed serum samples from confirmed leptospirosis patients from the Netherlands (N = 14), blood donor sera from Switzerland (N = 20), and sera from a cross-sectional study in Colombia (N = 321). All leptospirosis ELISA-positive, and a random of negative samples from Colombia were tested by the MAT for confirmation. The ELISA performed with a sensitivity of 100% (95% CI 77% - 100%) and a specificity of 100% (95% CI 83% - 100%) based on MAT confirmed Leptospira spp. positive and negative samples. In the cross-sectional study in Colombia, the ELISA performed with a sensitivity of 100% (95% CI 2-100%) and a specificity of 21% (95% CI 15-28%). Assuming a 5% Leptospira spp. seroprevalence in this population, the positive predictive value was 6% and the negative predictive value 100%. The Leptospira spp. seroprevalence in the Wiwas tested by the ELISA was 39%; however, by MAT only 0.3%. The ELISA is suitable to diagnose leptospirosis in acutely ill patients in Europe several days after onset of disease. For cross-sectional studies it is not recommended due to its low specificity. Despite the evidence of a high leptospirosis prevalence in other study areas and populations in Colombia, the Wiwa do not seem to be highly exposed to Leptospira spp.. Nevertheless, leptospirosis should be considered and tested in patients presenting with febrile illness.

Prevalence and molecular identification of zoonotic Anisakis and Pseudoterranova species in fish destined to human consumption in Chile.

Zoonotic larvae of the family Anisakidae found in several fish species represent a serious risk in public health since they may cause food-borne anisakidosis in humans. Chile has culinary preferences including eating raw fish in many traditional preparations. In the present study, a total of 180 fish specimens representing three different fish species, i.e., Chilean hake (Merluccius gayi), snoek (Thyrsites atun), and sea bream (Brama australis), were caught at central coast of Chile. Parasitological examination was performed on musculature and abdominal cavity for subsequent extraction and quantification of anisakid larvae. Estimation of infection parameters, such as prevalence, was performed indicating 100% (CI: 0.94-1.0) prevalence of anisakid L3 in Chilean hakes and snoeks. Moreover, sea breams reached a prevalence of 35% (CI: 0.23-0.48). Prevalence of anisakid larvae in muscle was also analyzed showing values of 18.6% (CI: 0.097-0.309) in Chilean hakes, 15% (CI: 0.07-0.26) in snoeks, and 1.7% (CI: 0-0.089) in sea breams. Meanwhile, prevalence of anisakid larvae in internal organs showed highest values for peritoneum (100% and 83.3%) for snoeks and Chilean hakes, respectively, for liver (96.7%) and gonads (86.6%) in Chilean hakes, and for intestine (98.3%) in snoeks. Molecular analysis of collected anisakid L3 unveiled presence of two potentially zoonotic nematode species, i.e., Pseudoterranova cattani and Anisakis pegreffii. P. cattani was found in Chilean hakes and snoeks being the first molecular host species report for Chilean snoeks. Besides, A. pegreffii was also identified in these species being the first molecular report on this regard. These findings are relevant for better understanding of epidemiology of anisakiasis in Chilean coasts and for public health issues considering potential risk of human population due to its culinary preferences in eating raw fish.

High prevalence of urinary schistosomiasis in a desert population: results from an exploratory study around the Ounianga lakes in Chad.

BACKGROUND: Researching a water-borne disease in the middle of the Sahara desert might not seem the most relevant concern. However, nomadic Sahelian pastoralists health concerns regarding their livestock and anecdotal reports about trematode infections of Fasciola spp. and Schistosoma spp. in desert-raised animals justified an exploratory study focusing on the lakes of Ounianga in Northern Chad. The aim was to test whether trematode parasites such as Schistosoma spp. occur in human populations living around the Sahara desert lakes of Ounianga Kebir and Ounianga Serir in northern Chad. METHODS: The study was carried out in January 2019 and comprised of three components. First, a cross sectional survey based on a random sample drawn from the population to detect infections with S. haematobium and S. mansoni; second, focus group discussions exploring disease priorities, access to health and health seeking behaviour; and third, surveying water contact sites for intermediate host snails. Samples of trematode parasites and snails were confirmed on species level by molecular genetic methods. For parasitological and malacological surveys descriptive statistics were performed. Qualitative data analysis included the full review of all transcripts, followed by a descriptive and explorative thematic analysis. RESULTS: Among 258 participants, the overall S. haematobium prevalence using urine filtration was 39.2% [95% confidence interval (CI): 33.5-45.1%], with 51.5% of the infected suffering from heavy infection. The intermediate host snail of S. haematobium (Bulinus truncatus) occurred at water contact sites near both study villages, revealing the potential for local transmission. Although a positive S. mansoni point-of-care circulating cathodic antigen (POC-CCA) test result was obtained from 8.6% (95% CI 5.7-12.8%) of the samples, no intermediate host snails of S. mansoni were found, and the relevance of S. mansoni remains uncertain. Qualitative findings underline the importance of morbidity caused by urinary schistosomiasis, and the lack of access to diagnostics and treatment as a major health concern. CONCLUSIONS: This research revealed a high prevalence of urinary schistosomiasis in the population living around the lakes of Ounianga in the Sahara, a United Nations Educational, Scientific and Cultural Organization (UNESCO) world heritage site in Chad. Despite the high public health importance of the associated morbidity expressed by the population, there is no access to diagnostics and treatment. Further work is needed to develop and test a context-adapted intervention.

TIDE Analysis of Cryptosporidium Infections by gp60 Typing Reveals Obscured Mixed Infections.

BACKGROUND: Cryptosporidiosis is a parasitic disease associated with potentially fatal diarrhea. The most used method in Cryptosporidium subtyping is based on the glycoprotein gene gp60. Each infection can represent a parasite population, and it is important to investigate the influence on transmission and virulence, as well as any impact on public health investigations. However, an easy-to-use method for detection is lacking. METHODS: Here we report on the use of the bioinformatic program TIDE for deconvolution of gp60 chromatograms. A combination of single oocyst analysis and cloning successfully confirmed the within-sample parasite population diversity. Retrospective sample analysis was conducted on archived chromatograms. RESULTS: For Cryptosporidium parvum, 8.6% multistrain infections (13 of 152) obscured by currently used consensus base calling were detected. Importantly, we show that single oocysts can harbor a mixed population of sporozoites. We also identified a striking dominance of unappreciated polymerase stutter artefacts in all 218 chromatograms analyzed, challenging the uncritical use of gp60 typing. CONCLUSIONS: We demonstrate the value of a new, easy-to-use analytical procedure for critical characterization of C. parvum and Cryptosporidium hominis in epidemiological investigations, also applicable retrospectively. Our findings illuminate the hidden parasite diversity with important implications for tracing zoonotic and person-to-person transmissions.

Molecular Evidence for Flea-Borne Rickettsiosis in Febrile Patients from Madagascar.

Rickettsiae may cause febrile infections in humans in tropical and subtropical regions. From Madagascar, no molecular data on the role of rickettsioses in febrile patients are available. Blood samples from patients presenting with fever in the area of the capital Antananarivo were screened for the presence of rickettsial DNA. EDTA (ethylenediaminetetraacetic acid) blood from 1020 patients presenting with pyrexia > 38.5 °C was analyzed by gltA-specific qPCR. Positive samples were confirmed by ompB-specific qPCR. From confirmed samples, the gltA amplicons were sequenced and subjected to phylogenetic analysis. From five gltA-reactive samples, two were confirmed by ompB-specific qPCR. The gltA sequence in the sample taken from a 38-year-old female showed 100% homology with R. typhi. The other sample taken from a 1.5-year-old infant was 100% homologous to R. felis. Tick-borne rickettsiae were not identified. The overall rate of febrile patients with molecular evidence for a rickettsial infection from the Madagascan study site was 0.2% (2/1020 patients). Flea-borne rickettsiosis is a rare but neglected cause of infection in Madagascar. Accurate diagnosis may prompt adequate antimicrobial treatment.

Evaluating Different Storage Media for Identification of Taenia saginata Proglottids Using MALDI-TOF Mass Spectrometry.

Taenia saginata is a helminth that can cause taeniasis in humans and cysticercosis in cattle. A species-specific diagnosis and differentiation from related species (e.g., Taenia solium) is crucial for individual patient management and disease control programs. Diagnostic stool microscopy is limited by low sensitivity and does not allow discrimination between T. saginata and T. solium. Molecular diagnostic approaches are not routinely available outside research laboratories. Recently, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) was proposed as a potentially suitable technique for species-specific helminth diagnosis. However, standardized protocols and commercial databases for parasite identification are currently unavailable, and pre-analytical factors have not yet been assessed. The purpose of this study was to employ MALDI-TOF MS for the identification of T. saginata proglottids obtained from a human patient, and to assess the effects of different sample storage media on the technique's diagnostic accuracy. We generated T. saginata-specific main spectral profiles and added them to an in-house database for MALDI-TOF MS-based diagnosis of different helminths. Based on protein spectra, T. saginata proglottids could be successfully differentiated from other helminths, as well as bacteria and fungi. Additionally, we analyzed T. saginata proglottids stored in (i) LC-MS grade water; (ii) 0.45% sodium chloride; (iii) 70% ethanol; and (iv) 37% formalin after 2, 4, 6, 8, 12, and 24 weeks of storage. MALDI-TOF MS correctly identified 97.2-99.7% of samples stored in water, sodium chloride, and ethanol, with log-score values ≥2.5, thus indicating reliable species identification. In contrast, no protein spectra were obtained for samples stored in formalin. We conclude that MALDI-TOF-MS can be successfully employed for the identification of T. saginata, and that water, sodium chloride, and ethanol are equally effective storage solutions for prolonged periods of at least 24 weeks.

No hints for abundance of Bacillus anthracis and Burkholderia pseudomallei in 100 environmental samples from Cameroon.

BACKGROUND: Little is known on the abundance of the pathogens Bacillus anthracis and Burkholderia pseudomallei in environmental samples in Cameroon. Therefore, 100 respective samples were assessed in a proof-of-principle assessment. METHODS: DNA residuals from nucleic acid extractions of 100 environmental samples, which were collected between 2011 and 2013 in the Mapé Basin of Cameroon, were screened for B. anthracis and B. pseudomallei by real-time PCR. The samples comprised soil samples with water contact (n = 88), soil samples without water contact (n = 6), plant material with water contact (n = 3), water (n = 2), and soil from a hospital dressing room (n = 1). RESULTS: B. anthracis and B. pseudomallei were detected in none of the samples assessed. CONCLUSION: The results indicate that at least a quantitatively overwhelming, ubiquitous occurrence of B. anthracis and B. pseudomallei in the environment in Cameroon is highly unlikely. However, the number and choice of the assessed samples limit the interpretability of the results.

Low Sensitivity of Real Time PCRs Targeting Retrotransposon Sequences for the Detection of Schistosoma japonicum Complex DNA in Human Serum.

While hybridization probe-based real-time PCR assays targeting highly repetitive multi-copy genome sequences for the diagnosis of S. mansoni complex or S. haematobium complex from human serum are well established, reports on the evaluation of respective assays for the identification of S. japonicum complex DNA in human serum are scarce. Here, we assessed the potential use of the retrotransposon sequences SjR2 and SjCHGCS19 from S. japonicum, S. mekongi and S. malayensis for the diagnosis of Asian Schistosoma infections. Based on available S. japonicum sequences and newly provided S. mekongi and S. malayensis sequences, hybridization probe-based real-time PCRs targeting SjR2 and SjCHGCS19 of the S. japonicum complex were designed both as consensus primer assays as well as multi-primer assays for the coverage of multiple variants of the target sequences. The assays were established using plasmids and S. mekongi DNA. While the consensus primer assays failed to detect S. mekongi DNA in human serum samples, the multi-primer assays showed positive or borderline positive results but only in 9.8% (6/61) of serum samples from patients with confirmed S. mekongi infections. Some cross-reactions with samples positive for S. mansoni or S. haematobium were observed but with the SjCHGCS19-PCR only. In spite of the low sensitivity, the presented experience may guide future evaluations of S. japonicum-complex-specific PCRs from human serum.

Comparison of Three In-House Real PCR Assays Targeting Kinetoplast DNA, the Small Subunit Ribosomal RNA Gene and the Glucose-6-Phosphate Isomerase Gene for the Detection of Leishmania spp. in Human Serum.

To perform PCR from serum for the diagnosis of visceral leishmaniasis is convenient and much less invasive than the examination of deeper compartments such as bone marrow. We compared three Leishmania-specific real-time PCRs with three different molecular targets (kinetoplast DNA, the small subunit-ribosomal RNA-(ssrRNA-)gene, the glucose-6-phosphate isomerase-(gpi-)gene) regarding their sensitivity and specificity in human serum. Residual sera from previous diagnostic assessments at the German National Reference Center for Tropical Pathogens Bernhard Nocht Institute for Tropical Medicine Hamburg and the Swiss Tropical and Public Health Institute were used. The sensitivities of kinetoplast DNA-PCR, ssrRNA-gene PCR, and gpi-PCR were 93.3%, 73.3%, and 33.3%, respectively, with 15 initial serum samples from visceral leishmaniasis patients, as well as 9.1%, 9.1%, and 0.0%, respectively, with 11 follow-up serum samples taken at various time points following anti-leishmanial therapy. Specificity was 100.0% in all assays as recorded with 1.137 serum samples from deployed soldiers and migrants without clinical suspicion of visceral leishmaniasis. Kinetoplast-DNA PCR from serum was confirmed as a sensitive and specific approach for the diagnosis of visceral leishmaniasis. The results also indicate the suitability of serum PCR for diagnostic follow-up after therapy, in particular regarding therapeutic failure in case of persisting positive PCR results.