Molecular detection of Leishmania (Sauroleishmania) adleri (Trypanosomatida: Trypanosomatidae) in Sergentomyia sp. sand flies (Diptera: Psychodidae) in Mali and Niger.

Phlebotomine sand flies of the genus Sergentomyia are considered to be of minor importance as vectors of Leishmania parasites pathogenic to humans, but are known to transmit lizard parasites of the subgenus Sauroleishmania, including L. (S.) adleri. However, knowledge on the geographic distribution of Sauroleishmania spp. and the infection rates in the vectors is very limited. Therefore, our study aimed (1) to further elucidate the distribution and prevalence of Sauroleishmania spp. in their respective vectors and (2) to assess the potential risk for occasional transmission of Leishmania parasites to international military personnel deployed in camps in Mali and Niger. A total of 1,482 wild-caught sand flies (Sergentomyia spp. and closely related Grassomyia spp.) were screened by real-time PCR for the presence of Leishmania DNA. Thirty-two sand fly pools were tested positive, with six from Mali and 26 from Niger. The DNA of four representative isolates was sequenced. The resulting sequences revealed a homology to L. adleri, which leads to the first report of this species from Mali and Niger to the best of our knowledge. The results suggest that Sergentomyia (Sintonius) clydei might be the natural sand fly vector, while Grassomyia spp. appear to be refractory. No Leishmania sp. pathogenic to humans was detected in these sand flies.

A case of SARS-CoV-2 pneumonia with successful antiviral therapy in a 77-year-old man with a heart transplant.

The SARS-CoV-2 infection can be seen as a single disease, but it also affects patients with relevant comorbidities who may have an increased risk of a severe course of infection. In this report, we present a 77-year-old patient with a heart transplant receiving relevant immunosuppressive therapy who tested positive for SARS-CoV-2 after several days of dyspnea, dry cough, and light general symptoms. Computed tomography confirmed interstitial pneumonia. The patient received antiviral therapy with hydroxychloroquine and showed no further deterioration of the clinical state. After 12 days of hospitalization, the patient was released; he was SARS-CoV-2 negative and completely asymptomatic.

Identification of Campylobacter fetus by fluorescence in situ hybridization (FISH).

Two new DNA FISH-probes for Campylobacter fetus were designed, in silico checked for cross-reactions and successfully evaluated in a multi-centric approach with 41 Campylobacter fetus isolates including isolates of all three know subspecies: Campylobacter fetus ssp. fetus, Campylobacter fetus ssp. venerealis, and Campylobacter fetus ssp. testudinum and 40 strains of five non-target Campylobacter species.

Molecular detection of spotted fever group rickettsiae in ticks from Cameroon.

In western and eastern Africa, rickettsioses are one cause of fever in humans. Little is known regarding the presence of Rickettsia sp. in northern Cameroon. The present work was conducted in order to identify potential tick-borne spotted fever group Rickettsia in the Adamawa region of northern Cameroon, which may contribute filling some of the knowledge gaps of these pathogens. Ticks were collected from cattle in the municipal slaughterhouse of Ngaoundere in the Adamawa region of northern Cameroon. After morphological identification of tick species, extracted DNA was analyzed by PCR targeting the rickettsial ompB gene and the intergenic spacers dksA-xerC, mppA-purC and rpmE-tRNAfMet. Of the 316 adult ticks collected, 149 (47.1%) were Amblyomma variegatum, 92 (29%) Rhipicephalus spp. and 75 (23.7%) Hyalomma spp. Through the use of conventional PCR assays for the rickettsial ompB gene, rickettsial DNA was detected in 104 (32.9%) samples (85 Amblyomma sp., 14 Hyalomma spp. and 5 Rhipicephalus spp.). The ompB gene and the three intergenic were sequenced for 10 ticks in order to determine the rickettsial species. Rickettsia africae was detected in Amblyomma variegatum, Rickettsia aeschlimannii in Hyalomma rufipes and Hyalomma truncatum, Rickettsia sibirica in H. truncatum, Rickettsia massiliae in Rhipicephalus lunulatus and Candidatus Rickettsia barbariae in R. lunulatus. To the best of the author's knowledge, this report represents the first molecular evidence of rickettsial infection in ticks in the Adamawa region of northern Cameroon, which suggests a possible exposure of the human population in this region.

More Pathogenicity or Just More Pathogens?-On the Interpretation Problem of Multiple Pathogen Detections with Diagnostic Multiplex Assays.

Modern molecular diagnostic approaches in the diagnostic microbiological laboratory like real-time quantitative polymerase chain reaction (qPCR) have led to a considerable increase of diagnostic sensitivity. They usually outperform the diagnostic sensitivity of culture-based approaches. Culture-based diagnostics were found to be insufficiently sensitive for the assessment of the composition of biofilms in chronic wounds and poorly standardized for screenings for enteric colonization with multi-drug resistant bacteria. However, the increased sensitivity of qPCR causes interpretative challenges regarding the attribution of etiological relevance to individual pathogen species in case of multiple detections of facultative pathogenic microorganisms in primarily non-sterile sample materials. This is particularly the case in high-endemicity settings, where continuous exposition to respective microorganisms leads to immunological adaptation and semi-resistance while considerable disease would result in case of exposition of a non-adapted population. While biofilms in chronic wounds show higher pathogenic potential in case of multi-species composition, detection of multiple pathogens in respiratory samples is much more difficult to interpret and asymptomatic enteric colonization with facultative pathogenic microorganisms is frequently observed in high endemicity settings. For respiratory samples and stool samples, cycle-threshold-value-based semi-quantitative interpretation of qPCR results has been suggested. Etiological relevance is assumed if cycle-threshold values are low, suggesting high pathogen loads. Although the procedure is challenged by lacking standardization and methodical issues, first evaluations have led to promising results. Future studies should aim at generally acceptable quantitative cut-off values to allow discrimination of asymptomatic colonization from clinically relevant infection.

Saccharomyces boulardii to Prevent Antibiotic-Associated Diarrhea: A Randomized, Double-Masked, Placebo-Controlled Trial.

Background. Antibiotic-associated diarrhea (AAD) and Clostridium difficile-associated diarrhea (CDAD) are common complications of antibiotic use. Data on the efficacy of probiotics to prevent AAD and CDAD are unclear. We aimed to evaluate the efficacy of Saccharomyces boulardii to prevent AAD and CDAD in hospitalized adult patients. Methods. We conducted a multicenter, phase III, double-masked, randomized, placebo-controlled trial in hospitalized patients who received systemic antibiotic treatment in 15 hospitals in Germany between July 2010 and October 2012. Participants received Perenterol forte 250 mg capsules or matching placebo twice per day within 24 hours of initiating antibiotic treatment, continued treatment for 7 days after antibiotic discontinuation, and were then observed for 6 weeks. Results. Two thousand four hundred forty-four patients were screened. The trial was stopped early for futility after inclusion of 477 participants. Two hundred forty-six patients aged 60.1 ± 16.5 years and 231 patients aged 56.5 ± 17.8 were randomized to the S boulardii group and the placebo group, respectively, with 21 and 19 AADs in the respective groups (P = .87). The hazard ratio of AAD in the S boulardii group compared with the placebo group was 1.02 (95% confidence interval, .55-1.90; P = .94). Clostridium difficile-associated diarrhea occurred in 0.8% of participants (4 of 477). Nine serious adverse events were recorded in the S boulardii group, and 3 serious adverse events were recorded in the placebo group. None were related to study participation. Conclusions. We found no evidence for an effect of S boulardii in preventing AAD or CDAD in a population of hospitalized patients without particular risk factors apart from systemic antibiotic treatment. ClinicalTrials.gov Identifier. NCT01143272.

PCR for enteric pathogens in high-prevalence settings. What does a positive signal tell us?

BACKGROUND: Molecular methods, in particular PCR, are increasingly used for the diagnosis of enteric pathogens in stool samples. In high-endemicity settings, however, asymptomatic carriage or residual DNA from previous infections will hamper the interpretation of positive test results. We assessed the quantitative dimension of this problem in schoolchildren in the rural highlands of Madagascar. METHODS: Stool samples were collected from 410 apparently healthy Madagascan schoolchildren and analysed by multiplex real-time PCR for enteroinvasive bacteria (Salmonella spp., Shigella spp./enteroinvasive Escherichia coli (EIEC), Campylobacter jejuni, Yersinia spp.), enteric protozoa (Entamoebea histolytica, Giardia duodenalis, Cryptosporidium spp., Cyclospora spp.), and helminths (Ascaris lumbricoides, Ancylostoma spp., Necator americanus, Strongyloides stercoralis). Symptoms of gastrointestinal disease were assessed. RESULTS: Among the 410 samples, we detected Giardia duodenalis in 195, Campylobacter jejuni in 91, Ascaris lumbricoides in 72, Cyclospora cayetanenesis in 68, Shigella spp./EIEC in 56, and Strongyloides stercoralis and Cryptosporum spp. in 1 case each. Salmonella spp., Yersinia spp. and hookworms were not observed. Relative risk assessment suggested few and incoherent associations with pathogen detections, indicating asymptomatic carriage or DNA residuals. Only 26.1% of the schoolchildren were tested negative for all analysed pathogens. CONCLUSIONS: The very high risk of detecting traces of asymptomatic carriage or residual DNA from previous infections limits the value of highly sensitive PCR for the causal attribution of detected enteric pathogens from stool samples to an infectious gastrointestinal disease in the high-endemicity setting. Evaluated standards for the interpretation of such results are needed both for the diagnostic routine and for epidemiological assessments.

Schistosoma mansoni in schoolchildren in a Madagascan highland school assessed by PCR and sedimentation microscopy and Bayesian estimation of sensitivities and specificities.

Madagascar is an endemic area for schistosomiasis, but recent prevalence data are scarce. We investigated stool samples of 410 children aged 4-18 years from a combined primary and secondary school in a Madagascan highland village near Ambositra in order to assess the prevalence of Schistosoma mansoni using microscopy and real-time polymerase chain reaction (PCR). A high prevalence of S. mansoni of 77.1% was detected by PCR, while only 15.2% of microscopic examinations of sedimentation-enriched stools were positive. We estimated the sensitivity and specificity of stool sedimentation microscopy (19.7% and 98.8%) and of PCR (98.9% and 89.3%) using a Bayesian approach for two dependant tests in one population without a reference standard. Our Bayesian posterior estimate of the prevalence is 80.2%. Simple sedimentation technique misses about 4/5 of all PCR-confirmed infections and is insufficient to determine the prevalence of S. mansoni. A survey comparing PCR with a classical standard technique (KatoKatz) is desirable.

Is rapid hepatitis C virus testing from corpses a screening option for index persons who have died after mass-casualty incidents in high-prevalence settings in the field?

INTRODUCTION: We tested a commercially available rapid hepatitis C virus (HCV) test assay for its potential use for analyses of corpses as a screening option for index persons who have died after mass-casualty incidents in high-prevalence settings in the field. MATERIALS AND METHODS: 50 blood samples were drawn from 16 recently deceased confirmed HCV-positive patients whose corpses were stored at 4°C in the mortuary and were analysed at admission and up to 48 h post mortem by rapid serological testing using the ImmunoFlow HCV test (Core Diagnostics, Birmingham, UK) in comparison with automated serological assays and PCR. Samples from 50 HCV-negative corpses were also analysed. RESULTS: The blood of only four of the 16 HCV-positive corpses reacted clearly with the ImmunoFlow HCV test, while in five cases the result was only weakly reactive and three cases showed very weak reactivity. Four of the infected corpses showed initially negative results, three of which became very weakly reactive 48 h post mortem. 49 out of 50 samples (98%) from HCV-negative corpses tested negative. DISCUSSION: The rapid test system we investigated showed insufficient sensitivity regarding the identification of HCV positivity. Automated serological testing or PCR should be preferred if it is realistically available in the deployed military setting.

Influence of parasite density and sample storage time on the reliability of Entamoeba histolytica-specific PCR from formalin-fixed and paraffin-embedded tissues.

We report on the reliability of polymerase chain reaction (PCR) for the detection of Entamoeba histolytica from formalin-fixed, paraffin-embedded tissue in comparison with microscopy and have determined predictors that may influence PCR results. E. histolytica-specific and Entamoeba dispar-specific real-time PCR and microscopy from adjacent histologic sections were performed using a collection of formalin-fixed, paraffin-embedded tissue specimens obtained from patients with invasive amebiasis. Specimens had been collected during the previous 4 decades. Association of sample age, parasite density, and reliability of PCR was analyzed. E. histolytica PCR was positive in 20 of 34 biopsies (58.8%); 2 of these 20 were microscopically negative for amebae in neighboring tissue sections. PCR was negative in 9 samples with visible amebae in neighboring sections and in 5 samples without visible parasites in neighboring sections. PCR was negative in all specimens that were older than 3 decades. Low parasite counts and sample ages older than 20 years were predictors for false-negative PCR results. All samples were negative for E. dispar DNA. PCR is suitable for the detection of E. histolytica in formalin-fixed, paraffin-embedded tissue samples that are younger than 2 decades and that contain intermediate to high parasite numbers. Negative results in older samples were due to progressive degradation of DNA over time as indicated by control PCRs targeting the human 18S rRNA gene. Moreover, our findings support previous suggestions that only E. histolytica but not E. dispar is responsible for invasive amebiasis.

Food and drinking water hygiene and intestinal protozoa in deployed German soldiers.

This report analyzes the occurrence of Cryptosporidium spp., E. histolytica, and G. intestinalis in stool of returnees from military deployments and the impact of hygiene precautions. Between 2007 and 2010, stool samples of 830 returnees that were obtained 8-12 weeks after military deployments in Afghanistan, Uzbekistan, the Balkans, Democratic Republic of the Congo/Gabonese Republic, and Sudan and 292 control samples from non-deployed soldiers were analyzed by PCR for Cryptosporidium spp., E. histolytica, G. intestinalis, and the commensal indicator of fecal contamination E. dispar. Data on hygiene precautions were available. The soldiers were questioned regarding gastrointestinal and general symptoms. Among 1122 stool samples, 18 were positive for G. intestinalis, 10 for E. dispar, and no-one for Cryptosporidium spp. and E. histolytica. An increased risk of acquiring chronic parasitic infections in comparison with non-deployed controls was demonstrated only for G. intestinalis in Sudan, where standardized food and drinking water hygiene precautions could not be implemented. Standard food and drinking water hygiene precautions in the context of screened military field camps proved to be highly reliable in preventing food-borne and water-borne chronic infections and colonization by intestinal protozoa, leading to detection proportions similar to those in non-deployed controls.

Fluorescence in situ hybridization (FISH) for rapid identification of Salmonella spp. from agar and blood culture broth--an option for the tropics?

BACKGROUND: Salmonella enterica is an important cause of diarrhea with the potential to cause systemic infection including sepsis, particularly in the tropics. Sepsis in particular requires quick and reliable identification to allow a rapid optimization of antibiotic therapy. We describe the establishment and evaluation of fluorescence in situ hybridization (FISH) as a rapid and easy-to-perform molecular identification procedure from agar and blood culture broths. METHODS: Two newly developed FISH probes with specificity for Salmonella spp. were evaluated with 10 reference strains, 448 clinical isolates of Gram-negative bacteria from Germany and Ghana including 316 Salmonella spp. strains, and 39 environmental Salmonella spp. isolates from rivers and streams in Ghana. One FISH probe was further tested with 207 pre-incubated blood culture broths from Germany with Gram-negative rod-shaped bacteria in Gram stain. RESULTS: Evaluation of the newly designed FISH probes demonstrated sensitivity of 99.2% and specificity of 98.4% for clinical isolates, sensitivity of 97.4% for environmental Salmonella spp. isolates, and sensitivity of 100% and specificity of 99.5% for blood culture materials. CONCLUSIONS: FISH proved to be highly reliable for a rapid identification of Salmonella spp. directly from pre-incubated blood culture broths as well as after growth on agar. The inexpensive and easy-to-perform procedure is particularly suitable for resource-limited areas where more sophisticated procedures are not available.

Increased detection of invasive enteropathogenic bacteria in pre-incubated blood culture materials by real-time PCR in comparison with automated incubation in Sub-Saharan Africa.

BACKGROUND: Invasive enteropathogenic bacteria can cause systemic infections. Data from studies with PCR detection suggest, at least for Salmonella enterica, that blood culture may lead to underestimation in the tropics. Corresponding data are lacking for other invasive enteropathogenic bacteria. We compared classical blood culture and molecular methods for the diagnosis of blood infections. METHODS: A real-time multiplex PCR for Salmonella spp., Shigella spp./entero- invasive Escherichia coli (EIEC), Yersinia spp., and Campylobacter jejuni was applied to 2321 retained blood culture samples from Ghanaian patients, after enrichment by automated culture. RESULTS: PCR detected Salmonella DNA in 56 out of 58 pre-incubated Ghanaian blood cultures with growth of S. enterica. In 2 samples molecular diagnosis was only possible after 1:10 dilution. Twenty-two samples negative by blood culture and 1 positive with Micrococcus spp. were PCR-positive for Salmonella spp. In addition, 3 Shigella spp./EIEC, 2 Yersinia spp., and 1 C. jejuni were detected by PCR but not by culture growth. CONCLUSIONS: Real-time PCR was more sensitive in identifying invasive enteropathogenic bacteria than automated blood culture, which is hampered by a lack of evidence-based standardization of pre-analytic conditions in the tropics. Primary agar culture and Gram-staining prior to automated blood culture is advisable in cases where transportation times are long.

Serological survey of HIV and syphilis in pregnant women in Madagascar.

OBJECTIVES: Peripartal transmission of human immunodeficiency virus (HIV) and Treponema pallidum, the causative agent of syphilis, leads to severe consequences for newborns. Preventive measures require awareness of the maternal infection. Although HIV and syphilis testing in Madagascar could be theoretically carried out within the framework of the national pregnancy follow-up scheme, the required test kits are rarely available at peripheral health centres. In this study, we screened blood samples of pregnant Madagascan women for HIV and syphilis seroprevalence to estimate the demand for systemic screening in pregnancy. METHODS: Retrospective anonymous serological analysis for HIV and syphilis was performed in plasma samples from 1232 pregnant women that were taken between May and July 2010 in Ambositra, Ifanadiana, Manakara, Mananjary, Moramanga and Tsiroanomandidy (Madagascar) during pregnancy follow-up. Screening was based on Treponema pallidum haemagglutination tests for syphilis and rapid tests for HIV, with confirmation of positive screening results on line assays. RESULTS: Out of 1232 pregnant women, none were seropositive for HIV and 37 (3%) were seropositive for Treponema pallidum. CONCLUSIONS: Our findings are in line with previous studies that describe considerable syphilis prevalence in the rural Madagascan population. The results suggest a need for screening to prevent peripartal Treponema pallidum transmission, while HIV is still rare. If they are known, Treponema pallidum infections can be easily, safely and inexpensively treated even in pregnancy to reduce the risk of transmission.

Rapid identification of Leishmania spp. in formalin-fixed, paraffin-embedded tissue samples by fluorescence in situ hybridization.

OBJECTIVE: To describe and validate fluorescence in situ hybridization (FISH), a new method of Leishmania spp. identification. FISH allows for a rapid detection of target organisms by specific binding of fluorescently labelled oligonucleotide probes to ribosomal RNA. METHODS: Two genus-specific, fluorescently labelled Leishmania spp. FISH probes were designed and evaluated with a panel of 18 Leishmania spp. and six Trypanosoma spp. including well-defined strains and clinical isolates. In addition, the FISH probes were tested in comparison with Giemsa staining in formalin-fixed, paraffin-embedded tissues of five mice that had been artificially infected with Leishmania major strains, leading to concordant results. Finally, 11 tissue samples of patients with cutaneous leishmaniasis, four tissue samples of patients with visceral leishmaniasis, and one native bone marrow sample of a patient with visceral leishmaniasis were analysed with FISH and Giemsa staining. RESULTS: Concordant results were achieved by FISH and Giemsa staining in 15/16 specimens. CONCLUSION: This analysis provides proof of principle that FISH is a suitable method for the rapid and easy detection of Leishmania spp. in formalin-fixed, paraffin-embedded tissue samples. Because of the good contrast of Leishmania spp. in tissue, FISH facilitates the identification of these organisms in tissue samples even by less experienced investigators.

Rapid identification of Burkholderia pseudomallei and Burkholderia mallei by fluorescence in situ hybridization (FISH) from culture and paraffin-embedded tissue samples.

We evaluated newly developed probes for rapid identification of Burkholderia (B.) pseudomallei and B. mallei and differentiation from B. thailandensis by fluorescence in situ hybridization (FISH). FISH correctly identified 100% of the tested B. pseudomallei (11), B. mallei (11), and B. thailandensis (1) strains, excluded 100% of all tested negative controls (61), and allowed demonstration of B. pseudomallei infection in a paraffin-embedded spleen tissue sample of an experimentally infected mouse.

Systemic bacteraemia in children presenting with clinical pneumonia and the impact of non-typhoid salmonella (NTS).

BACKGROUND: The diagnosis and antimicrobial treatment of pneumonia in African children in the absence of diagnostic means such as x-ray facilities or microbiological laboratories relies primarily on clinical symptoms presented by the patients. In order to assess the spectrum of bacterial pathogens, blood cultures were performed in children fulfilling the clinical criteria of pneumonia. METHODS: In total, 1032 blood cultures were taken from children between 2 months and 5 years of age who were admitted to a rural hospital in Ghana between September 2007 and July 2009. Pneumonia was diagnosed clinically and according to WHO criteria classified as "non-severe pneumonia" and "severe pneumonia" ("severe pneumonia" includes the WHO categories "severe pneumonia" and "very severe pneumonia"). RESULTS: The proportion of bacteriaemia with non-typhoid salmonella (NTS) was similar in children with pneumonia (16/173, 9.2%) compared to children hospitalized for other reasons (112/859, 13%). NTS were the predominant organisms isolated from children with clinical pneumonia and significantly more frequent than Streptococcus pneumoniae (8/173, 4.6%). Nine percent (9/101) of children presenting with severe pneumonia and 10% (7/72) of children with non-severe pneumonia were infected with NTS. Nineteen out of 123 NTS isolates (15%) were susceptible to aminopenicillins (amoxycillin/ampicillin), 23/127 (18%) to chlorampenicol, and 23/98 (23%) to co-trimoxazole. All NTS isolates were sensitive to ceftriaxone and ciprofloxacin. CONCLUSION: In Sub-saharan Africa, sepsis with NTS should be considered in children with symptoms of pneumonia and aminopenicillins might often not be the adequate drugs for treatment.

Short communication: first record of Aedes albopictus in Gabon, Central Africa.

The Asian tiger mosquito Aedes albopictus is an important arbovirus vector. Originating in East Asia, the species has been introduced to the Americas, the Indo-Pacific and Australasian regions as well as Europe and Africa, mostly during the past 30 years and probably by transportation in used tires. We report Ae. albopictus for the first time from Gabon (Libreville). In addition, the yellow fever mosquito Ae. aegypti ssp. formosus and 16 other culicid species were detected throughout the city, four of which are also new records for Gabon.

Brucella endocarditis in prosthetic valves.

Human brucellosis is a multiple organ disease that presents with fever and is most often transmitted via contaminated, unpasteurized goat milk and cheese. In chronic cases, focal complications (eg, spondylitis, neurobrucellosis and endocarditis) are frequently seen. Although the disease may be severely debilitating, the mortality rate is low. Fatal cases are often due to endocarditis. Because Brucella endocarditis is a rare complication (2% to 5%), therapeutic considerations are based on single-case experiences only. Therapy includes long-term antibiotic treatment using combinations of various antimicrobial drugs and surgical valve replacement when required. A case of Brucella endocarditis complicated by the infection of two valvular prostheses implanted after involvement of the mitral and aortic valve due to rheumatic fever is described. The patient was successfully treated by a medical and surgical approach. Therapeutic strategies in Brucella endocarditis are discussed in light of the current literature.

Growth characteristics of Bacillus anthracis compared to other Bacillus spp. on the selective nutrient media Anthrax Blood Agar and Cereus Ident Agar.

Anthrax Blood Agar (ABA) and Cereus Ident Agar (CEI) were evaluated as selective growth media for the isolation of Bacillus anthracis using 92 B. anthracis and 132 other Bacillus strains from 30 species. The positive predictive values for the identification of B. anthracis on ABA, CEI, and the combination of both were 72%, 71%, and 90%, respectively. Thus, less than 10% of all species were misidentified using both nutrient media. Species which might be misidentified as B. anthracis were B. cereus, B. mycoides, and B. thuringiensis. Particularly, 30% of B. weihenstephanensis strains were misidentified as B. anthracis.