Suitability of current typing procedures to identify epidemiologically linked human Giardia duodenalis isolates.

BACKGROUND: Giardia duodenalis is a leading cause of gastroenteritis worldwide. Humans are mainly infected by two different subtypes, i.e., assemblage A and B. Genotyping is hampered by allelic sequence heterozygosity (ASH) mainly in assemblage B, and by occurrence of mixed infections. Here we assessed the suitability of current genotyping protocols of G. duodenalis for epidemiological applications such as molecular tracing of transmission chains. METHODOLOGY/PRINCIPAL FINDINGS: Two G. duodenalis isolate collections, from an outpatient tropical medicine clinic and from several primary care laboratories, were characterized by assemblage-specific qPCR (TIF, CATH gene loci) and a common multi locus sequence typing (MLST; TPI, BG, GDH gene loci). Assemblage A isolates were further typed at additional loci (HCMP22547, CID1, RHP26, HCMP6372, DIS3, NEK15411). Of 175/202 (86.6%) patients the G. duodenalis assemblage could be identified: Assemblages A 25/175 (14.3%), B 115/175 (65.7%) and A+B mixed 35/175 (20.0%). By incorporating allelic sequence heterozygosity in the analysis, the three marker MLST correctly identified 6/9 (66,7%) and 4/5 (80.0%) consecutive samples from chronic assemblage B infections in the two collections, respectively, and identified a cluster of five independent patients carrying assemblage B parasites of identical MLST type. Extended MLST for assemblage A altogether identified 5/6 (83,3%) consecutive samples from chronic assemblage A infections and 15 novel genotypes. Based on the observed A+B mixed infections it is estimated that only 75% and 50% of assemblage A or B only cases represent single strain infections, respectively. We demonstrate that typing results are consistent with this prediction. CONCLUSIONS/SIGNIFICANCE: Typing of assemblage A and B isolates with resolution for epidemiological applications is possible but requires separate genotyping protocols. The high frequency of multiple infections and their impact on typing results are findings with immediate consequences for result interpretation in this field.

Increasing metronidazole and rifampicin resistance of Helicobacter pylori isolates obtained from children and adolescents between 2002 and 2015 in southwest Germany.

BACKGROUND: Increasing antibiotic resistance has been reported for Helicobacter pylori, but data on the prevalence of antibiotic resistance of H. pylori in pediatric patients and the development of resistance over time are sparse. METHODS: Data for 610 H. pylori isolates obtained between 2002 and 2015 from gastric biopsies of 582 (mainly treatment-naïve) pediatric patients from southwest Germany were analyzed retrospectively regarding the antibiotic susceptibility determined by Etest and patients' characteristics. RESULTS: Overall resistance to metronidazole, clarithromycin, and rifampicin was 28.7%, 23.2%, and 13.3%, respectively, while resistance to amoxicillin was rare (0.8%). Simultaneous resistance to metronidazole and clarithromycin was observed for 7.7% of the isolates, and 2.3% were resistant to metronidazole, clarithromycin, and rifampicin. Differences between primary vs secondary resistance existed for metronidazole (24.7% vs 38.8%, P=.01) and clarithromycin (17.2% vs 54.1%, P=.0001). From 2002-2008 to 2009-2015, resistance to metronidazole increased from 20.8% to 34.4% (P=.003) and to rifampicin from 3.9% to 18.8% (P=.0001); this was not associated with increased numbers of patients previously treated for H. pylori infection in the second study period. In contrast, resistance to clarithromycin did not change significantly over time. Resistance was not associated with age, sex, or family origin in Europe. CONCLUSIONS: The considerable antibiotic resistance of H. pylori isolates argues for standard antibiotic susceptibility testing of H. pylori in pediatric patients prior to the initiation of antibiotic therapy.

Antimicrobial resistance of Neisseria gonorrhoeae isolates in south-west Germany, 2004 to 2015: increasing minimal inhibitory concentrations of tetracycline but no resistance to third-generation cephalosporins.

Increasing antimicrobial resistance of Neisseria gonorrhoeae, particularly to third-generation cephalosporins, has been reported in many countries. We examined the susceptibility (determined by Etest and evaluated using the breakpoints of the European Committee on Antimicrobial Susceptibility Testing) of 434 N. gonorrhoeae isolates collected from 107 female and 327 male patients in Stuttgart, south-west Germany, between 2004 and 2015. During the study period, high proportions of isolates were resistant to ciprofloxacin (70.3%), tetracycline (48.4%; increasing from 27.5% in 2004/2005 to 57.7% in 2014/2015; p = 0.0002) and penicillin (25.6%). The proportion of isolates resistant to azithromycin was low (5.5%) but tended to increase (p = 0.08). No resistance and stable minimum inhibitory concentrations were found for cefixime, ceftriaxone, and spectinomycin. High-level resistance was found for ciprofloxacin (39.6%) and tetracycline (20.0%) but not for azithromycin; 16.3% of the isolates produced betalactamase. Thus, cephalosporins can still be used for the treatment of gonorrhoea in the study area. To avoid further increasing resistance to azithromycin, its usage should be limited to patients allergic to cephalosporins, or (in combination with cephalosporins) to patients for whom no susceptibility testing could be performed or those co-infected with chlamydiae.

Highly specific detection of Cryptosporidium spp. oocysts in human stool samples by undemanding and inexpensive phase contrast microscopy.

To compare phase contrast microscopy (PCM) of unstained slides for the detection of Cryptosporidium spp. oocysts with a commercially available enzyme immunoassay (EIA) for the detection of cryptosporidial antigen in human stool samples, we prospectively analysed by both methods 463 fresh human stool samples obtained from diarrhoeic patients between July and October 2014. Compared with the EIA, the sensitivity, specificity, positive and negative predictive value of PCM were 88.9 % (95 % confidence interval (CI), 66.0-98.1 %), 100 % (95 % CI, 99.0-100 %), 100 % (95 % CI, 77.3-100 %) and 99.6 % (95 % CI, 98.3-100 %), respectively. Additionally, we retrospectively examined with PCM 65 fixed stool samples that had been collected in 2010 from mostly asymptomatic Rwandan children <5 years of age; 14 of these samples had previously yielded positive results with a highly sensitive real-time (RT)-PCR. PCM detected cryptosporidia in 5/14 RT-PCR-positive samples, and notably, also in one of 51 RT-PCR-negative samples, which was subsequently confirmed by acid-fast staining. Positive and negative percent agreement of PCM with RT-PCR were 35.7 % (95 % CI, 16.2-61.4 %) and 98.0 % (95 % CI, 88.7-100 %), respectively. Positive PCM results were associated with higher RT-PCR cycle threshold values (p = 0.044). In conclusion, PCM offers a highly specific, undemanding and inexpensive method for the laboratory diagnosis of acute human cryptosporidiosis independent of the causative Cryptosporidium species.

[Rat-bite fever--two cases of infection with Streptobacillus moniliformis within two months]. / Rattenbissfieber--zwei Fälle von Infektionen mit Streptobacillus moniliformis innerhalb von zwei Monaten.

Medical history and clinical findings | Two cases of the rarely diagnosed Streptobacillus moniliformis infection (rat bite or haverhill fever) emerged within two months in Stuttgart. The first patient presented with typical symptoms, i.e., fever and arthralgia. The second patient, however, was afebrile with severe back pain and fatigue as only symptoms. None of the patients reported rat bites or other animal contacts. Examinations | Physical examination did not reveal any focus of infection in the first patient. Further examinations could not be completed. Suspected diagnosis was therefore "unclear, most likely viral infection". In the case of the second patient, ultrasound revealed an engorged right kidney and urinary obstruction. Upon concomitant detection of Escherichia coli in the urine, pyelonephritis was suspected. Laboratory parameters were not indicative in neither case. Detection of the infectious agents was accomplished by blood cultures and subsequent identification by mass spectrometry, albeit after discharge of the patients. Treatment and course | The first patient left the hospital against the doctors' advice the day after his admission. The second patient improved under ciprofloxacin and metamizole therapy and was discharged after five days with the recommendation to continue the antibiotic therapy. Conclusion | Cases of rat bite or haverhill fever are difficult to diagnose when no rat bites are recognized. Seroprevalence data of S. moniliformis infection would be desirable to estimate how often atypical or subclinical cases of this potentially lethal infection go undiagnosed.

Accurate detection of Campylobacter spp. antigens by immunochromatography and enzyme immunoassay in routine microbiological laboratory.

Campylobacter spp. are fastidious microorganisms, and their detection by culture depends on the freshness of the stool sample and the skills of the laboratory staff. To improve laboratory diagnosis, assays for the detection of specific antigens have been developed. Here, we evaluated two assays for the detection of Campylobacter spp.-specific antigens, i.e., one immunochromatographic test and one enzyme-linked immunosorbent assay (EIA), in 38 frozen Campylobacter spp.-positive specimens and prospectively in 533 fresh stool samples with a conventional enzyme immunoassay (EIA) and culture. Both assays were positive for 36 samples with Campylobacter jejuni and one with Campylobacter coli among 38 Campylobacter spp.-positive frozen samples. One Campylobacter lari-positive sample was identified by the immunochromatographic assay (ICA) only. In a prospective study performed within the course of routine microbiology, both assays were positive for 24/25 C. jejuni culture-positive samples (positive percent agreement, 96.0% [95% CI: 78.9-100%]). ICA and EIA also were positive for 14 and 10 culture-negative samples, respectively (negative percent agreement: ICA, 97.2% [95% CI: 95.4-98.4%]; EIA, 98.0% [95% CI: 96.4-99.0%]). In conclusion, the high agreement between both antigen-detection assays and culture indicates that both assays may be initially performed followed by culture only upon a positive test result.

Prevalence of erythromycin and clindamycin resistance among clinical isolates of the Streptococcus anginosus group in Germany.

Members of the Streptococcus anginosus group (SAG) are frequently involved in pyogenic infections in humans. In the present study, the antimicrobial susceptibility of 141 clinical SAG isolates to six antimicrobial agents was analysed by agar dilution. All isolates were susceptible to penicillin, cefotaxime and vancomycin. However, 12.8 % displayed increased MIC values (0.12 mg l(-1)) for penicillin. Resistance to erythromycin was detected in eight (5.7 %) isolates. Characterization of the erythromycin-resistant isolates with the double-disc diffusion test revealed Macrolide-Lincosamide-Streptogramin(B) and M-type resistance in six and two isolates, respectively. The erythromycin-resistant isolates were further characterized by PCR for the resistance genes ermA, ermB and mefA. Resistance and intermediate resistance to ciprofloxacin were detected in two and six isolates, respectively. Molecular typing by PFGE revealed a high genetic heterogeneity among the SAG isolates and no evidence for a clonal relationship between the erythromycin-resistant isolates. Our data show that resistance to erythromycin, clindamycin and ciprofloxacin has emerged among SAG isolates in Germany. The implications of these findings for susceptibility testing and antimicrobial therapy of SAG infections are discussed.

Prevalence of Pseudomonas aeruginosa in households of patients with cystic fibrosis.

Colonization of airways with Pseudomonas aeruginosa (PA) is known to be a risk factor for infection and adverse clinical outcome in patients with cystic fibrosis (CF). Whereas infections with PA have been linked to contaminated medical devices in hospitals, sources of colonization and infection with PA in households of patients with CF remain to be identified. Using a standardized sampling protocol, we prospectively examined the presence of PA in 102 households of patients with CF in Germany. PA was detected in 73 (71.6%) of 102 households. PA was detected most frequently in drains of showers (39.6%), drainpipes of hand-basins in kitchens (35.0%) and bathrooms (34.7%), and drainpipes of toilets (26.5%). Toilet seats and dish-clothes did not show PA in any household. The frequency and intensity of cleaning measures did not impact the detection rate of PA. Results of the present study for the first time determinate the rate of contamination with PA in households of patients with CF. Future studies will determine the risk of transmission of PA from households locations to patients with CF.