Prevalence and Genotyping of Water- and Food-Borne Parasitic Protozoans (Giardia duodenalis and Cryptosporidium spp.) in Hospitalized Patients from Northwestern Romania.

Giardia duodenalis and Cryptosporidium spp. are important zoonotic protozoan pathogens that infect the gastro-intestinal tract of numerous vertebrates, including humans, and both parasites are responsible for water- or food-borne outbreaks of disease worldwide. Although, globally, both parasites are highly prevalent, particularly in developing countries, epidemiological data from Romania are scarce, and genotyping has rarely been performed. The aims of the present study were to investigate the occurrence and genetic diversity of G. duodenalis and Cryptosporidium spp. in patients hospitalized in Northwestern Romania in relation to clinical and paraclinical presentation and to identify the relative frequency of non-specific symptoms and potential risk factors. Between June 2022 and January 2024, 426 fecal samples were screened for gastro-intestinal parasites by rapid tests and microscopical examination, further confirmed by PCR and sequencing. Giardia duodenalis was detected and characterized in 12 samples (2.82%), while Cryptosporidium parvum was confirmed in four samples (0.94%). A majority of positive patients were symptomatic and reported nausea and vomiting with a significantly higher frequency compared to negative ones. This study provides new insights into the epidemiological status and clinical implications of gastro-intestinal parasite species and genospecies in Romania that are necessary for an in-depth understanding of the potential zoonotic transmission and improvement of patient care.

Changes in Epidemiology and Antibiotic Prescription of Influenza: Before and after the Emergence of COVID-19.

BACKGROUND: The appearance of COVID-19 had a major impact on healthcare and the epidemiology of other diseases. Following the cessation of non-pharmacologic interventions destined to limit the spread of COVID-19, influenza reemerged. The aim of this study was to compare the pre-pandemic influenza seasons with the influenza seasons after the emergence of the COVID-19 pandemic, and to identify differences in terms of clinical characteristics, risk factors, complications, outcomes, and antiviral and antibiotic treatments. METHODS: We conducted a retrospective cohort study from the Teaching Hospital of Infectious Diseases database in Cluj-Napoca, Romania. We analyzed four pre-pandemic seasons and the seasons after the onset of COVID-19. We included adult patients hospitalized with confirmed influenza between October 2016 and August 2023. Variables such as age, sex, duration of hospitalization, severity, clinical manifestations, comorbidities, and Charlson comorbidity index were assessed. RESULTS: A total of 941 patients were included in the analysis. The percentage of severe influenza was similar in both groups, but mortality from influenza was significantly lower after 2022. Virtually all patients were prescribed antivirals; antibiotic prescriptions decreased in the post-COVID-19 influenza seasons. CONCLUSION: The present study suggests that influenza seasons after 2022 had lower mortality and attenuated clinical presentation.

Toxoplasmosis Screening during Pregnancy in a Romanian Infectious Diseases Tertiary Center: Results of a 15 Years Follow-Up Program.

Maternal infection with Toxoplasma gondii during pregnancy may have serious consequences for the fetus. In Romania, screening for toxoplasmosis is included in the first antenatal visit. A retrospective study was performed on all toxoplasmosis antenatal screening patients between May 2008 and February 2023. Twenty-seven thousand one hundred sixty-nine (27,169) pregnant women presented for prenatal screening once (22,858) or several times: during the same pregnancy (209) or during multiple pregnancies (4102). Thirty-one thousand six hundred fifty-eight (31,658) tests for IgM and IgG antibodies were performed. Nine thousand eighty-three (9083) tests (28.69%), corresponding to 7911 women (29.12%), were positive for IgG antibodies. The seroprevalence increased with patients' age, decreased in time intervals, and was more frequently associated with rural residence. At risk for acquiring the infection during the pregnancy were women with negative anti-Toxoplasma IgG antibodies (70.88%), but only 0.9% of them presented for rescreening during the same pregnancy. Acute Toxoplasma infection (ATI) was suspected in 44 patients (0.16%) due to IgG seroconversion and/or low or borderline IgG avidity. A questionnaire follow-up interview was performed, and no congenital toxoplasmosis was identified in children born from mothers with probable ATI. Our study demonstrates poor compliance with the screening program in the Romanian population.

Retrospective Survey of Human Trichinellosis in a Romanian Infectious Diseases Hospital over a Thirty-Year Interval-The Never-Ending Story.

Trichinellosis remains a food-safety risk in Romania due to cultural traditions and food behavior. The aim of the present study was to evaluate the epidemiological, clinical and therapeutical data of all human trichinellosis cases in patients admitted to an Infectious Diseases Hospital from northwestern Romania during a thirty-year interval. Between 1 January 1988 and 31 December 2018, a total of 558 patients were hospitalized with the diagnosis of trichinellosis. The number of cases/year varied between 1 and 86. The source of infection was known for 524 patients, represented by domestic pig meat (n = 484; 92.37%) and wild boar (n = 40; 7.63%). Most patients (410; 73.48%) presented were part of family or group outbreaks. Demographical and clinical data of patients will be presented. Antiparasitic therapy was prescribed in 99.46% of cases, and corticosteroids were prescribed in 77.06% of patients. In total, 48 patients (8.6%) presented complications of trichinellosis: 44 for a single complication (neurological, cardiovascular or respiratory); the others multiple complications. Pregnancy was documented in five patients. No fatalities occurred during the study period. Although the number of hospitalized patients has decreased in the last years, trichinellosis still remains an important public health problem in northwestern Romania.

Serological Survey of Mosquito-Borne Arboviruses in Wild Birds from Important Migratory Hotspots in Romania.

In the context of climate change, globalization, and enhanced human traveling, arboviruses continue to represent a threat to public health. West Nile and Usutu viruses are mosquito-borne viruses belonging to the Flaviviridae family (Flavivirus genus) and members of the Japanese encephalitis virus serocomplex. Included in the Togaviridae family (Alphavirus genus), the Sindbis virus is also vectored by mosquitoes. In the present study, we aimed to analyze the presence of antibodies concerning the abovementioned viruses in migratory and resident birds in the South-Eastern region of Romania, as avian hosts represent the main reservoir for human infection. Blood samples were collected from wild birds between May 2018 and October 2019 in nine locations from three counties. All the samples were serologically tested by ELISA and a serum neutralization test. Overall, a seroprevalence of 8.72% was registered for the West Nile virus, 2.71% for the Usutu virus, and 0% for the Sindbis virus. To our best knowledge, this is the first large-scale comprehensive study to assess the West Nile virus seropositivity in wild birds and the first serological confirmation of the Usutu virus in wild birds in Romania. Moreover, this is the only follow-up study reviewing the current seroprevalence of the Sindbis virus in Romania since 1975.

Longitudinal determination of BNT162b2 vaccine induced strongly binding SARS-CoV-2 IgG antibodies in a cohort of Romanian healthcare workers.

Mass vaccination against the disease caused by the novel coronavirus (COVID-19) was a crucial step in slowing the spread of SARS-CoV-2 in 2021. Even in the face of new variants, it still remains extremely important for reducing hospitalizations and COVID-19 deaths. In order to better understand the short- and long-term dynamics of humoral immune response, we present a longitudinal analysis of post-vaccination IgG levels in a cohort of 166 Romanian healthcare workers vaccinated with BNT162b2 with weekly follow-up until 35 days past the first dose and monthly follow-up up to 6 months post-vaccination. A subset of the patients continued with follow-up after 6 months and either received a booster dose or got infected during the Delta wave in Romania. Tests were carried out on 1694 samples using a CE-marked IgG ELISA assay developed in-house, containing S1 and N antigens of the wild type virus. Participants infected with SARS-CoV-2 before vaccination mount a quick immune response, reaching peak IgG levels two weeks after the first dose, while IgG levels of previously uninfected participants mount gradually, increasing abruptly after the second dose. Overall higher IgG levels are maintained for the previously infected group throughout the six month primary observation period (e.g. 36-65 days after the first dose, the median value in the previously infected group is 5.29 AU/ml, versus 3.58 AU/ml in the infection naïve group, p less than 0.001). The decrease of IgG levels is gradual, with lower median values in the infection naïve cohort even 7-8 months after vaccination, compared to the previously infected cohort (0.7 AU/ml versus 1.29 AU/ml, p = 0.006). Administration of a booster dose yielded higher median IgG antibody levels than post second dose in the infection naïve group and comparable levels in the previously infected group.

The Diagnostic Challenges and Clinical and Serological Outcome in Patients Hospitalized for Suspected Lyme Neuroborreliosis.

The aim of our study was to evaluate the differential diagnosis and clinical/serological outcome to antibiotic treatment in patients hospitalized for suspected Lyme neuroborreliosis (LNB). A prospective study included patients hospitalized in a Romanian hospital between March 2011 and October 2012 with neurological symptoms, positive laboratory tests for Borrelia burgdorferi, cerebrospinal fluid (CSF) analysis, and no previous treatment for LNB. A questionnaire was completed for each patient at admission, at the end of treatment, and 3 months later. Patients were treated with antibiotic therapy (ceftriaxone/cefotaxime), irrespective of CSF analysis results. A symptomatic scoring scale was used for the follow-up. Out of the 42 patients included, no patient fulfilled criteria for definite LNB; 7 patients were classified as possible LNB; and in 33 patients, LNB was excluded. Two patients could not be classified (insufficient amount of CSF). Clinical follow-up suggested a better response to therapy in the group of patients with possible LNB than in the group with LNB excluded. The patients' differential diagnosis and serological follow-up are presented. Patients investigated for suspected LNB present diverse clinical manifestations and comorbidities that complicate differential diagnosis. LNB may be misdiagnosed if CSF analysis is not performed.

Seroprevalence Rates against West Nile, Usutu, and Tick-Borne Encephalitis Viruses in Blood-Donors from North-Western Romania.

INTRODUCTION: West Nile virus (WNV), Usutu virus (USUV), and the tick-borne encephalitis virus (TBEV) are all arboviruses belonging to Flaviviridae family. All are characterized by vectorial transmission and sometimes associated with neuroinvasive infections. The circulation of these viruses is considered endemic in parts of Europe, with human cases reported in many countries. Among hosts, the viruses are vectored by hematophagous arthropods, such as mosquitoes (WNV, USUV) and ticks (TBEV). Considering the currently outdated knowledge regarding the epidemiology of these viruses in Romania, the aim of our study was to assess the seroprevalence rates of WNV, USUV, and TBEV among healthy blood donors in north-western Romania. METHODS: Human blood samples from healthy donors were collected between November 2019 and February 2020 in six counties from the north-western region of Romania. The samples were serologically tested by ELISA and serum neutralization test. RESULTS: Overall, we obtained a seroprevalence of 3.17% for WNV, 0.08% for TBEV, and 0% for USUV. CONCLUSION: Despite the low seroprevalence of WNV, USUV, and TBEV in our study, we highlight the need for continuous nationwide vector and disease surveillance and implementation of control measures. Further research is required for an optimal overview of the epidemiological status of the Romanian population regarding these flaviviruses together with countrywide awareness campaigns.

Favipiravir in Kidney Transplant Recipients With COVID-19: A Romanian Case Series.

BACKGROUND: Favipiravir (FPV) is an orally administrable antiviral drug that selectively inhibits RNA-dependent RNA polymerase and has been repurposed for COVID-19 treatment. There is limited information on the use of FPV in kidney transplant recipients (KTx), who often have multiple comorbidities and run a higher risk for death from COVID-19. METHODS: We retrospectively reviewed all KTx at our institution who got sick with COVID-19 between March 1, 2020, and May 31, 2021, and who received FPV (loading dose of 1800 mg × 2 on day 1, maintenance dose 2  ×  800 mg/d for 5-14 days) as part of their COVID treatment. We analyzed demographics, clinical course, laboratory data, management, and outcome. RESULTS: Nine KTx with COVID-19 received FPV; all were hospitalized. The median age was 52 years (range, 32-60 years), and women were predominant (77.7%). Eight KTx had pulmonary involvement on chest radiograph. On admission 1 patient had mild, 5 had moderate, 2 had severe, and 1 had critical disease. Leukopenia and increased creatinine were universally noted. Three patients had disease progression under treatment. Seven patients (77.7%) required additional oxygen, and 4 (57.1%) needed intensive care unit admission. Three KTx died, resulting in an overall mortality of 33.3%. Survivors did not show increased transaminases or creatinine during or after FPV treatment; leukocytes, neutrophils, and platelets improved on discharge compared with admission values. CONCLUSIONS: FPV appears well tolerated by KTx with COVID-19, but its clinical benefit remains unclear. Larger analyses are needed.

Seroprevalence of antibodies against Borrelia burgdorferi sensu lato in healthy blood donors in Romania: an update.

BACKGROUND: The Borrelia burgdorferi sensu lato (s.l.) genogroup is the causative agent responsible for Lyme borreliosis, a common tick-borne infectious disease in some temperate regions of the Northern Hemisphere. In humans, the clinical manifestations of Lyme borreliosis vary from dermatological infection to severe systemic manifestations. In Romania, data on the seroprevalence of Lyme borreliosis and associated risk factors are scarce and outdated, as the only seroprevalence study with a large dataset was published more than 20 years ago. Therefore, the aim of the present study was to evaluate the seroprevalence for Borrelia burgdorferi s.l. in healthy blood donors from six Romanian counties and identify the associated risk factors. METHODS: The study was conducted among 1200 healthy blood donors aged between 18 and 65 years during November 2019 and September 2020 from six counties in the northwestern and central parts of Romania. A two-tiered testing strategy was applied. Positive and equivocal immunoenzymatic test results for IgG and IgM antibodies were further confirmed by Western blot. RESULTS: Serum samples from 20% of the blood donors had positive or equivocal IgG and IgM ELISA index values. In total, 2.3% of the serum samples for IgG and 1.8% for IgM were positive by Western blot. The seroprevalence for both antibodies varied between 1.5% (Satu-Mare) and 6.5% (Bistrița-Nasaud) in the six counties investigated. The highest seroprevalence was observed in men (4.7%), in blood donors performing their professional activities outdoors (4.2%), and in those aged ≥ 56 years (8%). CONCLUSIONS: These findings confirm the presence of specific IgG and IgM antibodies to B. burgdorferi s.l. among healthy blood donors from Romania. Furthermore, potential risk factors, such as gender, age, and behavior, associated with the presence of positive B. burgdorferi s.l. antibodies among healthy blood donors were identified.

Post-infectious irritable bowel syndrome after a laboratory-proven enteritis.

There are scarce data on risk factors for post-infectious irritable bowel syndrome (PI-IBS). The objective of this study was to determine the risk factors of developing PI-IBS following an acute infectious gastroenteritis (AGE) episode in which, by laboratory tests, the etiological agent was isolated. The study was conducted on patients admitted to a tertiary center of infectious diseases during three consecutive years. The patients were divided into two groups: a group consisting of patients admitted with AGE (with an isolated etiological agent) and a control group consisting of patients admitted for an acute upper respiratory tract infection (URTI). The subjects were recalled in our center 6 months after the admission and were evaluated with Rome III IBS diagnostic questionnaire and Bristol Stool Form Scale. The questionnaires were paper printed and directly filled in by the subjects. The response rate in the case group was 5% and in the control group 100%. The prevalence of PI-IBS was higher in patients with AGE, presenting a relative risk (RR) of 4.16 [95% confidence interval (CI), 1.89-9.17], statistically significant (P<0.001) vs. URTI. From 28 female patients, 22 patients (79%) developed PI-IBS and from 17 male patients, 3 patients (18%) had developed PI-IBS with a risk of 4.4 (95% CI, 1.56-12.65), P<0.001. Regarding the infectious etiology of the AGE, Campylobacter jejuni had the highest risk of developing PI-IBS, RR=1.2 (95% CI, 0.13-3.11), P=0.04 compared with the other agents with a lower risk. The risk to develop PI-IBS after AGE infection is 4.16 higher than after URTI. Female sex is a risk factor for PI-IBS, 79% of the female patients developed PI-IBS after AGE. The incidence of PI-IBS is highest in patients with Campylobacter jejuni AGE compared with the other agents.

Multiple Tick-Borne Pathogens in Ixodes ricinus Ticks Collected from Humans in Romania.

Ticks are medically important vectors of infectious diseases that are able to transmit pathogens to humans and animals. Tick-borne diseases represent a major health concern, posing an increasing risk to the public health during the last century and affecting millions of people. The aim of the current study was to provide epidemiological data regarding the presence of certain tick-borne pathogens in ticks feeding on humans in Romania. Overall, 522 Ixodes ricinus ticks collected from humans were screened for six pathogens: Borrelia spp., Neoehrlichia mikurensis, Babesia spp., Coxiella spp., Bartonella spp., and Francisella tularensis. Ticks attached to humans were collected between 2013-2015 in Cluj County, Romania. Conventional, nested and quantitative PCR were used to detect specific genetic sequences of each pathogen. For identifying the infectious agents, positive samples were sequenced. The infection prevalence was 21.07% from which 8.18% were mixed infections. The detected agents were Borrelia spp., N. mikurensis and Babesia spp. The present data reveal the endemic occurrence of potentially zoonotic pathogens in Romania. Revealing the current distribution of tick-borne pathogens in ticks collected from humans may provide new insights in understanding the complex ecology of tick-borne diseases and enlightens current knowledge about the infection prevalence at local, regional and national levels.

Clinical and serological one-year follow-up of patients after the bite of Ixodes ricinus ticks infected with Borrelia burgdorferi sensu lato.

BACKGROUND: The risk of developing Lyme borreliosis (LB) after the bite of a Borrelia (B.) burgdorferi sensu lato (s.l.) infected tick in Romania is unknown. METHODS: The present prospective study, performed in 2010-2011 in a hospital in Romania, has followed-up clinical and serological outcome of patients that presented with B. burgdorferi positive Ixodes (I.) ricinus bite. A second group of patients, including age, sex and residence-matched individuals bitten by B. burgdorferi negative ticks, was followed-up as a control group. The subjects' outcome was evaluated one year after the tick bite. RESULTS: Forty-three out of 389 ticks detached from patients were positive by hbb Real-Time PCR (RT-PCR) for B. burgdorferi s.l. (mainly B. afzelii, but also B. garinii, B. burgdorferi sensu stricto, B. spielmanii/B. valaisiana and B. lusitaniae). Twenty patients bitten by B. burgdorferi positive ticks and twenty matched control patients returned for the one year follow-up. Two patients from the B. burgdorferi positive group developed clinical manifestations of acute LB (erythema migrans) and 5 patients seroconverted (two from the B. burgdorferi positive group and three from the B. burgdorferi negative group). Borrelia afzelii was identified in ticks collected from persons that developed erythema migrans (EM). Comparing the two groups of patients, no statistical significant differences were found regarding presence of clinical symptoms or seroconversion. CONCLUSIONS: No outcome differences were found between the group of patients bitten by B. burgdorferi positive ticks and the group of patients bitten by B. burgdorferi negative ticks.