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MANAGEMENT OF HUMAN CRYPTOSPORIDIOSIS IN BULGARIA, A COUNTRY WITH LOW ENDEMICITY

Introduction. La cryptosporidiose est une infection protozoaire, chez l‘homme elle est le plus souvent causée par deux espèces – Cryptosporidium parvum et C. hominis. La maladie est le plus souvent accompagnée de diarrhée et a été définie par L‘OMS comme une infection liée au VIH. Si en Bulgarie des tests de cryptosporidiose sont pratiqués depuis près de trente ans, la maladie n’est pas encore suffisamment connue des professionnels de santé. L’objectif de cette étude est de déterminer l‘incidence et certaines caractéristiques démographiques de la cryptosporidiose humaine en Bulgarie sur une période de 10 ans, de 2009 à 2018. Matériel et méthodes. Pendant la période de l’étude, un total de 2706 patients a été examiné pour la cryptosporidiose par des méthodes microscopiques et immuno-chromatographiques, dont 2618 patients étaient immunocompétents et 88 étaient infectés par le VIH. ABSTRACT


ABSTRACT
Introduction. Cryptosporidiosis is a protozoan infection, and in humans is most often caused by two species, Cryptosporidium parvum and C. hominis. The disease most often presents with diarrhea, and it has been defined by the World Health Organization (WHO) as a human immunodeficiency virus (HIV)-related infection. Although in Bulgaria the tests for cryptosporidiosis have been performed for almost thirty years, the disease is still insufficiently known to healthcare professionals. The objective of the study was to determine the incidence and demographic characteristics of human cryptosporidiosis in Bulgaria for a 10-year period, from 2009 to 2018. Material and methods. During the study period, a total of 2706 patients were examined for cryptosporidiosis by microscopic and immunochromatographic methods, of whom 2618 were immunocompetent and 88 were HIV-infected. Results The infection in humans most often presents with diarrhea, which in immunocompromised patients is profuse and cholera-like. Human cryptosporidiosis has been identified by the World Health Organization (WHO) as an acquired immunodeficiency syndrome (AIDS) indicator [3][4][5][6] . Vulnerable groups are also premature babies, children in nursery homes, travelers to tropical countries, livestock breeders, veterinarians, zootechnicians, workers in the meat industry.
Over the years, epidemic outbreaks of cryptosporidiosis related to drinking water 7-9 , recreational waters and swimming pools [10][11][12][13] , contaminated food consumption, animal contact and outdoor activities 14,15 have been reported from various countries all over the world. Notwithstanding that to date epidemic outbreaks of cryptosporidiosis associated with different types of water sources have not been registered in Bulgaria, a 2007 study found that out of 135 samples from different water sources, 33 (24%) were contaminated with Cryptosporidium oocysts, with the largest number of positive samples (n = 21) from rivers (n = 21 out of 42 samples), followed by sewage water (n = 6 out of 12 samples), springs (n = 5 out of 18 samples) and water pumps (n = 1 out of 3 samples) 16 .
The average incidence of cryptosporidiosis in the European Union (EU) countries for the period 2015-2017 was 3.37 per 100 000 population. Among them, countries with the highest morbidity were the United Kingdom, Ireland, Germany, the Netherlands. Children aged 0-4 years were most affected (incidence of 12.5 cases per 100 000 population in 2017) 17 .
Mots-clés: cryptosporidiose, infection parasitaire associée au SIDA, méthode de diagnostic microscopique and 7 immunocompromised. The average annual incidence of the disease for the studied period was 0.03 per 100 000 population, and the level of this indicator is similar in some European Union countries such as Poland, Czech Republic, Latvia, Norway. The data from our study showed that the most affected were the age groups from 0 to 4 years and the female patients, and that the disease prevails in the summer months of the year. Conclusions. In Bulgaria, human cryptosporidiosis has a low incidence rate, but in our opinion, this is mostly due to the fact that, currently, only a small number of laboratories perform tests, that hinder the determination of the actual prevalence among the population.
Keywords: cryptosporidiosis, AIDS-associated parasitic infection, microscopic diagnostic method. In Bulgaria, more systematic research on cryptosporidiosis began in the 1990s. The disease has been diagnosed among people returning from tropical countries, children from rural areas, livestock breeders 18,19 . Familial cryptosporidiosis among immunocompetent individuals (husband and wife) as a result of consumption of fresh goat milk during a trip to Greece was described in 2014 20 . In a microscopic examination of samples from HIV-infected individuals and AIDS patients over a 15-year period (1986-2000), Cryptosporidium oocysts were found in 6 (5.26%) of 114 subjects. In one patient with AIDS, both intestinal and pulmonary cryptosporidiosis was found 21 . In a more recent study by Yancheva et al three cases of cryptosporidiosis were diagnosed among 33 patients with HIV infection suffering from watery diarrhea and malnutrition 22 .
THE OBJECTIVE OF THE STUDY was to conduct a retrospective analysis of the prevalence, the incidence and some demographic characteristics of cryptosporidiosis in Bulgaria over a 10-year period (2009-2018).

Study design
The study was conducted retrospectively, from data obtained for clinical purposes and epidemiological investigations in persons with diarrheal syndrome, with or without compromised immunity, and in accordance with the Helsinki Accords and the requirements of Bulgarian legislation.

Data collection
In Bulgaria, cryptosporidiosis is a subject to mandatory notification and registration, according to Ordinance No 21/2005 on the procedure for registration, notification and reporting of communicable diseases of Ministry of Health. The annual analyses of parasitic diseases in the country, prepared by the Department of Parasitology and Tropical Medicine at the National Centre of Infectious and Parasitic Diseases (NCIPD), Sofia (Bulgaria) were used to conduct the retrospective study, for the period between January 2009 and December 2018. Also, data from the annual reports of the registered cases of parasitic diseases of the Regional Health Inspectorates (28 in number), and data of the National Centre for Public Health and Analyses for the diseases, subject to obligatory reporting and registration in the country, were used. Data from the European Union's annual summary reports on trends and sources of zoonotic diseases, zoonotic agents and foodborne outbreaks for the specified period were also used.

Groups of patients
Immunocompetent persons: in the study, there were examined fecal samples of 2618 people from different age groups (from 0 to 69 years) with diarrhea and other symptoms of the gastrointestinal tract.
Immunocompromised patients: the results include studies of 88 individuals (28 to 67 years) with HIV infection and diarrheal syndrome from the AIDS ward of the Specialized Hospital for Infectious and Parasitic Diseases in Sofia, Bulgaria. The samples were tested in the National Reference Laboratory "Diagnosis of Parasitic Diseases" in the Department of "Parasitology and Tropical Medicine" at the National Centre of Infectious and Parasitic Diseases (NCIPD). All patients included in the study were grouped by age and sex.

Methods for parasitological diagnosis
Microscopy: modified Ziehl-Neelsen (MZN) method, for presence of coccidia oocysts: a) dried fecal smears (fresh and preserved) were fixed for 3 minutes with 96º methanol; b) stained with cold concentrated carbol fuchsin for 10 minutes; c) rinsed with tap water; d) differentiated with 3% acid-alcohol (3% HCl in 96º ethyl alcohol); e) rinsed for 30 sec with 0.25% malachite green; f) washed, rinsed and dried; g) microscopy was performed at 400x magnification and at 1000x with immersion. On a green background, the red or pink Cryptosporidium oocysts containing dark brown or dark gray intra-cystic bodies may be seen selectively 23,24 .
Immunochromatographic test: RIDA®QUICK C r y p t o s p o r i d i u m / G i a r d i a C o m b i Immunochromatographic Test (ICT) -the test was carried out according to the manufacturer's instructions.

Microscopic analysis
The study included a total of 2706 individuals (2618 immunocompetent and 88 infected with HIV) with diarrhea and/or other gastrointestinal complaints and with suspected Cryptosporidium spp. infection. Of these, 2658 individuals were examined by microscopic methods alone, 31 individuals were examined by immunochromatographic test alone, and a combination of the two tests was used in 17 subjects. Microscopic examination of fecal smears stained by MZN method revealed 22 positive samples (0.81%) with presence of Cryptosporidium oocysts. Of the surveyed individuals, 15 (68%) of the immunocompetent persons and 7 (32%) of the HIV-infected patients were positive. Patients with laboratory-confirmed cryptosporidiosis were tested three times and/or until negative results were obtained after treatment in HIV-infected persons (Azithromycin 500 mg/day for 14 days, with more severe immunosuppression for up to 21 days, in combination with antiretroviral therapy). In one of the patients with compromised immunity, a negative result was obtained after treatment and 5-fold study for a month.

Immunochromatographic Test (ICT)
A rapid immunochromatographic test for the qualitative determination of Cryptoporidium parvum/or Giardia duodenalis in stool samples (RIDA®QUICK Cryptosporidium/Giardia Combi Immunochromatographic Test (ICT), R-Biopharm, Darmstadt, Germany) was applied. The test is used mainly in the National Reference Laboratory "Diagnosis of Parasitic Diseases" and for the period of the study 48 of all patients were tested, of whom 43 were immunocompetent and 5 HIV-infected. A positive result was found in 15 (71.43%) of them from a total of 22 persons diagnosed by MZN method. In two of the cases, in whom a combination of the two methods was applied, ICT was negative. Both cases were HIV-infected patients.
For the period of study, the highest number of immunocompetent and immunocompromised indi-  (Table 1).
The largest number of positive for cryptosporidiosis for the study period was found in Varna district in 2016 and 2017 -8 cases among children with diarrheal syndrome aged 0 to 9 years. In 2012, in Burgas district there were diagnosed 4 cases among women with diarrheal syndrome, aged 25-29 years. The remaining 10 cases diagnosed with cryptosporidiosis  were reported by Regional Health Inspectorates of Sofia-city, one case annually: Despite the significant number of tested subjects in Pernik district for the years of the study no positive cases of cryptosporidiosis were found.

Seasonality
The seasonal distribution of confirmed (n = 22) cases of cryptosporidiosis for the study period was as follows: in January, February, March, April, October one case was diagnosed, 4 cases in June, July and August, 5 cases in September, while in May, November and December no case of infection with Cryptosporidium sp. was found (Fig. 3). These data outline the prevailing summer seasonality of the prevalence of cases of cryptosporidiosis in Bulgaria.

DISCUSSION
Cryptosporidium is one of the major pathogens causing diarrhea in immunocompromised individuals and children 25 . The timely and accurate diagnosis and subsequent etiological treatment are crucial for a positive outcome of the disease. In Europe, as elsewhere, the severity of the disease is difficult to measure due to the lack of appropriate, standardized surveillance and control systems 26 . According to the Bulgarian legislation, which is harmonized with the Directives of the European Union, cryptosporidiosis in humans is a subject to mandatory registration and notification. In this regard, we believe that the data from our study show the real state of the problem with some stipulations. Due to the need for well-trained staff to perform microscopic diagnosis, targeted tests for cryptosporidiosis are performed in a few laboratories and cases of the disease may remain undiagnosed.
For the studied period, only sporadic cases of the disease were recorded in the country, with no data on epidemic outbreaks related to water or food.
The annual incidence rates range from 0. Bulgaria cases seem negligible (0.02% of cases). Nevertheless, cases of the disease are registered almost annually, although exception of one case of familial cryptosporidiosis no epidemiological link has been established between them, making it difficult to determine the source of infection.
Of the registered persons with cryptosporidiosis in Bulgaria, 32% (n = 7) were HIV-infected and 68% (n = 15) in patients with diarrhea syndrome, without evidence of compromised immunity. The prevalence of cryptosporidiosis among HIV-infected individuals was 7.95% and 0.57% in immunocompetent people. In this respect, our data are similar in the literature 29 . The distribution of cases by age shows that in persons without data of compromised immunity, the most affected was the age group of children from 0 to 4 years (n = 7.32%), and these data are identical to the literature 26 , while in HIV-infected persons the cases of co-infection were distributed almost evenly in the age groups from 25 to 69 years and no dominant age group was established.
The distribution by gender shows a significantly greater involvement of females (77%) than males (23%). It is difficult to give any explanation for this fact, but the data presented in Table 1 show that all 8 cases of cryptosporidiosis diagnosed in children and adolescents were in females. The data of Painter et al, from a study in the USA covering the period from 2010 to 2012, are similar 30 .
Cryptosporidiosis among humans in Bulgaria shows marked seasonality, with over 76% of all cases registered in the months of June to September (n = 17). During the rest of the year, except for May, November and December, one case per month was registered.
Regarding the diagnosis of the disease, although limited in volume, the data from our study indicated a relatively good sensitivity and specificity of ICT for antigen detection. In only two cases of HIV-positive individuals the test had a false-negative result. In the remaining over 88% of the cases of cryptosporidiosis there is a complete coincidence of the results of microscopic examination and ICT. Due to their ease of implementation and lack of need for specially trained personnel, they are particularly applicable in routine practice in persons with diarrhea syndrome, but still microscopic detection of the parasite remains the main method for the diagnosis of the disease. Of course, the merits of molecular diagnostic methods should not be ruled out, but they also require special laboratory equipment and trained staff.
The data from our study are similar with those of other studies conducted in Europe and the rest of the world, which show that the epidemiological characteristics of the disease in humans caused by Cryptosporidium spp. do not differ much in terms of territory and climate conditions. Of course, the present study gives us grounds to assume that cryptosporidiosis in Bulgaria has a low endemicity, although there are suspicions of undiagnosed or misdiagnosed cases. This is probably because that the tests for the presence of Cryptosporidium oocysts are performed only in a small number of laboratories in the country, which makes it difficult to determine the actual incidence.

CONCLUSIONS
Our observations over the 10-year study period revealed an insufficient focus of general practitioners and specialists for conducting parasitological tests among people with diarrhea syndrome. In most cases, their focus is on a bacterial and/or viral infection. All these data underline the need to expand and deepen environmental research and targeted, active research of people with gastrointestinal complaints (especially children under 5 years of age, travelers to the tropics, people with compromised immune systems, veterinarians, breeders, etc.), as well as in the event of water epidemics for presence of Cryptosporidium oocysts.