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Musabaev Erkin, DSc., in medicine, professor, Head of Scientific Research Institute of Virology Scientific Research Institute of Virology E-mail: drmusabaev@rambler.ru Vafakulov Sadullo, DSc. in medicine, professor, department of infections, epidemiology and dermatovenerology Samarkand State Medical Institute E-mail: sadullo_vafakulov@mail.ru Nargiza Yarmukhamedova, Ph D., in medicine, department of infections, epidemiology and dermatovenerology Samarkand State Medical Institute E-mail: nargiza_ya@inbox.ru Mamatova Rano, Scientific Research Institute of Virology, E-mail: m.ranokhon@gmail.com
THE CHALLENGE OF EMERGING AND RE-EMERGING INFECTIOUS DISEASES IN UZBEKISTAN: STUDY OF RICKETTSIOSIS USING PCR DIAGNOSTIC METHOD
Abstract: In recent years, in many countries around the world, a noticeable increase in the incidence of coxiosis has been registered. In epidemiological terms, coxiosis is a kind of zoonotic infection, reservoirs of the pathogen in nature and, therefore, more than 96 species of wild warm-blooded animals can serve as a source of infection. In recent years, the study of kokieilez began to pay attention in our Republic, this is due to the general noticeable increase in the incidence of coxiosis in the whole world, and the proximity to the endemic zone of coxeella - North Asia. Thus, a number of works were performed on the search for C. Burnetii in the blood of patients hospitalized with acute fever of unclear etiology with the help of ELISA. At the same time, the true number of cases in the region could not be determined, since in many patients the infection is asymptomatic, and the search for C. Burnetii in the main carriers - mites, was also not performed to confirm the presence of endemic zones according to the incidence of fever in Ku. In connection with the above, the frequency of occurrence of C. Burnetii in mites collected in the area of the Samarkand region by means of PCR diagnostics was studied.
Keywords: coxiellosis, rickettsia, zoonotic infection.
Relevance. In the recent years, in many countries around the world, there has been registered a noticeable increase in the morbidity coxiellosis. The true number of cases in the world cannot be established, as in many patients the infection is asymptomatic. We have to state the fact that medical workers, due to lack of awareness, do not show proper vigilance regarding this infection, which leads to diagnostic errors and untimely treatment of patients. Such a development of the disease in people often leads to disability and even lethality [1, 2, 5, 11].
In epidemiological terms, coxiellosis is a kind of zoo-notic infection with the presence of two types of focuses: primarily (natural) and secondary - anthropurgic (usually
agricultural). The pathogenic agent reservoir in nature and, hence, the source of infection can be more than 96 species of wild warm-blooded animals [9, 11,17]. The last ones, being donors of the pathogenic agent for ticks, secrete it to the external environment with urine and feces. The main source and carrier of C. burnetii in nature are mites that are able to feed on infected animals. More than 70 types of bloodsucking ticks (mostly ixodes, less often - gamazoic and argasoic) have been identified. In their organism, the coxiella survive for life, without losing pathogenic features for humans and warm-blooded animals. In terms of the number of tick bacilli involved in the circulation, coxiellosis ranks first place among other transmissible infections. This is the main reason why the
Section 11. Medicine
natural focuses of this disease have the greatest spread of natural focuses among all infections [3, 5, 6, 12, 14]. In the recent years in our Republic began to pay attention, for study of coxi-ellosis, it is associated with a general noticeable increase in the incidence of coxiellosis in the whole world, and proximity to the endemic zone of coxiellosis - North Asia. For example, a number of works were carried out on the search for C. Burnetii in the blood of patients hospitalized with acute fever of unknown etiology in the regional infectious diseases hospital in Samarkand, with the help of immune-enzyme analysis (IEA) Qfever was detected in blood of some of these patients [8]. At the same time, the true number of cases in the region could not be determined, cause of infection in many patients passes asymptomatic, and the search for C. Burnetii in the main carriers - mites to confirm the presence of endemic zones according to the incidence of Qfever was also not performed.
Detection and characterization of specific fragments of DNA amplified in PCR is a quick method of identifying rick-ettsia, even if they are present in relatively small amounts in clinical or field samples. In the presence of several dozen fully sequenced genomes of different species of rickettsia, specialists have almost unlimited possibilities for selecting unique sites for detection of different species of rickettsia. Several sites and primers most commonly used in diagnostic practice were chosen relatively randomly, so they do not represent the most specific or most sensitive combinations [10, 13]. Among these sites, several sections of 17 kDa protein, citrate synthesize and OmpA and OmpB surface proteins, which were among the first genes, are sequenced for rickettsia, and, as it turned out, they have enough unique and common sites to differentiate almost all species of rickettsia, except R. bellii and R. canadensis [10, 12, 13]. The use of conservative primers designed to amplify the 16 rRNA of the eubacterial gene has made it possible to identify new representatives of the genus rickettsia circulating outside the classical ecological systems typical of pathogenic rickettsia, including rickettsia found in leeches, ladybirds and other insects. In the absence of clinical samples, PCR testing of ticks and other ectoparasites collected from the skin or clothes of the patient is a good indicator of potential infection when the pathogenic agent is found in these samples [11]. Also, the presence of rickettsia in mites of a certain range may indicate the contribution of this infection to the development of fevers of the unknown genesis of this region.
Purpose of the research: In connection with the above, we set the aim of the investigation: by using PCR diagnostics to determine the frequency of occurrence of C. Burnetii in mites collected in the area of the Samarkand region.
Materials and methods. The material for the investigation was field material - mites in the amount of 1200 pieces collected in the areas of the Samarkand region (Jambay,
Tailak, Ishtikhan, Narpay, Urgut). These mites, after parsing by species composition, were pooled and prepared suspensions. The mites were ground in chilled mortars, because of the high sensitivity of rickettsia to antibiotics, we didn't use them, instead of it a physiological solution was added to the mortar in the amount of 1-1,5 ml per pool. After centrifug-ing the obtained suspensions; the supernatant fluid was taken for PCR testing. Methods of research. By using "Ample Prime ®RIBO Sorb" from the prepared suspensions was DNA isolated, then was the determination of rickettsia (in particular, C. burnetii) by PCR on a RotorGene apparatus.
Results and Discussion. The inhalation and alimentary method of transferring coxiellosis has a big influence in spreading ofthis infection, but the main source and carrier of C. burnetii in nature are mites that can feed on the infected animals. The results of the study showed that the main types of imago - mites distributed in the Samarkand region were the following: the most common H. Asiaticum, H. Detritum, H. An. Anatolicum, were common in all areas of the region, whereas Argas persicus was found in the Narpay and Urgut regions, and H. Plymbium only in the Jambay, B. Calcaratus in Narpay, and B. Anullatus, R. Turanicu in the Ishtikhan districts.
It should be noted that the highest occurrence of mites species H. An. Anatolicum and H. As. Asiaticum, in this study should be connected in the widespread occurrence of these mites on the entire territory of Central Asia and Uzbekistan in particular. According to the results of this investigation, DNA of C. burneti was detected in 9,1% of samples in mites in the Samarkand region. The results of PCR were positive in the testing of 155 Argas persicus specimens (13 pools), 308 H. Asiaticum specimens (14 pools) collected in the Narpay region. In the Urgut region of the PCR reaction to the detection of coxiellosis was positive only in 96 specimens H. Asiaticum mites. In the Ishtikhan region C. Burnetii was identified in 96 specimens of H. An. Anatolicum mites and in 54 specimens of H. Detritum mites. At the same time, the greatest number of positive pools was detected in H. Asiaticum mites (4 positive from the 6 pools) of the Urgut district and H. An. Anatolicum of Ishtikhan district (out of 17 pools, 6 are positive).
Simultaneously with the study of mites, PCR study was conducted among patients with acute fever admitted to the regional clinical infectious diseases hospital in Samarkand. Thus, from the 56 collected blood serums, the Q-fever was established in 21,4% of cases, while 8,9% had positive results for mite-borne typhus, tsutsugamushi fever was observed in 1,4%. Mixt forms were noted in 3,8% of patients. The geographic area of these diseases is described in the Samarkand region. So the tsutsugamushi fever was registered in 6 districts of the Samarkand region and in one district of the Djizak region. The
material for the study was blood serums from patients, suffer with fever of 5 or more days [8].
The last circumstance is especially significant in our conditions, given that according to the data of the Veterinary Research Institute of the Republic of Uzbekistan, 26 species of ecto- and endoparasites have been identified on the territory of the Samarkand region, among which there are carriers of various transmissible diseases [8].
During the search for a chain of relationships of infection with rickettsial infection, it was revealed that in the vast maj ority
of patients with rickettsiosis were a rural population, and also had close contact with domestic animals (large and small cattle, dogs and cats), and used raw milk products. It is important that the sample of patients with a positive result was predominantly young people aged 19 to 30 (50%), there were also cases of positive response in children 4, 13 and 15 years old.
Conclusions. Thus, the using of new molecular-biological technologies will make possible to identify rickettsiosis and will help increase the effectiveness of epidemiological surveillance of this pathology in the Republic of Uzbekistan.
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