Anna Brylak-Błaszków1, Karol Szeszko1, Monika Dmitrzak-Węglarz1
1GenXone Laboratory for Molecular Diagnostics SA
Keywords: diagnostics, Real-Time PCR, digestive system infections
Digestive tract infections caused by bacteria, viruses and parasites are responsible for significant pathogenicity and mortality in all age groups. Both adults and children, staying in large groups, travelling around the world, staying in hotels with a low level of cleanliness, biting nails, people with immunological deficiencies and not following proper hand hygiene are at risk of contracting the infection [1]. Dirty hands are an excellent means of transport for microorganisms and parasite eggs that can survive outside the host’s body for up to several days. Factors contributing to infection include: staying in public places, eating unwashed fruit, vegetables, raw fish, and pork meat, drinking unboiled water, and swimming in natural water reservoirs. Digestive system infections are manifested by several symptoms such as diarrhoea, abdominal pain, vomiting, and fever. In the case of parasitic infections, the symptoms may be less severe and of a chronic nature, which is why patients often ignore them and do not associate them with infection. In most cases, the symptoms of a digestive system infection pass spontaneously after a few to several hours, without any significant impact on the patient’s health. Sometimes, however, a harmless infection leaves behind long-term and serious health complications and even poses a direct threat to life. Dehydration is the most common consequence of acute diarrhoea and vomiting. Failure to replenish fluids, with increased fluid loss, may cause anuria which is directly related to kidney dysfunction and poisoning. Moreover, long-term dehydration of the body affects the cells of the nervous system and the functioning of the brain. In extreme cases, a high degree of dehydration leads to the death of the patient. Some strains of bacteria that cause food poisoning can cause so-called haemorrhagic enteritis and toxic distension of the large intestine. These pathological conditions may cause intestinal obstruction, leading to their necrosis and, consequently, to excision of the affected part of the intestine. Bacterial infections, especially in children, can also cause the so-called hemolytic uremic syndrome. It is a multi-organ disease that can lead to acute renal failure. Campylobacter bacteria are one of the most common causes of bacterial diarrhoea and can cause a neurological disease – Guillain-Barre syndrome. In most patients, the syndrome resolves without leaving any permanent consequences, but in some patients, some neurological symptoms remain, and in rare cases, death is due to respiratory failure. Quick and precise identification of gastrointestinal pathogens is necessary for the implementation of targeted and effective causal treatment and appropriate epidemiological control to limit or prevent the spread of pathogens, often responsible for serious health complications, and in extreme cases even for the threat to the patient’s life.
Possibilities and diagnostic problems of digestive system infections
The clinical symptoms of gastrointestinal infections are often similar, therefore diagnostic tests play an important role in identifying the infection factor. Laboratory diagnostics offers both conventional techniques such as microscopic examinations, breeding and serological methods as well as modern molecular techniques. The range of laboratory methods available often has limitations due to the sensitivity of the method, specificity, execution time and cost.
Breeding methods are still the gold standard in the diagnosis of bacterial pathogens. The media used enable the growth of various bacteria and only allow for their initial differentiation. The incubation time for most bacteria is 18-24 hours, for anaerobic bacteria and fungi 48 hours. Further identification of the obtained colonies is carried out based on biochemical activity, the ability to metabolize various substances, e.g. carbohydrates or the production of specific enzymes, e.g. oxidase. Classical microbiological methods often do not allow unambiguous identification of bacterial strains due to common biochemical features. In addition, this method is time-consuming, which is its greatest limitation in the situation of immediate treatment necessity. For the identification of pathogenic bacteria, immunological methods are also used, using the reaction of bacterial antigens with specific antibodies and mass spectrometry-based on the analysis of ribosomal proteins. Despite obtaining reliable results, mass spectrometry is characterized by a very high cost of testing and the inability to determine susceptibility to antibiotics [3,4].
There is still no gold standard in the diagnosis of intestinal parasites, which translates into a large variety of laboratory techniques used. Macroscopic, microscopic and immunological methods are used, allowing the detection of specific antibodies in the serum (IgG, IgM, IgA, IgE) or testing for the presence of parasite antigens. The most frequently used biological material for the identification of gastrointestinal parasites is feces. In the case of detection of protozoa (Giardia intestinalis, Cryptosporidium spp.) and roundworms (Strongylodes stercoralis), the diagnostic material is also the content of the duodenum. Collecting bile with a probe is an invasive procedure and is associated with the risk of complications [2].
In the case of the identification of viral pathogens in the digestive system, immunochromatographic methods are used to detect virus antigens. Serological methods are used to identify rotavirus, adenovirus and norovirus infections.
The advantage of molecular over biochemical and microscopic research
Molecular methods are invaluable in identifying pathogens in the digestive system and in making a proper diagnosis. They are indispensable when the cultivation of microorganisms is impossible, technically difficult or long-term. Molecular methods allow the detection of the genetic material of microorganisms, which is particularly important in the case of the identification of viruses, slow-growing bacteria, bacterial strains with similar biochemical characteristics, parasites or other atypical microorganisms. The classic PCR reaction allows the duplication of unique DNA fragments characteristic of specific pathogens, i.e. their amplification with the use of the polymerase enzyme. The amplified DNA fragments are made visible with special dyes and then analyzed. The Real-Time PCR (real-time PCR) method, which is a modification of the classic PCR reaction with the use of fluorescent probes, allows for the detection of the amplification product during the reaction. The method makes it possible to assess the number of copies of pathogens, which allows for monitoring the course of the infection and the effectiveness of the patient’s treatment. The time it takes to perform the test is relatively short, usually several hours. Regarding the culture methods, the Real-Time PCR technique shows both higher sensitivity and specificity. When the test sample has a low viral load or the excretion period is short, the viral concentration may drop rapidly. Consequently, the virus may be undetectable by means other than Real-Time PCR. Due to the high sensitivity of Real-Time PCR, the analysis can be performed based on a small amount of collected material. A revolution in laboratory diagnostics is the Real-Time PCR multiplex reaction that identifies genetic material from several to several dozen pathogens during one reaction. Thanks to this, it is possible to detect co-infections (detect a greater number of causative pathogens) and select the appropriate treatment. In the case of classical methods, the detection of one pathogenic factor often leads to the cessation of further diagnosis, which does not necessarily cure the infection.
Purpose of the study: Assessment of the structure of commissioned studies and identified pathogens on the example of own experience
Methodology
The study material consisted of 184 stool samples suspended in 1 mL of eNAT ™ transport medium (Copan). The samples were delivered to the genXone S.A. Molecular Diagnostics Laboratory between 12.2019 and 11.2020. Before the initiation of the test procedure, biological material was stored at 4 °C. All samples were subjected to RNA and DNA isolation based on the column method using GeneAll® RiboSpin™ vRDII reagent kits. The stages of preparation of samples for isolation and its course were carried out according to the manufacturer’s instructions, using the protocol for stool samples. Amplification and detection of sequences specific for viruses causing gastrointestinal infections were performed using a one-step Real-Time RT-PCR multiplex reaction. Sequence detection of selected bacterial and parasitic pathogens was performed using the Real-Time PCR multiplex technique.
Allplex™ Gastrointestinal Panel Assays (Seegene) and Allplex™ GI-Helminth I Assay were used for the amplification step. The composition of the reaction mixture and the temperature profile of the reaction was in line with the manufacturer’s recommendations. Each PCR reaction was performed in parallel for the test samples and the positive and negative controls. The conducted research was retrospective and did not bear the characteristics of an experiment.
1.1. Territorial structure of commissioned research and obtained positive results.
A total of 184 tests were commissioned to detect and identify pathogens responsible for gastrointestinal infections. The orders came from all 16 voivodships in Poland, most of which came from the Mazowieckie (55 orders), Pomorskie (25) and Wielkopolskie (21) voivodships. Positive results were obtained in 41 patients from all over the country. In the case of the division into voivodships, the greatest number was found in patients from Mazowieckie (14 positive results), Wielkopolskie (6) and Lubuskie (3).
The analysis of the structure of commissioned studies in terms of the size of the place of residence of patients showed that most orders came from large and very large cities with a population of, respectively: 100,000 to 500,000 – 52 orders (28% of all orders) and over 500,000. – 46 (25%). The lowest number of orders was recorded from cities with 50,000-100,000. – 11 (6%) and up to 10,000 – 4 (2%). Further analyses narrowed down the division of towns into: large (> 100,000), medium (<100,000) and small (<10,000). Applying the following criteria, 98 (53%) of orders came from patients from large towns, 47 (26%) from medium and 39 (21%) from small ones.
1.2. Structure of the studied patients by sex and age.
The gender distribution of the examined patients was comparable, 93 (51% of all orders) women and 91 (49%) men were examined. In the case of the analysis of the structure of commissioned studies in terms of the age of the patients, it was shown that adults (124 orders) were ordered almost twice as often as compared to children (60).
1.3. The structure of the examined patients according to the ordered tests and detected pathogens.
Among the available 4 panels allowing for detection of the genetic material of pathogens constituting the most common cause of digestive system infections, the most recommended panel was HELMINTY (flatworms, roundworms, microsporidia; GI-H) – 113 orders (61% of all panels ordered). Less popular in terms of the tests ordered were diagnostic panels that simultaneously detected BACTERIA, VIRUSES AND PRIMEROUS (GI-FULL) – 37 orders (20%) and the PRIMARY PANEL (GI-P) of 33 ordered tests (18%). The BACTERIA diagnostic panel (GI-B) was the least frequently ordered – 1 order (Fig. 7).
A total of 184 tests were commissioned to detect and identify pathogens responsible for gastrointestinal infections. In 145 studies, none of the tested pathogens were detected, and in one case the result was non-diagnostic. One pathogen was detected in 33 studies, while in 5 studies, 2 pathogens were detected simultaneously. In total, 9 types of pathogens were identified, including 4 bacteria, 3 protozoa and 2 helminths. The most commonly diagnosed pathogens were protozoa, with a total of 27 positive results. In this group, Blastocystis hominis was identified 14 times, Dientamoeba fragilis – 12 and Giardia lamblia – 1 time. Bacteria were diagnosed in 13 cases in total and they were: Aeromonas spp. and enteropathogenic Escherichia coli with the eaeA genes (5 times), Clostridium difficile strain with the gene coding for toxin B (2 times) and enteroaggregating Escherichia coli with the aggR 1 gene ( 1 time). Helminths were the group of the least diagnosed pathogens. In total, they were identified in 3 cases, including Enterobius vermicularis (2 times) and Enterocytozoon spp./Encephalitozoon spp. (1 time).
Discussion
Diagnostic laboratories performing tests for microbial infections are obliged by the Regulation of the Minister of Health to report the results of tests for biological pathogens in humans (the last ones published in the Journal of Laws of the Republic of Poland on June 26, 2020; item 1118). The list includes a total of 63 pathogens that require measures to be taken by the bodies of the State Sanitary Inspection to protect public health. This list is not exhaustive and is subject to updating. Unfortunately, to make such an update, it is necessary to carry out systematic monitoring of all diagnosed pathogens, in particular those not included in the list. Therefore, the aim of this study is an attempt to assess the frequency and type of pathogens causing gastrointestinal infections in the Polish population. The results obtained by us indicate that patients all over Poland, both men and women, struggle with the symptoms of digestive tract infection throughout their lives. The age of the patients ranged from 1 to 96 years. Two times more studies were commissioned in adults than in children. This is an interesting result because children, especially preschoolers, are much more likely to be at risk of developing gastrointestinal infections. Most of the parents of these children have witnessed symptoms such as abdominal pain, diarrhoea and vomiting at least once in their lives, sometimes with a high fever. These symptoms are most often caused by rotavirus infections, but also by other enterotropic viruses, bacteria and protozoa. Of the bacteria, the most common infections are paradura (Salmonellosis) and dysentery (Shigella). Staphylococci or anaerobic bacteria (after eating contaminated foods, such as ice cream) can cause mass poisoning in children. Loose stools and vomiting can lead to dehydration and loss of electrolytes, which can directly cause life-threatening complications. Meanwhile, the number of ordered tests in children may, on the one hand, indicate a low awareness of parents regarding the diagnosis of causative pathogens resulting from the rapid cessation of symptoms and the lack of the need to implement pharmacological treatment. On the other hand, it seems that the observed high percentage (67%) of referrals by adults may indicate a lack of satisfactory results of treatment in primary care and/or the chronic nature of symptoms. Unfortunately, the lack of access to information on the initial diagnosis and treatment performed so far does not allow for concluding.
Among the available 4 panels of digestive system infections, which allowed the detection of the genetic material of pathogens, the most common (61%) was a panel identifying 8 multicellular endoparasites (flatworms, roundworms) and 1 endoparasite in three samples, Enterobius vermicularis (human pinworm – 2 times) and once Enterocytozoon spp./Encephalitozoon spp. (1 time). Together, they accounted for less than 8% of all detected pathogens. Data from the WHO indicate that about 24% of people in the world are infected with helminths, although their distribution is not even and results from the prevailing sanitary and hygienic conditions. Based on this information, patients’ interest in various types of tests for pathogens causing helminthiasis and the “deworming fashion” has increased. However, it should be acknowledged that the independent use of preparations/drugs to “prophylactically get rid of worms” is unjustified and may be associated with harmful health effects. Any symptoms indicating parasitic diseases should be consulted with a doctor and preceded by appropriate diagnostic tests.
Among the detected pathogens, the most numerous group was the protozoan Blastocystis hominis, which is the cause of a diarrheal disease called blastocystosis (32%). This protozoan lives in the digestive tract of humans and many animals. Human infection occurs via the faecal-oral route (“dirty hands” disease) or the oral route through food or water contaminated with protozoa cysts. The disease is usually mild and self-limiting. The second most frequently detected pathogen is one of the smaller colon parasites, Dientamoeba fragilis (28%), which causes a disease called dientoamebosis. How the infection is transmitted is still unknown. In turn, E. coli sticks accounted for 16% of positive results. In the bulletin, “Infectious diseases and poisonings in Poland in 2019” available on the website of the National Institute of Public Health – National Institute of Hygiene, a total of 586 cases of E. coli infection throughout Poland were recorded. It should be borne in mind that these data mainly concern hospitalized patients, i.e. those with the most severe course of the disease. All statistics elude relatively mild infections, so they are not diagnosed or diagnosed individually/privately. Hence, it seems that we are far from knowing the scale of the contribution of individual pathogens in causing gastrointestinal infections.
Therefore, it seems advisable to keep such statistics from diagnostic laboratories and make them public. However, the presented studies have their limitations resulting from the relatively small number of results, which makes it extremely important to continue the already started observations. It is equally important to emphasize the need to constantly and reliably inform doctors and parents about the possibilities and benefits of diagnosing pathogenic agents with the use of more and more commonly available, modern molecular techniques, such as multiplex Real-Time PCR. Based on the presented results, it can also be concluded that the greatest diagnostic value in the case of infections of the digestive system of unknown origin is offered by commissioning tests in all 4 diagnostic panels at the same time.
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