WIESŁAW PIECHOTA1
RUBELLA SEROPOSITIVITY IN PATIENTS HOSPITALIZED
IN THE MILITARY INSTITUTE OF MEDICINE IN 2017
SEROPOZYTYWNI PACJENCI W KIERUNKU RÓŻYCZKI HOSPITALIZOWANI W WOJSKOWYM INSTYTUCIE
MEDYCZNYM W 2017 ROKU
1 Department of Laboratory Diagnostics, Military Institute of Medicine, Warsaw 2 Sysmex Poland Ltd., Warsaw 3 Department of Epidemiology,
National Institute of Public Health – National Institute of Hygiene, Warsaw
} AGNIESZKA WOŹNIAKKOSEK
Department of Laboratory Diagnostics, Military Institute of Medicine, Warsaw, 128, Szaserów Str., PL-04141 Warsaw, e-mail: awozniak-kosek@wim.mil.pl Received: 12.12.2018 Accepted: 03.01.2019 DOI: dx.doi.org/10.15374/FZ2018060 *according to the order on the list of Authors
ABSTRACT: Rubella is a viral disease caused by a virus of the genus Rubivirus. A person with rubella disease can be the source of the infection a few days before the appearance of a rash. The infection is transferred via droplets, by direct contact with excretions from the nasopharyn-geal cavity of the affected person or through the placenta during pregnancy. Since 1989, vac-cinations against rubella are mandatory in Poland. They were initially performed only among 13-year-old girls. From January to September 2017, 377 cases of suspected rubella were repor-ted. From this group, 112 samples were sent to the Reference Laboratory (29.7%), and the po-sitive result for rubella was obtained only in 6 samples (that is 5.4% of samples sent for the ana-lysis and 1.6% of all cases). The low rate of laboratory-confirmed cases was caused by repor-ting based on clinical symptoms. As a result of this procedure, various rashes were reported as rubella. The aim of the study is to evaluate the presence of class IgG and IgM antibodies in the serum samples of patients hospitalized in the Military Institute of Medicine (MIM) between Ja-nuary and August 2017. Forty two serum anti-rubella antibody tests were performed in the De-partment of Laboratory Diagnostics of MIM. In all cases, the results were positive and indicated the presence of antibodies in the IgG class, while the negative results were noted for IgM an-tibodies. The largest number of orders for testing was received from the Gynecology Depart-ment (23.8%), followed by the Gastroenterology and Cardiology/Cardiac Surgery (11.9%), He-matology (9.5%), and Neurology Departments (14.3%). A limited number of samples (28.5%) was received from the Dermatology, Oncology, Ophthalmology, Laryngology and Paediatrics Departments. The obtained results suggest that hospitalized MIM patients have the immuni-ty that is likely a result of the vaccination. The mean age of hospitalized patients was 30–35 years. Currently, rubella vaccination is mandatory and is performed in the 13–14th month of life
and then in 10-year-old children irrespective of sex. The laboratory sign of a new infection wo-uld be the presence of the specific antibodies in the IgM class, which has not been reported in any case in this study.
KEY WORDS: infections, rubella, serologic testing, viral infections
STRESZCZENIE: Różyczka jest chorobą wirusową wywoływaną przez wirus z rodzaju Rubi-virus, który charakteryzuje się wysoką zakaźnością. Źródłem zakażenia jest chory na różycz-kę człowiek już kilkanaście dni przed pojawieniem się wysypki. Zakażenie przenosi się dro-gą kropelkową poprzez bezpośredni kontakt z wydzielinami z jamy nosowo-gardłowej oso-by chorej. Innym przykładem jest zakażenie wewnątrzpłodowe – w przypadku kiedy kobie-ta w ciąży zakazi się wirusem różyczki. Sytuacja kobie-taka niesie za sobą szereg komplikacji dla pło-du. W Polsce od 1989 roku prowadzone są szczepienia obowiązkowe przeciw różyczce, począt-kowo tylko wśród 13-letnich dziewcząt. W 2017 roku, od stycznia do połowy września, zgło-szono 377 przypadków/podejrzeń różyczki, z czego do laboratorium pełniącego funkcję refe-rencyjnego wpłynęło 112 próbek (tj. 29,7%), a wynik pozytywny w kierunku różyczki uzyska-no jedynie w przypadku 6 próbek, tj. 5,4% w stosunku do przesłanych do analizy próbek i 1,6%
w stosunku do wszystkich przypadków. Przyczyną niskiego odsetka potwierdzeń laboratoryj-nych różyczki jest zgłaszanie przypadków na podstawie rozpoznania klinicznego. W rezulta-cie takiego postępowania jako różyczka zgłaszane są różne choroby wysypkowe. Celem pracy jest ocena występowania przeciwciał w klasie IgG i IgM w surowicy pacjentów hospitalizowa-nych w Wojskowym Instytucie Medycznym (WIM) w okresie od stycznia do sierpnia 2017 roku, u których wykonano badania laboratoryjne w tym kierunku. W Zakładzie Diagnostyki Labora-toryjnej WIM przeprowadzono 42 badania surowicy na obecność przeciwciał przeciwko wiru-sowi różyczki. Nie wykonywano testu awidności przeciwciał IgG. We wszystkich przypadkach wynik analiz był pozytywny i wskazywał na obecność przeciwciał w klasie IgG, natomiast ne-gatywny wynik był notowany dla oznaczenia przeciwciał w klasie IgM. Najwięcej zleceń od-notowano z Oddziału Ginekologicznego (23,8%), następnie z: Oddziału Gastroenterologiczne-go i KardiologiczneGastroenterologiczne-go/KardiochirurgiczneGastroenterologiczne-go (po 11,9%), HematologiczneGastroenterologiczne-go (9,5%), Neurolo-gicznego/Neurochirurgicznego (14,3%) oraz pojedyncze przypadki z dermatologii, onkologii, okulistyki i laryngologii oraz pediatrii, co w sumie stanowiło 11,9%. Na podstawie uzyskanych wyników można wnioskować, że hospitalizowani pacjenci WIM, u których wykonano bada-nia przy użyciu platformy immunochemicznej, posiadają odporność nabytą prawdopodobnie w wyniku wykonanych szczepień. Średni wiek hospitalizowanych mieścił się w zakresie 30–35 lat. Obecnie szczepienie przeciwko różyczce należy do szczepień obowiązkowych i jest wyko-nywane w 13.–14. miesiącu życia, a następnie w 10. roku życia bez względu na płeć. Wykład-nikiem świeżego zakażenia byłaby obecność przeciwciał w klasie IgM, której nie odnotowano.
SŁOWA KLUCZOWE: diagnostyka serologiczna, infekcje wirusowe, różyczka, zakażenie
INTRODUCTION
Viral and bacterial infections present a serious problem for in-patient care. Viral diseases, including infections with the rubella virus, can seriously affect patients’ health, espe-cially that of a pregnant women’s, and pose a threat to the fe-tus. A diagnostic laboratory rule in such cases is to use ap-propriate analytical methods confirming or ruling out the occurrence of infection. In rubella virus diagnostics, sero-logical tests are of crucial importance. The presence of IgM antibodies directed against the rubella virus in the patient’s blood can be detected several days after the appearance of the rash. An additional confirmation of the presence of the virus in the material can be achieved through PCR (poly-merase chain reaction) determining the presence of rubella virus RNA in the tested material or the confirmation of the presence of the virus in the tissue culture cell lines particu-larly dedicated for this purpose.
However, the key role of prophylactics in this aspect sho-uld always be remembered. The advancement taking pla-ce in medicine, the introdupla-ced recommendations, vaccina-tions, new diagnostic and therapeutic methods make it po-ssible to significantly reduce the risk of the occurrence of many disease processes, including those caused by viral in-fections.
Rubella is an infectious disease, typical especially of chil-dhood. The etiological factor of the disease is RNA virus of the genus Rubivirus belonging to the Togaviridae family.
Only one serotype of the rubella virus is known, and hu-mans are its only reservoir. The infection is transferred via droplets, by direct contact with excretions from the naso-pharyngeal cavity of the affected person or through the pla-centa during pregnancy. The incubation period is 18 days on average. Rubella is infectious 7 days prior and two we-eks after the appearance of a rash. Initially, the virus attacks the respiratory system, then it is transmitted to lymph no-des, which in turn leads to the infection of other tissues and the appearance of a mild rash [13].
In Poland, reporting rubella has been obligatory since 1966, and since 1997 it has also been required to report CRS (congenital rubella syndrome), which is a challenge to pu-blic health. Taking care of patients with rubella poses a se-rious problem due to possible complications such as deaf-ness (60%), heart disorders such as pulmonary artery ste-nosis, ventricular septal defect and patent ductus arterio-sus (45%) as well as abnormalities of the eye (25%). Pro-phylactic vaccination is the best method of prevention of rubella. Rubella infections, apart from isolated communi-ties, is observed all over the world. In countries where vac-cinations started in the 1970s the incidence of rubella drop-ped by even 99% in comparison to the time period befo-re vaccinations [11]. In 1989, a prophylactic program has been introduced in Poland, which involves vaccinating all girls, who have not reached their reproductive age, against rubella [2]. The most important argument in favour of in-troducing such vaccinations was the elimination of rubella
infections in pregnant women, and consequently preven-ting the occurrence of congenital rubella. In Poland, there are combination vaccines available, which contain attenu-ated strains of measles, mumps and rubella viruses. Since November 2003, obligatory vaccination of 13/14-month-old children has been included in the Prophylactic Vaccinations Program. In Central and Eastern Europe, such vaccinations are also obligatory at the age of 2. Since 2006, an obligato-ry second dose for 10-year-old children has been included in the Prophylactic Vaccination Schedule in Poland [7]. In the vaccination calendar for 2019 the time of administration of the second dose of rubella vaccine from the age of 10 to 6 years of age was changed. This is important to ensure that children are protected before they start school. A booster vaccination against measles, mumps and rubella will be car-ried out twice at the childrens 6 years and the age of 10. This will result in no need for compensatory vaccinations in 7, 8 and 9 years of age [16].
The aim of this study is to evaluate the presence of an-ti-rubella antibodies of the IgG and IgM class in the serum of patients hospitalized in MIM. The analysis was based on laboratory tests performed in the Department of Laboratory Diagnostics of Military Institute of Medicine (MIM) betwe-en January and August 2017.
MATERIAL AND METHODS
Forty two samples of serum obtained from patients ho-spitalized in MIM were used for testing. The patients had no symptoms typical of rubella which could strongly suggest infection, and thus for them the tests mainly served the pur-pose of excluding the possibility of the occurrence of rubella as a disease entity. Among 42 patients, 19% (8 subjects) were men with a mean age of 39 and 81% (34 subjects) were wo-men with a mean age of 33 (Table 1).
Blood samples were taken from the ulnar vein into stan-dard test-tubes containing separating gel. After centrifuga-tion, the serum was analyzed with the automatic immuno-chemistry Cobas® e601 analyzer with a electrochemilumi-nescence method. Then the diagnostic material was kept at the temperature of 4 centigrade above zero so that the sts could potentially be repeated within 24 hours. In the te-sted material, the level of specific IgM antibodies was deter-mined using qualitative tests (Elecsys® Rubella IgM) and the level of IgG antibodies using quantitative tests (Elecsys® Ru-bella IgG). The level of IgM anti-ruRu-bella virus immunoglo-bulins was expressed in the form of the cut-off index (COI). The level of IgM antibodies higher than or equal to 1.0 COI was treated as a positive result and the level below 0.8
№ Clinical department % of the overall num-ber of rubella tests performed
Clinical diagnosis
1 Urology 2.38 Chronic kidney disease (peritoneal dialysis),
undetermined erythematous rashes, allergy
2 Infectious Diseases 4.76 General exanthema induced by drugs or other
medicinal products
3 Emergency Room 9.46 Examination and observation due to the
re-asons related to a high-risk pregnancy
4 Dermatology 2.38 Undetermined erythematous rashes, allergy
5 Oncology 2.38 Pigmented nevi, benign mammary dysplasia,
neoplasms of an uncertain or unknown cha-racter and of different and undetermined lo-cations
6 Laryngology 4.76 Embedded and impacted teeth, oral cavity
cancer of an uncertain or unknown character, paranasal sinusitis
7 Paediatrics 2.38 Enlargement of liver, not classified elsewhere
8 Hematology 9.5 Hematopoietic disorders
9 Neurology/Neuro-surgery
14.4 Other unclassified disorders of the nervous
sys-tem, idiopathic and hereditary neuropathy 10
Cardiology/Cardio-surgery
11.9 Idiopathic undefined acute pericarditis, heart
rhythm disorder, Dressler syndrome, bacterial pneumonia
11 Gastroenterology 11.9 Other intestinal diseases, pain in the
abdomi-nal and pelvic region, enlargement of liver and spleen, not classified elsewhere, multiorgan sarcoidosis, acute pancreatitis, gastrointestinal cancer of an uncertain or unknown character
12 Gynecology 23.8 Androgen excess (hyperandrogenism),
obste-tric care because of pregnancy-related
condi-Table 1. The percentage of orders for rubella te-sts from individual clinical departments of MIM along with clinical diagnosis.
COI as a negative result. In case of specific anti-rubella IgG antibodies, the concentration was expressed in international units per milliliter (IU/mL). The level of IgG antibodies hi-gher than or equal to 10 IU/mL was a positive result and the level lower than 10 IU/mL was considered to be a negative result. The tests were performed according to the manufac-turer’s recommendations.
MEASURING RUBELLA VIRUS IGG ANTIBODY LEVELS
10 μL serum samples were incubated with biotinyla-ted monoclonal antibody against human IgG, rubella vi-rus-like particles RLP and monoclonal fragments of ruthe-nium-labeled antibodies. A biotinylated, recombinant anti-gen E1 specific for rubella virus and ruthenium-labeled E1 were also added. In the resulting mixture, due to a speci-fic antibody binding, a sandwich complex was formed (anti-gen-antibody). In the other incubation, streptavidin-coated particles were added. The reaction mixture was then trans-ferred into the measuring chamber of the analyzer, where the particles were attracted to the surface of the electrode with a magnet. Next, the excess of the unbound substances was removed using a special washing solution. The voltage applied to the electrode-induced photon emission and elec-trochemiluminescence reaction, which was measured with the use of a photomultiplier. The obtained results were read from the calibration curve prepared for the particular ana-lyzer on the basis of a 2-point calibration and the reference curve included in the barcode of the reagent.
MEASURING RUBELLA VIRUS IGM ANTIBODY LEVELS
ΜCAPTURE
10 μL serum samples after automatic dilution 1:20 with an appropriate solvent were incubated with biotinylated monoc-lonal antibody against specific IgM, recombinant antigen spe-cific for rubella in order to create a complex. In the other in-cubation, ruthenium-labeled antibodies specific for rubella and streptavidin-coated particles were added. A complex was created which bound with the solid phase owing to the affi-nity between biotin and streptavidin. Reaction mixture was then transferred into the measuring chamber of the analyzer, where the particles were attracted to the surface of the elec-trode with a magnet. Next, the excess of the unbound sub-stances was removed using a special washing solution. The
voltage applied to the electrode induced photon emission and electrochemiluminescence reaction, which was measured with the use of a photomultiplier. The obtained results were read from the calibration curve prepared for the particular analyzer on the basis of a 2-point calibration and the referen-ce curve included in the bar code of the reagent.
RESULTS
THE RESULTS OF SEROLOGIC TESTS FOR THE
PRESENCE OF ANTIRUBELLA VIRUS ANTIBODIES
The tests showed the presence of rubella virus antibodies in the IgG class in all tested samples and a lack of antibodies in the IgM class. The level of rubella virus IgG antibodies de-termined in 42 serum samples taken from patients was ≥10 IU/mL, which indicates a positive result and an interpreta-tion of the analyses results as reactive. The level of IgM an-tibodies determined in the same 42 samples was <0.8 COI, which indicates a negative and non-reactive IgM result.
The obtained results may suggest that patients hospita-lized in MIM, whose blood was tested for rubella using the immunochemistry platform, belong to a group which was earlier exposed to the rubella virus via infection or prophy-lactic vaccination. However, it is more likely that they were previously immunized by vaccination. In 2017, from Janu-ary to the middle of September, there were 377 potential ca-ses of rubella reported in whole Poland, from which the Re-ference Laboratory in National Institute of Public Health (NIPH-NIH) received 122 samples, i.e. 29.7% [7]. A positi-ve rubella result was obtained only in 6 samples, i.e. 5.4% of the samples sent for analysis and 1.6% of all reported cases. The low rate of laboratory confirmed cases is caused by re-porting based on clinical diagnosis. As a result of this pro-cedure, various rashes are reported as a symptom of rubel-la. In the Department of Laboratory Diagnostics of MIM, 42 serum samples were tested. The table includes a list of clini-cal departments which ordered tests with the corresponding clinical diagnosis. The highest number of orders was recor-ded from the Cardiology/Cardiac Surgery Department and only a few cases were received from, for instance, Dermato-logy, OncoDermato-logy, OphthalmoDermato-logy, LaryngoDermato-logy, and Paedia-trics Departments.
in Poland ge by voivodeship sessment
Rubella/measles/ mumps
96.7% 94.2–99.00% Coverage of the third
year of life
Rubella 99.6% 99.3–100.0% Girls in the 15th year
of life
DISCUSSION
Patients once infected with rubella have IgG antibodies flowing in their blood till the end of their lives. Infection in people who suffered from or were vaccinated against rubella is confirmed on the basis of an increase of the concentration of IgG antibodies and a lack of antibodies in the IgM class. In patients with a fresh infection, there are IgM and IgA an-tibodies present. It is possible to determine the time of in-fection by assessing rubella specific IgG avidity. The asses-sment of intrauterine infection is possible thanks to the use of the reverse transcription polymerase chain reaction (RT- -PCR) in order to evaluate amniotic fluid [6, 14]; however, these methods are not the subject of this study.
The risk that a perinatal rubella infection carries is the reason why the World Health Organization (WHO) intro-duced programs which purpose is to eliminate infections through stopping virus transmission and preventing cases of congenital rubella syndrome. The incidence of rubella in recent years has remained at the level of 4–6 thousand cases per year, which is much higher than the EU avera-ge [15]. In 2013, there was a six-fold increase in the num-ber of rubella cases, which was regarded as a compensato-ry epidemic [1]. The observed epidemic increase in the in-cidence of rubella mostly affected young males aged 15–19 and 20–24 who were born in the years when MMR (Me-asles, Mumps, Rubella) vaccinations were not obligatory. After the period of compensatory epidemic in 2015, the-re has been a drop in the number of rubella cases by 2017 with the incidence at the level of 5.3 per 100 000 people, which was almost three times lower than in 2014 [12, 15]. Presently, the epidemiological situation in Poland with re-gard to the incidence of rubella is favourable. Vaccination coverage in Poland (for the year 2015) is presented in the Table 2.
In Poland, for the last 14 years both girls and boys have undergone preventive vaccination. In 2017, from Janu-ary to the middle of September, there have been 377 po-tential cases of rubella reported out of which only 6 cases (i.e. 1.6%) were laboratory confirmed as positive. Looking back at the earlier epidemiological data for instance from the year 2014, it can be seen that in that period there were 5891 cases of rubella reported, which was a drop in inci-dence by 85% in comparison to the year 2013. Such a decli-ning trend is observed in all voivodeships and, what is im-portant, no cases of congenital rubella syndrome were no-ted [9]. The further decline of rubella cases in Poland has occurred recently: between October 2017 and September 2018 there were 472 cases mostly in children (only 3 labo-ratory-confirmed) [3]. The results we obtained confirm the epidemiological data presented above because IgG antibo-dies signifying acquired immunity were found in 100% of
females tested in MIM. Similar results in women were ob-tained by Pasternak et al. [10]. It shows that vaccinations have been successful in preventing rubella. Moreover, at-tention should be drawn to the fact that vaccinations not only among females, but also among males play an impor-tant role in preventing rubella virus infections as they re-strict the circulation of the virus in population reducing the risk of transfer onto pregnant women [5, 8]. Nevertheless, in Poland there are still subpopulations susceptible to infec-tions due to avoiding vaccinainfec-tions. These are various eth-nic groups and children of the supporters of anti-vaccina-tion movements [4].
CONCLUSIONS
1. All studied patients had anti-rubella IgG antibodies le-vels securing their immunity against infection with rubella virus.
2. None of the patients was positive for anti-rubella IgM antibodies which ruled out current rubella infection. 3. The most probable cause of anti-rubella IgG
positivi-ty is vaccination.
CONFLICT OF INTEREST: All Authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.
REFERENCES
1. Abramczuk E, Częścik A, Pancer K, Gut W. Problem różyczki w Polsce po epide-mii wyrównawczej w 2013 roku. Przegl Epidemiol 2016;70(4):549–554. 2. Dulny G. Szczepionka przeciwko różyczce. In: Magdzik W, Naruszewicz-Lesiuk D,
Zieliński A (eds). Wakcynologia. α-medica Press, Bielsko-Biała, 2007, pp. 440–447. 3. European Centre for Disease Prevention and Control. Monthly measles and ru-bella monitoring report. November 2018. ECDC (online) 2018; https://ecdc. europa.eu/en/publications-data/monthly-measles-and-rubella-monitoring-report-november-2018
4. Gut W. Program eliminacji odry i różyczki w Europie a sytuacja w Polsce. Me-dycyna Praktyczna (online) 2012; https://www.mp.pl/szczepienia/aktua-lnosci/70970,program-eliminacji-odry-i-rozyczki-w-europie-a-sytuacja-w-polsce 5. Kelly H, Worth L, Karapanagiotidis T et al. Interruption of rubella virus transmis-sion in Australia may require vaccination of adult males: evidence from a Vic-torian sero-survey. Commun Dis Intell 2004;28(1):69–73.
6. Macé M, Cointe D, Six C et al. Diagnostic value of reverse transcription-PCR of amniotic fluid for prenatal diagnosis of congenital rubella infection in pre-gnant women with confirmed primary rubella infection. J Clin Microbiol 2004;42(10):4818–4820.
7. National Institute of Public Health – National Institute of Hygiene (NIPH – NIH), Chief Sanitary Inspectorate. Dwutygodniowe meldunki o zachorowaniach na choroby zakaźne, zakażeniach i zatruciach w Polsce. NIPH – NIH (online) 2013; http://www.pzh.gov.pl/oldpage/epimeld/2013/index_mp.html
8. Paradowska-Stankiewicz I, Czarkowski MP, Derrough T, Stefanoff P. Ongoing outbreak of rubella among young male adults in Poland: increased risk of con-genital rubella infections. Euro Surveill 2013;18(21).
9. Paradowska-Stankiewicz I, Rogalska J, Polkowska A. Rubella in Poland in 2014. Przegl Epidemiol 2016;70(3):341–348.
10. Pasternak J, Rajtar B, Rybacka E et al. Ocena częstości występowania przeciw-ciał przeciw wirusowi cytomegalii i różyczki u kobiet w wieku rozrodczym. Medycyna Ogólna i Nauk o Zdrowiu 2017;23(3):185–188.
news-room/fact-sheets/detail/rubella
12. Sadkowska-Todys M, Zieliński A, Czarkowski MP. Infectious diseases in Poland in 2016. Przegl Epidemiol 2018;72(2):129–141.
13. Tesini BL. Rubella. Merck Manual Professional Version (online) 2018; https:// www.merckmanuals.com/home/children-s-health-issues/viral-infections-in-infants-and-children/rubella
and convenient method for determination of rubella virus – specific immuno-globulin G avidity. Clin Vaccine Immunol 2007;14(11):1416–1419.
15. Wojtyniak B, Goryński P. Sytuacja zdrowotna ludności Polski i jej uwarunkowa-nia. NIZP-PZH, Warszawa, 2016.
16. Komunikat Głównego Inspektora Sanitarnego w sprawie Programu Szcze-pień Ochronnych na rok 2019. Dziennik Urzędowy Ministra Zdrowia z dnia 25 października 2018 r., poz. 104.
