Clinical and laboratory predictors of antitoxic immunity against diphtheria and tetanus in adults with HIV infection

Clinical and laboratory predictors of antitoxic immunity against diphtheria and tetanus in adults with HIV infection. Revenko H.O., Mavrutenkov V.V., Chykarenko Z.O. Antiretroviral therapy has made HIV infection a chronic controlled disease, where aspects of the immunoprophylaxis of infectious diseases have acquired important clinical significance. The goal of the study was to determine the clinical and laboratory predictors of antitoxic immunity against diphtheria and tetanus in HIV-infected adults. The study included 90 HIV-infected patients aged 22 to 60 years (main group). The control group consisted of 49 immunocompetent volunteers of the corresponding age. The levels of anti-diphtheria and anti-tetanus antibodies were determined by ELISA using the diagnostic test systems RIDASCREEN Diphtheria IgG and RIDASCREEN Tetanus IgG (R-Biopharm AG, Germany). Statistical processing was performed using the licensed software product STATISTICA v.6.1. Significant differences were found between the titers of antitoxic antibodies in HIV-infected and immunocompetent adults. According to the correlation analysis, the decrease in the titers of antidiphtheria antibodies was revealed with an increase in the age of the HIV-infected patient (rs=-0.21; p=0.05). The fact of smoking (rs=-0.31; p=0.003), lowered body weight (BMI<18.5 kg/m) (rs=-0.29; p=0.006), the presence of arterial hypertension (rs=-0.38; p<0.001), a history of bone fractures over the past 5 years (rs=-0.38; p<0.001), anemia (rs=-0.21; p=0.049), thrombocytopenia (rs=-0.44; p<0.001), accelerated ESR (rs=-0.61; p<0.001), the presence of hairy leukoplakia of the tongue (rs=-0.23; p=0.027), frequent infections caused by herpes simplex (rs=0.52; p=0.003) and varicella zoster virus (rs=-0.34; p=0.013) are associated with low levels of antidiphtheria antibodies. A direct relationship was found between the intensity of anti-diphtheria immunity and patients receiving OST (rs=+0.54; p=0.003) and with a history of injuries with impaired skin integrity (rs=+0.31; p=0.003). Decreased anti-tetanus immunity in HIV-infected patients was also associated with smoking (rs=-0.48; p<0.001), decreased BMI (rs=-0.71; p<0.001), anemia (rs=-0.33; p=0.002), thrombocytopenia (rs=-0.75; p<0.001), a history of bone fractures over the past 5 years (rs=-0.67; p<0.001); the total number of HIV-associated opportunistic diseases (rs=-0.42; p<0.001), including the presence of oropharyngeal candidiasis (rs=-0.23; p=0.032) and hairy leukoplakia of the tongue (rs=-0.57; p<0.001), history of Herpes Zoster in the past (rs=-0.48; p<0.001), with frequent relapses of diseases caused by herpes simplex viruses (rs=-0.78; p<0.001) and repeated episodes of herpes zoster (rs=-0.74; p<0.001), as well as with pathology of the skin (rs=-0.55; p<0.001). Protective predictors of anti-tetanus antitoxic immunity strength were established: male gender (rs=+0.22; p=0.039), parenterally acquired HIV infection (rs=+0.21; p=0.05), HIV-infected patients receiving OST (rs=+0.40; p=0.041). A direct relationship was found with the decreased level of hemoglobin (rs=+0.41; p<0.001), the increase of relative number of lymphocytes in the blood (rs=+0.21; p=0.05), as well as with living in rural areas (rs=+0.40; p<0.001) and the presence of injuries with impairment of skin integrity (rs=+0.84; p<0.001). Clinical and laboratory predictors of strength of antitoxic immunity against diphtheria and tetanus in HIV-infected adults were identified, which allows us to create an individual “vaccination roadmap” for patients in this category. Реферат. Клініко-лабораторні предиктори напруженості антитоксичного імунітету проти дифтерії та правця в дорослих осіб з ВІЛ-інфекцією. Ревенко Г.О., Маврутенков В.В., Чикаренко З.О. Антиретровірусна терапія привела до того, що ВІЛ-інфекція стала хронічною керованою хворобою, де аспекти імунопрофілактики інфекційних захворювань набули важливого клінічного значення. Метою дослідження було визначити клінічні та лабораторні предиктори напруженості антитоксичного імунітету проти дифтерії та правця у ВІЛ-інфікованих дорослих осіб. Обстеження проведено в 90 ВІЛ-інфікованих пацієнтів віком від 22 до 60 років (основна група). Групу контролю становили 49 імунокомпетентних добровольців відповідного віку.

HIV-infected persons are at increased risk of any infectious disease. Risk lowering strategies for the potential development of preventable infectious diseases should regard vaccine prevention as a leading component in providing support and management of all HIV-infected individuals. Reducing the incidence of infections that can be prevented by immunoprophylaxis is one of the priorities for healthcare personnel in the new era of HIV infection [1,7,4,11].
Uncertainty about vaccine safety significantly impedes immunization. However, evidence suggests that inactivated vaccines, namely, diphtheria and tetanus toxoid, have a similar safety profile both among HIV-infected and HIV-uninfected persons [3,6]. Recent studies show that the progression of HIV infection was not observed during the immunization with the above vaccines in ART recipients. Only in a cohort of HIV-infected individuals who did not receive ART, transient decreases in CD4 + T lymphocyte counts and increases in HIV RNA levels can be observed, but these rates normalize within 2-4 weeks after vaccination [3,12]. Carrying out safe, timely and effective immunizations is the most effective method of avoiding diseases that can be prevented in HIV-infected individuals [1,9].
Studies of antitoxic immunity against diphtheria and tetanus in HIV-infected adults are very limited in the world, and have not been conducted in Ukraine, which makes the selected topic relevant.
The purpose of the study was to determine the clinical and laboratory predictors of the intensity of antitoxic immunity against diphtheria and tetanus in HIV-infected adults.

MATERIALS AND METHODS OF RESEARCH
The study involved 90 patients with HIV between the ages of 22 and 60, with an average age of 40. Copying of clinical and laboratory data was carried out from medical records at the aforementioned healthcare institutions.
Laboratory study of the levels of antitoxins in the serum against diphtheria (anti-DT) and tetanus (anti-TT) toxins in the observation group was carried out at the Diagnostic Center of the Dnepropetrovsk Medical Academy of the Ministry of Health of Ukraine. Immunoassay enzyme-linked immunosorbent assay (ELISA) was used to assess RIDASCREEN Diphtheria IgG and RIDASCREEN Tetanus IgG (R-Biopharm AG, Germany) diagnostic test systems. The examination was carried out according to the manufacturer's instructions. The status of diphtheria and tetanus immunity was assessed by determining the concentration of antibodies in IU/ml. The assessment of the intensity of antitoxic immunity was carried out according to the following criteria (table 1). The determination of HIV RNA in the blood was performed by polymerase chain reaction (PCR) with Real-time PCR detection using standardized technology with automated preparation. The quantification of lymphocyte subpopulations in peripheral blood was determined by flow cytometry using monoclonal antibodies.
The control group included 49 healthy immunocompetent volunteers of the relevant age group -mean age -39.0±1.2 years (p=0.44 by t-test). The results of serological monitoring were copied from the materials of the State Institution "Dnipropetrovsk Regional Laboratory Center of the Ministry of Health of Ukraine", conducted on the basis of the Ministry of Health Order No 545 of 24.11.2003 "On the state of immunity of the population of Ukraine to diphtheria and tetanus".
Ethical aspects of the work were approved at the meeting of the Committee on Biomedical Ethics of the SE "Dnipropetrovsk medical academy of Health Ministry of Ukraine" (Protocol No 1 of 20.01.2016).
Statistical processing of the results was performed using the licensed computer program STATISTICA v.6.1 (Statsoft Inc., USA, Serial No. AGAR909E415822FA). Taking into account the law of distribution of quantitative data estimated by the Shapiro-Wilk criterion, parametric and non-parametric characteristics and methods of analysis were used: for the normal law -arithmetic mean (M), standard error (m), Student's t-test (t), Fisher (F); in other cases, the median (Me), the interquartile range (25%-75%), the Mann-Whitney test (U). The relationship between traits was estimated by the Spearman rank correlation coefficient (r s ) using the following criteria to evaluate the link strength: |r s | 0.1 to 0.29 is weak, 0.3 to 0.7 is moderate, and more than 0.7 is strong. The critical level of statistical significance (p) was assumed to be ≤0.05 [10].

RESULTS AND DISCUSSION
The main route of infection in 70.0% of cases (n=63) was sexual, which is generally in concurrence with the current trend, in 30.0% (n=27) the infection occurred parenterally among injecting drug users (IDUs). Among IDUs, methadone or buprenorphine 51.9% (n=14) people were receiving opioid substitution therapy (OST) with methadone or buprenorphine. By clinical stages patients with stage III-IV (according to WHO clinical classification, 2006) -72.2% (n=65) dominated, clinical stages I-II were diagnosed in 27.8% (n=25) of HIVinfected persons. 76.7% (n=69) of those surveyed received ART and 23.3% (n=21) did not receive therapy. ART experience ranged from 1 to 11 years and averaged 2.97±0.24 years. Immediately after the diagnosis of HIV infection, ART was administered to 42.0% (n=29) of patients, 27.6% (n=19) after 1-3 years and 30.4% after 4 years or more (n=21). A high adherence to ART was observed in 69.6% (n=48) of subjects versus 30.4% (n=21) with low adherence.
Analysis of antitoxic immunity indicators revealed that HIV-infected adults did not have sufficient levels of both anti-DT and anti-TT. It was found that the median of anti-DT titers in the main group was 0.17 (0.09-0.38) IU/ml, compared to the corresponding indicator in the control group -1.03 (0.56-1.27) IU/ml (p<0.001 by U-criterion). A similar situation was found with anti-TT titers, namely: the median was 0.59 (0.28-1.09) IU/ml in HIV-infected individuals, in the control group -1.33 (1.13-1.45) IU/ml (p<0.001 by U-criterion), which reflects the low level of seroprevalence to diphtheria and tetanus toxin. Overall, the unprotected layer against diphtheria among the main group was 93.3% (n=84), and against the tetanus -52.2% (n=47).
In more in-depth analysis of immunological and clinical and laboratory parameters that can affect the intensity of antitoxic immunity, the following data were obtained. Thus, according to the correlation analysis, there was established a decrease in the titers of anti-DT with increasing age of the HIVinfected person (r s = -0.21; p=0.05), which is a consequence of immunoscence, ie "depletion" of the immune system caused by age itself and the inability to develop long-term immune memory, especially during vaccination [8]. Similarly, the fact of smoking (r s = -0.31; p=0.003), body weight deficit (BMI<18.5 kg/m 2 ) (r s = -0.29; p=0.006); as well as the presence of such serious somatic pathology as arterial hypertension (r s = -0.38; p<0.001) and the presence of a history of bone fracture over the past 5 years (r s = -0,38; p<0.001) are associated with low titers of antitoxic anti-diphtheria antibodies (Fig. 1).

05) between clinical-laboratory parameters and the intensity of antitoxic anti-diphtheria immunity in HIV-infected adults
Laboratory indicators with low levels of protection against diphtheria include the presence of anemia in patients (r s = -0.21; p=0.049), thrombocytopenia (r s = -0.44; p<0.001), increased ESR (r s = -0.61; p<0.001). Among HIV-related diseases, it should be noted that hairy leukoplakia of the tongue (r s = -0.23; p=0.027), frequent recurrences (2 times a year and more) of infections caused by herpes simplex viruses (r s = -0.52; p=0.003) and varicella zoster virus (r s = -0.34; p=0.013) correlate with low levels of anti-diphtheria antibodies. Conversely, a moderate correlation was obtained between the intensity 20/ Том XXV / 3 of antidiphtheric immunity with taking of OST by the patients (r s = +0.54; p=0.003) and with a history of injuries with impaired skin integrity (r s = +0.31; p=0.003).

05) between clinical laboratory parameters and the intensity of antitoxic immunity in HIV-infected adults
Moreover, in contrast to the correlations of the above indicators that reduce anti-diphtheria immunity (body weight deficiency, thrombocytopenia, frequent relapses of herpetic infection), the tightness of correlation with seroprevalence to tetanus was stronger (|r s |>0.7).
Protective predictors of strength of anti-toxic antitetanus immunity have been established -male (r s = +0.22; p=0.039), parenteral route of HIV infection (r s = +0.21; p=0.05), taking of OST by HIVpositive persons (r s = +0.40; p=0.041). There was a direct relationship with hemoglobin level (r s = +0.41; p<0.001) and relative lymphocyte count in the blood (r s = +0.21; p=0.05). It is also worth pointing out the interesting fact obtained regarding the direct correlation between the intensity of anti-toxic antitetanus immunity and living in the countryside (r s = +0.40; p<0.001), various injuries with impaired skin integrity (r s = +0.84; p<0.001). One of the possible assumptions that explains this immunological paradox is episodes of unintentional infection with minor (booster) doses of tetanus toxin at home, На умовах ліцензії CC BY 4.0 which, in the end, helps maintain the intensity of specific immunity. But in our opinion, such "wild" immunization is unacceptable, because sooner or later it can provoke disease. That is, an immunocompromised macroorganism is able to synthesize humoral antibodies. Also in favor of this is the fact that individuals who received antitetanus toxoid (n=25; 27.8%) in the last five years, due to trauma, have higher antitetanus immunity (r s = +0.52; p<0.001).
The similarity of trends in the rates of antidiphtheria and anti-tetanus immunity in HIV-positive individuals is due to the direct moderate relationship between them -r s = +0.392; p=0.0002, which is explained by the combined content of toxoids in the composition of the vaccine, since, as a rule, the prevention of these diseases is carried out at the same time (Fig. 3).

Fig. 3. Correlation between the indicators of antitoxic immunity against diphtheria and tetanus
It is significant that some discoordination has been established between the main indicative laboratory parameters for HIV infection and the intensity of anti-diphtheria and antitetanus immunity.
2. The consistency of predictors and indicators of antitoxic immunity against diphtheria and tetanus (rs=+0.392; p=0.0002) allows us to extrapolate the results of the studies to each other.
3. It is possible to predict the intensity of antitoxic immunity against diphtheria and tetanus without additional specific studies, which is of economic and practical importance.
4. The identified clinical and laboratory predictors of antitoxic immunity may serve as important indicators for further development of a "roadmap for vaccination" of HIV-infected patients.
The work is a fragment of the scientific research work of the Department of Infectious Diseases of the State Institution "Dnipropetrovsk Medical Academy of the Ministry of Health of Ukraine": "Epigenetic factors of developement of the diseases associated with persistent infections in children and adults", number of state registration 0117U004785.
Conflict of interests. The authors declare no conflict of interest.