Method of early tuberculosis diagnostics in hiv-infection

FIELD: medicine.

SUBSTANCE: method of early tuberculosis diagnostics in HIV-infection involving immunologic marker monitoring by immune-enzyme assay is used to describe a complex of serum cytokines: tumour necrosis factor alpha, interleukin - 10 and a soluble receptor interleukin - 6 to fix the level, and while observing certain values of immunological markers, tuberculosis is diagnosed in the HIV-infected patients.

EFFECT: method allows higher quality and reduced length of early diagnostics of tuberculosis infection in HIV-infection.

1 dwg, 4 tbl

 

The invention relates to medicine, in particular to the section of infectious diseases, HIV infection, tuberculosis.

TB can occur at any stage of HIV infection and its clinical manifestations clearly correspond to the immune status of the patient. In the early stages of HIV infection when the immune status of the patient is only partially broken, of a tuberculous process appears more typical pattern. The process affects mainly the upper lungs, formed caverns, and in General, the TB seems to be the same as in the period before the appearance of HIV.

With increasing immunodeficiency manifestations of tuberculosis are becoming more atypical. There are signs, characteristic for the primary period TB infection are becoming more frequent defeat of intrathoracic lymph nodes, proximal localization process, extrapulmonary and disseminated processes. In cases of pronounced immunodeficiency (CD<0,2×109/l) in individuals who are already sick with tuberculosis may occur dissemination process, which in turn often leads to generalization of tuberculosis and even death. Analysis of data on patients with TB with HIV co-infection, has shown that often in this category of patients develop disseminated tuberculosis was regarded as a manifestation which begins later stages of HIV infection (stage 4B, 4B and 5), and therefore, it was wrongly assigned the appropriate late stages of HIV treatment and clinical supervision (V.V. Pokrovsky, 2006).

Thus, as the development of immunodeficiency and HIV in late-stage clinical-morphological TB symptoms significantly worse. Given the above, in order to timely detect tuberculosis, appropriate after diagnosis of HIV infection and to the development stages pronounced immunodeficiency identify patients belonging to the group of high risk of TB, for subsequent dynamic monitoring of TB and the timely appointment of chemoprophylaxis or treatment of tuberculosis. To this end we use the following diagnostic methods: inspection, physical examination, radiology (including CT scan), tuberculin, serological (ELISA for antibodies to Mycobacterium tuberculosis, bacteriological (fluorescent smear, bacteriophase), histological. For screening are used fluoroscopic examination of the chest (or R-graphy), Mantoux test.

Each of these methods has its advantages and disadvantages. For example, active tuberculosis, in combination with HIV infection, has many clinical is reavley and can hide under the guise of other diseases. Pulmonary tuberculosis proceeds with the formation of cavities, infiltrates in the upper lobes, accompanied by pneumonia and fibrotic changes in the lungs. In HIV-infected patients with stage 4B on the background of deeper damage the immune system begins to develop acquired immunodeficiency syndrome. In the structure of tuberculosis during this period disappear forms with predominantly productive processes. Tuberculous process in the lungs becomes more widespread, and the morphological examination begin increasingly to register areactively picture. But the greatest clinical and morphological changes appear in 4B and 5 stages at which the structure of TB is dominated by disseminated processes and tuberculosis of intrathoracic lymph nodes, making a total of 61.4%. In 36% of patients in this period took place in the generalization of tuberculosis (Michailidis, S. et al., 2005). On average was amazed with 6 bodies, most of this light (97.8 per cent), spleen, liver, kidney (13.6%), lymph nodes (11,6%), meninges and brain tissue (5,8%). Clinical manifestations of pulmonary tuberculosis usually remain the same in patients with HIV infection and those without. However, complaints of cough are observed less frequently, which may be associated with less frequent development of cavities, attenuation of inflammatory reactions and rasaraj is of the bronchial mucosa by reducing cellular immunity. Similarly become more rare hemoptysis due to the weak intensity of the processes of caseous necrosis of the bronchial arteries in HIV-infected patients. The diagnosis can be difficult in patients with advanced stages of HIV infection, due to the fact that instead of the upper lobe cavitary infiltrates typical of reactivation of the disease, they can show the signs more typical of primary tuberculosis with intrathoracic lymphadenopathy proximal infiltrates, pleural effusion, or even a normal x-ray picture. Usually these patients have symptoms: cough, fatigue, weight loss, night sweats. It was also established that tuberculosis is the most frequent cause of prolonged fever in HIV-infected patients. Cavity decay at late stages of HIV infection occurred only in 20-36% of cases, which is associated with a sharp decline in exudative-proliferative processes on the background of severe immunodeficiency, which, in turn, leads to rapid dissemination of tuberculosis. Disseminated TB is often manifested by the appearance in all lung fields small polymorphic or monomorphic lesions moderate intensity and in some cases bright clinical picture was ahead at 4-14 weeks the appearance of dissemination. Among the clinical manifestations predominate t is placed severe intoxication, painful cough, bringing relief, scarce viscous sputum and one-third of patients find cachexia. Twice as often in HIV-positive patients meet extrapulmonary forms of tuberculosis. Extrapulmonary tuberculosis in the context of HIV infection most commonly manifests itself in the form of lymphadenopathy, serous effusions (in the cavity of the pleura, peritoneum and into the cavity of the pericardium), and disseminated or miliary TB (Karachunskii M.A., 2000). Extrapulmonary TB can infect the Central nervous system, gastrointestinal tract, spine, and bones and joints (Frolov OP, 2002).

The smear examination remains the basic method of diagnostics of tuberculosis, even in areas with a high prevalence of HIV infection. However, the number of cases with negative results of sputum smears among patients with pulmonary tuberculosis combined with HIV infection is slightly higher than among TB patients only (Gebrekristos H.T. et al., 2005). In addition, sputum smears in HIV-infected patients typically find fewer organisms than in HIV-negative patients. Because of this, acid-fast bacilli can remain unnoticed if you have not viewed the entire area of the smear under a microscope with high magnification. Cavity decay at late stages of HIV infection marked Neche is that due to a sharp decline in exudative-proliferative processes on the background of severe immunodeficiency. Perhaps, therefore, the number of positive cases among patients with advanced HIV infection is only 19%.

Tuberculin skin tests are of limited value in the diagnosis of tuberculosis in adults, although they are very informative when studying the prevalence of TB in the community, especially in the early stages of HIV infection. With active tuberculosis skin reaction to tuberculin may be negative. Tuberculin tests in the later stages of HIV infection in most cases are uninformative, while in the early stages of the frequency of their detection does not differ from that in TB patients without HIV infection.

Chest x-ray should be performed in all patients with suspected pulmonary tuberculosis with negative results of sputum smears and no effect on the purpose of the broad-spectrum antibiotics. Bronchitis and pneumonia caused by Streptococcus pneumoniae and Haemophilus influenzae, and other common pathogens, quite often develop in HIV-infected persons Radiographic signs, pathognomonic only for tuberculosis, does not exist, although the classical references in this disease remain is pechalnoe location process, the formation of cavities, the fibrosis and shrinkage of the lung, as well as calcification. Similar to the typical pattern observed in HIV-infected patients is still relatively preserved immune system. With increasing immunodeficiency picture changes in the lungs becomes more atypical. Infiltrative changes more often localized in the lower lung, increased intrathoracic lymph nodes. Often x-ray picture of light remains normal. Non-tuberculous lung disease in these patients may also manifest as typical and atypical radiological picture. This fact must be considered in the differential diagnosis, especially in patients with negative results of sputum smears. The most frequent lesions resembling tuberculosis in HIV-infected patients are bacterial pneumonia and pneumonia caused by Pneumocystis carinii, sarcoma Galoshes, fungal infections and Nocardia. Particularly difficult are the cases in which dissemination in the lungs at all radiographically is not registered. This often is not clearly marked on the x-ray tuberculosis of intrathoracic lymph nodes. And in some cases and complaints of cough absent. Prospective on ludena for this category of patients, and often retrospective analysis of these cases showed that many of the patients, to detect tuberculosis (including posthumous), for several months complained only on a periodic rises in temperature to 39 degrees and above, sudden sweating and weakness (Frolov OP, 2006). Analysis of causes of death in individuals who had active tuberculous process in the later stages of HIV infection, showed that he was the leading cause of death in 38.5% of cases.

It is known that the development of active tuberculosis infection on the background of HIV infection exacerbates immune disorders. Tuberculosis causes disorders of the immune system itself, as evidenced by changes in immune status in HIV-negative patients with tuberculosis (Chereshnev V.A. Immunology inflammation: role of cytokines. /Waiteresses, Eyisi//Honey. immunology. - 2002. - P.88-92). Studies of cytokine regulation mechanisms of development of tuberculosis infection in HIV-infected patients are scarce and often contradictory. Therefore, of particular interest is the study of the effects of both agents on the immune system and their mutual influence is very difficult. As already mentioned, the protection from tuberculosis is associated with a Thl response. The combination of TB and HIV marked increase in the level of proinflammatory cytokines in the serum, which is determined by the t degree of immune activation in individuals with HIV infection and tuberculosis, especially with a reduced number of CD4+ (Ala-Menkalinan, Vinegrove, 2001). Magarachski (Karachunskii M.A. Tubercules for HIV infection. // Problems of tuberculosis. - 2000 - No. 1. P.47 - 52)) have shown that under the influence of HIV infection in TB patients increased production of IL-4 and IL-8 and changed the entire profile of cytokine secretion. These changes were not dependent on CD4+ and CD8+lymphocytes, but directly correlated with the progression of AIDS. It is noted that the levels of IFN-γ and TNF-α significantly reduced in patients coinfected with HIV/TB.

Thus, the immunopathogenesis of HIV infection and tuberculosis as monoinfected well understood. The understanding of the mechanisms underlying the synergistic interaction of HIV and tuberculosis, can lead to improved control strategies for both infections as additional diagnostic criteria.

The objective of the invention is development of a method for early diagnosis of tuberculosis with regard to the incidence of clinical and laboratory parameters in HIV-infected patients according to the materials of prognostic studies of comparative analysis.

The technical result - improving the quality and reducing the time of early diagnosis of tuberculosis infection in HIV-infection.

The inventive method for early diagnosis of TB in HIV-infection has no prototypes. If p is avidinii analysis of the available scientific and patent information is not detected information, related to the use of the method of early diagnosis of TB in HIV-infected patients.

To achieve the technical result in the way of early diagnosis of TB in HIV infection, including the monitoring of immunological markers, conduct an assessment of immunological parameters enzyme-linked immunosorbent assay, determine the range of cytokines: tumor necrosis factor - alpha (TNF-α), interleukin-10 (IL-10) and soluble receptor of interleukin-6 (RR IL-6) and at intervals of measure TNF-α of 19.72 to 23,87 PG/ml, IL-10, 15,48 to 21,86 PG/ml and PP IL-6 from 770,76 to 1800,31 PG/ml when viral load (VL) more than 10,000 COP/ml and CD4+ less than 500 diagnose tuberculosis in patients with HIV infection, with the duration of preventive treatment of tuberculosis less than three months.

The essence of the proposed method for early diagnosis of TB in HIV is that determine the usefulness of serum levels (system) cytokines and their soluble receptors as markers for early diagnosis of tuberculosis in patients with HIV-associated TB.

The choice of the studied cytokines was not accidental and was based on comparison of their biological effects and pathomorphological phenomena (Yarylo A.A. fundamentals of immunology./All//M.: Medicine, 1999. - 607 C.) Range of cytokines involved in the group of proinflammatory cytokines, produced in response to a direct effect of infectious agents - TNF-α, IL-6 and their soluble receptors (RRR TNF-α, RRR TNF-α), IL-6) and anti-inflammatory mediator - activator predominantly Th2-lymphocytes and immune regulator of inflammation - IL-10.

The object of the study were 90 patients: 40 patients with HIV-associated tuberculosis (group 1) at the age from 19 to 53 years (average of 27.3±3.3 years; 85% men) and 50 patients with HIV without TB (group 2) from 20 to 55 years (average of 26.3±4.8 years; 80% male). While all patients included in the 1st group, TB is diagnosed in the context of HIV infection. The estimated duration of HIV infection, defined by the period of time from the moment of occurrence of the risk factor, ranged from 5 to 7 years. In 10% of patients to determine the time of infection was not possible. In 85% of cases the infection was parenterally. The diagnosis of tuberculosis in patients of the first group exhibited based on a comprehensive clinical, radiological, bacterioscopic and bacteriological studies. Prevailed infiltrative form 17 patients (42.5 per cent) and disseminated - 13 (33,7%). Focal tuberculosis was detected in 10 patients (25%). The majority of patients with disseminated TB (9), and 2 patients with infiltrative tuberculosis of the lungs was in advanced IVB stage of HIV and the infection.

The survey studied the levels of the cytokines (TNF-α, IL-6, IL-10 and soluble receptors RRR TNF-α (type I), RRR TNF-α (type II), PP IL-6) in serum of patients by the method of solid-phase ELISA using diagnostic kits (R&D Diagnostics Inc., USA) with a sensitivity of 1 PG/ml

Obtaining serum to determine the concentration of cytokines.

For this purpose, peripheral blood (5 ml) were taken by syringe from the cubital vein, centrifuged at 3000 rpm./min in the cold for 10 minutes, the Serum was poured into 0.5 ml in Eppendorf frozen and stored until use at -76°C. the Calculations of a number of cytokines was performed by constructing a calibration curve using a computer program and expressed in PG/ml.

The control group consisted of 50 healthy persons of the same age and gender.

As shown by our study revealed significant differences in the cytokine profile of patients analyzed groups of indicators of control. Installed hyperproduction as early (TNF-α)and late (IL-6) Pro-inflammatory cytokines, whereas the content of anti-inflammatory mediator IL-10 has changed little (table 1). The level of TNF-α in patients with HIV infection (44,2±10,32 PG/ml)and HIV-TB co (108,6±18,62 PG/ml) statistically significantly different from performance in healthy individuals (0,95±0,32 PG/ml, p<, 001). This shows the important role of this cytokine in the implementation of mechanisms of anti-infective protection, which is consistent with literature data that TNF-α is a major mediator of inflammation and cellular immune responses (Khaitov R.M R.M. Physiology of the immune system. / Rmitv - M.: Medicine, 2001. - 289 C.).

Table 1
The level of cytokines and their soluble receptors in the serum of patients of different groups and healthy people
The indices M±mHealthy (n=50)HIV infection (n=50)HIV/tuberculosis (n=40)
PG/ml
TNF-α0,95±0,3244,2±10,32***
p2-3<0,01
108,6±18,62***
p1-3<0,01
RRR
TNF-α
390,1±38,0770,1±20,0***
p2-3>0,05
766,6±10,32***
p1-3<0,001
RRR
TNF-α
2673,4±58,0 8421,2±971,0*
p2-3>0,05
10887,5±851,3**
p1-3<0,001
IL-61,99±0,5318,46±7,35*
p2-3>0,05
30,57±8,66**
p1-3>0,05
PP
IL-6
3120,0±18,02812,0±30,7**
p2-3<0,001
2010,80±39,44**
p1-3<0,001
IL-1014,43±1,2518,84±2,60
p2-3>0,05
26,39±3,11***
p1-3>0,05
Note: statistical significance of differences with the control group: p<0,05 - *; p<0,01 - **; p<0,001 - ***; p1,2,3- the comparison group.

Note, however, that the protective activity of TNF-α in terms of the persistence of infectious process seems inadequate. Thus among the analyzed groups of patients the rate of change of the indicated cytokine was different. The highest level of TNF-α was detected in patients with HIV-TB co, and exceed 50-100 times the normal value. In patients with HIV without TB, this figure is only 10 times differed from the results in healthy. When statistics is practical analysis of serum levels of TNF-α in the analyzed groups were detected reliably distinguishable differences (p< 0.001 to 0.01). These results support the majority opinion of the authors that when HIV infection is damage to alveolar macrophages (because they carry the marker CD4), which also plays a negative role in co-infection (Freidlin, I.S., 2001; Kapustin, Siennica, 2004). It can be assumed that co-infected patients the decrease in CD4 lymphocytes, which play a key role in TB immunity, and their functional disability is accompanied by increased reproduction in the lungs of Mycobacterium tuberculosis (MBT) and the dissemination of Mycobacterium tuberculosis. Along with this office and their products activate the replication of HIV. So, mononuclear cells from peripheral blood of patients with co-infection produce more TNF-α than is observed in patients with only TB or only HIV-infection. TNF-α is required for TB to limit the inflammatory process, combined infection promotes more rapid multiplication of the virus, resulting in the aggravation of immunodeficiency due to active development of the two infections.

The content of IL-6 in the serum of patients was in 5-30 times higher than in the control group. However, no statistically significant differences in the content of IL-6 in the serum of patients with different gruppu the same time the level is ü IL-10 was the lowest in patients with HIV infection. However, the last value of IL-10 did not differ from that of the control group (18,84±2,60 PG/ml against 14,43=1,25 PG/ml, respectively; p>0,05).

As is known, the implementation of functional activity of cytokines occurs as a result of their interaction with specific receptors on target cells (Simbirtsev A.S., 2004). Receptors of cytokines can be separated from the cells by organic proteolysis (shedding) with their appearance in biological fluids. There are two types of receptor for TNF-α. Receptor type 1 (CD 120a) has a molecular mass of about 55 kDa (RRR TNF-α), the receptor of the 2nd type (CD 120V) - about 75 kDa (RRR TNF-α). The cytoplasmic domains of these receptors are deprived of similarity that indicates their different signaling functions. Receptors TNF-α type I and TNF-α induce type II cells different answer. Through receptor 75 kDa TNF-α stimulates the proliferation of T-cells via receptor 55 kDa - induces death of cells by apoptosis. Soluble receptors bind TNF-α and act as inhibitors of its biological activity.

In our study the increase in the number of soluble receptor of TNF-α type II (RRR) in the serum of patients of all groups compared with their level in the control. The highest values recorded during HIV-associated tuberculosis (10887,5±851,3 PG/ml). Patients of both groups you who go the higher level RRR TNF-α and reduced PP IL-6 (p< 0,001).

In additional analysis, we also confirmed the differences between groups of patients with HIV-infection and HIV-associated tuberculosis in the level of the RR type I TNF-α and PP IL-6. In patients with HIV-associated tuberculosis revealed a relatively lower level RR IL-6 (χ2=7,652 and exceeds χ2St, p=0.005, 1 tswoboda) and a higher level of PP type II TNF-α (χ2=4,160 and exceeds χ2St, p=0,041, 1 tbsp. of freedom). At the same time, no differences in the level of the RR type I TNF-α in patients with HIV these groups (χ2=0,007 p=0,935, 1 tbsp. of freedom).

Studied the dependence of the probability of detection of tuberculosis in HIV-infected patients from baseline immunological parameters at different stages of the disease, both individually and collectively and in combination with other factors. For risk of TB detection was calculated 95%confidence interval (95% CI), and the comparison was done using the criterion χ2or two-sided Fisher's exact test, and continuous variables using student's criterion or the ranking criterion of Wilcoxon signed. The relationship between levels of systemic cytokines, their soluble receptors and development of tuberculous process was evaluated using stratified analysis. We assessed the validity of the assumption of linear dependence of TB on stage Zab the diseases and levels of the studied mediators using generalized linear models (package S-plus), estimated using a smoothing cubic spline. Was also performed logistic regression analysis with step-by-step with the exception of predictors and detection of tuberculosis infection as the dependent variable. The critical level of statistical significance when deleting predictors was taken equal to p=0.25, the results of the test of credibility. The initial model consisted of four groups noncollinear (independent) variables. In the first group consisted of components of a strategy for the prevention of tuberculosis: the presence or absence of prophylactic regimens, duration of the scheme. In addition, the analyzed schema art, if it was administered, the period of its beginning and duration of use, number of changes in treatment, however, they were not included in the final model because of their collinearity. The second group of variables included stage of HIV infection and recent indicators of the level of viral RNA and the number of lymphocytes CD4+. The third group consisted of age and sex, while in the fourth performance of cytokine status before and after the detection of tuberculosis. We then performed step-by-step logistic regression in two subgroups: after tuberculosis and in its absence at different stages of HIV infection based on indicators of immunological parameters. For analysis, we used the software package STATA. The results of the prize which was avalis statistically significant at p< 0,05.

Univariate analysis showed that the risk of tuberculosis infection largely depends on the level of HIV RNA (p<0,001), the number of lymphocytes CD4+ (p<0.001) and duration of preventive treatment for tuberculosis (p<0,001). In General, the level of viral RNA<400 copies/ml, the risk of tuberculosis infection was 0.6% (95% CI 0.4 to 0.9 per cent), with 1000-10000 copies/ml and 1.5% (95% CI of 0.8-2.5%), and at the level of more than 10000 copies/ml to 6.8% (95% CI 4.6 to 9.6 percent). It was also revealed the relationship between the frequency of detection of tuberculosis and HIV infection. As to the advanced stage of the disease increased the probability of detecting tuberculosis (p=0.001). The risk of infection was not influenced by age (p=0,19), gender (p=0,12), the scheme of art (p=0.15).

In the final model step-by-step logistic regression remained independent influence levels of viral RNA, CD4+lymphocytes and stage of HIV infection on the risk of tuberculosis infection (table 2). The adjusted odds ratio for patients with diagnosed tuberculosis with immunodeficiency (CD4+-lymphocytes < 500 cells/ml) was 1.92 (95% CI of 0.98-3,79). When the level of viral RNA less than 400 copies/ml it amounted to 1.14 (95% CI of 0.33-3,90), and at the level of more than 400 copies/ml increased to 9.82 (95% CI 5,24-18,37). Planned preventive TB treatment in the initial model almost no influence on the risk of tuberculosis infection, however, in the final model significantly nigalo him with a duration of more than three months (p=0.001).

Conducted studies to determine the cytokine status in patients with HIV infection and HIV-associated TB has allowed to reveal a number of features that reflect the vector of changes in the immunoreactivity of the subject. It has been found that as the progression of HIV infection increased the level of TNF-α, PP type II TNF-α, IL-6 and decreased (IV and IVB stages) PP IL-6, IL-10 was increased only in the terminal stage (table 3). In patients with HIV-associated tuberculosis in the progression of infection to IVB stage increased the content of TNF-alpha, its soluble receptor type II, IL-6, IL-10 and PP IL-6. While in the terminal stages of HIV-associated tuberculosis registered relative reduction in soluble receptor of TNF-α type II, IL-10 and soluble receptor of IL-6 compared with patients with HIV infection is similar to the stage. Differences in immune reactivity of patients of different groups give rise to the development of additional criteria for the diagnosis of tuberculosis in combination with HIV-infection.

Table 2
Early diagnosis of tuberculosis in patients with HIV infection using analysis multiple logistic regression
IndicatorsThe initial modelThe final modelp
The odds ratio95% confidence intervalThe odds ratio95% confidence interval
Stage III of HIV infection0,840,35-2,030,850,35-2,030,019
Stage IV of HIV infection3,251,32-8,033,371,40-8,11
NR HIV > 10000 COP/ml9,364,90-17,879,825,24-18,370,001
NR HIV 400-9999 COP/ml2,461,19-5,092,521,25-5,11
NR HIV < 400 COP/ml1,130,33-3,901,14 0,33-3,90
CD4+/ul > 5001,150,61-2,181,220,65-2,270,017
CD4+/µl of < 5001,790,88-3,621,920,98-3,79
TNF-α1,700,94-3,101,610,92-2,830,049
PP IL-60,560,29 was 1.060,490,26-0,89
IL-100,900,46-1,760,810,42-1,56
Preventive treatment for TB to 3 months6,541,32-32,318,531,93-37,590,010
Preventive TB treatment for more than 3 monthsand 5.301,36-20,72 between 6.081,66-22,26

Table 3
Dynamics of cytokines in patients with HIV infection and HIV-TB co depending on the stage
Group of patients and stage of HIVTNF-αPPp55
α
RRR
α
IL-6PP
IL-6
IL-10
HIV (n=50)III10↑2↑5↑2↑2↓2↑
IVA13↑3↑7↑3↑2↓4↑
IV20↑3↑8↑7↑2↑6↑
IVB5↑10↑10↑2↑4↑
V30↑5↑3↑10↑2↓2↑
HIV/TB (n=40)III25↑2↑-2↑3↓3↑
IVA40↑2↑2↑3↑3↓5↑
IV55↑3↑3↑10↑2↑6↑
IVB60↑3↑4↑15↑3↑8↑
V 100↑3↑2↑20↑1,5↑1,5↑

Multifactor analysis of indicators of cytokines in the serum factors of poor prognosis (TB screening in asymptomatic stage of HIV infection) were the levels of TNF-α, IL-10 and PP IL-6 (p=0,049).

Defined within the parameters of descriptive statistics the data intervals immunological parameters associated with tuberculosis detection in asymptomatic HIV infection (table 4).

Table 4
The parameters of descriptive statistics immunological parameters used with prognostic purposes in patients with HIV infection for TB detection in asymptomatic stages
StatisticsImmunological parameters (PG/ml)
TNF-αIL-10PP IL-6
The number of observations606060
Average21,3918,561231,37
The standard deviation2,073,45464,28
Standard error1,872,37396,87
Median21,2819,071261,02
Minimum (10% percentile)18,7214,98782,86
Maximum (90% percentile)23,9322,261906,96

To measure TNF-α is a match of 19.72-23,87 PG/ml for IL-10 - 15,48-21,86 PG/ml for PP IL-6 - 770,76-1800,31 PG/ml (n=60, 80% interpretationem range). Therefore, these intervals immunological markers can be considered as a prognostic criteria of tuberculosis infection in asymptomatic stages of HIV infection.

The results of our research allow us to offer the diagnostic algorithm of search of early detection of TB in HIV-infected, inwhich with no complaints, clinical data, in the absence of radiographic changes recommended to introduce monitoring of the above immunological markers and their identification in certain intervals will indicate the risk of the likelihood of possible infection and repeated in-depth research to identify tuberculosis (drawing). A higher degree of risk increases with NR > 10000 COP/ml, CD4+<500 and duration of preventive treatment for tuberculosis, if any, less than 3 months.

Clinical case using the algorithm of screening for TB patients with HIV infection:

Patient I., 33 years old, first came to the health center at the place of residence 17.11.04, with complaints of cough with a small amount of light sputum, stuffiness in the chest, increased body temperature up to 37.5-38°C for 1 week. The local doctor diagnosed acute bronchitis and was prescribed Biseptol-480 2 tablets 2 times a day, mukaltin. In the survey drew attention to the increase in ESR (27 mm/h)that were observed in the future (and characteristic of Pneumocystis pneumonia). X-ray examination of lungs 18.11.03, pathology has not revealed. Again in the clinic were not addressed. Apparently Biseptol, appointed in therapeutic dose (too small for the treatment of Pneumocystis pneumonia in HIV-infection), helped the patient (is it possible at the beginning of the disease), as he re-applied for medical assistance only in the beginning of December to the clinic complaining of a feeling of lack of air, worse on exertion, cough with sputum for 2 weeks, weight loss. During the examination (examination, radiography of the thoracic cavity, ECG, complete blood count) pathological changes, in addition to a high erythrocyte sedimentation rate (55 mm/h), was not detected. The patient was recommended consulting pulmonologist, allergist, immunologist, abdominal ultrasound, fibrogastroduodenoscopy. However, the examination is not passed and a week later went to the polyclinic with the same complaints. Her primary care physician noted a decrease in body mass, increased supraclavicular and axillary lymph nodes to the size of a bean, somewhat weakened breath in the lungs and after the examination (General analysis of blood, urine, ultrasound of the abdominal organs, increased erythrocyte sedimentation rate, enlargement of the spleen, is detected in the right lobe of the liver of education 28×20 mm) with a diagnosis of Hodgkin's disease?, education right lobe of the liver?" sent the patient for a consultation with a gastroenterologist, who was diagnosed with liver hemangiomas. Pulmonologist after the x-ray showed negative dynamics of the pattern in the lungs compared with the study from 18.11.04 - increased pulmonary pattern, soft model the roots of the lungs). After the conclusion of "congestion in the lungs? systemic pathology? myocarditis?" the patient is recommended consulting a rheumatologist, hematologist, cardiologist to exclude systemic disease. Next the patient are fibrobronchoscopy (endoscopic picture without pathological phenomena), ECG, Echocardiography and consultation of the cardiologist (prolapse of the front door of the mitral valve of 0.3 cm systole), cycle ergometry, but due to severe shortness of breath this manipulation was terminated.

16.12.04, computed tomography of the chest were determined strengthening and deformation pulmonary pattern, in the middle and lower lung fields - infiltration of lung tissue interstitial character, located mainly subpleural. Was suspected idiopathic fibrosing alveolitis, and after re-consulting pulmonologist to this diagnosis was added and miliary tuberculosis?".

20.12.04, the patient was consulted by a specialist. Description the TB picture is fully consistent picture, often characteristic of Pneumocystis pneumonia - "on direct review radiographs of the lungs in both lung lung picture more in the basal zones of excessive strain, net-spotted by compacting and interstitial infiltration of lung tissue; infiltration is more pronounced perednik departments; cortical lungs, the area of the tops of the emphysematous. The roots of the patterns, the pulmonary artery is not increased". The diagnosis of TB: "disseminated in the lungs of unknown etiology, presumably idiopathic fibrosing alveolitis". The study of sputum for Mycobacterium tuberculosis (the result of the analysis is negative). Conducted Mantoux test with 2 Tu - "Yolochka reaction". The patient was consulted by a rheumatologist and to avoid systemic pathology assigned to a scheduled examination, but the patient is no longer treated. The patient's health continued to deteriorate and 19.12.04 was hospitalized in Railway hospital, where he stayed for 4 days with a diagnosis of idiopathic fibrosing alveolitis?". In the statement contains important information on the available clinical symptoms (corresponding PCP): "he Admitted with complaints expressed shortness of breath mixed in peace, aggravated by the slightest physical activity, raising the temperature to 39°C for 3 days, cough with scanty sputum. Loss of body weight over 15 kg Radiograph of the lungs (19.12.04): diffuse malosetti picture with lesions of interstitial tissue. Emphysema... ESR 17 mm/h...". Patient treatment was conducted (gentamicin and penicillin, prednisone, heparin), against which celebrated the ü some improvement. By agreement was transferred to city hospital, where he was with 23.12.04, 06.01.05, and received prednisolone, anti-TB therapy, Biseptol-480 2 tablets 2 times a day, furosemide. At this time, marked cyanosis of the lips, frequency of 32 breaths per minute, tachycardia (heart rate 100 / min), there were changes on the ECG (sinus arrhythmia, violation of repolarization processes in the myocardium, local violations vnutriepreserdnaya conductivity). Increased changes in x-ray pictures of the lungs (pulmonary figure greatly increased and deformed; visible fine mesh enlightenment, the changes are more in the lower divisions; the roots of mesostructure", and in the study through week - "strengthening and deformation pulmonary pattern progressing with melkouzelkova inclusions"). Thyroid ultrasound: in the left lobe detected education on the left. Was re-observed by TB, suspected of TB and prescribes treatment. 28.12.04 for the first time treatment of a patient in a medical institution, a survey was conducted it for HIV and received positive results (enzyme-linked immunosorbent assay and immune blotting). Was invited a specialist from the centre for the prevention and control of AIDS for advice, which is fully collected anamnesis (optional whom was identified, what the patient considers himself to be HIV-infected about 2 years, but maybe more a long-standing infection, over the last 3-4 years, said recurrence of herpetic lesions of the lips). Was appointed as additional tests (immune status, viral load, repeated consultation of TB, sputum for Mycobacterium tuberculosis, the number of opportunistic diseases and the study of cytokine status with the study of the content on the examination algorithm TNF-α), IL-6 and IL-10), suggested the possibility of Pneumocystis pneumonia and recommended therapy exjuvantibus - intravenous Biseptol-480 (20 mg/kg per day), and antiviral therapy. 05.01.05, was re-examined by infectious center for prevention and fight against AIDS: the observed severe condition, on the mucous membrane of the oral cavity appeared white curd raids, atty on the lower surface of the tongue, and therefore assigned to antifungal therapy (diflucan). Indicators of these cytokines was outside the limits of the intervals for the tuberculous process, which enabled to exclude tuberculous process (TNF-α of 10.21 PG/ml) IL-6 3003,15 PG/ml and IL-10 27,11 PG/ml).

06.01.05 patient, accompanied by the relatives of unauthorized resigned from office and was at home, almost nepalica no cure, and 18.01.05 was re-admitted to the hospital with symptoms of respiratory insufficiency III level, where the next day the death occurred.

Pathological diagnosis: the disease caused by HIV (positive reaction of the immune blotting from 28.12.04 year). Bilateral total Pneumocystis pneumonia. Cachexia. The patient had typical clinical picture of Pneumocystis pneumonia characterized by signs of respiratory distress and chest x-ray changes, ended in death. The disease was not recognized in time due to the fact that despite the presence of severe lung disease in a young person and significant weight loss in a short period of time, none of treating and advising him of specialists in various medical institutions do not have any suspicion in a patient with HIV infection, indicating the lack of awareness of physicians in terms of HIV infection. The example illustrates the fact that the erroneous diagnosis of tuberculosis was more common in patients suffering from other infections caused by HIV-related disorders.

Literature:

1. Clinical recommendations. HIV infection and AIDS. Ed. Was. - M.: GEOTAR-Media, 2006. - 128 S.

2. Michailidis, S., Pozniak A.L., Mandalia s, Basnayake, S., Nelson M.R., B.G. Gazzard Clinical characteristics of IRIS syndrome i patients with HIV and tuberculosis // Antivir Ther. 2005; 10(3):417-22.

3. Karachunskii M.A. Tuberculosis in HIV infection. // Problems of tuberculosis. - 2000 - No. 1. P.47 - 52.

4. Frolov OP, Jakubowiak Century, Kravchenko A.V. and other Recommendations for reducing the incidence of tuberculosis among the population with a high prevalence of HIV infection. - M., 2004. - 104 S.

5. Gebrekristos H.T., and growth M.N., Mthethwa N., Q.A. Karim Knowledge and acceptability of HAART among TV patients in Durban, South Africa. // AIDS Care. - 2005. - Aug; 17(6):767-72.

6. Chereshnev V.A. Immunology inflammation: role of cytokines. / Waiteresses, Bigpaw // Honey immunology. - 2001. - V.3, №3. - S-368.

7. Karachunskii, M.A. Tuberculosis in HIV infection. / Magarachskii // VIII Russian national Congress "Man and medicine": Lectures for practitioners. - M., 2002. - P.88-92.

8. Chereshnev, V.A. Immunology inflammation: role of cytokines. / Waiteresses, Eyisi // Honey immunology. - 2001. - So 3, No. 3. - S-368.

9. The Yarylo A.A. fundamentals of immunology. / All. // M: Medicine, 1999. - 607 S.

10. Khaitov R.M R.M., Physiology of the immune system. / Rmitv - M.: Medicine, 2001. - 289 C.

11. Freidlin, I.S. cells of the immune system. V.3: Lymphocytes / Istration, Tan - SPb.: Science, 2001.

12. System cytokines: Theoretical and clinical aspects. Ed. Wageslave, Siennica. - Novosibirsk: Nauka, 2004. - 324 S.

13. Simbirtsev A.S. cytokines: classification and biological functions. / Assemblies // cytokines and inflammation. - 2004. - V.3, №2. - P.1-22.

Method for early diagnosis of tuberculosis in HIV infection, including the monitoring of immunological markers, wherein assessing immunological markers enzyme-linked immunosorbent assay, determine the complex of serum cytokines: tumor necrosis factor - alpha, interleukin - 10 and soluble receptor of interleukin - 6 and set their levels, and indicators of tumor necrosis factor - alpha of 19.72 to 23,87 PG/ml, interleukin - 10 from 15,48 to 21,86 PG/ml and soluble receptor of interleukin - 6 from 770,76 to 1800,31 PG/ml in viral load over 10,000 COP/ml and CD4+less than 500 diagnose TB in HIV-infected patients.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to a method of human blood serum analysis for a soluble form of antigen CD50 dimer involving the use of CD50-specific monoclonal antibodies whereat a tray reaction includes tetramethyl benzidine as a substratum.

EFFECT: invention provides detecting the soluble form of antigen CD50 dimer in human blood serum.

1 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science, namely to microbiology and immunology. The method involves cultivation of Brucella abortus I-206 L-strain on a dense nutrient medium for L-brucellas at 37°C for 3-5 days. Thereafter, a microbial mass is washed of with buffered 0.9% normal saline of pH 7.2±0.2 and inactivated by adding 2.5% formalin. A bacterial suspension is kept for one day at 37°C with specific sterility being controlled. The bacterial suspension is reduced to concentration 4.5·1010-5·10 m.c. in ml with buffered 0.9% normal saline (pH 7.2±0.2) and thermally treated on a water bath at 100°C for 45-50 minutes. The suspension is centrifuged at 7000 rpm for 50-60 minutes; the supernatant is separated and frozen-dried. The method allows producing the preparation exhibiting high specificity and activity, available for preparing based immunobiological preparations and a test-system for human and animal blood serum examination for L-brucella antibodies.

EFFECT: method is technological, accessible, does not require using expensive devices and equipment.

2 ex

FIELD: medicine.

SUBSTANCE: substance of the invention involves a method of detecting an antibody in a sample being tested containing a body fluid of a specified mammal where said antibody is a biological marker of a disease state or propensity for a disease where the method involves (a) contacting said sample being tested with a set of various amounts of an antigen specific to said antibody, (b) evaluating specific binding of said antibody and said antigen, (c) drawing a diagram or calculating a curve of said specific binding to the amount of the antigen for each amount of the antigen used at the stage (a), and (d) stating the presence or absence of said disease state or propensity for the disease by the specific binding of said antibody and said antigen for each individual antigen concentration used where the presence of said disease state or propensy for the disease is detected by screening a titration curve starting with the stage (c) for observing an S-shaped or sigmoid curve.

EFFECT: higher sensitivity of the diagnostic technique.

23 cl, 7 ex, 6 tbl, 15 dwg

FIELD: medicine.

SUBSTANCE: method for typing lepromatous leprosy consists in evaluating neutrophilic granulocyte (phagocytes) recovered from peripheral blood, incubated with lepromin; a chemoluminescence (CL) level is specified, and maximum intensity lepromin activated CL enables to type lepromatous leprosy.

EFFECT: method provides higher accuracy and information value of typing lepromatous leprosy.

1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to infectious disease, and can be used for prediction of antiretroviral therapy efficiency in case of HIV-infection. For this purpose by method of solid-phase immunoenzyme assay level of cytokines is determined. If indices of soluble tumour necrosis factor alpha receptor protein 75 are from 4335.48 to 6001.86 pg/ml, soluble tumour necrosis factor alpha receptor protein 55 - from 768.72 to 1323.87 pg/ml and soluble interleukin-6 receptor from 1770.77 to 3800.31 pg/ml, favourable clinic course of HIV-infection after 1-3 months since beginning of antiretroviral therapy is predicted.

EFFECT: method ensures increase of accuracy of antiretroviral therapy efficiency prediction due to selection of certain immunological criteria.

1 dwg, 7 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: offered is a method for selection of a contingent indicated for diphtheria prophylactic immunisation: vaccination or revaccination. Venous blood serum of a person being tested and a reference venous blood serum sample are examined for levels of specific IgG to diphtheria antitoxin, IgGTDA and IgGRDA respectively. Saliva of the person being tested and a reference saliva sample are examined for levels of specific secretory IgA to diphtheria dialysate antigen, slgATDDA and slgARDDA respectively. The derived values slgATDDA and slgARDDA are compared, and the produced values are also compared thereby. Provided IgGTDA≤IgGRDA and slgATDDA≤slgARDDA, diphtheria prophylactic immunisation is considered to be indicated.

EFFECT: method presents more reliable determination of indications for diphtheria prophylactic immunisation.

FIELD: medicine.

SUBSTANCE: admission laboratory blood plasma examination in the patients with acute calculous cholecystitis is added with evaluating a cholecystokinin level both preoperative one, and on the 9th day following cholecystectomy, and if the examination shows decreasing concentration more than in 2 times, function-type Oddi sphincter dyssynergia is predicted.

EFFECT: invention enables early prediction of developing function-type Oddi sphincter dyssynergia following cholecystectomy.

2 ex, 5 dwg

FIELD: medicine.

SUBSTANCE: in newborn babies blood is sampled from umbilical artery and concentration of IL-6, IL-8 and NO2 in umbilical blood serum is determined by ELISA. After that diagnostic index (DI) is calculated by formula and if DI is lower than 0, it is concluded that laboratory indices of perinatal affection of nervous system in newborn babies are absent, and if DI is higher than 0, it is concluded that laboratory indices of perinatal affection of nervous system in newborn babies are present.

EFFECT: application of the method makes it possible to diagnose perinatal affection of nervous system in a newborn baby at pre-clinic stage.

3 ex

FIELD: medicine.

SUBSTANCE: in order to diagnose reproductive disorders in men, concentration of cytokines IL-8, IL-10 and IL-1RA in blood serum is determined by ELISA, after which diagnostic index DI is calculated. If DI is higher than 0, early reproductive disorders in men are diagnosed, if DI is lower than 0, it is concluded that laboratory indices of reproductive disorders in men are absent.

EFFECT: application of the method makes it possible to increase accuracy of diagnostics of early impaired fertility in men with normal indices of spermogram and to take decision about necessity of carrying out therapeutic correction of detected disorders in due time.

2 ex

FIELD: medicine.

SUBSTANCE: claimed is method of diagnosing phenotype of multiple medication resistance. Unicellular suspensions are prepared from tumour tissue and comparison cultures and fixed, after which incubation with primary and secondary antibodies and double washing are carried out. D values of statistical test of Kholmogorov-Smirnov are calculated and on the basis of obtained data determined is coefficient of level of expression of multiple medication resistance markers, showing phenotype expression. Coefficient of expression level makes it possible to point out three degrees of its expression: low, medium and high. On the basis of coefficient it is possible to determine tumour sensitivity to chemical preparations.

EFFECT: method makes it possible to increase accuracy of determining multiple medication resistance markers and carry out analysis in short terms.

2 dwg

FIELD: medicine, ophthalmology.

SUBSTANCE: in lacrimal liquid one should detect the content of interleukin 8 (IL-8) and that of interleukin 1 beta (IL-1β) to calculate prognostic coefficient (PC) due to dividing the first value by the second one by the following formula: At PC value being below 10.0 one should predict favorable disease flow, and at PC value being above 10.0 - unfavorable flow.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, medicinal microbiology.

SUBSTANCE: method involves growing microorganism culture to be studied in solid nutrient medium followed by preparing microbial suspension and its incubation in the presence of lactoferrin. Control sample is prepared in parallel series. Control and experimental samples are incubated, supernatant is removed from bacterial cells and lactoferrin concentration is determined in supernatant of experimental and control sample by immunoenzyme analysis. Then anti-lactoferrin activity is calculated by difference of concentrations of residual lactoferrin in experimental and control samples. This method provides enhancing the sensitivity and precision in carrying out the quantitative evaluation of anti-lactoferrin activity in broad spectrum of microorganisms that is urgent in diagnosis and prognosis of diseases with bacterial etiology. Invention can be used in determination of persistent indices of microorganisms for assay of their etiological significance in pathological processes.

EFFECT: improved assay method.

3 tbl, 3 ex

FIELD: medicine, biology.

SUBSTANCE: invention relates to nutrient medium used for accumulation of cells for the following cytological and/or immunocytochemical analysis carrying out. Invention relates to medium containing salts NaCl, KCl, anhydrous CaCl2, MgSO4 x 6 H2O, MgCl2 x 6 H2O, Na2HPO4 x 2 H2O, KHPO4, NaHCO3, and also glucose and Henx's solution, 10% albumin solution and polyglucin taken in the ratio 1:1:1. Invention provides enhancing the preservation of cells.

EFFECT: improved an valuable properties of nutrient medium.

3 ex

FIELD: medicine, cardiology.

SUBSTANCE: in peripheral blood one should detect the level of CD95(+) and CD16(+) neutrophilic granulocytes and at combination of increased level of CD95(+) neutrophilic granulocytes by 4 times and more and CD16(+) neutrophilic granulocytes by 0.6 times against the norm with ECG signs of myocardial infarction one should predict lethal result of large-focal myocardial infarction.

EFFECT: higher accuracy of prediction.

FIELD: medicine, parasitology.

SUBSTANCE: one should carry out immunoenzymatic assay to detect diagnostic optic density and that of labeled immune complex in a plot's hole with tested serum measured in conventional units at wave length being 492 nm. One should calculate coefficient of antibodies concentration measured in conventional units by the following formula: CAC = (Odtsh - Odd) x 100, where CAC - coefficient of antibodies concentration, Odtsh - optic density of the hole with tested serum, Odd - diagnostic value of optic density, 100 - coefficient of serumal dilution. By CAC value one should detect the titer of antibodies to Lamblia intestinalis antigens to interpret results of the trial. The method enables to study the dynamics of disease flow.

EFFECT: higher efficiency and accuracy of diagnostics.

1 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: the present innovation deals with studying and treating diseases of inflammatory, autoimmune and degenerative genesis. One should perform sampling of heparinized blood followed by its sedimentation to obtain blood plasma with leukocytes and centrifuging to isolate the latter which are washed against erythrocytic and serumal admixtures, and, also, it deals with calculating the number of cells in samples out of leukocytic suspension after incubation (B) for 1.5 h at 37 C in holes of plastic microplotting board, out of leukocytic suspension one should additionally prepare two samples, one should be applied to calculate total number of leukocytes before incubation (A), the second sample undergoes incubation at the same mode at addition of autoserum to calculate the number of cells remained after incubation (C). One should state upon adhesive properties of leukocytes by the index of spontaneous adhesion (D), where D=(A-B)/B.100%, and effect for enhanced cellular adhesion under the impact of autoserum should be detected by the value of K=(B-C)/C.100% at K ≥ 30%, where B - C - the number of cells undergone additional adhesion after addition of autoserum. The present innovation widens functional possibilities of the suggested method due to obtaining additional values depicting adhesive properties of blood leukocytes.

EFFECT: higher accuracy of detection.

FIELD: medicine, immunology.

SUBSTANCE: one should carry out reaction of blast-transformation, detect proliferation of T-lymphocytes activated with antibodies to CD3 in the presence of interleukin-7 (ACT IL-7) and in the presence of interleukin-7 and dexametazone (ACT IL-7 D), calculate the index for dexametazone action as the ratio of ACT IL-7 to ACT IL-7 D, moreover, the value of dexametazone action index being above 1.2 indicates increased production of cytokins that suppress T-lymphocytes in neonatals. The method enables to detect functional defect of immune system that characterizes neonatal period.

EFFECT: higher efficiency of detection.

2 ex

FIELD: medicine.

SUBSTANCE: method involves measuring forced exhalation volume per 1 s (FEV1) in l, full right ventricle evacuation time (RVE) in ms and angiotensin II value (AII) in ng/l. Discriminant relationship is built as D=0.504·RVE+3.038·FEV1 - 2.0·AII. D being less than 83.88, pulmonary hypertension occurrence is predicted within 1 year. D being equal to or greater than 83.88, no pulmonary hypertension is predicted to occur.

EFFECT: enhanced accuracy of prediction.

FIELD: medicine, medicinal immunology.

SUBSTANCE: method involves determination of heterophilic antibodies in human serum blood by the Paul-Bunnel's method relatively the level of circulating immune complexes, complement-activating properties of heterophilic antibodies by incubation of standardized ram erythrocytes with 0.8% serum for 30 ± 5 min and the following measurement of the erythrocytes lysis degree. The measurement of the effector function coefficient of heterophilic antibodies is carried out by the complement system Keff.f.h.a.-c.s. by the formula: Keff.f.h.a.-c.s. = Y/Tg.a. wherein Y means a lysis degree, %; Tg.a. means a reverse titer of heterophilic antibodies to ram erythrocytes. The damage assay is carried out by comparison of the immune status with the relative level of circulating immune complexes in serum. Method provides detection of preclinic from of immunodeficiency and autoimmune diseases that opens the possibility for their prophylaxis at most early stages of development. Invention can be used for assay of damage in the immune status in human serum blood.

EFFECT: improved method for assay.

5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: method involves concurrently examining anti-inflammatory IL-4 level in blood serum and lacrimal fluid. The value being within the limits of 60-70 pg/l in blood serum and 5-15 pg/l in lacrimal fluid, disease prognosis is considered to be unfavorable. The IL-4 concentration being within the limits of 90-100 pg/l in blood serum and 20-30 pg/l in lacrimal fluid, disease prognosis is considered to be favorable.

EFFECT: high accuracy of diagnosis.

Up!