RU2847711C1 - Method for diagnosing bacterial and fungal infections of the bloodstream - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely medical microbiology, and can be used to diagnose bacterial and fungal infections of the bloodstream. If a patient is suspected of having a bloodstream infection, blood is taken from the elbow vein into a single test tube to obtain human blood serum. At the same time, blood is taken into two vials for haemocultivation of aerobic and anaerobic microflora, which are placed in an analyser until a growth signal is received. If a growth signal is present, the culture is inoculated to identify the pathogen and determine its antibiotic sensitivity. If no signal is present, blood is sampled again every 24 hours. The concentration of procalcitonin and (1-3)-β-D-glucan is determined in the blood serum. If procalcitonin is positive and (1-3)-β-D-glucan is negative, bacterial endotoxin is additionally determined in the blood serum, and if it is positive, the presence of a Gram-negative bacterial infection is confirmed, and if it is negative, the presence of a Gram-positive bacterial infection is confirmed. If the procalcitonin result is negative and the (1-3)-β-D-glucan result is positive, Candida mannan antigen is additionally determined in the blood serum, and if the result is positive, the presence of a yeast infection is confirmed, and if the result is negative, an additional test for the presence of another fungal infection is prescribed. If the results for procalcitonin, (1-3)-β-D-glucan, endotoxin, and Candida mannan antigen are positive, venous blood is additionally collected into a vial with a bacterial growth inhibitor for haemocultivation of yeast fungi and placed in an analyser until a growth signal is expected. and if a growth signal is present, a culture is taken from this vial to identify the pathogen and determine its antibiotic and antimycotic sensitivity, and if there is no growth in the blood culture, blood is sampled again every 24 hours. If the results for procalcitonin, (1-3)-β-D-glucan, and Candida mannan antigen are positive and the endotoxin test is negative, Gram-positive bacterial and fungal infections are diagnosed, and if the results for procalcitonin, (1-3)-β-D-glucan, and Candida mannan antigen are negative and the endotoxin test is positive, Gram-negative bacterial and fungal infections are diagnosed. If procalcitonin and (1-3)-β-D-glucan are negative, repeat tests are performed every 24 hours. In the absence of blood culture growth, but with positive procalcitonin and (1-3)-β-D-glucan and Candida mannan antigen results, repeated blood cultures are recommended every 24 hours until culture growth is obtained.

EFFECT: enables the diagnosis of bacterial and fungal bloodstream infections by determining the concentration of procalcitonin, (1-3)-β-D-glucan, endotoxin, and Candida mannan antigen in the blood.

1 cl, 6 dwg, 7 ex

Description

The invention relates to medicine, namely to medical microbiology, and can be used in clinical practice for the diagnosis of bacterial and fungal infections of the bloodstream.

In the etiology of life-threatening infectious complications, gram-negative and gram-positive bacteria are most often encountered, but yeast fungi, including those of the genus Candida, play a significant role (see Barros N., Rosenblatt R.E., Phipps M.M., Fomin V., Mansour M.K. Invasive fungal infections in liver diseases. Hepatol Commun. 2023; 7(9): e0216. DOI: 10.1097/HC9.00000000000000216; see Klyasova G.A., Fedorova A.V., Frolova I.N., Khrulnova S.A., Vetokhina A.V., Kaporskaya T.S., et al. Antibiotic resistance of hospital strains of Enterococcus spp. isolated from blood cultures of patients with tumors of the system blood: results of a multicenter study. Clinical Microbiology and Antimicrobial Chemotherapy. 2018; 20 (2): 142–149; see Klyasova G. A., Malchikova A., Maschan M., Molchanova I., Kutsevalova O., Chernenkaya T., et al. In vitro activity of echinocandins and azoles against Candida spp. isolated from hematological (hem) and non-hematological (non-hem) patients in 11 centers of Russia. Mycoses. 2017; 60; see Kutsevalova O. Yu., Pokudina I. O., Rozenko D. A., Martynov D. V., Kaminsky M. Yu. Current problems of antibiotic resistance in gram-negative pathogens of nosocomial infections in the Rostov Region. Medical Bulletin of the South of Russia. 2019. Vol. 10. No. 3. P. 91-96; see Kutsevalova O. Yu., Kit O. I., Panova N. I., Rozenko D. A., Yakubenko S. V. et al. Current trends in antibiotic therapy of gram-negative pathogens of nosocomial infections in the Rostov region. Antibiotics and chemotherapy. 2018. Vol. 63. No. 11-12. P. 24-30).

Fungal diagnosis remains challenging due to their slow growth and the need for specialized knowledge. Mixed bacterial-fungal infections further complicate diagnosis and prolong the time to result, which is vital for effective treatment (see Ferngren G, Yu D, Unalan-Altintop T, Dinnétz P, Özenci V. Epidemiological patterns of candidaemia: A comprehensive analysis over a decade. Mycoses. 2024 May;67(5):e13729. doi: 10.1111/myc.13729. PMID: 38682399).

The clinical features of candidemia are nonspecific and indistinguishable from those of bacterial sepsis (see McCarty TP, White CM, Pappas PG. Candidemia and Invasive Candidiasis. Infect Dis Clin North Am. 2021 Jun;35(2):389-413. doi: 10.1016/j.idc.2021.03.007. PMID: 34016283).

Blood culture remains the gold standard for diagnosis; however, the difficulties of cultivation make this method insufficiently reliable (see Kutsevalova O.Yu., Yankovskaya G.V., Astvatsaturyan E.I. Characteristics of invasive candidiasis pathogens. Problems of Medical Mycology. 2014. Vol. 16. No. 2. P. 97; see Krylov V.B., Solovev, A.S., Puchkin, I.A., Yashunsky, D.V., Antonets, A.V. et. al. Reinvestigation of Carbohydrate Specificity of EBCA-1 Monoclonal Antibody Used for the Detection of Candida Mannan. Journal of Fungi. 2021. Vol. 7. No. 7. P. 504-512. DOI: 10.3390/jof7070504; see Kutsevalova O. Yu., Kozel Yu. Yu., Alaverdyan A. I., Gusak D. A. Analysis of the etiological structure of bloodstream infections using the automatic bacteriological analyzer JUNONA® LABSTAR 100. Clinical laboratory diagnostics. 2022; 67 (2): 101-105. DOI: 10.37489/0235-2990-2022-67-5-6-30-38).

The rate of positive results in microbiological blood testing is 21–71% and depends on the patient profile, sampling frequency, and the volume of blood collected (see Haydour Q, Hage CA, Carmona EM, Epelbaum O, Evans SE, Gabe LM, et al. Diagnosis of Fungal Infections. A Systematic Review and Meta-Analysis Supporting the American Thoracic Society Practice Guideline. Ann Am Thorac Soc. 2019; 16(9):1179–1188. DOI:10.1513/AnnalsATS.201811-766OC).

New diagnostic methods are needed to complement traditional culture tests, particularly to identify the “missing percentage” of patients with negative blood cultures (see Clancy CJ, Nguyen MH. Finding the “missing 50%” of invasive candidiasis: how nonculture diagnostics will improve understanding of the disease spectrum and transform patient care. Clin Infect Dis. 2013; 56(9):1284-92. DOI:10.1093/cid/cit006).

The use of biomarkers that assess the risk of developing yeast invasion may facilitate the development of an initial antifungal strategy. Negative biomarker values may also be useful to exclude an invasive process and discontinue unnecessary antifungal therapy (see Lass-Flörl C, Kanj S.S., Govender N.P., Thompson G.R. 3rd, Ostrosky-Zeichner L., Govrins M.A. Invasive candidiasis. Nat Rev Dis Primers. 2024; 10(1):20. DOI: 10.1038/s41572-024-00503-3; see Fabre V., Sharara S.L., Salinas A.B., Carroll K.C., Desai S., Cosgrove S.E. Does This Patient Need Blood Cultures? A Scoping Review of Indications for Blood Cultures in Adult Nonneutropenic Inpatients. Clin Infect Dis. 2020; 71(5):1339-1347. DOI:10.1093/cid/ciaa039).

In each specific case, if a generalized infectious process is suspected, an individual diagnostic search algorithm should be developed, which includes a targeted examination using the most informative methods in a given situation (see Kit O.I., Kutsevalova O.Yu., Lysenko I.B., Dmitrieva V.V., Shalashnaya E.V., Panova N.I. Method for determining the etiology of fever of unknown genesis in cancer patients. Patent for invention RU 2647456 C1, 15.03.2018. Application No. 2017112212 dated 10.04.2017).

The technical result of the proposed method is the development of a method that allows for the diagnosis of bacterial and fungal bloodstream infections.

The technical result is achieved in that, if a bloodstream infection is suspected, blood is collected from the patient's cubital vein into one test tube to obtain human blood serum, and in parallel, blood is collected into two vials for hemoculture of aerobic and anaerobic microflora, which are placed in the analyzer until a growth signal is expected, and if a growth signal is present, seeding is performed to identify the pathogen and determine its antibiotic sensitivity, and if there is no signal, repeated blood sampling is performed every 24 hours, and the concentration of procalcitonin and (1-3)-β-D-glucan is determined in the blood serum, and if the result for procalcitonin is positive and (1-3)-β-D-glucan is negative, bacterial endotoxin is additionally determined in the blood serum, and if its result is positive, the presence of a gram-negative bacterial infection is ascertained, and if its result is negative, the presence of a gram-positive bacterial infection is ascertained; If the procalcitonin result is negative and (1-3)-β-D-glucan is positive, the mannan antigen is additionally determined.Candidain the blood serum and if its result is positive, the presence of a yeast infection is confirmed, and if its result is negative, an additional study is prescribed for the presence of another fungal infection; if the results are positive, procalcitonin, (1-3)-β-D-glucan, endotoxin and mannan antigenCandidaAdditionally, venous blood is collected in a vial with a bacterial growth inhibitor for hemoculture of yeast fungi and placed in the analyzer until a growth signal is detected, and if a growth signal is detected, cultures are taken from this vial to identify the pathogen and determine its antibiotic and antifungal sensitivity; and if there is no growth in the hemoculture, repeat blood sampling is performed every 24 hours; if procalcitonin, (1-3)-β-D-glucan, and mannan antigen results are positiveCandidaand negative endotoxin indicates the presence of a gram-positive bacterial and yeast infection; if procalcitonin and (1-3)-β-D-glucan results are negative, repeated studies are carried out dynamically every 24 hours, in the absence of blood culture growth, but if procalcitonin and (1-3)-β-D-glucan and mannan antigen results are positiveCandidaIt is recommended to repeat blood cultures every 24 hours until culture growth is obtained.

The novelty of the method is that the use of a certain sequence of a complex of biomarkers: procalcitonin, (1-3)-β-D-glucan, Candida mannan antigen and bacterial endotoxin and a vial with a bacterial growth inhibitor for hemoculture of yeast fungi allows for the diagnosis of bacterial and fungal infections of the bloodstream in a short time, without waiting for the standard 5 days of cultivation, thereby prescribing early adequate empirical therapy.

The method was carried out at the clinical microbiology laboratory of the National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation, Rostov-on-Don, from 01.01.2022 to 30.06.2024.

Venous blood samples served as the material for the study.

The blood of patients with suspected generalized infection was examined using the JUNONA® Labstar 100 automatic blood culture analyzer (SCENKER Biological Technology Co., Ltd., China).

The study of biomarkers was carried out using reagent kits for determining the level of procalcitonin (PCT) (Vector-Best, Russia), Candida mannan antigen using PLATELIA™ Candida Ag PLUS kits (Bio-Rad Laboratories, France), a test for bacterial endotoxin and determination of (1-3)-β-D-glucan (Era Biology Group, China).

The method is performed as follows.

If a bloodstream infection is suspected, blood is drawn from the patient's cubital vein into one test tube to obtain human blood serum, and blood is cultured in parallel, according to the current Methodological Guidelines MU 4.2.2039-05 "Technique for collecting and transporting biomaterials to microbiological laboratories" (see Methodological Guidelines MU 4.2.2039-05 "Technique for collecting and transporting biomaterials to microbiological laboratories" (approved and put into effect by the Chief State Sanitary Doctor of the Russian Federation on December 23, 2005) into vials with a nutrient medium with antibiotic neutralizers for cultivating aerobes and anaerobes JUNONA®, which are placed in the analyzer until a growth signal is expected, and if a growth signal is present, culture is performed to identify the pathogen and determine its antibiotic sensitivity, and if there is no signal, blood is re-drawn every 24 hours, and the concentration of PCT and (1-3)-β-D-glucan.

If the PCT result is positive and (1-3)-β-D-glucan is negative, bacterial endotoxin is additionally determined in the blood serum, and if it is positive, the presence of a gram-negative bacterial infection is confirmed, and if it is negative, the presence of a gram-positive bacterial infection is confirmed; if the PCT result is negative and (1-3)-β-D-glucan is positive, Candida mannan antigen is additionally determined in the blood serum, and if it is positive, the presence of a yeast infection is confirmed, and if it is negative, an additional study is prescribed for the presence of another fungal infection.

If the results of PCT, (1-3)-β-D-glucan, endotoxin and Candida mannan antigen are positive, venous blood is additionally collected into a vial with a bacterial growth inhibitor for hemoculture of yeast fungi and placed in the analyzer until a growth signal is detected. If a growth signal is detected, culture from this vial is performed to identify the pathogen and determine its antibiotic and antifungal sensitivity; if there is no growth in the hemoculture, repeat blood sampling is performed every 24 hours.

With positive results of PCT, (1-3)-β-D-glucan, mannan antigenCandidaand negative endotoxin is used to confirm the presence of gram-positive bacterial and yeast infections; if the results of PCT and (1-3)-β-D-glucan are negative, repeated studies are carried out dynamically every 24 hours.

If there is no blood culture growth, but the results of PCT and (1-3)-β-D-glucan and Candida mannan antigen are positive, repeat blood cultures are recommended every 24 hours until culture growth is obtained.

Preparation of chloramphenicol solution (Sigma-Aldrich) for hemoculture of yeast fungi .

Dissolve 50 mg of chloramphenicol in 2 ml of ethyl alcohol. Mix thoroughly and aseptically dispense 150 µl into sterile conical eppendorf tubes. Next, transfer the contents of each eppendorf tube (through a puncture in the rubber stopper) using a syringe into a vial containing Juno® aerobic culture medium. Prepared vials with the inhibitor are tested for the absence of contamination by microorganisms by incubation in an aerobic atmosphere at 35°C for 72 hours (see Menshikov V.V., Kozlov R.S., Polyak M.S., Mikhailova V.S., Inozemtseva L.O., Shulyak B.F. et al. Clinical guidelines Intralaboratory quality control of nutrient media for clinical microbiological studies. 2014. URL: https://fedlab.ru/upload/medialibrary/3ca/kochetov-ag.-klin.-rek._-kld_-2014_-vlkk-pitatelnykh-sred.pdf). An optical sensor at the bottom of the vial will allow one to judge its sterility.

We provide examples of the method's application.

Example 1. Patient A., 48 years old, was admitted to the abdominal department of the National Medical Research Center of Oncology with a diagnosis of stage 3 gastric malignancy. After a repeat operation for anastomotic leak, she was transferred to the anesthesiology and intensive care department. Heart rate >90/min, respiratory rate >20/min, pulse 95 beats per minute, low blood pressure (80/65 mmHg). General weakness, confusion, fever, temperature 38.9°C. Laboratory parameters of a complete blood count (CBC): Hb - 76 g/l, c.p - 0.80, erythrocytes 2.89×1012/l, leukocytes 25×10 ⁹ /l, ESR 50 mm/h.

The patient's blood was collected from the cubital vein into one test tube to obtain human blood serum and, in parallel, for blood seeding into two vials for hemoculture of aerobic and anaerobic microflora, which were placed in the analyzer until a growth signal was detected.

In parallel, procalcitonin (PCT) was determined in the blood serum - a concentration of 12.8 ng/ml and (1-3)-β-D-glucan - a concentration of 50.9 pg/ml. Additionally, since PCT was positive, an endotoxin test was performed - a concentration of 0.62.

A gram-negative infection was diagnosed. Based on PCT and bacterial endotoxin concentrations, empirical antibacterial therapy was prescribed, taking into account the gram-negative pathogen.

After 19 hours, a growth signal was obtained in a flask with a nutrient medium with antibiotic neutralizers for cultivating JUNO® aerobes (see Fig. 1).

After another 38 hours, the identification results and antibiogram were obtained: multidrug-resistant P. aeruginosa producing carbapenemases. Targeted adjustments to antibacterial therapy were made.

It was concluded that the patient had a confirmed Gram-negative bloodstream infection. Biomarker results allowed for immediate initiation of appropriate therapy, and the antibiogram results were adjusted.

Example 2. Patient P., 56 years old, was admitted to the therapy department of Regional Clinical Hospital No. 1 with a preliminary diagnosis of pneumonia and sepsis. On admission: heart rate 96 beats per minute, respiratory rate 25 beats per minute, pulse 112 beats per minute, blood pressure 75/60 mmHg. General weakness, fever, body temperature within 37.8-38.2°C. Shortness of breath and sweating with minimal physical exertion. Laboratory parameters of the complete blood count: Hb - 110 g/l, c.p - 0.82, erythrocytes 3.0×10 ¹² /l, leukocytes 12×10 ⁹ /l, ESR 30 mm/h.

The patient's blood was collected from the cubital vein into one test tube to obtain human blood serum and, in parallel, for blood seeding into two vials for hemoculture of aerobic and anaerobic microflora, which were placed in the analyzer until a growth signal was detected.

In parallel, PCT was determined in the blood serum - a concentration of 8 ng/ml, a test for bacterial endotoxin - a concentration of 0.30, and the concentration of (1-3)-β-D-glucan - 45.5 pg/ml.

A gram-positive infection was diagnosed. Based on positive PCT and negative bacterial endotoxin tests, empirical antibacterial therapy was prescribed, taking into account the gram-positive pathogen.

After 23 hours, a growth signal was detected in a vial of JUNONA® aerobic culture medium containing antibiotic neutralizers. Microscopic examination revealed gram-positive cocci.

After another 38 hours, the final S. pneumoniae result was obtained. The pathogen was identified and its antibiotic susceptibility was determined. By the time the culture grew, positive dynamics were observed. The culture results and antibiogram allowed for adjustment of therapy.

Example 3. Patient M., 21 years old. Admitted to the surgical department of Regional Clinical Hospital No. 1 with a diagnosis of acute appendicitis. Five days after appendectomy, the patient complained of abdominal pain and nausea. On examination: functional intestinal obstruction, abdominal muscle tension, severe intoxication. The condition is severe. HR 95/min, RR 25/min, pulse 110 beats per minute, low blood pressure (75/60 mmHg). Laboratory parameters of the complete blood count: Hb - 95 g / l, c.p - 0.80, erythrocytes 2.80 × 10 ¹² / l, leukocytes 25 × 10 ⁹ / l, ESR 35 mm / h. General weakness, fever, elevated temperature (37.8-38.5 ° C).

The patient's blood was collected from the cubital vein into one test tube to obtain human blood serum and, in parallel, for blood seeding into two vials for hemoculture of aerobic and anaerobic microflora, which were placed in the analyzer until a growth signal was detected.

In parallel, the following were determined in the blood serum: PCT concentration - 0.2 ng/ml, (1-3)-β-D-glucan - 80 pg/ml. Concentration of Candida spp. mannan antigen - 350 pg/ml.

A yeast infection was diagnosed. Based on the results of PCT and (1-3)-β-D-glucan concentrations and Candida spp. mannan antigen, empirical antifungal therapy was prescribed.

After 41 hours, a growth signal was detected in a vial of JUNONA® aerobic nutrient medium containing antibiotic neutralizers. Microscopic examination revealed yeast cells. After another 2 days, Candida glabrata growth was observed. The pathogen was identified and its antifungal susceptibility was determined (see Fig. 2).

Thus, using the proposed method, an adequate initial therapy result was achieved more than 40 hours earlier. The final therapy was adjusted based on the microbiological test results.

Example 4. Patient F., 59, was admitted to the abdominal department of the National Medical Research Center of Oncology with a diagnosis of colon malignancy. After a repeat operation for intestinal anastomotic leak, she was transferred in a moderate condition to the anesthesiology and intensive care department. Heart rate 92 bpm, respiratory rate 28 bpm, pulse 95 bpm, blood pressure 70/90 mmHg. General weakness, confusion, fever, temperature up to 39.2°C. Laboratory parameters of the complete blood count: Hb - 75 g/l, c.p - 0.80, erythrocytes 2.67×10 ¹² /l, leukocytes 19×10 ⁹ /l, ESR 32 mm/h.

The patient's blood was collected from the cubital vein into one test tube to obtain human blood serum and, in parallel, for blood seeding into two vials for hemoculture of aerobic and anaerobic microflora, which were placed in the analyzer until a growth signal was detected.

In parallel, the following were determined in the blood serum: PCT concentration - 12.8 ng/ml, endotoxin test - concentration 0.65, (1-3)-β-D-glucan concentration - 83 pg/ml. Concentration of Candida spp. mannan antigen - 500 pg/ml.

A bacterial and fungal infection was diagnosed. Additionally, venous blood was collected into a vial containing a bacterial growth inhibitor for blood culture of yeast fungi and placed in the analyzer until a growth signal was detected.

Empirical therapy was prescribed taking into account mixed infection (possible gram-negative pathogen and yeast fungi).

After 12 hours, a growth signal was obtained in a vial of JUNONA® aerobic nutrient medium containing antibiotic neutralizers. Microscopy revealed gram-negative bacteria. The identification result was K. pneumoniae (see Fig. 3).

The pathogen was identified and its antibiotic sensitivity was determined.

After another 50 hours, a growth signal was detected in a vial of JUNONA® aerobic nutrient medium with antibiotic neutralizers and a bacterial growth inhibitor. The identification result was Candida glabrata (see Fig. 4).

The pathogen was identified and its antifungal sensitivity was determined.

The proposed algorithm allowed us to identify the most problematic bacterial-candidal association and promptly initiate appropriate empirical therapy for the bacterial-fungal mixed infection based on biomarker results. An additional fungal flagellant, in turn, enabled the growth of C. glabrata in association with K. pneumoniae .

It should be noted that K. pneumoniae is poorly responsive to empirical therapy due to its high resistance rate. C. glabrata fungi are also difficult to treat, as they are naturally resistant to major antifungal agents. An additional vial of bacterial growth inhibitor allowed us to isolate the pathogens by association, confirm candidal infection, and conduct a test.

Example 5. Patient M., 30, was admitted to the surgical department of Rostov State Medical University with a diagnosis of pancreatic necrosis. On examination, she complained of severe girdle-like pain in the left abdomen and lower back. Body temperature was 38.0°C and higher. Intoxication. An emergency operation was performed. A few days later, a repeat pancreatic surgery was performed due to anastomotic and suture leakage. The patient was transferred to the intensive care unit in a moderate condition. Heart rate was 90/min, respiratory rate 22/min, white blood cell count was 12×10 ⁹ /L. General weakness, fever, temperature above 38.0°C. Laboratory parameters of the complete blood count: Hb – 90 g/l, c.p – 0.80, erythrocytes 2.6×1012/l, leukocytes 15×10 ⁹ /l, ESR 35 mm/h.

The patient's blood was collected from the cubital vein into one test tube to obtain human blood serum and, in parallel, for blood seeding into two vials for hemoculture of aerobic and anaerobic microflora, which were placed in the analyzer until a growth signal was detected.

In parallel, the following were determined in the blood serum: PCT concentration - 12.8 ng/ml, endotoxin test - 0.28, (1-3)-β-D-glucan - 116.8 pg/ml. Concentration of Candida spp. mannan antigen - 250 pg/ml.

Based on the results of the proposed algorithm, antibacterial (taking into account the gram-positive pathogen) and antifungal therapy were prescribed.

After 14 hours, a growth signal was observed; microscopic examination revealed gram-positive chains of cocci (see Fig. 5).

The pathogen was identified by growth of E. faecium , and its antibiotic susceptibility was determined. A signal was detected in a vial of JUNONA® aerobic culture medium with a bacterial growth inhibitor after 25 hours of final identification. Microscopic examination revealed yeast cells. Two days later, the final result was C. albicans (see Fig. 6).

The pathogen was identified - the growth of C. albicans - and its antifungal sensitivity was determined.

A bacterial and fungal infection was diagnosed. Additionally, venous blood was collected into a vial containing a bacterial growth inhibitor for blood culture of yeast fungi and placed in the analyzer until a growth signal was detected.

Biomarker results enabled timely and adequate empirical therapy for the bacterial-fungal mixed infection. The (1-3)-β-D-glucan and mannan antigen results allowed for the detection of a yeast infection and early initiation of antifungal therapy.

Using a bottle with a bacterial growth inhibitor for culturing yeast fungi, a culture was obtainedC. albicans.Identification has been carried out and determination of the antimycoticogram. The involvement of yeast fungi in the etiology of the infectious process was confirmed.

Example 6. Patient E., 47 years old, was admitted to the oncohematology department of the National Medical Research Center of Oncology with a diagnosis of acute lymphoblastic leukemia. Upon examination, the patient complained of general weakness and an increase in body temperature to 37.8°C. Heart rate was 92/min, respiratory rate was 22/min. Laboratory parameters of the complete blood count: Hb - 45 g/l, c.p - 0.80, erythrocytes 0.9x10 ¹² /l, lymphocytes 73.7%, monocytes 15.9%, leukocytes 20.9x10 ⁹ /l, platelets - 84 thousand, ESR 66 mm/h.

The patient's blood was collected from the cubital vein into one test tube to obtain human blood serum and, in parallel, for blood seeding into two vials for hemoculture of aerobic and anaerobic microflora, which were placed in the analyzer until a growth signal was detected.

In parallel, the following were determined in the blood serum: PCT concentration - 1.8 ng/ml, (1-3)-β-D-glucan concentration - 48.5 pg/ml.

After 24 hours, there was no growth in blood culture; given the severity of the condition, repeat studies were prescribed according to the algorithm in dynamics after 24 hours.

Example 7. Patient E., 17, was admitted to the pediatric department of the National Medical Research Center of Oncology with a diagnosis of acute lymphoblastic leukemia, type B-II, for planned chemotherapy. After the next round of chemotherapy, the patient complained of general weakness, fatigue, and a fever of 37.5-37.8°C.

Heart rate - 94/min, respiratory rate 26/min. Laboratory parameters of the complete blood count: Hb - 27 g/l, c.p - 0.80, erythrocytes 0.9×10 ¹² /l, lymphocytes 73.7%, monocytes 15.9%, leukocytes 20.9×10 ⁹ /l, platelets - 84 thousand, ESR 80 mm/h.

The patient's blood was collected from the cubital vein into one test tube to obtain human blood serum and, in parallel, for blood seeding into two vials for hemoculture of aerobic and anaerobic microflora, which were placed in the analyzer until a growth signal was detected.

In parallel, the following were determined in the blood serum: PCT concentration - 38.4 ng/ml, endotoxin test - concentration 0.28, (1-3)-β-D-glucan concentration - 186.3 pg/ml, Candida spp. mannan antigen concentration - 250 pg/ml.

The presence of a bacterial-fungal infection was confirmed.

Additionally, venous blood was collected into a vial with a bacterial growth inhibitor for hemoculture of yeast fungi and placed in the analyzer until a growth signal was received.

Blood culture results after 24 hours showed no growth. Repeat blood cultures were ordered every 24 hours to obtain a blood culture. Empirical therapy showed improvement after 3 days. On day 5, PCT levels were 5.8 ng/ml, and (1-3)-β-D-glucan levels were 59 pg/ml.

Using this method, the prognosis for 120 patients was calculated.

The technical and economic efficiency of the method lies in the fact that the use of a complex of biomarkers in a certain sequence: procalcitonin, (1-3) - β-D-glucan, Candida mannan antigen and bacterial endotoxin and a vial with a bacterial growth inhibitor for hemoculture of yeast fungi allows for the diagnosis of bacterial and fungal infections of the bloodstream.

Claims (1)

A method for diagnosing bacterial and fungal bloodstream infections, which consists in the fact that, if a bloodstream infection is suspected, blood is collected from the cubital vein of a patient into one test tube to obtain human blood serum, blood is simultaneously collected into two vials for hemoculture of aerobic and anaerobic microflora, which are placed in an analyzer until a growth signal is expected, and if a growth signal is present, seeding is carried out to identify the pathogen and determine its antibiotic sensitivity, and if there is no signal, blood is re-sampled every 24 hours, and the concentration of procalcitonin and (1-3)-β-D-glucan is determined in the blood serum, and if the result for procalcitonin is positive and (1-3)-β-D-glucan is negative, bacterial endotoxin is additionally determined in the blood serum and, if the result is positive, the presence of a gram-negative bacterial infection is ascertained, and if the result is negative, the presence of a gram-positive bacterial infection is ascertained; if the procalcitonin result is negative and (1-3)-β-D-glucan is positive, the Candida mannan antigen is additionally determined in the blood serum, and if it is positive, the presence of a yeast infection is confirmed, and if it is negative, an additional study is prescribed for the presence of another fungal infection; if the results of procalcitonin, (1-3)-β-D-glucan, endotoxin, and Candida mannan antigen are positive, venous blood is additionally collected into a vial with a bacterial growth inhibitor for hemoculture of yeast fungi and placed in the analyzer until a growth signal is detected, and if a growth signal is present, culture is performed from this vial to identify the pathogen and determine its antibiotic and antifungal sensitivity; and if there is no growth in the hemoculture, blood is re-sampled every 24 hours; If the results of procalcitonin, (1-3)-β-D-glucan, and Candida mannan antigen are positive and endotoxin is negative, gram-positive bacterial and fungal infections are diagnosed, and if endotoxin is positive, gram-negative bacterial and fungal infections are diagnosed; if the results of procalcitonin and (1-3)-β-D-glucan are negative, repeated studies are carried out dynamically every 24 hours, in the absence of blood culture growth, but if the results of procalcitonin and (1-3)-β-D-glucan and Candida mannan antigen are positive, repeated blood culture is recommended every 24 hours until culture growth is obtained.