- Email: [email protected]
Cryoglobulinemic purpura in visceral leishmaniasis
Letters to the Editor
271 *Corresponding author.
E-mail address: [email protected] Accepted 20 March 2015 http://dx.doi.org/10.1016/j.jinf.2015.03.005 ª 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Cryoglobulinemic purpura in visceral leishmaniasis
KEYWORDS Cryoglobulinemia; Leishmaniasis; Purpura
Dear Editor, We read the spotlessly review on leishmaniasis1 published in your journal recently and would like to present an uncommon clinical manifestation of visceral leishmaniasis. An 85 year-old man was admitted to the emergency room of the hospital due to a 10-day history of macroscopic hematuria, as well as thrombocytopenia which had been found by a laboratory test. Moreover, he reported fever, perspiration during the night, fatigue, 10 kg weight loss during the last month, and appearance of a rash on his legs which had already regressed at the time of his admission to the hospital. His medical record includes coronary disease and coronary artery bybass surgery, hypertension, hypertrophy of the prostate gland and appendectomy. He was under medication, which included carvedilol 75 mg, isosorbite-5-mononitrate 60 mg, acetylsalicylic acid 100 mg and metoprolol 100 mg. He is neither a smoker nor a drinker, and he is the owner of a dog. On admission, physical examination disclosed splenomegaly, as well as hepatomegaly and lymphadenia of the left axilla. Abdominal sonography and the computed tomography scan confirmed the hepatic and splenic enlargement, both of which were measured to be 19 cm. Moreover, a small infarct was found in the middle of the spleen. Major laboratory findings included pancytopenia, hypoalbuminemia, elevated levels of C-reactive protein (CRP), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and g-gloutamyltraspeptidase (gGT), hypergammaglobulinemia, increased IgG globulin and b2-microglobulin and a positive rheumatoid factor (RF). Urine microscopy proved hematuria and proteinuria. The patient tested positive for antibodies against the K39 antigen of Leishmania and antibodies against Leishmania (1/320) in the serum. Furthermore, polymerase
chain reaction (PCR) was positive for the presence of protozoa of Leishmania in the blood, but negative for their presence at the bone marrow. However, contrary to expectations, Leismania was not found in the bone marrow biopsy. Because of the above findings, we initiated treatment with liposomal amphotericin B (3 mg/kg days 1e5, 14 and 21). However, on the 9th day of his hospitalization, the patient developed palpable purpura on both legs (Fig. 1). We checked for cryoglobulins, and a cryocrit of 3.5% was detected. Moreover, a remarkable renal dysfunction was noticed (glomerular filtration rate (GFR) was 69 ml/min on the day the patient was admitted to the hospital, but was found to be 34 ml/min on the 8th day of hospitalization). The patient tested negative for hepatitis B and C and human immunodeficiency virus infection. By the 14th day of the hospitalization, the purpura had completely regressed without any additional therapy. GFR was stable at 35 ml/min. We measured again the cryocrit and found out that it had noticeably decreased. Moreover, thrombopenia and leukopenia had regressed. The patient was discharged in a good performance status, after having taken 1500 mg of liposomal amfotericin B. He was advised to visit the hospital again, in order to receive two more doses of the treatment (on days 14 and 21 of the treatment). On the last day of the treatment (day 21) we noticed improved renal function (GFR: 51 ml/min), and also a significant reduction of the spleen (15 cm at the abdominal ultrasound). No purpura was detected (Table 1). In general, patients suffering from visceral leishmaniasis may exhibit fever, fatigue, anorexia, nausea, weight loss, pallor, abdominal discomfort, joint pain, arthralgia or cough, vomiting, diarrhea and bleeding. The clinical signs are hepatomegaly, splenomegaly, and more rarely lymphadenopathy and cutaneous vasculitis. Laboratory findings include pancytopenia, hypergammaglobulinaemia and absence of detectable cell-mediated immunity. More rarely, VL is also associated with manifestations of autoimmune diseases. These include low serum complement levels, increased titers of RF, antinuclear antibodies (ANA), anti-glomerular basement-membrane antibodies (GBM), anti-striated muscle antibodies (AMA) and anti-smooth
Figure 1
Cryoglobulinemic purpura.
272 Table 1
Letters to the Editor Evolution of serum laboratory and immunological parameters of the patient, on admission and during follow-up.
Laboratory variables
On admission
5th day of therapy
Followeup (9 days after the consummation of the treatment)
Hemoglobin (12e18 g/dl) White blood cell count (5.2e12.4 109/l) Platelet count (130e140 109/l) C-reactive protein (<5 mg/l) g-gloutamyltraspeptidase (10e75 IU/l) Alkaline phosphatase (20e130 IU/l) Creatinine (0.7e1.5 mg/dl) Serum albumin (3.5e5 g/dl) Rheumatoid factor (<15 IU/ml) Antinuclear antibodies titer Cryoglobulin cryocrit (%) C3c (10e40 mg/dl) C4 (<15 mg/dl) IgG (700e1600 mg/dl) Red blood cell in urine test
13.2 3.91 65 70 79 77 1.2 2.5 603 þ(1/360) 3.5 91 17.9 2833 12e15
9.8 4.49 159 18 87 88 1.9 2.5 612
10.4 4.48 181 1.28
muscle antibodies (ASMA), and rarely presence of monoclonal gammopathy or cryoglobulins.1e7 Cryoglobulinemia is the formation of cryoglobulins in the serum. It can accompany various diseases and is classically grouped into three types. Type I is characterized by a monoclonal cryoglobulin component (usually IgM paraprotein), and is associated with lymphoproliferative and myeloproliferative disorders. On the other hand, cryoglobulinemia of type II and III (mixed cryoglobulinemia) is associated with lymphoproliferative and connective tissue disorders, along with viral (mostly hepatitis C) and bacterial infections. Type-II cryoglobulins are immunocomplexes that have a polyclonal component, usually IgG and a monoclonal component, usually IgM or IgA, which has an RF function. Type-III cryoglobulins are composed of polyclonal IgM and IgG molecules. The main clinical manifestations of cryoglobulinemia are purpura, arthralgia and weakness. In addition, cryoglobulinemic vascuilitis may affect various organs, and cause peripheral neuropathy, acute nephritic or nephrotic syndromes, skin ulcers and diffuse vasculitis.8e10 Although in the case of our patient, we were not able to determine the type of cryoglobulinemia, the bacterial nature of the infection points to mixed cryoglobulinemia. Various explanations for the formation of cryoglobulins have been proposed. They include genetic factors, activation of protooncogenes, prolonged antigenic stimulation, infectious agents, polyclonal activation of B lymphocytes, reduced clearance of immune complexes in the liver, and cross-reactivity of various antigens. However, the phenomenon of molecular mimicry that has been associated with the formation of lupus-related autoantibodies, such as antinucleoprotein, anti-Ro, anti-La and anti-Sm, can perhaps explain better the induction of cryoglobulinemia. Patients with cryoglobulinemia secondary to VL or hepatitis C have high titers of autoantibodies toward a common epitope of the lymphocyte activation gene 3 (LAG-3.1) protein product. These autoantibodies are not found in patients who suffer from hepatitis C and have no cryoglobulinemia. This suggests that the same mechanism of molecular mimicry may be responsible for the ability of both HCV and
traces 78 12.1 3427 70
1.04
15e20
Leishmania to produce mixed cryoglobulinemia, due to anti e LAG 3.1 autoantibody.2,3,6e8 To summarize, a patient with VL may be misdiagnosed as having an autoimmune disease, due to the common clinical and laboratory findings. If so, the potential treatment with immune-suppressive drugs may have fatal consequences. Therefore, it is important that the detection of antibodies for Leishmania is taken seriously into consideration, even if the parasite is not found in the bone marrow biopsy.
References 1. Pace D. Leishmaniasis. J Infect 2014;69(1):10e8. 2. Liberopoulos E, Kei A, Apostolou F, Elisaf M. Autoimmune manifestations in patients with visceral leishmaniasis. J Microbiol Immunol Infect 2013;46(4):302e5. 3. Casato M, de Rosa FG, Pucillo LP, Ilardi I, di Vico B, Zorzin LR, et al. Mixed cryoglobulinemia secondary to visceral Leishmaniasis. Arthritis Rheum 1999;42(9):2007e11. ~o-Castro B, Sa Ferreira JA, Marzochi KF, Marzochi MC, 4. Galva Coutinho SG, Lambert PH. Polyclonal B cell activation, circulating immune complexes and autoimmunity in human american visceral leishmaniasis. Clin Exp Immunol 1984;56(1): 58e66. 5. Voulgari PV, Pappas GA, Liberopoulos EN, Elisaf M, Skopouli FN, Drosos AA. Visceral leishmaniasis resembling systemic lupus erythematosus. Ann Rheum Dis 2004;63(10):1348e9. 6. Argov S, Jaffe CL, Krupp M, Slor H, Shoenfeld Y. Autoantibody production by patients infected with Leishmania. Clin Exp Immunol 1989;76(2):190e7. 7. Makaritsis KP, Gatselis NK, Ioannou M, Petinaki E, Dalekos GN. Polyclonal hypergammaglobulinemia and high smooth-muscle autoantibody titers with specificity against filamentous actin: consider visceral leishmaniasis, not just autoimmune hepatitis. Int J Infect Dis 2009;13(4):e157e60. 8. Rizos E, Dimos G, Liberopoulos EN, Elisaf MS, Drosos AA. Cryoglobulinemic purpura in visceral leishmaniasis. Rheumatol Int 2005;25(6):469e71. 9. Lamprecht P, Gause A, Gross WL. Cryoglobulinemic vasculitis. Arthritis Rheum 1999;42(12):2507e16. 10. Ferri C, Zignego AL, Pileri SA. Cryoglobulins. J Clin Pathol 2002; 55(1):4e13.
Letters to the Editor
273
Adamantia Petraki* Naso Kittou Emilia Hadziyannis Spyros P. Dourakis 2nd Department of Medicine, University of Athens Medical School, Hippokration General Hospital, Athens, Greece *Corresponding author. 2nd Department of Medicine, University of Athens Medical School, Hippokration General Hospital, 114 Vasilissis Sophias Avenue, 11 527 Athens, Greece. Tel.: þ30 6977508102; fax: þ30 2132088639.
E-mail address: [email protected] (A. Petraki) Accepted 7 March 2015 http://dx.doi.org/10.1016/j.jinf.2015.03.002 ª 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Increased IL-9 mRNA expression as a biomarker to diagnose childhood tuberculosis in a high burden settings
KEYWORDS Mycobacterium tuberculosis; IL-9; Cytokine; Biomarker; Diagnosis
We read with interest the paper by Armand et al. who evaluated the cytokine responses to Mtb antigens in pediatric tuberculosis,1 in which 5 of 19 cytokines/chemokines were showed to be different between LTBI/TB disease and non-infected controls, but none of them, including IFN-g was able to discriminate between LTBI and TB disease.1 The diagnosis of TB disease in young children remains a challenging because the infant immune system has deficits that delay the adaptive immune response to TB. The efforts have been made recently to look for novel markers for the immunodiagnostic potential. These markers included interferon-g-induced-protein-10 (IP-10)2 and a combination of four markers (IFN-a2, IL-RA, sCD40L and VEGF), which potentially detected tuberculosis in children.3 We evaluated the potential roles of 7 immunerelated markers which represent Th1 cells (IFN-g, IL-12b), Th2 cells (IL-9), Treg cells (FOXP3, IL-10) and inflammatory
responses (IL-6, IL-8), respectively in pediatric TB in an endemic region. In this prospective study, 39 patients (age 0e5 years old) with tuberculosis were recruited from TB Clinic at Chest Hospital of Xinjiang, Urumqi, China. PBMCs were obtained from all the patients before anti-TB therapy started. Except 3 smear culture positive, other 36 culture negative patients were classified with TB disease based on clinical characteristics. Diagnosis of 39 TB patients includes 13 with primary pulmonary TB, 26 with extrapulmonary TB. Congenital TB was excluded because all the mothers of the children with TB were heathy. HIV infection and immunosuppressive regimens were excluded for all the TB patients. Healthy subjects (n Z 25) were recruited among the children when visited for regular check-up at child health unit in People’s Hospital of Xinjiang as normal controls. We did not separate normal subjects into Mtb infected or non-infected individuals because all the normal subjects were 0e5 years old, BCG vaccinated and live in an endemic region. This study was approved by the institutional review boards of People’s Hospital of Xinjiang and Chest Hospital of Xinjiang, China. After 15 h stimulation by ESAT-6 protein, mRNA expression of 7 gene markers was measured in PBMC by using qPCR (Bio-Rad, Hercules, CA). Results were expressed as fold change. The difference of gene expression was analyzed with the KruskaleWallis test and ANOVA for multiple group comparison and ManneWhitney test for two group comparison. As shown in Fig. 1, compared to normal individuals, no difference of IFN-g expression was found in patients with TB disease (Fig. 1A), including pulmonary TB and extrapulmonary TB (Fig. 1C). However, significantly increased IL-9 expression in response to ESAT6 stimulation was observed in patients with TB disease (p < 0.0001) (Fig. 1B), particularly in pulmonary TB but not in extrapulmonary TB (Fig. 1D). The discrimination performance was assessed using ROC. With the cut-off of 1.74, maximum discrimination was reached between patients with TB disease and normal individuals (AUC Z 0.8313, 95% CI, 0.73e0.92), with 74.36% sensitivity, 84% specificity, and 78.13% of cases correctly classified (Fig. 2A). With the cut-off 9.85, maximum discrimination was reached between patients with pulmonary TB and normal individuals (AUC Z 0.9200, 95% CI, 0.83e1), with 69.23% sensitivity, 96% specificity, and 86.84% of cases correctly classified (Fig. 2B). All other genes remain no difference between patients with TB disease and normal controls. In this study, we found an increased IL-9 expression in children with pulmonary TB, which represents a potential to distinguish patients with TB disease from normal individuals in high burden settings where most individuals are exposed to environmental Mtb or nontuberculous mycobacteria. Th1 response provides an immune protection against Mtb infection for the host. An initiation of efficient Th1 response in Mtb infection relies on fully functional mature dendritic cells. Since dendritic cells in
271 *Corresponding author.
E-mail address: [email protected] Accepted 20 March 2015 http://dx.doi.org/10.1016/j.jinf.2015.03.005 ª 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Cryoglobulinemic purpura in visceral leishmaniasis
KEYWORDS Cryoglobulinemia; Leishmaniasis; Purpura
Dear Editor, We read the spotlessly review on leishmaniasis1 published in your journal recently and would like to present an uncommon clinical manifestation of visceral leishmaniasis. An 85 year-old man was admitted to the emergency room of the hospital due to a 10-day history of macroscopic hematuria, as well as thrombocytopenia which had been found by a laboratory test. Moreover, he reported fever, perspiration during the night, fatigue, 10 kg weight loss during the last month, and appearance of a rash on his legs which had already regressed at the time of his admission to the hospital. His medical record includes coronary disease and coronary artery bybass surgery, hypertension, hypertrophy of the prostate gland and appendectomy. He was under medication, which included carvedilol 75 mg, isosorbite-5-mononitrate 60 mg, acetylsalicylic acid 100 mg and metoprolol 100 mg. He is neither a smoker nor a drinker, and he is the owner of a dog. On admission, physical examination disclosed splenomegaly, as well as hepatomegaly and lymphadenia of the left axilla. Abdominal sonography and the computed tomography scan confirmed the hepatic and splenic enlargement, both of which were measured to be 19 cm. Moreover, a small infarct was found in the middle of the spleen. Major laboratory findings included pancytopenia, hypoalbuminemia, elevated levels of C-reactive protein (CRP), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and g-gloutamyltraspeptidase (gGT), hypergammaglobulinemia, increased IgG globulin and b2-microglobulin and a positive rheumatoid factor (RF). Urine microscopy proved hematuria and proteinuria. The patient tested positive for antibodies against the K39 antigen of Leishmania and antibodies against Leishmania (1/320) in the serum. Furthermore, polymerase
chain reaction (PCR) was positive for the presence of protozoa of Leishmania in the blood, but negative for their presence at the bone marrow. However, contrary to expectations, Leismania was not found in the bone marrow biopsy. Because of the above findings, we initiated treatment with liposomal amphotericin B (3 mg/kg days 1e5, 14 and 21). However, on the 9th day of his hospitalization, the patient developed palpable purpura on both legs (Fig. 1). We checked for cryoglobulins, and a cryocrit of 3.5% was detected. Moreover, a remarkable renal dysfunction was noticed (glomerular filtration rate (GFR) was 69 ml/min on the day the patient was admitted to the hospital, but was found to be 34 ml/min on the 8th day of hospitalization). The patient tested negative for hepatitis B and C and human immunodeficiency virus infection. By the 14th day of the hospitalization, the purpura had completely regressed without any additional therapy. GFR was stable at 35 ml/min. We measured again the cryocrit and found out that it had noticeably decreased. Moreover, thrombopenia and leukopenia had regressed. The patient was discharged in a good performance status, after having taken 1500 mg of liposomal amfotericin B. He was advised to visit the hospital again, in order to receive two more doses of the treatment (on days 14 and 21 of the treatment). On the last day of the treatment (day 21) we noticed improved renal function (GFR: 51 ml/min), and also a significant reduction of the spleen (15 cm at the abdominal ultrasound). No purpura was detected (Table 1). In general, patients suffering from visceral leishmaniasis may exhibit fever, fatigue, anorexia, nausea, weight loss, pallor, abdominal discomfort, joint pain, arthralgia or cough, vomiting, diarrhea and bleeding. The clinical signs are hepatomegaly, splenomegaly, and more rarely lymphadenopathy and cutaneous vasculitis. Laboratory findings include pancytopenia, hypergammaglobulinaemia and absence of detectable cell-mediated immunity. More rarely, VL is also associated with manifestations of autoimmune diseases. These include low serum complement levels, increased titers of RF, antinuclear antibodies (ANA), anti-glomerular basement-membrane antibodies (GBM), anti-striated muscle antibodies (AMA) and anti-smooth
Figure 1
Cryoglobulinemic purpura.
272 Table 1
Letters to the Editor Evolution of serum laboratory and immunological parameters of the patient, on admission and during follow-up.
Laboratory variables
On admission
5th day of therapy
Followeup (9 days after the consummation of the treatment)
Hemoglobin (12e18 g/dl) White blood cell count (5.2e12.4 109/l) Platelet count (130e140 109/l) C-reactive protein (<5 mg/l) g-gloutamyltraspeptidase (10e75 IU/l) Alkaline phosphatase (20e130 IU/l) Creatinine (0.7e1.5 mg/dl) Serum albumin (3.5e5 g/dl) Rheumatoid factor (<15 IU/ml) Antinuclear antibodies titer Cryoglobulin cryocrit (%) C3c (10e40 mg/dl) C4 (<15 mg/dl) IgG (700e1600 mg/dl) Red blood cell in urine test
13.2 3.91 65 70 79 77 1.2 2.5 603 þ(1/360) 3.5 91 17.9 2833 12e15
9.8 4.49 159 18 87 88 1.9 2.5 612
10.4 4.48 181 1.28
muscle antibodies (ASMA), and rarely presence of monoclonal gammopathy or cryoglobulins.1e7 Cryoglobulinemia is the formation of cryoglobulins in the serum. It can accompany various diseases and is classically grouped into three types. Type I is characterized by a monoclonal cryoglobulin component (usually IgM paraprotein), and is associated with lymphoproliferative and myeloproliferative disorders. On the other hand, cryoglobulinemia of type II and III (mixed cryoglobulinemia) is associated with lymphoproliferative and connective tissue disorders, along with viral (mostly hepatitis C) and bacterial infections. Type-II cryoglobulins are immunocomplexes that have a polyclonal component, usually IgG and a monoclonal component, usually IgM or IgA, which has an RF function. Type-III cryoglobulins are composed of polyclonal IgM and IgG molecules. The main clinical manifestations of cryoglobulinemia are purpura, arthralgia and weakness. In addition, cryoglobulinemic vascuilitis may affect various organs, and cause peripheral neuropathy, acute nephritic or nephrotic syndromes, skin ulcers and diffuse vasculitis.8e10 Although in the case of our patient, we were not able to determine the type of cryoglobulinemia, the bacterial nature of the infection points to mixed cryoglobulinemia. Various explanations for the formation of cryoglobulins have been proposed. They include genetic factors, activation of protooncogenes, prolonged antigenic stimulation, infectious agents, polyclonal activation of B lymphocytes, reduced clearance of immune complexes in the liver, and cross-reactivity of various antigens. However, the phenomenon of molecular mimicry that has been associated with the formation of lupus-related autoantibodies, such as antinucleoprotein, anti-Ro, anti-La and anti-Sm, can perhaps explain better the induction of cryoglobulinemia. Patients with cryoglobulinemia secondary to VL or hepatitis C have high titers of autoantibodies toward a common epitope of the lymphocyte activation gene 3 (LAG-3.1) protein product. These autoantibodies are not found in patients who suffer from hepatitis C and have no cryoglobulinemia. This suggests that the same mechanism of molecular mimicry may be responsible for the ability of both HCV and
traces 78 12.1 3427 70
1.04
15e20
Leishmania to produce mixed cryoglobulinemia, due to anti e LAG 3.1 autoantibody.2,3,6e8 To summarize, a patient with VL may be misdiagnosed as having an autoimmune disease, due to the common clinical and laboratory findings. If so, the potential treatment with immune-suppressive drugs may have fatal consequences. Therefore, it is important that the detection of antibodies for Leishmania is taken seriously into consideration, even if the parasite is not found in the bone marrow biopsy.
References 1. Pace D. Leishmaniasis. J Infect 2014;69(1):10e8. 2. Liberopoulos E, Kei A, Apostolou F, Elisaf M. Autoimmune manifestations in patients with visceral leishmaniasis. J Microbiol Immunol Infect 2013;46(4):302e5. 3. Casato M, de Rosa FG, Pucillo LP, Ilardi I, di Vico B, Zorzin LR, et al. Mixed cryoglobulinemia secondary to visceral Leishmaniasis. Arthritis Rheum 1999;42(9):2007e11. ~o-Castro B, Sa Ferreira JA, Marzochi KF, Marzochi MC, 4. Galva Coutinho SG, Lambert PH. Polyclonal B cell activation, circulating immune complexes and autoimmunity in human american visceral leishmaniasis. Clin Exp Immunol 1984;56(1): 58e66. 5. Voulgari PV, Pappas GA, Liberopoulos EN, Elisaf M, Skopouli FN, Drosos AA. Visceral leishmaniasis resembling systemic lupus erythematosus. Ann Rheum Dis 2004;63(10):1348e9. 6. Argov S, Jaffe CL, Krupp M, Slor H, Shoenfeld Y. Autoantibody production by patients infected with Leishmania. Clin Exp Immunol 1989;76(2):190e7. 7. Makaritsis KP, Gatselis NK, Ioannou M, Petinaki E, Dalekos GN. Polyclonal hypergammaglobulinemia and high smooth-muscle autoantibody titers with specificity against filamentous actin: consider visceral leishmaniasis, not just autoimmune hepatitis. Int J Infect Dis 2009;13(4):e157e60. 8. Rizos E, Dimos G, Liberopoulos EN, Elisaf MS, Drosos AA. Cryoglobulinemic purpura in visceral leishmaniasis. Rheumatol Int 2005;25(6):469e71. 9. Lamprecht P, Gause A, Gross WL. Cryoglobulinemic vasculitis. Arthritis Rheum 1999;42(12):2507e16. 10. Ferri C, Zignego AL, Pileri SA. Cryoglobulins. J Clin Pathol 2002; 55(1):4e13.
Letters to the Editor
273
Adamantia Petraki* Naso Kittou Emilia Hadziyannis Spyros P. Dourakis 2nd Department of Medicine, University of Athens Medical School, Hippokration General Hospital, Athens, Greece *Corresponding author. 2nd Department of Medicine, University of Athens Medical School, Hippokration General Hospital, 114 Vasilissis Sophias Avenue, 11 527 Athens, Greece. Tel.: þ30 6977508102; fax: þ30 2132088639.
E-mail address: [email protected] (A. Petraki) Accepted 7 March 2015 http://dx.doi.org/10.1016/j.jinf.2015.03.002 ª 2015 The British Infection Association. Published by Elsevier Ltd. All rights reserved.
Increased IL-9 mRNA expression as a biomarker to diagnose childhood tuberculosis in a high burden settings
KEYWORDS Mycobacterium tuberculosis; IL-9; Cytokine; Biomarker; Diagnosis
We read with interest the paper by Armand et al. who evaluated the cytokine responses to Mtb antigens in pediatric tuberculosis,1 in which 5 of 19 cytokines/chemokines were showed to be different between LTBI/TB disease and non-infected controls, but none of them, including IFN-g was able to discriminate between LTBI and TB disease.1 The diagnosis of TB disease in young children remains a challenging because the infant immune system has deficits that delay the adaptive immune response to TB. The efforts have been made recently to look for novel markers for the immunodiagnostic potential. These markers included interferon-g-induced-protein-10 (IP-10)2 and a combination of four markers (IFN-a2, IL-RA, sCD40L and VEGF), which potentially detected tuberculosis in children.3 We evaluated the potential roles of 7 immunerelated markers which represent Th1 cells (IFN-g, IL-12b), Th2 cells (IL-9), Treg cells (FOXP3, IL-10) and inflammatory
responses (IL-6, IL-8), respectively in pediatric TB in an endemic region. In this prospective study, 39 patients (age 0e5 years old) with tuberculosis were recruited from TB Clinic at Chest Hospital of Xinjiang, Urumqi, China. PBMCs were obtained from all the patients before anti-TB therapy started. Except 3 smear culture positive, other 36 culture negative patients were classified with TB disease based on clinical characteristics. Diagnosis of 39 TB patients includes 13 with primary pulmonary TB, 26 with extrapulmonary TB. Congenital TB was excluded because all the mothers of the children with TB were heathy. HIV infection and immunosuppressive regimens were excluded for all the TB patients. Healthy subjects (n Z 25) were recruited among the children when visited for regular check-up at child health unit in People’s Hospital of Xinjiang as normal controls. We did not separate normal subjects into Mtb infected or non-infected individuals because all the normal subjects were 0e5 years old, BCG vaccinated and live in an endemic region. This study was approved by the institutional review boards of People’s Hospital of Xinjiang and Chest Hospital of Xinjiang, China. After 15 h stimulation by ESAT-6 protein, mRNA expression of 7 gene markers was measured in PBMC by using qPCR (Bio-Rad, Hercules, CA). Results were expressed as fold change. The difference of gene expression was analyzed with the KruskaleWallis test and ANOVA for multiple group comparison and ManneWhitney test for two group comparison. As shown in Fig. 1, compared to normal individuals, no difference of IFN-g expression was found in patients with TB disease (Fig. 1A), including pulmonary TB and extrapulmonary TB (Fig. 1C). However, significantly increased IL-9 expression in response to ESAT6 stimulation was observed in patients with TB disease (p < 0.0001) (Fig. 1B), particularly in pulmonary TB but not in extrapulmonary TB (Fig. 1D). The discrimination performance was assessed using ROC. With the cut-off of 1.74, maximum discrimination was reached between patients with TB disease and normal individuals (AUC Z 0.8313, 95% CI, 0.73e0.92), with 74.36% sensitivity, 84% specificity, and 78.13% of cases correctly classified (Fig. 2A). With the cut-off 9.85, maximum discrimination was reached between patients with pulmonary TB and normal individuals (AUC Z 0.9200, 95% CI, 0.83e1), with 69.23% sensitivity, 96% specificity, and 86.84% of cases correctly classified (Fig. 2B). All other genes remain no difference between patients with TB disease and normal controls. In this study, we found an increased IL-9 expression in children with pulmonary TB, which represents a potential to distinguish patients with TB disease from normal individuals in high burden settings where most individuals are exposed to environmental Mtb or nontuberculous mycobacteria. Th1 response provides an immune protection against Mtb infection for the host. An initiation of efficient Th1 response in Mtb infection relies on fully functional mature dendritic cells. Since dendritic cells in