Diagnosis of Pneumocystis jirovecii pneumonia (PCP) is conventionally based on direct staining and visualization. Challenges in obtaining alveolar samples have stimulated interest in techniques for detection of Pneumocystis DNA in non-invasive samples, which can give good sensitivity and specificity. Robust diagnosis is key to ensuring appropriate therapy.
by Dr Farnaz Dave, Dr Ashley Horsley, Dr Thomas Whitfield
and Dr Clare van Halsema
Introduction
Pneumocystis jirovecii (previously Pneumocystis carinii) is a pathogen capable of causing life threatening Pneumocystis pneumonia (PCP) in the immunocompromised with case fatality rates among those hospitalized of around 10% [1]. PCP typically occurs in individuals with hematological malignancies on chemotherapy or with other causes of acquired cellular immunodeficiency or, most frequently, in human immunodeficiency virus (HIV)-positive individuals with CD4 T-cell counts <200 cells/µL or <14% of total white cell count [2, 3]. First-line treatment is co-trimoxazole, a combination of the antibiotics sulfamethoxazole and trimethoprim, at high dose for 3 weeks, which has the clinically significant potential side effects of bone marrow suppression, rash and bronchial hypersensitivity. Unfortunately the classical clinical presentation of PCP of progressive dry cough, dyspnoea and malaise is non-specific and chest examination and radiographs are often normal or near normal [4]. Oxygen desaturation on exercise is a helpful clinical sign in the right patient population [5]. Furthermore, many individuals with PCP do not produce sputum, so laboratory confirmation can be challenging. Given the serious nature of the illness and the possible side effects of treatment, accurate diagnosis is key to making an informed treatment decision.
Incidence of PCP among HIV-positive individuals has declined since the widespread availability of antiretroviral therapy (ART) and has also declined as a cause of hospitalization of HIV-positive individuals [6]. In a study in a large HIV centre in London, mortality for all hospitalizations was around 10% during the first 10 years of availability of effective ART [1]. In our unit a decrease in mortality among those admitted to intensive care with PCP was seen between the periods of 1986–1995 and 1996–2004. This improvement is thought to be due to advances in critical care rather than treatment of PCP itself [7].
Reaching a diagnosis
Traditional methods
P. jirovecii is a fungus that cannot be cultured in vitro and so the organism is identified using histochemical staining techniques of fluid samples. Grocott-Gomori methenamine silver nitrate or direct immunofluorescence monoclonal antibody (IFA) stains on deep respiratory samples are generally regarded as the gold standard in diagnosis [8]. The life cycle of P. jirovecii is demonstrated by trophic, pre-cystic and cystic forms by morphological criteria. Diagnosis through microscopy of the cystic stage requires significant technical expertise and can still lead to false-negative results. In florid disease, P. jirovecii is present throughout the bronchial tree, from the upper respiratory tract down to the alveolar surface. Induced sputum samples are recommended by most guidelines, if routinely available, as spontaneously expectorated sputum is not considered an adequate alveolar sample and microscopy could be falsely negative. If the results of testing on induced sputum are not conclusive then a bronchoalveolar lavage (BAL) is recommended (sensitivity 86–98%) [9, 10]. In clinical practice, induced sputum is often not readily available, and BAL may not be possible due to hypoxia or may lead to a delay in sampling of several days; therefore, in clinical practice spontaneously expectorated samples are often processed. The disadvantages of this are twofold: a lower yield of cystic forms for visualization and potential for false-positive results due to colonization. Only in rare cases would a lung biopsy be appropriate (sensitivity 95–98%) in circumstances of poor response to empirical treatment and negative initial testing [9].
Molecular techniques
Molecular testing of lower respiratory tract secretions and blood is an alternative and operator-independent method for confirming the presence of P. jirovecii. Nucleic acid amplification techniques (NAAT) can be used with a number of primers targeting different substrates – most commonly the major surface glycoprotein (MSG), mitochondrial large subunit (MTLSU) rRNA and internal transcribed spacer (ITS) region genes [11]. One potential pitfall with these techniques is that the detection of specific nucleic acid sequence does not distinguish between colonization and disease or between viable and non-viable organisms [12]. P. jirovecii RNA is less stable and rapidly degraded after cell death so is a more reliable marker of viable organisms. Modification of standard PCR protocols with quantitative methods (e.g. quantitative touch-down PCR) may help to differentiate between colonization and infection through the selection of thresholds to maximize sensitivity [13]. An added advantage of these molecular techniques is they may provide information on molecular epidemiology and resistance-associated mutations in the gene encoding dihydropteroate synthase (DHPS), the target of sulfamethoxazole, though the benefit of this is controversial [14]. One caveat to this is the potential for point mutations in DNA paired with primer sequences and risk of false negatives as a result.
These molecular tests are said to have increased sensitivity compared with cyst staining techniques but variable specificity depending on the specimens used, the primer chosen and whether treatment has been started [15]. The three most commonly assessed specimen groups are sputa (ideally induced), oropharyngeal washes (OPW) and blood. The clinical relevance of the known detectability of P. jirovecii DNA in whole blood has not been fully established but could represent colonization as well as disease [16]. The use of cycle threshold values has been proposed as a method to distinguish colonization from disease using BAL samples, although further studies are needed to validate cut offs on different samples [17].
Although there has been a widespread adoption of NAATs the current British HIV Association guidance, and that of similar professional bodies, still suggests combining them where available with a traditional visualization technique as described previously and performing them on alveolar specimens where possible to increase sensitivity and specificity [10, 18].
PCP diagnosis by detection of DNA in non-respiratory samples
Due to the variability in sampling methods, the challenges in obtaining ideal samples and the need for prompt diagnosis research has been conducted on the use of NAATs on OPW and blood. Samples are relatively non-invasive, collection is straight-forward and no special equipment or preparation is required. A study in our unit compared NAATs on OPW and blood with sputum, spontaneously expectorated or induced, using primers for the P. jirovecii MTLSU rRNA gene [19].
All patients were consenting adults presenting to a regional infectious disease unit who were being investigated for PCP as part of routine care. A spectrum of patients was included of different pre-test probabilities to allow estimates of sensitivity and specificity. Each participant was asked to provide sputum (spontaneous or induced), OPW and blood for analysis. OPW was obtained by gargling of normal saline for 10 to 30 seconds without any additional preparation.
Forty-five participants were included, 41 male (91%), 38 Caucasian (84%) with a median age of 39 years. One participant was an HIV-negative renal transplant recipient. Forty-four were HIV-positive with a median CD4 count of 64 cells/mL. Thirty-five of the 44 were not on ART with a median HIV RNA of 164 550 copies/mL. Thirty-nine of the 45 started empirical treatment for PCP a median of 2 days before sampling. We compared the sensitivity and specificity of tests on blood and OPW compared with sputum. Sputum PCR was positive in 60% of participants and in this group 47% of OPW and 50% of blood PCRs were positive. None with negative sputum PCR had positive OPW or blood PCR. A diagnosis of PCP could be reached in 14 of 16 patients with positive NAAT on sputum using these non-respiratory specimens.
Among those with P. jirovecii DNA detected in sputum a sensitivity of 47% for OPW was increased to 80% when considering only OPW samples taken within 48 hours of starting treatment. When this was combined with blood sample testing in the same time frame the sensitivity increased to 88%, which is comparable to that quoted in previous similar studies [12, 13, 15]. There were no false positives based on no OPW or blood PCR positives in those with negative PCR on sputum. As the laboratory techniques used were routine, few additional skills or resources were required.
Overall, using molecular tests on non-respiratory samples was of diagnostic benefit and show potential for savings in time and resources. The molecular tests provide excellent specificity and good sensitivity comparable with sputum without proceeding to time-consuming and invasive tests [20]. However, in view of uncertainty regarding the specificity of testing these non-invasive samples at all, results must be interpreted with care and in the right clinical context.
Conclusions
PCP diagnosis remains a combination of clinical suspicion and physical examination, supported by radiological and microbiological investigations. Using a combination of traditional microscopy with staining and NAAT on appropriate specimens, plus interpretation of results in the clinical context a clear diagnosis can be reached in most cases and this may prevent unnecessary treatment. Using non-respiratory specimens taken early to maximize sensitivity could reduce the requirement for invasive testing or diagnostic uncertainty.
Future developments
We expect the use of NAATs to become even more widely available and useful diagnostic aids alongside traditional techniques. With a plethora of protocols the sensitivity, specificity and utility of these will improve further over time. Combination with other laboratory techniques such as β-D-glucan may be similarly useful. Given the inability to culture the organism and so look for in vitro susceptibility to sulfamethoxazole-based treatment, molecular methods for detecting mutations and potential resistance may develop as a routinely used test.
References
1. Walzer P, Evans H, Copas A, Edwards S, Grant A, Miller R. Early predictors of mortality from Pneumocystis jirovecii pneumonia in HIV-infected patients: 1985–2006. Clin Infect Dis. 2008; 46: 625–633.
2. Phair J, Munoz A, Detels R, Kaslow R, Rinaldo C, Saah A. The risk of Pneumocystis carinii pneumonia among men infected with human immunodeficiency virus type 1. Multicenter AIDS Cohort Study Group. N Engl J Med. 1990; 322: 161–165.
3. Kaplan J, Hanson D, Navin T, Jones J. Risk factors for primary Pneumocystis carinii pneumonia in human immunodeficiency virus-infected adolescents and adults in the United States: reassessment of indications for chemoprophylaxis. J Infect Dis 1998; 178: 1126–1132.
4. Opravil M, Marincek B, Fuchs WA, Weber R, Speich R, Battegay M, Russi EW, Lüthy R. Shortcomings of chest radiography in detecting Pneumocystis carinii pneumonia. J Acquir Immune Defic Syndr. 1994; 7: 39–45.
5. Smith D, McLuckie A, Wyatt J, Gazzard B. Severe exercise hypoxaemia with normal or near normal X-rays: a feature of Pneumocystis carinii infection. Lancet 1988; 2: 1049–1051.
6. Grubb J, Moorman A, Baker R, Masur H. The changing spectrum of pulmonary disease in patients with HIV infection on antiretroviral therapy. AIDS 2006; 20:1095–1107.
7. Travis J, Hart E, Helm J, Duncan T, Vilar J. Retrospective review of Pneumocystis jirovecii pneumonia over two decades. Int J STD AIDS 2009; 20: 200-201.
8. Thomas J, Limper A. Pneumocystis pneumonia. N Engl J Med. 2004; 350: 2487–2498.
9. Broaddus C, Dake MD, Stulbarg MS, Blumenfeld W, Hadley WK, Golden JA, Hopewell PC. Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann Intern Med. 1985; 102: 747–752.
10. Nelson M, Dockrell D, Edwards S; BHIVA Guidelines Subcommittee, Angus B, Barton S, Beeching N, Bergin C, Boffito M, et al. British HIV Association and British Infection Association guidelines for the treatment of opportunistic infection in HIV-seropositive Individuals 2011. HIV Med. 2011; 12(Suppl 2): 1–140.
11. Lu J, Chen C, Bartlett M, Smith J, Lee C. Comparison of six different PCR methods for detection of Pneumocystis carinii. J Clin Microbiol. 1995; 33: 2785–2788.
12. Huggett J, Taylor M, Kocjan G, Evans H, Morris-Jones S, Gant V, Novak T, Costello A, Zumla A, Miller R. Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients. Thorax 2008; 63: 154–159.
13. Larsen H, Huang L, Kovacs J, Crothers K, Silcott V, Morris A, Turner J, Beard C, Masur H, Fischer S. A prospective, blinded study of quantitative touch-down polymerase chain reaction using oral-wash samples for diagnosis of Pneumocystis pneumonia in HIV-infected patients. J Infect Dis. 2004; 189: 1679–1683.
14. Durand-Joly I, Chabé M, Fabienne Soula F, Delhaes L, Camus D, Dei-Cas E. Molecular diagnosis of Pneumocystis pneumonia. FEMS Immunol Med Microbiol. 2005; 45: 405–410.
15. Olsson M, K. Strålin K, Holmberg H. Clinical significance of nested polymerase chain reaction and immunofluorescence for detection of Pneumocystis carinii pneumonia. Clin Microbiol Infect. 2001; 7: 492–497.
16. Rabodonirina M, Cotte L, Boibieux A, Kaiser K, Mayencon M, Raffenot D, Trepo C, Peyramond D, Picot S. Detection of Pneumocystis carinii DNA in blood specimens from human immunodeficiency virus-infected patients by nested PCR J. Clin Microbiol. 1999; 37: 27–131.
17. Fauchier T, Hasseine L, Gari-Toussaint M, Casanova V, Marty P, Pomares C. Detection of Pneumocystis jirovecii by quantitative PCR to differentiate colonization and pneumonia in immunocompromised HIV-positive and HIV-negative patients. J Clin Micro. 2016; 54: 1487–1495.
18. Centers for Disease Control and Prevention, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America. Guidelines for the prevention and treatment of opportunistic infections in HIV-infected adults and adolescents AIDSinfo 2013. (https://aidsinfo.nih.gov/contentfiles/lvguidelines/adult_oi.pdf)
19. van Halsema C, Johnson L, Baxter J, Douthwaite S, Clowes Y, Guiver M, Ustianowski A. Diagnosis of Pneumocystis jirovecii pneumonia by detection of DNA in blood and oropharyngeal wash, compared with sputum. AIDS Res Hum Retroviruses 2016; 32: 463–466.
20. de Oliveira A, Unnasch T, Crothers K, Eiser S, Zucchi P, Moir J, Beard C, Lawrence G, Huang L. Performance of a molecular viability assay for the diagnosis of Pneumocystis pneumonia in HIV-infected patients. Diagn Microbiol Infect Dis. 2007; 57: 169–176.
The authors
Farnaz Dave MBChB, MRCP; Ashley Horsley MBChB, MRCP; Thomas Whitfield MBChB, MSc, MRCP; Clare van Halsema* MBChB, MRCP, MD, DipHIVMed
North West Infectious Diseases Unit, North Manchester
General Hospital, Manchester M8 5RB, UK
*Corresponding author
E-mail: clare.vanhalsema@pat.nhs.uk
Randox Respiratory Multiplex Array
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, /in Featured Articles /by 3wmediaAn ideal method for colorectal cancer screening?
, /in Featured Articles /by 3wmediaColorectal cancer (CRC) is the third most frequent malignant neoplasm globally. In Europe CRC is the second most common cause of cancer death in women (breast cancer is the most common), and the third most common in men (after prostate and lung cancer). Data are similar from other Western countries. However if the condition is diagnosed very early via effective screening programmes, mortality can be greatly reduced, and various screening options are available.
The US guidelines advise screening people every ten years from the age of 50 via colonoscopy, or every five years via virtual colonoscopy, flexible sigmoidoscopy or double-contrast barium enema. The reported mortality reduction ranges from 60–70% in the participating population but, because of the nature of the procedures, screening adherence is problematical. The EU recommends biennial screening using the fecal occult blood test (FOBT). Such tests are usually mailed to older European residents to carry out at home, with both instructions on use and where to return tests on completion. Although this population screening method is easy and convenient it is not very reliable; specificity is low. Ingesting certain foods and drugs prior to testing, as well as conditions such as haemorrhoids, gastrointestinal ulcers and inflammations can all give false positive results. Sensitivity is also very low: various trials indicate that around 50% of tumours are not detected, and that reduction in mortality as a result of these screening programmes ranges from only 15–21%.
Although population screening utilizing CRC tumour markers would seem to be an ideal approach, in practice previously developed tests incorporating CEA combined with CA 19-9 have not provided high enough sensitivity. However, a project carried out by several Russian centres may well have found the solution. The researchers have developed a 3D hydrogel-based biochip that utilizes autoantibodies to detect specific, tumour-associated glycans in serum. This test format, which allows a more equal distribution of the molecular probes than planar systems, simultaneously measures protein-based tumour markers, the autoantibodies-to-glycans ratio, and immunoglobulin levels. By testing healthy controls, patients with CRC and patients with inflammatory bowel disease, it was possible to define both prognostic and diagnostic signatures. The prototype allowed diagnosis of CRC with a specificity of 95% and a sensitivity of 87%. The testing system is predicted to reach clinical laboratories in Russia in the near future. Hopefully the end result will be a simple, highly specific and sensitive test for CRC that can be performed at home.
Diagnosis of Pneumocystis jirovecii pneumonia
, /in Featured Articles /by 3wmediaDiagnosis of Pneumocystis jirovecii pneumonia (PCP) is conventionally based on direct staining and visualization. Challenges in obtaining alveolar samples have stimulated interest in techniques for detection of Pneumocystis DNA in non-invasive samples, which can give good sensitivity and specificity. Robust diagnosis is key to ensuring appropriate therapy.
by Dr Farnaz Dave, Dr Ashley Horsley, Dr Thomas Whitfield
and Dr Clare van Halsema
Introduction
Pneumocystis jirovecii (previously Pneumocystis carinii) is a pathogen capable of causing life threatening Pneumocystis pneumonia (PCP) in the immunocompromised with case fatality rates among those hospitalized of around 10% [1]. PCP typically occurs in individuals with hematological malignancies on chemotherapy or with other causes of acquired cellular immunodeficiency or, most frequently, in human immunodeficiency virus (HIV)-positive individuals with CD4 T-cell counts <200 cells/µL or <14% of total white cell count [2, 3]. First-line treatment is co-trimoxazole, a combination of the antibiotics sulfamethoxazole and trimethoprim, at high dose for 3 weeks, which has the clinically significant potential side effects of bone marrow suppression, rash and bronchial hypersensitivity. Unfortunately the classical clinical presentation of PCP of progressive dry cough, dyspnoea and malaise is non-specific and chest examination and radiographs are often normal or near normal [4]. Oxygen desaturation on exercise is a helpful clinical sign in the right patient population [5]. Furthermore, many individuals with PCP do not produce sputum, so laboratory confirmation can be challenging. Given the serious nature of the illness and the possible side effects of treatment, accurate diagnosis is key to making an informed treatment decision. Incidence of PCP among HIV-positive individuals has declined since the widespread availability of antiretroviral therapy (ART) and has also declined as a cause of hospitalization of HIV-positive individuals [6]. In a study in a large HIV centre in London, mortality for all hospitalizations was around 10% during the first 10 years of availability of effective ART [1]. In our unit a decrease in mortality among those admitted to intensive care with PCP was seen between the periods of 1986–1995 and 1996–2004. This improvement is thought to be due to advances in critical care rather than treatment of PCP itself [7]. Reaching a diagnosis
Traditional methods
P. jirovecii is a fungus that cannot be cultured in vitro and so the organism is identified using histochemical staining techniques of fluid samples. Grocott-Gomori methenamine silver nitrate or direct immunofluorescence monoclonal antibody (IFA) stains on deep respiratory samples are generally regarded as the gold standard in diagnosis [8]. The life cycle of P. jirovecii is demonstrated by trophic, pre-cystic and cystic forms by morphological criteria. Diagnosis through microscopy of the cystic stage requires significant technical expertise and can still lead to false-negative results. In florid disease, P. jirovecii is present throughout the bronchial tree, from the upper respiratory tract down to the alveolar surface. Induced sputum samples are recommended by most guidelines, if routinely available, as spontaneously expectorated sputum is not considered an adequate alveolar sample and microscopy could be falsely negative. If the results of testing on induced sputum are not conclusive then a bronchoalveolar lavage (BAL) is recommended (sensitivity 86–98%) [9, 10]. In clinical practice, induced sputum is often not readily available, and BAL may not be possible due to hypoxia or may lead to a delay in sampling of several days; therefore, in clinical practice spontaneously expectorated samples are often processed. The disadvantages of this are twofold: a lower yield of cystic forms for visualization and potential for false-positive results due to colonization. Only in rare cases would a lung biopsy be appropriate (sensitivity 95–98%) in circumstances of poor response to empirical treatment and negative initial testing [9].
Molecular techniques
Molecular testing of lower respiratory tract secretions and blood is an alternative and operator-independent method for confirming the presence of P. jirovecii. Nucleic acid amplification techniques (NAAT) can be used with a number of primers targeting different substrates – most commonly the major surface glycoprotein (MSG), mitochondrial large subunit (MTLSU) rRNA and internal transcribed spacer (ITS) region genes [11]. One potential pitfall with these techniques is that the detection of specific nucleic acid sequence does not distinguish between colonization and disease or between viable and non-viable organisms [12]. P. jirovecii RNA is less stable and rapidly degraded after cell death so is a more reliable marker of viable organisms. Modification of standard PCR protocols with quantitative methods (e.g. quantitative touch-down PCR) may help to differentiate between colonization and infection through the selection of thresholds to maximize sensitivity [13]. An added advantage of these molecular techniques is they may provide information on molecular epidemiology and resistance-associated mutations in the gene encoding dihydropteroate synthase (DHPS), the target of sulfamethoxazole, though the benefit of this is controversial [14]. One caveat to this is the potential for point mutations in DNA paired with primer sequences and risk of false negatives as a result.
These molecular tests are said to have increased sensitivity compared with cyst staining techniques but variable specificity depending on the specimens used, the primer chosen and whether treatment has been started [15]. The three most commonly assessed specimen groups are sputa (ideally induced), oropharyngeal washes (OPW) and blood. The clinical relevance of the known detectability of P. jirovecii DNA in whole blood has not been fully established but could represent colonization as well as disease [16]. The use of cycle threshold values has been proposed as a method to distinguish colonization from disease using BAL samples, although further studies are needed to validate cut offs on different samples [17].
Although there has been a widespread adoption of NAATs the current British HIV Association guidance, and that of similar professional bodies, still suggests combining them where available with a traditional visualization technique as described previously and performing them on alveolar specimens where possible to increase sensitivity and specificity [10, 18].
PCP diagnosis by detection of DNA in non-respiratory samples
Due to the variability in sampling methods, the challenges in obtaining ideal samples and the need for prompt diagnosis research has been conducted on the use of NAATs on OPW and blood. Samples are relatively non-invasive, collection is straight-forward and no special equipment or preparation is required. A study in our unit compared NAATs on OPW and blood with sputum, spontaneously expectorated or induced, using primers for the P. jirovecii MTLSU rRNA gene [19].
All patients were consenting adults presenting to a regional infectious disease unit who were being investigated for PCP as part of routine care. A spectrum of patients was included of different pre-test probabilities to allow estimates of sensitivity and specificity. Each participant was asked to provide sputum (spontaneous or induced), OPW and blood for analysis. OPW was obtained by gargling of normal saline for 10 to 30 seconds without any additional preparation.
Forty-five participants were included, 41 male (91%), 38 Caucasian (84%) with a median age of 39 years. One participant was an HIV-negative renal transplant recipient. Forty-four were HIV-positive with a median CD4 count of 64 cells/mL. Thirty-five of the 44 were not on ART with a median HIV RNA of 164 550 copies/mL. Thirty-nine of the 45 started empirical treatment for PCP a median of 2 days before sampling. We compared the sensitivity and specificity of tests on blood and OPW compared with sputum. Sputum PCR was positive in 60% of participants and in this group 47% of OPW and 50% of blood PCRs were positive. None with negative sputum PCR had positive OPW or blood PCR. A diagnosis of PCP could be reached in 14 of 16 patients with positive NAAT on sputum using these non-respiratory specimens.
Among those with P. jirovecii DNA detected in sputum a sensitivity of 47% for OPW was increased to 80% when considering only OPW samples taken within 48 hours of starting treatment. When this was combined with blood sample testing in the same time frame the sensitivity increased to 88%, which is comparable to that quoted in previous similar studies [12, 13, 15]. There were no false positives based on no OPW or blood PCR positives in those with negative PCR on sputum. As the laboratory techniques used were routine, few additional skills or resources were required.
Overall, using molecular tests on non-respiratory samples was of diagnostic benefit and show potential for savings in time and resources. The molecular tests provide excellent specificity and good sensitivity comparable with sputum without proceeding to time-consuming and invasive tests [20]. However, in view of uncertainty regarding the specificity of testing these non-invasive samples at all, results must be interpreted with care and in the right clinical context.
Conclusions
PCP diagnosis remains a combination of clinical suspicion and physical examination, supported by radiological and microbiological investigations. Using a combination of traditional microscopy with staining and NAAT on appropriate specimens, plus interpretation of results in the clinical context a clear diagnosis can be reached in most cases and this may prevent unnecessary treatment. Using non-respiratory specimens taken early to maximize sensitivity could reduce the requirement for invasive testing or diagnostic uncertainty.
Future developments
We expect the use of NAATs to become even more widely available and useful diagnostic aids alongside traditional techniques. With a plethora of protocols the sensitivity, specificity and utility of these will improve further over time. Combination with other laboratory techniques such as β-D-glucan may be similarly useful. Given the inability to culture the organism and so look for in vitro susceptibility to sulfamethoxazole-based treatment, molecular methods for detecting mutations and potential resistance may develop as a routinely used test.
References
1. Walzer P, Evans H, Copas A, Edwards S, Grant A, Miller R. Early predictors of mortality from Pneumocystis jirovecii pneumonia in HIV-infected patients: 1985–2006. Clin Infect Dis. 2008; 46: 625–633.
2. Phair J, Munoz A, Detels R, Kaslow R, Rinaldo C, Saah A. The risk of Pneumocystis carinii pneumonia among men infected with human immunodeficiency virus type 1. Multicenter AIDS Cohort Study Group. N Engl J Med. 1990; 322: 161–165.
3. Kaplan J, Hanson D, Navin T, Jones J. Risk factors for primary Pneumocystis carinii pneumonia in human immunodeficiency virus-infected adolescents and adults in the United States: reassessment of indications for chemoprophylaxis. J Infect Dis 1998; 178: 1126–1132.
4. Opravil M, Marincek B, Fuchs WA, Weber R, Speich R, Battegay M, Russi EW, Lüthy R. Shortcomings of chest radiography in detecting Pneumocystis carinii pneumonia. J Acquir Immune Defic Syndr. 1994; 7: 39–45.
5. Smith D, McLuckie A, Wyatt J, Gazzard B. Severe exercise hypoxaemia with normal or near normal X-rays: a feature of Pneumocystis carinii infection. Lancet 1988; 2: 1049–1051.
6. Grubb J, Moorman A, Baker R, Masur H. The changing spectrum of pulmonary disease in patients with HIV infection on antiretroviral therapy. AIDS 2006; 20:1095–1107.
7. Travis J, Hart E, Helm J, Duncan T, Vilar J. Retrospective review of Pneumocystis jirovecii pneumonia over two decades. Int J STD AIDS 2009; 20: 200-201.
8. Thomas J, Limper A. Pneumocystis pneumonia. N Engl J Med. 2004; 350: 2487–2498.
9. Broaddus C, Dake MD, Stulbarg MS, Blumenfeld W, Hadley WK, Golden JA, Hopewell PC. Bronchoalveolar lavage and transbronchial biopsy for the diagnosis of pulmonary infections in the acquired immunodeficiency syndrome. Ann Intern Med. 1985; 102: 747–752.
10. Nelson M, Dockrell D, Edwards S; BHIVA Guidelines Subcommittee, Angus B, Barton S, Beeching N, Bergin C, Boffito M, et al. British HIV Association and British Infection Association guidelines for the treatment of opportunistic infection in HIV-seropositive Individuals 2011. HIV Med. 2011; 12(Suppl 2): 1–140.
11. Lu J, Chen C, Bartlett M, Smith J, Lee C. Comparison of six different PCR methods for detection of Pneumocystis carinii. J Clin Microbiol. 1995; 33: 2785–2788.
12. Huggett J, Taylor M, Kocjan G, Evans H, Morris-Jones S, Gant V, Novak T, Costello A, Zumla A, Miller R. Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients. Thorax 2008; 63: 154–159.
13. Larsen H, Huang L, Kovacs J, Crothers K, Silcott V, Morris A, Turner J, Beard C, Masur H, Fischer S. A prospective, blinded study of quantitative touch-down polymerase chain reaction using oral-wash samples for diagnosis of Pneumocystis pneumonia in HIV-infected patients. J Infect Dis. 2004; 189: 1679–1683.
14. Durand-Joly I, Chabé M, Fabienne Soula F, Delhaes L, Camus D, Dei-Cas E. Molecular diagnosis of Pneumocystis pneumonia. FEMS Immunol Med Microbiol. 2005; 45: 405–410.
15. Olsson M, K. Strålin K, Holmberg H. Clinical significance of nested polymerase chain reaction and immunofluorescence for detection of Pneumocystis carinii pneumonia. Clin Microbiol Infect. 2001; 7: 492–497.
16. Rabodonirina M, Cotte L, Boibieux A, Kaiser K, Mayencon M, Raffenot D, Trepo C, Peyramond D, Picot S. Detection of Pneumocystis carinii DNA in blood specimens from human immunodeficiency virus-infected patients by nested PCR J. Clin Microbiol. 1999; 37: 27–131.
17. Fauchier T, Hasseine L, Gari-Toussaint M, Casanova V, Marty P, Pomares C. Detection of Pneumocystis jirovecii by quantitative PCR to differentiate colonization and pneumonia in immunocompromised HIV-positive and HIV-negative patients. J Clin Micro. 2016; 54: 1487–1495.
18. Centers for Disease Control and Prevention, the National Institutes of Health, and the HIV Medicine Association of the Infectious Diseases Society of America. Guidelines for the prevention and treatment of opportunistic infections in HIV-infected adults and adolescents AIDSinfo 2013. (https://aidsinfo.nih.gov/contentfiles/lvguidelines/adult_oi.pdf)
19. van Halsema C, Johnson L, Baxter J, Douthwaite S, Clowes Y, Guiver M, Ustianowski A. Diagnosis of Pneumocystis jirovecii pneumonia by detection of DNA in blood and oropharyngeal wash, compared with sputum. AIDS Res Hum Retroviruses 2016; 32: 463–466.
20. de Oliveira A, Unnasch T, Crothers K, Eiser S, Zucchi P, Moir J, Beard C, Lawrence G, Huang L. Performance of a molecular viability assay for the diagnosis of Pneumocystis pneumonia in HIV-infected patients. Diagn Microbiol Infect Dis. 2007; 57: 169–176.
The authors
Farnaz Dave MBChB, MRCP; Ashley Horsley MBChB, MRCP; Thomas Whitfield MBChB, MSc, MRCP; Clare van Halsema* MBChB, MRCP, MD, DipHIVMed
North West Infectious Diseases Unit, North Manchester
General Hospital, Manchester M8 5RB, UK
*Corresponding author
E-mail: clare.vanhalsema@pat.nhs.uk
Proteomic approach to investigate ALL biomarkers for early diagnosis and treatment evaluation
, /in Featured Articles /by 3wmediaThe aim of this study was to perform proteomic analysis of serum from pediatric patients with B-cell acute lymphoblastic leukemia (B-ALL) to identify candidate biomarker proteins, for use in early diagnosis and evaluation of treatment. This approach is an alternative to traditional techniques that can investigate the disease from another perspective. Acute lymphoblastic leukemia is the most common malignant cancer in childhood and the symptoms of childhood cancer are difficult to recognize.
by Dr M. de S. Cavalcante, Prof. A. E. Vieira-Neto,
Dr R. de A. Moreira and Dr A. C. de O. Monteiro-Moreira
Background and significance
Acute lymphoblastic leukemia (ALL) is the most common malignant cancer in childhood, and is responsible for approximately 25% of all childhood cancers and 72% of all cases of pediatric leukemia [1]. The current standards for diagnosis of ALL integrate the study of cell morphology, immunophenotyping and genetics/cytogenetics, as described in the classification of lymphoid cancers published by the World Health Organization (WHO) in 2008 [2]. Of lymphoid cancers, as designated using the most recent WHO classification, the purely leukemic presentation, B-lineage ALL (85 %) is the most common [3], and will be addressed in this study. The signs and symptoms of childhood cancer are very challenging to identify, as it is not the first diagnosis to be considered for nonspecific complaints, leading to potential uncertainty in diagnosis. Moreover, children showing the first signs of cancer frequently do not appear severely ill, which may delay diagnosis. In addition, childhood cancer can mimic other common childhood diseases and even normal developmental physiological processes [4]. In the specific case of ALL, early diagnosis and treatment increase the chances of a cure [4].
Future prospects
A label-free proteomic approach was used for the quantitative analysis. Other approaches could also be used in the future, for example it is possible to find studies using RNA interference, mainly silencing expression of specific genes [5]. In our proteomic approach, for each protein, the program ExpressionE selected all corresponding peptides from the samples and compared the intensities of these for relative protein quantification. Using the intensity of a peptide of known quantity, alcohol dehydrogenase (ADH), the program performed self-standardization of data sets. Lists of proteins were then filtered to show only those present in all three repeated injections of each sample, from which an output table was created. This table showed the names, access codes, and expression levels of the proteins, and indicated whether they were upregulated ≥2-fold, downregulated ≤0.5-fold, or whether they did not show significant differences between the groups (unchanged), 0.5 < expression level < 2. The list of proteins generated from three injections of samples in MS, coupled with broad limits used for protein expression levels and serum samples used the controls (non-leukemic pediatric patients) may suggest that the panel of candidate protein biomarkers is clearly increased in the disease state. Biotechnological resources
Affinity chromatography with α-D-galactose-binding lectin from Artocarpus incisa [6] immobilized on a SepharoseTM 4B gel, combined with identification and quantification of glycoproteins by mass spectrometry, are excellent tools for comparative serum studies. The biomarker pipeline is commonly viewed as a series of preclinical phases: biomarker discovery, and verification before the final clinical evaluation. The comparative analysis results in a list of hundreds of proteins that are differentially expressed between healthy and diseased samples [7]. In this study, the preclinical phase of biomarker discovery was applied and a proteomic analysis of serum samples from pediatric patients with B-ALL was performed, to analyse levels of glycoprotein expression, with the aim of identifying biomarkers to aid in the early diagnosis of B-ALL and to assess the response to induction therapy.
The depletion of high-abundance proteins in serum, human serum albumin (HSA) and IgG, followed by affinity chromatography with the plant lectin Frutalin immobilized on SepharoseTM 4B (Fig. 1), reduced the dynamic range and increased the capacity to identify lower-abundance proteins. The retained fraction (FR) peak containing the protein of interest was concentrated and digested, for later analysis by nano-LC-MS/MS.
Proteomic approach
The study population was composed mainly of children from the lower middle class, who attended a reference hospital for the diagnosis and treatment of childhood cancers in the State of Ceará, Brazil. The study was conducted with the approval of the Research Ethics Committee at the Hospital Infantil Albert Sabin, associated with the Secretary of Health of the State of Ceará. The demographic and clinical data for the patients are summarized in Table 1. The pediatric patients were evaluated at two different times: at diagnosis (B-ALL group; n = 10) and after induction therapy (AIT group; n = 10). Samples of healthy children (Control group; n = 10) were obtained for comparison.
The differentially expressed proteins were used for pathway analysis. Swiss-Prot accession numbers were inserted into the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) software, version 9.05 (available at http://string.embl.de/), with the following analysis parameters: Homo sapiens, confidence level 0.400–0.900, using the active prediction method [8].
Biomarker panel for ALL diagnosis
A panel of protein biomarker candidates has been developed for pre-diagnosis of B-ALL and also provide information that would indicate a favourable response to treatment after induction therapy. In the proteomic analysis, a total of 96 proteins were identified. Leucine-rich alpha-2-glycoprotein 1 (LRG1), Clusterin (CLU), thrombin (F2), heparin cofactor II (SERPIND1), alpha-2-macroglobulin (A2M), alpha-2-antiplasmin (SERPINF2), Alpha-1 antitrypsin (SERPINA1), Complement factor B (CFB) and Complement C3 (C3) were over-expressed in the B-ALL compared to the Control and AIT groups, and were, therefore, identified as candidate biomarkers for early diagnosis of B-ALL. The AIT group showed no significant differences in the expression levels of these proteins compared to the Control group, and did not show any significant change in the level of expression of these proteins, a fact that further reaffirms the presence of these potential biomarkers in a disease state, as all patients achieved complete remission after treatment (Fig. 2). Our results also confirm the important relationship between cancer and phenomena associated with blood coagulation. Several studies have reported that approximately 50% of patients with malignant disease and more than 90% of those that evolve to metastasis present evidence of abnormalities in coagulation and/or fibrinolysis [9–13].
Conclusion
Acute lymphoblastic leukemia is the most common malignant cancer in childhood and this proteomic approach is an alternative to traditional techniques, since the signs and symptoms of childhood cancer are very challenging to identify. LRG1, CLU, F2, SERPIND1, A2M, SERPINF2, SERPINA1, CFB, and C3 were identified as candidate biomarkers for early diagnosis of B-ALL; all were over-expressed in the B-ALL group compared to the Control and AIT groups. The AIT group did not display any significant changes in the expression levels of these proteins, compared to the Control group. All patients in the AIT group achieved complete remission after treatment; this indicates that these biomarkers are only present in the disease state. These candidate biomarkers may improve the pre-diagnosis of B-ALL, which is currently difficult to diagnose in the early stages; the biomarkers may also provide key information on the response to treatment after induction therapy. Further clinical and genomic studies will be important to improve the survival of children with this disease.
Acknowledgements
FINEP, CNPq, RENORBIO-UNIFOR, ALBERT SABIN HOSPITAL
This article is a summary of a paper first published in Biomarker Research: Cavalcante Mde S, Torres-Romero JC, Lobo MD, Moreno FB, Bezerra LP, Lima DS, Matos JC, Moreira Rde A, Monteiro-Moreira AC. A panel of glycoproteins as candidate biomarkers for early diagnosis and treatment evaluation of B-cell acute lymphoblastic leukemia. Biomarker Research 2016; 4: 1 (doi: 10.1186/s40364-016-0055-6) [14].
References
1. Scheurer ME, Bondy ML, Gurney JG. Epidemiology of Childhood Cancer. In: Pizzo PA, Poplack DG, editors. Principles and practice of pediatric oncology, 6th ed, pp2–16. Lippincott Williams and Wilkins 2011.
2. Vardiman JW, Thiele J, et al. The 2008 revision of the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia: rationale and important changes. Blood 2009; 114: 937–951.
3. Chiaretti S, Zini G, Bassan R. Diagnosis and subclassification of acute lymphoblastic leukemia. Mediterr J Hematol Infect Dis. 2014; 6: e2014073.
4. Rodrigues KE, Camargo B. Diagnóstico precoce do câncer infantil: responsabilidade de todos. Rev Assoc Med Bras. 2003; 49: 29–34 (in Portuguese).
5. Trougakos IP, So A, et al. Silencing expression of the clusterin/apolipoprotein j gene in human cancer cells using small interfering RNA induces spontaneous apoptosis, reduced growth ability, and cell sensitization to genotoxic and oxidative stress. Cancer Res. 2004; 64: 1834–1842.
6. Monteiro-Moreira ACO, Pereira HD, et al. Crystallization and preliminary x-ray diffraction studies of Frutalin, an α-D-galactose-binding lectin from Artocarpus incisa seeds. Acta Crystallographica Session F, 2015.
7. Parker CE, Borchers CH. Mass spectrometry based biomarker discovery, verification, and validation–quality assurance and control of protein biomarker assays. Mol Oncol. 2014; 8(4): 840–858.
8. Jensen LJ, Kuhn M, et al. STRING 8–a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res. 2009; 37: D412–416.
9. Kwon H-C, Oh SY, et al. Plasma levels of prothrombin fragment F112, D-dimer and prothrombin time correlate with clinical stage and lymph node metastasis in operable gastric cancer patients. Jpn J Clin Oncol. 2008; 38: 2–7.
10. Bick RL. Coagulation abnormalities in malignancy: a review. Semin Thromb Hemost. 1992; 18: 353–372.
11. Luzzatto G, Schafer Al. The prethrombotic state in cancer. Semin Oncol. 1990; 17: 147–159.
12. Nigel O’Connor, Gozzard DI, et al. Haemostatic abnormalities and malignant disease. Lancet 1986; 8: 303–304.
13. Hillen HF. Thrombosis in cancer patients. Ann Oncol. 2000; 11: 273–276.
14. Cavalcante Mde S, Torres-Romero JC, et al. A panel of glycoproteins as candidate biomarkers for early diagnosis and treatment evaluation of B-cell acute lymphoblastic leukemia. Biomarker Research 2016; 4: 1 (doi: 10.1186/s40364-016-0055-6).
The authors
Márcio de Souza Cavalcante1, Antonio Eufrásio Vieira-Neto², Renato de Azevedo Moreira3, Ana Cristina de Oliveira Monteiro-Moreira3*
1Northeast Network of Biotechnology (RENORBIO), State University of Ceará, Ceará, Brazil.
2Center of Experimental Biology (NUBEX), University of Fortaleza (UNIFOR), Ceará, Brazil.
3Department of Biochemistry and Molecular Biology, Federal University of Ceará, Ceará, Brazil.
4Development and Technological Innovation in Drug Program, Federal University of Ceará, Ceará, Brazil
5Reference Center at Children’s Cancer Diagnosis and Adolescents Dr. Murilo Martins, Albert Sabin Hospital, Ceará, Brazil.
*Corresponding author
E-mail: acomoreira@unifor.br