Diagnostic accuracy of serum (1-3)-ß-D-glucan for Pneumocystis jirovecii pneumonia: a systematic review and meta-analysis.

BACKGROUND: Pneumocystis jirovecii pneumonia (PJP) can be a life-threatening opportunistic infection in immunocompromised hosts. The diagnosis can be challenging, often requiring semi-invasive respiratory sampling. The serum 1,3-ß-D-glucan (BDG) assay has been proposed as a minimally invasive test for the presumptive diagnosis of PJP. METHOD: We carried out a systematic review and meta-analysis using articles in the English language published between January 1960 and September 2019. We estimated the pooled sensitivity and specificity of BDG testing using a bivariate random effects approach and compared test performance in human immunodeficiency virus (HIV) and non-HIV subgroups with meta-regression. Data from the pooled sensitivity and specificity were transformed to generate pre- and post-test probability curves. RESULTS: Twenty-three studies were included. The pooled sensitivity and specificity of serum BDG testing for PJP were 91% (95%CI 87-94%) and 79% (95%CI 72-84%) respectively. The sensitivity in patients with HIV was better than in patients without (94%, 95%CI 91-96%) versus 86% (95%CI 78-91%) (p 0.02), with comparable specificity (83%, 95%CI 69-92% versus 83%, 95%CI 72-90%) (p 0.10). A negative BDG was only associated with a low post-test probability of PJP (≤5%) when the pre-test probability was low to intermediate (≤20% in non-HIV and ≤50% in HIV). CONCLUSIONS: Among patients with a higher likelihood of PJP, the pooled sensitivity of BDG is insufficient to exclude infection. Similarly, for most cases, the pooled specificity is inadequate to diagnose PJP. Understanding the performance of BDG in the population being investigated is therefore essential to optimal clinical decision-making.

Diagnostic accuracy of (1→3)-ß-D-glucan to predict Pneumocystis jirovecii pneumonia in non-HIV-infected patients.

Background Pneumocystis jirovecii pneumonia (PCP) is a common and potentially fatal opportunistic infection in immunocompromised non-HIV individuals. There are problems with clinical and diagnostic protocols for PCP that lack sensitivity and specificity. We designed a retrospective study to compared several methods that were used in diagnostics of PCP. Patients and methods One hundred and eight immunocompromised individuals with typical clinical picture for PCP and suspicious radiological findings were included in the study. Serum samples were taken to measure the values of (1→3)-ß-D-glucan (Fungitell, Associates of Cape Cod, USA). Lower respiratory tract samples were obtained to perform direct immunofluorescence (DIF, MERIFLUOR® Pneumocystis, Meridian, USA) stain and real-time PCR (qPCR). Results Fifty-four (50%) of the 108 patients in our study had (1→3)-ß-D-glucan > 500 pg/ml. Patients that had (1→3)-ß-D-glucan concentrations < 400 pg/ml in serum, had mean threshold cycles (Ct) 35.43 ± 3.32 versus those that had (1→3)-ß-D-glucan concentrations >400 pg/mL and mean Ct of 28.97 ± 5.27 (P < 0.001). If we detected P. jirovecii with DIF and qPCR than PCP was proven. If the concentration of (1→3)-ß-D-glucan was higher than 400 pg/ml and Ct of qPCR was below 28.97 ± 5.27 than we have been able be certain that P. jirovecii caused pneumonia (odds ratio [OR] 2.31, 95% confidence interval [CI] 1.62-3.27, P < 0.001). Conclusions Measurement of (1→3)-ß-D-glucan or qPCR alone could not be used to diagnose PCP. Diagnostic cut-off value for (1→3)-ß-D-glucan > 400pg/ml and qPCR below 30 Ct, allow us to conclude that patient has PCP. If the values of (1→3)-ß-D-glucan are < 400 pg/ml and qPCR is above 35 Ct than colonization with P. jirovecii is more possible than PCP.

[Severe hypoxemic respiratory failure caused by Pneumocystis jirovecii in a late kidney transplant recipient].

Pneumonia caused by Pneumocystis jirovecii is an uncommon infection in kidney transplant patients that can have an acute and rapid progression to respiratory failure and death. The period of greatest risk occurs in the first six months after the transplant, and it relates to the high doses of immunosuppression drugs required by patients. However, it may occur late, associated with the suspension of prophylaxis with trimethoprim-sulfamethoxazole.We present two cases of renal transplant patients who had severe hypoxemic respiratory failure due to P. jirovecii six years after transplantation. In addition to steroids, they received treatment with trimethoprim-sulfamethoxazole. One patient died, while the other had clinical recovery, with preservation of the renal graft function.

Insuficiencia respiratoria hipoxémica grave por Pneumocystis jirovecii después de trasplante renal / Severe hypoxemic respiratory failure caused by Pneumocystis jirovecii in a late kidney transplant recipient

Resumen La neumonitis por Pneumocystis jirovecii es una infección infrecuente en pacientes con trasplante de riñón, que se presenta de forma aguda y puede progresar rápidamente hasta la insuficiencia respiratoria y la muerte. El período de mayor riesgo es el de los primeros seis meses después del trasplante, y se asocia con las altas dosis de medicamentos inmunosupresores que reciben los pacientes. La condición también puede presentarse de manera tardía, asociada con la suspensión de la profilaxis con trimetoprim-sulfametoxazol. Se reportan dos casos de pacientes con trasplante renal que presentaron insuficiencia respiratoria hipoxémica grave por P. jirovecii pasados seis años del trasplante, y que fueron tratados con trimetoprim-sulfametoxazol y esteroides. Uno de los pacientes murió y el otro se recuperó sin que hubiera efectos en la función del injerto renal.

Abstract Pneumonia caused by Pneumocystis jirovecii is an uncommon infection in kidney transplant patients that can have an acute and rapid progression to respiratory failure and death. The period of greatest risk occurs in the first six months after the transplant, and it relates to the high doses of immunosuppression drugs required by patients. However, it may occur late, associated with the suspension of prophylaxis with trimethoprim-sulfamethoxazole. We present two cases of renal transplant patients who had severe hypoxemic respiratory failure due to P. jirovecii six years after transplantation. In addition to steroids, they received treatment with trimethoprim-sulfamethoxazole. One patient died, while the other had clinical recovery, with preservation of the renal graft function.

An evaluation of the performance of the Dynamiker® Fungus (1-3)-ß-D-Glucan Assay to assist in the diagnosis of Pneumocystis pneumonia.

The Dynamiker® Fungus (1-3)-ß-D-Glucan Assay (D-BDG) has recently become available in the Western Hemisphere for the diagnosis of invasive fungal disease (IFD). Evaluations of its performance for Pneumocystis pneumonia (PcP) are limited. A retrospective evaluation of D-BDG diagnosis of PcP was performed (23 PcP cases and 23 controls). Sensitivity and specificity were 87% and 70%, respectively, reducing the positivity threshold to 45 pg/ml increased sensitivity (96%), whereas a threshold of 300 pg/ml increased specificity (96%). The performance of D-BDG for the detection of PcP is comparable to other IFD, but sensitivity is below that required to confidently exclude PcP.

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii.

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different.IMPORTANCEPneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens.

Structure-activity correlations for three pyrido[2,3-d]pyrimidine antifolates binding to human and Pneumocystis carinii dihydrofolate reductase.

To further define the interactions that enhance the selectivity of binding and to directly compare the binding of the most potent analogue {N(6)-methyl-N(6)-(3,4,5-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine; compound 26} in the series of bicyclic pyrido[2,3-d]pyrimidine analogues of piritrexim (PTX) with native human (h), Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) dihydrofolate reductase (DHFR) enzymes, the crystal structures of hDHFR complexed with N(6)-methyl-N(6)-(4-isopropylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 22), of hDHFR complexed with compound 26 and of pcDHFR complexed with N(6)-methyl-N(6)-1-naphthylpyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 24) are reported as ternary complexes with NADPH. This series of bicyclic pyrido[2,3-d]pyrimidines were designed in which there was a transposition of the 5-methyl group of PTX to the N9 position of the pyrido[2,3-d]pyrimidine. It was hypothesized that the N9-methyl group would preferentially interact with Ile123 of pcDHFR (and Ile123 of pjDHFR), but not with the shorter Val115 in hDHFR. Structure-activity data for this series of antifolates revealed that a trifluoro derivative (26) was the most selective against pjDHFR compared with mammalian DHFR (h/pj = 35.7). Structural data for the hDHFR-26 complex revealed that 26 binds in a different conformation from that observed in the pcDHFR-26 complex. In the hDHFR-26 complex the trifluorophenyl ring of 26 occupies a position near the cofactor-binding site, with close intermolecular contacts with Asp21, Ser59 and Ile60, whereas this ring in the pcDHFR-26 complex is positioned away from the cofactor site and near Ile65, with weaker contacts with Ile65, Phe69 and Ile123. Comparison of the intermolecular contacts between the N9-methyl group with Val115/Ile123 validates the hypothesis that the N9-methyl substituent preferentially interacts with Ile123 compared with Val115 of hDHFR, as the weaker contact with Val115 in the hDHFR structure is consistent with its weaker binding affinity compared with pcDHFR. The results for the structures of hDHFR-22 and pcDHFR-24 show that their inhibitor-binding orientation is similar to that observed in pcDHFR-26 and the pcDHFR variant (F69N) reported previously. The naphthyl moiety of 24 makes several intermolecular contacts with the active-site residues in pcDHFR that help to stabilize the binding, resulting in a more potent inhibitor.

Evidence for Proinflammatory ß-1,6 Glucans in the Pneumocystis carinii Cell Wall.

Inflammation is a major cause of respiratory impairment during Pneumocystis pneumonia. Studies support a significant role for cell wall ß-glucans in stimulating inflammatory responses. Fungal ß-glucans are comprised of d-glucose homopolymers containing ß-1,3-linked glucose backbones with ß-1,6-linked glucose side chains. Prior studies in Pneumocystis carinii have characterized ß-1,3 glucan components of the organism. However, recent investigations in other organisms support important roles for ß-1,6 glucans, predominantly in mediating host cellular activation. Accordingly, we sought to characterize ß-1,6 glucans in the cell wall of Pneumocystis and to establish their activity in lung cell inflammation. Immune staining revealed specific ß-1,6 localization in P. carinii cyst walls. Homology-based cloning facilitated characterization of a functional P. carinii kre6 (Pckre6) ß-1,6 glucan synthase in Pneumocystis that, when expressed in kre6-deficient Saccharomyces cerevisiae, restored cell wall stability. Recently synthesized ß-1,6 glucan synthase inhibitors decreased the ability of isolated P. carinii preparations to generate ß-1,6 carbohydrate. In addition, isolated ß-1,6 glucan fractions from Pneumocystis elicited vigorous tumor necrosis factor alpha (TNF-α) responses from macrophages. These inflammatory responses were significantly dampened by inhibition of host cell plasma membrane microdomain function. Together, these studies indicate that ß-1,6 glucans are present in the P. carinii cell wall and contribute to lung cell inflammatory activation during infection.

Combined quantification of pulmonary Pneumocystis jirovecii DNA and serum (1->3)-ß-D-glucan for differential diagnosis of pneumocystis pneumonia and Pneumocystis colonization.

This study assessed a quantitative PCR (qPCR) assay for Pneumocystis jirovecii quantification in bronchoalveolar lavage (BAL) fluid samples combined with serum (1→3)-ß-d-glucan (BG) level detection to distinguish Pneumocystis pneumonia (PCP) from pulmonary colonization with P. jirovecii. Forty-six patients for whom P. jirovecii was initially detected in BAL fluid samples were retrospectively enrolled. Based on clinical data and results of P. jirovecii detection, 17 and 29 patients were diagnosed with PCP and colonization, respectively. BAL fluid samples were reassayed using a qPCR assay targeting the mitochondrial large subunit rRNA gene. qPCR results and serum BG levels (from a Fungitell kit) were analyzed conjointly. P. jirovecii DNA copy numbers were significantly higher in the PCP group than in the colonization group (1.3 × 10(7) versus 3.4 × 10(3) copies/µl, P < 0.05). A lower cutoff value (1.6 × 10(3) copies/µl) achieving 100% sensitivity for PCP diagnosis and an upper cutoff value (2 × 10(4) copies/µl) achieving 100% specificity were determined. Applying these two values, 13/17 PCP patients and 19/29 colonized patients were correctly assigned to their patient groups. For the remaining 14 patients with P. jirovecii DNA copy numbers between the cutoff values, PCP and colonization could not be distinguished on the basis of qPCR results. Four of these patients who were initially assigned to the PCP group presented BG levels of ≥100 pg/ml. The other 10 patients, who were initially assigned to the colonization group, presented BG levels of <100 pg/ml. These results suggest that the combination of the qPCR assay, applying cutoff values of 1.6 × 10(3) and 2 × 10(4) copies/µl, and serum BG detection, applying a 100 pg/ml threshold, can differentiate PCP and colonization diagnoses.

Test performance of blood beta-glucan for Pneumocystis jirovecii pneumonia in patients with AIDS and respiratory symptoms.

OBJECTIVE: The objective of this study was to define the test characteristics of plasma beta-glucan for diagnosis of Pneumocystis jirovecii pneumonia (PCP) in AIDS patients with respiratory symptoms. DESIGN: Analysis of baseline blood samples in a randomized strategy study of patients with acute opportunistic infections, limited to participants with respiratory symptoms. METHODS: Participants in the 282-person ACTG A5164 trial had baseline plasma samples assayed for beta-glucan testing. As part of A5164 trial, two study investigators independently adjudicated the diagnosis of PCP. Respiratory symptoms were identified by investigators from a list of all signs and symptoms with an onset or resolution in the 21 days prior to or 14 days following study entry. Beta-glucan was defined as positive if at least 80 pg/ml and negative if less than 80 pg/ml. RESULTS: Of 252 study participants with a beta-glucan result, 159 had at least one respiratory symptom, 139 of whom had a diagnosis of PCP. The sensitivity of beta-glucan for PCP in participants with respiratory symptoms was 92.8% [95% confidence interval (CI) 87.2-96.5], and specificity 75.0% (95% CI 50.9-91.3). Among 134 individuals with positive beta-glucan and respiratory symptoms, 129 had PCP, for a positive predictive value of 96.3% (95% CI 91.5-98.8). Fifteen of 25 patients with a normal beta-glucan did not have PCP, for a negative predictive value of 60% (95% CI 38.7-78.9). CONCLUSION: Elevated plasma beta-glucan has a high predictive value for diagnosis of PCP in AIDS patients with respiratory symptoms. We propose an algorithm for the use of beta-glucan as a diagnostic tool on the basis of the pretest probability of PCP in such patients.

Association between circulating DNA, serum (1->3)-ß-D-glucan, and pulmonary fungal burden in Pneumocystis pneumonia.

Circulating Pneumocystis jirovecii DNA and (1→3)-ß-d-glucan determined in 70 serum samples from immunocompromised patients were compared to fungal load in bronchoalveolar lavage fluids assessed using quantitative polymerase chain reaction. Both serum biomarkers are influenced by pulmonary fungal load, which should be taken into account when diagnosing Pneumocystis infection.

Protein pharmacophore selection using hydration-site analysis.

Virtual screening using pharmacophore models is an efficient method to identify potential lead compounds for target proteins. Pharmacophore models based on protein structures are advantageous because a priori knowledge of active ligands is not required and the models are not biased by the chemical space of previously identified actives. However, in order to capture most potential interactions between all potentially binding ligands and the protein, the size of the pharmacophore model, i.e. number of pharmacophore elements, is typically quite large and therefore reduces the efficiency of pharmacophore based screening. We have developed a new method to select important pharmacophore elements using hydration-site information. The basic premise is that ligand functional groups that replace water molecules in the apo protein contribute strongly to the overall binding affinity of the ligand, due to the additional free energy gained from releasing the water molecule into the bulk solvent. We computed the free energy of water released from the binding site for each hydration site using thermodynamic analysis of molecular dynamics (MD) simulations. Pharmacophores which are colocalized with hydration sites with estimated favorable contributions to the free energy of binding are selected to generate a reduced pharmacophore model. We constructed reduced pharmacophore models for three protein systems and demonstrated good enrichment quality combined with high efficiency. The reduction in pharmacophore model size reduces the required screening time by a factor of 200-500 compared to using all protein pharmacophore elements. We also describe a training process using a small set of known actives to reliably select the optimal set of criteria for pharmacophore selection for each protein system.

Quantitative real-time PCR and the (1→3)-ß-D-glucan assay for differentiation between Pneumocystis jirovecii pneumonia and colonization.

We evaluated whether quantitative PCR (qPCR) and (1 → 3)-ß-d-glucan assays could be used to differentiate Pneumocystis pneumonia (PCP) from Pneumocystis jirovecii colonization in immunocompromised patients with pulmonary infiltrates. A total of 40 bronchoalveolar lavage samples and 107 induced sputum samples from 147 patients who were suspected of having PCP were obtained for PCR detection of P. jirovecii. Diagnoses of definite PCP, probable PCP, pneumonia with P. jirovecii colonization (colonization) and pneumonia without colonization (non-colonization) were made in 11, 42, 15 and 60 patients, respectively. A PCP diagnosis was undetermined in 19 patients. The copy numbers, determined using qPCR, were significantly higher in definite PCP and probable PCP patients than in colonized patients. The area under the receiver-operating characteristic curve (AUC), sensitivity and specificity for discriminating definite PCP from colonization were 0.96, 100.0% and 80.0%, respectively, at a cut-off value of 1300 copies/mL. The values for discriminating probable PCP from colonization were 0.71, 66.7% and 73.3%, respectively, at a cut-off value of 340 copies/mL. ß-d-glucan levels were significantly higher in patients with both definite PCP and probable PCP than in colonized patients. The AUC, sensitivity and specificity for discriminating definite PCP were 0.91, 100.0% and 80.0%, respectively, at a cut-off value of 15.6 pg/mL. The values for discriminating probable PCP were 0.78, 76.2% and 73.3%, respectively, at a cut-off value of 6.0 pg/mL. Both qPCR and the ß-d-glucan assay displayed high accuracy for discriminating colonization from definite PCP and displayed moderate accuracy for discriminating colonization from probable PCP.

Substrate analysis of the Pneumocystis carinii protein kinases PcCbk1 and PcSte20 using yeast proteome microarrays provides a novel method for Pneumocystis signalling biology.

Pneumocystis carinii (Pc) undergoes morphological transitions between cysts and trophic forms. We have previously described two Pc serine/threonine kinases, termed PcCbk1 and PcSte20, with PcSte20 belonging to a family of kinases involved in yeast mating, while PcCbk1 is a member of a group of protein kinases involved in regulation of cell cycle, shape, and proliferation. As Pc remains genetically intractable, knowledge on specific substrates phosphorylated by these kinases remains limited. Utilizing the phylogenetic relatedness of Pc to Saccharomyces cerevisiae, we interrogated a yeast proteome microarray containing >4000 purified protein based peptides, leading to the identification of 18 potential PcCbk1 and 15 PcSte20 substrates (Z-score > 3.0). A number of these potential protein substrates are involved in bud site selection, polarized growth, and response to mating α factor and pseudohyphal and invasive growth. Full-length open reading frames suggested by the PcCbk1 and PcSte20 protoarrays were amplified and expressed. These five proteins were used as substrates for PcCbk1 or PcSte20, with each being highly phosphorylated by the respective kinase. Finally, to demonstrate the utility of this method to identify novel PcCbk1 and PcSte20 substrates, we analysed DNA sequence data from the partially complete Pc genome database and detected partial sequence information of potential PcCbk1 kinase substrates PcPxl1 and PcInt1. We additionally identified the potential PcSte20 kinase substrate PcBdf2. Full-length Pc substrates were cloned and expressed in yeast, and shown to be phosphorylated by the respective Pc kinases. In conclusion, the yeast protein microarray represents a novel crossover technique for identifying unique potential Pc kinase substrates.

Blood (1->3)-beta-D-glucan as a diagnostic test for HIV-related Pneumocystis jirovecii pneumonia.

UNLABELLED: (See the editorial commentary by Morris and Masur, on pages 203-204.) BACKGROUND: Improved noninvasive diagnostic tests for Pneumocystis jirovecii pneumonia (PCP) are needed. We evaluated the test characteristics of plasma (1 → 3)-ß-D-glucan (ß-glucan) for HIV-related PCP among a large group of patients presenting with diverse opportunistic infections (OIs). METHODS: The study population included all 282 participants in AIDS Clinical Trials Group A5164, a study of early versus deferred antiretroviral therapy in conjunction with initial therapy of acute OIs. Baseline plasma samples were assayed for ß-glucan, with standard assay reference values defining ≥ 80 pg/mL as positive. Before this analysis, diagnosis of PCP was independently adjudicated by 2 study investigators after reviewing reports from study sites. RESULTS: A total of 252 persons had a ß-glucan result that could be analyzed, 173 (69%) of whom had received a diagnosis of PCP. Median ß-glucan with PCP was 408 pg/mL (interquartile range [IQR], 209-500 pg/mL), compared with 37 pg/mL (IQR, 31-235 pg/mL) without PCP (P < .001). The sensitivity of ß-glucan dichotomized at 80 pg/mL for the diagnosis of PCP was 92% (95% confidence interval [CI], 87%-96%), and the specificity was 65% (95% CI, 53%-75%); positive and negative predictive values were 85% (95% CI, 79%-90%) and 80% (95% CI, 68%-89%) respectively, based on the study prevalence of 69% of patients with PCP. Rates of abnormal lactate dehyrogenase levels did not differ significantly between those with and without PCP. CONCLUSIONS: Blood (1 → 3)-ß-D-glucan is strongly correlated with HIV-related PCP. In some clinical centers, this may be a more sensitive test than the induced sputum examination and could reduce the need for both bronchoscopy and empirical therapy of PCP.

ß-d-Glucan kinetics for the assessment of treatment response in Pneumocystis jirovecii pneumonia.

Serum (1→3)-ß-D-Glucan (BG) is a biomarker for Pneumocystis jirovecii pneumonia (PJP). However, information concerning its usefulness for monitoring the clinical course is lacking. We conducted a retrospective study to investigate whether consecutive BG-measurements can be used to assess treatment response in PJP. Analysis of sera from 18 patients during PJP therapy shows that decreasing BG-levels strongly correlate with a favourable clinical course. In contrast, increasing BG-levels were associated with treatment failure or fatal outcome is only 44% of patients. As a consequence, BG-kinetics might be used to confirm treatment success but seem to be of limited value for the identification of treatment failure.