Background The wealth of information on protein structure has led to a variety of statistical analyses of the role played by individual amino acid types in the protein fold. In particular, the contact propensities between the various amino acids can be converted into folding energies that have proved useful in structure prediction. The present study addresses the relationship of protein folding propensities to the evolutionary relationship between residues.
Background
The wealth of information on protein structure has led to a variety of statistical analyses of the role played by individual amino acid types in the protein fold. In particular, the contact propensities between the various amino acids can be converted into folding energies that have proved useful in structure prediction. The present study addresses the relationship of protein folding propensities to the evolutionary relationship between residues.Results The contact preferences of residue types observed in a representative sample of protein structures are converted into a residue similarity matrix or inter-residue distance matrix. Remarkably, these distances correlate excellently with evolutionary substitution costs. Residue vectors are derived from the distance matrix. The residue vectors give a concrete picture of the grouping of residues into families sharing properties crucial for protein folding.
Results
The contact preferences of residue types observed in a representative sample of protein structures are converted into a residue similarity matrix or inter-residue distance matrix. Remarkably, these distances correlate excellently with evolutionary substitution costs. Residue vectors are derived from the distance matrix. The residue vectors give a concrete picture of the grouping of residues into families sharing properties crucial for protein folding.Conclusions Inter-residue distances have proved useful in showing the explicit relationship between contact preferences and evolutionary substitution rates. It is proposed that the distance matrix derived from structural analysis may be useful in aligning proteins where remote homologs share structural features. Residue vectors derived from the distance matrix illustrate the spatial arrangement of residues and point to ways in which they can be grouped.
Conclusions
Inter-residue distances have proved useful in showing the explicit relationship between contact preferences and evolutionary substitution rates. It is proposed that the distance matrix derived from structural analysis may be useful in aligning proteins where remote homologs share structural features. Residue vectors derived from the distance matrix illustrate the spatial arrangement of residues and point to ways in which they can be grouped.

Background The large number of protein crystal structures available has naturally led to statistical analyses of protein folding and protein interaction in the hope that these will point to intrinsic residue characteristics and therefore serve as aids in protein fold and interaction prediction. The first such analysis was performed by Miyazawa and Jernigan [ 1 - 3 ], where a statistical protein folding potential, the MJ matrix, was deduced from residue contact propensities in a set of monomeric protein crystal structures. The MJ matrix has been used in various  in silico  folding experiments, reviewed by Jernigan et al [ 4 ], and shown to point to the essentially hydrophobic nature of folding interactions [ 5 ]. An analysis of the MJ matrix has enabled the reduction in sequence complexity by grouping residues into families [ 6 ]. A more detailed study of crystal interactions focusing on hydrogen bond distributions has resulted in mean force potentials that have been successfully used in ligand prediction [ 7 ]. It is reasonable therefore to conclude that the statistical approach has pointed to an intrinsic residue:residue potential. In this study we will show that crystal contact statistics can be used to define an inter-residue similarity score that is strongly correlated with an evolutionary substitution cost. As this score is not based on aligning homologous proteins it can serve as a complement to similarity scores derived from substitution matrices when faced with the problem of aligning remotely homologous but structurally similar proteins. The observation that evolutionarily close residues appear to have similar contact propensities leads us to postulate that the extent of similarity between the contact propensities corresponding to two particular amino acids is related to the ease with which these amino acids can be mutated into each other. We define the contact propensity as  , where  N ij  is the number of possible pairings between residue type  i  and residue type  j  and  C ij  is the number of these parings corresponding to residues in contact. Only non-neighbouring residues on the protein chain are considered and a pair of residues is defined to be in contact if any of their side chain atoms are within a given distance of each other. The difference in contact propensities for two amino acid types can be measured by their rms difference and we define   as our amino acid difference measure or distance matrix. If we have really got a measure of the distances between residue types then it should follow that residues sharing physical properties are close together. More crucially, we expect that residues that are distant according to  D(P)  will be difficult to mutate into one another and vice versa. This is because the factors involved in determining mutation rates are dominated by those affecting the structural integrity of the protein. Such factors are residue hydropathy, size, charge and etc. Substitution matrices such as PAM and Blosum are determined from mutation rates in aligned protein sequences [ 8 , 9 ]. We can define an amino acid distance matrix in a similar way to  D ij  above. That is,  , where  S  is the substitution rate matrix. We show below that  D ( P ) is indeed strongly correlated with  D ( S ). It must be stressed that  D ( P ) and  D ( S ) are independently derived, with one based on structure and the other on sequence alignment. Their strong correlation is indicative of the validity of our definition of inter-residue distance. Relating amino acids through a structurally defined distance measure should provide a useful tool for aligning remotely homologous protein sequences. Also, a distance measure naturally leads us to look for a vector representation of the amino acids. In much the same way as average hydropathy plots are useful in structural analysis we expect that average vector profiles will also pick out various structural features. Given a vector for each residue type we can visualise the residues in some abstract space and look for natural groupings of residues and thereby find ways of reducing the effective number of residues.
Background
The large number of protein crystal structures available has naturally led to statistical analyses of protein folding and protein interaction in the hope that these will point to intrinsic residue characteristics and therefore serve as aids in protein fold and interaction prediction. The first such analysis was performed by Miyazawa and Jernigan [ 1 - 3 ], where a statistical protein folding potential, the MJ matrix, was deduced from residue contact propensities in a set of monomeric protein crystal structures. The MJ matrix has been used in various  in silico  folding experiments, reviewed by Jernigan et al [ 4 ], and shown to point to the essentially hydrophobic nature of folding interactions [ 5 ]. An analysis of the MJ matrix has enabled the reduction in sequence complexity by grouping residues into families [ 6 ]. A more detailed study of crystal interactions focusing on hydrogen bond distributions has resulted in mean force potentials that have been successfully used in ligand prediction [ 7 ]. It is reasonable therefore to conclude that the statistical approach has pointed to an intrinsic residue:residue potential. In this study we will show that crystal contact statistics can be used to define an inter-residue similarity score that is strongly correlated with an evolutionary substitution cost. As this score is not based on aligning homologous proteins it can serve as a complement to similarity scores derived from substitution matrices when faced with the problem of aligning remotely homologous but structurally similar proteins.in silicoThe observation that evolutionarily close residues appear to have similar contact propensities leads us to postulate that the extent of similarity between the contact propensities corresponding to two particular amino acids is related to the ease with which these amino acids can be mutated into each other. We define the contact propensity as  , where  N ij  is the number of possible pairings between residue type  i  and residue type  j  and  C ij  is the number of these parings corresponding to residues in contact. Only non-neighbouring residues on the protein chain are considered and a pair of residues is defined to be in contact if any of their side chain atoms are within a given distance of each other. The difference in contact propensities for two amino acid types can be measured by their rms difference and we define   as our amino acid difference measure or distance matrix.N ijijC ijIf we have really got a measure of the distances between residue types then it should follow that residues sharing physical properties are close together. More crucially, we expect that residues that are distant according to  D(P)  will be difficult to mutate into one another and vice versa. This is because the factors involved in determining mutation rates are dominated by those affecting the structural integrity of the protein. Such factors are residue hydropathy, size, charge and etc. Substitution matrices such as PAM and Blosum are determined from mutation rates in aligned protein sequences [ 8 , 9 ]. We can define an amino acid distance matrix in a similar way to  D ij  above.D(P)D ijThat is,  , where  S  is the substitution rate matrix. We show below that  D ( P ) is indeed strongly correlated with  D ( S ). It must be stressed that  D ( P ) and  D ( S ) are independently derived, with one based on structure and the other on sequence alignment. Their strong correlation is indicative of the validity of our definition of inter-residue distance.SDPDSDPDSRelating amino acids through a structurally defined distance measure should provide a useful tool for aligning remotely homologous protein sequences. Also, a distance measure naturally leads us to look for a vector representation of the amino acids. In much the same way as average hydropathy plots are useful in structural analysis we expect that average vector profiles will also pick out various structural features. Given a vector for each residue type we can visualise the residues in some abstract space and look for natural groupings of residues and thereby find ways of reducing the effective number of residues.Results A representative set of crystal structures was compiled from the PDBselect25 database [ 10 ], which contains structures sharing at most 25% sequence homology. We made sure that side chain coordinates were defined and restricted chain lengths to be greater than 50 and less than 500 residues long. In short we arrived at 1073 structures and performed the statistical analysis on these. Residues are held to be in contact if any of their respective side chain atoms are within a given distance of each other. Only residue pairs that are not neighbours along the chain are considered in the analysis of intra-molecular contacts. As explained above the contact propensities can be converted to a distance matrix  D ( P ). If this matrix is really a measure of residue similarity then we should be able to correlate it with an equivalent matrix constructed from an evolutionary substitution rate matrix. In what follows we will take PAM250 as the substitution matrix. In Figure  1a  we show the contour plots of  D ( S ) in the top triangle and  D ( P ) in the bottom triangle for a contact cut-off of 4.5Å, where the pearson correlation (r) is maximal, with  r  = 0.82. See  additional table 1  for explicit values of  P  and  D ( S ). The correlation can be seen explicitly in Figure  2b . The extent of correlation is roughly constant over a large range of cut-offs (4~8Å) and only drops when the cut-off is small and contacts are rare or when the cut-off is so big that non-interacting residues are scored, see Figure  1c . We expect that, due to the wide range of side chain sizes, a full atom representation is more accurate than a centroid representation and we find that the centroid  D ( P ) is consistently less well correlated with  D ( S ), peaking at  r  = 0.64 for a cutoff of 8Å, see Figure  1c . We have defined inter-residue distances and this implies that there must be a vector representation for the residues. In this case the distance matrix will be  , where   are the residue vectors. Explicitly, the vectors are defined such that   is minimal. When these vectors are derived it becomes clear that Cysteine is quite separate from the other residues in this property space and this maybe due to the unique role played by Cysteine in stabilising folds. Though it must be made clear that the distance matrix is independent of the frequency of an amino acid contacting its own kind and therefore does not count Cysteine bridges in the structures. Without Cysteine the distance matrix can be projected onto a plane i.e. the vectors can be taken to be two-dimensional and this vector space is illustrated in Figure  2a . It is a reasonable postulate that neighbouring residues share physical characteristics and we see similar residue groupings in a standard amino acid Venn diagram [ 11 ]. Indeed the vector grouping may serve as a way of reducing the effective amino acid number [ 6 ]. It is illuminating to compare vector spaces derived from other statistical analyses. The substitution rate vector space Figure  2b  is, as expected, similar to that of the contact propensity vector space, though in  D ( P ) residues with opposite hydropathies tend to be further apart. This is consistent with hydropathy playing a pivotal role in protein folding. In contrast, the MJ energy matrix vector space is shown in Figure  2c  and here the residues effectively lie on a line, which is in accordance with Li et al [ 5 ], where the MJ matrix was shown to be dominated by its principal eigenvector reduction. However, for the contact propensity and evolutionary substitution rate spaces, a lot of information is lost in such a linear projection and our analysis clearly points to a higher dimensional representation of the residues. Nonetheless, to make a concrete comparison of our vectors with existing scalar representations of amino acid properties we are forced to project our vectors onto a line. See  additional table 2  for the explicit vector components of the contact propensity, substitution rate and MJ energy matrices. The dominant driving force of folding, at least in defining the crude fold, is hydrophobicity and it is apparent that residues with similar hydrophobicities are grouped together. It also seems that residues of similar size tend to be close in this space. To make a direct comparison between existing residue scales and our vectors we can project the residue vectors onto a line. Here the amino acid scalars, one-dimensional vectors,  d i  are defined such that   is minimal. We find that these distance matrix derived scalars have a correlation of 0.65 with the Kyte-Doolittle hydrophobicity scale [ 12 ] and a correlation of 0.53 with an amino acid volume scale [ 13 ]. It is clear therefore that the residue vectors capture a combination of factors determining protein folding. It is worth noting that a scalar reduction of the distance matrix can be got by a principal eigenvector analysis. In a principal eigenvector reduction of the contact propensity matrix we have  P ij  =  λ e i e j , where  λ  is the principal eigenvalue and  e i  is the principal eigenvector, and consequently our distance matrix has a scalar representation,  . It is not surprising that the eigenvector is closely related to our scalar, in fact  r ( e , d ) = 0.98. There are many hydrophobicity scales in the literature [ 14 ] and some are remarkably similar to our scalar amino acid representation, for example  r  = 0.95 for Wertz & Scheraga scale [ 15 ]. However, the highly correlated scales are derived from residue burial statistics in protein structures and are therefore not independent of our statistic.
Results
A representative set of crystal structures was compiled from the PDBselect25 database [ 10 ], which contains structures sharing at most 25% sequence homology. We made sure that side chain coordinates were defined and restricted chain lengths to be greater than 50 and less than 500 residues long. In short we arrived at 1073 structures and performed the statistical analysis on these. Residues are held to be in contact if any of their respective side chain atoms are within a given distance of each other. Only residue pairs that are not neighbours along the chain are considered in the analysis of intra-molecular contacts.As explained above the contact propensities can be converted to a distance matrix  D ( P ). If this matrix is really a measure of residue similarity then we should be able to correlate it with an equivalent matrix constructed from an evolutionary substitution rate matrix. In what follows we will take PAM250 as the substitution matrix. In Figure  1a  we show the contour plots of  D ( S ) in the top triangle and  D ( P ) in the bottom triangle for a contact cut-off of 4.5Å, where the pearson correlation (r) is maximal, with  r  = 0.82. See  additional table 1  for explicit values of  P  and  D ( S ). The correlation can be seen explicitly in Figure  2b . The extent of correlation is roughly constant over a large range of cut-offs (4~8Å) and only drops when the cut-off is small and contacts are rare or when the cut-off is so big that non-interacting residues are scored, see Figure  1c . We expect that, due to the wide range of side chain sizes, a full atom representation is more accurate than a centroid representation and we find that the centroid  D ( P ) is consistently less well correlated with  D ( S ), peaking at  r  = 0.64 for a cutoff of 8Å, see Figure  1c .DPDSDPrPDSDPDSrWe have defined inter-residue distances and this implies that there must be a vector representation for the residues. In this case the distance matrix will be  , where   are the residue vectors. Explicitly, the vectors are defined such that   is minimal. When these vectors are derived it becomes clear that Cysteine is quite separate from the other residues in this property space and this maybe due to the unique role played by Cysteine in stabilising folds. Though it must be made clear that the distance matrix is independent of the frequency of an amino acid contacting its own kind and therefore does not count Cysteine bridges in the structures. Without Cysteine the distance matrix can be projected onto a plane i.e. the vectors can be taken to be two-dimensional and this vector space is illustrated in Figure  2a . It is a reasonable postulate that neighbouring residues share physical characteristics and we see similar residue groupings in a standard amino acid Venn diagram [ 11 ]. Indeed the vector grouping may serve as a way of reducing the effective amino acid number [ 6 ]. It is illuminating to compare vector spaces derived from other statistical analyses. The substitution rate vector space Figure  2b  is, as expected, similar to that of the contact propensity vector space, though in  D ( P ) residues with opposite hydropathies tend to be further apart. This is consistent with hydropathy playing a pivotal role in protein folding. In contrast, the MJ energy matrix vector space is shown in Figure  2c  and here the residues effectively lie on a line, which is in accordance with Li et al [ 5 ], where the MJ matrix was shown to be dominated by its principal eigenvector reduction. However, for the contact propensity and evolutionary substitution rate spaces, a lot of information is lost in such a linear projection and our analysis clearly points to a higher dimensional representation of the residues. Nonetheless, to make a concrete comparison of our vectors with existing scalar representations of amino acid properties we are forced to project our vectors onto a line. See  additional table 2  for the explicit vector components of the contact propensity, substitution rate and MJ energy matrices.DPThe dominant driving force of folding, at least in defining the crude fold, is hydrophobicity and it is apparent that residues with similar hydrophobicities are grouped together. It also seems that residues of similar size tend to be close in this space. To make a direct comparison between existing residue scales and our vectors we can project the residue vectors onto a line. Here the amino acid scalars, one-dimensional vectors,  d i  are defined such that   is minimal. We find that these distance matrix derived scalars have a correlation of 0.65 with the Kyte-Doolittle hydrophobicity scale [ 12 ] and a correlation of 0.53 with an amino acid volume scale [ 13 ]. It is clear therefore that the residue vectors capture a combination of factors determining protein folding.diIt is worth noting that a scalar reduction of the distance matrix can be got by a principal eigenvector analysis. In a principal eigenvector reduction of the contact propensity matrix we have  P ij  =  λ e i e j , where  λ  is the principal eigenvalue and  e i  is the principal eigenvector, and consequently our distance matrix has a scalar representation,  . It is not surprising that the eigenvector is closely related to our scalar, in fact  r ( e , d ) = 0.98. There are many hydrophobicity scales in the literature [ 14 ] and some are remarkably similar to our scalar amino acid representation, for example  r  = 0.95 for Wertz & Scheraga scale [ 15 ]. However, the highly correlated scales are derived from residue burial statistics in protein structures and are therefore not independent of our statistic.Pijλeiejλe iredrDiscussion We have generated full atom residue:residue contact propensity profiles for intra-molecular interactions from a non-redundant crystal structure database. Recasting the contact propensity matrix as a distance matrix we see that close residues are those with a low evolutionary substitution cost. The structure derived distance measures can serve as additional scores when aligning proteins where remote homologs share structural features. The distance matrix led us naturally to derive effective residue vectors. We found that residues sharing similar physical characteristics, such as hydrophobicity and volume, are grouped together. In contrast to the MJ matrix analysis, we find that a scalar representation for the residues is inadequate to capture the complexity of the propensity distance matrix. The most successful scalar representation for the amino acid residues has been the hydropathy scale. Representing a sequence as a smoothed hydropathy profile through wavelet analysis or simple averaging has resulted in many effective analytical tools, such as periodic structure prediction [ 16 ], remote homology analysis, helix prediction [ 14 ], transmembrane prediction [ 17 ] etc. It is then probable that a higher dimensional vector representation of the amino acids may lead to a more subtle sequence analysis. The distance matrix may also serve as an additional tool in sequence alignment as it gives one a measure of the structural cost of residue mutation and this is an idea we hope to pursue in a future study.
Discussion
We have generated full atom residue:residue contact propensity profiles for intra-molecular interactions from a non-redundant crystal structure database. Recasting the contact propensity matrix as a distance matrix we see that close residues are those with a low evolutionary substitution cost. The structure derived distance measures can serve as additional scores when aligning proteins where remote homologs share structural features. The distance matrix led us naturally to derive effective residue vectors. We found that residues sharing similar physical characteristics, such as hydrophobicity and volume, are grouped together. In contrast to the MJ matrix analysis, we find that a scalar representation for the residues is inadequate to capture the complexity of the propensity distance matrix. The most successful scalar representation for the amino acid residues has been the hydropathy scale. Representing a sequence as a smoothed hydropathy profile through wavelet analysis or simple averaging has resulted in many effective analytical tools, such as periodic structure prediction [ 16 ], remote homology analysis, helix prediction [ 14 ], transmembrane prediction [ 17 ] etc. It is then probable that a higher dimensional vector representation of the amino acids may lead to a more subtle sequence analysis. The distance matrix may also serve as an additional tool in sequence alignment as it gives one a measure of the structural cost of residue mutation and this is an idea we hope to pursue in a future study.Conclusions In this study we have shown that inter-residue distance matrices and residue vectors allow us to make an explicit connection between amino acid interaction preferences observed in protein structures and amino acid evolutionary substitution costs. When problems are encountered with aligning structurally related proteins that are remote homologs then the structurally defined distance matrix may prove to be an effective supplement to existing substitution rate derived matrices. The distance matrix leads naturally to an amino acid vector representation. Projecting the vectors onto a two-dimensional plane illustrates ways in which the amino acids can be grouped and their effective number thereby reduced.
Conclusions
In this study we have shown that inter-residue distance matrices and residue vectors allow us to make an explicit connection between amino acid interaction preferences observed in protein structures and amino acid evolutionary substitution costs. When problems are encountered with aligning structurally related proteins that are remote homologs then the structurally defined distance matrix may prove to be an effective supplement to existing substitution rate derived matrices. The distance matrix leads naturally to an amino acid vector representation. Projecting the vectors onto a two-dimensional plane illustrates ways in which the amino acids can be grouped and their effective number thereby reduced.Methods The database used in the present study was compiled from the PDBselect25 [ 10 ] list of representative proteins with known crystal structure that share less than 25% sequence homology. The structural coordinates were downloaded by automated ftp from the NCBI protein data bank. All programmes were written in C, compiled with Metrowerks CodeWarrior and run on a PC. In brief, the contact propensity statistics were compiled by reading the amino acid sequence and atomic coordinates for the specified chain of each pdb structure file in turn. The number of possible pairings of amino acid type  i  with amino acid type  j ,  N ij  were counted together with the number of these pairings corresponding to a pair with side chain atoms within a given distance of each other,  C ij . The contact propensity matrix is given by  . The residue vectors were defined such that   is minimal. The minimisation was carried out by a standard Newton-Raphson steepest descent iteration.
Methods
The database used in the present study was compiled from the PDBselect25 [ 10 ] list of representative proteins with known crystal structure that share less than 25% sequence homology. The structural coordinates were downloaded by automated ftp from the NCBI protein data bank. All programmes were written in C, compiled with Metrowerks CodeWarrior and run on a PC. In brief, the contact propensity statistics were compiled by reading the amino acid sequence and atomic coordinates for the specified chain of each pdb structure file in turn. The number of possible pairings of amino acid type  i  with amino acid type  j ,  N ij  were counted together with the number of these pairings corresponding to a pair with side chain atoms within a given distance of each other,  C ij . The contact propensity matrix is given by  . The residue vectors were defined such that   is minimal. The minimisation was carried out by a standard Newton-Raphson steepest descent iteration.ijN ijC ijSupplementary Material Additional table 1 Contact propensities and distance matrix derived from the structural database with contact cut-off set at 4.5Å. Click here for file Additional table 2 Two dimensional residue vector components derived from the contact propensity distance matrix, the PAM250 distance matrix and the MJ distance matrix. Click here for file
Supplementary Material
Additional table 1
Contact propensities and distance matrix derived from the structural database with contact cut-off set at 4.5Å.Click here for fileAdditional table 2
Two dimensional residue vector components derived from the contact propensity distance matrix, the PAM250 distance matrix and the MJ distance matrix.Click here for file


Background Pneumocystis jiroveci pneumonia (PCP) is an important opportunistic infection among immunosuppressed patients, especially in those infected with human immunodeficiency virus (HIV). The clinical presentation of PCP in immunosuppressed patients have been well-reported in the literature. However, the clinical importance of PCP manifesting in the setting of an immunorestitution disease (IRD), defined as an acute symptomatic or paradoxical deterioration of a (presumably) preexisting infection, which is temporally related to the recovery of the immune system and is due to immunopathological damage associated with the reversal of immunosuppressive processes, has received relatively little attention until recently.
Background
Pneumocystis jiroveci pneumonia (PCP) is an important opportunistic infection among immunosuppressed patients, especially in those infected with human immunodeficiency virus (HIV). The clinical presentation of PCP in immunosuppressed patients have been well-reported in the literature. However, the clinical importance of PCP manifesting in the setting of an immunorestitution disease (IRD), defined as an acute symptomatic or paradoxical deterioration of a (presumably) preexisting infection, which is temporally related to the recovery of the immune system and is due to immunopathological damage associated with the reversal of immunosuppressive processes, has received relatively little attention until recently.Case presentation We aim to better define this unique clinical syndrome by reporting two cases of PCP manifesting acutely with respiratory failure during reversal of immunosuppression in non-HIV infected patients, and reviewed the relevant literature. We searched our databases for PCP cases manifesting in the context of IRD according to our predefined case definition, and reviewed the case notes retrospectively. A comprehensive search was performed using the Medline database of the National Library of Medicine for similar cases reported previously in the English literature in October 2003. A total of 28 non-HIV (excluding our present case) and 13 HIV-positive patients with PCP manifesting as immunorestitution disease (IRD) have been reported previously in the literature. During immunorestitution, a consistent rise in the median CD4 lymphocyte count (28/μL to 125/μL), with a concomitant fall in the median HIV viral load (5.5 log 10  copies/ml to 3.1 log 10  copies/ml) was observed in HIV-positive patients who developed PCP. A similar upsurge in peripheral lymphocyte count was observed in our patients preceding the development of PCP, as well as in other non-HIV immunosuppressed patients reported in the literature.
Case presentation
We aim to better define this unique clinical syndrome by reporting two cases of PCP manifesting acutely with respiratory failure during reversal of immunosuppression in non-HIV infected patients, and reviewed the relevant literature. We searched our databases for PCP cases manifesting in the context of IRD according to our predefined case definition, and reviewed the case notes retrospectively. A comprehensive search was performed using the Medline database of the National Library of Medicine for similar cases reported previously in the English literature in October 2003. A total of 28 non-HIV (excluding our present case) and 13 HIV-positive patients with PCP manifesting as immunorestitution disease (IRD) have been reported previously in the literature. During immunorestitution, a consistent rise in the median CD4 lymphocyte count (28/μL to 125/μL), with a concomitant fall in the median HIV viral load (5.5 log 10  copies/ml to 3.1 log 10  copies/ml) was observed in HIV-positive patients who developed PCP. A similar upsurge in peripheral lymphocyte count was observed in our patients preceding the development of PCP, as well as in other non-HIV immunosuppressed patients reported in the literature.Conclusions PCP manifesting as IRD may be more common than is generally appreciated. Serial monitoring of total lymphocyte or CD4 count could serve as a useful adjunct to facilitate the early diagnosis and pre-emptive treatment of this condition in a wide range of immunosuppressed hosts, especially in the presence of new pulmonary symptoms and/or radiographic abnormalities compatible with the diagnosis.
Conclusions
PCP manifesting as IRD may be more common than is generally appreciated. Serial monitoring of total lymphocyte or CD4 count could serve as a useful adjunct to facilitate the early diagnosis and pre-emptive treatment of this condition in a wide range of immunosuppressed hosts, especially in the presence of new pulmonary symptoms and/or radiographic abnormalities compatible with the diagnosis.

Background Pneumocystis jiroveci  (Pj) (previously known as  Pneumocystis carinii  f. sp.  hominis ) was first identified as a pathogen in premature infants suffering from interstitial plasma cell pneumonia in European countries during and after World War II, occasionally occurring in epidemics [ 1 - 3 ]. Since then Pneumocystis pneumonia (PCP) had only been reported sporadically in patients with malignancies and solid organ transplantations until the HIV epidemic [ 4 ]. The incidence of PCP increased significantly after the emergence of human immunodeficiency virus (HIV) infection. However, with the identification of CD4 T lymphocyte depletion as an independent risk factor for the development of PCP [ 5 ], widespread use of antimicrobial prophylaxis [ 4 ], and the introduction of highly active antiretroviral therapy (HAART), there has been a steady decline in the incidence of PCP among HIV-infected patients [ 7 , 8 ]. Nevertheless, with the rising number of patients receiving immunosuppressive therapies for malignancies, solid organ transplantations and autoimmune diseases, PCP has been increasingly recognized in non-HIV immunosuppressed hosts [ 9 - 15 ]. For instance, PCP occurs in 3.4% to 43% of solid organ transplant recipients [ 16 ], and it is particularly prevalent among those patients who are put on long-term steroids. In a non-HIV immunosuppressed cohort with PCP, the use of steroids was found to be a contributing factor in 87% of patients [ 17 ]. In another similar cohort of immunosuppressed patients, steroids had been administered systemically in 90.5% within one month before the diagnosis of PCP. Although a median daily dose equivalent to 30 mg of prednisone was administered in most of these patients prior to the development of PCP, up to 25% had received as little as 16 mg of prednisone daily [ 18 ]. Interestingly, PCP has also been reported in patients with endogenous steroid excess due to Cushing's disease [ 19 , 20 ]. Paradoxically, the clinical symptoms of PCP were often unmasked in HIV-negative immunosuppressed patients during the reversal of immunosuppression, often at the time when the dose of steroids was tapered [ 11 , 17 , 21 - 24 ], or when the endogenous steroid production was reduced [ 25 - 27 ]. However, serial changes in the absolute lymphocyte count before and during reversal of immunosuppression were not mentioned in these patients. Recently, paradoxical worsening of clinical symptoms and signs of PCP after initiation of HAART has also been reported in HIV-positive patients [ 28 - 31 ]. The onset of clinical deterioration was associated with an upsurge in the CD4 lymphocyte count and a reduction in the HIV viral load [ 28 - 31 ]. Tissue damage is thought to occur as a result of immune reconstitution in HIV-positive patients. Here, we report two cases and review the literature on this topic from the perspective of immunorestitution disease.
Background
Pneumocystis jiroveci  (Pj) (previously known as  Pneumocystis carinii  f. sp.  hominis ) was first identified as a pathogen in premature infants suffering from interstitial plasma cell pneumonia in European countries during and after World War II, occasionally occurring in epidemics [ 1 - 3 ]. Since then Pneumocystis pneumonia (PCP) had only been reported sporadically in patients with malignancies and solid organ transplantations until the HIV epidemic [ 4 ]. The incidence of PCP increased significantly after the emergence of human immunodeficiency virus (HIV) infection. However, with the identification of CD4 T lymphocyte depletion as an independent risk factor for the development of PCP [ 5 ], widespread use of antimicrobial prophylaxis [ 4 ], and the introduction of highly active antiretroviral therapy (HAART), there has been a steady decline in the incidence of PCP among HIV-infected patients [ 7 , 8 ].Pneumocystis jiroveciPneumocystis cariniihominisNevertheless, with the rising number of patients receiving immunosuppressive therapies for malignancies, solid organ transplantations and autoimmune diseases, PCP has been increasingly recognized in non-HIV immunosuppressed hosts [ 9 - 15 ]. For instance, PCP occurs in 3.4% to 43% of solid organ transplant recipients [ 16 ], and it is particularly prevalent among those patients who are put on long-term steroids. In a non-HIV immunosuppressed cohort with PCP, the use of steroids was found to be a contributing factor in 87% of patients [ 17 ]. In another similar cohort of immunosuppressed patients, steroids had been administered systemically in 90.5% within one month before the diagnosis of PCP. Although a median daily dose equivalent to 30 mg of prednisone was administered in most of these patients prior to the development of PCP, up to 25% had received as little as 16 mg of prednisone daily [ 18 ]. Interestingly, PCP has also been reported in patients with endogenous steroid excess due to Cushing's disease [ 19 , 20 ].Paradoxically, the clinical symptoms of PCP were often unmasked in HIV-negative immunosuppressed patients during the reversal of immunosuppression, often at the time when the dose of steroids was tapered [ 11 , 17 , 21 - 24 ], or when the endogenous steroid production was reduced [ 25 - 27 ]. However, serial changes in the absolute lymphocyte count before and during reversal of immunosuppression were not mentioned in these patients. Recently, paradoxical worsening of clinical symptoms and signs of PCP after initiation of HAART has also been reported in HIV-positive patients [ 28 - 31 ]. The onset of clinical deterioration was associated with an upsurge in the CD4 lymphocyte count and a reduction in the HIV viral load [ 28 - 31 ]. Tissue damage is thought to occur as a result of immune reconstitution in HIV-positive patients. Here, we report two cases and review the literature on this topic from the perspective of immunorestitution disease.Case presentation Case 1 This is a fifty-one year old female patient with history of diabetes mellitus and systemic lupus erythematoses (SLE) complicated by lupus nephritis. Although we have included her case in our previous publication [ 32 ], we have not reported her clinical details at that time. She was put on prednisolone 30 mg and azathioprine 100 mg daily since end of June and mid-July 2002, respectively. She was admitted to Queen Mary Hospital on 11 th  August 2002 for investigation of jaundice. Investigations revealed deranged liver function tests with cholestatic pattern. A diagnosis of drug-induced hepatotoxicity was entertained, and azathioprine was stopped after admission. As her autoimmune disease was under control, her steroid dosage was reduced from 25 mg to 15 mg daily within the next 14 days. Her CXR taken on admission was normal. Soon after her immunosuppressive therapy was tapered, she developed fever and non-productive cough. A repeat CXR performed on 9 th  Sept revealed new infiltrate over the left mid-zone, suggestive of pneumonia. She was started on intravenous ceftazidime 1 gram eight hourly and oral clarithromycin 500 mg twice daily. Serial CXR performed three days later showed increasing bilateral pulmonary infiltrates and worsening hypoxemia. There was an upsurge of total lymphocyte count from 0.7 × 10 9 /L (total white cell count 7.2 × 10 9 /L) at the time of admission to 5.6 × 10 9 /L (total white cell count 10.8 × 10 9 /L) at the time of clinical deterioration. Bronchoscopy with transbronchial biopsy performed on the same day revealed  Pneumocystitis jiroveci  by methenamine sliver stain. Workup for other opportunistic pathogens including cytomegalovirus and aspergillus was negative. She was commenced on intravenous pentamidine (4 mg/kg/day) and corticosteroids for severe PCP infection. Despite active treatment she developed progressive respiratory failure and required admission to intensive care unit. She subsequently recovered after a stormy hospital course, and upon discharge from hospital, her total lymphocyte count had returned to her baseline of 0.86 × 10 9 /L. Case 2 A thirty-three year old gentleman initially presented to Prince of Wales Hospital with a diagnosis of SLE/dermatomyositis overlap syndrome. He was treated with steroid and hydroxychloroquine 200 mg twice daily since 1997. He had a flare up of disease in May 1998 with active vasculitis and myositis, for which he was put on prednisolone and azthioprine 50 mg and 100 mg daily respectively. Upon reassessment one month later, disease activity was under control, and the dosage of prednisolone was reduced to 45 mg daily. Twelve day after reducing the immunosuppressive regimen, he was admitted to hospital for treatment of left buttock abscess. The CXR taken on admission was unremarkable. An aspirate of the pus from the lesion grew methicillin-sensitive  staphylococcus aureus ; he was treated with cloxacillin 1 g intravenously every 6 hourly, together with incision and drainage of the buttock abscess. In view of the underlying active pyogenic infection, the steroid dosage was rapidly tapered from 45 mg to 15 mg daily within the next four days. However, he was noted to have persistent fever associated with mild unproductive cough. A repeat chest radiograph showed new infiltrates over the right upper and left lower zones, and he was empirically treated with intravenous ceftazidime 1 gram every 8 hours, cloxacillin 1 gram every 6 hours and netimicin 100 mg every 8 hours. As there was no clinical response after 5 days of treatment, bronchoscopy and bronchoalveolar lavage (BAL) was performed, which was positive for  Pneumocystis jiroveci . Investigation for the presence of co-existing opportunistic pathogens such as cytomegalovirus and aspergillus was negative. On the day after bronchoscopy, he was commenced on intravenous cotrimoxazole 1.3 grams every 6 hours. He remained stable initially with fever on downward trend. However, on the 3 rd  day of treatment, he developed sudden desaturation with resurgence of high fever, and required supplemental oxygen therapy. Repeat chest radiograph showed increased perihilar hazziness in both lung fields. There was also an upsurge of total lymphocyte count from 0.6 × 10 9 /L (total white cell count 11.2 × 10 9 /L) on admission, to 1.3 × 10 9 /L (total white cell count 10.4 × 10 9 /L) at the time of clinical deterioration. He was treated with high dose prednisolone (80 mg daily), and his condition improved promptly afterwards. He was subsequently discharged, and on follow up at the clinic one month later, his total lymphocyte count had returned to his baseline level of 0.6 × 10 9 /L. Immunorestitution disease (IRD) has been described in both HIV and non-HIV immunosuppressed hosts previously [ 27 - 31 ]. In the setting of PCP, it is defined as an acute symptomatic presentation of the disease that is related temporally to the recovery of the immune system, associated with reversal of immunosuppressive processes such as reduction in the dosage of corticosteroids and/or cytotoxic agents or a reduction of HIV viral load due to HAART, which results in the development of immunopathological damage. The preexisting microbial infection could be either asymptomatic or mildly symptomatic. Using this case definition, we attempted to review the English literature for other reported cases of PCP manifesting as IRD. The English-language literature (1966 – 2003) was searched in the Medline database of the National Library of Medicine in October 2003. The keywords " Pneumocystis carinii ", " Pneumocystis jiroveci ", "HIV", immunosuppression", "immunosuppressive", "steroid", and "corticosteroid" were used to select cases. All the case reports and case series with clinical details were included in this study if they fulfilled the above definition of IRD. When appropriate, the cited bibliographies were also retrieved for further analysis. As for statistical analysis, we used the Wilcoxon Signed Rank test, a non-parametric test for comparing paired samples, to analyze the serial changes in lymphocyte counts and HIV viral loads before and during the development of IRD. A two-tailed p-value of less than 0.05 was considered significant. All statistical analyses were performed using SPSS version 11.5 for Windows. Including our present case, a total of 29 cases of PCP in non-HIV immunosuppressed hosts fulfilling our definition of IRD have been reported in the literature (table  1 ) [ 22 - 27 , 32 ]. There were altogether 13 males and 8 females, with a median age of 38 years (range 2 to 75 years). The age and sex were not mentioned in 8 cases. Their underlying immunosuppressive conditions included solid organ tumours (13 cases), haematological diseases (8), autoimmune diseases (4), endogenous Cushing's disease (3), and a solid organ transplant recipient (1). All patients had received steroids or had excessive endogenous steroid production, whereas 18 (62.1%) of them had concomitant cytotoxic therapy for the underlying diseases. The median duration between steroid tapering and clinical manifestations of PCP was 21 days (range 1 to 83 days). Steroids were completely withdrawn at a median of 7.5 days (range 1 to 21 days) before the onset of symptoms in eight patients. Serial lymphocyte counts were only available in eight patients. An upsurge of the absolute lymphocyte counts was observed from the time of reduction of immunosuppression (median 300/μL, range 290 to 600/μL at baseline) to the time of occurrence of IRD (median 1200/μL, range 600 to 5620/μL); the median increase in total lymphocyte count was 800/μL, with a range of 300 to 4880/μL. Comparing the lymphocyte counts before and after reversal of immunosuppressive therapy, the difference was statistically significant (Wilcoxon Signed Rank Test for paired samples; p = 0.012). In addition to our patient, reintroduction or increasing doses of steroids were required in 7 (53.8%) of 13 patients in the acute management of PCP in the literature, at the time when they developed clinical deterioration during antimicrobial therapy [ 24 - 27 ]. Seven (53.8%) of 13 cases had respiratory failure requiring mechanical ventilation. Among these 29 cases, 13 (44.8%) subsequently died of PCP. Among HIV-positive patients, 13 cases with newly diagnosed PCP were reported in the literature, in which IRD occurred shortly after the introduction of HAART (table  2 ) [ 28 - 31 ]. Seven (53.8%) out of 13 cases received steroids as adjunctive therapy in addition to antimicrobials. HAART was given in all cases at a median 18 days (range 1 to 35 days) after the initiation of treatment for PCP. During IRD, recurrence of fever (100%), dyspnoea (100%), and paradoxical worsening of pulmonary infiltrates (58.3%) were observed in these patients [ 28 - 31 ]. IRD occurred at a median of 14 days (range 5 to 17 days) after HAART. An upsurge of the CD4 lymphocyte count was observed before (median 28/μL, range 4 to 290/μL) and during IRD (median 125/μL, range 30 to 564/μL); this was associated with a concomitant reduction of the median HIV viral load from 5.5 log 10  copies/ml (range 5.0 to 5.9 log 10  copies/ml) to 3.1 log 10  copies/ml (range 2.9 to 4.5 log 10  copies/ml) before and during IRD respectively, and the differences observed in both the CD4 counts and viral loads before and during IRD reached statistical significance (Wilcoxon Signed Rank Test for related samples; p = 0.001 and 0.017, respectively). Antimicrobials, steroids, or both for PCP were reintroduced for IRD in 4, 1, and 6 cases respectively. Only 2 cases were treated conservatively. One case required mechanical ventilation for severe respiratory distress. None of the patients died. PCP manifesting as a form of IRD is not a rare phenomenon. As shown in our previous study, it happens in 7 out of 10 (70%) of HIV-negative immunosuppressed hosts infected with Pj [ 32 ]. However, the diagnosis of PCP is usually delayed in this group of patients because of atypical presentation. In this clinical setting, PCP manifesting as IRD often runs an acute and fulminant course, with nonspecific lesions on chest radiographs, and high absolute lymphocyte counts [ 32 ]. In our own reported series, despite the administration of steroid therapy to suppress the immunopathological damage, more than 80% of patients developed acute respiratory failure and required mechanical ventilation. Patients who developed PCP during reversal of immunosuppressive therapy in our series tended to be older, and this might partially explain the increased mortality observed in this group [ 32 ]. Rapid reduction of immunosuppressive therapy such as steroids has been implicated as a predisposing factor for the development of PCP in HIV-negative patients [ 11 , 17 , 23 , 24 ]. In one study, PCP occurred in 79 (70%) of 113 patients during steroid tapering [ 17 ]. Another study suggested that 8 (72.2%) out of 11 episodes of PCP developed when steroid therapy was tapered [ 23 ]. A subsequent study also demonstrated that 43% of patients had a rapid reduction of steroid dosing before the clinical manifestations of PCP [ 11 ]. A similar experience was reported in children, and 17 (89.5%) out of 19 children were diagnosed to have PCP during steroid tapering according to a previous report [ 21 ]. Another series revealed that 7 of 11 patients experienced acutely symptomatic PCP when the dose of steroids was decreased or terminated 5 days to 3 weeks before the diagnosis of PCP [ 22 ]. However, all these cases were not analyzed from a perspective of IRD. Serial changes of the absolute lymphocyte counts or their subsets were either not noted or reported [ 11 , 17 , 21 - 24 ]. Hence we have not included these cases for further analysis in this review. Among HIV positive patients, PCP manifesting acutely during the initiation anti-retroviral therapy is a well-recognized phenomenon. The underlying immunopathological nature of this condition, which is reminiscent to IRD occurring in non-HIV infected patients, has been confirmed by histological examination of the lungs and transbronchial biopsy specimens, which demonstrated mixed inflammatory infiltrates including macrophages, neutrophils, lymphocytes, and plasma cells. Almost all infiltrating lymphocytes found in the tissues were of the T cell lineage, shown by immunophenotyping to be predominantly CD4 and CD8 cells [ 28 ]. In another study [ 30 ], the BAL fluid obtained from one of six patients with an IRD-type presentation of PCP was analyzed. Infiltration of predominantly CD4 and CD8 lymphocytes with the proliferative marker (Ki67) and perforin-positive cell were seen in the BAL specimen. Therefore, it is likely that the phagocytosed Pj is presented by alveolar macrophage to T cells, which trigger the inflammatory response [ 30 ]. In our own experience, as well as from the review of published literature, it appears that a surge of absolute lymphocyte count, especially the CD4 lymphocyte count in HIV-positive patients, could potentially act as a surrogate marker for immunopathological damage during IRD in both HIV-negative and HIV-positive patients. In our recent publication [ 32 ], 7 out of 10 non-HIV immunosuppressed patients demonstrated a consistent rise in the absolute lymphocyte count during tapering of immunosuppression prior to the onset of symptomatic PCP. In this group of patients, the surge in lymphocyte count is likely the result of withdrawal of lymphocytotoxic immunosuppressants such as corticosteroids. Similarly, a rising trend of the CD4 lymphocyte count, consistent with immune reconstitution after HAART, was also observed in 13 HIV-positive cases before and during the development of symptomatic PCP [ 28 - 31 ]. In fact, an upsurge in the absolute lymphocyte count has been shown to be a marker of IRD in our previous publications involving viral and tuberculous infections [ 33 - 36 ]. However, it must be emphasized that the number of circulating lymphocytes may not always correlate with their number in the affected tissues or their in vivo functional activity. This can be exemplified by a case of PCP occurring during steroid withdrawal, in which the lymphocyte counts surged to a very high level and then rapidly dropped to a low level within one day. The migration of lymphocytes from the circulation to tissue might explain this rapid drop in lymphocyte count and the resulting immunopathological damage [ 27 ]. In the future, further studies on lymphocyte subsets and cytokine profiles of susceptible hosts during the development of IRD should be performed to elucidate the underlying immunopathological mechanisms behind this interesting phenomenon. From the result of this review, it appears that HIV-positive patients with PCP are at risk of clinical deterioration due to IRD if HAART therapy is started within 1 to 2 weeks after the initiation of treatment for PCP (table  2 ). With a better understanding of the pathogenetic mechanisms resulting in IRD, we may be able to prevent the occurrence of IRD by delaying the initiation of HAART in HIV-positive patients with PCP. However, in non-HIV immunosuppressed patients, it is even more important to recognize the atypical presentations of PCP in the context of IRD. Since the clinical and/or radiological features alone may not be sufficient for diagnosis, analysis of serial changes in lymphocyte counts in patients undergoing a reduction of immunosuppression can alert the clinician to the possibility of IRD due to occult pathogens such as Pj. To prevent IRD in non-HIV immunosuppressed patients, the use of prophylactic antibiotics against Pj to reduce the microbial load in selected patients remains an important issue. Recently, a multi-center study showed that the CD4 lymphocyte count may be a useful marker to monitor the risk of development of PCP in non-HIV immunosuppressed hosts [ 37 ], and patients with low CD4 lymphocyte counts of less than 300 or 400 may require prophylaxis. In fact, asymptomatic colonization of Pj has been demonstrated in HIV-negative patients when the CD4 lymphocyte count was less than 400 [ 38 ]. Nested polymerase chain reaction (PCR) identified a significant percentage of clinically silent Pj colonization in 20% of non-HIV immunosuppressed patients [ 39 ]. Therefore, early detection of asymptomatic infection of Pj in blood and respiratory specimens before, and during intense immunosuppression may enable selection of cases for pre-emptive treatment of Pj infection in order to prevent the development of IRD during reversal of immunosuppression [ 40 , 41 ].
Case presentation
Case 1 This is a fifty-one year old female patient with history of diabetes mellitus and systemic lupus erythematoses (SLE) complicated by lupus nephritis. Although we have included her case in our previous publication [ 32 ], we have not reported her clinical details at that time. She was put on prednisolone 30 mg and azathioprine 100 mg daily since end of June and mid-July 2002, respectively. She was admitted to Queen Mary Hospital on 11 th  August 2002 for investigation of jaundice. Investigations revealed deranged liver function tests with cholestatic pattern. A diagnosis of drug-induced hepatotoxicity was entertained, and azathioprine was stopped after admission. As her autoimmune disease was under control, her steroid dosage was reduced from 25 mg to 15 mg daily within the next 14 days. Her CXR taken on admission was normal. Soon after her immunosuppressive therapy was tapered, she developed fever and non-productive cough. A repeat CXR performed on 9 th  Sept revealed new infiltrate over the left mid-zone, suggestive of pneumonia. She was started on intravenous ceftazidime 1 gram eight hourly and oral clarithromycin 500 mg twice daily. Serial CXR performed three days later showed increasing bilateral pulmonary infiltrates and worsening hypoxemia. There was an upsurge of total lymphocyte count from 0.7 × 10 9 /L (total white cell count 7.2 × 10 9 /L) at the time of admission to 5.6 × 10 9 /L (total white cell count 10.8 × 10 9 /L) at the time of clinical deterioration. Bronchoscopy with transbronchial biopsy performed on the same day revealed  Pneumocystitis jiroveci  by methenamine sliver stain. Workup for other opportunistic pathogens including cytomegalovirus and aspergillus was negative. She was commenced on intravenous pentamidine (4 mg/kg/day) and corticosteroids for severe PCP infection. Despite active treatment she developed progressive respiratory failure and required admission to intensive care unit. She subsequently recovered after a stormy hospital course, and upon discharge from hospital, her total lymphocyte count had returned to her baseline of 0.86 × 10 9 /L.
Case 1
This is a fifty-one year old female patient with history of diabetes mellitus and systemic lupus erythematoses (SLE) complicated by lupus nephritis. Although we have included her case in our previous publication [ 32 ], we have not reported her clinical details at that time. She was put on prednisolone 30 mg and azathioprine 100 mg daily since end of June and mid-July 2002, respectively. She was admitted to Queen Mary Hospital on 11 th  August 2002 for investigation of jaundice. Investigations revealed deranged liver function tests with cholestatic pattern. A diagnosis of drug-induced hepatotoxicity was entertained, and azathioprine was stopped after admission. As her autoimmune disease was under control, her steroid dosage was reduced from 25 mg to 15 mg daily within the next 14 days. Her CXR taken on admission was normal.Soon after her immunosuppressive therapy was tapered, she developed fever and non-productive cough. A repeat CXR performed on 9 th  Sept revealed new infiltrate over the left mid-zone, suggestive of pneumonia. She was started on intravenous ceftazidime 1 gram eight hourly and oral clarithromycin 500 mg twice daily. Serial CXR performed three days later showed increasing bilateral pulmonary infiltrates and worsening hypoxemia. There was an upsurge of total lymphocyte count from 0.7 × 10 9 /L (total white cell count 7.2 × 10 9 /L) at the time of admission to 5.6 × 10 9 /L (total white cell count 10.8 × 10 9 /L) at the time of clinical deterioration. Bronchoscopy with transbronchial biopsy performed on the same day revealed  Pneumocystitis jiroveci  by methenamine sliver stain. Workup for other opportunistic pathogens including cytomegalovirus and aspergillus was negative. She was commenced on intravenous pentamidine (4 mg/kg/day) and corticosteroids for severe PCP infection. Despite active treatment she developed progressive respiratory failure and required admission to intensive care unit. She subsequently recovered after a stormy hospital course, and upon discharge from hospital, her total lymphocyte count had returned to her baseline of 0.86 × 10 9 /L.Pneumocystitis jiroveciCase 2 A thirty-three year old gentleman initially presented to Prince of Wales Hospital with a diagnosis of SLE/dermatomyositis overlap syndrome. He was treated with steroid and hydroxychloroquine 200 mg twice daily since 1997. He had a flare up of disease in May 1998 with active vasculitis and myositis, for which he was put on prednisolone and azthioprine 50 mg and 100 mg daily respectively. Upon reassessment one month later, disease activity was under control, and the dosage of prednisolone was reduced to 45 mg daily. Twelve day after reducing the immunosuppressive regimen, he was admitted to hospital for treatment of left buttock abscess. The CXR taken on admission was unremarkable. An aspirate of the pus from the lesion grew methicillin-sensitive  staphylococcus aureus ; he was treated with cloxacillin 1 g intravenously every 6 hourly, together with incision and drainage of the buttock abscess. In view of the underlying active pyogenic infection, the steroid dosage was rapidly tapered from 45 mg to 15 mg daily within the next four days. However, he was noted to have persistent fever associated with mild unproductive cough. A repeat chest radiograph showed new infiltrates over the right upper and left lower zones, and he was empirically treated with intravenous ceftazidime 1 gram every 8 hours, cloxacillin 1 gram every 6 hours and netimicin 100 mg every 8 hours. As there was no clinical response after 5 days of treatment, bronchoscopy and bronchoalveolar lavage (BAL) was performed, which was positive for  Pneumocystis jiroveci . Investigation for the presence of co-existing opportunistic pathogens such as cytomegalovirus and aspergillus was negative. On the day after bronchoscopy, he was commenced on intravenous cotrimoxazole 1.3 grams every 6 hours. He remained stable initially with fever on downward trend. However, on the 3 rd  day of treatment, he developed sudden desaturation with resurgence of high fever, and required supplemental oxygen therapy. Repeat chest radiograph showed increased perihilar hazziness in both lung fields. There was also an upsurge of total lymphocyte count from 0.6 × 10 9 /L (total white cell count 11.2 × 10 9 /L) on admission, to 1.3 × 10 9 /L (total white cell count 10.4 × 10 9 /L) at the time of clinical deterioration. He was treated with high dose prednisolone (80 mg daily), and his condition improved promptly afterwards. He was subsequently discharged, and on follow up at the clinic one month later, his total lymphocyte count had returned to his baseline level of 0.6 × 10 9 /L. Immunorestitution disease (IRD) has been described in both HIV and non-HIV immunosuppressed hosts previously [ 27 - 31 ]. In the setting of PCP, it is defined as an acute symptomatic presentation of the disease that is related temporally to the recovery of the immune system, associated with reversal of immunosuppressive processes such as reduction in the dosage of corticosteroids and/or cytotoxic agents or a reduction of HIV viral load due to HAART, which results in the development of immunopathological damage. The preexisting microbial infection could be either asymptomatic or mildly symptomatic. Using this case definition, we attempted to review the English literature for other reported cases of PCP manifesting as IRD. The English-language literature (1966 – 2003) was searched in the Medline database of the National Library of Medicine in October 2003. The keywords " Pneumocystis carinii ", " Pneumocystis jiroveci ", "HIV", immunosuppression", "immunosuppressive", "steroid", and "corticosteroid" were used to select cases. All the case reports and case series with clinical details were included in this study if they fulfilled the above definition of IRD. When appropriate, the cited bibliographies were also retrieved for further analysis. As for statistical analysis, we used the Wilcoxon Signed Rank test, a non-parametric test for comparing paired samples, to analyze the serial changes in lymphocyte counts and HIV viral loads before and during the development of IRD. A two-tailed p-value of less than 0.05 was considered significant. All statistical analyses were performed using SPSS version 11.5 for Windows. Including our present case, a total of 29 cases of PCP in non-HIV immunosuppressed hosts fulfilling our definition of IRD have been reported in the literature (table  1 ) [ 22 - 27 , 32 ]. There were altogether 13 males and 8 females, with a median age of 38 years (range 2 to 75 years). The age and sex were not mentioned in 8 cases. Their underlying immunosuppressive conditions included solid organ tumours (13 cases), haematological diseases (8), autoimmune diseases (4), endogenous Cushing's disease (3), and a solid organ transplant recipient (1). All patients had received steroids or had excessive endogenous steroid production, whereas 18 (62.1%) of them had concomitant cytotoxic therapy for the underlying diseases. The median duration between steroid tapering and clinical manifestations of PCP was 21 days (range 1 to 83 days). Steroids were completely withdrawn at a median of 7.5 days (range 1 to 21 days) before the onset of symptoms in eight patients. Serial lymphocyte counts were only available in eight patients. An upsurge of the absolute lymphocyte counts was observed from the time of reduction of immunosuppression (median 300/μL, range 290 to 600/μL at baseline) to the time of occurrence of IRD (median 1200/μL, range 600 to 5620/μL); the median increase in total lymphocyte count was 800/μL, with a range of 300 to 4880/μL. Comparing the lymphocyte counts before and after reversal of immunosuppressive therapy, the difference was statistically significant (Wilcoxon Signed Rank Test for paired samples; p = 0.012). In addition to our patient, reintroduction or increasing doses of steroids were required in 7 (53.8%) of 13 patients in the acute management of PCP in the literature, at the time when they developed clinical deterioration during antimicrobial therapy [ 24 - 27 ]. Seven (53.8%) of 13 cases had respiratory failure requiring mechanical ventilation. Among these 29 cases, 13 (44.8%) subsequently died of PCP. Among HIV-positive patients, 13 cases with newly diagnosed PCP were reported in the literature, in which IRD occurred shortly after the introduction of HAART (table  2 ) [ 28 - 31 ]. Seven (53.8%) out of 13 cases received steroids as adjunctive therapy in addition to antimicrobials. HAART was given in all cases at a median 18 days (range 1 to 35 days) after the initiation of treatment for PCP. During IRD, recurrence of fever (100%), dyspnoea (100%), and paradoxical worsening of pulmonary infiltrates (58.3%) were observed in these patients [ 28 - 31 ]. IRD occurred at a median of 14 days (range 5 to 17 days) after HAART. An upsurge of the CD4 lymphocyte count was observed before (median 28/μL, range 4 to 290/μL) and during IRD (median 125/μL, range 30 to 564/μL); this was associated with a concomitant reduction of the median HIV viral load from 5.5 log 10  copies/ml (range 5.0 to 5.9 log 10  copies/ml) to 3.1 log 10  copies/ml (range 2.9 to 4.5 log 10  copies/ml) before and during IRD respectively, and the differences observed in both the CD4 counts and viral loads before and during IRD reached statistical significance (Wilcoxon Signed Rank Test for related samples; p = 0.001 and 0.017, respectively). Antimicrobials, steroids, or both for PCP were reintroduced for IRD in 4, 1, and 6 cases respectively. Only 2 cases were treated conservatively. One case required mechanical ventilation for severe respiratory distress. None of the patients died. PCP manifesting as a form of IRD is not a rare phenomenon. As shown in our previous study, it happens in 7 out of 10 (70%) of HIV-negative immunosuppressed hosts infected with Pj [ 32 ]. However, the diagnosis of PCP is usually delayed in this group of patients because of atypical presentation. In this clinical setting, PCP manifesting as IRD often runs an acute and fulminant course, with nonspecific lesions on chest radiographs, and high absolute lymphocyte counts [ 32 ]. In our own reported series, despite the administration of steroid therapy to suppress the immunopathological damage, more than 80% of patients developed acute respiratory failure and required mechanical ventilation. Patients who developed PCP during reversal of immunosuppressive therapy in our series tended to be older, and this might partially explain the increased mortality observed in this group [ 32 ]. Rapid reduction of immunosuppressive therapy such as steroids has been implicated as a predisposing factor for the development of PCP in HIV-negative patients [ 11 , 17 , 23 , 24 ]. In one study, PCP occurred in 79 (70%) of 113 patients during steroid tapering [ 17 ]. Another study suggested that 8 (72.2%) out of 11 episodes of PCP developed when steroid therapy was tapered [ 23 ]. A subsequent study also demonstrated that 43% of patients had a rapid reduction of steroid dosing before the clinical manifestations of PCP [ 11 ]. A similar experience was reported in children, and 17 (89.5%) out of 19 children were diagnosed to have PCP during steroid tapering according to a previous report [ 21 ]. Another series revealed that 7 of 11 patients experienced acutely symptomatic PCP when the dose of steroids was decreased or terminated 5 days to 3 weeks before the diagnosis of PCP [ 22 ]. However, all these cases were not analyzed from a perspective of IRD. Serial changes of the absolute lymphocyte counts or their subsets were either not noted or reported [ 11 , 17 , 21 - 24 ]. Hence we have not included these cases for further analysis in this review. Among HIV positive patients, PCP manifesting acutely during the initiation anti-retroviral therapy is a well-recognized phenomenon. The underlying immunopathological nature of this condition, which is reminiscent to IRD occurring in non-HIV infected patients, has been confirmed by histological examination of the lungs and transbronchial biopsy specimens, which demonstrated mixed inflammatory infiltrates including macrophages, neutrophils, lymphocytes, and plasma cells. Almost all infiltrating lymphocytes found in the tissues were of the T cell lineage, shown by immunophenotyping to be predominantly CD4 and CD8 cells [ 28 ]. In another study [ 30 ], the BAL fluid obtained from one of six patients with an IRD-type presentation of PCP was analyzed. Infiltration of predominantly CD4 and CD8 lymphocytes with the proliferative marker (Ki67) and perforin-positive cell were seen in the BAL specimen. Therefore, it is likely that the phagocytosed Pj is presented by alveolar macrophage to T cells, which trigger the inflammatory response [ 30 ]. In our own experience, as well as from the review of published literature, it appears that a surge of absolute lymphocyte count, especially the CD4 lymphocyte count in HIV-positive patients, could potentially act as a surrogate marker for immunopathological damage during IRD in both HIV-negative and HIV-positive patients. In our recent publication [ 32 ], 7 out of 10 non-HIV immunosuppressed patients demonstrated a consistent rise in the absolute lymphocyte count during tapering of immunosuppression prior to the onset of symptomatic PCP. In this group of patients, the surge in lymphocyte count is likely the result of withdrawal of lymphocytotoxic immunosuppressants such as corticosteroids. Similarly, a rising trend of the CD4 lymphocyte count, consistent with immune reconstitution after HAART, was also observed in 13 HIV-positive cases before and during the development of symptomatic PCP [ 28 - 31 ]. In fact, an upsurge in the absolute lymphocyte count has been shown to be a marker of IRD in our previous publications involving viral and tuberculous infections [ 33 - 36 ]. However, it must be emphasized that the number of circulating lymphocytes may not always correlate with their number in the affected tissues or their in vivo functional activity. This can be exemplified by a case of PCP occurring during steroid withdrawal, in which the lymphocyte counts surged to a very high level and then rapidly dropped to a low level within one day. The migration of lymphocytes from the circulation to tissue might explain this rapid drop in lymphocyte count and the resulting immunopathological damage [ 27 ]. In the future, further studies on lymphocyte subsets and cytokine profiles of susceptible hosts during the development of IRD should be performed to elucidate the underlying immunopathological mechanisms behind this interesting phenomenon. From the result of this review, it appears that HIV-positive patients with PCP are at risk of clinical deterioration due to IRD if HAART therapy is started within 1 to 2 weeks after the initiation of treatment for PCP (table  2 ). With a better understanding of the pathogenetic mechanisms resulting in IRD, we may be able to prevent the occurrence of IRD by delaying the initiation of HAART in HIV-positive patients with PCP. However, in non-HIV immunosuppressed patients, it is even more important to recognize the atypical presentations of PCP in the context of IRD. Since the clinical and/or radiological features alone may not be sufficient for diagnosis, analysis of serial changes in lymphocyte counts in patients undergoing a reduction of immunosuppression can alert the clinician to the possibility of IRD due to occult pathogens such as Pj. To prevent IRD in non-HIV immunosuppressed patients, the use of prophylactic antibiotics against Pj to reduce the microbial load in selected patients remains an important issue. Recently, a multi-center study showed that the CD4 lymphocyte count may be a useful marker to monitor the risk of development of PCP in non-HIV immunosuppressed hosts [ 37 ], and patients with low CD4 lymphocyte counts of less than 300 or 400 may require prophylaxis. In fact, asymptomatic colonization of Pj has been demonstrated in HIV-negative patients when the CD4 lymphocyte count was less than 400 [ 38 ]. Nested polymerase chain reaction (PCR) identified a significant percentage of clinically silent Pj colonization in 20% of non-HIV immunosuppressed patients [ 39 ]. Therefore, early detection of asymptomatic infection of Pj in blood and respiratory specimens before, and during intense immunosuppression may enable selection of cases for pre-emptive treatment of Pj infection in order to prevent the development of IRD during reversal of immunosuppression [ 40 , 41 ].
Case 2
A thirty-three year old gentleman initially presented to Prince of Wales Hospital with a diagnosis of SLE/dermatomyositis overlap syndrome. He was treated with steroid and hydroxychloroquine 200 mg twice daily since 1997. He had a flare up of disease in May 1998 with active vasculitis and myositis, for which he was put on prednisolone and azthioprine 50 mg and 100 mg daily respectively. Upon reassessment one month later, disease activity was under control, and the dosage of prednisolone was reduced to 45 mg daily.Twelve day after reducing the immunosuppressive regimen, he was admitted to hospital for treatment of left buttock abscess. The CXR taken on admission was unremarkable. An aspirate of the pus from the lesion grew methicillin-sensitive  staphylococcus aureus ; he was treated with cloxacillin 1 g intravenously every 6 hourly, together with incision and drainage of the buttock abscess. In view of the underlying active pyogenic infection, the steroid dosage was rapidly tapered from 45 mg to 15 mg daily within the next four days. However, he was noted to have persistent fever associated with mild unproductive cough. A repeat chest radiograph showed new infiltrates over the right upper and left lower zones, and he was empirically treated with intravenous ceftazidime 1 gram every 8 hours, cloxacillin 1 gram every 6 hours and netimicin 100 mg every 8 hours. As there was no clinical response after 5 days of treatment, bronchoscopy and bronchoalveolar lavage (BAL) was performed, which was positive for  Pneumocystis jiroveci . Investigation for the presence of co-existing opportunistic pathogens such as cytomegalovirus and aspergillus was negative. On the day after bronchoscopy, he was commenced on intravenous cotrimoxazole 1.3 grams every 6 hours. He remained stable initially with fever on downward trend. However, on the 3 rd  day of treatment, he developed sudden desaturation with resurgence of high fever, and required supplemental oxygen therapy. Repeat chest radiograph showed increased perihilar hazziness in both lung fields. There was also an upsurge of total lymphocyte count from 0.6 × 10 9 /L (total white cell count 11.2 × 10 9 /L) on admission, to 1.3 × 10 9 /L (total white cell count 10.4 × 10 9 /L) at the time of clinical deterioration. He was treated with high dose prednisolone (80 mg daily), and his condition improved promptly afterwards. He was subsequently discharged, and on follow up at the clinic one month later, his total lymphocyte count had returned to his baseline level of 0.6 × 10 9 /L.staphylococcus aureusPneumocystis jiroveciImmunorestitution disease (IRD) has been described in both HIV and non-HIV immunosuppressed hosts previously [ 27 - 31 ]. In the setting of PCP, it is defined as an acute symptomatic presentation of the disease that is related temporally to the recovery of the immune system, associated with reversal of immunosuppressive processes such as reduction in the dosage of corticosteroids and/or cytotoxic agents or a reduction of HIV viral load due to HAART, which results in the development of immunopathological damage. The preexisting microbial infection could be either asymptomatic or mildly symptomatic. Using this case definition, we attempted to review the English literature for other reported cases of PCP manifesting as IRD. The English-language literature (1966 – 2003) was searched in the Medline database of the National Library of Medicine in October 2003. The keywords " Pneumocystis carinii ", " Pneumocystis jiroveci ", "HIV", immunosuppression", "immunosuppressive", "steroid", and "corticosteroid" were used to select cases. All the case reports and case series with clinical details were included in this study if they fulfilled the above definition of IRD. When appropriate, the cited bibliographies were also retrieved for further analysis. As for statistical analysis, we used the Wilcoxon Signed Rank test, a non-parametric test for comparing paired samples, to analyze the serial changes in lymphocyte counts and HIV viral loads before and during the development of IRD. A two-tailed p-value of less than 0.05 was considered significant. All statistical analyses were performed using SPSS version 11.5 for Windows.Pneumocystis cariniiPneumocystis jiroveciIncluding our present case, a total of 29 cases of PCP in non-HIV immunosuppressed hosts fulfilling our definition of IRD have been reported in the literature (table  1 ) [ 22 - 27 , 32 ]. There were altogether 13 males and 8 females, with a median age of 38 years (range 2 to 75 years). The age and sex were not mentioned in 8 cases. Their underlying immunosuppressive conditions included solid organ tumours (13 cases), haematological diseases (8), autoimmune diseases (4), endogenous Cushing's disease (3), and a solid organ transplant recipient (1). All patients had received steroids or had excessive endogenous steroid production, whereas 18 (62.1%) of them had concomitant cytotoxic therapy for the underlying diseases. The median duration between steroid tapering and clinical manifestations of PCP was 21 days (range 1 to 83 days). Steroids were completely withdrawn at a median of 7.5 days (range 1 to 21 days) before the onset of symptoms in eight patients. Serial lymphocyte counts were only available in eight patients. An upsurge of the absolute lymphocyte counts was observed from the time of reduction of immunosuppression (median 300/μL, range 290 to 600/μL at baseline) to the time of occurrence of IRD (median 1200/μL, range 600 to 5620/μL); the median increase in total lymphocyte count was 800/μL, with a range of 300 to 4880/μL. Comparing the lymphocyte counts before and after reversal of immunosuppressive therapy, the difference was statistically significant (Wilcoxon Signed Rank Test for paired samples; p = 0.012). In addition to our patient, reintroduction or increasing doses of steroids were required in 7 (53.8%) of 13 patients in the acute management of PCP in the literature, at the time when they developed clinical deterioration during antimicrobial therapy [ 24 - 27 ]. Seven (53.8%) of 13 cases had respiratory failure requiring mechanical ventilation. Among these 29 cases, 13 (44.8%) subsequently died of PCP.Among HIV-positive patients, 13 cases with newly diagnosed PCP were reported in the literature, in which IRD occurred shortly after the introduction of HAART (table  2 ) [ 28 - 31 ]. Seven (53.8%) out of 13 cases received steroids as adjunctive therapy in addition to antimicrobials. HAART was given in all cases at a median 18 days (range 1 to 35 days) after the initiation of treatment for PCP. During IRD, recurrence of fever (100%), dyspnoea (100%), and paradoxical worsening of pulmonary infiltrates (58.3%) were observed in these patients [ 28 - 31 ]. IRD occurred at a median of 14 days (range 5 to 17 days) after HAART. An upsurge of the CD4 lymphocyte count was observed before (median 28/μL, range 4 to 290/μL) and during IRD (median 125/μL, range 30 to 564/μL); this was associated with a concomitant reduction of the median HIV viral load from 5.5 log 10  copies/ml (range 5.0 to 5.9 log 10  copies/ml) to 3.1 log 10  copies/ml (range 2.9 to 4.5 log 10  copies/ml) before and during IRD respectively, and the differences observed in both the CD4 counts and viral loads before and during IRD reached statistical significance (Wilcoxon Signed Rank Test for related samples; p = 0.001 and 0.017, respectively). Antimicrobials, steroids, or both for PCP were reintroduced for IRD in 4, 1, and 6 cases respectively. Only 2 cases were treated conservatively. One case required mechanical ventilation for severe respiratory distress. None of the patients died.PCP manifesting as a form of IRD is not a rare phenomenon. As shown in our previous study, it happens in 7 out of 10 (70%) of HIV-negative immunosuppressed hosts infected with Pj [ 32 ]. However, the diagnosis of PCP is usually delayed in this group of patients because of atypical presentation. In this clinical setting, PCP manifesting as IRD often runs an acute and fulminant course, with nonspecific lesions on chest radiographs, and high absolute lymphocyte counts [ 32 ]. In our own reported series, despite the administration of steroid therapy to suppress the immunopathological damage, more than 80% of patients developed acute respiratory failure and required mechanical ventilation. Patients who developed PCP during reversal of immunosuppressive therapy in our series tended to be older, and this might partially explain the increased mortality observed in this group [ 32 ].Rapid reduction of immunosuppressive therapy such as steroids has been implicated as a predisposing factor for the development of PCP in HIV-negative patients [ 11 , 17 , 23 , 24 ]. In one study, PCP occurred in 79 (70%) of 113 patients during steroid tapering [ 17 ]. Another study suggested that 8 (72.2%) out of 11 episodes of PCP developed when steroid therapy was tapered [ 23 ]. A subsequent study also demonstrated that 43% of patients had a rapid reduction of steroid dosing before the clinical manifestations of PCP [ 11 ]. A similar experience was reported in children, and 17 (89.5%) out of 19 children were diagnosed to have PCP during steroid tapering according to a previous report [ 21 ]. Another series revealed that 7 of 11 patients experienced acutely symptomatic PCP when the dose of steroids was decreased or terminated 5 days to 3 weeks before the diagnosis of PCP [ 22 ]. However, all these cases were not analyzed from a perspective of IRD. Serial changes of the absolute lymphocyte counts or their subsets were either not noted or reported [ 11 , 17 , 21 - 24 ]. Hence we have not included these cases for further analysis in this review.Among HIV positive patients, PCP manifesting acutely during the initiation anti-retroviral therapy is a well-recognized phenomenon. The underlying immunopathological nature of this condition, which is reminiscent to IRD occurring in non-HIV infected patients, has been confirmed by histological examination of the lungs and transbronchial biopsy specimens, which demonstrated mixed inflammatory infiltrates including macrophages, neutrophils, lymphocytes, and plasma cells. Almost all infiltrating lymphocytes found in the tissues were of the T cell lineage, shown by immunophenotyping to be predominantly CD4 and CD8 cells [ 28 ]. In another study [ 30 ], the BAL fluid obtained from one of six patients with an IRD-type presentation of PCP was analyzed. Infiltration of predominantly CD4 and CD8 lymphocytes with the proliferative marker (Ki67) and perforin-positive cell were seen in the BAL specimen. Therefore, it is likely that the phagocytosed Pj is presented by alveolar macrophage to T cells, which trigger the inflammatory response [ 30 ].In our own experience, as well as from the review of published literature, it appears that a surge of absolute lymphocyte count, especially the CD4 lymphocyte count in HIV-positive patients, could potentially act as a surrogate marker for immunopathological damage during IRD in both HIV-negative and HIV-positive patients. In our recent publication [ 32 ], 7 out of 10 non-HIV immunosuppressed patients demonstrated a consistent rise in the absolute lymphocyte count during tapering of immunosuppression prior to the onset of symptomatic PCP. In this group of patients, the surge in lymphocyte count is likely the result of withdrawal of lymphocytotoxic immunosuppressants such as corticosteroids. Similarly, a rising trend of the CD4 lymphocyte count, consistent with immune reconstitution after HAART, was also observed in 13 HIV-positive cases before and during the development of symptomatic PCP [ 28 - 31 ]. In fact, an upsurge in the absolute lymphocyte count has been shown to be a marker of IRD in our previous publications involving viral and tuberculous infections [ 33 - 36 ]. However, it must be emphasized that the number of circulating lymphocytes may not always correlate with their number in the affected tissues or their in vivo functional activity. This can be exemplified by a case of PCP occurring during steroid withdrawal, in which the lymphocyte counts surged to a very high level and then rapidly dropped to a low level within one day. The migration of lymphocytes from the circulation to tissue might explain this rapid drop in lymphocyte count and the resulting immunopathological damage [ 27 ]. In the future, further studies on lymphocyte subsets and cytokine profiles of susceptible hosts during the development of IRD should be performed to elucidate the underlying immunopathological mechanisms behind this interesting phenomenon.From the result of this review, it appears that HIV-positive patients with PCP are at risk of clinical deterioration due to IRD if HAART therapy is started within 1 to 2 weeks after the initiation of treatment for PCP (table  2 ). With a better understanding of the pathogenetic mechanisms resulting in IRD, we may be able to prevent the occurrence of IRD by delaying the initiation of HAART in HIV-positive patients with PCP. However, in non-HIV immunosuppressed patients, it is even more important to recognize the atypical presentations of PCP in the context of IRD. Since the clinical and/or radiological features alone may not be sufficient for diagnosis, analysis of serial changes in lymphocyte counts in patients undergoing a reduction of immunosuppression can alert the clinician to the possibility of IRD due to occult pathogens such as Pj. To prevent IRD in non-HIV immunosuppressed patients, the use of prophylactic antibiotics against Pj to reduce the microbial load in selected patients remains an important issue. Recently, a multi-center study showed that the CD4 lymphocyte count may be a useful marker to monitor the risk of development of PCP in non-HIV immunosuppressed hosts [ 37 ], and patients with low CD4 lymphocyte counts of less than 300 or 400 may require prophylaxis. In fact, asymptomatic colonization of Pj has been demonstrated in HIV-negative patients when the CD4 lymphocyte count was less than 400 [ 38 ]. Nested polymerase chain reaction (PCR) identified a significant percentage of clinically silent Pj colonization in 20% of non-HIV immunosuppressed patients [ 39 ]. Therefore, early detection of asymptomatic infection of Pj in blood and respiratory specimens before, and during intense immunosuppression may enable selection of cases for pre-emptive treatment of Pj infection in order to prevent the development of IRD during reversal of immunosuppression [ 40 , 41 ].Conclusions PCP occurring in the context of IRD is not a rare phenomenon and is likely to be under-reported in the literature. In this setting, it may be more common for PCP to manifest acutely with a fulminant clinical course. Clinicians caring for immunosuppressed patients should be alert to this unique phenomenon so as to initiate timely and appropriate investigations and treatment for their patients. Serial monitoring of lymphocyte count, or if possible CD4 count, could serve as a useful adjunct to facilitate the diagnosis and management of this condition in a wide range of immunosuppressed hosts, especially in the presence of new pulmonary symptoms and/or radiographic abnormalities compatible with the diagnosis.
Conclusions
PCP occurring in the context of IRD is not a rare phenomenon and is likely to be under-reported in the literature. In this setting, it may be more common for PCP to manifest acutely with a fulminant clinical course. Clinicians caring for immunosuppressed patients should be alert to this unique phenomenon so as to initiate timely and appropriate investigations and treatment for their patients. Serial monitoring of lymphocyte count, or if possible CD4 count, could serve as a useful adjunct to facilitate the diagnosis and management of this condition in a wide range of immunosuppressed hosts, especially in the presence of new pulmonary symptoms and/or radiographic abnormalities compatible with the diagnosis.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions RAL and DSH were involved in the clinical evaluation and treatment of patients. BST and IFH helped with literature searching and review. AKW and VCC drafted and refined the manuscript. KYY conceived the study, participated in its design and coordination, and supervised the preparation of the manuscript. All authors have read and approved the final draft of the manuscript before submission.
Authors' contributions
RAL and DSH were involved in the clinical evaluation and treatment of patients. BST and IFH helped with literature searching and review. AKW and VCC drafted and refined the manuscript. KYY conceived the study, participated in its design and coordination, and supervised the preparation of the manuscript. All authors have read and approved the final draft of the manuscript before submission.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: