immunodeficient individuals , either acquired ( e.g. hiv / aids ) or genetic ( e.g. severe combined immunodeficiency ) , are exquisitely susceptible to developing pneumocystis pneumonia , . accordingly , hiv / aids patients are routinely given anti - pneumocystis prophylaxis , trimethoprim - sulfamethoxazole , when cd4t - cell counts fall below 200 cells / ul . however , pneumocystis is re - emerging in the clinical context of individuals receiving immunosuppressive drug therapy for hematologic malignancies or management of post - transplantation rejection . hiv - negative patients with pneumocystis tend to have a more fulminant presentation with fever , dyspnea , and chills , and have a greater extent of morbidity and mortality compared to hiv - positive patients . trimethoprim - sulfamethoxazole , in addition to use in prophylaxis , is the first line therapy for pneumocystis , although treatment failure rates are greater than 20% . although the microbiology of pneumocystis is difficult to elucidate due to the lack of a reliable culture method , much has been gleaned about the multiphasic life cycle of pneumocystis using microscopy of the organism in the alveoli . the asci are circular with a thick -1,3-glucan shell and house eight ascospores that will be released as trophs . much remains unknown about the pneumocystis life in vivo , such as the factors that lead to sexual reproduction , trophic release , and how the balance between asci and trophs is maintained in vivo . gms staining , the primary diagnostic stain used on bronchoalveolar lavage fluid , preferentially stains the asci . similarly , echinocandins , which inhibit the synthesis of -1,3-glucan , have been shown in murine models to deplete the ascus form of infection , demonstrating that chemical modification of the pneumocystis life cycle is possible in vivo 
 . a 5-year - old male with trisomy 21 , wolf - parkinson - white syndrome , and known pre - b cell acute lymphoblastic leukemia ( all ) completing maintenance chemotherapy ( with 6-mercaptopurine , methotrexate , and the final 5-day course of oral dexamethasone therapy completed 1 month prior ) was hospitalized with a 2-week history of rhinorrhea , 3 days of non - productive cough , fever , and 24  h of increased work of breathing . clinical examination on hospital day 0 revealed a child in severe respiratory distress with hypoxemia and shock requiring immediate intubation for mechanical ventilation and vasopressor support . laboratory evaluation demonstrated leukopenia ( an absolute lymphocyte count of 54  cells / mm and 15% eosinophils ) and thrombocytopenia ( table 1 ) . 1(a ) ) . given continued hypoxemia , the patient underwent a bronchoalveolar lavage ( bal ) on hospital day + 2 and intravenous trimethoprim / sulfamethoxazole ( t / s , 20  mg / kg / day ) and systemic intravenous corticosteroids were empirically started due to concern of pneumocystis jirovecii pneumonia ( pcp ) , despite compliance with oral t / s prophylaxis . bal histopathology revealed mixed inflammation and the grocott 's methenamine silver ( gms ) stain demonstrated abundant asci of pneumocystis , confirming the diagnosis of pcp ( fig . a real time polymerase - chain - reaction ( rt - pcr ) assay of the bal sample also detected pneumocystis jirovecii , quantifiable at 62 million copies / ml ( focus diagnostics , san juan capistrano , california ) . the patient initially defervesced and became hemodynamically stable , but continued to require significant supplementary oxygen and ventilatory support including high positive end - expiratory pressures . with recrudescence of fevers , blood cultures were again obtained and empirical antimicrobials , including micafungin , were initiated . a computed tomography ( fig . 1(c ) ) on hospital day + 17 revealed patchy airspace opacities superimposed upon diffuse ground - glass opacities bilaterally . the gms staining on this bal was negative , although the qrt - pcr quantified 111 million copies / ml of p. jirovecii in the bal fluid ( fig . 1(d ) ) . given the lack of clinical response to t / s , the possibility of a resistant strain of pneumocystis was explored . genomic dna from the bal sample from hospital day + 17 , which was gms - negative with rare , morphologically altered pneumocystis , was isolated and the dihydropterate synthetase ( dhps ) and the dihydrofolate reductase genes were cloned . the patient did not have any mutations within either gene that accounted for clinical non - response . rna from the bal from hospital day + 17 was also isolated and evaluated for pneumocystis burden using qrt - pcr for the mitochondrial large subunit rrna ( lsu ) ( fig . the patient had detectable lsu copies by qrt - pcr , corroborating the quantitative results obtained during the hospital course . however , further analyses were performed to evaluate the possible changes in pneumocystis life - forms ( e.g. ascus and trophozoites ) . -1,3-glucan synthase ( gsc-1 ) , an ascus - enriched protein involved in fungal cell wall assembly , had decreased transcript expression in the patient by qrt - pcr compared to the lsu transcript . by comparison , a positive control sample from a rhesus macaque infected with siv and pneumocystis had the opposite transcriptional profile , as gsc-1 transcript levels were substantially higher than that of lsu ( fig . similar analyses were performed on the deep endotracheal aspirate collected on hospital day + 47 , but pneumocystis rna was not detectable at this time point . the patient ultimately required placement of a tracheostomy tube due to prolonged intubation , and the patient received t / s for a total of 70 days . the patient was treated with a prolonged corticosteroid taper , and was ultimately able to be discharged home . an outpatient follow - up visit four months later revealed that the child was doing well , had successfully transitioned to t / s prophylaxis , had improvement in lymphopenia , and did not require additional respiratory support . pcp remains an opportunistic infection with high morbidity and mortality in immunocompromised patients . with advances in cancer chemotherapy , more aggressive immunosuppressive regimens , and increasing survival , a growing proportion of patients may be at risk for pcp . breakthrough pcp in patients receiving t / s prophylaxis is unusual , but has been reported , occurring most frequently in patients with underlying hematologic malignancies . when infection does occur , hiv - negative patients tend to have lower organism burdens , but have a more robust inflammatory response in the lungs than hiv - positive patients . importantly , mortality rates from pcp are higher in non - hiv patients compared with hiv - positive patients and are highest in patients with cancer , with a mortality of 36% among children with all , , . furthermore , this patient had trisomy 21 as an underlying genetic condition , which presents its own challenges in terms of defects in immune response and drug metabolism . such immune defects have been postulated to explain the higher incidence and severity of acute lung injury and ards after pneumonia compared with children without trisomy 21 . one of the biggest concerns in our patient was the possibility of t / s resistance given compliance with prophylaxis and lack of clinical response despite 2 weeks of t / s therapy . though studies have demonstrated an association between the use of sulfa drugs for pcp prophylaxis and dhps / dhfr gene mutations , differences in clinical outcome with dhps mutations remains an area of active investigation . while t / s resistance remains an important consideration on a differential diagnosis of difficult - to - treat pneumocystis , the current patient was found to have no molecular evidence of decreased t / s response . diagnosis of pcp is largely based on the detection of pneumocystis jirovecii by conventional microscopic examination and staining methods of respiratory fluids or tissue samples with gms , as pneumocystis remains unculturable . while the thick glucan cell wall of the ascus form of pneumocystis will stain with gms , the irregularly shaped trophic form will not consistently stain gms - positive . this limitation of gms was evident in the current patient , as the first bal sample had an abundant number of gms - positive , circular asci with a thick cell wall . the second bal on hospital day + 17 , appeared to have scarce pneumocystis asci on gms staining . this was discordant with the pcr from the same bal , which showed increased copies of pneumocystis rna compared to the bal performed on hospital day + 2 . without the concomitant pneumocystis real - time pcr , it would have been difficult to determine if the pneumocystis had resolved . one study found that a real - time pcr assay in hiv - uninfected immunocompromised hosts with clinical suspicion of pcp had a diagnostic sensitivity of 87% and specificity of 92% and a high negative predictive value . while pcr assays can have utility , as in this patient , further research on the reliability and threshold of pneumocystis nucleic acid levels needs to be performed . the possible increase in pneumocystis burden by real - time pcr findings in conjunction with decreasing asci observed on gms staining also raises the interesting issue of changes in the pneumocystis life cycle over the course of infection . one possible explanation is that unknown host - pathogen interactions over the course of infection favored the trophozoite life form over the ascus form . as described above , trisomy 21 patients have immunologic defects , and as such such conditions have been determined experimentally , as pharmacologic manipulation of the life cycle of pneumocystis has been well documented with the use of echinocandin class of antifungals , such as micafungin . micafungin has been shown to inhibit synthesis of 1,3--d - glucan and thus selectively inhibit asci , but not trophozoites , in a rodent model of pcp . while the selective effect on the asci has yet to be studied in humans , cases of progression of pneumocystis therefore the utility of molecular diagnostics may have the greatest impact in patients receiving echinocandins . ultimately , a giemsa stain was requested on the second bal specimen and was found to be negative for trophozoites . the possibility of immune reconstitution inflammatory syndrome ( iris ) was also raised in this patient . however , the patient had a protracted course , not a paradoxical worsening of symptoms while receiving systemic corticosteroids . further , there was no concrete evidence of immune recovery and microbiological improvement ; in fact the patient continued to have severe lymphopenia in both peripheral blood and bal specimens and aside from the negative gms staining , could not explain the rising pneumocystis pcr . while micafungin therapy in the current patient was initiated simultaneously with the second gms - negative bronchoscopy , these studies highlight the importance that different clinical scenarios may have on the pneumocystis life cycle , and thus diagnosis . in our patient , the transcriptional profile was vastly different compared to an archived sample from a siv - infected rhesus macaque and demonstrated a decrease in gsc-1 , an ascus enriched protein , compared to a lsu probe . while underlying host - pathogen interactions differ between these samples , the lack of gsc-1 signal compared to lsu in our patient corroborates the reduction in asci burden by gms . these findings highlight the importance of including pneumocystis on a differential diagnosis in the presence of questionable or negative gms , particularly if an echinocandin is included in the therapeutic regimen . in summary , this current case of pneumocystis has several unique aspects and teaching points . first , this young child with trisomy 21 and underlying all had breakthrough pneumocystis despite compliance with oral t / s prophylaxis and had a prolonged and severe course . ultimately , additional molecular diagnostics allowed confirmation that the clinical specimen had no mutations conferring t / s resistance and t / s remained the therapy of choice . this patient had a negative gms staining on bal due to selective pressure on the ascus form of pneumocystis , further highlighting the importance of understanding the plasticity of the pneumocystis life cycle and selectivity of diagnostic stains . this case exemplifies the necessity of considering pneumocystis in the differential diagnosis in an immunocompromised patient requiring respiratory support , even in the setting of a gms - negative bronchoalveolar lavage .
we report a case of pneumocystis pneumonia in a 5-year - old male with trisomy 21 and acute lymphoblastic leukemia . the lack of response to trimethoprim - sulfamethoxazole raised concerns for antimicrobial resistance . 
 further , diagnosis of pneumocystis in this patient was complicated by a gms - negative bronchoalveolar lavage despite molecular evidence of pneumocystis infection .