False Negative Biopsy in Pneumocystis carinii Pneumonia* William A. Demicco, M.D.; Avn~m Stein, D.O.; JohnS. Urbanetti, M.D., F.C.C.P.; and Batty L. Fanburg, M.D.
Unlike most pneumonlas, the diagnosis of Pneumocystis carinii pneumonia is based solely on identifying organisms by stain, usually with methenamine-silver. Because of tecbnical problems involved with adequate staining, control samples usually are done concurrent with tissue specimens to be examined. Lung containing fnngi often is used as a controL We recently observed false-negative biopsy specimens In a case of P carinii pneumonia where the Pneumocystis orpnism failed to stain .with methenamine-silver on several octasions, although fungal controis were positive. This report emphasizes the importance of using P ccwinii as a control whenever attempting to diagnose P ccwinii pneumonia.
Dneumocystis carinii has come to be widely recog-
C nized as a cause of pneumonia in the immunocompromised host. 1 In these patients, the disease is fatal unless identified and treated promptly. The methenamine-silver nitrate method of Gomori and Grocott remains the standard method of identifying the cysts of P carinii in both smears and tissue section. 2 •8 It is important to utilize a simultaneous positive P carinii control slide when employing the methenamine-silver stain. The practice of relying on a fungal control, as is often used, may be inadequate and misleading.
CASE REPoRT A 36-year-old Portuguese woman was admitted to the New England Medical Center with a progressive interstitial pneumonia. She had been well until 16 months prior to admission when she was admitted to another haspital because of weakness, fever, and a right apical infiltrate. Cultures and smears for tuberculosis were negative, but the patient responded to therapy with isoniazid and ethambutol hydrochloride. Five months later, these medications were discontinued because of a suspected drug reaction. Six months before admission, the patient was readmitted to the other hospital with abdominal pain. Laparotomy revealed tuberculous portal lymph nodes, and a liver biopsy specimen showed caseating granulomas. A sputum culture was positive for Mycobacteria tuberculom (sensitive to all drugs tested). Regimens of isoniazid ( 300 mg/ day), ethambutol ( 1000 mg/day), and streptomycin sulfate 1 gm three times weekly) were started. One month prior to admission, herpes zoster developed over the right anterior and lateral chest wall. Prednisone, 40 mg/day, was started. Two weeb later, the patient developed a non-productive cough and fever. A chest x-ray film revealed diffuse interstitial infiltrates. Rifampin and erythromycin were added, and prednisone was continued. Multiple sputum smears for acid-fast °From the Department of Medicine, Tufts University School of Medicine, and the New England Medical Center Hospital, Boston. &print requut8: Dr. Fanburg, New England Medical Center
HOifJital, 171 Hamson, BOBton 0!111
CHEST, 75: 3, MARCH, 1979
organisms were negative, and bacterial, fungal, and mycobacterial cultures of sputa, blood, and urine grew no pathogens. The patient failed to respond to medication and was transferred to the New England Medical Center. She complained of cough and dyspnea. Physical examination showed cyanosis, clubbing, and bilateral chest crackles. Chest x-ray film revealed progression of the bilateral interstitial pneumonia. White blood cell count was 5000/cu mm with 85 percent neutrophils. The patient sequentially underwent a transtracheal aspiration, fl.beroptic bronchoscopy with brushings and transbronchial biopsy, and an open lung biopsy. No pathogens were identified on smears or isolated by culture. Methenamine-silver stains of bronchial brushings, transbronchial biopsy, and open lung biopsy specimens, done with simultaneous control of lung containing Candida albictmS, were interpreted as negative for P ctJrinii. The patient's condition deteriorated with progressive pulmonary consolidation and evidence of cavitation by chest x-ray film. Respiratory failure ensued requiring mechanical ventilation. She died on the eighteenth hospital day. At post mortem, the lungs showed an alveolar space-fl.lling pneumonitis with multiple cavitary abscesses. Methenaminesilver stains were markedly positive for numerous P ctJrinii organisms (although initial staining of the autopsy material with fungal control was only faintly positive and almost missed, and repeat staining was necessary to demonstrate the numerous organisms clearly). The original transbronchial and open lung biopsy specimens were then recut and stained using the patient's own post mortem lung tissue as the control, and both specimens were positive for P carinii. DISCUSSION
The Grocott modification of Gomori's methenaminesilver technique involves the liberation of aldehyde groups from polysaccharides as the result of oxidation with chromic acid. Subsequently, the aldehyde oxidation products reduce silver nitrate to metallic silver rendering them visible. In addition, the chromic acid also tends to oxidize the newly released aldehyde groups to breakdown products that will not combine with silver.' This feature has the advantage of suppressing weaker back~ ground reactions of collagen and basement membranes while leaving reactive only those substances with large quantities of polysaccharides such as glycogen, mucin, fungal walls, melanin, and insoluble calcium salts. The intensity of staining, ie, deposition of metallic silver, depends to some degree on the length of time of pretreatment with chromic acid, in addition to the freshness of reagents and the thickness of tissue. To assure adequately stained slides, a known positive specimen must be simultaneously stained to verify that organisms have picked up sufficient silver deposition. For P carinii, understaining will fail to demonstrate organisms whereas overstaining will obliterate the important internal morphologic details characteristic of the cysts, as well as cause silver deposition on other cells, notably macrophages and RBCs. a The importance of proper staining technique in the diagnosis of P carinii pneumonia was discussed by Walzer et al. 8 In their experience at the Center for Disease Control, 194 patients with P carinii pneumonia, largely drawn from university centers in the United States, were studied, and the authors reported identi-
FALSE NEGATIVE BIOPSY IN P CARINII PNEUMONIA 389
lying organisms in six cases initially misdiagnosed as negative by the referring hospital. Most errors in diagnoses resulted from poor staining methods and lack of familiarity with the appearance of the organisms. The authors then emphasized the need for simultaneous controls in methenamine-silver staining for P carinil. However, they failed to explicitly state the need for the control to be P carinii. Following this case, we conducted a telephone survey of 15 hospitals in the greater Boston area. All hospitals employed the methenamine-silver stain for the identification of suspected P carlnU. However, only three hospitals were using a positive P carinfi specimen as a control at the time of our patient's hospitalization. Subsequently, our own and one other hospital have adopted the practice of routinely using P carlnfi controls. Pneumocystia carinii bas become a frequent cause of pneumonia as the population of immunocompromised hosts has increased. Pneumocystia carinfi pneumonia also has been reported to occur in otherwise healthy adults 5 •8 and has been reported to be a frequent cause of infection in premature infants in Europe. 7 Cases can now be expected to be seen outside of academic centers. Careful attention to the proper histologic preparation and staining of specimens is required if the diagnosis of P carinii is to be made. On the basis of our case, we conclude that fungal controls do not guarantee adequate staining of P carinii and that controls during methenamine-silver staining should be of P carlnfi itself. An additional advantage to the implementation of this procedure will be the greater familiarity pathologists will acquire for the morphologic details of P carinil organisms.
1 Doppman JL, Geelhoed GW and DeVita VT: Atypical radiographic features' in PneumocystiB carinU pneumonia. Radiology 114:39-44, 1975 2 Hughes W: Pfi8UmOC!Jiti8 carinH pneumonia. N Engl J Med 297:1381-1383, 1977 3 Walzer PD, Perl DP, Krogstad, DJ, et al: Pneumocyltil carlnii pneumonia in the United States. Ann Intem Med 80:83-93, 1974 . 4 Grocott RG: A stain in tissue sections and smears using Gomori's methenamine-silver nitrate technic. Am J Clin Pathol25:975-979, 1955 5 Lyons HA, Vinijchaikul K, Hennigar GR: PneumocystiB carlnii pneumonia unassociated with other disease. Arch lntem Med 108:929-936, 1961 6 Watanbe JM, Chincbinian H. Weitz C: Pneumocylti8 C.. lrUi pneumonia in a family. JAMA 193:685-686, 1965 7 lvady G, Paldy L, Koltay M, et al: Pneumocyltil carinU pneumonia. Lancet:616-617, 1967
380 CASAROnO ET AL
Surgical Removal of a Left Atrial Myxoma During Pregnancy* Dino Ct~~arotto, M.D.; Ubedo Bortolottl, M.D.; Rosario Russo, M.D.; DarlO Betti, M.D.; Luciano SchWaz.appa, M.D.; and Gaetano Thiefle, M.D.
Saeeessfol removal of a left atrial mymma In a prepant woman bas not been previously reported. The patient postoperatively bad an 11DC011lplleated prepaney, deBvering at term a normal, healthy baby. first case reported by Leyse and colleagues, Afterheartthesurgery, with the aid of cardiopulmonary 1
bypass ( CPB), can currently be accomplished during pregnancy with minimal hazards both for the mother and the fetus. We describe the first successful removal of a left atrial myxoma in a pregnant woman.
A 39-year-old woman, who previously had had two wcomplicated pregnancies, experienced easy fatigability, weight loss, and occasional palpitation two years prior to admission. Recently, some episodes of noctumal dyspnea occurred, and signs of congestive heart failure appeared. She was referred to our department for further evaluation. On admission, May 5, 1977, she was in the 21st week of pregnancy, the uterine enlargement being compatible with the gestational age. Blood pressure was 120/80 mm Hg. Pulse rate was 120 beats per minute and regular, and fetal heart rate was 140 beats per minute. The intensity of the first sound was increased; a protodiastolic sound, interpreted as an opening snap of the mitral valve, and a grade 3/6 diastolic-presystolic murmur at the apex were noted; the auscultatory 6ndings were apparently not related to positional changes. Bilateral pulmonary rales were present, and the liver was not enlarged. Pertinent laboratory findings revealed a moderate hypochromic anemia and a high erythrocyte sedimentation rate. The ECG showed sinus tachycardia and the chest x-ray film, pulmonary venous congestion and a moderate enlargement of the left atrium. · Since her condition progressively deteriorated despite medical treatment, a right cardiac catheterization was accomplished on May 12. The capillary pulmonary wedge pressure was markedly increased (average 28 mm Hg), and moderate pulmonary hypertension was present. During the levophase, an angiopneumocardiogram disclosed a roundshaped 6lling defect in the left atrial cavity which moved during the cardiac cycle from the atrium into the ventricle (Fig 1). The diagnosis of a floating left atrial mass was made and emergency surgery was performed on the same day. From the Departments of Cardiovascular Surgery, CardiolOIY,. and Patliology, University of Padova Medical School, Paaova, Italy. Reprint reqt.IB8t8: Dr. Ct~~arotto, Clmica ChitVrgica Genertils, Centro di Canliochifvgia, Via GiulejnitJni 2, 35100, Padooti, Italy 0
CHEST, 75: 3, MARCH, 1979
Unlike most pneumonlas, the diagnosis of Pneumocystis carinii pneumonia is based solely on identifying organisms by stain, usually with methenamine-silver. Because of tecbnical problems involved with adequate staining, control samples usually are done concurrent with tissue specimens to be examined. Lung containing fnngi often is used as a controL We recently observed false-negative biopsy specimens In a case of P carinii pneumonia where the Pneumocystis orpnism failed to stain .with methenamine-silver on several octasions, although fungal controis were positive. This report emphasizes the importance of using P ccwinii as a control whenever attempting to diagnose P ccwinii pneumonia.
Dneumocystis carinii has come to be widely recog-
C nized as a cause of pneumonia in the immunocompromised host. 1 In these patients, the disease is fatal unless identified and treated promptly. The methenamine-silver nitrate method of Gomori and Grocott remains the standard method of identifying the cysts of P carinii in both smears and tissue section. 2 •8 It is important to utilize a simultaneous positive P carinii control slide when employing the methenamine-silver stain. The practice of relying on a fungal control, as is often used, may be inadequate and misleading.
CASE REPoRT A 36-year-old Portuguese woman was admitted to the New England Medical Center with a progressive interstitial pneumonia. She had been well until 16 months prior to admission when she was admitted to another haspital because of weakness, fever, and a right apical infiltrate. Cultures and smears for tuberculosis were negative, but the patient responded to therapy with isoniazid and ethambutol hydrochloride. Five months later, these medications were discontinued because of a suspected drug reaction. Six months before admission, the patient was readmitted to the other hospital with abdominal pain. Laparotomy revealed tuberculous portal lymph nodes, and a liver biopsy specimen showed caseating granulomas. A sputum culture was positive for Mycobacteria tuberculom (sensitive to all drugs tested). Regimens of isoniazid ( 300 mg/ day), ethambutol ( 1000 mg/day), and streptomycin sulfate 1 gm three times weekly) were started. One month prior to admission, herpes zoster developed over the right anterior and lateral chest wall. Prednisone, 40 mg/day, was started. Two weeb later, the patient developed a non-productive cough and fever. A chest x-ray film revealed diffuse interstitial infiltrates. Rifampin and erythromycin were added, and prednisone was continued. Multiple sputum smears for acid-fast °From the Department of Medicine, Tufts University School of Medicine, and the New England Medical Center Hospital, Boston. &print requut8: Dr. Fanburg, New England Medical Center
HOifJital, 171 Hamson, BOBton 0!111
CHEST, 75: 3, MARCH, 1979
organisms were negative, and bacterial, fungal, and mycobacterial cultures of sputa, blood, and urine grew no pathogens. The patient failed to respond to medication and was transferred to the New England Medical Center. She complained of cough and dyspnea. Physical examination showed cyanosis, clubbing, and bilateral chest crackles. Chest x-ray film revealed progression of the bilateral interstitial pneumonia. White blood cell count was 5000/cu mm with 85 percent neutrophils. The patient sequentially underwent a transtracheal aspiration, fl.beroptic bronchoscopy with brushings and transbronchial biopsy, and an open lung biopsy. No pathogens were identified on smears or isolated by culture. Methenamine-silver stains of bronchial brushings, transbronchial biopsy, and open lung biopsy specimens, done with simultaneous control of lung containing Candida albictmS, were interpreted as negative for P ctJrinii. The patient's condition deteriorated with progressive pulmonary consolidation and evidence of cavitation by chest x-ray film. Respiratory failure ensued requiring mechanical ventilation. She died on the eighteenth hospital day. At post mortem, the lungs showed an alveolar space-fl.lling pneumonitis with multiple cavitary abscesses. Methenaminesilver stains were markedly positive for numerous P ctJrinii organisms (although initial staining of the autopsy material with fungal control was only faintly positive and almost missed, and repeat staining was necessary to demonstrate the numerous organisms clearly). The original transbronchial and open lung biopsy specimens were then recut and stained using the patient's own post mortem lung tissue as the control, and both specimens were positive for P carinii. DISCUSSION
The Grocott modification of Gomori's methenaminesilver technique involves the liberation of aldehyde groups from polysaccharides as the result of oxidation with chromic acid. Subsequently, the aldehyde oxidation products reduce silver nitrate to metallic silver rendering them visible. In addition, the chromic acid also tends to oxidize the newly released aldehyde groups to breakdown products that will not combine with silver.' This feature has the advantage of suppressing weaker back~ ground reactions of collagen and basement membranes while leaving reactive only those substances with large quantities of polysaccharides such as glycogen, mucin, fungal walls, melanin, and insoluble calcium salts. The intensity of staining, ie, deposition of metallic silver, depends to some degree on the length of time of pretreatment with chromic acid, in addition to the freshness of reagents and the thickness of tissue. To assure adequately stained slides, a known positive specimen must be simultaneously stained to verify that organisms have picked up sufficient silver deposition. For P carinii, understaining will fail to demonstrate organisms whereas overstaining will obliterate the important internal morphologic details characteristic of the cysts, as well as cause silver deposition on other cells, notably macrophages and RBCs. a The importance of proper staining technique in the diagnosis of P carinii pneumonia was discussed by Walzer et al. 8 In their experience at the Center for Disease Control, 194 patients with P carinii pneumonia, largely drawn from university centers in the United States, were studied, and the authors reported identi-
FALSE NEGATIVE BIOPSY IN P CARINII PNEUMONIA 389
lying organisms in six cases initially misdiagnosed as negative by the referring hospital. Most errors in diagnoses resulted from poor staining methods and lack of familiarity with the appearance of the organisms. The authors then emphasized the need for simultaneous controls in methenamine-silver staining for P carinil. However, they failed to explicitly state the need for the control to be P carinii. Following this case, we conducted a telephone survey of 15 hospitals in the greater Boston area. All hospitals employed the methenamine-silver stain for the identification of suspected P carlnU. However, only three hospitals were using a positive P carinfi specimen as a control at the time of our patient's hospitalization. Subsequently, our own and one other hospital have adopted the practice of routinely using P carlnfi controls. Pneumocystia carinii bas become a frequent cause of pneumonia as the population of immunocompromised hosts has increased. Pneumocystia carinfi pneumonia also has been reported to occur in otherwise healthy adults 5 •8 and has been reported to be a frequent cause of infection in premature infants in Europe. 7 Cases can now be expected to be seen outside of academic centers. Careful attention to the proper histologic preparation and staining of specimens is required if the diagnosis of P carinii is to be made. On the basis of our case, we conclude that fungal controls do not guarantee adequate staining of P carinii and that controls during methenamine-silver staining should be of P carlnfi itself. An additional advantage to the implementation of this procedure will be the greater familiarity pathologists will acquire for the morphologic details of P carinil organisms.
1 Doppman JL, Geelhoed GW and DeVita VT: Atypical radiographic features' in PneumocystiB carinU pneumonia. Radiology 114:39-44, 1975 2 Hughes W: Pfi8UmOC!Jiti8 carinH pneumonia. N Engl J Med 297:1381-1383, 1977 3 Walzer PD, Perl DP, Krogstad, DJ, et al: Pneumocyltil carlnii pneumonia in the United States. Ann Intem Med 80:83-93, 1974 . 4 Grocott RG: A stain in tissue sections and smears using Gomori's methenamine-silver nitrate technic. Am J Clin Pathol25:975-979, 1955 5 Lyons HA, Vinijchaikul K, Hennigar GR: PneumocystiB carlnii pneumonia unassociated with other disease. Arch lntem Med 108:929-936, 1961 6 Watanbe JM, Chincbinian H. Weitz C: Pneumocylti8 C.. lrUi pneumonia in a family. JAMA 193:685-686, 1965 7 lvady G, Paldy L, Koltay M, et al: Pneumocyltil carinU pneumonia. Lancet:616-617, 1967
380 CASAROnO ET AL
Surgical Removal of a Left Atrial Myxoma During Pregnancy* Dino Ct~~arotto, M.D.; Ubedo Bortolottl, M.D.; Rosario Russo, M.D.; DarlO Betti, M.D.; Luciano SchWaz.appa, M.D.; and Gaetano Thiefle, M.D.
Saeeessfol removal of a left atrial mymma In a prepant woman bas not been previously reported. The patient postoperatively bad an 11DC011lplleated prepaney, deBvering at term a normal, healthy baby. first case reported by Leyse and colleagues, Afterheartthesurgery, with the aid of cardiopulmonary 1
bypass ( CPB), can currently be accomplished during pregnancy with minimal hazards both for the mother and the fetus. We describe the first successful removal of a left atrial myxoma in a pregnant woman.
A 39-year-old woman, who previously had had two wcomplicated pregnancies, experienced easy fatigability, weight loss, and occasional palpitation two years prior to admission. Recently, some episodes of noctumal dyspnea occurred, and signs of congestive heart failure appeared. She was referred to our department for further evaluation. On admission, May 5, 1977, she was in the 21st week of pregnancy, the uterine enlargement being compatible with the gestational age. Blood pressure was 120/80 mm Hg. Pulse rate was 120 beats per minute and regular, and fetal heart rate was 140 beats per minute. The intensity of the first sound was increased; a protodiastolic sound, interpreted as an opening snap of the mitral valve, and a grade 3/6 diastolic-presystolic murmur at the apex were noted; the auscultatory 6ndings were apparently not related to positional changes. Bilateral pulmonary rales were present, and the liver was not enlarged. Pertinent laboratory findings revealed a moderate hypochromic anemia and a high erythrocyte sedimentation rate. The ECG showed sinus tachycardia and the chest x-ray film, pulmonary venous congestion and a moderate enlargement of the left atrium. · Since her condition progressively deteriorated despite medical treatment, a right cardiac catheterization was accomplished on May 12. The capillary pulmonary wedge pressure was markedly increased (average 28 mm Hg), and moderate pulmonary hypertension was present. During the levophase, an angiopneumocardiogram disclosed a roundshaped 6lling defect in the left atrial cavity which moved during the cardiac cycle from the atrium into the ventricle (Fig 1). The diagnosis of a floating left atrial mass was made and emergency surgery was performed on the same day. From the Departments of Cardiovascular Surgery, CardiolOIY,. and Patliology, University of Padova Medical School, Paaova, Italy. Reprint reqt.IB8t8: Dr. Ct~~arotto, Clmica ChitVrgica Genertils, Centro di Canliochifvgia, Via GiulejnitJni 2, 35100, Padooti, Italy 0
CHEST, 75: 3, MARCH, 1979
