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Clinical Investigations: INFECTION |

Low Sensitivity of a Nested Polymerase Chain Reaction in Oropharyngeal Washings for the Diagnosis of Pneumocystis Pneumonia in HIV-Infected Patients* FREE TO VIEW

Kennedy Nyamande, MBChB, FCP; Umesh G. Lalloo, FRCP(London), FCCP; Dennis York, PhD; Mogambal Naidoo, BTech; Elvis M. Irusen, FCP; Runjan Chetty, FFPath, FRCPath, DPhil
Author and Funding Information

*From the Departments of Medicine (Drs. Nyamande, Lalloo, and Irusen), Pathology (Dr. Chetty), and Virology (Dr. York and Mr. Naidoo), Faculty of Health Sciences, Nelson R Mandela School of Medicine, University of KwaZulu-Natal, Durban, South Africa.

Correspondence to: Umesh G. Lalloo, FRCP(London), FCCP, University of KwaZulu-Natal, Nelson R Mandela School of Medicine, Department of Medicine, Pulmonology Unit, Private Bag X7, Congella, Durban 4013, South Africa; e-mail: lalloo@ukzn.ac.za



Chest. 2005;128(1):167-171. doi:10.1378/chest.128.1.167
Text Size: A A A
Published online

Study objective: To compare the relative yield and diagnostic utility of the polymerase chain reaction (PCR) analysis for Pneumocystis jirovecii DNA in oropharyngeal washings using transbronchial biopsy (TBBx) and BAL as “gold standards.”

Design: Prospective study.

Setting: Academic tertiary center.

Patients: Oropharyngeal washes were obtained in 50 consecutive patients with clinical pneumocystis pneumonia (PCP). Because of varying clinical severity, not all patients tolerated bronchoscopy. Thirty-five patients underwent TBBx, and 48 patients underwent BAL.

Methods: DNA extracted from oropharyngeal washings and BAL was subjected to a nested PCR test using primers for the large subunit mitochondrial ribosomal RNA of P jirovecii. Oropharyngeal washings were compared with BAL PCR and TBBx.

Results: Sixteen of the 35 TBBx procedures had positive results for PCP (46%). Oropharyngeal washings yielded positive results for pneumocystis in 7 of the 16 patients (sensitivity, 44%; specificity, 79%). Thirty-five of 48 patients (73%) had positive PCR results on BAL analysis. The relative yield of the PCR in oropharyngeal washes compared with BAL fluid was 40% (14 of 35 washes), giving a sensitivity of 40% and specificity of 77%.

Conclusion: PCR DNA amplification of oropharyngeal washings in HIV-seropositive subjects has a low sensitivity and specificity for the diagnosis of PCP.

Pneumocystis pneumonia (PCP) is caused by a fungus that is now called Pneumocystis jirovecii (previously known as Pneumocystis carinii). Sub-Saharan Africa is currently the epicenter of the HIV pandemic. In 2003, the World Health Organization estimated the number of people living with HIV/AIDS in the region at 29.4 million. PCP is the most frequent, opportunistic lung infection in AIDS patients,14 with a reported mortality rate of 10 to 30%. Before the era of highly active antiretroviral therapy, PCP developed in as many as 75% of individuals infected with HIV.1,3 Although infection with pneumocystis was initially reported as rare in sub-Saharan Africans with AIDS,1,56 the incidence seems to be on the increase as the HIV pandemic progresses and more severe cases of infection present to hospitals.711 Despite notable advances in the treatment of the virus, PCP remains a significant cause of morbidity and mortality in those with limited access to health care, and those who cannot afford expensive, specific antiretroviral therapy or chemoprophylaxis.

Transbronchial biopsy (TBBx), open-lung biopsy, fiberoptic bronchoscopy with BAL, and sputum induction have been the four established methods by which samples are obtained for the diagnosis of PCP. This is the current approach in the United States and Western Europe. The former two methods are invasive and carry the risk of significant morbidity and mortality and are not routinely available in resource-poor settings. All four procedures are contraindicated in seriously ill patients. These procedures are unavailable in the vast majority of resource-limited settings and, as a result, patients are treated empirically.

The etiology of pulmonary infiltrates in HIV is a major diagnostic challenge.34 It is important to distinguish between PCP, bacterial pneumonia, and pulmonary tuberculosis, particularly in sub-Saharan countries, which have experienced a phenomenal increase in HIV-associated tuberculosis12 that may mimic PCP. In addition, polymicrobial pulmonary infections are well recognized in HIV. An erroneous or missed diagnosis has important implications in terms of cost, drug side effects, morbidity, and mortality. Therefore, it is crucial to develop diagnostic procedures that can be utilized easily in resource-poor developing countries ravaged by HIV/AIDS. The aim of this study was to compare the relative yield and diagnostic utility of PCR as a potentially noninvasive and rapid screen for P jirovecii DNA in oropharyngeal samples compared with histology and BAL fluid, which are the “gold standards.”

Site

The study was performed at King Edward VIII Hospital, a tertiary teaching institution of the Nelson R Mandela School of Medicine of the University of KwaZulu-Natal (Durban, South Africa). The hospital is an 800-bed referral center for patients from the province of KwaZulu-Natal, which has a population of 9 million. It serves a predominantly black population. Ethical approval for the study was obtained from the local Ethics Committee.

Patient Selection

Consecutive HIV-seropositive subjects admitted to the medical wards from March to September 2000 and presenting with respiratory symptoms together with pulmonary infiltrates on chest radiographs suggestive of PCP were studied prospectively. None of the patients were receiving highly active antiretroviral therapy or trimethoprim-sulfamethaxazole prophylaxis for PCP. In our institution, the usual standard of care for patients with PCP is empiric treatment for the majority. This is partly due to limited technical, financial, and human resources, and because patients are too sick for invasive or semi-invasive procedures. Signed informed consent for fiberoptic bronchoscopy was obtained. Oropharyngeal samples and BAL from 16 HIV-seronegative subjects with respiratory symptoms were used as negative controls subjects. We believed it was important to have HIV-negative patients not at risk for PCP as negative control subjects. We anticipated this to be a very sensitive test.

After an overnight fast, each subject gargled with 10 to 20 mL of normal saline solution to obtain oropharyngeal washings. Patients gargled for approximately 5 s. We did not measure the volume of oropharyngeal washes returned into the container. No repeat gargles were performed. This was followed immediately by fiberoptic bronchoscopy for BAL and TBBx.

Fiberoptic Bronchoscopy and BAL

Fiberoptic bronchoscopy and BAL were performed in standard fashion. Briefly, after an overnight fast, patients were nebulized with salbutamol for 10 min. Two milliliters of robinul and 2 mg of midazolum were then administered IV, and the vocal cords anesthetized with a 2% anesthetic spray. An Olympus fiberoptic bronchoscope was then passed via the oral route, and the vocal cords visualized. Two percent liquid parenteral-local anesthetics was then instilled into the trachea via the open cords. The trachea, carina, and bronchi were inspected. BAL of the most affected lung segments (on chest radiograph) was performed with 50 mL of 0.9% saline solution per lavage. A maximum of three lavages were performed. Transbronchial samples were obtained for biopsy from the same segments using a flexible biopsy forceps. Four to six pieces of lung tissue were obtained per patient and suspended in 5% formal saline solution (41% formaldehyde/0.9% sodium chloride [1:8, volume per volume]).

Oropharyngeal samples were obtained in all 50 patients. Forty-eight patients were able to tolerate fiberoptic bronchoscopy, TBBx was performed in 35 patients, and BAL was performed in 48 patients.

Oropharyngeal Washings and BAL Fluid Analysis

One milliliter of sample was used to extract DNA. The procedure for DNA extraction and nested PCR was performed as described previously.13 The primers used in the PCR reaction were based on the gene encoding the large subunit (LSU) mitochondrial ribosomal RNA (rRNA).13 The details of the primers are as shown as follows: pAZ102-E, -5′-GATGGCTGTTTCCAAGCCCA-3′; pAZ102-H, -5′-GTGTACGTTGCAAAGTACTC-3′; P1, -5′-CTAGGATATAGCTGGTTTTC-3′; and P2, -5′-TCGACTATCTAGCTTATCGC-3′.

Nested PCR Method

Nested PCR method was performed as described by Khan et al.13 Oropharyngeal and BAL fluids were collected as described previously and stored at 4°C until processing. One hundred microliters of fluid were added to 200 μL 10% Chelex (Bio-Rad; Hercules, CA) and incubated at 56°C for 30 min. The sample was then boiled at 100°C for 10 min. After a brief microcentrifugation, 10 μL were removed and added to 40 μL PCR master mix containing primers pAZ102-E and pAZ102-H that target the gene encoding the LSU mitochondrial rRNA. After 40 cycles of amplification (94°C for 20 s, 55°C for 20 s, and 72°C for 20 s) and a 7-min soak at 72°C, 1 μL was removed and added to the second nested step PCR master mix, which contained primers P1 and P2. Fifteen microliters of the nested step were analyzed on a 2% agarose gel containing ethidium bromide. The presence of a 205 base-pair product, when the gel was visualized on an ultraviolet transilluminator, was regarded as positive for PCP. Positive and negative control subjects were included in each run. No correction was made for the fluid differences between BAL and oropharyngeal fluid. This assay has a theoretical sensitivity limit of detecting 300 genome equivalents per milliliter of fluid.

Histopathologic Examination

In addition to standard hematoxylin-eosin stains, a silver stain (Gomori methanamine) was performed on all cases together with Gram, periodic acid-Schiff, and Ziehl-Neelsen stains. Several sections on each case were examined.

It was not possible to obtain a TBBx and BAL in all of the patients because 2 patients had desaturation or intolerance of the bronchoscope and, in 13 patients, a TBBx was not performed because of thrombocytopenia and/or low hemoglobin. Positive histology results for PCP were obtained in 16 of the 35 subjects (46%). The remaining 19 PCP-negative reports were as follows: normal lung (n = 4); lymphocytic interstitial pneumonitis (n = 1); nonspecific mixed inflammation (n = 11); undifferentiated carcinoma (n = 1); chronic inflammation with bilharzia (n = 1); and fibro-fatty tissue (n = 1).

Oropharyngeal washings yielded positive results for P jirovecii in 7 of the 16 patients who had histologically proven PCP, giving a sensitivity of 44%. Four patients who had negative histology results for PCP had positive PCR results in oropharyngeal samples. Compared with histology, the PCR test for P jirovecii DNA in oropharyngeal washes had a specificity of 79%, a positive predictive value of 64%, and a negative predictive value of 63% (Table 1 ).

Bronchoscopy and BAL performed was performed on 48 patients. Thirty-five patients (73%) had a positive result for PCP on lavage fluid analysis by PCR. The relative yield of oropharyngeal washings compared with BAL fluid was 40%. Compared with BAL fluid, oropharyngeal washes had a sensitivity of 40%, specificity of 77%, a positive predictive value of 82%, and a negative predictive value of 32% (Table 2 ). The number of PCP-positive subjects by either BAL PCR or TBBx is shown in Table 3 .

Fifteen HIV-seronegative control subjects had fiberoptic bronchoscopy and BAL and were negative for P jirovecii DNA by PCR. One subject had sarcoidosis, one had squamous cell carcinoma, nine had nonspecific inflammation and or interstitial fibrosis and, in the remaining four patients, no diagnosis was made from TBBx. None were positive for P jirovecii in oropharyngeal washes.

PCR-based assays have been used as diagnostic tests and clinical tools and for epidemiologic studies.1422 If a PCR test had good sensitivity and specificity in oropharyngeal washings, it could be a useful substitute for sputum induction in resource-limited settings and where fiberoptic bronchoscopy is either unavailable or contraindicated. However, the procedure is potentially useful. This study has shown that the application of this set of primers for PCP had a low sensitivity and specificity when compared with lung biopsy, as a “gold standard” for the diagnosis of PCP in our resource-limited setting. In addition, the low sensitivity and specificity was also demonstrated for BAL.

The use of PCR has resulted in increased detection of P jirovecii from BAL fluid, induced sputum, and expectorated sputum.23The utility of DNA amplification to detect P jirovecii in oropharyngeal washes has previously been described by Wakefield et al,25 who reported a sensitivity of 56%26 in comparison to the standard investigation of microscopy after methenamine silver staining of the bronchoscopic lavage sample. To date, no study has compared the sensitivity and specificity of oropharyngeal washes to histology.

Sampling the oropharynx in the HIV-seropositive patient is appealing. The technique is simple, has no risk to the patient, and is quick. One hundred percent of subjects were able to provide an oropharyngeal sample in this study. Thus, the procedure can be easily performed in the outpatient setting. This is not possible with sputum induction, BAL, and TBBx. These require special equipment and expertise. PCR is now widely available and readily accomplished.

Wakefield et al25 have shown improved sensitivity of up to 78% with oligoblotting. Refinements of the methods of sample collection, storage, and laboratory techniques can be expected to result in higher sensitivities. Empiric treatment for PCP was started in all patients before oropharyngeal washes and bronchoscopy. This was done for logistical and ethical reasons because the patients were quite sick. Sampling the oropharynx before empiric treatment as well as obtaining more than one sample may improve the sensitivity. Significant prolongation of the duration of gargling is unlikely, because most of these patients are dyspnoeic. Concern has been raised regarding problems with false-positive results for control patients,13 which might limit the applicability of this method.

The diagnosis of PCP generally requires the demonstration of the organism in lower respiratory tract secretions from BAL or TBBx. Both procedures are invasive and possible only in stable patients. The sensitivities of the two methods have been reported to be between 98% and 100% in some series.2,2728 Induced sputum, a noninvasive method of diagnosing PCP, has a sensitivity of approximately 50%2 but is also contraindicated in seriously ill patients.

Expectorated sputum is inadequate for the diagnosis of PCP, with a sensitivity of only 34%.24,27 Empiric treatment is not always desirable, and some patients have atypical presentations.

Our study has shown the sensitivity of DNA amplification in oropharyngeal washes to be 44% and 40% using histology and BAL, respectively, as the “gold standards.” Unlike previous studies, oropharyngeal washes were compared with more sensitive diagnostic techniques, and this may account for the lower sensitivities obtained.

We chose to utilize primers that target the gene encoding the LSU mitochondrial rRNA. They have been used in some previous studies.13 We had the primers in our laboratory. There are other genetic loci for which PCR primers are available, and we could have used these, if they had been available to us.

Positive oropharyngeal washings were obtained in four subjects; all four had negative results for PCP. These could be false-positive results, but it is more likely that these were true-positive results, where the histology could have been negative due to a sampling error as blind biopsies were obtained.

Although an oropharyngeal PCR-positive result is of value, a diagnosis of PCP cannot be excluded if the result is negative. Although PCP is predominantly an alveolar disease, the detection of organisms in oropharyngeal samples could be explained on the basis of some organisms reaching the upper airways during coughing or invasion of the upper airways with severe disease. In a study29 of 150 HIV-infected African children with pneumonia from Malawi, P jirovecii was isolated from nasopharyngeal aspirates in 16 cases; thus, the organisms are capable of colonizing the upper airways. The probability of isolating organisms from the upper airways and mouth may depend on the depth and frequency of the cough, the severity of the disease, as well as the degree of immunocompromise.

The notion that PCP is rare in sub-Saharan Africans afflicted with AIDS is no longer true. Our study has shown that 46 to 73% of HIV-infected subjects presenting with respiratory symptoms and pulmonary infiltrates may have PCP. This has to be taken into account in drawing up treatment protocols for the region.

In conclusion, the use of nested PCR, under the above conditions, to detect PCP in oropharyngeal washings from HIV-seropositive subjects has a low sensitivity and specificity for the diagnosis of PCP. The low sensitivity could be due to the sensitivity limits imposed by the extraction method and sample collection. The low specificity could be due to the lower sensitivity of the reference diagnostic standard. However, the potential utility of PCR in oropharyngeal washings in resource-limited settings should not be undervalued.

Abbreviations: LSU = large subunit; PCP = pneumocystis pneumonia; PCR = polymerase chain reaction; rRNA = ribosomal RNA; TBBx = transbronchial biopsy

Table Graphic Jump Location
Table 1. Two-by-Two Table for Oropharyngeal Washes vs Histology in 35 Subjects*
* 

Data are presented as No. Sensitivity = 44%; specificity = 79%.

Table Graphic Jump Location
Table 2. Two-by-Two Table for Comparison of BAL with Oropharyngeal Washes*
* 

Data are presented as No. Sensitivity = 40%; specificity = 77%.

Table Graphic Jump Location
Table 3. Comparison of BAL PCR with TBBx*
* 

Data are presented as No.

McLeod, DT, Neill, P, Robertson, VJ, et al (1989) Pulmonary diseases in patients infected with the human immunodeficiency virus in Zimbabwe, Central Africa.Trans R Soc Trop Med Hyg83,694-697. [CrossRef] [PubMed]
 
Pitchenik, AE, Ganjei, P, Torres, A, et al Sputum examination for the diagnosis ofPneumocystis cariniipneumonia in the acquired immunodeficiency syndrome.Am Rev Respir Dis1986;133,226-229. [PubMed]
 
Levine, SJ Pulmonary complications of HIV.Clin Chest Med1996;17,665-695. [CrossRef] [PubMed]
 
Acquired immunodeficiency syndrome and the lung. Albert, RK Spiro, SG Jett, JR eds.Comprehensive respiratory medicine1999;32,1-22 Harcourt Brace
 
Abouya, YL, Beaumel, A, Lucas, S, et al Pneumocystis cariniipneumonia: an uncommon cause of death in African patients with the acquired immunodeficiency syndrome.Am Rev Respir Dis1992;145,617-620. [PubMed]
 
Elvin, KM, Lumbwe, CM, Luo, NP, et al Pneumocystis cariniiis not a major cause of pneumonia in HIV infected patients in Lusaka, Zambia.Trans R Soc Trop Med Hyg1989;83,553-555. [CrossRef] [PubMed]
 
Ruffini, DD, Madhi, SA The high burden ofPneumocystis cariniipneumonia in African HIV-1-infected children hospitalized for severe pneumonia.AIDS2002;16,105-112. [CrossRef] [PubMed]
 
Madhi, SA, Cutland, C, Ismail, K, et al Ineffectiveness of trimethoprim-sulfamethoxazole prophylaxis and the importance of bacterial and viral coinfections in African children withPneumocystis cariniipneumonia.Clin Infect Dis2002;35,1120-1126. [CrossRef] [PubMed]
 
Nathoo, KJ, Gondo, M, Gwanzura, L, et al FatalPneumocystis cariniipneumonia in HIV-seropositive infants in Harare, Zimbabwe.Trans R Soc Trop Med Hyg2001;95,37-39. [CrossRef] [PubMed]
 
Chintu, C, Mudenda, V, Lucas, S, et al Lung diseases at necropsy in African children dying from respiratory illnesses: a descriptive necropsy study.Lancet2002;360,985-990. [CrossRef] [PubMed]
 
Ansari, NA, Kombe, AH, Kenyon, TA, et al Pathology and causes of death in a series of human immunodeficiency virus-positive and -negative pediatric referral hospital admissions in Botswana.Pediatr Infect Dis J2003;22,43-47. [CrossRef] [PubMed]
 
Raviglione, MC, Snider, DE, Kochi, A Global epidemiology of tuberculosis: morbidity and mortality of a worldwide epidemic.JAMA1995;273,220-226. [CrossRef] [PubMed]
 
Khan, MA, Farrag, N, Butcher, P Diagnosis ofPneumocystis cariniipneumonia: immunofluorescence staining, simple PCR or nPCR.J Infect1999;39,77-80. [CrossRef] [PubMed]
 
Atzori, C, Agostoni, F, Angeli, E, et al Combined use of blood and oropharyngeal samples for noninvasive diagnosis ofPneumocystis cariniipneumonia using the polymerase chain reaction.Eur J Clin Microbiol Infect Dis1998;17,241-246. [PubMed]
 
Helweg-Larsen, J, Jensen, JS, Benfield, T, et al Diagnostic use of PCR for detection ofPneumocystis cariniiin oral wash samples.J Clin Microbiol1998;36,2068-2072. [PubMed]
 
Tsolaki, AG, Miller, RF, Wakefield, AE Oropharyngeal samples for genotyping and monitoring response to treatment in AIDS patients withPneumocystis cariniipneumonia.J Med Microbiol1999;48,897-905. [CrossRef] [PubMed]
 
Matos, O, Costa, MC, Lundgren, B, et al Effect of oral washes on the diagnosis ofPneumocystis cariniipneumonia with a low parasite burden and on detection of organisms in subclinical infections.Eur J Clin Microbiol Infect Dis2001;20,573-575. [CrossRef] [PubMed]
 
Fischer, S, Gill, VJ, Kovacs, J, et al The use of oral washes to diagnosePneumocystis cariniipneumonia: a blinded prospective study using a polymerase chain reaction-based detection system.J Infect Dis2001;184,1485-1488. [CrossRef] [PubMed]
 
Lishimpi, K, Kasolo, F, Chintu, C, et al Identification ofPneumocystis cariniiDNA in oropharyngeal mouth washes from AIDS children dying of respiratory illnesses.AIDS2002;16,932-934. [CrossRef] [PubMed]
 
Larsen, HH, Masur, H, Kovacs, JA, et al Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosingPneumocystis cariniipneumonia.J Clin Microbiol2002;40,490-494. [CrossRef] [PubMed]
 
Huang, L, Crothers, K, DeOliveira, A, et al Application of an mRNA-based molecular viability assay to oropharyngeal washes for the diagnosis of Pneumocystis pneumonia in HIV-infected patients: a pilot study.J Eukaryot Microbial2003;50(suppl),618-620
 
Larsen, HH, Huang, L, Kovacs, JA, et al A prospective, blinded study of quantitative touch-down polymerase chain reaction using oral-wash samples for diagnosis of Pneumocystis pneumonia in HIV-infected patients.J Infect Dis2004;189,1679-1683. [CrossRef] [PubMed]
 
Tuncer, S, Erguven, S, Kocagov, S, et al Comparison of cytochemical staining, immunofluorescence and PCR for diagnosis ofPneumocystis cariniion sputum samples.Scand J Infect Dis1998;30,125-128. [CrossRef] [PubMed]
 
Wakefield, AE, Pixley, FJ, Barneji, S, et al Detection ofPneumocystis cariniiwith DNA amplification.Lancet1990;336,451-453. [CrossRef] [PubMed]
 
Wakefield, AE, Miller, RF, Guiver, LA, et al Oropharyngeal samples for detection ofPneumocystis cariniiby DNA amplification.Q J Med1993;86,401-406. [PubMed]
 
Miller, R Clinical aspects ofPneumocystis cariniipneumonia in HIV-infected patients.FEMS Immunol Med Microbiol1997;22,103-105. [CrossRef]
 
Leigh, TR, Hume, C, Gazzard, B, et al Sputum induction for diagnosis ofPneumocystis cariniipneumonia.Lancet1989;2,205-206. [PubMed]
 
Metersky, ML, Aslenzadeh, J, Stelmach, P A comparison of induced and expectorated sputum for the diagnosis ofPneumocystis cariniipneumonia.Chest1998;113,1555-1559. [CrossRef] [PubMed]
 
Graham, SM, Mtitimila, EI, Kamanga, HS, et al Clinical presentation and outcome ofPneumocystis cariniipneumonia in Malawian children.Lancet2000;355,850
 

Figures

Tables

Table Graphic Jump Location
Table 1. Two-by-Two Table for Oropharyngeal Washes vs Histology in 35 Subjects*
* 

Data are presented as No. Sensitivity = 44%; specificity = 79%.

Table Graphic Jump Location
Table 2. Two-by-Two Table for Comparison of BAL with Oropharyngeal Washes*
* 

Data are presented as No. Sensitivity = 40%; specificity = 77%.

Table Graphic Jump Location
Table 3. Comparison of BAL PCR with TBBx*
* 

Data are presented as No.

References

McLeod, DT, Neill, P, Robertson, VJ, et al (1989) Pulmonary diseases in patients infected with the human immunodeficiency virus in Zimbabwe, Central Africa.Trans R Soc Trop Med Hyg83,694-697. [CrossRef] [PubMed]
 
Pitchenik, AE, Ganjei, P, Torres, A, et al Sputum examination for the diagnosis ofPneumocystis cariniipneumonia in the acquired immunodeficiency syndrome.Am Rev Respir Dis1986;133,226-229. [PubMed]
 
Levine, SJ Pulmonary complications of HIV.Clin Chest Med1996;17,665-695. [CrossRef] [PubMed]
 
Acquired immunodeficiency syndrome and the lung. Albert, RK Spiro, SG Jett, JR eds.Comprehensive respiratory medicine1999;32,1-22 Harcourt Brace
 
Abouya, YL, Beaumel, A, Lucas, S, et al Pneumocystis cariniipneumonia: an uncommon cause of death in African patients with the acquired immunodeficiency syndrome.Am Rev Respir Dis1992;145,617-620. [PubMed]
 
Elvin, KM, Lumbwe, CM, Luo, NP, et al Pneumocystis cariniiis not a major cause of pneumonia in HIV infected patients in Lusaka, Zambia.Trans R Soc Trop Med Hyg1989;83,553-555. [CrossRef] [PubMed]
 
Ruffini, DD, Madhi, SA The high burden ofPneumocystis cariniipneumonia in African HIV-1-infected children hospitalized for severe pneumonia.AIDS2002;16,105-112. [CrossRef] [PubMed]
 
Madhi, SA, Cutland, C, Ismail, K, et al Ineffectiveness of trimethoprim-sulfamethoxazole prophylaxis and the importance of bacterial and viral coinfections in African children withPneumocystis cariniipneumonia.Clin Infect Dis2002;35,1120-1126. [CrossRef] [PubMed]
 
Nathoo, KJ, Gondo, M, Gwanzura, L, et al FatalPneumocystis cariniipneumonia in HIV-seropositive infants in Harare, Zimbabwe.Trans R Soc Trop Med Hyg2001;95,37-39. [CrossRef] [PubMed]
 
Chintu, C, Mudenda, V, Lucas, S, et al Lung diseases at necropsy in African children dying from respiratory illnesses: a descriptive necropsy study.Lancet2002;360,985-990. [CrossRef] [PubMed]
 
Ansari, NA, Kombe, AH, Kenyon, TA, et al Pathology and causes of death in a series of human immunodeficiency virus-positive and -negative pediatric referral hospital admissions in Botswana.Pediatr Infect Dis J2003;22,43-47. [CrossRef] [PubMed]
 
Raviglione, MC, Snider, DE, Kochi, A Global epidemiology of tuberculosis: morbidity and mortality of a worldwide epidemic.JAMA1995;273,220-226. [CrossRef] [PubMed]
 
Khan, MA, Farrag, N, Butcher, P Diagnosis ofPneumocystis cariniipneumonia: immunofluorescence staining, simple PCR or nPCR.J Infect1999;39,77-80. [CrossRef] [PubMed]
 
Atzori, C, Agostoni, F, Angeli, E, et al Combined use of blood and oropharyngeal samples for noninvasive diagnosis ofPneumocystis cariniipneumonia using the polymerase chain reaction.Eur J Clin Microbiol Infect Dis1998;17,241-246. [PubMed]
 
Helweg-Larsen, J, Jensen, JS, Benfield, T, et al Diagnostic use of PCR for detection ofPneumocystis cariniiin oral wash samples.J Clin Microbiol1998;36,2068-2072. [PubMed]
 
Tsolaki, AG, Miller, RF, Wakefield, AE Oropharyngeal samples for genotyping and monitoring response to treatment in AIDS patients withPneumocystis cariniipneumonia.J Med Microbiol1999;48,897-905. [CrossRef] [PubMed]
 
Matos, O, Costa, MC, Lundgren, B, et al Effect of oral washes on the diagnosis ofPneumocystis cariniipneumonia with a low parasite burden and on detection of organisms in subclinical infections.Eur J Clin Microbiol Infect Dis2001;20,573-575. [CrossRef] [PubMed]
 
Fischer, S, Gill, VJ, Kovacs, J, et al The use of oral washes to diagnosePneumocystis cariniipneumonia: a blinded prospective study using a polymerase chain reaction-based detection system.J Infect Dis2001;184,1485-1488. [CrossRef] [PubMed]
 
Lishimpi, K, Kasolo, F, Chintu, C, et al Identification ofPneumocystis cariniiDNA in oropharyngeal mouth washes from AIDS children dying of respiratory illnesses.AIDS2002;16,932-934. [CrossRef] [PubMed]
 
Larsen, HH, Masur, H, Kovacs, JA, et al Development and evaluation of a quantitative, touch-down, real-time PCR assay for diagnosingPneumocystis cariniipneumonia.J Clin Microbiol2002;40,490-494. [CrossRef] [PubMed]
 
Huang, L, Crothers, K, DeOliveira, A, et al Application of an mRNA-based molecular viability assay to oropharyngeal washes for the diagnosis of Pneumocystis pneumonia in HIV-infected patients: a pilot study.J Eukaryot Microbial2003;50(suppl),618-620
 
Larsen, HH, Huang, L, Kovacs, JA, et al A prospective, blinded study of quantitative touch-down polymerase chain reaction using oral-wash samples for diagnosis of Pneumocystis pneumonia in HIV-infected patients.J Infect Dis2004;189,1679-1683. [CrossRef] [PubMed]
 
Tuncer, S, Erguven, S, Kocagov, S, et al Comparison of cytochemical staining, immunofluorescence and PCR for diagnosis ofPneumocystis cariniion sputum samples.Scand J Infect Dis1998;30,125-128. [CrossRef] [PubMed]
 
Wakefield, AE, Pixley, FJ, Barneji, S, et al Detection ofPneumocystis cariniiwith DNA amplification.Lancet1990;336,451-453. [CrossRef] [PubMed]
 
Wakefield, AE, Miller, RF, Guiver, LA, et al Oropharyngeal samples for detection ofPneumocystis cariniiby DNA amplification.Q J Med1993;86,401-406. [PubMed]
 
Miller, R Clinical aspects ofPneumocystis cariniipneumonia in HIV-infected patients.FEMS Immunol Med Microbiol1997;22,103-105. [CrossRef]
 
Leigh, TR, Hume, C, Gazzard, B, et al Sputum induction for diagnosis ofPneumocystis cariniipneumonia.Lancet1989;2,205-206. [PubMed]
 
Metersky, ML, Aslenzadeh, J, Stelmach, P A comparison of induced and expectorated sputum for the diagnosis ofPneumocystis cariniipneumonia.Chest1998;113,1555-1559. [CrossRef] [PubMed]
 
Graham, SM, Mtitimila, EI, Kamanga, HS, et al Clinical presentation and outcome ofPneumocystis cariniipneumonia in Malawian children.Lancet2000;355,850
 
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