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 pa-tients, 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. Although infection with pneumocystis was initially reported as rare in sub-Saharan Africans with AIDS, the incidence seems to be on the increase as the HIV pandemic progresses and more severe cases of infection present to hospi-tals. 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. 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 tuberculosis 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.” To know more about diseases is rather easy you may check out the website of Canadian Neighbor Pharmacy and find the necessary information for you.
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.
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. The primers used in the PCR reaction were based on the gene encoding the large subunit (LSU) mitochondrial ribosomal RNA (rRNA). The details of the primers are as shown as follows: pAZ102-E, -5′-GATGGCTGTT-TCCAAGCCCA-3′; pAZ102-H, -5′-GTGTACGTTGCAAAG-TACTC-3′; P1, -5′-CTAGGATATAGCTGGTTTTC-3′; and P2, -5′-TCGACTATCTAGCTTATCGC-3′.
Nested PCR Method
Nested PCR method was performed as described by Khan et al. 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.
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.