Increased Sensitivity in the Diagnosis of Tuberculosis in HIV- positive Patients. through Small Membrane Filter Method of Microscopy

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1 JCM Accepts, published online ahead of print on 26 June 2013 J. Clin. Microbiol. doi: /jcm Copyright 2013, American Society for Microbiology. All Rights Reserved Increased Sensitivity in the Diagnosis of Tuberculosis in HIV- positive Patients through Small Membrane Filter Method of Microscopy 3 4 Microscopy processing methods sensitivity Patrícia Quincó 1 #, Samira Bührer-Sékula 2,3, Walber Brandão 2, Rossiclea Monte 2, Silvia Leopoldina Souza 2, Valeria Saraceni 2,4, Moises Palaci 5, Reynaldo Dietze 5, Marcelo Cordeiro-Santos 1,2. 1. Universidade do Estado do Amazonas/Pós-Graduação em Medicina Tropical, Manaus, Amazonas, Brazil. 2. Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Manaus, Amazonas, Brazil. 3. Universidade Federal de Goiás, Instituto de Patologia Tropical e Saúde Pública.Goiânia, Goiás, Brazil. 4. Secretaria Municipal de Saúde do Rio de Janeiro, RJ, Brazil 5. Núcleo de Doenças Infecciosas, Universidade Federal do Espírito Santo, Vitória, ES, Brazil. Corresponding author (#): Samira Bührer samira@buhrer.net Fundação de Medicina Tropical Dr. Heitor Vieira Dourado, Pós-Graduação em Medicina Tropical. Avenida Pedro Teixeira, 25, Dom Pedro, Manaus, Amazonas, Brazil. Phone:

2 Summary The sensitivity of microscopy for the diagnosis of tuberculosis is around 50%, but decreases by about 15% in TB suspects co-infected with HIV. We compared the accuracy of three processing methods of sputum microscopy (concentration by centrifugation with or without NALC and concentration by filtration on polycarbonate membrane) to culture on Ogawa-Kudoh medium as the gold standard method. Sputa were obtained from 432 patients with suspected pulmonary TB, 60% were HIV-infected. Analysis was performed using the first specimen. Compared to the gold standard culture, the small membrane filter (SMF) method was the most sensitive microscopic method. In TB HIV-infected patients, sensitivity for SMF was significantly higher than after centrifugation of sputa treated with or without NALC (61.9%, 47.6% and 45.2%; p = 0.001) respectively. Similarly, in TB patients without HIV, the SMF sensitivity was significantly higher (81.8%, 63.6% and 57.5%; p = 0.001) respectively. In both study groups, TB patients with or without HIV, no significant differences between the specificities of the three methods were observed. Handling of the second sputum sample similarly by centrifugation with or without NALC and SMF showed an increase of positivity by 13%, 11% and 4% respectively. The overall agreement between microscopy and culture was above 90% for all groups. Microscopic evaluation of sputum treated with NALC compared to sputum without NALC did not show any increase sensitivity. Altogether, the sensitivity of SMF method is higher compared to the other two microscopic methods studied without loss of specificity. Palavras-chaves: Diagnosis. Filtration. Bacilloscopy. 59

3 3 60 INTRODUCTION Diagnosing TB in HIV-infected patients still presents a major challenge since clinical and radiological findings are often atypical (14, 17) and many cases of smearnegative pulmonary TB are undiagnosed (6). Sputum smear microscopy, a low-cost method, is important in resource-limited scenarios where culture is not available but presents a lower sensitivity in suspected HIV-infected TB patients (8, 10, 16). The filtration on polycarbonate membrane or small membrane filtration (SMF) method has proven to have a greater sensitivity and specificity than direct microscopy, even after centrifugation of sputum (7, 18), but HIV status of the population was not reported. In the routine conditions of a bacteriology lab, we evaluated this method in TB suspects, mostly HIV-infected patients, along with bacilloscopy after centrifugation, with or without decontamination. The three methods were compared to culture on solid media (Ogawa-Kudoh), considered the gold standard for TB diagnosis in our setting, and sensitivity, specificity and Cohen s kappa coefficient were calculated.

4 4 76 MATERIALS AND METHODS Design and study setting. A prospective study was conducted between September 2011 and January 2012 in Manaus, Amazonas, Brazil, recruiting patients from Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD) and also from the regional reference center for TB, Policlínica Cardoso Fontes. Enrollment. Patients suspected of pulmonary TB were eligible. The inclusion occurred consecutively as they attended the services. As the collection of clinical and demographic data was conducted prospectively using a standardized questionnaire. A pulmonary tuberculosis case was defined as patients with positive sputum microscopy or/and positive culture or patients with suggestive clinical and radiological findings treated as TB. Sample Collection. Two sputum samples on two consecutive days were requested from each patient, but patients were included even if they had only one sputum sample. Five hundred and eight patients provided the first sample but the second was provided by 227 patients. Sputa samples with a volume equal or higher than 1.5 ml were included in the study. In the case of HIV-positive subjects, blood was drawn for CD4 T-cell counts and HIV plasma viral load tests via the routine laboratory service of FMT-HVD. Sample size calculations were based on an estimated prevalence of 13.1%, sensibility of 89.0%, specificity of 99.6%. The precision of the estimate was based on 95% confidence intervals and beta of 20%. For a power of 80% we would need 451 samples. These expectations were based on the previous Small Membrane Filter study (7). Centrifugation Sputum Smear. The smears were prepared using a 500 μl aliquot after centrifugation at 3000gx at 4 º C for 15 minutes and stained by the Ziehl- Neelsen (ZN) method (Figure 1).

5 Centrifugation Sputum Smear with NALC. The second aliquot was digested with NALC using a solution of 5% NaOH / 2.9% sodium citrate in a volume corresponding to 10% of the sputum sample; it was left to rest for 15 minutes at 20 C. Later, the sample was divided into two digested aliquot (9). In the first digested aliquot, phosphate buffer ph 6.8 was added up to 50 ml and after centrifugation at 3000 gx the solution was left to rest for 15 minutes at 4 C. The smear was then stained by the ZN method. Characteristics of the filtration system. The system described by Fennelly et al.(7) was used with minor changes: 1) ten vacuum system stainless steel filtration points controlled by manual shut-off valves. 2) pre-filter nylon membrane of 25 mm with a pore size of 30 μm. 3) filter with white polycarbonate membrane of 13 mm with a pore size of 0.8 μm (Millipore). 4) electric vacuum pump for applying negative pressure (5 mmhg). SMF method. Using the second digested aliquot, twice the aliquot volume of NaOCl 5% was added. The sample was homogenized for 15 minutes, 95% ethanol/triton1% in volume equal to the volume of the mixture was added and again homogenized for 15 seconds. After the passage of sputum though filter systems, the valves were closed and the membranes still in the filter were stained with the Kinyoun method(1). The membranes were then transferred to microscope slides, and a coverslip was placed on each slide for microscopic reading. Reading and interpretation of microscopy. The slides were observed under a bright field microscope with 100X immersion objective to detect Acid Fast Bacilli (AFB) in 100 fields and interpreted as a semi-quantitative scale. Ogawa Kudoh (OK) culture medium Used as the gold standard method. The first sputa aliquots were decontaminated by the method of OK (13) and plated onto

6 solid culture medium. The cultures were incubated at 37 C and read for up to 8 weeks after which culture was reported as negative. The culture results were reported as positive when the growth of a colony forming unit occurred, with the presence of AFB confirmed by microscopy. Identification of Mycobacteria isolates: Isolates of M. tuberculosis were identified using the criteria of morphology, pigmentation of colonies, microscopy, niacin test and growth inhibition test in medium with para-nitrobenzoic and in medium with thiophen-2-carboxylic hydrazide. Statistical analysis. The study population was characterized with simple descriptive statistics and patients with and without tuberculosis were compared with the Wilcoxon rank-sum test, t- test, χ 2 square test, and Fisher s exact test accordingly. The sensitivity, specificity and predictive values of the three tests were estimated with 95% confidence interval and were determined by comparing those individually with the culture results of the first sputum sample. McNemar test was used to compare the sensitivity of the different microscopy methods. To calculate the sensitivity increase when using two samples, positive results of the second sample from patients whose first sputum was negative were used as numerator and the total number of patients diagnosed as TB was the denominator. The Cohen s Kappa coefficient was used to determine the agreement between culture and 3 other diagnostic methods. Data were analyzed with EpiInfo (version 3.5.3). Ethical aspects. The study was approved by the Research Ethics Committee of the FMT-HVD, protocol 1845/2011 of 09/09/11. All patients signed the informed consent form.

7 RESULTS Population and samples: Figure 1 shows that from 508 eligible TB suspected patients and 76 were excluded from the analysis: 1.8% (9/508) had a sample volume less than 1.5 ml, 9.6% (49/508) the system failed to filter and 3.5% (18/508) had a contaminated culture, Table 1 summarizes the characteristics of 432 patients included in the analysis. The mean age was 39 years and 60% (261/432) were HIVinfected patients. Based on all specimen culture results available (on 1st or 2nd specimen), the prevalence of pulmonary tuberculosis in the eligible group of patients was 19.4% (95% CI 15.9% %; n=84/432). From those, 50% (42/84) were HIV positive. The prevalence of tuberculosis in HIV infected patients was 16.1% (95% CI 12.0% %; n=42/261). Moreover, in 9 cases the patients had a positive bacilloscopy, although a negative culture. HIV infected patients with positive TB cultures had lower mean CD4 counts than patients with negative culture (mean CD4 count 65 versus 279, p <0.001). Cohen s Kappa coefficient between the gold standard and microscopy methods: Table 2 shows the kappa coefficient between culture and sputum smear performed with 3 different methods, In the HIV-infected group the kappa result was considered moderate, k = 0.55 and k = 0.578, respectively. Microscopy methods performance: As observed in Table 3, when computing results obtained testing only the first specimen, the sensitivity obtained using the SMF method was higher than the sensitivity when using methods of centrifugation treated or not with NALC, (70.6% vs. 54.6%, p < 0.001) and (70.6% vs. 50.6%, p < 0.001), respectively. The SMF method presented the highest sensitivity for the HIVnegative patients group and for the HIV-positive patients group, 81.8% and 61.4% (p

8 <0.001), respectively. There was no statistically significant difference between the sensitivity of microscopy after centrifugation with and without NALC (p = 0.246). When analyzing the performance of all three processing methods using results from specimen 1 and 2 (table 4), similar results were obtained. Loss of sensitivity for NALC and SMF was observed but no significant difference was observed (p= 0.05). A second specimen was provided by 227 patients, 47 of these had a positive culture. The second specimen showed an increase of 13% (6/47) positivity for microscopy after centrifugation with NALC, 11% (5/47) positivity for microscopy after centrifugation without NALC, and 4% (2/47) positivity in SMF method. Table 5 shows the performance of microscopy methods for 227 patients that delivered 1 st and 2 nd specimen. DISCUSSION In general all TB diagnostic methods have a poorer performance, reduced sensitivity, among TB/HIV coinfected patients. A rapid diagnostic tool to assist the TB diagnosis for HIV-infected patients is needed in order to improve the prognosis of such patients. In our study the sensitivity of sputum smear microscopy treated with NALC in the HIV-infected patients group was lower (47.6%) than the sensitivity of 61.8% in a study that used as eligibility criterion a CD4 cell count > 200 cells/mm 3 (15), and also lower than the 72% found in a study using fluorescence microscopy (3). On the other hand, we found a similar sensitivity to a study that used the hot Ziehl-Neelsen method (4), and yet again, similar to a study with TB suspected outpatients in a reference laboratory in South Africa (20), a country with a high TB and HIV burden. The differences observed are likely to be due to variation in patient populations as well as the methodologies used in each study.

9 In our study the specificity of smear microscopy after centrifugation with and without NALC was virtually the same in both infected and uninfected HIV patients groups. The value of the specificity of smear after centrifugation with NALC among infected and non-infected HIV group (99% and 99.3%, respectively) are consistent with results from other studies (4, 12, 19) The SMF method had a sensitivity of 61.9% for HIV infected patients in our study higher than the other microscopic methods used. The sensitivity of 81.8% in HIVnegative subjects was similar to the 80% and 89% reported by Smithwick & Stratigos (18) and Fennelly et al.(7), respectively. The Xpert MTB/RIF assay (Cepheid Inc., Sunnyvale, CA, USA) is a rapid molecular diagnostic that has been endorsed by the WHO. A recent systematic review found for this assay had a sensitivity of 80% in a HIV+ population(11), higher than the sensitivity of 67% for SMF in our study. However, the Xpert is considerably more expensive than other methods used for tuberculosis diagnosis and cannot be used to monitor treatment because it does not distinguish between viable and nonviable microorganisms. SMF could be an important alternative especially in peripheral health care settings. The SMF method may have presented a better performance if a more sensitive culture medium was used. Limitations of the culture used probably decreased the measured specificity of the SMF method. Additional information of the microscopy positive culture negative cases was sought. From them, 3 were also positive in sputum after centrifugation with and without NALC and, from these group, 2 were HIV-infected patients who died of sepsis and one was diagnosed with pulmonary TB on a 3 rd positive sputum culture, and was under treatment for TB. Of the 6 patients

10 who only had a positive SMF, 4 were diagnosed with pulmonary TB based on clinical and radiological findings and responded to TB treatment, two were diagnosed by culture of the 3 rd specimen, one with pulmonary and extrapulmonary TB and one with pulmonary TB. These findings suggested that sputum cultures performed in the first sample in this study weren t sensitive enough to detect TB in all cases, due to intrinsic limitations with solid media and TB culture in general. The Brazilian Ministry of Health suggests that 2 samples should be submitted to bacilloscopy (without centrifugation or decontamination)(2), because of microscopy s limited sensitivity. Due to the higher sensitivity of SMF method in the first sample, our results suggest that the second sputum sample, which was often missing, may be less important when using this method. The study was performed in routine settings and some limitations might have influenced the sensitivity and specificity of the studied microscopy methods. One of them could be the use of solid culture medium which has a lower sensitivity than liquid media such as MGIT(5). Secondly, quality control of SMF reading was not performed mainly because the bacilli staining of the SMF fades away after 24 hours. As the two staining methods use the same stain, it might be possible that the remaining hypochlorite and/or Triton in the membrane would fade the stain. The SMF process takes double time compared to the 15 minutes of microscopy after centrifuging; concerning safety issues handling samples is similar to microscopy. SMF requires higher technical expertise than the XpertMTB/Rif but is more affordable for places where the amount of specimen to be tested does not allow investments in costly equipment.

11 In respect to technical difficulties, the cleaning of SMF material used in this study was laborious because disposable components weren t available, and due to technical problems, 10% of sputa samples could not be filtered. The clogged membranes might have occurred due to lack of some air in the filter holder. It was not due to thickness of samples as most of them were salivary samples. The observed pitfalls led to improvements on the filtration system, with the use of disposable components and adjustment to the filtration process. Conclusion The sensitivity of the SMF method was superior to all other microscopic techniques applied without any loss of specificity and may be a useful approach for the diagnosis of TB in HIV-infected suspects, reducing morbidity and improving patients' prognosis. Treatment of specimens with NALC did not increase the sensitivity of smear microscopy with centrifugation, indicating that the addition of such procedures is not necessary. We believe the SMF method has considerable advantages for the detection of TB infection in HIV positive patients especially considering that the GeneXpert system might not appropriated for monitoring patient s treatment. ACKNOWLEDGMENTS We would like to thank Dr. Richard Antony for his comments and English revision. This study was partially supported by contracts, CNPq/INCT number / and PPSUS/FAPEAM 05/2010.

12 References de Dezembro 2012, posting date. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Departamento de Vigilância Epidemiológica. Manual Nacional de Vigilância Laboratorial da Tuberculose e outras Micobactérias.Brasília. [Online.] de Dezembro 2012, posting date. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Departamento de Vigilância Epidemiológica. Manual de recomendações para o controle da Tuberculose no Brasil. [Online.] 3. Cattamanchi, A., J. L. Davis, W. Worodria, S. den Boon, S. Yoo, J. Matovu, J. Kiidha, F. Nankya, R. Kyeyune, P. Byanyima, A. Andama, M. Joloba, D. H. Osmond, P. C. Hopewell, and L. Huang Sensitivity and specificity of fluorescence microscopy for diagnosing pulmonary tuberculosis in a high HIV prevalence setting. Int J Tuberc Lung Dis 13: Cattamanchi, A., D. W. Dowdy, J. L. Davis, W. Worodria, S. Yoo, M. Joloba, J. Matovu, P. C. Hopewell, and L. Huang Sensitivity of direct versus concentrated sputum smear microscopy in HIV-infected patients suspected of having pulmonary tuberculosis. BMC infectious diseases 9: Chew, W. K., R. M. Lasaitis, F. A. Schio, and G. L. Gilbert Clinical evaluation of the Mycobacteria Growth Indicator Tube (MGIT) compared with radiometric (Bactec) and solid media for isolation of Mycobacterium species. Journal of medical microbiology 47: Davis, J. L., W. Worodria, H. Kisembo, J. Z. Metcalfe, A. Cattamanchi, M. Kawooya, R. Kyeyune, S. den Boon, K. Powell, R. Okello, S. Yoo, and L. Huang Clinical and radiographic factors do not accurately diagnose smear-negative tuberculosis in HIV-infected inpatients in Uganda: a cross-sectional study. PLoS One 5:e Fennelly, K. P., C. G. Morais, D. J. Hadad, S. Vinhas, R. Dietze, and M. Palaci The small membrane filter method of microscopy to diagnose pulmonary tuberculosis. Journal of clinical microbiology 50: Getahun, H., M. Harrington, R. O'Brien, and P. Nunn Diagnosis of smear-negative pulmonary tuberculosis in people with HIV infection or AIDS in resource-constrained settings: informing urgent policy changes. Lancet 369: Hadad, D. J., C. G. Morais, S. A. Vinhas, K. P. Fennelly, R. Dietze, C. P. Nascimento, and M. Palaci Evaluation of processing methods to equitably aliquot sputa for mycobacterial testing. Journal of clinical microbiology 50: Hargreaves, N. J., O. Kadzakumanja, C. J. Whitty, F. M. Salaniponi, A. D. Harries, and S. B. Squire 'Smear-negative' pulmonary tuberculosis in a DOTS programme: poor outcomes in an area of high HIV seroprevalence. Int J Tuberc Lung Dis 5: Karen R Steingart, H. S., Ian Schiller, Lorie A Kloda, Catharina C Boehme, Madhukar Pai, Nandini Dendukuri Xpert MTB/RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. The Cochrane Library: Kivihya-Ndugga, L. E., M. R. van Cleeff, W. A. Githui, L. W. Nganga, D. K. Kibuga, J. A. Odhiambo, and P. R. Klatser A comprehensive comparison of Ziehl-Neelsen and fluorescence microscopy for the diagnosis of tuberculosis in a resource-poor urban setting. Int J Tuberc Lung Dis 7: Kudoh, S., and T. Kudoh A simple technique for culturing tubercle bacilli. Bull World Health Organ 51: Lawn, S. D., and R. Wood Tuberculosis in antiretroviral treatment services in resource-limited settings: addressing the challenges of screening and diagnosis. J Infect Dis 204 Suppl 4:S Matee, M., L. Mtei, T. Lounasvaara, W. Wieland-Alter, R. Waddell, J. Lyimo, M. Bakari, K. Pallangyo, and C. F. von Reyn Sputum microscopy for the diagnosis of HIV-associated pulmonary tuberculosis in Tanzania. BMC Public Health 8: Perkins, M. D., and J. Cunningham Facing the crisis: improving the diagnosis of tuberculosis in the HIV era. J Infect Dis 196 Suppl 1:S15-27.

13 Schutz, C., G. Meintjes, F. Almajid, R. J. Wilkinson, and A. Pozniak Clinical management of tuberculosis and HIV-1 co-infection. Eur Respir J 36: Smithwick, R. W., and C. B. Stratigos Acid-fast microscopy on polycarbonate membrane filter sputum sediments. J Clin Microbiol 13: Steingart, K. R., V. Ng, M. Henry, P. C. Hopewell, A. Ramsay, J. Cunningham, R. Urbanczik, M. D. Perkins, M. A. Aziz, and M. Pai Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. Lancet Infect Dis 6: Wilkinson, D., and A. W. Sturm Diagnosing tuberculosis in a resource-poor setting: the value of sputum concentration. Trans R Soc Trop Med Hyg 91: Downloaded from on May 2, 2018 by guest

14 FIG. 1. Study population and samples Downloaded from on May 2, 2018 by guest

15 FIG. 2: Processing of Sputum Specimens Downloaded from on May 2, 2018 by guest

16 1 Table 1: Characteristics of patients by TB culture results Characteristics Culture Positive Negative TOTAL p-value (n=84) (n=348) (n=432) Age, years (mean, SD) 34.3 (11.4) 40.1 (14.5) 39 ( 14.2) <0.001 Gender (male, %) Outpatient clinic (%) <0.001 New TB case (%) HIV-positive (%) CD4 cells (mean, SD), if HIV-positive SD= standard deviation; 65 (54) 279 (231) 239(226) <0.001 TABLE 2: Microscopy methods compared to solid culture in HIV-infected and uninfected patients groups Centrifugation Centrifugation - NALC SMF Culture POS NEG POS NEG POS NEG HIV infected POS N=261 NEG Agreement Kappa 0,555 0,578 0,692 HIV uninfected POS N=171 NEG Agreement Kappa 0,669 0,720 0,737 Total POS N=432 NEG Agreement Kappa 0,607 0,643 0,714

17 2 TABLE 3: Performance of microscopy methods compared to solid culture in HIV-infected and uninfected patients groups when computing results of the first specimen Microscopy Centrifugation % (95% CI) Sensitivity Specificity PPV NPV Centrifugation with NALC % (95% CI) Sensitivity Specificity PPV NPV SMF % (CI 95%) Sensitivity Specificity PPV NPV HIV + patients (N=261) 45.2( ) 99( ) 90.4( ) 89.6( ) 47.6( ) 99( ) 90.9( ) 90.0( ) 61.9( ) 98.5( ) 89.6( ) 92.5( ) HIV- patients (N=171) 57.5( ) 99.3( ) 95.00( ) 91.7( ) 63.6( ) 99.3( ) 95.4( ) 92.8( ) 81.8( ) 96.7( ) 84.3( ) 96.1( ) TOTAL (N=432) 50.6 ( ) 99.1 ( ) 92.6( ) 90.5( ) 54.6( ) 99.1( ) 93.1( ) 91.2( ) 70.6( ) 97.4( ) 85.4( ) 94.0( ) Abbreviations: CI=Confidence Interval; PPV=Positive Predictive Value, NPV= Negative Predictive Value; HIV = human immunodeficiency virus TABLE 4: Performance of microscopy methods compared to solid culture in HIV-infected and uninfected patients groups for 432 patients, when considering postivity of the 1 st and 2 nd specimen Microscopy Centrifugation % (95% CI) Sensitivity Specificity PPV NPV Centrifugation with NALC % (95% CI) Sensitivity Specificity PPV NPV SMF % (CI 95%) Sensitivity Specificity PPV NPV HIV + patients (N=261) 46.9( ) 99.1( ) 92( ) 89.0( ) 49.0( ) 98.6( ) 88.9( ) 89.3( ) 57.1( ) 98.6( ) 90.3( ) 90.9( ) HIV- patients (N=171) 57.1( ) 99.3( ) 95.2( ) 90.0( ) 62.9( ) 99.3( ) 95.7( ) 91.2( ) 74.3( ) 94.9( ) 78.8( ) 93.5( ) TOTAL (N=432) 51.2 ( ) 99.1 ( ) 93.5( ) 89.4( ) 54.8( ) 98.9( ) 92.0( ) 90.1( ) 64.3( ) 97.1( ) 84.4( ) 91.9( ) Abbreviations: CI=Confidence Interval; PPV=Positive Predictive Value, NPV= Negative Predictive Value; HIV = human immunodeficiency virus

18 3 TABLE 5: Performance of microscopy methods compared to solid culture in HIV-infected and uninfected patients groups for 227 patients that delivered 1 st and 2 nd specimen Microscopy Centrifugation % (95% CI) Sensitivity Specificity PPV NPV Centrifugation with NALC % (95% CI) Sensitivity Specificity PPV NPV SMF % (CI 95%) Sensitivity Specificity PPV NPV HIV + patients (N=163) 31.3( ) 99.2( ) 90.9( ) 85.5( ) 49.0( ) 98.5( ) 87.5( ) 87.8( ) 53.1( ) 98.5( ) 89.5( ) 89.6( ) HIV- patients (N=64) 60.0( ) 98.0( ) 90.0( ) 89.0( )) 73.3( ) 98.0( ) 91.7( ) 92.3( ) 73.3( ) 93.9( ) 78.6( ) 92.0( ) TOTAL (N=227) 40.4( )) 98.9 ( ) 90.5( ) 86.4( ) 53.2( ) 98.3( ) 89.3( ) 88.9( ) 59.6( ) 97.2( ) 84.9( ) 90.2( ) Abbreviations: CI=Confidence Interval; PPV=Positive Predictive Value, NPV= Negative Predictive Value; HIV = human immunodeficiency virus Downloaded from on May 2, 2018 by guest