Diagnostic performance of multiplex polymerase chain reaction test in identifying pneumonia causing micro-organisms on sputum samples of HIV positive patients accessing clinical care at Kiruddu and Naguru hospitals

Abstract

Pneumonia is one of the leading causes of morbidity and mortality among HIV infected people in sub-Saharan Africa. A disease with such a burden requires an appropriate diagnostic modality to allow a logical approach to guided treatment hence preventing the upsurge of antimicrobial resistance strains. Conventional diagnostic methods although available in Uganda are not fully utilized by clinicians due to associated diagnostic limitations, and as such present challenges in the successful management of pneumonia. To overcome the above diagnostic limitations, we set out to assess the performance of a new diagnostic modality, the Multiplex PCR assay that is rapid, sensitive and specific in evaluation of pneumonia. The Multiplex PCR assay is a commercial PCR test capable of detecting 18 bacteria (11 Gram negative, 4 Gram positive and 3 atypical), 7 antibiotic resistance markers, and 9 viruses that cause lower respiratory tract infections (pneumonia). Objective: To evaluate the diagnostic performance of Multiplex PCR test using a composite reference standard consisting of Gram stain test and sputum culture in detecting pneumonia causing micro-organisms in sputum samples of adult HIV positive patients at Naguru and Kiruddu referral hospitals. Materials and Methods: A diagnostic cross-sectional study design was employed. The study was conducted between November 2019 and February 2020 at inpatients wards and out-patients department of Kiruddu and Naguru referral hospitals. Adult HIV positive patients with cough, fever, dyspnea, infiltrates on chest x-ray and a negative gene-expert result for tuberculosis were consecutively sampled, instructed on sputum collection and two sputum samples obtained from each study participant was transported to the microbiology and infectious diseases institute translational laboratory Mulago. Sputum samples sent to microbiology laboratory were analyzed using gram staining and sputum culture tests, while sputum samples sent to translational laboratory were analyzed using multiplex PCR test. Data was analyzed using STATA version 14.0 software. Socio-demographic variables were summarized using descriptive statistics (frequency, median IQR) to see their distribution. A 2x2 contingency table and Receiver Operating Characteristic curves were employed to calculate the primary outcomes of the study i.e. sensitivity, specificity and area under curve. Administrative clearance was obtained from the respective hospital Institutional Review Board (IRB) and approval by School of Medicine Research and Ethics Committee (SOMREC). Results A total of 88 adult HIV positive patients meeting the inclusion criteria were enrolled into the study. Over half 47 (53.4%) were female, the median age was 45 years (IQR 35-53 years). 21.6% (23) had CD4 T cell count <100 cells/ul, 14.8% (13) were on antiretroviral treatment for a duration of <6 months. Of the 79 (89.7%) taking antibiotics prior to hospital visit, majority 36 (45.5%) had taken Ampiclox antibiotics. Pneumonia cases confirmed using the composite reference standard were 23 patients. Of the 23 confirmed pneumonia cases, multiplex PCR detected 21 cases out of 23 cases giving a sensitivity of 91.3%. 65 patients did not have pneumonia using the composite reference standard but multiplex PCR was negative in 29 of them 65 non diseased cases giving a specificity of 44.6%. Area under the curve was 0.68 (68%), the positive and negative predictive value were 37% and 93.5% respectively. Conclusions. Multiplex PCR test demonstrated a high sensitivity and low specificity which makes the test a good screening test but not a good confirmatory test for detection of pneumonia organisms in sputum. These results show the importance of utilizing rapid screening diagnostic tests with high sensitivity for identifying pneumonia causing micro-organisms in sputum samples.