Pneumocystis pneumonia (PCP) is an opportunistic infection that commonly occurs in immunocompromised individuals. A definite diagnosis of PCP can be made only when the organism is identified in a respiratory specimen. It remains unclear whether qPCR can differentiate patients with PCP from those with Pneumocystis jirovecii colonization. In this study, we retrospectively collected data from HIV and non-HIV patients during 2013–2019. A diagnosis of definite, probable PCP, or PCP excluded was made based on clinical criteria, radiological reports, and three standard laboratory staining methods with blinding to qPCR data. Data from qPCR that was performed to determine the fungal burden (DNA copies/μl) in the BAL specimens of 69 HIV and 286 non-HIV patients were then obtained and reviewed. Receiver Operating Characteristic (ROC) curve analysis was performed to determine the upper and lower cut-off values for PCP diagnosis in HIV and non-HIV groups. In the non-HIV group, the lower cut-off value of 1,480 DNA copies/μl yielded a sensitivity of 100% (95% confidence interval [CI], 91.0–100), specificity of 72.9% (95% CI, 64.0–80.7), a positive predictive value (PPV) of 54.9% (95% CI, 47.6–62.1), and a negative predictive value (NPV) of 100% with Youden index of 0.73 for PCP diagnosis. In this group, the upper cut-off value of 9,655 DNA copies/μl showed the sensitivity of 100% (95% CI, 91.0–100) and specificity of 95.8% (95% CI, 90.4–98.6) with PPV of 88.6% (95% CI, 76.8–94.8) and a NPV of 100% with Youden index of 0.96 for PCP diagnosis. Regarding the HIV group, the lower cut-off value of 1,480 DNA copies/μl showed the sensitivity of 100% (95% CI, 92.5–100%) and specificity of 91.7% (95% CI, 61.5–99.8) with PPV of 97.9% (95% CI, 87.8–99.7) and a NPV of 100% with Youden index of 0.92 for PCP diagnosis. The sensitivity and specificity of the upper cut-off value of 12,718 DNA copies/μl in this group were 97.9% (95%CI, 88.7–100) and 100% (95%CI, 73.5–100), respectively. The values above the upper cut-off point had a PPV of 100% (95% CI, N/A) and a NPV of 92.3% (95% CI, 63.3–98.8) with Youden index of 0.98 for PCP diagnosis in the HIV group.
Lymphatic filariasis (LF) is a neglected major tropical disease that is a leading cause of permanent and long-term disability worldwide. Significant progress made by the Global Programme to Eliminate Lymphatic Filariasis (GPELF) has led to a substantial decrease in the levels of infection. In this limitation, DNA detection of lymphatic filariae could be useful due to it capable of detecting low level of the parasites. In the present study, we developed a diagnostic assay that combines a miniPCR with a duplex lateral flow dipstick (DLFD). The PCR primers were designed based on the HhaI and SspI repetitive noncoding DNA sequences of Brugia malayi and Wuchereria bancrofti, respectively. The limits of detection and crossreactivity of the assay were evaluated. In addition, blood samples were provided by Thais living in a brugian filariasis endemic area. The miniPCR-DLFD assay exhibited a detection limit of 2 and 4 mf per milliliter (mL) of blood for B. malayi as well as W. bancrofti, respectively, and crossamplification was not observed with 11 other parasites. The result obtained from the present study was in accordance with the thick blood smear staining for the known cases. Thus, a miniPCR-DLFD is an alternative tool for the diagnosis of LF in point-of-collection settings with a modest cost (~USD 5) per sample.
Objectives To determine the performance of droplet digital polymerase chain reaction (ddPCR) assays in diagnosing Pneumocystis pneumonia (PCP), and to survey the sulfamethoxazole-trimethoprim (SMX-TMP) resistant mutations in our PCP cohort.Methods A prospective study was conducted from January 2017 to June 2018. Adult immunocompromised subjects with pneumonia were enrolled. Bronchoalveolar lavage fluid samples were obtained for standard microscopic testing and ddPCR to quantify the Pneumocystis MSG gene. DHPS and DHFR gene sequencings were performed to detect SMX-TMP resistance.Results Of 54 subjects, 12 had definite PCP, 7 had probable PCP, and 35 were non-PCP. In the PCP cohort, 10 (53%) had HIV infections. Using a cutoff value of ≥ 1.94 copies/μL, the ddPCR exhibited an overall sensitivity of 91.7% (61.5-99.8%) and specificity of 88.1% (74.4-96%). It showed a better performance when different cutoff values were used in subjects with HIV ( ≥ 1.80 copies/μL) and non-HIV ( ≥ 4.5 copies/μL). ROC curves demonstrated an AUC of 0.80 (95% CI, 0.56-1.0) for the HIV group, and 0.99 (95% CI, 0.95-1.0) for the non-HIV group. Of 16 PCP samples tested for DHPS -and DHFR -mutations, only DHPS mutations were detected (2). Most of the subjects, including those with DHPS mutations, demonstrated favorable outcomes.Conclusions The ddPCR exhibited a satisfactory diagnostic performance for PCP. Based on very limited data, the treatment outcomes of PCP did not seem to be affected by the DHPS mutations.
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