Demographic and clinical features of study participants
The present study included 200 study subjects. Among them, 100 were suspected cases of PCP, and 100 were healthy controls. Among the suspected cases of PCP, 59% were males, and 41% were females, while among the healthy controls, 55% were males, and 45% were females. Two age group were determined based on the mean age of all study participant, <50 years of age group cases were 45% while >50 years of age group cases were 55% while in healthy controls <50 years of age group healthy controls were 48% while >50 years of the age group of healthy controls were 52%. As shown in Table 2, all the suspected cases had cough (100%), 92% had fever, 80% had weight loss, 76% had dyspnea, and 54% had night sweats, which was the most common clinical manifestations of PCP cases. 62% and 75% suspected cases of PCP had crept and wheezing, respectively. Chest X-ray showed multiple pulmonary findings, and the most common was pulmonary infiltrates (Figure 1).
Cytokine IL-2 mRNA expression
Increased mRNA expression of IL-2 (5.72 fold) was observed in suspected cases of PCP compared to healthy controls (Table 2). Significantly increased expression of IL-2 mRNA was associated with crept, wheezing, and chest X-ray findings. Patients who had a clinical feature like crept showed 6.30 fold IL-2 mRNA expression, while patients with no crept showed 4.68 fold IL-3 mRNA expression, and differences among them were found to be significant (p=0.0007). Patients finding like wheezing showed 6.30 fold IL-3 mRNA expression, while patients without wheezing showed 3.96 fold IL-2 mRNA expression (p=0.001). Patient’s chest X-ray findings showed central perihilar infiltrates (7.59 fold), patchy infiltrates (9.98 fold), consolidation (5.73 fold), hilar lymphadenopathy (6.62 fold), pneumothorax (5.28 fold), pleural effusion (6.54 fold) showed higher expression of IL-2 mRNA expression compared to those patients did not show any such findings in X-ray (4.61 fold), and differences among them were found to be statistically significant (p<0.0001).
Cytokine IL-4 mRNA expression
IL-4 mRNA expression was 4.35 fold higher among the cases compared to healthy controls (Table 2). Higher expression of IL-4 mRNA was found to be significantly associated with the weight loss (p=0.002), dyspnea (p=0.003), crept (p=0.01), and chest X-ray findings (p<0.0001). Cases with weight loss, dyspnea, crept had 4.77 fold, 4.94 fold, 5.0 fold IL-4 mRNA expression, while cases with no weight loss, dyspnea, crept showed 2.66 fold, 2.49 fold, 3.28 fold IL-4 mRNA expression, respectively, which is lower compared to counterpart. Cases with different findings in X-rays such as central perihilar infiltrates, patchy infiltrates, consolidation, hilar lymphadenopathy, pneumothorax, pleural effusion showed 7.55 fold, 3.46 fold, 2.94 fold, 5.87 fold, 1.08 fold, 14.22 fold, respectively.
Cytokine IL-10 mRNA expression
Overall, 3.77 fold increased IL-10 mRNA expression was observed among the cases compared to healthy controls (Table 2). Significantly higher expression of IL-10 mRNA was found to be associated with weight loss (p=0.02), dyspnea (p=0.01), night sweats (p=0.006), wheezing (p=0.007), and different findings of chest X-ray (p<0.0001). Patients who had weight loss had 3.99 fold IL-10 mRNA expression, dyspnea had 4.15 fold IL-10 mRNA expression, night sweats had 4.62 fold IL-10 mRNA expression, and wheezing had 4.06 fold IL-10 mRNA which is higher compared to counterpart. Patient’s chest X-ray findings such as central perihilar infiltrates, patchy infiltrates, consolidation, hilar lymphadenopathy had higher IL-10 mRNA expression comparatively other findings.
Cytokine IL-13 mRNA expression
Increased mRNA expression of IL-13 (7.59 fold) was observed among the cases compared to healthy controls (Table 2). A significant association of IL-13 mRNA was observed with cases with fever (p=0.02). Cases with fever showed 7.88 fold higher IL-13 mRNA expressions, while cases without fever showed 4.34 fold IL-13 mRNA expression while no such association was observed with other clinical findings.
PCP detection by IFAT and cytokines mRNA expression
Suspected cases of PCP were detected by the IFAT stain, and 35% were observed to be positive, and 65% were negative (Figure 2). All the 35% positive cases showed 7.78 fold IL-2 mRNA expression, 5.51 fold IL-10 mRNA expression, 6.08 fold mRNA expression, and 8.78 fold IL-13 mRNA expression. In comparison, 65% of negative cases showed 4.61 fold IL-2 mRNA expression, 2.83 fold IL-10 mRNA expression, 3.41 fold mRNA expression, and 6.95 fold IL-13 mRNA expression, respectively lower compared to its counterpart and differences among cytokines were found to be significant except IL-13 mRNA expression.
PCP detection by GMSS and cytokines mRNA expression
GMSS stain showed 15 (42.86%) positive cases out of 35 positive cases confirmed by IFAT for PCP, and others were negative (Figure 3). Cytokines mRNA expression such as IL-2 (p=0.001) and IL-10 (p=0.004) showed a significant difference among the groups while IL-4 and IL-13 did not show any significant difference. PCP positive cases by GMSS stain showed 8.37 fold IL-2 mRNA expressions, 5.56 fold IL-10 mRNA expression, while negative cases showed 5.25 fold IL-2 mRNA expressions, 3.45 fold IL-10 mRNA expression. Positive cases by GMSS had 5.23 fold IL-4 mRNA expression, 8.43 fold IL-13 mRNA expression, while negative cases for PCP by GMSS had 4.19 fold IL-4 mRNA expression and 7.45 fold IL-13 mRNA expression.
PCP detection by Giemsa and cytokines mRNA expression
Giemsa stain showed 8 (17.6%) positive cases out of 35 positive cases confirmed by IFAT for PCP, and others were negative (Figure 4). No such significant differences were reported in cytokines mRNA expression among the groups except IL-4 mRNA expression (p=0.04). Positive cases for PCP by Giemsa stain showed 6.21 fold IL-4 mRNA expression, while negative patients showed 4.16 fold IL-4 mRNA expression.
PCP detection by TBO and cytokines mRNA expression
TBO stain showed 1 (2.8%) positive cases out of 35 positive cases confirmed by IFAT for PCP, and others were negative (Figure 5). No significant differences were found in the expression of IL-2, IL-4, IL-10, and IL-13 mRNA expression between the positive and negative detected cases of PCP.
PCP detection by RT-PCR and cytokines mRNA expression
Detection of PCP was done by RT-PCR method, 39 (39%) of patients have PCP, and 59% suspected cases were negative for PCP (Figure 5). PCP confirmed cases showed significantly higher Th1 (IL-2 and IL-10) and Th2 (IL-4 and IL-13) cytokines mRNA expression compared to negative cases. PCP positive cases showed 7.86 fold IL-2, 5.15 fold IL-10, 5.89 fold IL-4, 8.77 fold IL-13 mRNA expression while negatives cases showed 4.38 fold IL-2, 2.89 fold IL-10, 3.36 fold IL-4, 6.84 fold IL-13 mRNA expression and differences among them was found to be significant (p<0.0001, p<0.0001, p<0.0001, p=0.04), respectively.