|Year : 2020 | Volume
| Issue : 3 | Page : 338-343
Chlamydia pneumoniae infection in patients hospitalised for community-acquired pneumonia in Southern Jordan
Tahany Saleem Al-Hajaya1, Wael Ali Al-Zereini1, Hesham Mahmoud Al-Younes2
1 Department of Biological Sciences, Faculty of Science, Mutah University, Al-Karak, Jordan
2 Department of Biological Sciences, Faculty of Science, The University of Jordan, Amman, Jordan
|Date of Submission||15-Feb-2020|
|Date of Decision||20-Jun-2020|
|Date of Acceptance||30-Jul-2020|
|Date of Web Publication||4-Nov-2020|
Dr. Wael Ali Al-Zereini
Department of Biological Sciences, Faculty of Science, Mutah University, Al-Karak 61710
Source of Support: None, Conflict of Interest: None
Purpose: Chlamydia pneumoniae is associated with human respiratory diseases. Few reports examined the involvement of this bacterium in community-acquired pneumonia (CAP) in Jordan. This study investigates for the first time the role of C. pneumoniae in the establishment of CAP among nationals residing the southern part of Jordan. Materials and Methods: Nasopharyngeal and sera samples were collected from 70 hospitalised CAP patients and 63 healthy controls from Al-Karak Governorate and examined using the microimmunofluorescence and polymerase chain reaction techniques. The overall prevalence of C. pneumoniae infection was estimated by detecting the chlamydial immunoglobulin G (IgG) antibodies at a titre of 1:16. Rate of acute infection was estimated by detecting chlamydial DNA in nasopharyngeal samples and IgG and IgM at titres of 1:512 and 1:16, respectively. Results: A higher overall seroprevalence of C. pneumoniae IgG was detected in CAP patients than controls (44.3% vs. 30.2%). The rate of acute infection in the entire CAP patients, based on IgG titre of 1:512, was 7.14% compared to 1.58% in the controls. Approximately, three-fold increase in the rate of acute infection was observed in CAP cases, seropositive at IgG titre of 1:16, compared to seropositive controls (16.1% vs. 5.3%). Interestingly, chlamydial IgM antibodies were detectable in 27.1% compared to only 3.2% of the controls. The presence of chlamydial nucleic acids was confirmed in 40% of CAP patients and in 7.9% of controls. Conclusions: The present findings clearly suggest a role of C. pneumoniae in the aetiology of CAP in Southern Jordan. However, coinfections with other respiratory pathogens should not be excluded.
Keywords: Chlamydia pneumoniae, community-acquired pneumonia, Jordan, microimmunofluorescence, polymerase chain reaction
|How to cite this article:|
Al-Hajaya TS, Al-Zereini WA, Al-Younes HM. Chlamydia pneumoniae infection in patients hospitalised for community-acquired pneumonia in Southern Jordan. Indian J Med Microbiol 2020;38:338-43
|How to cite this URL:|
Al-Hajaya TS, Al-Zereini WA, Al-Younes HM. Chlamydia pneumoniae infection in patients hospitalised for community-acquired pneumonia in Southern Jordan. Indian J Med Microbiol [serial online] 2020 [cited 2020 Nov 28];38:338-43. Available from: https://www.ijmm.org/text.asp?2020/38/3/338/299847
| ~ Introduction|| |
Chlamydia pneumoniae, a primarily human pathogen, is associated with many respiratory and non-respiratory diseases, including community-acquired pneumonia (CAP), bronchitis, chronic obstructive pulmonary disease and atherosclerosis.,,, In general, anti-C. pneumoniae immunoglobulin G (IgG) seropositivity was demonstrated in 40%–86% of apparently healthy adults.,, Previous reports indicated that this pathogen could be responsible for 1%–22% of CAP cases worldwide., The incidence of C. pneumoniae-associated CAP is two- to five-folds less in adult patients younger than 65 years compared to persons ageing 65 years or older, males being more vulnerable than females to infection.
To the best of our knowledge, few investigations have analysed the seroprevalence and the medical significance of C. pneumoniae in the Jordanian population. In 2009, a research demonstrated that approximately 61% of apparently healthy male adults were IgG seropositive to C. pneumoniae. In a subsequent population-based study, an IgG detection rate of 54.9% was observed in the individuals of both genders ageing between 2 and 86 years. On the other hand, reports investigating the C. pneumoniae role in human diseases, particularly CAP, bronchitis and atherosclerosis focused on Jordanians inhabiting the central and northern parts of Jordan.,, However, in Southern Jordan, the overall C. pneumoniae seroprevalence and the involvement of this pathogen in causing human diseases have not yet been verified. Therefore, this study aimed at unveiling the possible causal role of C. pneumoniae in CAP based on the detection of the bacterial DNA and C. pneumoniae-specific IgG and IgM antibodies in clinical samples collected from hospitalised CAP adults inhabiting Southern Jordan, particularly Al-Karak Governorate.
| ~ Materials and Methods|| |
This study was performed during the period from March 2014 to May 2015 and included 133 individuals (63 controls and 70 patients). Patients were hospitalised at Al-Karak Governmental Hospital and Prince Ali Bin Al-Hussein Military Hospital, Al-Karak, Jordan, with a clinical and radiological diagnosis of CAP. Patients included 36 males and 34 females and aged between 17 and 90 years (mean age 47.3 ± 25.5 years). Demographics, clinical data and co-morbidities were documented. Controls were apparently healthy hospital workers, laboratory personnel at Al-Karak Governmental Hospital and blood donors. The control group included 33 males and 30 females with ages ranging from 17 to 85 years (mean age 34.5 ± 13.9 years). When possible, controls were age- and sex-matched with patients. Participating controls did not suffer from respiratory diseases and did not take antibiotics during the past 3 months before enrolment in this study. Controls and CAP cases were divided into four age groups: 17–32 years, 33–48 years, 49–64 years and ≥65 years.
The study was approved by the Scientific Research Committees at the Department of Biological Sciences and the School of Post-Graduate Studies and the Scientific Ethics Committee at the Department of Medicine, Mutah University, Al-Karak. Before enrollment, a signed informed consent was obtained from the participants.
Collection and transport of specimens
Clinical specimens from CAP patients were collected within 48 h of admission. Venous blood samples (3–4 ml each) from CAP cases and controls were withdrawn into gel-containing plain tubes (AMPulab™, Düsseldorf, Germany), left to clot at the room temperature and then centrifuged at 3,500 rpm for 10 min (Combi-514R, Hanil, South Korea). Sera were then transferred into sterile Eppendorf tubes and frozen at −20°C until tested for the presence of C. pneumoniae IgM and IgG antibodies. Moreover, nasopharyngeal samples from the participants were obtained using sterile Dacron-tipped swabs that were immediately placed in 1 ml transport medium-containing tubes (Vircell MICROBIOLOGISTS, Santa Fe, Spain) and then stored at −70°C until analysed for the presence of chlamydial DNA.
A previously described indirect microimmunofluorescence test (MIF) was followed to detect human C. pneumoniae-specific IgG antibodies using fluorescein-isothiocyanate-labelled goat anti-human IgG (Bio-Rad, USA). For the detection of IgM antibodies in the collected sera, Chlamydophila pneumoniae IFA IgM kit (Vircell MICROBIOLOGISTS, Sana Fe, Spain) was used following the manufacturer's instructions. The overall prevalence of infection was estimated by screening IgG antibodies at a titre of 1:16. Sera reactive at this dilution was further tested at serial two-fold dilutions from 1:16 to1:512 for IgG antibody endpoint titres determination. C. pneumoniae-specific IgM antibodies in sera were screened at a titre of 1:16.
Nucleic acid extraction and polymerase chain reaction assay
DNA was extracted from the collected nasopharyngeal sample using the commercially available G-spin total DNA extraction kit (iNtRON Biotechnology, Gyeonggi-do, Korea). The polymerase chain reaction (PCR) assay targeting a fragment within C. pneumoniae 16S rRNA was performed. Forward and reverse oligonucleotide primers were 5'-TGACAACTGTAGAAATACAGC-3' and 5'-CGCCTCTCTCCTATAAAT-3', respectively (Midland Company Inc., Midland, USA). The amplification reaction mixture contained 500 ng of extracted DNA, 10 μl of 2x PCR master mix solution (i-Max II, iNtRON Biotechnology), 1.5 μl of each forward and reverse primer (10 pM/μl). Nuclease-free water was added to each reaction mixture to have a total volume of 20 μl. The cycling conditions were as follows: Denaturation for 5 min at 95°C, followed by 45 cycles, each of which consisted of a denaturation step for 1 min at 95°C, annealing for 1 min at 50°C, extension for 1 min at 72°C and a final elongation step for 7 min at 72°C. In every PCR run, negative (nuclease-free water) and positive (heat-lysed C. pneumoniae) controls were added instead of the DNA harvested from the clinical specimens. The presence of an amplicon size of 463 base pairs, analysed by agarose gel electrophoresis, was considered indicative of infection with the pathogen.
Criteria for etiological diagnosis
The IgG titre of 1:16 was considered a serological marker for C. pneumoniae positivity.,, A chlamydial IgG antibody titre of 1:16–1:256 was indicative of past-infection. The detection of IgG specific to the bacterium at a titre of 1:512 or IgM at a titre of 1:16 provides serological evidence for acute (current or recent) infection. Similarly, detection of chlamydial DNA in nasopharyngeal samples was indicative of acute infection.,
For data analysis, the Statistical Package for the Social Sciences software, version 14.0.1 (SPSS Inc., Chicago, IL, USA) was used. Calculation of the specificity, sensitivity, positive predictive value (PPV), and negative predictive value (NPV) was performed for clinically diagnosed CAP cases by MIF tests and PCR with acute infection. Data were statistically analysed using the Chi-squared test and Fisher's exact test. A P < 0.05 is considered statistically significant.
| ~ Results|| |
Medical profiles and sociodemographic data of enrolled individuals
A total of 70 hospitalised CAP cases (36 males and 34 females; mean age 47.3 ± 25.5 years) in addition to 63 apparently healthy controls (33 males and 30 females; mean age 34.5 ± 13.9 years) from Al-Karak Governorate, Southern Jordan, were included in this study. A significant difference between the ages of CAP patients and controls was detected (P = 0.004). Moreover, the differences between the numbers of individuals of each sex in both control and patient groups were insignificant (P = 0.94), indicating almost sex matching between controls and patients.
Among CAP patients, the frequency of fever was 82.9%, whereas cough was observed in 84.3% and 48.6% had sputum production. Approximately, 20%, 51.4% and 48.6% of CAP patients were suffering from rigors, dyspnea and chest pain, respectively. The radiographic findings showed pulmonary infiltrates in about 84.3% of CAP cases.
Overall seroprevalence of Chlamydia pneumoniae in controls and community-acquired pneumonia cases
The general prevalence of C. pneumoniae in CAP patients and controls was assessed at IgG antibody titre of 1:16. A significant increased detection rate (P = 0.03) of C. pneumoniae was observed in CAP patients (31/70; 44.3%) than in healthy controls (19/63; 30.2%).
Age-related prevalence of Chlamydia pneumoniae immunoglobulin G in seropositive individuals
The overall age-specific seropositives of C. pneumoniae IgG were estimated at a titre of 1:16 [Table 1]. In the control cohort, the overall prevalence was the lowest in the age group of 17–32 years (12.8%) and increased considerably in the two subsequent age brackets to reach a peak of 71.4% in the age group of 49–64 years. In the oldest controls (≥65 years), one out of two individuals (50%) was IgG seropositive. In contrast, IgG detection rates in CAP patients younger than 49 years were relatively low (<20%) and increased dramatically in the subsequent age groups to reach a peak of 76.2% in the oldest CAP patients [Table 1].
|Table 1: Age-based distribution of anti-Chlamydia pneumoniae IgG antibodies (at a titer of 1:16) in patients and controls|
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Determination of immunoglobulin G endpoint titres in seropositive community-acquired pneumonia and control individuals
To estimate the endpoint titres of C. pneumoniae-specific IgG, sera reactive at 1:16 in both control and patient groups were serially two-fold diluted to a titre of 1:512 and further examined by the IgG MIF assay. As shown in [Figure 1], most of seropositive controls (13/19; 68.4%) had 1:16 as the highest endpoint level, whereas <10% (3/31) of the seropositive CAP cases had the same IgG titre (P < 0.001). Percentages of control subjects with IgG endpoint titres higher than 1:16 sharply decreased and reached about 10% at the titre of 1:32 and about 5% at each of the remaining higher titres. In CAP patients, the number of individuals with detectable IgG gradually increased at titres of 1:32, 1:64 and 1:128 and then sharply decreased at titres of 1:256 and 1:512. Interestingly, the percentage of CAP patients with an endpoint titre of 1:128 reached 35.5%, which was 6.7-folds more than that in the control group demonstrating the same endpoint level (P < 0.001). Clearly, percentage of patients with IgG endpoint titre of 1:512, indicative of acute infection, was higher than that in the control group (16.1% vs. 5.3%; P = 0.01).
|Figure 1: Distribution of Chlamydia pneumoniae immunoglobulin G endpoint titres in sera of 31 community-acquired pneumonia patients and 19 control subjects that showed seropositivity by microimmunofluorescence; *P = 0.01, **P < 0.001|
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Immunoglobulin G-based evaluation of acute Chlamydia pneumoniae infection in study groups
Evaluation of acute C. pneumoniae infection at IgG titre of 1:512 revealed about three-fold increase in the rate of acute infection in CAP cases who were positive at IgG titre 1:16, compared to that in seropositive controls (16.1% vs. 5.3%). The etiologic role of C. pneumoniae in CAP was further determined by assessing the overall prevalence of C. pneumoniae acute infection in the entire populations of controls and CAP cases. Interestingly, the difference in the detection rate of acute infection in CAP patients and the control group was statistically insignificant (P = 0.08). Approximately, 7.1% of CAP patients (5/70) had an IgG titre of 1:512, indicative of acute infection, whereas only 1.58% of the control population (1/63) had the high IgG titre.
Anti-Chlamydia pneumoniae specific immunoglobulin M antibody in controls and CAP cases
The anti-C. pneumoniae IgM antibodies at a dilution of 1:16 is considered here as a criterion for the definition of acute infection. IgM antibodies were significantly detected in 27.1% (19/70) of CAP patients compared to only 3.2% (2/63) of the controls (P<0.001). Overall, the current findings reveal that more than one quarter of CAP patients had recent infection based on IgM seropositivity, strongly suggesting a role of C. pneumoniae in the etiology of CAP in Southern Jordan. In addition, these data also strongly suggest that IgM MIF is more credible in detecting C. pneumoniae acute infection, when compared to IgG MIF results shown above.
Detection rate of Chlamydia pneumoniae by polymerase chain reaction
Nasopharyngeal samples from the study individuals were analysed for the presence of C. pneumoniae DNA using the standard PCR. Intriguingly, a five-fold elevation in PCR positivity was detected in specimens from patients (28/70, 40%), compared to those from controls (5/63, 7.9%). This difference was statistically significant (P< 0.001). Collectively, these results corroborate the findings of IgM that strongly link between C. pneumoniae and CAP in Southern Jordan.
Age distribution of seropositive individuals with acute Chlamydia pneumoniae infection based on immunoglobulin G, immunoglobulin M, and on polymerase chain reaction findings
Here, age distribution of acute infection was analysed based on IgG positivity at a titre of 1:512, IgM positivity at a titre of 1:16, and DNA positivity. None of the patients within the youngest age group had IgG serological marker for an acute infection [Table 2]. However, IgG-based detection of acute infection was observed in other age groups of CAP patients with highest prevalence (25%) detected in the age bracket 49–64 years [Table 2]. In comparison, none of the participants within the control age groups had detectable IgG serological sign of acute infection, except one person out of seven in the age group of 49–64 years.
|Table 2: Age distribution of anti-Chlamydia pneumoniae IgG (at a titre of 512), IgM (at a titre of 1:16) antibodies and nasopharyngeal polymerase chain reaction positivity for the determination of acute infection in patients and controls|
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As shown in [Table 2], IgM positivity was detected in all age brackets of CAP cases with the lowest IgM seroprevalence in individuals aged between 17 and 32 years (9.5%) and the highest IgM detection rates in the age intervals 33–48 years (50%) and 49–64 years (41.7%). In the cohort of controls, C. pneumoniae-specific IgM was not detected in individuals belonging to the age groups of 17–32 years and ≥65 years. Whereas, only one individual with IgM positivity was found in each of the remaining age groups of controls.
Intriguingly, significant chlamydial nucleic acid positivity, ranging from 33.3–58.3%, were confirmed in all age groups of CAP patients, compared to the respective age groups in the control cohort (P < 0.001) [Table 2]. However, chlamydial DNA was detected in only two control age groups. Approximately, 10.2% of controls within the age group of 17–32 years in addition to 14.3% of participants with ages ranged from 49 to 64 years were DNA positive.
The comparison between IgG and IgM MIF and PCR for the estimation of C. pneumoniae acute infection and correlation of serological and molecular finding.
Out of 70 CAP cases, five individuals had IgG titre of 1:512 and 19 demonstrated IgM titre of 1:16 using MIF tests, whereas 28 of the entire patient population had the pathogen DNA in their nasopharyngeal samples [Table 3]. In comparison, one, two and five out of 63 healthy controls showed evidence of acute infection by IgG MIF, IgM MIF and PCR, respectively. Taken together, these data show a poor correlation of PCR and IgM findings with those of IgG MIF. Moreover, PPV, NPV, specificities and sensitivities of MIF and PCR were calculated for the diagnosis of acute infection using clinically diagnosed CAP cases as the gold standard. As shown in [Table 3], IgG MIF had low sensitivity in detecting acute infection (7.14%), compared to those for IgM MIF (27.1%) and PCR (40%). However, specificities for IgG and IgM MIFs were high and almost comparable (98.41% and 96.83%, respectively), while specificity for PCR in detecting acute infection was slightly lower (92.06%). Moreover, IgG MIF and PCR had PPV of 83.33% and 84.85%, respectively, values less than that for IgM MIF (90.48%).
|Table 3: Sensitivity, specificity, positive predictive value and negative predictive value of the serological assays (IgG and IgM microimmunofluorescence tests) and polymerase chain reaction for the determination of acute Chlamydia pneumoniae infection in patients suffering from community-acquired pneumonia|
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Considering the distribution of IgM- and PCR-positive controls and CAP cases based on their IgG serological findings, five (12.8%) and 17 (43.6%) out of 39 CAP IgG-negative patients were positive by IgM MIF and PCR, respectively. Whereas, out of the 44 controls with IgG negative sera, none was IgM positive and only four (8.9%) was PCR-positive. Twenty six CAP cases had moderate C. pneumoniae IgG titres (1:16-1:256). Eleven of them (42.3%) were IgM-seropositive and nine were reactive in PCR (34.6%). In contrast, only two controls with IgG seropositivity at titres ranging from 1:16-1:256 were positive either in IgM MIF or PCR. Sixty percent and 40% of CAP patients who had an IgG titre of 1:512 were positive by IgM MIF and PCR, respectively, whereas the single control individual that was IgG positive at 1:512 was IgM and PCR-negative.
| ~ Discussion|| |
Literature available from Jordan focused on the role of C. pneumoniae in CAP in residents of the Central and Northern Jordan.,,,, Therefore, this study was initiated to investigate, for the first time, the overall prevalence and the causal role of C. pneumoniae in CAP in Jordanians residing Southern Jordan (Al-Karak Governorate). Herein, infection prevalence in CAP patients, determined at an IgG titer of 1:16, was detected at a lower rate (44.3%) than that reported in adult patients from the central part of Jordan (70%) and in other countries (50%–70%)., Comparably, the overall IgG prevalence estimated here for healthy controls (30.2%) is also lower than previously estimated rates in healthy controls from Jordan (61%–62%), and other countries (40%–86%).,, Nevertheless, the increased overall IgG positivity in CAP population, noticed here, compared to healthy controls indicated a possible association between C. pneumoniae and CAP in Southern area of Jordan.
Epidemiological investigations have suggested that the detection of chlamydial IgG at a titre of ≥1:512 or IgM at a titre of ≥1:16 using single serum sample,,,, or four-fold rise in IgG titre using paired sera are the most convincing markers for acute infection. PCR is the second most widely used technique for the detection of acute infection after MIF. All of these diagnostic criteria, except seroconversion, were implemented here to screen for acute infection in the patient and control cohorts. Collectively, the MIF- and PCR-based results obtained here clearly demonstrated a higher rate of C. pneumoniae acute infection in CAP cases than controls, strongly suggesting a causal link between C. pneumoniae and CAP in Southern Jordan.
In a study conducted in the Northern Jordan, C. pneumoniae IgM was detected in 14% of children and 23% of adults hospitalised for CAP. Another report found C. pneumoniae DNA in 4.5% of Jordanian children diagnosed with acute respiratory infection. In contrast, C. pneumoniae DNA was not detected by multiplex PCR in throats and nasopharynxes of Jordanian children <2 years old and hospitalised with acute respiratory disease (Asem Shehabi, personal communication, 2014). Of note, all of these earlier studies lacked control participants and did not analyse C. pneumoniae IgG antibody. Assessed by an IgG titer of 1:512 in a case–control study, higher acute infection prevalence was noticed in the entire CAP (16.3%) and control (5.5%) populations, compared to only 7.14% of CAP patients and 1.58% of controls enrolled in the present study. The same study analysed the IgG titre of 1:512 in participants that were seropositive at an IgG of 1:16 and found that 25% and 8.8% of seropositive CAP cases and controls, respectively, had acute infection, compared to 16.1% of seropositive CAP cases and 5.3% of seropositive controls examined here. These differences could be due to the variations in study participants examined, population densities and geographic locations.
The overall 7.14% detection rate of acute infection obtained here based on the IgG titre of 1:512, correlates well with the available epidemiological findings indicating that C. pneumoniae accounts for 1%–22% of CAP cases worldwide., However, the frequency of acute infection observed here based on IgM and PCR exceeded these epidemiological data and reached 27.1% and 40%, respectively. The relatively vast difference between the rate of acute infection obtained by IgG MIF and that obtained by IgM MIF or PCR could be attributed to the lack of sensitivity of IgG MIF using a single serum sample. Importantly, the Centres for the Disease Control and Prevention recommended the use of a second (convalescent) serum sample to detect a four-fold increase in the IgG level as evidence for acute infection. Obtaining paired sera from the study participants was technically impossible.
IgM-based detection of C. pneumoniae acute infection in CAP patients was almost analogous to that reported in other countries which ranged from 2.4% to 30%, and to an uncontrolled study in Northern Jordan that reported acute infection in 23% of hospitalised CAP adults. Here, IgM positivity was significantly 8.6-fold higher in CAP cases than that in the control cohort (27.1% vs. 3.2%, P < 0.001), strongly suggesting a potential role of this pathogen in CAP establishment in Southern Jordan. Intriguingly, PCR used in this study seems to be the most sensitive, as it led to the significant detection of chlamydial DNA in nasopharyngeal samples collected from CAP patients, compared to controls (40% vs. 7.9%, P < 0.001). This finding is contradictory to that reported previously, where nested PCR assay failed to correlate C. pneumoniae with CAP aetiology. These inconsistent results may reflect the differences in examined populations, inconsistencies of PCR assays, differences in sample collection and processing and DNA extraction.
The general lack of correlation between IgG results and those for PCR and IgM in patients indicates that IgG MIF is insensitive with a single serological specimen, as enough time should elapse after exposure to pathogens for an IgG response to generate. Thus, if a second serum samples had been obtained, these results might have been improved. Not surprisingly, large percentage of CAP patients positive at IgG titres of 1:16–1:256 was PCR-negative, indicating past-infection or presence of mixed infection in these cases. Furthermore, three patients who showed IgG positivity at 1:512 were PCR-negative, which could be partly explained by the unsuccessful collection of nasopharyngeal specimens with optimal cellular material.
Despite the high specificities of used assays in detecting acute C. pneumoniae infection (>90%), they unfortunately exhibited weak to moderate sensitivities (7.14% for IgG MIF, 27.1% for IgM MIF and 40% for PCR) [Table 3]. IgM MIF was more sensitive in detecting acute infection than IgG MIF with comparable specificity and higher PPV and NPV values. Clearly, PCR assay showed a slight improvement in the sensitivity for the diagnosis of acute infection but had misleading negative results in CAP patients (low PPV value).
| ~ Conclusions|| |
This study combined three diagnostic methods to analyse for the first time the overall prevalence and the causal link between C. pneumoniae and CAP in nationals inhabiting the Southern part of Jordan, specifically Al-Karak Governorate. In general, used assays were all successful in providing evidence that C. pneumoniae may play a role in the establishment of CAP in the community of Southern Jordan.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]