Dengue and Chikungunya virus co-infection in major metropolitan cities of provinces of Punjab and Khyber Pakhtunkhwa: A multi-center study.

Dengue has become endemic in Pakistan with annual recurrence. A sudden increase in the dengue cases was reported from Rawalpindi in 2016, while an outbreak occurred for the first time in Peshawar in 2017. Therefore, a multi-center study was carried out to determine the circulating dengue virus (DENV) serotypes and Chikungunya virus (CHIKV) co-infection in Lahore, Rawalpindi, and Peshawar cities in 2016-18. A hospital-based cross-sectional study was carried out in Lahore and Rawalpindi in 2016-18, while a community-based study was carried out in Peshawar in 2017. The study participants were tested for dengue NS1 antigen using an immunochromatographic device while anti-dengue IgM/IgG antibodies were detected by indirect ELISA. All NS1 positive samples were used for DENV serotyping using multiplex real-time PCR assay. Additionally, dengue samples were tested for CHIKV co-infection using IgM/IgG ELISA. A total of 6291 samples were collected among which 8.11% were NS1 positive while 2.5% were PCR positive. DENV-2 was the most common serotype (75.5%) detected, followed by DENV-1 in 16.1%, DENV-3 in 3.9% and DENV-4 in 0.7% while DENV-1 and DENV-4 concurrent infections were detected in 3.9% samples. DENV-1 was the predominant serotype (62.5%) detected from Lahore and Rawalpindi, while DENV-2 was the only serotype detected from Peshawar. Comorbidities resulted in a significant increase (p-value<0.001) in the duration of hospital stay of the patients. Type 2 diabetes mellitus substantially (p-value = 0.004) contributed to the severity of the disease. Among a total of 590 dengue positive samples, 11.8% were also positive for CHIKV co-infection. Co-circulation of multiple DENV serotypes and CHIKV infection in Pakistan is a worrisome situation demanding the urgent attention of the public health experts to strengthen vector surveillance.

Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis.

Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N-termini of the antigens PE35, tnPstS1 and FbpC1, all the fusion molecules were expressed at high levels in E. coli in a soluble form. ELISA analysis of the plasma samples of TB patients against HSPX-tnPstS1 showed 57.7% sensitivity which is nearly the same as the expected combined value obtained after deducting the number of plasma samples (32) containing the antibodies against both the individual antigens. Likewise, the 54.4% sensitivity of HSPX-PE35 was nearly the same as that expected from the combined values of the contributing antigens. Structural analysis of all the fusion molecules by CD spectroscopy showed that α-helical and ß-sheet contents were found close to those obtained through molecular modeling. Molecular modeling studies of HSPX-tnPstS1 and HSPX-PE35 support the analytical results as most of the epitopes of the contributing antigens were found to be available for binding to the corresponding antibodies. Using these fusion molecules in combination with other antigenic molecules should reduce the number of antigenic proteins required for a more reliable and economical serodiagnosis of tuberculosis. Also, HSPX seems to have potential application in soluble expression of heterologous proteins in E. coli.

Fusion of selected regions of mycobacterial antigens for enhancing sensitivity in serodiagnosis of tuberculosis.

Serodiagnosis of tuberculosis requires detection of antibodies against multiple antigens of Mycobacterium tuberculosis, because antibody profiles differ among the patients. Using fusion proteins with epitopes from two or more antigens would facilitate in the detection of multiple antibodies. Fusion constructs tn1FbpC1-tnPstS1 and tn2FbpC1-tnPstS1 were produced by linking truncated regions of variable lengths from FbpC1 to the N-terminus of the truncated PstS1. Similarly a truncated fragment of HSP was linked to the N-terminus of a truncated fragment from FbpC1 to produce tnHSP-tn1FbpC1. ELISA analysis of the plasma samples of TB patients against tn2FbpC1-tnPstS1 showed 72.2% sensitivity which is nearly the same as the expected combined value for the two individual antigens. However, the sensitivity of tn1FbpC1-tnPstS1 was lowered to 60%. tnHSP-tn1FbpC1 showed 67.7% sensitivity which is slightly less than the expected combined value for the two individual antigens, but still significantly higher than that of each of the individual antigen. Data for secondary structure analysis by CD spectrometry was in reasonable agreement with the X-ray crystallographic data of the native proteins and the predicted structure of the fusion proteins. Comparative molecular modeling suggests that the epitopes of the constituent proteins are better exposed in tn2FbpC1-tnPstS1 as compared to those in tn1FbpC1-tnPstS1. Therefore, removal of the N-terminal non-epitopic region of FbpC1 from 34-96 amino acids seems to have unmasked at least some of the epitopes, resulting in greater sensitivity. The high level of sensitivity of tn2FbpC1-tnPstS1 and tnHSP-tn1FbpC1, not reported before, shows that these fusion proteins have great potential for use in serodiagnosis of tuberculosis.

Improving sensitivity for serodiagnosis of tuberculosis using TB16.3-echA1 fusion protein.

This study aimed at developing and assessing the fusion proteins with enhanced sensitivity to detect antibodies in plasma as a diagnostic method for tuberculosis. DNA fragments encoding TB16.3 and echA1 gene regions corresponding to proteins TB16.3 and echA1 from Mycobacterium tuberculosis were amplified through PCR. Through a series of restrictions and ligations two novel fusion constructs TB16.3-echA1 and TB16.3-tnPstS1 were produced and expressed in Escherichia coli. These were screened for detection of antibodies in human plasma. The individual antigens TB16.3, echA1 and tnPstS1 and the fusion protein TB16.3-tnPstS1 and TB16.3-echA1 showed sensitivities of 29%, 25.5%, 42.8%, 40.0% and 47.2%, respectively. Lower sensitivity in case of TB16.3-tnPstS1 seems to be due to the structural arrangement between the two proteins, which is likely to mask several of their epitopes. The higher sensitivity of TB16.3-echA1 appears to be due to lesser interaction between the two proteins thus allowing free availability of epitopes for binding antibodies. 64% of TB patients were found positive for either one of the two fusion proteins TB16.3-echA1 and TB16.3-tnPstS1. This study indicates that the novel fusion protein TB16.3-echA1 has a potential in serodiagnosis of TB with improved sensitivity and reliability.

Demographic and clinico-epidemiological features of dengue fever in Faisalabad, Pakistan.

This cross-sectional study was carried out to explore the epidemiological and clinical features of dengue fever in Faisalabad, Pakistan during 2011 and 2012. During the study period, anti-dengue IgM positive cases were reported in the post-monsoon period during the months of August-December. Certain hotspots for the dengue infection were identified in the city that coincide with the clusters of densely populated urban regions of the city. Out of total 299 IgM positive patients (male 218 and female 81); there were 239 dengue fever (DF) and 60 dengue hemorrhagic fever (DHF) patients. There was decrease in the median age of dengue patients from 31 years in 2011 to 21.5 years in 2012 (p<0.001). Abdominal pain was seen in 35% DHF patients followed by nausea in 28.3%, epistaxis in 25% and rash in 20% patients (p<0.05). Patients reported to be suffering from high-grade fever for an average of 8.83 days in DHF as compared to 5.82 days in DF before being hospitalized. Co-morbidities were found to be risk factor for the development of DHF in dengue patients. Clinical and laboratory features of dengue cases studied could be used for the early identification of patients at risk of severe dengue fever.

Truncation of PstS1 antigen of Mycobacterium tuberculosis improves diagnostic efficiency.

PstS1, also named 38-kDa antigen, is one of the earliest known immune-dominant antigens of Mycobacterium tuberculosis and it has been commonly used in serodiagnostic tests. We constructed a truncated version, tnPstS1, by removing 96 and 14 amino acid residues from the N- and C-terminals, respectively of the native PstS1. The native and the truncated 29.5 kDa proteins were expressed in insoluble forms in Escherichia coli to levels of 15% and 25% of the total cell proteins, respectively. Both the variant molecules reacted equally well with the antisera raised in rabbit against the native protein. PstS1 and tnPstS1 were evaluated through ELISA against plasma samples from 160 culture positive tuberculosis patients and 40 healthy controls. With tnPstS1 43% of the patient samples were detected positive for the antibody as compared to only 36% in the case of the native PstS1. Data for the secondary structures of the native and the truncated variants as obtained by circular dichroism agreed with the known 3-D structure of the native protein and the predicted structure of the truncated version, respectively. The results show that the truncated tnPstS1 is more efficient as compared to the native PstS1 for use as a serodiagnostic agent.