Evaluation of PCR-based approach for serotype determination of Streptococcus pneumoniae

Determination of Streptococcus pneumoniae serotypes is essential for epidemiological surveillance. Therefore accurate, reliable and cost effective serotyping method is crucial. In this study, we determined the serotypes of 41 pneumococcal isolates recovered from human anterior nares by multiplex Polymerase Chain Reaction (PCR) utilizing published primers. The data was then compared with conventional serology using latex agglutination (LA) and the Quellung reaction. Based on the PCR-approach, 8 different serogroups/serotypes were detected with one isolate classified as non-typeable (cpsA- negative). In reference to the serology-based data, the results were in agreement except for one isolate. For the latter isolate, the LA and Quellung tests failed to show a reaction but the PCR-approach and sequencing identified the isolate as serogroup 15B/C. Based on this experimental setting, we found that the PCR-approach for pneumococcal serotypes determination is reliable to serve as the alternative for determining the pneumococcal serotyping

Association between human cytomegalovirus related factors and development of the disease in renal and bone marrow transplant recipients in a tertiary hospital, Malaysia

Human cytomegalovirus (HCMV) infection is known to be a major infectious complication after transplantation which associated with significant morbidity and mortality in solid organ and bone marrow transplant recipients. We studied the viral factors of HCMV and correlate results with the development of HCMV disease. This aim of this study is to detect HCMV, their genotypes and co-infection with other herpesviruses namely Epstein-Barr virus (EBV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7) in post-solid organ and bone marrow transplant recipients and to correlate them with the clinical presentation and outcome of HCMV disease. In this study, 100 blood samples from renal transplant recipients and 100 bone marrow transplant recipients in Kuala Lumpur Hospital were included. All tests were carried out by real time polymerase chain reaction (qPCR). HCMV were detected in higher incidence compared to other herpes virus indicating that the virus was the most common virus infecting the immunosuppressed patients. The results revealed that the incidence of HCMV infection were 78% and 63% in renal and bone marrow transplant recipients respectively. We found that patients with high viral load show symptoms of HCMV disease, whereby fever being most common symptom. As Malaysia has multi-races citizens, we also demonstrate the incidence of HCMV infection among renal and bone marrow transplant recipients by ethnicity namely Malay, Chinese, Indian and other minority races as ‘others’. In renal transplant recipients, there was no significant difference between the ethnic. Nevertheless, we found that there was a significant HCMV positivity among races in bone marrow transplant recipients where Malays were the most infected. One of the pathogenesis of HCMV depends on the genes encoding envelope glycoprotein that associated with different clinical outcomes. Reactivation of latent viral infection by HCMV and other herpesviruses results in active viral infection after organ transplantation and may cause complications. HCMV genotyping analysis revealed that all three HCMV gB, gH and gN genotypes were presence in the population where gB1 strain being the most common gene detected in both renal (100%) and bone marrow (100%) transplant recipients. Mix infection by more than one HCMV genotypes was also detected with various percentages with the gB+gH+gN combination was the least type of mix infection. We also found that recipient with high HCMV viral load (>5,000 copies/mL) has increased risk of developing HCMV disease. No statistically significant difference was found between type of genotypes and the manifestation of HCMV disease (p>0.05). Co-infection with other herpesviruses with HCMV disease was significant in bone marrow transplant recipients but not significant in renal transplant recipient

Prevalence of human cytomegalovirus disease and its related factors in renal and bone marrow transplant recipients in a tertiary hospital, Malaysia

Human cytomegalovirus (HCMV) infection may cause substantial morbidity and mortality after renal and bone marrow transplantation [1]. There are 3 major consequences of HCMV infection: HCMV disease with a wide range of clinical illnesses; superinfection with opportunistic pathogens; and injury to the transplanted organ [2]. Other than serological method to diagnose HCMV infection, viral load quantitation by real time polymerase chain reaction has been widely appreciated to diagnose and monitor the progress of viral infections. The aims of this study were to determine the incidence of HCMV infection in renal and bone marrow transplant recipients and to investigate its associations with HCMV disease, gender, and races.
 This retrospective cohort analysis involved 1520 blood samples from transplant recipients (renal, n = 164 and bone marrow, n = 182) from January 2020 to December 2021 collected from Virology Unit, Hospital Kuala Lumpur (NMRR ethical approval: NMRR-20-993-53201(IIR). The samples were analysed with quantitative polymerase chain reaction for HCMV DNA and the demographic, clinical and paraclinical aspects were evaluated. HCMV infection was present if the patient had positive HCMV viraemia and HCMV disease was diagnosed if HCMV infection was followed by clinical signs and symptoms. Statistical comparisons of patient demographics were performed with Chi-square tests for the categorical variables.
 The overall incidence of HCMV infection in the study group was 65% (225/346) where renal and bone marrow transplants account for 78.2% (176/225) and 21.7% (49/225) respectively. The incidence of HCMV infection in renal transplantation differed significantly by sex (p<0.05) where it was higher in males (71.8%) than in females (28.2%) but there was not statistically significant by sex in bone marrow transplantation in which males and females account for 61.2% and 38.7% respectively.  The incidence of HCMV differed significantly (p<0.05) by races in both transplantation types as follows: 58% in Malay, 36% in Chinese, 5% in Indian and 1% in other indigenous races in renal transplantation while 59% in Malay, 29% in Chinese, 10% in Indian and 2% in other indigenous races in bone marrow transplantation.  The incidence of HCMV disease differed significantly (p<0.05) by type of transplantation where it is higher in renal transplantation (30.9%) than in bone marrow transplantation (20.2%). The most seen symptoms were fever, generalised lethargy, and headache. Viral load of HCMV has been shown to be a major determinant factor in the severity and the manifestation of the HCMV infection[3].
 It is significantly higher in patients who develop HCMV disease[3]. Various risk factors have been described for the progress of symptomatic HCMV infection in organ transplant recipient[3]. The incidence of HCMV infection was higher in renal transplant as compared to bone marrow transplant among Malaysian. This study has shown that HCMV viral load has a significant association with age, gender and HCMV disease. Various syndromes can be caused by HCMV ranging from a mild fever to severe end-organ diseases. Treatment with anti-HCMV therapy results in decline in HCMV load, usually to undetectable

Comparative Study of Wheatley’s Trichrome Stain and In-vitro Culture against PCR Assay for the Diagnosis of Blastocystis sp. in Stool Samples

Background: This study evaluated the performance of routine permanent stain and cultivation method in comparison with polymerase chain reaction assay as the reference technique to detect Blastocystis sp. Methods: A cross-sectional study was conducted among aboriginal populations that reside in Pahang, Peninsular Malaysia in Feb to Mar 2015. A total of 359 stool samples were examined using Wheatley’s trichrome stain, in-vitro cultivation in Jones’ medium and PCR assay. Positive amplicons were subjected to sequencing and phylogenetic analysis. Results: Fifty-six (15.6%) samples were detected positive with Blastocystis sp. by Wheatley’s trichrome stain and 73 (20.3%) by in-vitro culture, while PCR assay detected 71 (19.8%) positive samples. Detection rate of Blastocystis sp. was highest in combination of microscopic techniques (27.9%). The sensitivity and specificity of Wheatley’s trichrome staining and in-vitro culture techniques compared to PCR assay were 49.3% (95% CI: 37.2-61.4) and 92.7% (95% CI: 89.1-95.4) and 39.4% (95% CI: 28.0-51.8) and 84.4% (95% CI: 79.7-88.4), respectively. However, the sensitivity [60.6% (95% CI: 48.3-71.9)] of the method increased when both microscopic techniques were performed together. False negative results produced by microscopic techniques were associated with subtype 3. The agreement between Wheatley’s trichrome stain, in-vitro culture and combination of microscopic techniques with PCR assay were statistically significant by Kappa statistics (Wheatley’s trichrome stain: K = 0.456, P<0.001; in-vitro culture: K = 0.236, P<0.001 and combination techniques: K = 0.353, P<0.001). Conclusion: The combination of microscopic technique is highly recommended to be used as a screening method for the diagnosis of Blastocystis infection either for clinical or epidemiological study to ensure better and accurate diagnosis

Evaluation of PCR-based approach for serotype determination of streptococcus pneumoniae

Determination of Streptococcus pneumoniae serotypes is essential for epidemiological surveillance. Therefore accurate, reliable and cost effective serotyping method is crucial. In this study, we determined the serotypes of 41 pneumococcal isolates recovered from human anterior nares by multiplex Polymerase Chain Reaction (PCR) utilizing published primers. The data was then compared with conventional serology using latex agglutination (LA) and the Quellung reaction. Based on the PCR-approach, 8 different serogroups/serotypes were detected with one isolate classified as non-typeable (cpsAnegative). In reference to the serology-based data, the results were in agreement except for one isolate. For the latter isolate, the LA and Quellung tests failed to show a reaction but the PCR-approach and sequencing identified the isolate as serogroup 15B/C. Based on this experimental setting, we found that the PCR-approach for pneumococcal serotypes determination is reliable to serve as the alternative for determining the pneumococcal serotyping