Rekha Pai

Sequential Multiplex PCR Approach for Determining Capsular Serotypes of Streptococcus pneumoniae Isolates

ABSTRACT Accurate serotyping is essential to monitor the changes in the seroepidemiology of Streptococcus pneumoniae. We devised a simple and schematic sequence-based system of seven multiplex PCRs, in a sequence order based upon Active Bacterial Core surveillance (ABCs) serotype distribution during 2002 to 2003, to reliably deduce specific pneumococcal serotypes. A total of 421 isolates from ABCs were randomly chosen to evaluate this system. Two hundred twenty-nine of the isolates (54.3%) were specifically assigned 1 of 17 serotypes by the multiplex PCR system, with the results in complete concordance with conventional serotyping. One hundred seventy-two additional isolates (40.9%) were assigned to 11 specific sets of 2 to 4 serotypes that with one exception (serotypes 6A and 6B) consisted of the single frequently occurring targeted serotype and 1 to 3 additional rare serotypes primarily within the same serogroup as the targeted serotype. Only 20 isolates (4.8%) could not be assigned specific serotypes or serotype sets, since they were either of rare serotypes not included in the assay design or were nonserotypeable. Overall, we found this system to be highly reliable, with the potential to greatly reduce our reliance upon conventional serotyping. Especially important is the capability of this system to give serotype-determining potential to any facility that lacks the expensive typing sera and expertise needed for conventional serotyping yet has the modest equipment necessary for DNA amplification and electrophoresis.

Vaccine escape recombinants emerge after pneumococcal vaccination in the United States.

The heptavalent pneumococcal conjugate vaccine (PCV7) was introduced in the United States (US) in 2000 and has significantly reduced invasive pneumococcal disease; however, the incidence of nonvaccine serotype invasive disease, particularly due to serotype 19A, has increased. The serotype 19A increase can be explained in part by expansion of a genotype that has been circulating in the US prior to vaccine implementation (and other countries since at least 1990), but also by the emergence of a novel “vaccine escape recombinant” pneumococcal strain. This strain has a genotype that previously was only associated with vaccine serotype 4, but now expresses a nonvaccine serotype 19A capsule. Based on prior evidence for capsular switching by recombination at the capsular locus, the genetic event that resulted in this novel serotype/genotype combination might be identifiable from the DNA sequence of individual pneumococcal strains. Therefore, the aim of this study was to characterise the putative recombinational event(s) at the capsular locus that resulted in the change from a vaccine to a nonvaccine capsular type. Sequencing the capsular locus flanking regions of 51 vaccine escape (progeny), recipient, and putative donor pneumococci revealed a 39 kb recombinational fragment, which included the capsular locus, flanking regions, and two adjacent penicillin-binding proteins, and thus resulted in a capsular switch and penicillin nonsusceptibility in a single genetic event. Since 2003, 37 such vaccine escape strains have been detected, some of which had evolved further. Furthermore, two new types of serotype 19A vaccine escape strains emerged in 2005. To our knowledge, this is the first time a single recombinational event has been documented in vivo that resulted in both a change of serotype and penicillin nonsusceptibility. Vaccine escape by genetic recombination at the capsular locus has the potential to reduce PCV7 effectiveness in the longer term.

Pre- and Postvaccination Clonal Compositions of Invasive Pneumococcal Serotypes for Isolates Collected in the United States in 1999, 2001, and 2002

ABSTRACT Monitoring of serotypes and their clonal associations is critical as pneumococci adapt to the selective pressures exerted by the pneumococcal seven-valent conjugate vaccine (PCV7). We genotyped 1,476 invasive isolates from the Active Bacterial Core surveillance (705 [89.8%] of the isolates were obtained from children <5 years of age, and 771 [18.4%] of the isolates were obtained from individuals >5 years of age) in 2001 and 2002 (after the introduction of PCV7). The data were compared to the results for 1,168 invasive isolates (855 [83.9%] of the isolates were from children <5 years of age) collected in 1999. Among children <5 years of age, the incidence of invasive disease due to non-PCV7 serogroups together with serogroup 19A increased (P < 0.001). Eighty-three clonal sets, representing 177 multilocus sequence types (STs), were compiled from the 3-year isolate set. Among the non-PCV7 serogroups, newly emerging clones were uncommon; and a significant expansion of already established clones occurred for serotypes 3 (ST180), 7F (ST191), 15BCF (ST199), 19A (ST199), 22F (ST433), 33F (ST662), and 38 (ST393). However, additional minor clonal types within serotypes 1, 6A, 6B, 7C, 9N, 10A, 12F, 14, 15B/C, 17F, 19A, 19F, 20, 22F, and 33F that were absent in 1999 were found during 2001 and 2002. Although 23 clonal sets exhibited multiple serotypes, for most serotypes there were either no changes or modest changes in clonal compositions since the introduction of PCV7. The only example of an identical ST shared between non-PCV7 and PCV7 or PCV7-related serotypes was ST199; however, ST199 was prevalent within serotypes 15B/C and 19A before and after PCV7 introduction. Continued genotypic surveillance is warranted, since certain clones not targeted by PCV7 are expanding, and their emergence as significant pathogens could occur with maintained vaccine pressure.

Clonal Association between Streptococcus pneumoniae Serotype 23A, Circulating within the United States, and an Internationally Dispersed Clone of Serotype 23F

ABSTRACT Streptococcus pneumoniae is an important pathogen in the United States and is associated with significant morbidity and mortality. Since the introduction of the seven-valent conjugate vaccine, a significant decline in pneumococcal disease has been reported. However, surveillance for pneumococcal disease remains essential, as the extent of cross protection against vaccine-related serotypes is still unclear. Further, any increase in non-vaccine-related serotypes also needs monitoring. We report on a new clonal association between a vaccine-related serotype, serotype 23A, obtained as part of the Active Bacterial Core surveillance, with an established internationally dispersed Pneumococcal Molecular Epidemiology Network (PMEN) clone, clone Colombia23F-26. Sixty-two isolates of serotype 23A collected from sterile sites during a 2-year period (2002 and 2003) were characterized. Twenty-one (34%) isolates were penicillin nonsusceptible, although none were fully resistant. Pulsed-field gel electrophoresis and multilocus sequence typing analysis showed that 24 (39%) of the serotype 23A isolates shared either genetic identity or high genetic relatedness with PMEN clone Colombia23F-26. Extensive variability was noted within the sequenced region of pbp2b in two penicillin-nonsusceptible isolates as well as in PMEN clone Colombia23F-26, suggesting that these isolates probably acquired penicillin resistance independently. The emergence of such new serotype and genotype associations highlights the dynamic nature of the pneumococcal population, necessitating continuous monitoring in the post-vaccine era.

p.Arg82Leu von Hippel-Lindau (VHL) Gene Mutation among Three Members of a Family with Familial Bilateral Pheochromocytoma in India: Molecular Analysis and In Silico Characterization

Various missense mutations in the VHL gene have been reported among patients with familial bilateral pheochromocytoma. However, the p.Arg82Leu mutation in the VHL gene described here among patients with familial bilateral pheochromocytoma, has never been reported previously in a germline configuration. Interestingly, long-term follow-up of these patients indicated that the mutation might have had little impact on the normal function of the VHL gene, since all of them have remained asymptomatic. We further attempted to correlate this information with the results obtained by in silico analysis of this mutation using SIFT, PhD-SNP SVM profile, MutPred, PolyPhen2, and SNPs&GO prediction tools. To gain, new mechanistic insight into the structural effect, we mapped the mutation on to 3D structure (PDB ID 1LM8). Further, we analyzed the structural level changes in time scale level with respect to native and mutant protein complexes by using 12 ns molecular dynamics simulation method. Though these methods predict the mutation to have a pathogenic potential, it remains to be seen if these patients will eventually develop symptomatic disease.

Detection of large deletions in the VHL gene using a Real-Time PCR with SYBR Green

Mutation in VHL gene causes the von Hippel-Lindau (VHL) disease, a dominantly inherited familial cancer syndrome. The VHL mutation pattern includes point mutations, small deletions and large deletions. While most mutations can be identified during sequencing, large deletions often remain unnoticed in initial mutational screening. We evaluated the utility of a previously described real-time quantitative PCR (RQ-PCR) using SYBR Green for detection of larger deletions in the VHL gene and normalized the data using two reference genes with a normal copy number i.e., ZNF80 (3q13.31) and GPR15 (3q12.1). DNA sequencing was also done on all cases included in the study. SJNB-6 cell line demonstrating distal 3p loss was used as a positive control for deletion. Out of 21 individual cases included of VHL disease, 2 cases were found with partial deletion by RQ-PCR, with an exon 1 deletion, while PCR–sequencing identified 5 cases with base pair substitution and 1 with splice site variant which were not picked up by RQ-PCR. RQ-PCR proved to be fast, accurate and sensitive for identifying large deletions and can be incorporated into the routine work-up for detection of large deletions in VHL disease.

Usefulness of Succinate dehydrogenase B (SDHB) immunohistochemistry in guiding mutational screening among patients with pheochromocytoma‐paraganglioma syndromes

Genetic testing of pheochromocytomas (PCC) and paragangliomas (PGL), although expensive, is gradually becoming a part of the routine laboratory investigation for patients with PCC‐PGL syndrome. Recently, Succinate dehydrogenase B (SDHB) immunochemistry has been shown to be an excellent indicator of germline mutations in the SDH genes and could help significantly reduce cost. This study assesses the utility of SDHB immunohistochemical analysis when used to guide genetic analysis, with emphasis on cost benefits it could provide in a resource‐limited setting. Forty‐four cases of PCC/PGL characterized by genetic analysis were included to determine their SDHB expression pattern by immunohistochemistry. SDHB antibody expression was negative among three cases each, with SDHB and SDHD mutations. Immunohistochemistry results were positive for all three cases of RET, a single case of neurofibromatosis and for two cases with Von Hippel‐Lindau (VHL) mutations while the remaining two cases with VHL mutations showed a diffuse ‘cytoplasmic blush’. Thirty of the remaining 31 samples demonstrated positive staining and were negative for mutations, while a lone sample that was negative for staining and mutation was not included in the final analysis as the internal control for the sample was not adequately stained. Cost analysis in our settings showed that triaging with SDHB immunohistochemistry could potentially reduce costs by USD 320–500 per patient. SDHB immunohistochemistry, when used as a guide to genetic testing, can significantly reduce the effort, time and costs of testing among patients with PCC‐PGL, a huge benefit in resource limited settings.

Discriminating thyroid cancers from benign lesions based on differential expression of a limited set of miRNA using paraffin embedded tissues

BACKGROUND Micro-RNAs (miRNAs) are expressed in a tissue-specific manner and are known to demonstrate differential expression even among the various subtypes of a given tumor. This differential expression has been harnessed successfully in the development of diagnostic assays for various malignant tumors. These assays have been found to be relevant and of value as additional diagnostic tools even among thyroid tumors, particularly with regard to thyroid carcinomas of follicular morphology. MATERIALS AND METHODS A limited set of miRNA have been assessed as part of this study in an effort to use minimal number of miRNA markers (miR-187, miR-221, miR-222, and miR-224) to differentiate the benign from the malignant thyroid tumors using miRNA derived from paraffin embedded material. RESULTS While miR-221 and miR-222 were found to provide good accuracy as individual markers (86% and 84%), a combination of the two provided slightly better accuracy (91%). Both miR-221 and 222 were able to significantly differentiate malignant tumors from the benign samples (P< 0.001) individually and as a combination of markers. However, inclusion of miR-187 and miR-224 in the panel did not provide any additional benefit. CONCLUSION While a combination of miR-221 and 222 when used in a diagnostic panel could provide fairly good accuracy additional markers may need to be investigated to augment their diagnostic utility.

Clinicopathologic features of non-small cell lung cancer in India and correlation with epidermal growth factor receptor mutational status

INTRODUCTION We performed retrospective analysis of 106 patients with lung cancer for which formalin-fixed paraffin-embedded tissues was available. Their epidermal growth factor receptor (EGFR) mutation status and treatment outcomes are described. MATERIALS AND METHODS All patients with confirmed non-small cell lung cancer (NSCLC) during Jan 2008 to Dec 2010 were included. EGFR sequencing was performed with ABI PRISM 310 genetic analyzer. RESULTS Forty-two (39.6%) patients had mutation in one of the four exons characterized. Patients whose EGFR mutational status was not available at presentation before the start of treatment were started on chemotherapy, n = 46 (43.39%). If EGFR mutational analysis was available and mutations were present, the patients were started on either upfront tyrosine kinase inhibitor (TKI), n = 15 (14.15%) or if on chemotherapy arm were allowed to finish six cycles and then start with maintenance TKIs, n = 26 (24.52%). The median progression free survival for patients with and without mutations was 11 months (95% CI,7-14) and 9 months (95% CI,7-10) respectively. A median PFS of 14 months (95%CI, 12-16) was seen in the mutation-positive group that received both chemotherapy followed by switch maintenance with TKIs versus 8 months (95%CI, 7-8 months) in the group that received only TKI. CONCLUSION The prevalence of EGFR mutations in this population of NSCLC patients was 39.6% with exon 19 mutation being the most common. The observed benefit of addition of chemotherapy over TKI in EGFR mutation-positive group raises the question, can we offer the therapy of chemotherapy-TKI combination to EGFR mutation-positive lung cancer patients as shown in the present study.

Status of O 6 -methylguanine-DNA methyltransferase [MGMT] gene promoter methylation among patients with glioblastomas from India

BACKGROUND O⁶ -methylguanine DNA methyltransferase [MGMT] gene promoter methylation has emerged as a promising marker in determining resistance to temozolomide, used in the treatment of patients with glioblastomas. AIM To determine the frequency of MGMT promoter methylation among patients with glioblastomas using methylation-specific polymerase chain reaction (MSP) and compare it to the results obtained by bisulfite sequencing of a subset of samples. MATERIALS AND METHODS DNA obtained from the frozen tissue of 27 samples of glioblastomas and three other gliomas, were analyzed for MGMT promoter methylation using a nested MSP assay. Sixteen samples were also subjected to bisulfite sequencing to determine the methylation status of 27 CpG sites within the sequenced region of the MGMT promoter. Data with respect to radiation, chemotherapy and survival outcome was also collected. RESULTS MGMT promoter methylation was seen in 67% of the cases included in the study using frozen tissues by MSP analysis, while 62% were methylated among glioblastomas alone. There was a 100% concordance between the results obtained by MSP analysis and bisulfite sequencing. Clinical outcome was known among 67% of cases and methylation was higher among those patients who had no recurrence, though it was not statistically significant [P=0.44]. CONCLUSION The frequency of methylation seen in this study concurs with that reported earlier from the country. MSP was easy to perform and interpret. However, the utility of this testing system in a routine diagnostic setting is still being debated.