Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan

Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

Meta-analysis of variable-temperature PCR technique performance for diagnosising Schistosoma japonicum infections in humans in endemic areas

Background As China is moving onto schistosomiasis elimination/eradication, diagnostic methods with both high sensitivity and specificity for Schistosoma japonicum infections in humans are urgently needed. Microscopic identification of eggs in stool is proven to have poor sensitivity in low endemic regions, and antibody tests are unable to distinguish between current and previous infections. Polymerase chain reaction (PCR) technologies for the detection of parasite DNA have been theoretically assumed to show high diagnostic sensitivity and specificity. However, the reported performance of PCR for detecting S. japonicum infection varied greatly among studies. Therefore, we performed a meta-analysis to evaluate the overall diagnostic performance of variable-temperature PCR technologies, based on stool or blood, for detecting S. japonicum infections in humans from endemic areas. Methods We searched literatures in eight electronic databases, published up to 20 January 2021. The heterogeneity and publication bias of included studies were assessed statistically. The risk of bias and applicability of each eligible study were assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 tool (QUADAS-2). The bivariate mixed-effects model was applied to obtain the summary estimates of diagnostic performance. The hierarchical summary receiver operating characteristic (HSROC) curve was applied to visually display the results. Subgroup analyses and multivariate regression were performed to explore the source of heterogeneity. This research was performed following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines and was registered prospectively in PROSPERO (CRD42021233165). Results A total of 2791 papers were retrieved. After assessing for duplications and eligilibity a total of thirteen publications were retained for inclusion. These included eligible data from 4268 participants across sixteen studies. High heterogeneity existed among studies, but no publication bias was found. The pooled analyses of PCR data from all included studies resulted in a sensitivity of 0.91 (95% CI: 0.83 to 0.96), specificity of 0.85 (95% CI: 0.65 to 0.94), positive likelihood ratio of 5.90 (95% CI: 2.40 to 14.60), negative likelihood ratio of 0.10 (95% CI: 0.05 to 0.20) and a diagnostics odds ratio of 58 (95% CI: 19 to 179). Case-control studies showed significantly better performances for PCR diagnostics than cross-sectional studies. This was further evidenced by multivariate analyses. The four types of PCR approaches identified (convention PCR, qPCR, Digital droplet PCR and nested PCR) differed significantly, with nested PCRs showing the best performance. Conclusions Variable-temperature PCR has a satisfactory performance for diagnosing S. japonicum infections in humans in endemic areas. More high quality studies on S. japonicum diagnostic techniques, especially in low endemic areas and for the detection of dual-sex and single-sex infections are required. These will likely need to optimise a nested PCR alongside a highly sensitive gene target. They will contribute to successfully monitoring endemic areas as they move towards the WHO 2030 targets, as well as ultimately helping areas to achieve these goals.

A Real-Time PCR Assay for Detection of Low Pneumocystis jirovecii Levels

Here we report a new real-time PCR assay using SYBR Green which provides higher sensitivity for the specific detection of low levels of Pneumocystis jirovecii. To do so, two primer sets were designed, targeting the family of genes that code for the most abundant surface protein of Pneumocystis spp., namely the major surface glycoproteins (Msg), and the mitochondrial large subunit rRNA (mtLSUrRNA) multicopy gene, simultaneously detecting two regions. PCR methods are instrumental in detecting these low levels; however, current nested-PCR methods are time-consuming and complex. To validate our new real-time Msg-A/mtLSUrRNA PCR protocol, we compared it with nested-PCR based on the detection of Pneumocystis mitochondrial large subunit rRNA (mtLSUrRNA), one of the main targets used to detect this pathogen. All samples identified as positive by the nested-PCR method were found positive using our new real-time PCR protocol, which also detected P. jirovecii in three nasal aspirate samples that were negative for both rounds of nested-PCR. Furthermore, we read both rounds of the nested-PCR results for comparison and found that some samples with no PCR amplification, or with a feeble band in the first round, correlated with higher Ct values in our real-time Msg-A/mtLSUrRNA PCR. This finding demonstrates the ability of this new single-round protocol to detect low Pneumocystis levels. This new assay provides a valuable alternative for P. jirovecii detection, as it is both rapid and sensitive.

Disproportionate Distribution of HBV Genotypes A and D and the Recombinant Genotype D/E in the High and Low HBV Endemic Regions of Uganda: A Wake-Up Call for Regional Specific HBV Management

Background. Hepatitis B virus (HBV) is the leading cause of liver-related diseases. In Uganda, there is a regional disparity in the HBV burden. Our study was aimed at establishing the circulating genotypes in a low and a high endemic region to give plausible explanations for the differences in regional burden and guide the future management of the disease. Methods. A total of 200 HBsAg-seropositive subjects were recruited into the study by convenience sampling. The HBsAg Rapid Test Strip (Healgen Scientific Limited Liability Company, Houston, TX77047- USA) was used to screen for HBsAg while the Roche machine (Roche, Basel Switzerland/Abbot Technologies (USA)) was used to determine the viral load. The Chemistry Analyzer B120 (Mindray, China) was used for chemistry analysis. For HBV genotyping, total DNA was extracted from whole blood using the QIAamp® DNA extraction kit. Nested PCR amplification was performed using Platinum Taq DNA Polymerase (Invitrogen Corporation, USA) to amplify the 400 bp HBV polymerase gene. Purification of nested PCR products was performed using Purelink PCR product purification kit (Life Technologies, USA). Automated DNA sequencing was performed using BigDye Terminator v3.1 Cycle Sequencing Kit on 3130 Genetic Analyzer (Applied Biosystems, USA). The NCBI HBV genotyping tool (https://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) was used for determination of genotype for each HBV sequence. Pearson’s chi-square, multinomial logistic regression, and Mann–Whitney U tests were used for the analysis. All the analyses were done using SPSS version 26.0 and MedCalc software version 19.1.3 at 95% CI. A p < 0.05 was considered statistically significant. Results. Majority of our study subjects were female (64.5%), youth (51.0%), and married (62.0%). Overall, genotype A was the most prevalent (46%). Genotype D and the recombinant genotype D/E were proportionately more distributed in the high endemic (38.2%) and low endemic (36.5%) regions, respectively. Genotype D was significantly more prevalent in the high endemic region and among the elderly ( p < 0.05 ). Genotype D was significantly associated with elevated viral load and direct bilirubin ( p < 0.05 ). The recombinant genotype D/E was significantly associated with elevated viral load ( p < 0.05 ). Similarly, genotype A was significantly associated with elevated AST and GGT, lowered viral load, and normal direct bilirubin levels ( p < 0.05 ). Conclusion. There is disproportionate distribution of genotypes A and D and the recombinant genotype D/E in the low and high endemic regions of Uganda. This probably could explain the differences in endemicity of HBV in our country signifying the need for regional specific HBV management and control strategies.

Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols

Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi Sporothrix sp. The aims of the present study were to evaluate, standardize and validate a nested PCR technique using two DNA purification kits for the extraction of DNA from formalin fixed and paraffin-embedded tissues (FFPE) for Sporothrix sp. detection. FFPE mycological culture pellet samples of different Sporothrix species (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii) were used as positive controls and clinical FFPE tissue samples of animals positive for Cryptococcus sp., Leishmania infantum and Histoplasma sp. were used as negative controls. Ten clinical FFPE skin samples from cats with sporotrichosis were used to validate the nested PCR. These samples were cut into two distinct paraffin sectioning protocols (5 and 16 μm thick). The paraffin sections were subjected to two different DNA extraction kits (chemical and thermal extractions). A nested PCR was performed on the extracted DNA to identify the genus Sporothrix. The chemical extraction protocol with the 5 μm thick paraffin section was more effective in extracting DNA from Sporothrix sp. from FFPE samples and the nested PCR technique showed the highest sensitivities (100% in the positive controls and of 50% in the skin samples of cats) and specificity (100%). Therefore, the nested PCR using this protocol has great potential to be applied in Sporothrix sp. diagnosis in FFPE samples of cats.

Kesesuaian Uji Antigen Capture Enzyme Linked Immunosorbant Assay dan Nested Multiplex Polymerase Chain Reaction untuk Diagnosis Rutin Bovine Viral Diarrhea

Bovine Viral Diarrhea (BVD) is one of the main causes of impaired productivity and reproduction of cows. Antigen capture Elisa (ACE) is one of the serological technique that is sensitive, reliable and used regularly for detecting persistent BVD infection individually which simpler than multiplex nested PCR. The aim of this study was to determine the agreement between ACE and multiplex nested PCR as a routine laboratory diagnostic technique to detect the presence of BVD infection. A total of 128 cow serum samples consisting of 63 positive and 65 negative samples based on ACE were used in this study. The samples were collected from active and passive surveillance in dairy and beef cattle conducted by Balai Besar Veteriner (BBVet) Wates. The serum samples were then tested molecularly using multiplex nested PCR against BVD. The result showed 48 out of 63 BVDV-1 positive samples were found positive BVD antigen whereas 57 of 65 BVDV-1 negative samples were negative using multiplex nested PCR, . The agreement value between the two different assays based on statistic analysis using Kappa method was 0.64 and classified a good one. The result concluded that the ACE BVD assay was equally suitable as routine diagnosis to determine BVD infected cattle in the farm. Keywords: Antigen capture ELISA; Bovine viral diarrhea; Kappa; Multiplex nested PCR. Abstrak Bovine Viral Diarrhea (BVD) merupakan salah satu penyebab gangguan produktivitas dan reproduksi sapi. Antigen capture ELISA (ACE) merupakan salah satu teknik serologis yang sensitif, dapat diandalkan dan digunakan secara teratur untuk mendeteksi infeksi BVD persisten secara individual yang lebih sederhana daripada multiplex nested PCR. Tujuan penelitian ini adalah untuk mengetahui kesesuaian antara uji ACE dan multiplex nested PCR sebagai teknik diagnostik laboratorium rutin untuk mendeteksi adanya infeksi BVD. Sebanyak 128 sampel serum sapi yang terdiri dari 63 sampel positif dan 65 negatif berdasarkan ACE BVDV Antigen Test Kit/Serum Plus (Idexx®) digunakan dalam kajian ini. Sampel serum sapi merupakan koleksi dari surveilans aktif dan pasif pada sapi perah dan potong yang dilakukan Balai Besar Veteriner (BBVet) Wates. Sampel serum kemudian diuji secara molekuler menggunakan multiplex nested PCR terhadap BVD. Hasil penelitian menunjukkan bahwa dengan teknik multiplex nested PCR, 48 dari 63 sampel positif BVDV-1 ditemukan positif untuk antigen BVD sedangkan 57 dari 65 sampel negatif BVDV-1 negatif untuk antigen BVD. Analisis statitik berdasarkan perhitungan metoda Kappa menunjukkan nilai kesesuaian antara dua uji sebesar 0,64 dan tergolong bagus. Hasil penelitian menunjukkan kesimpulan bahwa uji ACE BVD sesuai sebagai diagnosis rutin untuk menentukan ternak yang terinfeksi BVD di peternakan. Kata kunci: Antigen capture ELISA; Bovine viral diarrhea; Kappa; Multiplex nested PCR.

PCR-RFLP Based genetic diversity of Plasmodium vivax genotypes in district Mardan, Pakistan

Plasmodium vivax is the most common human malaria parasite in Asian countries including Pakistan. Present study was designed to explore the genetic diversity of plasmodium vivax genotypes based on Pvmsp-3α and Pvmsp-3βgenes using allelic specific nested PCR and RFLP assays markers from field isolates in district Mardan, Pakistan. Blood samples of 200 P. vivax malarial patients were collected after taking their written informed consent. Genetic diversity in nested PCR products was determined by Restriction Fragment Length Polymorphism (RFLP) utilizing Alu1 and PstI restriction enzymes for alpha and beta gene products digestion, respectively. For analysis the genetic diversity of the sub allelic variants of Pvmsp3α and Pvmsp3β genes, Chi-Square test was performed by utilizing Minitab programming software 18. The P value 0.05 was considered as statistically significant. For Pvmsp-3α genes after gel electrophoresis of digested products, four distinct genotypes were obtained from total of 50 samples; type A: 35 (70%) (1.5-2.0 kb), 12 of type B (24%) (1.5-1.7 kb), 2 of type C (4%) (0.5-1.5) and one for type D (2%) (0.5-0.65 kb) which could be characterized into 9 allelic pattern (A1-A4, B1-B3, C1, D), in which A3 remained the most predominant. For Pvmsp-3βgenes, three distinct genotypes were obtained from 50 samples; 40(80%) of type A (1.5-2.5 kb), 9 (18%) of type B (1.0-1.5kb) and 1(2%) of type C (0.65 kb) which could be characterized into 6 allelic patterns (A1-A3, B1-B2, and C1). Most dominant one in Type A was A1 alleles which were noted (46%), while in Type B, the most dominant were B1 (10%).This study is the first ever report of molecular epidemiology and genetic variation in Pvmsp-3α and Pvmsp-3β genes of P. vivax isolates by using PCR/RFLP from District Mardan and showed a remarkable level of genetic diversity in the studied genes of circulating parasites in the study area. The results of this study will contribute in future studies about the genetic structure of parasite and vaccine development against the malaria.

Performance of CO1 and ITS2 nested PCR in molecular identification of ordinary scabies (Sarcoptes scabiei var. hominis)

Scabies is a global disease with a high prevalence, causing morbidity and even mortality, especially in poor and developing countries. However, it is often misdiagnosed due to varied and unspecified lesions. The gold standard technique for diagnosis is a microscopic examination, which requires experienced experts in finding mites, mainly in ordinary scabies. CO1 and ITS2 genes have been widely used in molecular identification to detect Sarcoptes scabiei and its variants. This study aimed to determine and compare the sensitivity and specificity of CO1 and ITS2 S. scabiei genes to the microscopic examination of scabies skin scrapings. The skin scrapings of 52 subjects with scabies diagnosed by anamnesis, physical examination, and dermoscopic examination were examined under a microscope and analyzed by nested PCR. The diagnostic test result showed that the sensitivity of nested PCR of both CO1 and ITS2 genes to micro‐ scope examination was 100%. However, the specificity of both CO1 and ITS2 nested PCR was poor (24% and 0%). Hence, CO1 and ITS2 nested PCR could be more suitable for screening ordinary scabies in humans than the microscopic examination.