Toward interpretable and generalized mitosis detection in digital pathology using deep learning.

Objective: The mitotic activity index is an important prognostic factor in the diagnosis of cancer. The task of mitosis detection is difficult as the nuclei are microscopic in size and partially labeled, and there are many more non-mitotic nuclei compared to mitotic ones. In this paper, we highlight the challenges of current mitosis detection pipelines and propose a method to tackle these challenges. Methods: Our proposed methodology is inspired from recent research on deep learning and an extensive analysis on the dataset and training pipeline. We first used the MiDoG'22 dataset for training, validation, and testing. We then tested the methodology without fine-tuning on the TUPAC'16 dataset and on a real-time case from Shaukat Khanum Memorial Cancer Hospital and Research Centre. Results: Our methodology has shown promising results both quantitatively and qualitatively. Quantitatively, our methodology achieved an F1-score of 0.87 on the MiDoG'22 dataset and an F1-score of 0.83 on the TUPAC dataset. Qualitatively, our methodology is generalizable and interpretable across various datasets and clinical settings. Conclusion: In this paper, we highlight the challenges of current mitosis detection pipelines and propose a method that can accurately predict mitotic nuclei. We illustrate the accuracy, generalizability, and interpretability of our approach across various datasets and clinical settings. Our methodology can speed up the adoption of computer-aided digital pathology in clinical settings.

Cell-free DNA as a potential diagnostic biomarker in academic stress: A case-control study in young adults.

Background: Stress is a pervasive issue in modern life, affecting both physical and mental health. Identifying biomarkers like cell-free DNA (cfDNA) could provide insights into stress response and help detect individuals at risk for stress-related disorders. Objective: The aim of this study is to investigate the potential use of cfDNA as a diagnostic biomarker in individuals experiencing stress. Methodology: A case-control analysis was conducted using convenient sampling on university participants (N = 285 cases, N = 500 controls) aged 18-24. The study assessed haematological and lipid profile parameters using the Sysmex XP-300TM automated analyzer and an automated biochemistry analyzer, and cfDNA was extracted using a standardized in house developed Phenol-Chloroform protocol and estimated using Agarose Gel Electrophoresis and Nanodrop. Statistical analysis was performed using SPSS ver. 21.0. Results: The results indicated a significant difference between stressed individuals and healthy controls in demographic, haematological and biochemical parameters. Specifically, stressed cases had significantly higher levels of cholesterol, LDL cholesterol, triglycerides, glucose, VLDL cholesterol, and lower levels of HDL compared to healthy controls. Stressed cases also showed significantly elevated levels of circulating cfDNA relative to healthy controls. Conclusion: These findings suggest that cfDNA may have potential as a diagnostic biomarker for stress.

Uropathogenic bacteria and deductive genomics towards antimicrobial resistance, virulence, and potential drug targets / Bacterias uropatógenas y genómica deductiva hacia la resistencia, virulencia y posibles objetivos farmacológicos a los antimicrobianos

Urinary tract infections (UTIs) are among the most prevalent bacterial infections affecting people in inpatient and outpatient settings. The current study aimed to sequence the genome of uropathogenic Escherichia coli strain CUI-B1 resourced from a woman having uncomplicated cystitis and pyelonephritis. Followed by deductive genomics towards potential drug targets using E. coli strain CUI-B1, strain O25b: H4-ST131, Proteus mirabilis strain HI4320, Klebsiella pneumoniae strain 1721, and Staphylococcus saprophyticus strain ATCC 15305 uropathogenic strains. Comparative genome analysis revealed that genes related to the survival of E. coli, P. mirabilis, K. pneumoniae, and S. saprophyticus, such as genes of metal-requiring proteins, defense-associated genes, and genes associated with general physiology, were found to be highly conserved in the genomes including strain CUI-B1. However, the genes responsible for virulence and drug resistance, mainly those that are involved in bacterial secretion, fimbriae, adherence, and colonization, were found in various genomic regions and varied from one species to another or within the same species. Based on the genome sequence, virulence, and antimicrobial-resistant gene dataset, the subtractive proteomics approach revealed 22 proteins mapped to the pathogen’s unique pathways and among them, entB, clbH, chuV, and ybtS were supposed to be potential drug targets and the single drug could be utilized for all above-mentioned strains. These results may provide the foundation for the optimal target for future discovery of drugs for E. coli-, P. mirabilis-, K. pneumoniae-, and S. saprophyticus-based infections and could be investigated further to employ in personalized drug development.(AU)

Uropathogenic bacteria and deductive genomics towards antimicrobial resistance, virulence, and potential drug targets.

Urinary tract infections (UTIs) are among the most prevalent bacterial infections affecting people in inpatient and outpatient settings. The current study aimed to sequence the genome of uropathogenic Escherichia coli strain CUI-B1 resourced from a woman having uncomplicated cystitis and pyelonephritis. Followed by deductive genomics towards potential drug targets using E. coli strain CUI-B1, strain O25b: H4-ST131, Proteus mirabilis strain HI4320, Klebsiella pneumoniae strain 1721, and Staphylococcus saprophyticus strain ATCC 15305 uropathogenic strains. Comparative genome analysis revealed that genes related to the survival of E. coli, P. mirabilis, K. pneumoniae, and S. saprophyticus, such as genes of metal-requiring proteins, defense-associated genes, and genes associated with general physiology, were found to be highly conserved in the genomes including strain CUI-B1. However, the genes responsible for virulence and drug resistance, mainly those that are involved in bacterial secretion, fimbriae, adherence, and colonization, were found in various genomic regions and varied from one species to another or within the same species. Based on the genome sequence, virulence, and antimicrobial-resistant gene dataset, the subtractive proteomics approach revealed 22 proteins mapped to the pathogen's unique pathways and among them, entB, clbH, chuV, and ybtS were supposed to be potential drug targets and the single drug could be utilized for all above-mentioned strains. These results may provide the foundation for the optimal target for future discovery of drugs for E. coli-, P. mirabilis-, K. pneumoniae-, and S. saprophyticus-based infections and could be investigated further to employ in personalized drug development.

Memory-type variance estimators using exponentially weighted moving average statistic in presence of measurement error for time-scaled surveys.

The present study suggested memory-type ratio and product estimators for variance estimation in the presence of measurement errors. We applied the exponentially weighted moving averages statistic which simultaneously utilizes the current and prior information for better estimation in surveys based on the time-scale. The expressions of approximate mean square errors of memory-type estimators are derived using Taylor series up to first order. Mathematical conditions are also obtained for which the suggested memory-type ratio and product estimators perform better than the conventional ratio and product estimators. The efficiency of the proposed estimators is observed using an extensive simulation study in the presence of measurement errors. A real data application is also carried out to support the mathematical expressions. From the results, it is shown that the use of prior sample information significantly increased the efficiency of the proposed estimators.

Genetic and antiretroviral drug resistance mutations analysis of reverse transcriptase and protease gene from Pakistani people living with HIV-1.

BACKGROUND: Antiretroviral therapy (ART) effectiveness is compromised by the emergence of HIV drug resistance mutations (DRM) and can lead to the failure of ART. Apart from intrinsic viral factors, non-compliance with drugs and/or the use of sub-optimum therapy can lead to the emergence of DRMs. In Pakistan HIV currently exists as a concentrated epidemic, however, ART coverage is very low, and drug adherence is poor. ART is selected assuming without baseline genotyping. Pakistan has recently seen a rise in treatment failures, but the country's actual burden of DRM is still unknown. In this study, we perform the genetic and drug resistance analysis of the pol gene from Pakistani HIV-positive ART-naïve and ART-experienced individuals. METHODS: In this study, HIV-1 pol was sequenced from 146 HIV-1 positive individuals, divided into ART-naïve (n = 37) and ART-experienced (n = 109). The sequences were also used to determine HIV-1 subtypes, the prevalence of DRM, and pol genetic variability. RESULTS: DRM analysis identified numerous DRMs against reverse transcriptase inhibitors in both ART-naïve and ART-experienced groups, including a few that are classified as rare. Additionally, the ART-experienced group showed mutations associated with resistance to protease inhibitors. Genetic analysis showed negative selection pressure in both groups, but a higher rate of evolution in the ART-naïve group. CONCLUSION: High prevalence of DRMs, especially against previous first-line treatment in ART- naïve and the accumulation of DRMs in ART-experienced groups is concerning and warrants that a more extensive DRM survey be carried out to inform first-line and second-line ART regimen recommendations.

Sublethal effects of spinetoram and emamectin benzoate on key demographic parameters of fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae) under laboratory conditions.

Fall armyworm (FAW) Spodoptera frugiperda, is a serious invasive pest of many crops that causes huge economic losses to agricultural commodities. Insecticides are employed for the management of S. frugiperda. In this study, we investigated the impact of sublethal concentration (LC10) and low lethal concentration (LC30) of spinetoram and emamectin benzoate on S. frugiperda using two-sex life table method. Bioassay results revealed that emamectin benzoate exhibited more toxicity on the 3rd instar of S. frugiperda (LC50 8.35 × 10-4 mgL-1) than spinetoram (LC50 2.6 × 10-2 mgL-1) after 48 h exposure. The total longevity, adult pre-ovipositional period (APOP) and total pre-ovipositional period (TPOP) were prolonged, while pre-adult survival rate and fecundity were reduced at both concentrations of spinetoram and emamectin benzoate. Moreover, the key demographic parameters, including the intrinsic rate of increase (r), finite rate of increase (λ), and net reproductive rate (R0), were significantly lower in insecticide treated groups as compared to untreated insects. Our findings revealed that the sublethal and low lethal concentrations of both insecticides reduce the survival and reproductive capability of S. frugiperda. These results would be useful to assess the overall effect of both insecticides on S. frugiperda and can provide important implications for the rational utilization of insecticides against S. frugiperda.

Structural vaccinology-based design of multi-epitopes vaccine against Streptococcus gordonii and validation using molecular modeling and immune simulation approaches.

Streptococcus gordonii is an oral bacterium colonizing the dental cavity and leading to plaque formation. This pervasive colonizer is also the etiologic agent of bacterial endocarditis and has a major role in infective endocarditis. The bacteria reach the heart through oral bleeding, leading to inflammation of cardiovascular valves. Over the past 50 years, it has shown a significant pathogenic role in immunocompromised and neutropenic patients. Since antibiotic resistance has created prophylaxis failure towards infective endocarditis, a potent therapeutic candidate is needed. Therefore, multi-epitopes vaccine offers advantages over the other approaches. Thus, herein, numerous molecular-omics tools were exploited to mine immunogenic peptides, i.e., T-cell and B-cell epitopes, and construct a vaccine sequence. Our findings revealed a total of 24 epitopes, including CTL, HTL, and B-cell are responsible for imparting immune responses, which were combined with the help of different linkers, and MEVC was constructed. Multifactorial validation of the candidate vaccine was performed to minimize the risk factors. The final sequence was docked with TLR2 to validate its conformation compatibility with receptor and long-term interactions stability. Our analysis revealed that the vaccine construct is immunogenic and non-allergenic. The construct also established various contacts with the immune receptor. Finally, the vaccine sequence was reverse-translated, optimized for codon usage, and analyzed for expression in the Escherichia coli K12 strain. Maximum expression was noted with a CAI score of 0.95. In silico immune simulation revealed that the antigen was neutralized on the 3rd day after injection. In conclusion, the current study warrants validation of the vaccine construct both in in vitro and in vivo models for accurate therapeutic intervention.

Characterization, Sensory and Oxidative Stability Analysis of Vegetable Mayonnaise Formulated with Olive Leaf Vinegar as an Active Ingredient.

Development of novel food products represents a basic meeting point for health and business requirements. Mayonnaise sauce is well-suited to be a healthy and tasty dressing. In this study, mayonnaise was formulated by using unconventional ingredients, such as olive leaf vinegar (OLV), soybean/high oleic sunflower oil blend, and soymilk (as an egg substitute). An 18% alcoholic vinegar was used as the control sample. OLV is a rich source of bioactive substances, especially polyphenols and represents a possible way to enhance the olive oil by-product valorisation. For this new typology of vinegar an high level of phenolic compounds (7.2 mg/mL GAE), especially oleuropein (6.0 mg/mL oleuropein equivalent) was found. OLV mayonnaise had 57% fat, composed of 11%, 64%, and 23% saturated, monounsaturated, and polyunsaturated fatty acids, while linolenic acid was up to 1.7%. The phenol and oleuropein contents were 68 and 52 mg/100 g, respectively. Sensory panellists expressed a moderate overall acceptability for both samples but attested more distinctive and positive sensations for the colour, odour, and taste attributes of OLV mayonnaise. Finally, oxidative stability and shelf life were better in OLV mayonnaise than in the control. Specifically, the peroxide value remained low (around 4.5 meqO2/kg) after 12 months of storage at room and low (4 °C) temperatures.

Assessment of sublethal and transgenerational effects of spirotetramat, on population growth of cabbage aphid, Brevicoryne brassicae L. (Hemiptera: Aphididae).

The cabbage aphid (Brevicoryne brassicae L.) is a devastating pest of cruciferous crops causing economic damage worldwide and notably owing to its increasing resistance to commonly used pesticides. Such resistance prompts the development of integrated pest management (IPM) programs that include novel pesticides being effective against the aphids. Spirotetramat is a novel insecticide used against sap-sucking insect pests, particularly aphids. This study evaluated the toxicity of spirotetramat to adult apterous B. brassicae after 72 h using the leaf dipping method. According to the toxicity bioassay results, the LC50 value of spirotetramat to B. brassicae was 1.304 mgL-1. However, the sublethal concentrations (LC5 and LC15) and transgenerational effects of this novel insecticide on population growth parameters were estimated using the age-stage, two-sex life table theory method. The sublethal concentrations (LC5; 0.125 mgL-1 and LC15; 0.298 mgL-1) of spirotetramat reduced the adult longevity and fecundity of the parent generation (F0). These concentrations prolonged the preadult developmental duration while decreasing preadult survival, adult longevity and reproduction of the F1 generation. The adult pre-reproductive period was also extended by spirotetramat treatment groups. Subsequently, the population growth parameters such as the intrinsic rate of increase r, finite rate of increase λ and net reproductive rate R 0 of the F1 generation were decreased in spirotetramat treatment groups whereas, the mean generation time T of the F1 generation was not affected when compared to the control. These results indicated the negative effect of sublethal concentrations of spirotetramat on the performance of B. brassicae by reducing its nymphal survival, extending the duration of some immature stages and suppressing the population growth of B. brassicae. Overall, we demonstrated that spirotetramat is a pesticide showing both sublethal activities, and transgenerational effects on cabbage aphid; it may be useful for implementation in IPM programs against this aphid pest.

The Case of the Young Male with a Longstanding History of Dyspeptic Symptoms: Peritoneal Tuberculosis.

Portal vein thrombosis associated with peritoneal tuberculosis is an uncommon manifestation of extrapulmonary tuberculosis. We report one such case of a 33-year-old male with a one-year history of dyspepsia, having been on proton pump inhibitors all this time with temporary relief. In view of ongoing symptoms, an endoscopy was done, which at first showed duodenal ulcer. On repeat endoscopy after an interval, there was evidence of mucosa-associated lymphoid tissue (MALT) lymphoma, which prompted a host of investigations in the patient. A positron emission tomography (PET) scan revealed extensive omento-peritoneal involvement along with a hypodense lesion in the liver with interval development of portal vein thrombosis on a CT scan of the abdomen. The biopsy of the hepatic lesion showed granulomatous inflammation. Faced with a diagnostic dilemma, finally, a laparoscopic biopsy was done, which confirmed the diagnosis of peritoneal TB with portal vein thrombosis. This case highlights the importance of keeping a high index of suspicion to include tuberculosis as a differential when presented with a case such as this and to conduct appropriate investigations to establish the correct diagnosis.

Comparative Potential of Zinc Sulfate, L-Carnitine, Lycopene, and Coenzyme Q10 on Cadmium-Induced Male Infertility.

The human exposure to toxic chemicals and heavy metals is one of the main predisposing factors contributing to male infertility. Acute exposure to cadmium chloride results in testicular damage and infertility. The purpose of the present study was to investigate and compare the curative effect of coenzyme Q10 (CoQ10), lycopene, L-carnitine (LC), and zinc sulfate against the cadmium-induced infertility in male Wistar rats. Cadmium chloride (0.4 mg/kg/day) was orally administered to rats for three consecutive days. Then, oral administration of different treatments (i.e., LC 100 mg/kg, CoQ10 20 mg/kg, lycopene 4 mg/kg, zinc sulfate 6 mg/kg, and a combination LC-CoQ10 at 500/50 mg/kg) was carried out for 30 days. The impact of different treatments on semen parameters, such as sperm count and motility, testicular antioxidants, and serum testosterone, was determined. Furthermore, the morphology of epididymis sperms and histopathology of rat testes were also assessed. Cadmium exposure decreased the sperm count, progressive sperm motility, testosterone, superoxide dismutase (SOD), and catalase and reduced glutathione (GSH). It also caused banana sperm tail, bent sperm head, vacuolization of seminiferous tubules, and oligospermia in rat testes. All treatments with nutraceuticals improved sperm count, sperm morphology, serum testosterone, vacuolization of seminiferous tubules, and oligospermia in diseased rats. Treatment with lycopene, LC, and LC-CoQ10 improved progressive sperm motility and other parameters and increased SOD, GSH, and CAT in the rat testes. CoQ10 also increased SOD activity in rat testes' tissue homogenates. It is concluded from the current study that all nutraceuticals partially improved reproductive toxicity of cadmium. The administration of lycopene and a high-dose combination of LC-CoQ10 were more efficacious in treating cadmium-induced infertility than other treatments. Treatment of cadmium-exposed rats with lycopene, LC, CoQ10, and LC-CoQ10 improved sperm count and motility through reduction of testicular oxidative stress and improving serum testosterone.

Influence of guar gum and chitosan enriched with lemon peel essential oil coatings on the quality of pears.

Pear is a typically climacteric fruit and highly perishable with a low shelf life owing to extreme metabolic activity after harvesting. The present study aimed to reduce weight loss and improve the firmness of pear during storage. The lemon peel essential oil (LPEO) has gained considerable attention due to being the richest source of bioactive compounds that behaved as a natural antioxidant agent, being cost-effective, and being generally recognized as safe. Edible coatings equipped with a natural antioxidant agent and renewable biopolymers have gained more research fame owing to their involvement in the direction of biodegradability and food safety. In this work, edible skin coating materials (ESCMs) embedded by chitosan (1%) and guar gum (2%) were fabricated, and afterward, five concentrations of LPEO (1, 1.5, 2, 2.5, and 3.0%) were incorporated individually into the ESCMs. Findings revealed that LPEO-ESCMs significantly reduced the weight loss and improved the firmness of pear up to 45 days of storage at 4 ± 2°C. Furthermore, the LPEO-ESCMs have enhanced the antioxidant capacity, antibacterial efficiency, and malondialdehyde level of pear during storage time. It was concluded that 3% of LPEO-ESCMs improved the overall acceptability of pear fruits. Taken together, the novel insights of guar gum and chitosan-based ESCMs entrapped with LPEO will remain a subject of research interest for researchers in the future.

Half circular modified burr-III distribution, application with different estimation methods.

The data related to many medical, environmental and ecological variables are often measured in terms of angles wherein its range is defined in [0,π). This type of data is referred to as axial or half circular data. Modeling based on half circular data has not received its due share of attention in statistical literature. In this paper, we introduce a new half circular distribution based on inverse stereographic projection technique on modified Burr-III distribution, called the half circular modified Burr-III (hcMB-III) distribution. The basic properties of the proposed distribution are derived. It is common observation that while estimating the parameters of a model, one usually adopts maximum likelihood estimation method as the starting point. In this paper, we consider seven frequentist methods of estimation, besides using maximum likelihood method for estimating the parameters of the hcMB-III distribution. Monte Carlo simulations are performed for investigating the performances of the considered methods in terms of their biases and mean square errors using small, medium and large sample sizes. Finally, one data set related to posterior corneal curvature of the eyes of 23 patients, is analyzed to check potentiality of the newly proposed model.

Prevalence and management of aphids (Hemiptera: Aphididae) in different wheat genotypes and their impact on yield and related traits.

Wheat (Triticum aestivum L.) production is significantly altered by the infestation of sucking insects, particularly aphids. Chemical sprays are not recommended for the management of aphids as wheat grains are consumed soon after crop harvests. Therefore, determining the susceptibility of different wheat genotypes and selecting the most tolerant genotype could significantly lower aphid infestation. This study evaluated the susceptibility of six different wheat genotypes ('Sehar-2006', 'Shafaq-2006', 'Faisalabad-2008', 'Lasani-2008', 'Millat-2011' and 'Punjab-2011') to three aphid species (Rhopalosiphum padi Linnaeus, Schizaphis graminum Rondani, Sitobion avenae Fabricius) at various growth stages. Seed dressing with insecticides and plant extracts were also evaluated for their efficacy to reduce the incidence of these aphid species. Afterwards, an economic analysis was performed to compute cost-benefit ratio and assess the economic feasibility for the use of insecticides and plant extracts. Aphids' infestation was recorded from the seedling stage and their population gradually increased as growth progressed towards tillering, stem elongation, heading, dough and ripening stages. The most susceptible growth stage was heading with 21.89 aphids/tiller followed by stem elongation (14.89 aphids/tiller) and dough stage (13.56 aphids/tiller). The genotype 'Punjab-2011' recorded the lower aphid infestation than 'Faisalabad-2008', 'Sehar-2006', 'Lasani-2008' and 'Shafaq-2006'. Rhopalosiphum padi appeared during mid-February, whereas S. graminum and S. avenae appeared during first week of March. Significant differences were recorded for losses in number of grains/spike and 1000-grain weight among tested wheat genotypes. The aphid population had non-significant correlation with yield-related traits. Hicap proved the most effective for the management of aphid species followed by Hombre and Husk among tested seed dressers, while Citrullus colocynthis L. and Moringa oleifera Lam. plant extracts exhibited the highest efficacy among different plant extracts used in the study. Economic analysis depicted that use of Hombre and Hicap resulted in the highest income and benefit cost ratio. Therefore, use of genotype Punjab-2011' and seed dressing with Hombre and Hicap can be successfully used to lower aphid infestation and get higher economic returns for wheat crop.

Laboratory induced selection of pyriproxyfen resistance in Oxycarenus hyalinipennis Costa (Hemiptera: Lygaeidae): Cross-resistance potential, realized heritability, and fitness costs determination using age-stage, two-sex life table.

Pyriproxyfen is a biorational insecticide from IGR family, used worldwide against several economic pests. To evaluate the risk of pyriproxyfen resistance in dusky cotton bug, Oxycarenus hyalinipennis Costa (Hemiptera: Lygaeidae), a major concern for cotton producers, and to formulate strategies effective to tackle resistance, a field collected population was selected with pyriproxyfen under laboratory conditions using seed-dip method. A resistant strain designated as Pyr-SEL (G18) was developed after repeatedly selecting O. hyalinipennis with pyriproxyfen over eighteen generations. Thereafter, fitness costs, realized heritability (h2) and cross-resistance were investigated. As a result of selection, Pyr-SEL (G18) developed a very high level of resistance (resistance ratio = 464.23-fold) compared with the susceptible strain unselected over twenty generations Un-SEL (G20). The Pyr-SEL (G18) conferred strong cross-resistance to bifenthrin (146.59-fold), lambda-cyhalothrin (132.96-fold) and fenoxycarb (91.06-fold), whereas showed moderate cross-resistance to diafenthiuron (28.86-fold) and fipronil (22.73-fold). The h2 estimate was 0.16 in Pyr-SEL (G18). The developmental duration of O. hyalinipennis pre-adult prolonged, but traits of λ, r and R0 reduced in Pyr-SEL (G18) compared with the Un-SEL (G20). Also, the population projection obtained lower population size for Pyr-SEL (G18) than Un-SEL (G20). Fitness studies revealed that high resistance development to pyriproxyfen lowered the relative fitness of Pyr-SEL (G18) (Rf = 0.38) compared with the Un-SEL (G20). These findings may be practically valuable in tackling O. hyalinipennis resistance for better pest management.

Correction to: Sublethal effects of a juvenile hormone analog, Pyriproxyfen demographic parameters of non-target predator, Hippodamia convergens Guerin-Meneville (Coleoptera: Coccinellidae).

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The emergence of an unassigned complex recombinant form in a Pakistani HIV-infected individual.

In Pakistan, the HIV situation has gone from an outbreak to a concentrated epidemic, and the virus has now crossed into the low-risk population. In addition, several new HIV outbreaks have occurred in different parts of the country. HIV-1 subtype A has been the major epidemic subtype in Pakistan; however, as the epidemic has grown, the emergence of several new subtypes and recombinant forms has been observed. Here, we present the first case and genetic analysis of an unassigned, complex recombinant form in a Pakistani HIV-infected individual with virological failure. Genetic analysis of the sequence indicated that this recombinant form is multi-drug resistant, harboring drug resistance mutations against more than one class of antiretroviral drugs.