A Rare Case of Internal Jugular Venous Malformation Treated by Surgical Excision.

This paper reports a case of an internal jugular venous malformation (IJVM) and route of treatment in a patient with limited symptoms. After history and imaging studies, a determination of surgical excision was made to rule out possible malignancy and future problems such as thrombosis. The mass was resected, and part of the IJVM was ligated. The mass had no identifiable malignancy, and the patient recovered fully with no complications. The paper highlights the importance of identifying venous malformations and highlights the reasoning behind the course of action.

Lung adenocarcinoma in a patient with Lynch syndrome: a case report and literature review.

This article presents a case of a 62-year-old Vietnamese woman with a history of Lynch syndrome (LS), who developed lung adenocarcinoma with EGFR L858R mutation. LS is an autosomal dominant cancer predisposition syndrome caused by a pathogenic germline variant in DNA mismatch repair genes, often leading to microsatellite instability. While LS is primarily associated with gastrointestinal, endometrial, ovarian, and urologic tract cancers, lung cancer accounts for less than 1% of LS-related cancers, with only six cases of LS-related lung cancer previously reported in the literature. The patient underwent multiple lines of treatment for her lung adenocarcinoma, including tyrosine kinase inhibitors, stereotactic body radiation therapy, pemetrexed and pembrolizumab, amivantamab, and fam-trastuzumab deruxtecan, but all resulted in only a partial response followed by a progressive disease. This case highlights the complex interplay of genetic cancer predisposition syndromes and the development of spontaneous driver mutations in the disease course and the subsequent management of tumors arising in these patients.

CD2-negative lymphoma-associated T-cells: a potential mechanism of immune-evasion in diffuse large B-cell lymphoma.

CD2 is a costimulatory protein expressed in all mature T/NK-cells, in particular memory T-cells. CD58 (or LFA-3) is the receptor for CD2 and is ubiquitously expressed. CD2-CD58 interaction has key functions in T-cell activation and organization of the immunological synapse between T- and antigen-presenting cells. Cancer cells have developed multiple mechanisms to evade immune surveillance. Loss of CD58 expression is one frequently reported in diffuse large B-cell lymphomas (DLBCL). On the other hand, in non-hematological neoplasms, tumor infiltrating lymphocytes (TILs) with reduced expression of CD2 have been associated with defective cytotoxicity and T-cell exhaustion. Here, we reported a case of DLBCL involving the jejunal mucosa associated with a rim of cytotoxic reactive T-cells with features of immune evasion (CD2- and TCR-) and T-cell exhaustion (PD1 + high). This case likely exemplifies a previously unrecognized immune evasion mechanism in lymphoma involving a decreased CD2 expression in the lymphoma-associated T-cells.

MYC and NOTCH1-positive postradiation cutaneous angiosarcoma of the breast.

Postradiation cutaneous angiosarcoma of the breast is a rare, delayed complication of adjuvant radiation treatment for breast carcinoma and is associated with a worse prognosis than the original primary cancer. Recent studies have characterized the diagnostic utility of MYC and NOTCH1 receptor expression as markers for secondary radiation-associated angiosarcomas. Herein, we report an exophytic secondary breast angiosarcoma with MYC and NOTCH1 immunoreactivity. This case illustrates the utility of these markers for the identification of radiation-associated angiosarcoma with MYC and NOTCH1 expression, potential for targeted therapy and need to identify patients for further studies of the clinicopathologic and prognostic significance.

Pathology Trainee Redeployment and Education During the COVID-19 Pandemic: An Institutional Experience.

Pathology training programs throughout the United States have endured unprecedented challenges dealing with the ongoing coronavirus disease 2019 pandemic. At Houston Methodist Hospital, the Department of Pathology and Genomic Medicine planned and executed a trainee-oriented, stepwise emergency response. The focus was on optimizing workflows among areas of both clinical and anatomic pathology, maintaining an excellent educational experience, and minimizing trainee exposure to coronavirus disease 2019. During the first phase of the response, trainees were divided into 2 groups: one working on-site and the other working remotely. With the progression of the pandemic, all trainees were called back on-site and further redeployed within our department to meet the significantly increased workload demands of our clinical laboratory services. Adjustments to trainee educational activities included, among others, the organization of a daily coronavirus disease 2019 virtual seminar series. This series served to facilitate communication between faculty, laboratory managers, and trainees. Moreover, it became a forum for trainees to provide updates on individual service workflows and volumes, ongoing projects and research, as well as literature reviews on coronavirus disease 2019-related topics. From our program's experience, redeploying pathology trainees within our department during the coronavirus disease 2019 pandemic resulted in optimization of patient care while ensuring trainee safety, and importantly, helped to maintain continuous high-quality education through active involvement in unique learning opportunities.

Mycobacterium tuberculosis diagnosed promptly by bronchial brushing cytology in an immunocompetent patient.

Respiratory cytology plays an important role in the diagnosis of lower respiratory tract infection. The timely diagnosis of pulmonary tuberculosis (TB) can be very challenging due to the nonspecific cytomorphologic features and limited number of organisms, especially in the immunocompetent patients. Here, we reported a case of TB diagnosed promptly by bronchial brushing cytology in a 51-year-old immunocompetent patient. She presented with a 4 cm fungating lesion involving right lower lobe of the lung and mediastinal lymphadenopathy with an initial concern for malignancy. Bronchial brushing showed scattered acute inflammatory cells in the background of necrosis. A cell block was prepared and acid-fast bacilli (AFB)-positive organisms were identified. Subsequent polymerase chain reaction (PCR) performed on the sputum detected Mycobacterium tuberculosis. This case highlights the importance of recognizing the cytomorphology of TB from a bronchial brushing specimen; and also emphasizes the potential utility of the cell block from respiratory cytology in the diagnosis of TB.

Acute myeloid leukaemia presenting as acute liver failure-a case report and literature review.

A 75-year-old woman presented with rapidly progressive fatigue, abdominal pain and jaundice. Physical examination revealed tender abdomen and splenomegaly. Magnetic resonance cholangiogram showed marked hepatomegaly, splenomegaly and scattered nodules or masses in the liver and spleen. The patient expired from multiorgan failure. Autopsy revealed infiltration of the liver, spleen and bone marrow by acute myeloid leukaemia.

Successful treatment of a stage IIIC small-cell carcinoma of the ovary hypercalcemic subtype using multi-modality therapeutic approach.

Small-cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare but highly undifferentiated, aggressive malignancy that primarily affects young women. Due to its early onset, unclear familial history and vague presenting symptoms, most SCCOHT patients present late with advanced disease. The prognosis is extremely poor, with <10% disease-free survival for advanced stages. Although several therapeutic regimens have been proposed, to date there is no consensus on the optimal strategy. Here, we describe a successful case of advanced-stage SCCOHT of the left ovary treated with cytoreductive surgery, semi-intense chemotherapy, high-dose consolidative chemotherapy, autologous hematopoietic stem cell transplantation and pelvic radiation with long-term survival. Given the almost universal mortality of advanced SCCOHT in long-term follow-up, we believe this case highlights the importance of prompt diagnosis when a young patient presents with abdominal swelling and hypercalcemia as well as early, aggressive, combined modality treatment. This case is also especially remarkable given the patient underwent fertility preservation surgery, which is not recommended by most of the current literature. However, as therapies improve and more young patients may survive SCCOHT, the question of fertility will increase in relevance. We believe the pros and cons of conservation should be discussed in detail with the patient.

Bioluminescence-based neuraminidase inhibition assay for monitoring influenza virus drug susceptibility in clinical specimens.

The QFlu prototype bioluminescence-based neuraminidase (NA) inhibition (NI) assay kit was designed to detect NA inhibitor (NAI)-resistant influenza viruses at point of care. Here, we evaluated its suitability for drug susceptibility assessment at a surveillance laboratory. A comprehensive panel of reference viruses (n = 14) and a set of 90 seasonal influenza virus A and B isolates were included for testing with oseltamivir and/or zanamivir in the QFlu assay using the manufacturer-recommended protocol and a modified version attuned to surveillance requirements. The 50% inhibitory concentrations (IC50s) generated were compared with those of NI assays currently used for monitoring influenza drug susceptibility, the fluorescent (FL) and chemiluminescent (CL) assays. To provide proof of principle, clinical specimens (n = 235) confirmed by real-time reverse transcription (RT)-PCR to contain influenza virus A(H1N1)pdm09 and prescreened for the oseltamivir resistance marker H275Y using pyrosequencing were subsequently tested in the QFlu assay. All three NI assays were able to discriminate the reference NA variants and their matching wild-type viruses based on the difference in their IC50s. Unless the antigenic types were first identified, certain NA variants (e.g., H3N2 with E119V) could be detected among seasonal viruses using the FL assays only. Notably, the QFlu assay identified oseltamivir-resistant A(H1N1)pdm09 viruses carrying the H275Y marker directly in clinical specimens, which is not feasible with the other two phenotypic assays, which required prior virus culturing in cells. Furthermore, The QFlu assay allows detection of the influenza virus A and B isolates carrying established and potential NA inhibitor resistance markers and may become a useful tool for monitoring drug resistance in clinical specimens.

Assays for monitoring susceptibility of influenza viruses to neuraminidase inhibitors.

Close monitoring of drug susceptibility among human influenza viruses was necessitated by widespread resistance to M2 inhibitors in influenza H1N1 (pre-pandemic and 2009 pandemic) and H3N2 viruses, and of oseltamivir resistance in pre-pandemic H1N1 viruses. The FDA-approved neuraminidase (NA) inhibitors (NAIs), oseltamivir and zanamivir, as well as investigational NAIs, peramivir and laninamivir, are currently the principal treatment options for managing influenza infection. However, there are challenges associated with assessing virus susceptibility to this class of drugs. Traditional cell culture-based assays are not reliable for phenotypic testing of NAI susceptibility due to complexity in interpretation. Two types of laboratory assays are currently available for monitoring NAI susceptibility, phenotypic such as the neuraminidase inhibition (NI) assay and genotypic. The NI assay's requirement for propagated virus lengthens testing turnaround; therefore, the need for timely detection of molecular markers associated with NAI resistance (e.g., H275Y in H1N1) has spurred the development of rapid, high-throughput assays, such as real-time RT-PCR and pyrosequencing. The high sensitivity of genotypic assays allows testing of clinical specimens thus eliminating the need for virus propagation in cell culture. The NI assays are especially valuable when a novel virus emerges or a new NAI becomes available. Modifications continue to be introduced into NI assays, including optimization and data analysis criteria. The optimal assay of choice for monitoring influenza drug susceptibility varies widely depending on the needs of laboratories (e.g., surveillance purposes, clinical settings). Optimally, it is desirable to combine functional and genetic analyses of virus isolates and, when possible, the respective clinical specimens.

A cluster of patients infected with I221V influenza b virus variants with reduced oseltamivir susceptibility--North Carolina and South Carolina, 2010-2011.

BACKGROUND: During 2010-2011, influenza B viruses with a novel neuraminidase substitution, denoted I221V (B/I221V), associated with reduced in vitro oseltamivir susceptibility were detected in North Carolina. METHODS: We determined the prevalence of I221V among B viruses submitted to the Centers for Disease Control and Prevention for antiviral resistance surveillance, including all B viruses submitted to North Carolina and South Carolina state laboratories, during October 2010-September 2011.We conducted chart reviews and telephone interviews to characterize North Carolina and South Carolina patients with B/I221V vs wild-type B virus infection (B/WT). RESULTS: We detected I221V in 45 (22%) of 209 B viruses from North Carolina and 8 (10%) of 82 B viruses from South Carolina. We detected I221V in 3 (0.3%) of 881 B viruses tested from 45 other states. B/I221V infection was not associated with differences in underlying conditions or illness severity, compared with B/WT infection. No patients with B/I221V infection received oseltamivir prior to specimen collection. Among patients who completed oseltamivir, those with B/I221V infection reported a longer duration until illness resolution (5 vs 3 days; P = .02). CONCLUSIONS: B/I221V cocirculated with B/WT in North Carolina and South Carolina during 2010-2011. I221V did not alter illness severity but may have reduced oseltamivir effectiveness. Thus, global surveillance for I221V is important.

Analysis of influenza viruses from patients clinically suspected of infection with an oseltamivir resistant virus during the 2009 pandemic in the United States.

During the 2009 influenza pandemic, the Centers for Disease Control and Prevention provided antiviral susceptibility testing for patients infected with suspected drug-resistant viruses. Specimens from 72 patients admitted to an intensive care unit or with a severe immunocompromising condition, who failed to clinically improve after oseltamivir treatment, were accepted for testing. Respiratory specimens were tested for the presence of the oseltamivir resistance-conferring H275Y substitution in the neuraminidase (NA) by pyrosequencing. Virus isolates propagated in MDCK cells were tested in phenotypic NA inhibition (NI) assays using licensed NA inhibitors (NAIs), zanamivir and oseltamivir, and investigational NAIs, peramivir and laninamivir. Conventional sequencing and plaque purification were conducted on a subset of viruses. Pyrosequencing data were obtained for 87 specimens collected from 58 of the 72 (81%) patients. Of all patients, 27 (38%) had at least one specimen in which H275Y was detected. Analysis of sequential samples from nine patients revealed intra-treatment emergence of H275Y variant and a shift from wildtype-to-H275Y in quasispecies during oseltamivir therapy. A shift in the H275Y proportion was observed as a result of virus propagation in MDCK cells. Overall, the NI method was less sensitive than pyrosequencing in detecting the presence of H275Y variants in virus isolates. Using the NI method, isolates containing H275Y variant at⩾50% exhibited resistance to oseltamivir and peramivir, but retained full susceptibility to zanamivir. H275Y viruses recovered from two patients had an additional substitution I223K or I223R that conferred a 38-52- and 33-97-fold enhancement in oseltamivir- and peramivir-resistance, respectively. These viruses also showed decreased susceptibility to zanamivir and laninamivir. These data suggest that pyrosequencing is a powerful tool for timely detection of NAI resistant viruses and that NI assays are needed for comprehensive testing to detect novel resistance substitutions.

Influenza B viruses with mutation in the neuraminidase active site, North Carolina, USA, 2010-11.

Oseltamivir is 1 of 2 antiviral medications available for the treatment of influenza B virus infections. We describe and characterize a cluster of influenza B viruses circulating in North Carolina with a mutation in the neuraminidase active site that may reduce susceptibility to oseltamivir and the investigational drug peramivir but not to zanamivir.

Detection of hemagglutinin variants of the pandemic influenza A (H1N1) 2009 virus by pyrosequencing.

For influenza viruses, pyrosequencing has been successfully applied to the high-throughput detection of resistance markers in genes encoding the drug-targeted M2 protein and neuraminidase. In this study, we expanded the utility of this assay to the detection of multiple receptor binding variants of the hemagglutinin protein of influenza viruses directly in clinical specimens. Specifically, a customized pyrosequencing protocol that permits detection of virus variants with the D, G, N, or E amino acid at position 222 in the hemagglutinin of the 2009 pandemic influenza A (H1N1) virus was developed. This customized pyrosequencing protocol was applied to the analysis of 241 clinical specimens. The use of the optimized nucleotide dispensation order allowed detection of mixtures of variants in 10 samples (4.1%) which the standard cyclic nucleotide dispensation protocol failed to detect. The optimized pyrosequencing protocol is expected to provide a more accurate tool in the analysis of virus variant composition.

Dual resistance to adamantanes and oseltamivir among seasonal influenza A(H1N1) viruses: 2008-2010.

Two distinct genetic clades of seasonal influenza A(H1N1) viruses have cocirculated in the recent seasons: clade 2B oseltamivir-resistant and adamantane-susceptible viruses, and clade 2C viruses that are resistant to adamantanes and susceptible to oseltamivir. We tested seasonal influenza A(H1N1) viruses collected in 2008-2010 from the United States and globally for resistance to antivirals approved by the Food and Drug Administration. We report 28 viruses with both adamantane and oseltamivir (dual) resistance from 5 countries belonging to 4 distinct genotypes. Because of limited options for antiviral treatment, emergence of dual-resistant influenza viruses poses a public health concern, and their circulation needs to be closely monitored.

Comprehensive assessment of 2009 pandemic influenza A (H1N1) virus drug susceptibility in vitro.

BACKGROUND: Antiviral drugs are an important option for managing infections caused by influenza viruses. This study assessed the drug susceptibility of 2009 pandemic influenza A (H1N1) viruses collected globally between April 2009 and January 2010. METHODS: Virus isolates were tested for adamantane susceptibility, using pyrosequencing to detect the S31N marker of adamantane resistance in the M2 protein and biological assays to assess viral replication in cell culture. To assess neuraminidase (NA) inhibitor (NAI) susceptibility, virus isolates were tested in chemiluminescent NA inhibition assays and by pyrosequencing to detect the H275Y (H274Y in N2 numbering) marker of oseltamivir resistance in the NA. RESULTS: With the exception of three, all viruses that were tested for adamantane susceptibility (n=3,362) were resistant to this class of drugs. All viruses tested for NAI susceptibility (n=3,359) were sensitive to two US Food and Drug Administration-approved NAIs, oseltamivir (mean ±sd 50% inhibitory concentration [IC(50)] 0.25 ±0.12 nM) and zanamivir (mean IC(50) 0.29 ±0.09 nM), except 23 (0.7%), which were resistant to oseltamivir, but sensitive to zanamivir. Oseltamivir-resistant viruses had the H275Y mutation in their NA and were detected in patients exposed to the drug through prophylaxis or treatment. NA activity of all viruses was inhibited by the NAIs peramivir, laninamivir (R-125489) and A-315675, except for H275Y variants, which exhibited approximately 100-fold reduction in peramivir susceptibility. CONCLUSIONS: This report provides data regarding antiviral susceptibility of 2009 pandemic influenza A (H1N1) surveillance viruses, the majority of which were resistant to adamantanes and sensitive to NAIs. These findings provide information essential for antiviral resistance monitoring and development of novel diagnostic tests for detecting influenza antiviral resistance.

Assessment of pandemic and seasonal influenza A (H1N1) virus susceptibility to neuraminidase inhibitors in three enzyme activity inhibition assays.

The neuraminidase inhibitors (NAIs) zanamivir and oseltamivir are currently the only antiviral drugs effective for the treatment and prophylaxis of 2009 pandemic influenza A (H1N1) virus infections. The proven potential of these viruses to acquire NAI resistance during treatment emphasizes the need to assess their NAI susceptibility. The 50% inhibitory concentrations (IC(50)s) are known to vary depending on the neuraminidase inhibition (NI) test used; however, few side-by-side comparisons of different NI assays have been done. In the present study, a panel of 11 isolates representing 2009 seasonal and pandemic influenza H1N1 viruses, including oseltamivir-resistant H275Y variants, were tested in three functional NI assays: chemiluminescent (CL), fluorescent (FL), and colorimetric (CM). The sensitivities of the viruses to zanamivir, oseltamivir, and three investigational NAIs (peramivir, R-125489, and A-315675) were assessed. All isolates with the exception of H275Y variants were sensitive to all five NAIs by all three NI assays. The H275Y variants showed substantially elevated IC(50)s against oseltamivir and peramivir. The three NI assays generally yielded consistent results; thus, the choice of NI assay does not appear to affect conclusions based on drug susceptibility surveillance. Each assay, however, offers certain advantages compared to the others: the CL assay required less virus volume and the FL assay provided the greatest difference in the IC(50)s between the wild type and the variants, whereas the IC(50)s obtained from the CM assay may be the most predictive of the drug concentrations needed to inhibit enzyme activity in humans. It would be desirable to develop an NI assay which combines the advantages of all three currently available assays but which lacks their shortcomings.

Detection of E119V and E119I mutations in influenza A (H3N2) viruses isolated from an immunocompromised patient: challenges in diagnosis of oseltamivir resistance.

The clinical use of the neuraminidase inhibitor (NAI) oseltamivir is associated with the emergence of drug resistance resulting from subtype-specific neuraminidase (NA) mutations. The influenza A/Texas/12/2007 (H3N2) virus isolated from an oseltamivir-treated immunocompromised patient exhibited reduced susceptibility to oseltamivir in the chemiluminescent neuraminidase inhibition (NI) assay (approximately 60-fold increase in its 50% inhibitory concentration [IC(50)] compared to that for a control virus). When further propagated in cell culture, the isolate maintained reduced susceptibility to oseltamivir in both chemiluminescent and fluorescent NI assays (approximately 50- and 350-fold increases in IC(50), respectively). Sequencing analysis of the isolate revealed a mix of nucleotides coding for amino acids at position 119 of the NA [E119(V/I)]. Plaque purification of the isolate yielded E119V and E119I variants, both exhibiting reduced susceptibility to oseltamivir. The E119I variant also showed decreased susceptibility to zanamivir and the investigational NAIs peramivir and A-315675. The emergence of E119V variants in oseltamivir-treated patients has been previously reported; however, the E119I mutation detected here is a novel one which reduces susceptibility to several NAIs. Both mutations were not detected in unpropagated original clinical specimens using either conventional sequencing or pyrosequencing, suggesting that these variants were present in very low proportions (<10%) in clinical specimens and gained dominance after virus propagation in MDCK cells. All virus isolates recovered from the patient were resistant to adamantanes. Our findings highlight the potential for emergence and persistence of multidrug-resistant influenza viruses in oseltamivir-treated immunocompromised subjects and also highlight challenges for drug resistance diagnosis due to the genetic instability of the virus population upon propagation in cell culture.

Neuraminidase inhibitor susceptibility testing in human influenza viruses: a laboratory surveillance perspective.

Neuraminidase inhibitors (NAIs) are vital in managing seasonal and pandemic influenza infections. NAI susceptibilities of virus isolates (n = 5540) collected during the 2008-2009 influenza season were assessed in the chemiluminescent neuraminidase inhibition (NI) assay. Box-and-whisker plot analyses of log-transformed IC(50)s were performed for each virus type/subtype and NAI to identify outliers which were characterized based on a statistical cutoff of IC(50) >3 interquartile ranges (IQR) from the 75(th) percentile. Among 1533 seasonal H1N1 viruses tested, 1431 (93.3%) were outliers for oseltamivir; they all harbored the H275Y mutation in the neuraminidase (NA) and were reported as oseltamivir-resistant. Only 15 (0.7%) of pandemic 2009 H1N1 viruses tested (n = 2259) were resistant to oseltamivir. All influenza A(H3N2) (n = 834) and B (n = 914) viruses were sensitive to oseltamivir, except for one A(H3N2) and one B virus, with D151V and D197E (D198E in N2 numbering) mutations in the NA, respectively. All viruses tested were sensitive to zanamivir, except for six seasonal A(H1N1) and several A(H3N2) outliers (n = 22) which exhibited cell culture induced mutations at residue D151 of the NA. A subset of viruses (n = 1058) tested for peramivir were sensitive to the drug, with exception of H275Y variants that exhibited reduced susceptibility to this NAI. This study summarizes baseline susceptibility patterns of seasonal and pandemic influenza viruses, and seeks to contribute towards criteria for defining NAI resistance.

Host cell selection of influenza neuraminidase variants: implications for drug resistance monitoring in A(H1N1) viruses.

The neuraminidase inhibitors (NAIs), oseltamivir and zanamivir, are essential for treatment and prevention of influenza A and B infections. Oseltamivir resistance among influenza A (H1N1) viruses rapidly emerged and spread globally during the 2007-2008 and 2008-2009 influenza seasons. Approximately 20% and 90% of viruses tested for NAI susceptibility at CDC during these seasons, respectively, were resistant to oseltamivir (IC(50) approximately 100-3000 time>those of sensitive viruses), based on the chemiluminescent NA inhibition assay. Pyrosequencing analysis confirmed H274Y mutation (H275Y in N1 numbering) in the neuraminidase (NA) gene of oseltamivir-resistant viruses. Full NA sequence analysis of a subset of oseltamivir-resistant and sensitive virus isolates from both seasons (n=725) showed that 53 (7.3%) had mutations at residue D151 (D-->E/G/N), while 9 (1.2%) had mutations at Q136 (Q-->K) and 2 (0.3%) had mutations at both residues. Viruses with very high IC(50) for oseltamivir and peramivir, and elevated IC(50) for zanamivir, had H274Y in addition to mutations at D151 and/or Q136, residues which can potentially confer NAI resistance based on recent N1 NA crystal structure data. Mutations at D151 without H274Y, did not elevate IC(50) for any tested NAI, however, Q136K alone significantly reduced susceptibility to zanamivir (36-fold), peramivir (80-fold) and A-315675 (114-fold) but not oseltamivir. Mutations at D151 and Q136 were present only in MDCK grown viruses but not in matching original clinical specimens (n=33) which were available for testing, suggesting that these variants were the result of cell culture selection or they were present in very low proportions. Our findings provide evidence that propagation of influenza virus outside its natural host may lead to selection of virus variants with mutations in the NA that affect sensitivity to NAIs and thus poses implications for drug resistance monitoring and diagnostics.